InteractiveFly: GeneBrief

Heat shock factor: Biological Overview | Regulation | Effect of mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Heat shock proteins (HSPs) serve multiple functions in a variety of organisms as evolutionarily distant as bacteria and humans. In the fly, there are many genes coding for multiple families of HSPs that function in regulatory pathways, both developmental and the response to heat stress. The developmental regulation is consonant with a role of HSPs as molecular chaperones. They are involved in protein folding: they aid in the establishment of native three dimensional structure for a variety of proteins that require cellular functions in order to attain such structure. HSPs function as partners for various signal transducing proteins, whose signaling dynamics are regulated by association with HSPs (Morimoto, 1994).

Organisms respond to elevated temperatures by rapidly inducing HSPs. The regulation of this response is mediated at the transcriptional level by a transcription factor called heat shock factor (HSF), which binds to the regulatory shock elements that are present upstream of all heat shock genes. Thus, HSF responds to the heat shock signal by binding to DNA and regulation of target gene transcriptional competence.

Stress induced binding of HSF to DNA is controlled by the transition of HSF from a monomer to a trimer. Trimerization increases the affinity for the heat shock elements of HSPs by several orders of magnitude, through the assembly of one trimeric protein complex containing three DNA-binding domains, one contributed from each HSF monomer (Wu, 1995).

How does the organism at the cellular level recognize heat shock duress and induce activation of heat shock factor? One idea is that a physical interaction between HSF and HSPs suppresses induction of the DNA-binding of HSF. HSPs may assist in constraining HSF in a latent, monomeric state. When the organism is subjected to heat stress, multiple protein species denature, increasing the demand on HSPs to refold proteins, thereby releasing HSF from cytoplasmic constraint, and allowing its transportation to the nucleus where it undergoes trimerization, and subsequent binding to the promoters of HSPs.

Although HSF associates with HSPs in vitro, and the interaction of HSF and hsp70 (Heat shock protein with a molecular weight of 70 kDa) has been documented, interaction of hsp70 with HSF does not correlate with the level of activation of HSF. Both latent and active forms of HSF interact with hsp70. Elevated concentrations of hsp70 alone do not significantly inhibit induction of the DNA-binding activity of endogenous HSF, and elevated concentrations of hsp70 do not alter the induction of HSPs (Rabindran, 1994).

Some clarification of the pathway followed during HSF activation comes from the study of the human HSF1 protein. A region of HSF1, located between the leucine repeat trimerization domain and the C-terminal transactivation domain functions as a regulatory domain. By itself, the regulatory domain can confer heat shock inducibility to chimeric proteins containing the activation domain, but it fails to function as a DNA binding domain. This function is served by the N-terminal sequence (Green, 1995).

Subsequent studies have shown that the regulatory domain of HSF1 is sufficient to sense heat stress. The regulatory domain can even function with a heterologous transcriptional activation domain, suggesting that the regulatory domain can function irrespective of the sequence of the C-terminal transactivation domain (Newton, 1996).

Phosphorylation has been thought of as a mechanism for modulation of heat shock factor activity. Phosphorylation of transcription factors can occur through a number of different pathways in the cell. For example, the Raf/ERK (extracellular-signal-regulated kinase) pathway is activated by FGF-receptor and EGF-receptor in Drosophila (See Rolled). Another example is the MEKK/SAP (JNK) (stress activated protein kinase/Jun N-terminal kinase) pathway, found in both mammals and Drosophila. Hemipterous and Basket/JNK are components of this pathway in Drosophila. In humans, phosphorylation serves to repress HSF 1 at control temperatures. Two specific proline-directed serine motifs in the regulatory domain are important for phosphorylation to function normally. Stimulation of the Raf/ERK pathway results in an increased level of phosphorylation at these motifs. The regulatory domain is an excellent substrate for the mitogen-activated MAPK/ERK. It has been concluded that phosphorylation of the regulatory domain of HSF1 decreases the activity of HSF1, and it is thought that this modification serves to control the response of the cell to growth control signals (Knauf, 1996).

Although such a mechanism may not function in Drosophila, its existence in humans points to the complex forces acting on HSF and its function. Studies of transcriptional activation by HSF in Drosophila have yielded insight into the process of chromatin remodeling required for gene activation (Tsukiyama, 1995a and b). This work is discussed in greater detail at the ISWI site.

Negative elongation factor accelerates the rate at which heat shock genes are shut off by facilitating dissociation of heat shock factor

Promoter-proximal pausing of RNA polymerase II (Pol II) occurs on thousands of genes in animal cells. This pausing often correlates with the rapid induction of genes, but direct tests of the relationship between pausing and induction rates are lacking. hsp70 and hsp26 in Drosophila are rapidly induced by heat shock. Contrary to current expectations, depletion of negative elongation factor (NELF), a key factor in setting up paused Pol II, reduced pausing but did not interfere with rapid induction. Instead, depletion of NELF delayed the time taken for these genes to shut off during recovery from heat shock. NELF depletion also delayed the dissociation of HSF from hsp70 and hsp26, and a similar delay was observed when cells were depleted of the histone acetyltransferase CBP. CBP has been reported to associate with Pol II, and acetylation of HSF by CBP has been implicated in inhibiting the DNA-binding activity of HSF. It is proposed that NELF-mediated pausing allows Pol II to direct CBP-mediated acetylation of HSF, thus causing HSF to dissociate from the gene. Activators are typically viewed as controlling Pol II. These results reveal a possible reciprocal relationship in which paused Pol II influences the activator (Ghosh, 2011).

Reduction of promoter proximal pausing on hsp70 does not alter the rate of heat shock induction. Correlations between the presence of paused Pol II and rapid induction of gene expression have led to the hypothesis that promoter proximal pausing provides the basis for rapid induction. However, none of these studies have actually tested if disruption of promoter proximal pausing delays expression of genes. The current analysis reveals that reduction of promoter proximal pausing at hsp70 does not detectably alter the rate of heat shock induction. This was evident by monitoring the association of Pol II at both the 5’ and 3’ ends of hsp70 using permanganate genomic footprinting and also by measuring synthesis of hsp70 RNA. The two types of assays are in good agreement with each other in that a substantial increase in the level of transcript and in the presence of Pol II at the 3’ end of the gene occurs between 2 and 4 minutes after heat shock. Also, the elongation rate of approximately 1.25 kb/min for transcription of hsp70 that can be deduced from permanganate data is in excellent agreement with other types of measurements, thus validating the use of permanganate footprinting as a means for analyzing the kinetics of transcription induction and elongation. It has been proposed that the establishment of paused Pol II at a promoter allows for rapid induction because steps that precede initiation such as chromatin remodeling and Pol II recruitment are bypassed. In the case of the heat shock genes, these steps are still likely to occur prior to heat shock induction even when promoter proximal pausing is impaired. TFIID, which provides the foundation for assembling a preinitiation complex, makes multiple sequence-specific contacts with hsp70 including the TATA box, the initiator, and downstream sequences. In addition, another DNA binding protein called GAGA factor maintains the promoter region in an accessible state even when TFIID binding is impaired. Since HSF associates with a transgenic hsp70 promoter deleted of its TATA box, the accessible state of the promoter in lieu of TFIID appears to be sufficient for HSF binding. Thus access of DNA by the general transcriptional machinery and HSF may not be limiting for hsp70 even when promoter proximal pausing is disrupted. The extent to which promoters retain an accessible state upon loss of paused Pol II is likely to vary. The Drosophila heat shock genes could represent one end of a spectrum where accessibility is retained without sustaining full occupancy of the promoter with paused Pol II because of the binding of GAGA factor and TFIID. At the other end of the spectrum may be genes that require the paused Pol II to maintain a potentially inducible state. Cases have been identified where the paused Pol II appears to contribute to induction by preventing a nucleosome from assembling over the promoter (Ghosh, 2011).

Depletion of NELF impacts the ability of hsp70 to shut-off during recovery from heat shock. Permanganate genomic footprinting provides a high-resolution view of the behavior of Pol II. Permanganate reactivity on hsp70 at positions +7 and +8 are indicative of newly initiated Pol II whereas reactivity downstream from +34 corresponds to Pol II that has read through the pause. Similarly for hsp26, permanganate reactivity at +9 and +10 are indicative of newly initiated Pol II whereas reactivity downstream from +45 corresponds to Pol II that has read through the pause. The patterns of permanganate reactivity that were observed indicate that both initiation and read-through persist for a longer time in NELF-depleted glands than in control glands during recovery from heat shock for both hsp70 and hsp26. Corroborating this conclusion is the demonstration that a brief heat shock causes these two genes to be more highly expressed in NELF-depleted glands than in control glands. Limiting the production of hsp70 protein to a level appropriate to the degree of stress could be critical for proper cell development, since ectopic expression of hsp70 in salivary glands under non-heat shock conditions inhibits growth of the cells in this tissue (Ghosh, 2011).

These results now expand the types of scenarios in which NELF and promoter proximal pausing serve to attenuate gene expression. In the case of the estrogen-mediated activation of the pS2 gene, direct interaction between the NELF-B subunit and the estrogen receptor causes estrogen-mediated association of NELF with the target gene. Depletion of NELF results in hyper-activation of the pS2 gene, indicating that the estrogen receptor is mediating both activation and repression to achieve a particular level of expression in the presence of stimuli. In the case of junB, depletion of NELF increased the expression of junB both before and after serum-mediated activation. The current results now reveal a role for NELF in shutting off gene expression upon withdrawal of the stimulus - this case being heat shock (Ghosh, 2011).

To investigate why the shut off of the heat shock genes was delayed by depletion of NELF, the association of HSF with hsp70 and hsp26 in salivary glands was measured using chromatin immunoprecipitation. Significantly more HSF was detected on the heat shock genes after 45 minutes of recovery in NELF-depleted than control glands indicating that the delay in shutting off the heat shock genes could be due to a delay in the dissociation of HSF and that NELF is somehow involved in this dissociation. It is unlikely that the dissociation of HSF is due to stress induced by depletion of NELF, since there is no evidence of this depletion inducing a stress response in salivary glands. Prior to heat shock, both control and NELF-depleted glands exhibit low levels of heat shock gene expression, low levels of HSF at heat shock gene promoters, and undetectable levels of HSF DNA-binding activity in amount of HSF detected in glands nor did it alter a heat shock-dependent shift in the mobility of HSF on SDS-PAGE. Approximately 2-fold higher HSF binding activity was detected in extracts from NELF-depleted glands than control glands after recovery from heat shock, but the standard deviations in the measurements indicate that this difference is not significant. Since the results showed that depletion of NELF resulted in a delay in the dissociation of HSF from heat shock genes during the recovery from heat shock, recent indirect evidence in human cells that acetylation of HSF by CBP regulates HSF binding activity was intriguing. The finding that depletion of CBP delays the dissociation of HSF from the heat shock genes provides the first direct evidence that CBP is involved in regulating HSF’s interaction with heat shock genes in vivo. Since depletion of CBP and NELF each delays the dissociation of HSF from the heat shock genes during recovery, it is proposed that their actions are linked. It is posited that NELF-mediated pausing of Pol II could position CBP to acetylate HSF, thus causing HSF to dissociate from the heat shock gene. Several observations provide support for this model. CBP associates with Pol IIa and not Pol IIo and the Pol II paused in the promoter proximal region is in the IIa state. Also, CBP associates with the heat shock genes in Drosophila during heat shock induction. Further investigation of this proposal will be aided by biochemical analyses of interactions between CBP and reconstituted paused elongation complexes. Multiple mechanisms can attenuate the activity of HSF during heat shock, so the possibility cannot be ruled out that the actions of NELF and CBP are independent of each other or that these proteins are acting indirectly on HSF by influencing other regulators of HSF. The heat shock proteins such as hsp70 produced during heat shock repress the activation domain of HSF thus providing a negative feedback loop that limits heat shock gene transcription. These chaperons have also been implicated in attenuating the DNA binding activity of HSF in Drosophila during extended periods of a moderate heat shock. Changes in phosphorylation of HSF have also been implicated in regulating its activity, but such changes do not appear to effect the DNA binding activity of HSF in Drosophila. The mechanisms by which heat shock genes are returned to their uninduced state during recovery from heat shock remain to be fully elucidated. The use of permanganate footprinting to directly monitor the behavior of Pol II at heat shock genes during recovery from heat shock in cells depleted of specific proteins provides a way to identify candidates involved in shutting off transcription of the heat shock genes. A significant advantage of this approach is that it allows one to directly monitor the reestablishment of the paused state (Ghosh, 2011).

Paused Pol II could function as a regulator of gene regulatory factors. Many stimulus-responsive and developmental genes are found to have paused Pol II, and attenuation of transcription of these genes is likely to be critical for establishing appropriate physiological or developmental programs of gene expression. The finding that paused Pol II influences dissociation of an activator from the promoter provides a mechanism for rapidly shutting off gene expression. The pausing of Pol II could function more broadly by allowing the Pol II to remain stationary at a promoter while it serves to recruit modulators of chromatin structure and gene expression (Ghosh, 2011).

REGULATION

cis-Regulatory Sequences and Functions

Functional analysis of Drosophila HSP70 promoter with different HSE numbers in human cells

The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38 degrees C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems (Kust, 2014; PubMed: 25101947).

Targets of Activity

The developmental and heat-shock-induced expression of two small heat-shock proteins (Hsp23 and Hsp27) was investigated during spermatogenesis in Drosophila melanogaster. Both of these Hsps are expressed in unstressed and stressed male gonads as shown by immunoblotting. Immunostaining of whole-mount organs and thin sections of testes showed that an anti-Hsp23 antibody specifically decorated cells of the somatic lineage, such as the cyst cells and the epithelial cells of the testis and of the seminal vesicle. Hsp27 is expressed in some somatic cells (cyst cells and epithelial cells of the accessory glands) and, in addition, is also visible in the maturing spermatocytes of the germline. The same cell-specific pattern of expression is observed after heat shock, and cells which do not express Hsp23 and Hsp27 in the absence of stress are similarly unable to mount a heat shock response for these s-Hsps. However other Hsps such as Hsp70 and Hsp22 are induced under heat-shock conditions in testes. Actinomycin D prevents the heat-induced accumulation of these Hsps indicating that the induction of Hsps is regulated at the transcriptional level. The heat shock transcriptional factor of Drosophila (HSF), present in significantly lower amount in testes when compared to other tissues such as the head, is shown to be required for the heat activation of Hsp22 and Hsp70. Immunostaining revealed that HSF expression is restricted to specific cells such as cyst cells, epithelial pigment cells, spermatogonia and spermatids but not the primary spermatocytes. These data show that the expression and induction of the different small Hsps is regulated in a cell-specific manner under both normal and heat shock conditions and suggest that factors other than the HSF are involved in this regulation in male gonads (Michaud, 1997).

Heat shock genes are activated by the binding of the heat shock transcription factor (HSF) to heat shock elements (HSEs), consisting of arrays of the 5-bp unit NGAAN arranged as inverted repeats. The interaction of the 5-bp unit with HSFs of Drosophila and Saccharomyces has been investigated. Mutations within the conserved, central trinucleotide GAA reduces the relative binding affinity of both HSFs. In addition, the base at position 1 (N1) also influences binding, with a strong preference for an A at this position. Methylation interference initially indicates that HSF contacts A1 in the minor groove, but interacts with the immediately adjacent base G2 in the major groove. Further characterization of this apparently abrupt minor to major groove transition by substitution of A1 with an inosine, shows that HSF contacts A1 in the major groove. Thus HSF recognizes the HSE primarily through contacts within the major groove of the DNA helix. Based on these observations and a re-evaluation of the base frequencies and criteria for consensus sequence assignment, it is proposed that the sequence AGAAN more accurately represents the consensus HSE motif (Fernandes, 1993).

Cooperative binding of HSF requires the close proximity of HSEs, rather than their helical alignment. Two or more trimeric HSEs organized as contiguous 5 bp units show much higher levels of cooperativity than multiple but separated HSEs. The DNA binding and trimerization domains alone may be sufficient for the full level of binding cooperativity between HSF trimers. This last result suggests that close proximity of HSEs for cooperative binding of HSF is a result of protein-protein interactions near the point of DNA contact (Fernandes, 1995).

The minimal promoter of the Drosophila hsp70 gene contains a TATA box and two nonidentical HSE sequences (HSEI and HSEII) that synergistically activate the promoter. Similar to deletion of HSEII, insertion in the spacer between the HSEs of 1 to 5 or 11 to 14 nucleotides (nt) reduces promoter activity to about 10% of that of the unaltered promoter. In contrast, HSEII is capable of contributing to promoter activity when the spacer is either shortened by 2 or 4 nt or extended by 6 to 10 or 16 or 18 nt. Hence, half of the possible helical arrangements of HSEs are compatible, whereas the other half are essentially incompatible with efficient promoter function. HSEII is ineffective when its distance to HSEI is increased by more than 18 nt. In vitro, HSEII is a weak binding site for heat shock transcription factor, while HSEI is a strong binding site; HSF binds to HSEII cooperatively. To find out whether the above periodicity reflects cooperative binding of HSF in vivo or represents the need of stereoalignment for synergistic activation of transcription, the weak HSF binding site HSEII was replaced with the strong binding site HSEI. This substitution greatly attenuated promoter periodicity, suggesting that the periodic effects are caused by cooperative binding of HSF to HSEII, and that stereoalignment of HSEs is not required for transcription activation. In agreement, in vitro assays using spacer mutants reveal cooperative binding of purified, recombinant HSF to HSEII with a similar periodicity as observed in vivo. Changing the distance between TATA and the HSEs does not produce promoter periodicity, indicating that stereoalignment of these elements is not important (Amin, 1994).

The parasitic helminth Schistosoma contains HSF activity that correlates with the pattern of hsp70 mRNA levels at different developmental stages in this organism. HSF activity revealed in extracts of Schistosoma mansoni (Sm) was further analyzed by competition experiments and compared with the well characterized HSF of Drosophila (Dm). The interactions of HSF in Sm extracts (SmHSF) and HSF of Dm (DmHSF) with 32P-labeled HSE probes, with and without unlabeled competitor DNA probes (HSE-related oligos), were analyzed by gel retardation assay. The binding and inhibition studies demonstrate that SmHSF and DmHSF differ in HSE sequence recognition: an array of three nGAAn inverted repeats according to the ideal consensus sequence (nGAAnnTTCnnGAAn) is recognized by DmHSF, but not by SmHSF. In the schistosome, binding is attained only when the third pentamer is a variant, composed of nGTAn instead of nGAAn. The presence of this variant in the promoter of the hsp70 gene of the parasite suggests coevolution of the variant sequence together with the SmHSF, which interacts efficiently with the variant, but not with the ideal HSE sequence. Further inhibition studies reveal additional differences between SmHSF and DmHSF in recognition of the first and second nGAAn pentamers of HSE. By analogy to other systems of ligand-protein interactions, it has been proposed that the complementarity between the HSE ligand and the HSF protein is higher in SmHSF, as compared to DmHSF (Levy-Holtzman, 1995).

The chromatin structure of the Drosophila hsp26 promoter is characterized by two DNAase hypersensitive (DH) sites harboring regulatory elements. Proximal and distal DH sites are separated by a positioned nucleosome. Both DH sites are readily reconstituted from extract components. They are separated by a nucleosome which is less strictly positioned than its in vivo counterpart. The interaction of GAGA factor (Trithorax-like) and Heat shock factor with their binding sites in chromatin occurs in two modes. Their interaction with binding sites in the nucleosome free regions does not require ATP. In the presence of ATP both factors also interact with nucleosomal binding sites, causing nucleosome rearrangements and a refinement of nucleosome positions. While chromatin remodeling upon transcription factor interaction has previously been interpreted to involve nucleosome disruption, the data suggest instead that energy-dependent nucleosome sliding is the main principle of chromatin reorganization (Wall, 1995).

Heat shock rapidly activates expression of some genes and represses others. The kinetics of changes in RNA polymerase distribution on heat shock-modulated genes was studied to provide a framework for evaluating the mechanisms of activation and repression of transcription. RNA polymerase transcription at the 5' end of the hsp70 gene can be detected within 30 to 60 s of induction, and by 120 s the first wave of polymerase can already be detected near the 3' end of the gene. A similar rapid induction is found for the small heat shock genes (hsp22, hsp23, hsp26, and hsp27). In contrast to this rapid activation, transcription of the histone H1 gene is found to be rapidly repressed, with transcription reduced by approximately 90% within 300 s of heat shock. The in vivo rate of movement of the first wave of RNA polymerase through the hsp70 gene is approximately 1.2 kb/min (O'Brien, 1993).

Synapses are critical sites of information transfer in the nervous system, and it is important that their functionality be maintained under stressful conditions to prevent communication breakdown. Synaptic transmission at the Drosophila larval neuromuscular junction is protected by prior exposure to heat shock, which strongly induces expression of heat shock proteins, in particular hsp70. Using a macropatch electrode to record synaptic activity at individual, visualized boutons, it has been found that prior heat shock sustains synaptic performance at high test temperatures through pre- and postsynaptic alterations. After heat shock, nerve impulses release more quantal units at high temperatures and exhibit fewer failures of release (presynaptic modification), whereas the amplitude of quantal currents remains more constant than does that in nonheat-shocked controls (postsynaptic modification). The time course of these physiological changes is similar to that of elevated hsp70. Thus, stress-induced neuroprotective mechanisms maintain function at synapses by modifying their properties (Karunanithi, 1999).

Since purified GAGA factor and TFIID interact similarly with the hsp70 and histone H3 promoters, the architecture of the endogenous H3 promoter has been analyzed to determine what interactions might be needed to establish a potentiated state containing a pause as seen in HSP70 promoter. Despite the detection of TFIID and GAGA on the H3 promoter, no paused polymerase as seen in the HSP70 promoter is evident. In addition, no proteins appear to interact with the transcription start. These results suggest that the GAGA factor and TFIID are not sufficient to establish a potentiated state containing paused polymerase and that TFIID interactions downstream from the TATA element could be important for pausing (Weber, 1995).

Three promoter sequences influence the access of HSF to its binding sites: the GAGA element, sequences surrounding the transcription start site, and a region in the leader of hsp70 where RNA polymerase II arrests during early elongation. The GAGA element has been shown to disrupt nucleosome structure. Because the two other critical regions include sequences that are required for stable binding of TFIID in vitro, the in vivo occupancy of the TATA elements were examined in the transgenic promoters. TATA occupancy correlates with HSF binding for some promoters. However, in all cases HSF accessibility correlates with the presence of paused RNA polymerase II. Thus, a complex promoter architecture is established by multiple interdependent factors, including GAGA factor, TFIID, and RNA polymerase II (Shopland, 1995).

There are two modes of regulation of the hsp26 promoter: one for promoters before heat shock, and another for promoters after heat shock. Efficient heat shock induction of Drosophila hsp26 gene transcription in vivo requires binding sites for heat shock factor (HSF) and GAGA factor (GAF) close to the TATA box (proximal elements) as well as 350 bp upstream of the start site of transcription (distal elements). Transcription in extracts from unstressed embryos relies solely on GAGA elements that efficiently counteract repression by abundant non-specific DNA-binding proteins. Transcription in extracts from heat shocked embryos depends only a little on GAGA elements, relying mainly on functional HSEs. These two modes of regulation in vitro may correspond to the two functional states of the promoter before and after heat shock in vivo (Sandaltzopoulos, 1995).

Transcription by RNA polymerase II is highly regulated at the level of initiation and elongation. Well-documented transcription activation mechanisms, such as the recruitment of TFIID and TFIIB, control the early phases of preinitiation complex formation. The heat shock genes provide an example of transcriptional regulation at a later step: in nuclei, TFIID can be detected at the TATA box prior to heat induction. Using cell-free systems for chromatin reconstitution and transcription, the mechanisms by which heat shock factor (HSF) increases transcription of heat shock genes in chromatin have been analyzed. HSF affects transcription of naked DNA templates in multiple ways: (1) by speeding up the rate of preinitiation complex formation (PIC), (2) by increasing the number of productive templates, and (3) by increasing the reinitiation rate. Under the more physiological conditions of potentiated chromatin templates, HSF affects only the reinitiation rate (Sandaltzopoulos, 1998)

In vivo, the first initiation of hsp26 transcription occurs in the absence of heat shock under conditions where HSF is unable to bind promoters with high specificity. Under such conditions, productive transcription from the hsp26 promoter requires the efficient release of the pausing polymerase as well as the recruitment of the transcription machinery to the TFIID-TFIIA complex for reinitiation. Conceivably, HSF may contact component(s) of the transcription machinery other than TFIID-TFIIA or a putative polymerase holo-enzyme complex. Such a recruitment would speed up the formation of the first PIC and subsequent reinitiation events and might also increase the template usage. In crude transcription systems an equilibrium between the formation of productive and nonproductive preinitiation complexes exists, brought about by competition between negative and positive regulators. HSF might increase the fraction of active templates by shifting this equilibrium towards the formation of productive complexes. It is, however, also possible that HSF increases the affinity of the committed complex for TFIIB by altering the conformation of a ternary TFIIA-TFIID-TATA complex (Sandaltzopoulos, 1998).

Reinitiation by newly recruited machinery is only possible if the previous polymerase has cleared the promoter. In heat shock genes, promoter clearance is a limiting step that can be overcome by activated HSF. Promoter clearance involves the phosphorylation of the polymerase CTD by the TFIIH kinase, a process that is integrated by TFIIE: TFIIE associates selectively with the hypophosphorylated form of the polymerase and stimulates the TFIIH kinase at a late step in PIC assembly. In vivo the paused polymerase at the hsp26 promoter should be in the elongation phase, but it has an unphosphorylated CTD. Since HSF induction leads to the release of this polymerase, it will be interesting to investigate whether HSF re-recruits TFIIE and TFIIH, as has been suggested, or an as yet unidentified heat shock-specific CTD kinase. Activator-dependent reinitiation of transcription, obviating the slow assembly of the TFIID-TFIIA complex on a promoter, may be especially crucial for genes requiring a fast response to inducers (Sandaltzopoulos, 1998).

The transition of heat shock factor to the induced state is accompanied by a chromosomal redistribution of HSF to the heat-shock puff sites. Over 150 additional chromosomal sites also accumulate HSF, including developmental loci that are repressed during heat shock. These findings suggest an unforeseen role for HSF as a repressor of normal gene activity during heat stress (Westwood, 1991).

Drosophila mediator complex is used by Heat shock factor

To decipher the mechanistic roles of Mediator proteins in regulating developmental specific gene expression and compare them to those of TATA-binding protein (TBP)-associated factors (TAFs), a multiprotein complex containing Drosophila Mediator (dMediator) homologs was isolated and analyzed. dMediator interacts with several sequence-specific transcription factors and basal transcription machinery and is critical for activated transcription in response to diverse transcriptional activators. The requirement for dMediator does not depend on a specific core promoter organization. By contrast, TAFs are preferentially utilized by promoters having a specific core element organization. Therefore, Mediator proteins are suggested to act as a pivotal coactivator that integrates promoter-specific activation signals to the basal transcription machinery (Park, 2001).

Previous studies in yeast and human cells have suggested that transcriptional activator proteins interact with Mediator complexes. The requirement of dMediator for the activated transcription in response to Gal4-VP16 indicates that dMediator may also serve as a binding target of transcriptional activators. Because several coactivators, such as TAFs and the GCN5 histone acetyltransferase (HAT) complex, have been suggested to interact directly with transcriptional activators, the relative binding affinities of these coactivator complexes with the VP16 protein were examined. After incubation of nuclear extracts with an excess of GST fusion protein beads containing either wild-type or mutant (Delta456FP442) VP16 activation domain, the supernatants were analyzed by immunoblotting with Abs against the components of the coactivator complexes. Almost all of the dMediator proteins in the nuclear extract (TRAP80, MED6, and Trfp) were removed by incubating with GST-VP16 but not with GST-VP16^{Delta456FP442}. However, the amounts of dGCN5, dTAF_II40, dTAF_II250, and dTBP in the extract were not reduced at all by the incubation. When the proteins bound to the beads were analyzed, a large amount of dMediator was retained only in the GST beads containing the functional VP16 activation domain. The TFIID and dGCN5 HAT complexes did bind to the wild-type VP16 beads, but the amounts were less than 2% of the total amounts present in the extract. These data indicate that, among known transcriptional coactivator complexes, Mediator is most strongly bound to and most readily recruited to the activation domain (Park, 2001).

In addition to the model VP16 activator derived from herpesvirus, dMediator interacts with Drosophila transcriptional activators Dorsal and heat shock factor (dHSF). When dMediator complex was incubated with FLAG-Dorsal or GST-dHSF fusion protein beads, more than 20% of the dMediator input was retained specifically on the beads even after extensive washing. To extend this study to other sequence-specific transcription factors important for Drosophila development, dMediator was immobilized on protein G-agarose beads through anti-dSOH1 Ab and the binding of diverse ³⁵S-labeled Drosophila transcription factors was examined. Bicoid, Krüppel, and Fushi-tarazu are retained specifically on the dMediator beads; Twist and Hunchback are not. Therefore, dMediator functions as a binding target for many, but not all, developmental specific transcription factors (Park, 2001).

To evaluate the requirement of dMediator for activated transcription in response to the Drosophila activator proteins that interact with dMediator, the ability of dMediator-deficient nuclear extracts to support transcriptional activation by the Dorsal and Gal4-dHSF proteins was examined. The addition of Dorsal or Gal4-dHSF to mock-depleted extract causes 20- and 25-fold increases, respectively, in transcription levels from the adenovirus early region 4 (E4) promoter linked to the appropriate DNA binding site. However, the level of transcriptional activation is reduced significantly (five- and three-fold activations, respectively) in nuclear extract that has been depleted by anti-dSOH1 Ab. Therefore, dMediator is absolutely required for transcriptional activation by all the activators tested. Addition of purified dMediator back to depleted extracts partially recovers activation by Dorsal and Gal4-dHSF in much the same way as it does in the case of Gal4-VP16. dMediator is not required for transcriptional repression by the sequence-specific transcription factor Even-skipped (Park, 2001).

dMediator is generally required for transcriptional activation from both TATA-containing and TATA-less promoters through direct communication with transcriptional activators. The function of dMediator seems to be exclusively related to sequence-specific transcription factors placed at upstream enhancer elements. However, the requirement of TAFs, or at least dTAF_II250, in activated transcription appears to be redundant in the in vitro transcription system used and affected by such factors as the core promoter organization or nucleosomal structure of transcriptional templates. Several TAF components in the TFIID complex indeed have biochemical activities and structural motifs adequate for the recognition of specialized settings of transcription templates. For example, certain TAFs recognize the Inr and DPE sequences located in many Drosophila core promoters and increase the stability of TFIID-promoter interactions. In addition, TFIID contains dTAF_II250, which has a HAT catalytic activity and also possesses a histone octamer-like module comprising the histone H2B-, H3-, and H4-like TAFs. Although not experimentally demonstrated, these TAFs may have some roles in the transcriptional regulation of nucleosomal templates (Park, 2001).

The sequence-specific transcription factors which interact physically with dMediator include VP16, Dorsal, dHSF, Bicoid, Krüppel, and Fushi-tarazu. These factors contain different types of activation domains (acidic and glutamine-rich domains). Most of these transcription factors have been shown to activate transcription either constitutively or inducibly. It is noteworthy that dHSF interacts with and requires dMediator for transcriptional activation because previous reports have shown that transcriptional activation by HSF in yeast does not require the function of the Mediator protein Srb4. However, the recent finding that activation by HSF depends on another Mediator protein, Rgr1 (Trap170), suggests that some function of Mediator is required for HSF-mediated transcriptional activation in yeast, as well. Since Rgr1, but not Srb4, is conserved between yeast and Drosophila, transcriptional activation by HSF might utilize the conserved Rgr1 components of the Mediator complexes (Park, 2001).

Although some human Mediator complexes appear to have a negative effect on activated transcription, dMediator does not exhibit such an activity in an in vitro transcription system reconstituted with Drosophila transcription factors. In addition, Even-skipped, a well-known Drosophila transcriptional repressor, does not interact with, or depend for its transcriptional repression on dMediator. Previous reports have shown that the repression domain of Even-skipped directly targets TBP. It has also been confirmed that the TFIID complex in the nuclear extract specifically interacts with Even-skipped under the same conditions in which Even-skipped fails to interact with dMediator. Although Krüppel has a well-characterized repressor function in Drosophila development, it can also act as a transcriptional activator under certain conditions. Therefore, it is more plausible that the dMediator-Krüppel interaction observed is a part of the mechanism for transcriptional activation rather than transcriptional repression. Taken together with the fact that dMediator is dispensable for basal transcription, the lack of defect of the dMediator-depleted nuclear extracts on transcriptional repression by Even-skipped protein suggests that dMediator is required mainly for the mediation of transcriptional activation signals to the basal transcription machinery. Very recently, developmental roles of certain dMediator proteins found in the Drosophila genome database have begun to be also identified in genetic studies. Genetic interactions between dMediator proteins and a homeotic regulator Sex combs reduced implicate dMediator proteins as a transcriptional activator-specific target critical for Drosophila development (Park, 2001).

Like yeast Mediator, dMediator bind with the CTD repeats of Drosophila Pol II. This implies that though dMediator was purified separately from Pol II, these two complexes indeed interact with each other and act together during transcriptional initiation. Besides the physical interaction with Pol II, dMediator also has some binding affinity for TBP, TFIIB, TFIIE, TFIIF, and TFIIS. Such interactions may be involved in the regulation of Pol II preinitiation complex assembly. Related with this idea, it has been reported that in yeast, recruitment of general transcription factors such as TBP, TFIIB, and TFIIH to active promoters requires the function of Mediator. Also, TFIIE interacts with the Mediator protein Gal11. Further analyses will be required to clarify whether these interactions, observed both in yeast and Drosophila, participate in the control of the stepwise preinitiation complex assembly in the course of transcription activation or simply reflect the affinities between the components of preassembled Pol II holoenzymeG (Park, 2001).

dMediator contains the protein kinase component Cdk8, which can phosphorylate serine residues in the CTD. This catalytic kinase subunit seems responsible, at least in part, for the Pol II phosphorylation by dMediator. In particular, dMediator and TFIIH synergistically phosphorylate the serine 5 residue of the carboxy-terminal Pol II repeats, suggesting the presence of a functional interaction between these complexes. Given that Pol II phosphorylation at serine 5 by TFIIH has been correlated with transcriptional activation processes, the synergy in the serine 5 phosphorylation by TFIIH and dMediator may be intimately linked with the regulatory effects that the Mediator complex exerts on Pol II transcription (Park, 2001).

Transcriptional elongation factors and HSF function on the hsp70 promoter

Transcriptional elongation regulators NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in extracts from human cells. A multifaceted approach was taken to investigate the potential role of these factors in promoter proximal pausing on the hsp70 gene in Drosophila. Immunodepletion of DSIF (FlyBase term: Spt5) from a Drosophila nuclear extract reduces the level of polymerase that pauses in the promoter proximal region of hsp70. Depletion of one Negative elongation factor E (NELF) subunit in salivary glands using RNA interference also reduces the level of paused polymerase. In vivo protein-DNA cross-linking shows that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborates the cross-linking result and shows that NELF, DSIF, and RNA polymerase IIa colocalize at the hsp70 genes, small heat shock genes, and many other chromosomal locations. Finally, following heat shock induction, DSIF and polymerase but not NELF are strongly recruited to chromosomal puffs harboring the hsp70 genes. It is proposed that NELF and DSIF cause polymerase to pause in the promoter proximal region of hsp70. The transcriptional activator, HSF, might cause NELF to dissociate from the elongation complex. DSIF continues to associate with the elongation complex and could serve a positive role in elongation (Wu, 2003).

It is proposed that promoter proximal pausing occurs when the nascent transcript emerges from the RNA exit channel of the Pol II and is grabbed by the NELF-E subunit. Tethering of the NELF-E to the elongation complex would generate a rigid body that could restrict the movement of the Pol IIa. This model is supported by several observations. The paused polymerase is in the Pol IIa state, and NELF and DSIF only inhibit elongation by Pol IIa. In vitro transcription analysis indicates that the elongation complex is not receptive to inhibition by NELF and DSIF until the nascent transcript is ~30 nucleotides long. This length coincides approximately to the distance polymerase elongates on hsp70 before it pauses. In vitro transcription analyses indicate that DSIF and NELF associate with polymerase shortly after initiation but probably before the polymerase reaches the region of pausing. Finally, NELF-E has an RNA-binding motif that is essential for its inhibitory action (Wu, 2003 and references therein).

Although NELF and DSIF are sufficient to slow the elongation rate of purified Pol IIa, it is suspected that additional proteins are involved in stably pausing Pol II on the hsp70 promoter. In cell-free transcription reactions done with other promoters, the pausing caused by DSIF and NELF appears to be transient -- the polymerase eventually moves forward if given enough time. In contrast, several observations indicate that the Pol II on hsp70 is stably paused. The paused Pol II remains associated with the hsp70 promoter when nuclei are isolated from uninduced cells, and sarkosyl or high salt must accompany addition of nucleotides to cause the Pol II to resume elongation. In a cell-free system, Pol II remains stably paused on the hsp70 promoter for at least 30 min. GAGA factor might be involved in stabilizing the pause because mutations in the GAGA element result in a loss of paused Pol II (Wu, 2003).

Heat shock rapidly induces transcription as a result of the association of HSF with sites located upstream from the TATA element. The data suggest that HSF may activate transcription in part by causing NELF to dissociate from the Pol II. How HSF might cause the release of NELF is unclear. Phosphorylation of Pol IIa is likely to be an important step because the Pol II found in the body of the gene during heat shock is hyperphosphorylated. Phosphorylation of DSIF is another possibility as this has been observed to occur early in elongation in vitro. It is also unclear which kinase might be responsible for phosphorylating the Pol II. P-TEFb is a candidate because it associates with the hsp70 gene during heat shock induction, and HSF can be bypassed by directing a Gal4/P-TEFb fusion protein to the hsp70 promoter. No interaction, however, has been detected between P-TEFb and HSF. Recent results show that HSF associates with the mediator. Drosophila mediator contains a kinase that phosphorylates the CTD, and phosphorylation can occur synergistically with the TFIIH kinase. Perhaps HSF recruits the mediator and in turn the mediator releases the paused polymerase by phosphorylating the CTD (Wu, 2003).

The strong immunofluorescence staining observed for DSIF at heat shock loci during heat shock indicates that DSIF is associated with many of the polymerase molecules transcribing the gene. RNA polymerase initiates at a rate of once every few seconds during heat shock resulting in a train of elongation complexes traversing the gene. In the absence of NELF, DSIF might act as a positive elongation factor. Shortly after DSIF was discovered, another investigation identified DSIF as a cofactor required for reconstituting tat-dependent transcription. In this situation, DSIF appears to be stimulating elongation. DSIF has been found in a complex with another positive elongation factor called Tat-SF1. Tat-SF1 was first identified as a stimulatory factor for Tat, but subsequent results indicate that Tat-SF1 may promote elongation on cellular genes. In yeast, DSIF appears to act as either a positive or negative regulator of elongation depending on circumstances. A hypothesis that unites the positive and negative activities of DSIF considers this factor an adaptor that connects other modulators to the elongation complex. In this regard, DSIF has been shown to bind on its own to Pol II, whereas the stable association of NELF with Pol II requires the presence of DSIF (Wu, 2003 and references therein).

NELF and DSIF appear to associate with several hundred interbands in polytene chromosomes. Each interband could contain many genes. The weak staining of interbands by Hoecsht suggests that the DNA in the interbands is in a decondensed state. Residing in these decondensed regions could be genes whose primary control mechanism does not involve a disruption of chromatin structure or even assembly of the initiation complex. Instead, alleviating repression by NELF and DSIF could underlie the mechanism of activation (Wu, 2003).

Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock

The uninduced Drosophila hsp70 gene is poised for rapid activation. The rapid changes upon heat shock in levels and location of heat shock factor (HSF), RNA polymerase II (Pol II) and its phosphorylated forms, and the Pol II kinase P-TEFb on hsp70 were examined in vivo by using both real-time PCR assays of chromatin immunoprecipitates and polytene chromosome immunofluorescence. These studies capture Pol II recruitment and progression along hsp70 and reveal distinct spatial and temporal patterns of serine 2 and serine 5 phosphorylation: in uninduced cells, the promoter-paused Pol II shows Ser5 but not Ser2 phosphorylation, and in induced cells the relative level of Ser2-P Pol II is lower at the promoter than at regions downstream. An early time point of heat shock activation captures unphosphorylated Pol II recruited to the promoter prior to P-TEFb, and during the first wave of transcription Pol II and the P-TEFb kinase can be seen tracking together across hsp70 with indistinguishable kinetics. Pol II distributions on several other genes with paused Pol II show a pattern of Ser5 and Ser2 phosphorylation similar to that of hsp70. These studies of factor choreography set important limits in modeling transcription regulatory mechanisms (Boehm, 2003).

Pol II is highly regulated both at the level of recruitment to promoters and in its progress through the stages of the transcription cycle. This regulation is executed through numerous associations with other proteins as Pol II enters the promoter, melts DNA, initiates transcription, begins early elongation, and eventually matures into a productive elongation complex. Pol II undergoes additional modifications, most notably phosphorylation of the CTD of its largest subunit as it progresses from its hypophosphorylated promoter entry form to the elongation phase, where it is highly phosphorylated at residues Ser2 and Ser5. These changes in phosphorylation are proposed to influence protein association, affecting not only Pol II's elongation properties but also its association with a variety of protein complexes that process pre-mRNA. Moreover, the pattern of phosphorylation is not stagnant during the elongation phases of the cycle and may be signaling specific associations. To define mechanisms involved in these processes in vivo, the rapidly and robustly activated hsp70 gene has been employed as a model (Boehm, 2003).

Technological advances of DNA-protein cross-linking and highly quantitative large-scale PCR assays were used to explore hsp70 activation kinetics of recruiting HSF, the critical Pol II kinase P-TEFb, and Pol II in vivo. The changes in Pol II were examined during the first and subsequent cycles of transcription that are triggered in response to an instantaneous heat shock of Drosophila cells. HSF recruitment occurs very rapidly, detectable at the earliest assay point of 5 s of heat shock, and reaches saturation within 75 s; this result is consistent with the rapid transcriptional response of heat shock genes and with previous, lower resolution assays of HSF recruitment. The recruitment of additional hypophosphorylated Pol II to the promoter occurs with rapidity similar to that of HSF recruitment but before an increase in Pol II phosphorylation at the promoter, which occurs by 75 s. All forms of Pol II achieve a maximal level on the promoter and gene by 5 min. The progress of Pol II across the gene can be observed, and its progress fits the known rate of elongation on Drosophila hsp70, 1.2 kb/min. Interestingly, total Pol II levels remain greater at the transcription start site than at the ORF, even during active transcription, consistent with the observation that promoter escape remains rate-limiting even during heat shock. P-TEFb, a major Pol II kinase, moves across the gene at a rate similar to that of Pol II during the first burst of transcription and thereafter remains distributed over the promoter and ORF during the full 20-min time course examined. This distribution supports a model where P-TEFb contributes to Pol II phosphorylation not only at the promoter but also during most or all of the elongation phase of transcription (Boehm, 2003).

The detection of Ser5 phosphorylation on a promoter-paused Pol II prior to gene induction corroborates a model where this phosphorylation is an early event involved in the transition from initiation of transcription to early phases of Pol II elongation. The mRNA associated with the paused Pol II molecule has previously been shown to be efficiently capped when long enough to allow access of the capping machinery. Since Ser5-P has been reported to enhance Pol II association with mRNA capping machinery and capping activity, the Ser5-P detected on paused Pol II might help to explain the efficient capping of paused RNAs. It is important to note that earlier analyses of the hsp70 gene, which determined that the paused Pol II CTD is hypophosphorylated prior to heat shock, were performed with antibodies different from those used in the present study. Importantly, the antibody generated to detect Pol IIo in those studies was directed against a peptide phosphorylated in vitro by CTK1, a yeast kinase thought to phosphorylate the CTD at serine 2. Thus, the previous analysis did not probe for the Ser5 phosphorylation reported here (Boehm, 2003).

While P-TEFb phosphorylates the CTD primarily at Ser2, it has also been shown to recognize Ser5 as a substrate. Present results suggest that the Ser5 detected in the uninduced state on hsp70 is not a result of P-TEFb activity, since P-TEFb is not detected prior to gene activation (as seen in this study). Ser5 is likely to be the substrate of the cdk7 component of TFIIH early in transcription. Indeed, cdk7 has been found in in vitro studies to be released earlier in the transcription cycle than P-TEFb. In vivo, Cdk7 is required very early in the transcription cycle and contributes to the generation of the paused Pol II on the promoter-proximal region of hsp70 (Boehm, 2003).

Analyses of the phosphorylation status of the CTD in other organisms have found Ser5-P levels to be higher at the promoter than at the ORF, a pattern similar to what was observed on hsp70 during active transcription. When total levels of Pol II are taken into account on hsp70, however, it appears that the level of Ser5-P remains constant along the gene. Comparatively, another study did not see a striking difference in total Pol II density along the genes analyzed. A third study detected more Ser5-P at the promoter than the 3' untranslated region of the human alpha-AT gene but also appeared to detect more total Pol II at the promoter region. Thus, it may be that in metazoans (or on some genes) the level of Ser5-P relative to Pol II is fairly constant along the gene. The possibility that the activity of a phosphatase may be system or gene specific is certainly plausible; for instance, heat shock of HeLa cells deactivates a CTD phosphatase (Boehm, 2003 and references therein).

Under non-heat shock conditions, total Pol II levels were greater at the 5' regions than at the ORFs for several genes that contain promoter-paused Pol II, while histone H1, which does not display a pause by nuclear run-on assay, shows no significant difference of 5' and ORF Pol II signals. Greater levels of Ser5-P were also detected at the 5' end of the genes containing paused Pol II, while levels on H1 were distributed evenly, indicating that this phosphorylation may be a general aspect of the regulatory status of a paused Pol II. This distribution of Ser5-P for the constitutively active genes Tub, GAP, and Actin5C is similar to the results of other studies which analyzed active transcription; however, Ser5-P levels on these genes are constant when standardized to total Pol II, similar to hsp70 in its active state. For these Drosophila genes, the higher level of Ser5-P at the promoter may be attributable to the presence and status of the paused Pol II, indicative of genes regulated at the level of elongation. Indeed, recent studies describe another constitutively active pause gene in human cells, dihydrofolate reductase, which shows a pattern similar to that of Ser5-P for these Drosophila genes (Boehm, 2003 and references therein).

Phosphorylation at Ser2 of the Pol II CTD may be important for processivity into active elongation and has been implicated in downstream events, including pre-mRNA splicing and 3' mRNA processing. Ser2-P levels are undetectable at +58 on hsp70 in the uninduced state, increase quickly at the 5' region upon heat shock, and appear constant through the gene in later time points (for example, 5 min). The increase in phosphorylation detected over time tracks with the recruitment of additional Pol II as well as the recruitment of P-TEFb. Taking into account total Pol II levels, there appears to be a slight increase in Ser2-P as Pol II progresses through the ORF. This correlates with the concomitant and approximately equivalent decrease in Pol IIa. Ser2-P patterns on additional genes containing a paused Pol II, when considered relative to levels of detectable total Pol II, are significantly higher in the ORF than are those in the 5' region. While these ratios may simply be a consequence of promoter-paused Pol II not being Ser2 phosphorylated, this result is similar to that of another study, where Ser2-P was only detected in the ORF. These observations led to speculation that an increase in Ser2-P may be important for cueing specific processes as Pol II proceeds through the gene. P-TEFb, the major kinase implicated in Ser2 phosphorylation, was detected concomitant with Pol II during active transcription on hsp70. While Pol II/P-TEFb ratios appear constant, a slight increase in Ser2-P occurs at the 3' end of the gene. As the presence of the kinase is not an indicator of its activity, work presently ongoing in the laboratory on P-TEFb kinase inactivation and hsp70 gene regulation should help to better understand this process (Boehm, 2003 and references therein).

Lastly, analysis of immunostaining of polytene chromosomes provides independent corroboration of the higher resolution and quantitative ChIP assays and provides insight into the formation and composition of the transcription puff. Paused Pol II on hsp70 was previously detected with this method, as was Pol II along hsp70 during heat shock. A modest detection of Ser5-P was observed on the promoter prior to heat shock. During the early stages of puff formation, Pol II resolves from promoter-bound HSF. Ser2-P and Ser5-P occupy the most decondensed regions of the puff forming a halo around the heat shock loci, while HSF is more concentrated at the chromosome core at one end of the puff. Taken together, these ChIP and immunofluorescence results provide a foundation for additional temporal and spatial assignments of specific factors relative to the phosphorylation events during the activation of transcription. Perturbation of the function or activity of specific factors using genetic and drug-based approaches will provide further insight into the mechanistic role of these factors in the recruitment and modification of transcriptional machinery and in the coupling of specific transcription processes and Pol II modifications to RNA processing events (Boehm, 2003).

Dynamics of heat shock factor association with native gene loci in living cells

Direct observation of transcription factor action in the living cell nucleus can provide important insights into gene regulatory mechanisms. Live-cell imaging techniques have enabled the visualization of a variety of intranuclear activities, from chromosome dynamics to gene expression. However, progress in studying transcription regulation of specific native genes has been limited, primarily as a result of difficulties in resolving individual gene loci and in detecting the small number of protein molecules functioning within active transcription units. This study reports that multiphoton microscopy imaging of polytene nuclei in living Drosophila salivary glands allows real-time analysis of transcription factor recruitment and exchange on specific native genes. After heat shock, this study has visualized the recruitment of RNA polymerase II (Pol II) to native hsp70 gene loci 87A and 87C in real time. Heat shock factor (HSF), the transcriptional activator of hsp70, is localized to the nucleus before heat shock and translocates from nucleoplasm to chromosomal loci after heat shock. Assays based on fluorescence recovery after photobleaching show a rapid exchange of HSF at chromosomal loci under non-heat-shock conditions but a very slow exchange after heat shock. However, this is not a consequence of a change of HSF diffusibility, as shown here directly by fluorescence correlation spectroscopy. The results provide strong evidence that activated HSF is stably bound to DNA in vivo and that turnover or disassembly of transcription activator is not required for rounds of hsp70 transcription. It is concluded that transcriptional activators display diverse dynamic behaviours in their associations with targeted loci in living cells. This method can be applied to study the dynamics of many factors involved in transcription and RNA processing, and in their regulation at native heat shock genes in vivo (Yao, 2006).

The rapid recovery pattern of HSF under non-heat shock (NHS) and slow recovery under heat shock (HS) corresponds in vivo to the marked difference in the DNA-binding affinity of HSF monomers (NHS) and trimers (HS). It is therefore proposed that a transcription activator's exchange dynamics on its targets may simply reflect the dissociation rate constant of the protein-promoter complex. The low affinity of some activators leads to their transient binding and has been suggested to cause the probabilistic assembly of transcriptional machinery. The high affinity of other activators leads to their stable binding, and this in turn is conducive to the formation of stable coactivator assemblies and the efficient recruitment of Pol II for repeated cycles of transcription. The exchange dynamics of some activators may involve other mechanisms; for instance, NF-kappaB, which has high affinity for DNA, was found to exchange rapidly at the tandem-repeat target gene loci. In addition, chromatin remodelling might have a function in these processes (Yao, 2006).

The slow exchange of activated HSF at the hsp70 promoter presents a sharp contrast with the rapid recruitment and elongation of RNA polymerase II at hsp70 genes during HS. During a 2-min transcription cycle (that is, the time it takes a Pol II molecule to transcribe the hsp70 gene, more than 20 Pol II molecules have begun the transcription of each hsp70 gene; however, very little new HSF has bound to the gene as shown by FRAP. Therefore, the data do not support the 'activation by destruction' hypothesis that the recruitment of new polymerase requires the ubiquitin-proteasome system (UPS) to turn over the 'spent' activator on the promoter. Moreover, more than the total amount of intracellular HSF would be degraded during a short period of heat shock if 'activation by destruction' were true for every round of heat shock gene transcription. HSF is an acidic, strong activator, like many positive regulatory factors, and hsp70 transcription resembles that of many other genes. Recent results on the yeast Gal4 activator have shown that it, too, is stably bound to its regulatory sites during gene activation. Therefore two independent and complementary approaches on the two widely studied acidic activators have revealed their stable binding to DNA during gene activation. Alternative models for activator function that propose activator recycling as a key component, such as hit and run, chaperone-assisted disassembly or UPS-mediated turnover, can apply to some but clearly not all transcriptional activators (Yao, 2006).

The stable binding of HS-activated HSF and the transient binding of ligand-activated GR collectively show the diverse 'action modes' of transcription activators: both stably bound and transiently bound activators can support gene transcription. How individual activators function in these two modes on their respective gene targets remains to be seen, with the underlying mechanisms yet to be determined. Importantly, the dynamic behaviour of coactivators, Pol II transcription and RNA-processing machinery at native mRNA genes is largely unknown in living cells, and the described experimental approach will be applicable to further investigations (Yao, 2006).

Chromatin landscape dictates HSF binding to target DNA elements

Sequence-specific transcription factors (TFs) are critical for specifying patterns and levels of gene expression, but target DNA elements are not sufficient to specify TF binding in vivo. In eukaryotes, the binding of a TF is in competition with a constellation of other proteins, including histones, which package DNA into nucleosomes. ChIP-seq assay was used to examine the genome-wide distribution of Drosophila Heat Shock Factor (HSF), a TF whose binding activity is mediated by heat shock-induced trimerization. HSF binds to 464 sites after heat shock, the vast majority of which contain HSF Sequence-binding Elements (HSEs). HSF-bound sequence motifs represent only a small fraction of the total HSEs present in the genome. ModENCODE ChIP-chip datasets, generated during non-heat shock conditions, were used to show that inducibly bound HSE motifs are associated with histone acetylation, H3K4 trimethylation, RNA Polymerase II, and coactivators, compared to HSE motifs that remain HSF-free. Furthermore, directly changing the chromatin landscape, from an inactive to an active state, permits inducible HSF binding. There is a strong correlation of bound HSEs to active chromatin marks present prior to induced HSF binding, indicating that an HSE's residence in 'active' chromatin is a primary determinant of whether HSF can bind following heat shock (Guertin, 2010).

Although bona fide HSF binding sites contain highly specific HSE motifs, only a small fraction of potential HSE motifs are occupied by HSF. To search for HSF-free binding sites, a conservative cut-off was used for conformity to the consensus HSE by using a p-value of 5×10^-6 or less, while ensuring that the flanking region is mappable. There are 708 HSF-free motifs that meet these criteria. Less than 15% (107/815) of the mappable HSE motifs with a p-value of 5×10^-6 or less are detectably bound by HSF after HS. Upon closer inspection, this study found that HSF-free motifs are absolutely HSF-free during non-heat shock (NHS), and these same motifs are either unoccupied or infrequently occupied after HS. In contrast, HSF-bound motifs are either very weakly occupied or unoccupied prior to HS, and show strong inducible binding after HS induction. Therefore, these two categories of motifs, HSF-free and HSF-bound, are distinct from one another (Guertin, 2010).

The distribution of HSF binding sites relative to annotated genes and promoter regions was determined. Annotated genes account for 60.6% of the Drosophila reference genome, however, 72% of the HSF-bound motifs are found within gene boundaries. HSF-bound motifs within promoters [500 bp upstream of a transcription start site (TSS)] were also enriched, accounting for 22% of the total bound motifs, while such promoter regions only account for 3.4% of the total reference genome. In contrast, the classification of the 708 HSF-free motifs is much closer to a background distribution; 63% HSF-free motifs are within genes and 5.5% are within promoters. These results indicate that HSE motifs are not simply enriched within gene and promoter boundaries, but that HSF preferentially interacts with HSEs that are present within genes and promoters (Guertin, 2010).

The ChIP-seq method is used routinely to determine genome-wide factor binding profiles; however, important controls and variations in the ChIP protocol more fully exploit this approach. The implementation of the control RNAi knockdown of HSF allowed elimination of the genome-wide set of false positive signals that were resistant to this knockdown, and prevented the elimination of many true positive binding sites. Another rigorous and complementary control for specificity includes performing independent ChIP experiments with multiple antiserum preparations, each of which is affinity purified with nonoverlapping antigens. The details of ChIP-seq chromatin preparation can also enhance peak detection. Additional crosslinking agents and crosslinkers that target particular types of protein/DNA interactions, such as exclusively probing direct protein/DNA interactions with UV light, can also augment the type and quality of information obtained by the basic ChIP-seq strategy (Guertin, 2010).

The non-sequence dependent specificity observed by TFs can be explained by non-mutually exclusive mechanisms: DNA binding is specifically inhibited by repressive chromatin, aided by active chromatin, or mediated by cooperative interactions with chromatin factors. This study report that repressive marks contribute minimally to restrict HSF binding, as only a small fraction of HSF-free motifs are associated with repressive chromatin. Additionally, it was observed that chromatin containing background levels of active and repressive marks is unfavorable to inducible HSF binding - the default state of an in vivo HSE can be considered inaccessible. In contrast, HSF inducibly binds to sites that contain TFs and marks of active chromatin prior to HS induction. This study has shown that the chromatin landscape can be modified to the permissive state and result in recognition and binding of a previously unbound HSE. This result suggests that HSF does not primarily function to bind DNA cooperatively with other factors, but simply co-occupies the same regions as other TFs, due to the accessible nature of the DNA. These results provide a framework for understanding the binding selectivity of HSF, and mechanistic studies can be anticipated that solidify the rules of in vivo binding specificity (Guertin, 2010).

Activators are generally thought to bind to promoters and recruit either Pol II or coactivators to produce productively elongating Pol II. HSF recruits the acetyltransferase CREB Binding Protein (CBP) and a methyltransferase, Trithorax, directly to HS genes. Paradoxically, this study shows that the chromatin landscape at HSF binding sites contains considerable histone acetylation and methylation prior to detectable HSF binding. HSF recruits these enzymes after HS to broaden the domain or increase the level of histone modifications. Another, non-mutually exclusive, possibility is that cofactors other than histones are the functional targets of recruited transferases. Although this study has describe the landscape at HSF binding sites prior to HS, it still remains unclear which factors are responsible for setting up or maintaining the accessibility of these motifs. Furthermore, many HSF-binding sites are probably passively occupied because they happen to be accessible and HSF binding is non-deleterious, but these sites likely have no function in the HS response. The global chromatin landscape is dynamic throughout development and environmental changes; therefore, it is expected that the HSF binding profile at non-functional sites is dynamic as well. Nonetheless, the HS response is a ubiquitous cellular response, so functional sites are likely to be evolutionarily constrained at the sequence level, and actively maintained in the accessible state at the level of chromatin organization (Guertin, 2010).

The maintenance of functional HSF binding sites may be occurring as a result of a specific class of activators. Non-traditional activators, such as GAF, are known to recruit cofactors that establish an accessible chromatin state, as opposed to directly activating transcription of the local gene. This general mechanism has been characterized at the phaseolin gene in Arabidopsis and at the PHO5 gene in yeast. Taken together, this suggests a step-wise process whereby a repressed site can be potentiated for activator binding and subsequently activated. Additionally, it has been shown that active marks are not simply a product of transcription, as the active marks that are associated with intergenic DNaseI hypersensitive sites and putative enhancers are not correlated with respective gene expression. The results suggest that the landscape may be marked with active histone modifications to allow binding of activators that can stimulate transcription; therefore, the presence of a modification would not be expected to correlate with gene expression if the activator has yet to bind. Further investigation of activator binding sites during non-induced conditions will determine the generality of this observation (Guertin, 2010).

Candidate gene analysis shows that HSF is not sufficient to activate local genes. Although inducibly activated genes are occupied by their cognate transcriptional activator near the TSS, it remains unclear how the majority of activators discriminate between locally bound genes to selectively activate. Strikingly, Caudal exhibits promoter element-specific activation, specifically activating genes that contain the Downstream Promoter Element (DPE). Previously, evidence is presented that the presence of a paused polymerase facilitates activation from an Hsp70 promoter, but it is unclear whether or not this is true for the majority HSF-inducible genes. Combinations of promoter features and gene properties are likely necessary for activation. One certainty, however, is that the recent emergence of genome-wide expression and binding data makes the characterization of complex regulatory mechanisms more exciting and promising than ever (Guertin, 2010).

RNAi screen in Drosophila larvae identifies histone deacetylase 3 as a positive regulator of the hsp70 heat shock gene expression during heat shock

Transcription regulation of the Drosophila hsp70 gene is a complex process that involves regulation of multiple steps including establishment of paused Pol II and release of Pol II into elongation upon heat shock activation. While the major players involved in regulation of gene expression have been studied in detail, additional factors involved in this process continue to be discovered. To identify factors involved in hsp70 expression, a screen was developed that capitalizes on a visual assessment of heat shock activation using a hsp70-beta galactosidase reporter and publicly available RNAi fly lines to deplete candidate proteins. The screen was validated by showing that depletion of HSF, CycT, Cdk9, Nurf 301, or ELL prevented full induction of hsp70 by heat shock. The screen also identified the histone deacetylase HDAC3 and its associated protein SMRTER as positive regulators of hsp70 activation. Additionally it was shown that HDAC3 and SMRTER contribute to hsp70 gene expression at a step subsequent to HSF-mediated activation and release of the paused Pol II that resides at the promoter prior to heat shock induction (Achary, 2014).

HSF interactions with chaperones

Heat shock transcription factors (HSFs) play important roles in the cellular response to physiological stress signals. To examine the control of HSF activity, a yeast two-hybrid screen was undertaken for proteins interacting with Drosophila HSF. DROJ1, the fly counterpart of the human heat shock protein HSP40/HDJ1, was identified as the dominant interacting protein (15 independent isolates from 58 candidates). The domain of HSF interacting in the two-hybrid screen has been mapped to the region between the N- and C-terminal leucine zippers, previously shown to modulate HSF activity. The corresponding interacting domain of DROJ1 maps to the C-terminal half of the protein and is involved in substrate binding. Overexpression of DROJ1 in Drosophila SL2 cells delays the onset of the heat shock response. Moreover, RNA interference involving transfection of SL2 cells with double-stranded droj1 RNA depletes the endogenous level of DROJ1 protein, leading to constitutive activation of endogenous heat shock genes. The induction level, modest when DROJ1 alone is depleted, reaches maximal levels when DROJ1 and HSP70/HSC70, or DROJ1 and HSP90, are depleted concurrently. Chaperone co-depletion is also correlated with strong induction of the DNA binding activity of HSF. These findings support a model in which synergistic interactions between DROJ1 and the HSP70/HSC70 and HSP90 chaperones modulate HSF activity by feedback repression (Marchler, 2001).

The Torso signaling pathway modulates a dual transcriptional switch to regulate tailless expression

The Torso (Tor) signaling pathway activates tailless (tll) expression by relieving tll repression. None of the repressors identified so far, such as Capicuo, Groucho and Tramtrack69 (Ttk69), bind to the tor response element (tor-RE) or fully elucidate tll repression. In this study, an expanded tll expression pattern was shown in embryos with reduced heat shock factor (hsf) and Trithorax-like (Trl) activities. The GAGA factor, GAF encoded by Trl, bound weakly to the tor-RE, and this binding was enhanced by both Hsf and Ttk69. A similar extent of expansion of tll expression was observed in embryos with simultaneous knockdown of hsf, Trl and ttk69 activities, and in embryos with constitutively active Tor. Hsf is a substrate of mitogen-activated protein kinase and S378 is the major phosphorylation site. Phosphorylation converts Hsf from a repressor to an activator that works with GAF to activate tll expression. In conclusion, the GAF/Hsf/Ttk69 complex binding to the tor-RE remodels local chromatin structure to repress tll expression and the Tor signaling pathway activate tll expression by modulating a dual transcriptional switch (Chen, 2009).

This study has shown that the interaction of GAF with Hsf and Ttk69 plays a critical role in regulation of tll expression. At locations where the Tor pathway is inactive, GAF, Hsf, and Ttk69 constitute a protein complex that binds to the tor-RE tightly. The protein complex recruits other co-repressors and chromatin remodeling factors containing Rpd3 to the organization of a high-order local chromatin structure. tll expression is off in this scenario (Chen, 2009).

Both Hsf and Ttk69 have been shown to be substrates of Mapk. At locations where the Tor pathway is active, activated Mapk phosphorylates both Hsf and Ttk69. The phosphorylated Ttk69 is degraded. The phosphorylation converts Hsf into an activator that leads to an increase in tll. In addition, the expanded lacZ expression patterns in hsf⁴ embryos at the nonpermissive temperature suggested that other activators, such as Stat, are apparently required for full activation of tll (Chen, 2009).

A protein–protein interaction network, including GAF, Hsf, Rpd3 and Ttk69, is required for tll repression. Studies show that Capicuo interacts with Groucho, which associates with Rpd3 and Sin3A to repress tll expression. Furthermore, CtBP is essential for Ttk69 to suppress neuronal cell fate. Therefore, these factors may also be recruited to repress tll expression (Chen, 2009).

Multiple GAFs and Ttk69s bind to the flanking regions of the tor-RE. The results from the DNaseI footprinting experiments showed that GAF binds to four sites in the tll-MRR, including the tor-RE. DNA sequences in footprints a and b match the consensus sequence bound by GAF, 3.5 GA repeats. Although DNA sequence in footprint c does not match the consensus sequence, this site contains a GAGA tandem repeat with 1-bp spacing. These three sites are well protected by GAF from DNaseI digestion, without or with little influence by Hsf. Therefore, GAF oligomer binds to these sites with high affinity and likely assists its own binding to the tor-RE. Similarly, binding of Even-skipped protein to low-affinity sites was assisted by its own binding to high-affinity sites at a distance (Chen, 2009).

Ttk69 acts as a co-repressor to increase GAF/Hsf binding to the tor-RE. In another EMSA experiment, a probe containing both the tor-RE and TC5 was used. Ttk69 binds to TC5 and assists GAF/Hsf/Ttk69 binding to the tor-RE. This is consistent with a previous report that base substitution a TCCT element (TC5) at the 3' flanking region of the tor-RE clearly affects the initiation of tll repression. Additionally, the binding of Ttk69 to TC2 might facilitate tll repression (Chen, 2009).

Results from the DNaseI footprintings showed different patterns over the tor-RE protected by either GAF/Hsf or GAF alone. Unexpectedly, these different tor-RE complexes showed the same mobility in results from EMSA experiments, which might be explained by the altered binding property of GAF in the presence of Hsf. BTB domains in GAF and human Promyelocytic Leukemia Zinc Finger (PLZF) proteins have been shown to belong to the Ttk subfamily, and BTB homodimer is a unit of PLZF oligomer. Since GAF oligomer presumably assisted itself in binding to the tor-RE, GAF homodimer could bind the 5'-end as well as the 3'-end of the tor-RE. When Hsf was added in addition to GAF, the interaction of GAF with Hsf influenced GAF binding to the tor-RE, leading to the alteration of the footprinting pattern. However, it remains unclear whether Hsf contacts DNA when it exists in the DNA–protein complex. Nevertheless, the results from shift-western blotting clearly demonstrated the presence of both GAF and Hsf in the DNA–protein complex (Chen, 2009).

Displacement of GAF monomer by Ttk69 to form a GAF/Hsf/Ttk69 complex might explain the unchanged band-shift patterns when the three proteins were added to the binding mixture. Molecular weights between GAF and Ttk69 are slightly different and BTB domains in both GAF and Ttk69 proteins belong to the same subfamily. Molecular modeling with crystal structure of the BTB domain in human BACH1 (PDB code: 2ihc) as a template was used to test the displacement hypothesis. Results showed that buried areas and free energies (ΔGs) of engaged interfaces among GAF and Ttk69 homodimers and GAF/Ttk69 heterodimer were similar, suggesting that formation of a GAF/Ttk69 heterodimer is possible. This explanation is partially supported by the detection of both Ttk69 and Hsf in the DNA–protein complex. In addition, Ttk69 has been shown to inhibit GAF activation in the absence of Ttk69 binding sites, and addition of Ttk69 significantly increases GAF binding to the 173-bp probe that contains multiple GAF binding sites. However, results from EMSA show that mobility of the DNA–protein complex is slightly affected. In conclusion, these data plus the existence of GAF, Hsf and Ttk69 in the DNA–protein complex indicate that the interaction of GAF with Hsf and Ttk69 form a protein complex binding to the tor-RE tightly (Chen, 2009).

In summary, multiple factors bind to the tor-RE and its flanking regions to form a large repression complex, consistent with the finding that the 240-bp cis-regulatory region of tll, but not the tor-RE itself, silences from a heterologous promoter (Chen, 2009).

Several studies on gene repression, i.e., bacterial bipA and eno genes, fly decapentaplegic and human Iε and GP91^phox genes, have revealed that multiple low-affinity sites are bound by a repressor to regulate gene expression. Likewise, multiple weak binding sites that cluster within a short range of a cis-regulatory region are reported to facilitate the cooperative binding of factors, leading to a sharp definition in expression patterns. Reduction of repressor concentration leads to a loss in the definition at the edge of expression domains. In this study, the binding affinity of GAF to the tor-RE is low, and multiple tor-REs are present in the tll cis-regulatory region. This explains the poorly-defined boundary of the tll expression patterns in embryos that have reduced hsf and Trl activities (Chen, 2009).

Footprint b was marginally affected by supplementing Hsf. The DNA sequence in this site contains one binding site each for GAF and Hsf (−130 GAGAGAG and −115 GAATCCTGCGGAA), located in regions O and P, respectively. The sequence for GAF binding matches the consensus sequence. Although this putative Hsf binding sequence does not match the consensus sequence, it has been shown that Hsf is able to bind a sequence with two GAAs spaced by 7 bp. Interestingly, in contrast to the role of tor-RE, deletion of either region O or P from the tll-MRR results in a drastic reduction of lacZ mRNA levels, but no changes in the expression patterns. These results not only further support the notion that Hsf and GAF are required for tll activation, but also provide an explanation for (1) the different tll expression patterns in embryos with reduced hsf and Trl activities by either removing one copy of the genes or using RNAi to knockdown activities of the genes, and (2) the different tll expression levels resulting from either base substitutions in the tor-RE or reduction of gene activities (Chen, 2009).

These results showed a moderate effect of Hsf on GAF binding to footprint c. In addition to the GAGA repeat, this site also contains two GAA repeats with the 7-bp spacing. Results from the DNaseI footprinting experiments showed that GAF bound to this site less efficiently. Furthermore, deletion of this site from the tll-MRR results in a low level and slightly expanded lacZ expression pattern. In addition, footprint d at the 5'-end of the tor-RE was a weak site bound by GAF and the footprint pattern protected by GAF was significantly affected by Hsf. Base substitutions to this site severely damaged tll repression. These data supported the notion that the low and high affinity sites bound by GAF are the major contributors to tll repression and activation (Chen, 2009).

All signaling pathways regulate, at least in part, specific factors to activate the expression of target genes. In most well-studied signal pathways, the signals directly activate factors. In some cases, the signals switch on expression of their target genes from a repressed state. For example, in the absence of Notch, Wnt, Hedgehog or nuclear receptor signaling, the expression of target genes is repressed. There is a common mechanism among these cases. A specific factor or complex, such as Su(H)/CBF1 in the Notch pathway, Lef/Tcf in the Wnt pathway, Ci/Gli in the Hh pathway or the nuclear receptors themselves, bind to a specific DNA sequence to prevent target genes from being transcribed. When the signaling pathway is active, the repression is relieved by the processed receptor, activated activator, co-activators or by the nuclear receptor itself. Results from this study indicated that both GAF-associated proteins, Hsf and Ttk69, constitute a dual al switch for tll expression that includes degradation of the Ttk69 co-repressor and conversion of the Hsf repressor into an activator after Mapk phosphorylation, where Mapk is a downstream effector of the Tor pathway (Chen, 2009).

Accurate prediction of inducible transcription factor binding intensities in vivo

DNA sequence and local chromatin landscape act jointly to determine transcription factor (TF) binding intensity profiles. To disentangle these influences, an experimental approach, called protein/DNA binding followed by high-throughput sequencing (PB-seq), was developed that allows the binding energy landscape to be characterized genome-wide in the absence of chromatin. These methods were applied to the Drosophila Heat Shock Factor (HSF), which inducibly binds a target DNA sequence element (HSE) following heat shock stress. PB-seq involves incubating sheared naked genomic DNA with recombinant HSF, partitioning the HSF-bound and HSF-free DNA, and then detecting HSF-bound DNA by high-throughput sequencing. PB-seq binding profiles were compared with ones observed in vivo by ChIP-seq and statistical models were developed to predict the observed departures from idealized binding patterns based on covariates describing the local chromatin environment. It was found that DNase I hypersensitivity and tetra-acetylation of H4 were the most influential covariates in predicting changes in HSF binding affinity. The extent was investigated to which DNA accessibility, as measured by digital DNase I footprinting data, could be predicted from MNase-seq data and the ChIP-chip profiles for many histone modifications and TFs. GAGA element associated factor (GAF), tetra-acetylation of H4, and H4K16 acetylation were found to be the most predictive covariates. Lastly, an unbiased model was generated of HSF binding sequences, which revealed distinct biophysical properties of the HSF/HSE interaction and a previously unrecognized substructure within the HSE. These findings provide new insights into the interplay between the genomic sequence and the chromatin landscape in determining transcription factor binding intensity (Guertin, 2010).

The PB-seq technique combined with EMSA and competition assays provides a straightforward, yet versatile and powerful framework for characterizing all potential binding sites in a genome, regardless of tissue specificity, developmental stage, or environmental conditions. Comparing in vitro and in vivo binding profiles, in the context of pre-induction genomic chromatin landscape, revealed DNase I hypersensitivity, H4 tetra-acetylation, and GAF as critical features that modulate cognate element binding intensity in vivo. Furthermore, DNase I sensitivity was found to be strongly influenced by high GAF occupancy and histone acetylation, while repressive factors were minimally influential in the statistical models. Finally, the full set of potential genomic binding sites provided a rich data set that was used to build more detailed sequence models, which tease apart substructure and features that are lost with traditional PSSM models (Guertin, 2010).

One initially surprising observation from this study was that 40% of the in vivo HSF peaks were not found in vitro. It is believed that the limited dynamic range for quantifying in vitro binding affinity may be responsible for the lack of detectable in vitro peaks. Although in vitro binding was quantified over an order of magnitude (40-400 pM), the experimental concentrations of HSF and genomic DNA and the depth of sequencing do not permit the detection of lower affinity HSF binding sites. For instance, only eleven sequence tags would be predicted to underlie a hypothetical 5 nM HSF binding site, and these would not be distinguishable from background. Upon further examination, it was found that the composite HSE representing those in vivo binding sites that were not found in vitro is more degenerate than those found using both assays. Moreover, the in vivo sites that were not found using PB-seq were also more accessible in vivo, in support of the hypothesis. Performing PB-seq at a range of protein and DNA concentrations, or increasing sequence coverage would expand the dynamic range of quantification by PB-seq (Guertin, 2010).

The notion that motif accessibility is driving inducible TF binding in vivo is supported by independent studies of distinct TFs: STAT1, HSF, glucocoticoid receptor (GR), and GATA1. These studies show that the chromatin landscape prior to TF binding influences inducible TF binding. In the one study, it was found that a large fraction of STAT1 induced binding sites contained H3K4me1/me3 marks prior to interferon-gamma (IFN-γ) induced STAT1 binding. Previously studies found that inducible HSF binding sites are marked by active chromatin compared to sites that remain HSF-free. A more recent study has shown that inducibly bound GR sites are marked by DNase I hypersensitive chromatin prior to GR binding. Likewise, the permissive chromatin state at GATA1 binding sites is established even in GATA1 knock out cells. While these correlations are instructive, no previous attempt has been made to model inducible TF binding using biological measurements of chromatin landscape present prior to TF binding. Recent models have successfully inferred TF binding profiles using DNA sequence and chromatin landscape data, generated at the same time the TF is bound. However, these models do not distinguish between the influence TFs have upon local chromatin and the chromatin features that permit TF binding. In contrast, this study modeled the changes between HSF in vitro binding (PB-seq) and in vivo binding (ChIP-seq) landscapes as a function of the non-heat shock chromatin state. This produced a quantitative model describing the important features that modulate the in vivo HSF binding intensity. Moreover, the use of a rules ensemble model enabled the capture of potential interactions between these chromatin features (Guertin, 2010).

This study reveals that DNase I hypersensitivity and acetylation of H4 and H3K9 are strong predictors of inducible HSF binding intensities, however the molecular events and factors that precede TF occupancy to maintain accessible chromatin remain poorly characterized. For instance, the degree to which pioneering factors or flanking DNA sequence, individually or in combination, maintain or restrict accessibility remains unclear. A recent study highlights the biological consequences of maintaining the inaccessibility of TF binding sites, in order to repress expression of tissue-specific transcription factors in the wrong tissues. The authors found that ectopic expression of CHE-1, a zinc-finger TF that directs ASE neuron differentiation, in non-native C. elegans tissue is not sufficient to induce neuron formation (Tursun, 2011). However, combining ectopic CHE-1 expression with knockdown of lin-53 did modify the expression patterns of CHE-1 target genes in non-native tissue, effectively converting germ line cells to neuronal cells (Tursun, 2011). LIN-53 has been implicated in recruitment of deacetylases, and deacetylase inhibitor treatment mimics lin-53 depletion, suggesting that LIN-53 is actively maintaining CHE-1 target sites inaccessible in germ cells (Guertin, 2010).

Alternatively, functional TF binding sites could be actively maintained in the accessible state. HSF binding within ecdysone genes has a functional role in shutting down their transcription, and activating ecdysone-inducible genes containing inaccessible HSEs causes chromatin changes that are sufficient to allow HSF binding. In this special case of HSF-bound ecdysone genes, active transcription and the corresponding histone marks are mediating access to HSEs, in order for HSF to bind and repress transcription upon heat shock. A more recent study has shown that activator protein 1 (AP1) actively maintains chromatin in the accessible state, so that Glucocorticoid receptor can bind to cognate elements (Guertin, 2010).

Although TF accessibility to critical genomic sites appears to be actively maintained, many binding sites may be a non-functional result of fortuitous TFBS recognition. It has long been hypothesized that the binding affinities for TF/DNA interactions are sufficiently strong to allow promiscuous binding at the cellular concentrations of TFs and DNA. There are roughly 32,000 HSF molecules per tetraploid S2 cell and the dissociation constants for trimeric-HSF/HSE interactions are in the picomolar range; therefore much of the in vivo HSF binding may be non-functional promiscuous binding. Additional investigation will further illuminate the role of chromatin context in TF binding and the mechanisms by which programmed developmental or environmental chromatin changes permit or deny TF binding (Guertin, 2010).

Elucidating the rules that govern accessibility is essential for predicting in vivo occupancy of TFs. Diverse transcription factors, from a broad spectrum of organisms, bind their sequences based on site accessibility. This study found that chromatin accessibility as measured by DNase I hypersensitivity could be inferred using ChIP-chip data for various histone modifications and transcription factors. Although the model can infer accessibility based on chromatin composition, the mechanism by which accessibility originates is not addressed. Previous studies have shown that activators, such as HSF, glucocorticoid receptor, and androgen receptor bind to their cognate sites and direct a concomitant increase in local acetylation, DNase I hypersensitivity, and nucleosome depletion. Androgen receptor also acts to position flanking nucleosomes marked by H3K4me2. These post-TF binding chromatin changes that occur are the result of acetyltransferase and nucleosome remodeler recruitment, both of which functionally interact with activators. For instance, both GR and GATA1 interact with the nucleosome remodeling complex Swi/Snf. Concomitant increases in locus accessibility likely allow large molecular complexes such as RNA Pol II and coactivators to access the region that in turn can reinforce and maintain active and accessible chromatin (Guertin, 2010).

Thorough biophysical characterization of TF binding site properties is critical for accurate predictions of TF binding sites, underscoring the need for more complete models of TF binding. While the commonly used position-specific scoring matrix model makes the assumption of base independence, recent work has revealed that richer models providing for interactions between positions are necessary. The current model captures critical features of the HSF/HSE interaction that are lost with simpler computational models, namely the interdependencies between the sub-binding sites of each HSF monomer. Consistent with this model, a series of in vitro experiments with S. cerevisiae, D. melanogaster, A. thaliana, H. sapien and D. rerio HSFs indicate that HSF from each of these species can bind to discontinuous HSEs containing canonical pentamers that contain intervening five base pair gaps; interestingly, however, C. elegans HSF strictly binds to continuous HSEs that do not contain gaps. The complex interactions between positions within a binding site are a critical aspect of inferring whether a polymorphism or mutation affects TF binding. These features should prove useful in providing degenerate HSE sequences for optimal co-crystallization of trimeric HSF and DNA and inferring changes in DNA sequence that affect HSF binding within and between species (Guertin, 2010).

In conclusion, the data and models presented in this study reinforce both the importance of chromatin landscape in modulating in vivo TF binding intensity and how genome wide, chromatin free, binding assays contribute to the understanding of TF sequence binding specificity (Guertin, 2010).

HSF multimerization

The heat shock transcription factor HSF activates expression of its target genes in response to elevated temperatures and chemical or physiological stress. A key step in the activation process involves the formation of HSF homotrimers, leading to high-affinity DNA binding. The mechanism by which HSF trimerization and DNA binding are regulated by stress signals has remained elusive. Trimerization and DNA binding of purified Drosophila HSF can be directly and reversibly induced in vitro by heat shock temperatures in the physiological range and by an oxidant, hydrogen peroxide. Other inducers of the heat shock response, including salicylate, dinitrophenol, ethanol, and arsenite, have no effect on HSF trimerization in vitro, indicating that these inducers act by indirect mechanisms. How might the direct action of heat of hydrogen peroxide enhance the formation of HSF trimers? Trimerization is mediated by the N-terminal heptad repeats whose propensity to associate as a three-stranded alpha-helical coiled-coil is repressed under non-shock conditions by the C-terminal region of HSF. The occurrence of a conserved hydrophobic heptad repeat in this region has led to the suggestion that repression could involve an intramolecular coiled-coil,formed between the N-terminal and C-terminal heptad repeats. Heat or oxidation is likely to induce a conformational change that unmasks the hydrophobic heptad repeats of the trimerization domain, thereby allowing assembly of the trimeric coiled-coil. The active sites of sensitivity to heat or oxidation on HSF are as yet undefined. These findings also imply that other heat shock inducers (such as heat shock proteins known to associate with HSF) exert secondary effects indirectly, perhaps by generating an intermediate redox signal, or by the action of cellular factors (Zhong, 1998).

The minimal DNA sequence required for the formation of a stable complex with Drosophila heat shock factor (HSF) in vitro is an inverted repeat of a 5 bp recognition unit, -GAA-. Surprisingly, both permutations of this 5 bp unit, head-to-head and tail-to-tail, bind to HSF with similar affinity and with striking 2-fold symmetry. HSF also binds to longer arrays of inverted 5 bp units, and the size of the HSF footprint increases with the addition of each 5 bp unit to these arrays. However, the electrophoretic mobility of the HSF-DNA complexes decreases most distinctly with the addition of every three 5 bp units. Cross-linking of purified HSF in the absence of DNA generates complexes with the sizes expected of HSF trimers. It is proposed that trimers of HSF bind to DNA and that the number of HSF subunits in direct contact with DNA is determined by the number of correctly positioned 5 bp recognition units (Perisic, 1989).

Activation of HSF occurs at two independent levels, DNA binding and the acquisition of transcriptional competence. The binding of HSF to DNA is accomplished by a stress-induced oligomeric switch of HSF protein. Calculation of the native molecular mass indicates that the two forms of Drosophila HSF are best described as a monomer and trimer, respectively, of the 77-kDa HSF polypeptide. The monomeric and trimeric states of HSF were verified by chemical cross-linking experiments. The finding of a monomeric composition for the latent form of HSF is incompatible with speculative models that suggest that molecular chaperones such as hsp70 feedback to inhibit trimerization of HSF by forming a stable heteromeric complex. Both HSF monomers and HSF trimers exhibit unusually high frictional ratios, indicating that they have asymmetric shapes. The degree of asymmetry is significantly greater for the HSF trimer, suggesting that the monomer undergoes a conformational change to a more extended structure upon trimerization. These findings are consistent with a model for the inert HSF protein that is based on a monomer constrained by intramolecular coiled-coil interactions between amino- and carboxy-terminal domains (Westwood, 1993).

The intracellular level of free heat shock proteins, in particular the 70-kDa stress protein family, has been suggested to be the basis of an autoregulatory mechanism by which the cell measures the level of thermal stress and regulates the synthesis of heat shock proteins. It has been proposed that the DNA-binding and oligomeric state of the heat shock transcription factor (HSF) is a principal step in the induction pathway that responds to the level of 70-kDa stress protein. The association between HSF and 70-kDa stress protein has been investigated by means of a coimmunoprecipitation assay. 70-kDa stress proteins associate to similar extents with both latent and active forms of HSF, although unlike other 70-kDa stress protein substrates, the association with HSF is not significantly disrupted in the presence of ATP. Gel mobility shift assays indicate that active HSF trimers purified from a bacterial expression system can not be substantially deactivated in vitro with purified 70-kDa stress protein and ATP. In addition, elevated concentrations of hsp70 alone can not significantly inhibit induction of the DNA-binding activity of endogenous HSF in cultured rat cells, and the induction is also not inhibited in cultured rat cells or Drosophila cells containing elevated levels of all members of the heat shock protein family. However, the deactivation of HSF to the non-DNA-binding state after prolonged heat stress or during recovery could be accelerated by increased levels of heat shock proteins. Hence, the level of heat shock proteins may affect the rate of disassembly of HSF trimers, but another mechanism, as yet undefined, appears to control the onset of the oligomeric transitions (Rabindran, 1994).

Heat shock factor (HSF), the transcriptional activator of eukaryotic heat shock genes, is induced to bind DNA by a monomer to trimer transition involving leucine zipper interactions. Although this mode of regulation is shared among many eukaryotic species, there is variation in the temperature at which HSF binding activity is induced. The response of a human HSF expressed in Drosophila cells and Drosophila HSF expressed in human cells was investigated. The temperature that induces DNA binding and trimerization of human HSF in Drosophila is decreased by approximately 10 degrees C to the induction temperature for the host cell, whereas Drosophila HSF expressed in human cells is constitutively active. The results indicate that the activity of HSF in vivo is not a simple function of the absolute environmental temperature (Clos, 1993).

The heat shock transcription factor (HSF) mediates the induction of heat shock gene expression. The activation of HSF involves heat shock-induced trimerization, binding to its cognate DNA sites, and the acquisition of transcriptional competence. In this study, the oligomeric properties of Drosophila HSF were analyzed by equilibrium analytical ultracentrifugation and gel filtration chromatography. Previous findings have shown that trimerization of purified Drosophila HSF is directly sensitive to heat and oxidation. Low pH, in the physiological range, also directly induces HSF trimerization and DNA binding in vitro. Furthermore, the induction of HSF trimerization by low pH is synergistic with the actions of heat and oxidation. Since heat or chemical stress leads to a moderate decrease of intracellular pH, it is suggested that intracellular acidification may contribute to activating the heat shock response in vivo (Zhong, 1999).

Protein interactions at heat shock gene promoters

A number of activators are known to increase transcription by RNA polymerase (pol) II through protein acetylation. While the physiological substrates for those acetylases are poorly defined, possible targets include general transcription factors, activator proteins and histones. Using a cell-free system to reconstitute chromatin with increased histone acetylation levels, a direct test was performed for a causal role of histone acetylation in transcription by RNA pol II. Chromatin, containing either control or acetylated histones, was reconstituted to comparable nucleosome densities and characterized by electron microscopy after psoralen cross-linking, as well as by in vitro transcription. Chromatin was reconstituted using histones from either TSA-treated CV-1 cells, which accumulates hyperacetylated histone isoforms, or from untreated cells, in which the histones are primarily non-acetylated onto a 7.75 kb plasmid containing an hsp26 minigene. This process involves the prior depletion of the endogenous histones present in the chromatin assembly extract such that chromatin is assembled quantitatively from the input, exogenous histones. The chromatin assembly reaction generates complex chromatin containing many non-histone proteins and enzymatic activities. While H1-containing control chromatin severely represses transcription of a model hsp26 gene, highly acetylated chromatin is significantly less repressive. Acetylation of histones, and particularly of histone H4, affects transcription at the level of initiation (Nightingale, 1998).

The regulatory elements of the hsp26 promoter are well-known from in vivo and in vitro studies. A proximal regulatory element includes the TATA box, proximal heat shock element (HSE) and an adjacent GAGA element, while a distal regulatory site corresponds to the distal HSE and GAGA binding sites. Monitoring the ability of the transcription machinery to associate with the promoter in chromatin, it was found that Heat shock factor, a crucial regulator of heat shock gene transcription, profits most from histone acetylation. Templates with mutated hsp26 promoters were assembled into control and acetylated chromatin and analysed for their transcription potential. A template bearing only the TATA box supports a very low level of transcription, even in the absence of chromatin; there is no discernible transcription from chromatinized templates even after prolonged exposure. Addition of the proximal HSEs however, results in significantly increased transcription from both mock assembled and chromatinized templates, confirming the important role of the activator HSF. This minimal promoter, containing only proximal HSEs and the TATA box, clearly shows increased transcription from the acetylated template. The addition of GAGA elements to the promoter enhances transcription significantly, but to a similar degree on control and acetylated chromatin templates. Interestingly, the GAGA elements do not increase transcription in the mock assembled control, confirming that GAGA factor is involved in overcoming chromatin-mediated transcriptional repression, but by a mechanism that does not profit from histone acetylation. These results suggest that the HSE and TATA box are the significant sequence elements for the increased transcription observed in acetylated chromatin. Thus histone acetylation can modulate activator access to their target sites in chromatin, and provide a causal link between histone acetylation and enhanced transcription initiation of RNA pol II in chromatin (Nightingale, 1998).

The generation of an accessible heat shock promoter in chromatin in vitro requires the concerted action of the GAGA transcription factor and NURF, an ATP-dependent nucleosome remodeling factor. NURF is composed of four subunits and is biochemically distinct from the SWI2/SNF2 multiprotein complex, a transcriptional activator that also appears to alter nucleosome structure. The 140 kDa subunit of NURF can be identified as ISWI, previously of unknown function but highly related to SWI2/SNF2, but only in the ATPase domain. The ISWI protein is localized to the cell nucleus and is expressed throughout Drosophila development at levels as high as 100,000 molecules/cell. The convergence of biochemical and genetic studies on ISWI and SWI2/SNF2 underscores these ATPases and their close relatives as key components of independent systems for chromatin remodeling (Tsukiyama, 1995a and b).

Genetic control elements are usually situated in local regions of chromatin that are hypersensitive to structural probes such as DNase I. Binding of the GAGA transcription factor on existing nucleosomes leads to nucleosome disruption, DNase I hypersensitivity at the TATA box and heat-shock elements, and rearrangement of adjacent nucleosomes. ATP hydrolysis facilitates this process, suggesting that an energy-dependent pathway is involved in chromatin remodeling (Tsukiyama, 1994).

Drosophila heat shock factor (HSF) binds to specific sequence elements of heat shock genes and can activate their transcription 200-fold. Though HSF has an acidic activation domain, the mechanistic details of heat shock gene activation remain undefined. HSF interacts directly with the general transcription factor TBP (TATA-box binding protein), and these two factors bind cooperatively to heat shock promoters. A third factor that binds heat shock promoters, GAGA factor, also interacts with HSF and further stabilizes HSF binding to heat shock elements (HSEs). The interaction of HSF and TBP is explored in some detail here and is shown to be mediated by residues in both the amino- and carboxyl-terminal portions of HSF. This HSF/TBP interaction can be disrupted specifically by competition with the potent acidic transcriptional activator VP16. The acidic domain of the largest subunit of Drosophila RNA polymerase II (Pol II) associates with TBP in vitro and is specifically displaced from TBP upon addition of HSF. The region of TBP that mediates both HSF and Pol II acidic domain binding maps to the conserved carboxyl-terminal repeats and depends on at least one of the TBP residues known to be contacted by VP16 and to be critical for transcription activation. It is thought that HSF triggers hsp70 transcription by freeing the hsp70 promoter-paused Pol II from the constraints on elongation caused by the affinity of Pol II for general transcription factors (Mason, 1997).

Heat shock regulates the level of phosphorylated histone H3

Posttranslational modifications of the N-terminal tails of the core histones within the nucleosome particle are thought to act as signals from the chromatin to the cell for various processes. The experiments presented here show that the acetylation of histones H3 and H4 in polytene chromosomes does not change during heat shock. In contrast, the global level of phosphorylated H3 decreases dramatically during a heat shock, with an observed increase in H3 phosphorylation at the heat shock loci. Additional experiments confirm that this change in phosphorylated H3 distribution is dependent on functional heat shock transcription factor activity. These experiments suggest that H3 phosphorylation has an important role in the induction of transcription during the heat shock response (Nowak, 2000).

The acetylation of the N-terminal tails is the best-studied modification of the core histones. Several transcription factors, such as GCN5, and the TAF_II250 subunit of TFIID, as well as subunits of the RNA polymerase complex show intrinsic histone acetyltransferase (HAT) activity, which suggests a potential role for histone acetylation in either the activation or maintenance of transcription. The acetylation of the N-terminal tail domains of core histones H3 and H4 at various lysine residues is essential for the normal implementation of various cellular processes, such as promoter-transcription factor association, gene transcription, and dosage compensation (Nowak, 2000 and references therein).

Phosphorylation of serine 10 of the N-terminal arm of histone H3 has been shown to be essential for proper mitotic chromosomal condensation and segregation. In addition, recent studies have outlined the possibility that histone H3 phosphorylation may have a role in the regulation of transcription. Ser 10 H3 phosphorylation is found to rapidly increase in quiescent cells during mitogenic stimulation, as well as during immediate-early gene induction via the epidermal growth factor (EGF)-signaling pathway. In addition, recent experiments performed in vitro have suggested that EGF-stimulated H3 phosphorylation may act as a signal for histone acetyltransferase binding and subsequent acetylation of a particular locus during transcription initiation (Nowak, 2000 and references therein).

Acetylation of core histones H3 and H4 at lysines 14 and 8, respectively, has been linked to gene transcription. In addition, deacetylation of core histones is thought to have a role in silencing specific loci. Because of the near-total repression of cellular gene products during a heat shock, it might be expected that the distribution of acetylated H3 and H4 would radically change during thermal stress in a manner reflective of the transcriptional profile of the cell. Because acetylation of H3 at Lys 14 of the N-terminal arm has been described as essential for transcription, the distribution of acetylated H3 was examined by staining polytene chromosomes with an antibody specific for Lys 14 acetylated histone H3. Lys 14 acetylated H3 staining is observed at the puffs, which are active sites of transcription in polytene chromosomes, and distributed throughout the chromosomes in discrete bands before heat shock. One locus, subdivision 62A, which becomes puffed during larval development in response to ecdysone, is intensely labeled with the Lys 14 acetylated H3 antibody. In addition, other chromosomal subdivisions such as 89B display Lys 14 acetylated H3 staining but are not puffed before heat shock. The chromosomal subdivision 93D, which is known to become puffed during heat shock, is Lys 14 acetylated but not puffed before heat shock. Examination of polytene chromosomes from larvae that were subjected to a 20-min heat shock shows that the 87A and 87C heat shock puffs, which contain the hsp70gene cluster, are stained by the anti-Lys 14 acetylated antibody, although the staining at these puffs appears to be less intense and rather diffuse. This might not represent a reduction in the level of acetylation, but rather a decrease in signal intensity due to the large puffing at the heat shock loci. After heat shock, the overall number of discrete stained bands does not appear to change significantly and regions that were stained before heat shock, such as 89B, remain acetylated. Loci with acetylated H3 staining that were puffed before heat shock, such as 62A, are no longer puffed after heat shock but remain acetylated. The observation that the heat shock genes are acetylated before heat shock, at a time when they are not transcribed, and non-heat shock genes, which are not transcribed during heat shock, are acetylated during heat shock, suggests that the presence of Lys 14-acetylated H3 does not necessarily denote an actively transcribed locus (Nowak, 2000).

Examination of H3 acetylation during EGF stimulation raises the issue that antibodies against Lys 14 acetylated H3 may show decreased recognition of their epitope when other modifications, such as phosphorylation, coexist on the same histone tail. This problem can be overcome by using antibodies against histone H3 acetylated at lysines 9 and 14. To ensure that these results were not caused by this potential artifact, the distribution of hyperacetylated H3 was examined using antibodies against H3 acetylated at lysines 9 and 14 on the N-terminal tail before and after heat shock. The results suggest that the distribution of diacetylated H3 is similar to the distribution of Lys 14 acetylated H3 before and after heat shock. Diacetylated H3 staining appears to be more widespread than monoacetylated staining, which is probably caused by the antibody's recognition of acetylation of H3 at lysine 9. The intensity of staining of the Lys 9,14-acetylated H3 antibody at several of the heat shock puffs examined appears to be similar to that observed with the Lys 14-acetylated H3 antibody. These results suggest that the diffuse staining at the heat shock puffs is not an artifact attributed to the masking of the acetylated Lys 14 epitope by Ser 10 phosphorylation (Nowak, 2000).

H4 acetylation was also examined using antibodies specific for Lys 8-acetylated histone H4 to stain polytene chromosomes isolated from third instar larvae. The distribution of Lys 8 acetylated histone H4 is similar to that of acetylated H3, with H4 acetylation observed in discrete bands in nonpuffed regions, such as subdivision 89B, and at ecdysone-induced puffed regions, such as 62A, before heat shock. Chromosomal subdivisions 87A and 87C, which contain the hsp70 heat shock genes, are acetylated before and after heat shock. Similar to acetylated H3, heat shock does not significantly affect the observed distribution of Lys 8 acetylated H4 in polytene chromosomes. Taken together, the above results suggest that the acetylation state of H3 and H4 does not change substantially during heat shock and that a gene locus can be acetylated when it is not actively transcribed (Nowak, 2000).

The absence of a drastic change in H3 acetylation during heat shock is rather surprising, given current models that indicate that H3 acetylation is a crucial step in transcription initiation. This would lead to the expectation that the heat shock loci would not be acetylated before heat shock and should become intensely acetylated during thermal stress. To determine if other histone modifications occur during the heat shock response, whether changes in histone H3 phosphorylation occur after temperature elevation was examined. Stimulation of quiescent cells with EGF leads to rapid and transient phosphorylation of histone H3 at Ser 10 of the N-terminal arm in vivo. This EGF-mediated phosphorylation of H3 is targeted to a small subpopulation of total histone H3 that is acetylated at the Lys 14 position. In addition, in vitro studies have shown that phosphorylated H3 may serve as an affinity-increasing substrate for HAT activity in H3 acetylation, which raises the possibility that phosphorylation may be tied to transcription. If histone phosphorylation were implicated in transcription, then the distribution of phosphorylated H3 might change in response to heat shock and would most likely be localized primarily to the heat shock puffs while disappearing from other loci after heat shock. Because histone H3 phosphorylation is a robust marker for mitotic cells, analysis of the distribution of phosphorylated H3 in polytene chromosomes, rather than isolation of phosphorylated H3 from whole cell extracts, allows for the examining of phosphorylation of H3 in a nonmitotic environment. To examine whether the heat shock-induced puffs contain N-terminal phosphorylated H3 molecules, polytene chromosomes were stained with antibodies specific for Ser 10 phosphorylated histone H3. Before heat shock, phosphorylated H3 staining is found in discrete bands throughout the chromosomes, with the most intense staining observed in the naturally occurring ecdysone-induced developmental puffs. After a 20-min heat shock at 37°C, phosphorylated H3 staining is not distributed throughout the chromosomes but is instead concentrated at a few specific sites. The most prominent of these regions corresponds to chromosomal divisions 63BC, 67B, and 87AC. These regions contain the hsp83 gene, the hsp22, hsp23, hsp26, and hsp27 gene cluster, and hsp70 gene clusters, respectively. These regions become reproducibly puffed during the heat shock response. Although in some chromosomes examined there are several non-heat shock loci that remain slightly phosphorylated during heat shock, the intensity of staining at these regions is much lower than the staining observed at the heat shock loci (Nowak, 2000).

The regions of the chromosome where the heat shock genes are located do not contain histone H3 phosphorylated at Ser 10 before heat shock. After temperature elevation, the only puffs that possess phosphorylated histone H3 are the heat shock puffs. The appearance of phosphorylated histone H3 in the heat shock puffs, accompanied by the nearly complete reduction of staining at all other loci during heat shock, leads to the conclusion that the presence of the Ser 10 phosphorylated isoform of histone H3 might be required for the transcriptional activation of the heat shock genes (Nowak, 2000).

Induction of the heat shock genes and cessation of normal gene expression is rapid and reproducible in response to heat shock. Transcription run-on assays reveal that after only 1 min at 37°C, the levels of many normal cellular gene transcripts have greatly diminished, with the heat shock gene transcripts dominating the population of total mRNA in the cell. Following a heat shock, the normal pattern of gene expression within the cell is restored gradually over time. Therefore an examination was made of the change in phosphorylated histone H3 staining over time during and after heat shock, to determine whether or not the appearance of phosphorylated H3 closely parallels the induction of transcription of the heat shock genes and whether or not the non-heat shocked H3 distribution might be restored following recovery from heat shock. After only 1 min at 37°C, there is a noticeable change in the distribution of Ser 10 phosphorylated H3. The level of global H3 phosphorylation decreases, with several regions remaining intensely phosphorylated. Within 5 min of incubation at 37°C, many of the less intense regions of staining have disappeared. After 10 min at 37°C, the only remaining intense regions of staining are those at the heat shock puffs. When larvae were allowed to recover at room temperature from a 20-min heat shock at 37°C, H3 phosphorylation reappears in several non-heat shock loci after 10 min of recovery. After 30 min of recovery from heat shock, the number and distribution of loci that contained phosphorylated H3 appears to be virtually indistinguishable from normal (i.e., non-heat shocked) chromosomes. This restoration of the normal (non-heat shocked) H3 phosphorylation pattern closely mimics previously described restoration of normal gene expression in cells experiencing thermal stress (Nowak, 2000).

During heat shock, the heat shock transcription factor (HSF) rapidly trimerizes in solution, localizes to the heat shock loci, binds to heat shock response promoter elements (HSEs), and induces the expression of the heat shock gene products. The appearance of phosphorylated H3 at the heat shock loci could therefore be due to HSF recruitment of a specific histone kinase on binding to the HSEs of the heat shock genes. To test this hypothesis, the staining pattern of phosphorylated H3 was examined in polytene chromosomes isolated from hsf⁴-mutant larvae, which lack functional HSF at restrictive temperatures and do not respond to thermal stress. Before heat shock, the distribution of phosphorylated H3 in hsf⁴-mutant chromosomes is similar to wild-type chromosomes, with staining observed in discrete bands and at the developmental puffs. In contrast to wild-type chromosomes, histone H3 at the heat shock loci does not become phosphorylated in hsf⁴-mutant chromosomes during heat shock, which suggests that phosphorylation of histone H3 at the heat shock loci depends on functional HSF activity. In addition, no H3 phosphorylation was detected in the rest of the genome during heat shock in hsf⁴ mutants, suggesting that repression of normal transcription and loss of H3 phosphorylation at non-heat shock loci does not require the presence of an active HSF protein (Nowak, 2000).

To determine if the loss of the HSF transcription factor could also alter the distribution of acetylated H3 and H4 during heat shock, acetylation of each of these histones was examined in hsf⁴-mutant polytene chromosomes. The distribution of Lys 14 acetylated histone H3 before and after heat shock in hsf⁴ mutants was indistinguishable from the wild-type distribution, with staining observed at both the developmental puffs and nonpuffed regions. H3 acetylation was observed at the 87A and 87C chromosomal subdivisions, which normally are puffed during heat shock but these regions do not become puffed in hsf⁴-mutant chromosomes. Examination of acetylated H3 using antibodies for Lys 9- and Lys 14-acetylated H3 shows a pattern similar to that observed for the Lys 14 acetylated H3 antibody. In addition, H4 acetylation does not change after heat shock in hsf⁴ mutants. Because the heat shock genes are not induced in hsf⁴ mutants during thermal stress and because hsf⁴-mutant chromosomes are acetylated, but not phosphorylated after heat shock, it is concluded that H3 phosphorylation, and not acetylation, depends on the presence of a functional heat shock transcription factor (Nowak, 2000).

How might acetylation and phosphorylation of histones H3 and H4 work together to promote transcription of a particular gene? The data suggest that acetylated histones might define a particular locus that is primed for possible phosphorylation and subsequent transcription. This acetylated locus would attract transcription factors that interact with the acetylated residues on histones H3 and H4, known to be essential for proper association of several transcription factors with their promoters. Once bound to this locus, the transcription factor would then recruit a particular histone, which phosphorylates Ser 10 of the N-terminal arm of histone H3. The most logical site of phosphorylation would be an H3 molecule with a Lys 14 acetylated N-terminal arm, a species that has been shown to exist in vivo. The presence of this dimodified H3 would define that locus as 'active' for transcription (Nowak, 2000).

There are several kinases known to localize to specific loci on polytene chromosomes that phosphorylate H3 in vitro, such as JIL-1 on the X chromosome and P-TEFb kinase at the heat shock loci (Lis, 2000). This raises the possibility that the specificity of a kinase for activation of a particular gene through H3 phosphorylation might be regulated by the specific transcription factors that control expression of this gene. It has yet to be determined whether phosphorylation of H3 is required for assembly of the RNA polymerase II complex or if phosphorylation is a by-product of complex formation and polymerase procession during transcription. If phosphorylation of H3 were indeed the critical step in activating gene transcription, then a reasonable hypothesis is that deactivation of a particular gene would be dependent on either regulated or unregulated phosphatase activity to remove the activating phosphate group from the N-terminal tails of H3. The disappearance of phosphorylated H3 at nontranscribing loci and appearance of phosphorylated H3 at actively transcribing loci during heat shock suggests that a functional transcription complex might actively maintain the phosphorylated state of histone H3, which would be subject to ready dephosphorylation by either passive or regulated phosphatase activity in a nontranscribing state (Nowak, 2000).

P-TEFb kinase promotes transcription at heat shock loci

P-TEFb kinase recruitment to heat shock loci during the heat shock response and functions to stimulate promoter-paused RNA polymerase II (Pol II) to enter into productive elongation. P-TEFb is located at >200 distinct sites on Drosophila polytene chromosomes. Upon heat shock, P-TEFb, like the regulatory factor heat shock factor (HSF), is rapidly recruited to heat shock loci, and this recruitment is blocked in an HSF mutant. Yet, HSF binding to DNA is not sufficient to recruit P-TEFb in vivo, and HSF and P-TEFb immunostainings within a heat shock locus are not coincident. Insight to the function of P-TEFb is offered by experiments showing that the direct recruitment of a Gal4-binding domain P-TEFb hybrid to an hsp70 promoter in Drosophila cells is sufficient to activate transcription in the absence of heat shock. Analyses of point mutants show this P-TEFb stimulation is dependent on Cdk9 kinase activity and on Cdk9's interaction with cyclin T. These results, coupled with the frequent colocalization of P-TEFb and the hypophosphorylated form of Pol II found at promoter-pause sites, support a model in which P-TEFb acts to stimulate promoter-paused Pol II to enter into productive elongation (Lis, 2000).

P-TEFb is required to produce full-length transcripts from a variety of cellular DNA templates in an in vitro transcription system that accurately recapitulates the normal DRB-sensitive transcription seen in cells (Marshall, 1995). These results suggest that P-TEFb may have a role in transcription of many cellular genes. If so, this kinase may localize to chromosomal loci that possess genes that are the target of its activity. The chromosomal distribution of P-TEFb was examined by staining salivary gland polytene chromosomes with a highly specific antibody to the cyclin T regulatory subunit. This cyclin T subunit binds tightly to Cdk9 and is a critical component of the P-TEFb activity (Peng, 1998a). Moreover, immunodepletion experiments show that the vast majority of Cdk9 is associated with a cyclin T subunit (Peng, 1998a), and probing of phosphocellulose fractions from Drosophila Kc cell nuclear extracts indicates that cyclin T is present only where P-TEFb activity is found. Therefore, the cyclin T antibody provides a good means of tracking the P-TEFb complex (Lis, 2000).

Heat shock causes a rapid and dramatic activation of transcription of heat shock genes and a concomitant reduction in transcription of many normally expressed genes. Immunofluorescence analysis of polytene chromosomes reveals that Pol II relocates to heat shock loci after a brief heat shock. P-TEFb distribution also changes dramatically following heat shock. In uninduced larvae, P-TEFb is undetectable at major heat shock loci 87A and 87C, which contain the five hsp70 genes, or at 59B, which, in this strain, contains an hsp70-lacZ transgene. After a 20-min heat shock, these and all the other major heat shock loci at 63B, 67B, 93D, and 95D are the prominent sites of labeling. Loci that had high levels of P-TEFb before heat shock now have a reduced level. Therefore, P-TEFb redistributes to heat shock loci following heat shock (Lis, 2000).

The Pol II level on the 5' end of the hsp70 gene begins to be elevated in as little as 70 sec following a very rapid heat shock induction (mixing cells with warm medium), and Pol II is detected beyond the pause region and in the middle of the gene in as little as 2 min. This rapid transcriptional activation leads to a very high density of hyperphosphorylated Pol II on these genes. Could P-TEFb be playing a role in the transition of Pol II to its hyperphosphorylated, elongationally competent mode? If so, then one might expect P-TEFb to be recruited as rapidly as Pol II to these newly activated heat shock sites (Lis, 2000).

The kinetics of localization to heat shock loci at 87A and 87C and to 59B, which in this strain contains an Hsp70-lacZ transgene, were examined. No P-TEFb is detected at the native or the transgenic sites before heat shock. However, within 2 min, staining is apparent at 87A and 87C, each of which contain multiple copies of hsp70. Some staining is also detectable at the transgenic copy of Hsp70-lacZ. By 5 min of heat shock, staining at all heat shock loci is strong and this high level persists and may even increase in the 10- and 15-min time points. The level remains high during heat shock measured out to 60 min. A shift back to normal fly culture temperature (e.g., a 60-min recovery) reduces heat shock gene transcription and the normal pattern transcription is largely re-established (Lis, 2000).

The recruitment of P-TEFb to heat shock loci is completely dependent on HSF. A Drosophila temperature-sensitive mutant HSF strain, hsf4, shows a much reduced induction of heat shock gene transcription and chromosome puffing. In this strain, heat shock fails to concentrate P-TEFb at heat shock loci. Additionally, heat shock does not lead to a dramatic loss of P-TEFb at the normally active chromosomal sites in the HSF mutant strain as exemplified at 88D (Lis, 2000).

Heat shock rapidly stimulates the trimerization and binding of HSF to the heat shock elements (HSEs) located upstream of every heat shock gene. HSF acquires strong DNA-binding activity and localizes to heat shock loci on polytene chromosomes within 2 min following heat shock. Therefore, the rapid induction of HSF binding is similar to the rapid recruitment of P-TEFb seen here. Could HSF itself be sufficient to recruit P-TEFb through a stable interaction? This hypothesis was tested in vivo using a transgenic line containing a polymer of native HSF-binding sites that are unlinked to the rest of the hsp70 promoter. Following heat shock, HSF is known to localize to sites on polytene chromosomes containing this polymer. This anti-HSF staining is more than an order of magnitude stronger than that seen at the regulatory region of a single hsp70 gene, and can be compared with the 87A and 87C loci that contain two and three copies of native hsp70, respectively. The 87C signal is considerably stronger than 87A (Lis, 2000).

If HSF is sufficient to recruit P-TEFb to heat shock loci in vivo, then one would expect to see high levels of P-TEFb at the polymer site. There is detectable P-TEFb at the polymer site, but the level is less than at the native heat shock loci 87A and 87C. Moreover, the ratio of P-TEFb to HSF staining is much higher at heat shock genes than at the polymer site. These results indicate that HSF does not on its own recruit P-TEFb, and other features of the heat shock promoters are required to provide P-TEFb's strong recruitment to heat shock genes (Lis, 2000).

P-TEFb appears to resolve from HSF at the 87A locus. In most extended chromosomes examined, it is observed that the P-TEFb label separates into a doublet with HSF overlapping and falling between the peaks of the P-TEFb doublet. This can be interpreted in terms of the known arrangement of hsp70 genes at 87A. The hsp70 genes are divergently transcribed and the regulatory DNA containing the binding sites for HSEs resides in this region between the genes. HSF binds these regulatory regions as was seen from a band of fluorescence in the middle of the puff. In contrast, the centers of P-TEFb staining appear to reside downstream of the HSEs on both copies of the hsp70 gene. The partial separation of P-TEFb and HSF is also consistent with the idea that P-TEFb does not derive its stable association with heat shock genes solely through interaction with HSF (Lis, 2000).

A biochemical assay of the interaction of HSF and P-TEFb adds further support to the conclusion that these proteins do not interact strongly. Plasmids that express HSF, Cdk9-Flag, and cyclin T-6His were cotransfected into Drosophila cells. Following a standard heat shock treatment, cleared lysates were prepared from these cells, and the lysates were then chromatographed over nickel-NTA beads, which bind the 6His-tag. Portions of the lysates and nickel-bound fractions were then examined by Western blotting using HSF or Flag antibodies. Whereas Cdk9 is efficiently recovered in the Ni-bound fraction, HSF is not recovered at levels exceeding the background from cells lacking cyclin T-6His. These results and the in vivo results indicate that the high levels of P-TEFb association with heat shock loci cannot be explained by an interaction of HSF with P-TEFb (Lis, 2000).

Does the redistribution of P-TEFb to heat shock loci influence transcription of the heat shock genes? The effects of directly recruiting P-TEFb subunits, Cdk9 or cyclin T, to the hsp70 promoter were tested. A pair of Gal4-binding sites (UASgal) was introduced upstream of a Drosophila hsp70-M reporter gene. The expression of this hybrid reporter gene can be distinguished from native hsp70 genes since it is marked by fusion to a bacterial DNA sequence. This reporter construct and copper-inducible expression vectors, which express the Gal4 DNA-binding alone (G4) or G4 fused to Cdk9, cyclin T and a variety of controls, were cotransfected into Drosophila cells. The inserted UASgal sequences are upstream of the regions critical for heat shock expression, so, as anticipated, transcription of this reporter gene is heat inducible, albeit at about a twofold lower level than the control containing no UASgal insert. The reporter gene containing UASgal sites is strongly activated without heat shock when cells are cotransfected with G4 fused to the activation domain of HSF (G4-HSF). The reporter gene carrying the UASgal sites is also strongly activated without heat shock when cells are cotransfected with plasmids expressing G4-Cdk9 or G4-cyclin T. A point mutation that disrupts the activity of the kinase subunit, Cdk9/D199N (Peng, 1998a), also disrupts the ability of the G4-Cdk9 hybrid protein to activate transcription from the hsp70 reporter. The levels of expression of wild-type and mutant G4-Cdk9 are similar. Also, a pair of mutations in cyclin T that disrupt its ability to interact with Cdk9, the double point mutant CycT/2XMut (Bieniasz, 1999), greatly impairs the ability of G4-cyclin T to activate transcription. These results demonstrate that artificially recruiting P-TEFb to the promoter by directly recruiting either of its two subunits is sufficient to strongly activate an hsp70 gene. A similar activation by G4-HSF, G4-Cdk9, and G4-cyclin T was observed with UASgal sequences inserted further upstream at -256, although the level of activation was reduced two to threefold (Lis, 2000).

A model is proposed in which P-TEFb acts on promoter-paused Pol II complexes to stimulate their escape into productive elongation. If P-TEFb is a major kinase that acts on the promoter-paused Pol II complex, its distribution should overlap at least some of the chromosomal sites that accumulate unphosphorylated RNA polymerase II (Pol IIa). However, the correlation need not be perfect, since the rate of formation of a promoter-paused Pol IIa is likely to be governed by mechanisms distinct from those that are responsible for recruiting P-TEFb. These mechanisms appear to be quite independent in an extreme case of heat shock genes, in which Pol IIa is present at full occupancy on the uninduced hsp70 promoter, and heat shock is needed to trigger both high levels of transcription and recruitment of P-TEFb. However, when a gene is active, Pol IIa is being continuously recruited to the promoter and maturing into a productive hyperphosphorylated polymerase II (Pol IIo) elongation complex. In the case of heat shock genes, the entry is fast enough to keep the pause region fully occupied with Pol II even when the gene is fully induced. Therefore, both the kinase responsible for phosphorylation and the Pol IIa would be expected to be present on active promoters, and their respective levels would be dictated by the relative rates of Pol entry and its maturation into a productive elongation complex (Lis, 2000 and references therein).

Chromosome were stained with antibodies to P-TEFb and Pol IIa. Most chromosomal sites in unstressed larvae that are labeled strongly by the P-TEFb (cyclin T) antibody are also labeled to various extents by the Pol IIa antibody; however, the ratio of labeling by these two antibodies varies at different sites. Therefore, the level of Pol IIa must be governed by factors that act at least somewhat independently from factors that govern the level of P-TEFb at specific sites. Nonetheless, the strong tendency of these proteins to colocalize is consistent with a model in which Pol IIa is a substrate for P-TEFb, and this phosphorylation serves to convert Pol II into a productive elongation complex (Lis, 2000).

The hyperphosphorylated form, PoI IIo, labels many more sites than does P-TEFb. Numerous sites are strongly labeled with antibody to Pol IIo, but not detectably labeled with antibody to P-TEFb. This pattern does not easily fit a model in which P-TEFb has a universal role in all Pol II transcription elongation. Presumably, there are distinct mechanisms (and other kinases) for producing Pol IIo that do not require the stable and continuous association of P-TEFb with a locus (Lis, 2000).

In contrast, there are few chromosomal sites that have P-TEFb, but no Pol IIo. A simple interpretation of this result, which is consistent with the known properties of P-TEFb, is that the recruitment of P-TEFb to a locus generally leads to efficient formation of transcription elongation complexes. These results also indicate that there is little recruitment of P-TEFb to sites that are not transcriptionally active (Lis, 2000).

P-TEFb is a kinase/cyclin heterodimer that was critical for overcoming an early block to transcriptional elongation (Marshall, 1995). Interestingly, the short transcripts of 20-40 nucleotides that are produced in the absence of P-TEFb are remarkably similar in size to those measured in vivo at genes that show promoter-associated pausing. Such pausing has been observed at a variety of genes; however, the heat shock promoters of Drosophila are perhaps the most thoroughly studied in eukaryotes. P-TEFb stimulates production of full-length transcripts in vitro (Marshall, 1995), and also from HIV templates in vivo (Mancebo, 1997). P-TEFb is normally located at >200 loci, but upon heat shock, it redistributes to native and transgenic heat shock loci with a robustness and rapidity that make it a good candidate for playing a critical role in the activation of heat shock gene transcription. The normally broad distribution of P-TEFb is simplified during heat shock, where the bulk of P-TEFb concentrates at all the major heat shock genes. The resolution of HSF and P-TEFb staining within the 87A locus is consistent with the long-held view that the DNA within this activated heat shock locus is in a very extended configuration. The two divergently transcribed hsp70 genes at this locus are separated by only 2 kb, and yet the P-TEFb staining resolves as two distinct bands. The major HSF staining resides between the two P-TEFb bands. HSF binding sites are known to reside in the region between the start sites of these genes. If the DNA in a highly decondensed puff approximates B-form DNA, that is, it has a chromatin packing ratio similar to that of highly transcribed ribosomal DNA, then the distance between the start sites of the two hsp70 genes would be ~0.7 µm. The centers of the two bands of staining are approximately twice that distance, implying that P-TEFb may be distributed over a region that extends downstream of the hsp70 start sites. Higher resolution biochemical methods will be required to precisely define the limits of the P-TEFb distribution. Nonetheless, the partial resolution of HSF and P-TEFb staining supports a view that these two components act at distinct points in the process of activating heat shock genes (Lis, 2000).

P-TEFb is not simply recruited by the hypophosphorylated Pol IIa. Pol IIa is the form of Pol II that is at the promoter pause region of hsp70 and other heat shock genes. Yet very little or no P-TEFb is detected at these sites prior the heat shock. It is speculated that a separate event must occur at these promoters to cause the association of P-TEFb, for example, another protein or proteins could recruit P-TEFb to these promoters. In the case of HIV, the Tat protein interacts with cyclin T to recruit the P-TEFb complex (Garber, 1998). For normal cellular genes, other host transcription factors may also play such a role; P-TEFb has been shown recently to be functionally recruited to MHC class II gene promoters by the CIITA activator (Kanazawa, 2000). Alternatively, transcription activation may normally allow a paused Pol IIa, a likely in vivo substrate of P-TEFb, to undergo a change or unmasking that now allows its association with P-TEFb (Lis, 2000).

P-TEFb is normally located at many chromosomal sites that are transcriptionally active. The chromosomal loci scored as positive with the cyclin T antibody may represent only a fraction of the genes that could be regulated by P-TEFb, owing to the dynamic developmental regulation of the Drosophila genome. Also, the existence of additional cyclin subunits that can couple with Cdk9 may produce a P-TEFb activity lacking cyclin T. Although whether P-TEFb activates transcription at all of the loci containing cyclin T cannot be evaluated, in the case of heat shock genes, the direct recruitment (via a Gal4 DNA-binding domain) of P-TEFb to an hsp70 promoter leads to an activation of this gene in the absence of heat shock. Although this activation is less than the very high level of activation caused by directly recruiting the activation domain of HSF, it is nonetheless clearly dependent on P-TEFb kinase activity. Interestingly, related kinases, Cdk2 and Cdk7, fail to activate this promoter and critical point mutations in the P-TEFb kinase or cyclin T disrupt the activation. The fact that Cdk7, the kinase of the TFIIH complex, fails to activate is worth noting, because it, like P-TEFb, can phosphorylate efficiently the CTD of Pol II. Perhaps these kinases have specificity for discrete steps in transcription. For example, P-TEFb may be capable of stimulating the elongation of the paused Pol II, whereas TFIIH kinase fulfills another role such as providing activity for an earlier step in transcription that does not necessarily lead to the full phosphorylation and maturation of elongationally competent Pol II. This specificity issue requires further, more focused investigation (Lis, 2000 and references therein).

Direct recruitment of P-TEFb to an HIV promoter has been shown to activate HIV transcription fully and bypasses the need for Tat (Bieniasz, 1999). Although the activation of hsp70 by directly recruiting P-TEFb that is observed in uninduced cells is strong, it is still less than that seen when the HSF activation domain is directly recruited. This fact suggests that HSF may be providing a function beyond triggering the events that lead to P-TEFb recruitment. The HSF activation domain is large enough to accommodate multiple interactions and functions (Lis, 2000 and references therein).

The colocalization of the hypophosphorylated Pol IIa with P-TEFb is intriguing, because the promoter-paused Pol II associated with all genes examined in Drosophila is hypophosphorylated. If the Pol IIa distribution is a general indicator of sites in which promoter-pausing is a part of the transcription mechanism, then P-TEFb may be stimulating the maturation of Pol II and its entry into productive elongation at a significant subset of active genes. Three of the four constitutively active genes that have been reported to have promoter-paused Pol II are at chromosomal sites that show significant P-TEFb. The fourth, Gapdh-2, is at 13F, a region that shows light P-TEFb staining. A higher resolution analysis will be required for a rigorous assignment of the P-TEFb signals to these specific genes (Lis, 2000 and references therein).

The failure to see a quantitative correlation of the intensity of staining of anti-Pol IIa and anti-P-TEFb at specific sites on polytene chromosomes is consistent with models in which the mechanism of generating paused Pol IIa is distinct from the mechanism that recruits P-TEFb. The extreme case of this is hsp70, in which, before heat shock, the promoter is fully occupied with Pol IIa, but has very little P-TEFb. Heat shock triggers the dramatic recruitment of P-TEFb, and the accumulation of Pol IIo on heat shock puffs. During heat shock, the paused Pol IIa still forms, but it escapes into productive elongation faster, once every 4 sec as compared with the uninduced level of once every 10 min. It is hypothesized that P-TEFb participates in this escape at heat shock genes and the subset of other genes that have promoter-paused Pol II (Lis, 2000).

DEVELOPMENTAL BIOLOGY

Oocyte and Embryonic development

Hsp70 is a broadly conserved thermotolerance factor, but inhibits growth at normal temperatures and cannot be induced in early embryos. In Drosophila embryos the temporal and spatial patterns of Hsp70 inducibility are unexpectedly complex, with striking differences between the soma and the germline. In both, regulation occurs at the level of transcription. During the refractory period for Hsp70 induction, HSF (heat-shock transcription factor) exhibits specific DNA-binding activity characteristic of activation in extracts of heated embryos. Remarkably, however, HSF is restricted to the cytoplasm in intact embryos even after heat shock. It was surprising to find HSF located in the cytoplasm of early embryos since in virtually all organisms and cell types studied to date HSF is constitutively nuclear. HSF moves from the cytoplasm to the nucleus in the absence of heat precisely when the capacity to induce Hsp70 is acquired (cycle 12 of the germline, cycle 13 in the soma). During oogenesis, Hsp70 inducibility is lost in nurse cells around stage 10, in a posterior-to-anterior gradient and HSF redistributes from nucleus to cytoplasm in the same spatiotemporal pattern. In general, HSF is nuclear before stage 10 and cytoplasmic after stage 11, but the precise timing of relocation varies. Notably, when relocalization occurs, it exhibits a sharp spatial gradient, with the nurse cells more proximal to the oocyte showing cytoplasmic localization first, while distal cells still display nuclear concentration. Oocyte nuclei seem to follow the same pattern as nurse cell nuclei, but because the vitelline membrane forms at stage 10, they could not be scored in all egg chambers. In a highly inbred derivative of the Samarkind strain, HSF moves into embryonic nuclei earlier than in the standard wild-type strain. Correspondingly, Hsp70 is inducible earlier, confirming that nuclear transport of HSF controls the inducibility of Hsp70 in early embryos. Also reported for the first time are the nuclear import patterns of two general transcription factors: RNA polymerase subunit Ilc and TATA binding protein (TBP). Both enter nuclei in a highly synchronous manner, independent of each other and of HSF. The import of TBP coincides with the first reported appearance of transcripts in the embryo. It is suggested that the potentiation of general and heat shock-specific transcription in Drosophila embryos is controlled by the developmentally programmed relocalization of general and heat shock-specific transcription factors. Restricted nuclear entry of HSF represents a newly described mechanism for regulating the heat-shock response. Therefore, hyper-phosphorylation is not an obligate precondition for the activation of Hsp70 in response to heat (Wang, 1998).

EFFECTS OF MUTATION

The single-copy Drosophila hsf is located between the genes staufen and Polycomblike at cytological position 55A. Mutations were isolated in hsf. All four hsf mutations isolated arrest at the 1st or 2nd larval instar stage of development. The temperature sensitive hsf4 allele fails to respond to high temperature. The inability to activate expression of hsp (heat shock protein) genes may be attributed to the attenuation of DNA-binding activity at temperatures that induce hsp genes with wild type hsf. The mutant HSF is able to respond to recovery from anoxia, an alternative inducer of the heat shock response. Inability of hsf4 to induce a hsp response also correlates with a compromised thermotolerance in the hsf4 mutant. The hsf4 mutant exhibits a single temperature-sensitive period from 1.5 to 2.5 days of development, corresponding to the 1st and 2nd larval instars. Upon eclosion, adults are viable and fertile at 29 degrees C. Thus, a requirement for HSF function, as defined by the hsf4 mutant, appears to be restricted to early larval development. Expression of HSP26, HSP70 and HSP83 is unaltered in hsf4 animals during the temperature sensitive period at the non-permissive temperature. These results are consistent with studies indicating that the developmental expression of HSPs is essentially independent of HSEs. It is concluded, therefore, that the essential developmental function of HSF may involve the regulation of novel, non-heat shock genes. There is no obvious morphological defect in mutant larvae (Jedlicka, 1997)

Oogenesis is arrested in hsf1, hsf2 and hsf3 mutant females, but not in hsf4 mutant females. Either the HSF4 mutant protein retains a residual amount of activity at higher temperature that is sufficient for normal oogenesis but not for larval development, or, alternatively, the hsf4 mutation disrupts a function only required in larval development. The hsf mutant phenotype resembles that of ovo phenotype. The hsf1 mutation affects neither germ-line stem cell divisions that yield the 16-cell cyst, nor the differentiation of this cyst into nurse cells and the oocyte. The only observable defect in hsf1 egg chambers is that the DNA content of the nurse cells appears somewhat low, suggesting a possible defect in the endoreplication that normally occurs in these cells. Thus, HSF is required in the female germ-line for development of the egg chamber, at a stage after formation and differentiation of the germ-line cyst, but prior to vitellogenesis. The requirement for HSF in oogenesis also does not appear to be related to the regulation of HSP expression. HSP83 is expressed in developing hsf1 mutant egg chambers. The timing of HSP26,and HSP28 expression during oogenesis does not coincide with the temporal requirement for HSF; moreover, the expression of HSP26 in the ovary has been shown to be HSE independent. It is concluded that the two developmental functions of HSF are genetically separable and appear not to be mediated through the induction of HSPs (Jedlicka, 1997).

EVOLUTIONARY HOMOLOGS

Yeast Heat shock factor

Heat shock elements (HSEs) are promoter sites that bind heat shock factor. An in vivo footprinting analysis of the S. cerevisiae HSP82 promoter has shown that yeast heat shock factor binds to strong HSEs in non-heat-shocked cells. Upon heat shock, however, additional binding of HSF becomes apparent at weak heat shock elements of the promoter as well. Recovery from heat shock results in a dramatic reduction in HSF binding at both strong and weak HSEs, consistent with a model in which HSF binding is subject to a negative feedback regulation by heat shock proteins. In vivo KMnO4 footprinting reveals that the interaction of the TATA-binding protein (TBP) with this promoter is also modulated: heat shock slightly increases TBP binding to the promoter and this binding is reduced upon recovery from heat shock. KMnO4 footprinting does not reveal a high density of polymerase at the promoter prior to heat shock, but a large open complex between the transcriptional start site and the TATA box is formed rapidly upon activation, similar to that observed in other yeast genes (Giardina, 1995a).

The anti-inflammatory drug sodium salicylate (aspirin) modulates the activity of specific transcription factors in humans. Salicylate and sorbate, another organic acid, stimulate DNA binding by yeast HSF in vivo. Surprisingly, salicylate inhibits heat shock gene transcription even in cells induced by a prior heat shock. This inhibition of transcription occurs at a step after HSF and transcription factor IID binding but before promoter melting by RNA polymerase. Salicylate appears to generate a tight binding but activation-impotent HSF by means of cytoplasmic acidification, since inhibiting proton efflux from cells triggers this same DNA binding and inhibition of heat shock gene expression (Giardina, 1995b).

In vitro DNA-binding assays demonstrate that the heat shock transcription factor (HSF) from the yeast Saccharomyces cerevisiae can adopt an altered conformation when stressed. This conformation, reflected in a change in electrophoretic mobility, requires that two HSF trimers be bound to DNA. Single trimers do not show this change, which appears to represent an alteration in the cooperative interactions between trimers. HSF isolated from stressed cells displays a higher propensity to adopt this altered conformation. Purified HSF can be stimulated in vitro to undergo the conformational change by elevating the temperature or by exposing HSF to superoxide anion. Mutational analysis maps a region critical for this conformational change to the flexible loop between the minimal DNA-binding domain and the flexible linker that joins the DNA-binding domain to the trimerization domain. The significance of these findings is discussed in the context of the induction of the heat shock response by ischemic stroke, hypoxia, and recovery from anoxia, all known to stimulate the production of superoxide (Lee, 2000).

The mutation M232V affects a highly conserved position within the DNA-binding domain of HSF. To assess whether the mutant might differ from wild-type HSF in DNA-binding ability, HSF binding to a synthetic HSE that accommodates only a single trimer was examined. There is no detectable difference in complex formation between wild-type and mutant HSF. Both form a single complex under nonshocked conditions. Although the inherent DNA-binding ability of HSFM232V appears normal, it might be possible that the lesion affects the ability of two bound trimers to interact. To test this possibility, the DNA-binding assays were repeated using a synthetic HSE that binds two HSF trimers (HSE6T). On this HSE, HSF forms three distinct complexes (I, II, and III), whose abundances vary depending on the source of HSF. Complex I has the identical mobility as a complex that forms on an HSE that can accommodate a single HSF trimer and therefore represents the binding of a single trimer to this HSE. Complex II has a mobility identical to that of a complex comprised of six HSF monomers and thus represents the binding of two trimers to HSE6T. Wild-type HSF from nonshocked cells forms almost exclusively complexes I and II, whereas HSF from heat-shocked cells forms a modest amount of complex III in addition to complexes I and II. Complex III becomes very abundant when HSFM232V is examined. The amount of complex III increases with HSF activity, showing quite clearly that the DNA-binding pattern of HSF is affected by its in vivo activity state. These observations suggest that complex III might represent an active species of HSF (Lee, 2000).

It has long been thought that the activation of HSF upon heat shock must involve a conformational change that 'unmasks' the transcriptional activation domains of HSF. There have been a number of analyses of HSF conformational changes, but these have generally examined metazoan HSF free in solution and have provided information primarily on the monomer-trimer transition. The observation that salicylate can induce human HSF to trimerize and bind DNA without concomitant activation of hsp70 expression suggests that there is likely to be a second aspect of HSF activation beyond trimerization. The observations presented here provide information about this second step, at least for Saccharomyces HSF (Lee, 2000).

The argument that complex III represents an active form of HSF is based on three kinds of evidence. The first is correlative, as it must be for an assay based on electrophoretic mobility. The propensity of wild-type HSF to form complex III in vitro depends, at least in part, on the physiological state of the cells from which the HSF is extracted. Heat shock favors the formation of complex III; recovery from heat shock disfavors it. Chemical stresses that affect the heat shock response also favor the formation of complex III. These correlations are imperfect, however. In part, this may reflect the fact that HSF can be induced to form complex III by exposure to superoxide, which is generated spontaneously in the DNA-binding reactions that are exposed to atmospheric oxygen. The imperfect correlation may also reflect the fact that reduced glutathione, present in whole-cell extracts, counteracts the induction of complex III in vitro (Lee, 2000).

The second line of evidence is that HSF can be induced to form complex III in vitro in response to agents that are known to induce HSF activity in vivo: heat and oxidation. If the mechanism of HSF activation were a direct effect of these stress agents, rather than a complex signal transduction cascade, this is the very result that would be predicted. The third line of evidence that complex III represents an active conformation of HSF is genetic. Several mutations that elevate HSF activity in vivo also increase the formation of complex III in vitro. Together, these three lines of evidence suggest that complex III may, indeed, represent a conformation that HSF can adopt upon activation (Lee, 2000).

The data do not reveal the nature of the conformational change that gives rise to complex III. However, they do provide two important pieces of information. First, the change in conformation is detectable only when two HSF trimers are bound cooperatively to DNA; singly bound trimers show no detectable differences in gel mobility shift assay. This suggests that the conformational change represents a change in the nature of the cooperative interaction between trimers. This is intriguing, because it helps explain how differences in the architecture of HSEs can exhibit differences in biological activity that are not easily explained simply by differences in binding affinity. For example, the HSEs of the yeast CUP1 gene and of the human IL1beta gene do not provide for cooperative interactions between trimers and display activities that are distinct from traditional HSEs. In contrast, traditional HSEs of natural heat shock genes typically have binding sites for two or more HSF trimers. In both yeast and Drosophila, these cooperative interactions have been shown to be either important (yeast) or critical (Drosophila) for normal heat shock-inducible transcription (Lee, 2000).

These data also map the regions that are important for complex III formation. The N-terminal "masking" domain, the flexible loop joining the DNA-binding domain to the linker domain, and residue Met₂₃₂ all seem to play roles in the transition between complexes II and III. Interestingly, these all share a certain physical proximity. The structural data for the DNA-binding domain indicate that the N-terminal domain joins the DNA-binding domain near the flexible loop, which in turn is capable of closely approaching Met₂₃₂. It is imagined that the flexible loop adopts one conformation in complexes I and II and a different conformation in complex III (Lee, 2000).

The data indicate that the transition to complex III can be induced in vitro by stimulation of purified HSF by heat or by the superoxide anion O₂^-. This indicates that HSF is directly responsive to the two stresses that are best known as inducers of the heat shock response: heat and oxidative stress. The difference that yeast HSF responds to O₂^- whereas Drosophila HSF responds to H₂O₂ may be caused by methodological differences or by differences in the physiology or in the sequences of HSF, but the similarity is striking. The production of O₂^- is coupled in vivo to the production of H₂O₂ via the dismutation of superoxide into H₂O₂. The major source of cellular O₂^- is mitochondrial metabolism, via which, it has been estimated, 1%-2% of electrons are 'spilled' into the generation of superoxide. The mitochondrial Mn-dependent superoxide dismutase rapidly converts much of this to H₂O₂. The generation of superoxide will increase during stresses that affect mitochondrial activity. Thus, the same basic physiological stress creates both species of reactive oxygen (Lee, 2000).

The finding that HSF is directly responsive to superoxide (or, in the case of Drosophila HSF, its byproduct H₂O₂) provides a mechanistic explanation for the finding that the heat shock response is induced by such stresses as recovery from anoxia, hypoxia, and ischemic stroke. For aerobic organisms, all of these stresses have the same result: a burst of superoxide production. Via the dismutation of O₂^- to H₂O₂ and the subsequent conversion to the hydroxyl radical (·OH), these treatments lead to significant cellular damage -- the classic 'reperfusion injury'. It has long been thought that the induction of a heat shock response was the result of the ·OH-induced protein damage, stimulating an unfolded protein response. These findings indicate that HSF is activated directly by the oxidative stress and may therefore occur before there is extensive protein damage. Thus, the induction of the heat shock response by anoxic stresses is immediate and potentially protective, rather than a mere reaction to damage previously done (Lee, 2000).

In analyses of protein damage by reactive oxygen species, it is usually the ·OH that causes damage after production of O₂^-. Hydroxylation of aromatic amino acid residues can usually be prevented by ·OH scavengers, but not by superoxide dismutase. It is therefore surprising that it is O₂^- itself that activates HSF. Superoxide seems to work directly, and probably reversibly, on yeast HSF. It is unlikely that O₂^- simply binds to HSF, because at least some HSF remains in its 'complex III-competent' form after extraction from stressed cells. More likely, O₂^- modifies one or more amino acid residues, in which case conversion of activated HSF back to the inactive form must entail the reversal of this modification (Lee, 2000).

It is possible that the inactivation of HSF during recovery from stress requires two activities: hsp70 proteins to help refold HSF and one or more glutathione-dependent enzymatic activities. It is striking that high concentrations of GSH can completely prevent nonshocked HSF from binding to DNA, as judged by gel mobility shift assay. This suggests that there may be an 'inactive' form of HSF that has a very low affinity for DNA, as is the case in metazoans. On the basis of these findings, it is suspected that this may occur via the GSH-dependent deactivation of HSF (Lee, 2000).

C. elegans Heat shock factor

In a screen for suppressors of activated GOA-1 (Galpha_o) under the control of the hsp-16.2 heat-shock promoter, three genetic loci were identified that affect heat-shock-induced GOA-1 expression. The cyl-1 (coding for an RS splice factor, Drosophila homolog: CG16903; vertebrate homolog Cyclin L) mutants are essentially wild type in appearance, while hsf-1 and sup-45 mutants have egg-laying defects. The hsf-1 mutation also causes a temperature-sensitive developmental arrest, and hsf-1 mutants have decreased life span. Western analysis indicates that mutations in all three loci suppress the activated GOA-1 transgene by decreasing its expression. Heat-shock-induced expression of hsp-16.2 mRNA is reduced in cyl-1 mutants and virtually eliminated in hsf-1 and sup-45 mutants, as compared to wild-type expression. The mutations could also suppress other transgenes under heat-shock control. cyl-1 and sup-45, but not hsf-1, mutations suppressed a defect caused by a transgene not under heat-shock control, suggesting a role in general transcription or a post-transcriptional aspect of gene expression. hsf-1 encodes the C. elegans homolog of the human heat-shock factor HSF1, and cyl-1 encodes a cyclin most similar to cyclin L. It is believed that HSF-1 acts in heat-shock-inducible transcription and CYL-1 acts more generally in gene expression (Hajdu-Cronin, 2004).

Integrin-linked kinase modulates longevity and thermotolerance in C. elegans through neuronal control of HSF-1

Integrin-signaling complexes play important roles in cytoskeletal organization and cell adhesion in many species. Components of the integrin-signaling complex have been linked to aging in both Caenorhabditis elegans and Drosophila, but the mechanisms underlying this function are unknown. This study investigated the role of Integrin-linked kinase (ILK), a key component of the integrin-signaling complex, in lifespan determination. Genetic reduction of ILK in both C. elegans and Drosophila increases resistance to heat stress, and leads to lifespan extension in C. elegans without majorly affecting cytoskeletal integrity. In C. elegans, longevity and thermotolerance induced by ILK depletion is mediated by the heat-shock factor-1 (HSF-1), a major transcriptional regulator of the heat-shock response (HSR). Reduction of ILK levels increases hsf-1 transcription and activation, and leads to enhanced expression of a subset of genes with roles in the HSR. Moreover, induction of HSR-related genes, longevity, and thermotolerance caused by ILK reduction required the thermosensory neuron AFD and interneuron AIY, which are known to play a critical role in the canonical HSR. Notably, ILK was expressed in neighboring neurons, but not in AFD or AIY, implying that ILK reduction initiates cell non-autonomous signaling through thermosensory neurons to elicit a non-canonical HSR. These results thus identify HSF-1 as a novel effector of the organismal response to reduced ILK levels, and show that ILK inhibition regulates HSF-1 in a cell non-autonomous fashion to enhance stress resistance and lifespan in C. elegans (Kumsta, 2013).

Xenopus Heat shock factor

There are distinct stress-inducible and developmentally regulated heat shock response element (HSE)-binding activities in Xenopus. The stress-induced HSE-binding complex is recognized by antiserum against mammalian HSF1, but not by HSF2 antiserum, suggesting that a Xenopus homolog of HSF1 is the major component of this activity. There is in addition an unusual constitutive HSE-binding complex in unstressed stage I and II oocytes, but not in later stage oocytes, eggs, or developing embryos. This constitutive complex is unaffected by heat or chemical treatments and is not recognized by either HSF1 or HSF2 antiserum. Appearance of the constitutive HSE-binding activity during oogenesis corresponds closely with peak levels of hsp70 mRNA. It is thought that the constitutive HSE-binding activity in early oocytes is formed by a unique developmentally regulated heat shock factor that may play a role in the expression of heat shock proteins during early stages of oogenesis (Gordon, 1997).

Stress-induced expression of the heat shock (hs) genes in eukaryotes is mediated by a transcription factor known as heat shock factor 1 (HSF1). HSF1 is present in a latent, monomeric form in unstressed metazoan cells and upon exposure to heat or other forms of stress is converted to an "active" trimeric form, which binds the promoters of hs genes and induces their transcription. The conversion of HSF1 to its active form is hypothesized to be a multistep process involving (1) oligomerization of HSF1, plus (2) additional changes in its physical conformation, (3) changes in its phosphorylation state, and for some species (4) translocation from the cytoplasm to the nucleus. Oligomerization of HSF appears to be essential for high affinity DNA binding, but it remains unclear what their mechanistic roles may be, or whether the other steps occur in all organisms. Germinal vesicles (nuclei) that are physically dissected from unshocked Xenopus laevis oocytes contain no HSF1 binding activity. Interestingly, in vitro heat shock treatments of isolated nuclei from unshocked oocytes activate HSF1 binding, indicating that HSF1 must have been present in the unshocked nuclei prior to isolation. Induction of HSF1 binding is not observed in enucleated oocytes. Western blot analysis using an affinity-purified polyclonal antibody made against X. laevis HSF1 shows that HSF1 is present in equal amounts in unshocked and shocked oocytes and isolated nuclei. HSF1 is not detected in enucleated oocytes. These results clearly demonstrate that HSF1 is a nuclear protein in oocytes prior to exposure to stress. In Xenopus oocytes, therefore, HSF1 translocation from the cytoplasm to the nucleus is not part of the multistep process of HSF1 activation. These results also imply that the signals and/or factors involved in HSF1 activation must have their effect in the nuclear compartment (Mercier, 1997).

Mammalian Heat shock factor

Cell and tissue injury activate the inflammatory response through the action(s) of arachidonic acid and its metabolites, leading to the expression of acute-phase proteins and inflammatory cytokines. At the molecular level, little is known how arachidonic acid regulates the inflammatory response. As inflammation is also associated with local increase in tissue temperatures, arachidonic acid has been examined to determine whether it is directly involved in the heat shock response. Extracellular exposure to arachidonic acid induces heat shock gene transcription in a dose-dependent manner via acquisition of DNA-binding activity and phosphorylation of heat shock factor 1 (HSF1). Exposure of cells to low concentrations of arachidonic acid, which by themselves does not induce HSF1 DNA-binding activity, reduces the temperature threshold for HSF1 activation from elevated temperatures that are not physiologically relevant (> 42 degrees C) to temperatures that can be attained during the febrile response (39-40 degrees C). These results indicate that elevated heat shock gene expression is a direct consequence of an arachidonic acid-mediated cellular response (Jurivich, 1994).

Promoter-proximal pausing during transcriptional elongation is an important way of regulating many diverse loci, including the human hsp70 gene. Pausing of RNA polymerase can be enhanced by chromatin structure. Activation of hsp70 leads to disruption of transcribed chromatin in front of RNA polymerase. In vivo, disruption of chromatin in the first 400 bp of the transcribed region of hsp70 following heat shock is resistant to the transcriptional inhibitor alpha-amanitin. Disruption of chromatin farther downstream also occurs following activation but is sensitive to alpha-amanitin, suggesting that polymerase movement is needed to disrupt distal portions of the hsp70 gene. In vitro, disruption of transcribed chromatin is dependent on the presence of the human heat shock factor 1 (HSF1) activation domains. These experiments demonstrate that HSF1 can direct disruption of chromatin in transcribed regions. It is suggested that this is one of the mechanisms used by HSF1 to facilitate transcriptional elongation. Previous studies have shown that SWI/SNF-containing fractions augment the readthrough of pausing on hsp70 in an activator-dependent fashion (Brown, 1997).

A preparation of mixed male germ cell types from mouse testis exhibit a lower temperature threshold for activation of HSF1 DNA binding relative to other mouse cell types. Is the phenomenon of reduced HSF1 activation temperature common to all testis cell types, both somatic and germ cell types, or is it a special property of male germ cells? A purified population of pachytene spermatocytes, one of the male germ cell types, exhibits a profile of reduced HSF1 activation temperature identical to that observed for the mixed germ cell preparation, with a threshold HSF1 activation temperature of 35 degrees C. Activation of HSF1 DNA binding in male germ cells by incubation at 38 degrees C is accompanied by the classic cellular stress response parameters of heat-induced HSF1 phosphorylation and increased expression of the hsp72 stress protein. In contrast, a preparation of somatic testis cell types exhibits HSF1 activation only at temperatures of 42 degrees C and above, a profile identical to that observed for mouse liver cells and mammalian cell lines. These results demonstrate that the phenomenon of reduced HSF1 activation temperature is a unique property of male germ cell types within the mammalian testis and demonstrate that HSF1 activated at this lower temperature threshold is fully capable of mediating a productive cellular stress response in these cell types (Sarge, 1995).

Heat shock factor 1 inhibits the expression of c-fos (See Drosophila Fos), an immediate early gene that controls responses to extracellular stimuli for growth and differentiation. Heat shock factor 1 inhibits the transcription of the c-fos gene and antagonizes the activating effects of the signal transducing protein Ras on the c-fos promoter and on the promoter of another Ras responsive gene, uPA. This property is specific for heat shock factor 1; c-fos repression is not seen with the structurally related protein heat shock factor 2. Repression involves different molecular mechanisms when compared with those involved in transcriptional activation by heat shock factor 1, and specifically does not require binding to the c-fos promoter. Thus, in addition to its known role as a transcriptional activator of the cellular heat shock response, heat shock factor 1 also antagonizes the expression of Fos, a key component of the ubiquitous AP-1 transcription factor complex; as such, it could influence multiple aspects of cell regulation (Chen, 1997).

Heat shock transcription factor 1 (HSF-1) activates the transcription of heat shock genes in eukaryotes. Under normal physiological growth conditions, HSF-1 is a monomer. Its transcriptional activity is repressed by constitutive phosphorylation. Upon activation, HSF-1 forms trimers, acquires DNA binding activity, increases transcriptional activity, and appears as punctate granules in the nucleus. In this study, using bromouridine incorporation and confocal laser microscopy, it has been demonstrated that newly synthesized pre-mRNAs colocalize to the HSF-1 punctate granules after heat shock, suggesting that these granules are sites of transcription. Evidence that glycogen synthase kinase 3beta (GSK-3beta) and extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) participate in the down regulation of HSF-1 transcriptional activity. Transient increases in the expression of GSK-3beta facilitate the disappearance of HSF-1 punctate granules and reduce hsp-70 transcription after heat shock. ERK is shown to be the priming kinase for GSK-3beta. Taken together, these results indicate that GSK-3beta and ERK MAPK facilitate the inactivation of activated HSF-1 after heat shock by dispersing HSF-1 from the sites of transcription (He, 1998).

GSK-3 consists of two isoforms: GSK-3alpha (51 kDa) and GSK-3beta (46 kDa). It was first identified as an enzymatic activity that phosphorylates and inactivates glycogen synthase. A second role of GSK-3 was found when studies showed that inhibition of phosphatase type I activity is relieved when GSK-3 phosphorylates phosphatase inhibitor 2. At least 15 other substrates have been reported to be phosphorylated by GSK-3, including the transcription factors c-Jun, JunD, c-myb, c-myc, L-myc, CREB, and NF-AT, most of which become inactivated when phosphorylated by GSK-3. GSK-3 tends to phosphorylate serine/threonine residues located next to a proline which, in turn, is near another serine residue that has been prephosphorylated by some other protein kinase (referred to as priming kinase). GSK-3 is constitutively active and, as a result, suppresses many of its substrates under normal physiological growth conditions. It appears that the activity of HSF-1 can be down regulated by protein kinases that are activated by diverse signal transduction pathways. The ERK MAPK pathway is activated during cell growth and development by multiple signaling pathways, which in turn are activated by growth factor receptors, G protein-coupled receptors, ceramide production, and a protein kinase C-dependent pathway. Recent evidence has suggested that ERK MAPK activation by heat shock may be through ceramide activation of protein kinase Raf-1. The pathways leading to GSK-3beta regulation are complex. GSK-3beta activity is down regulated by PKB/Akt, p70S6K, or p90rsk as a result of phosphorylation on serine residues. Activation of PKB/Akt leads to increased cell survival, as is the case with activation of ERK. The ability of ERK to mediate cell survival is dependent on the activation of transcription factors such as Elk1 and repair of damaged proteins. The ability of PKB/Akt to mediate cell survival is likely to be dependent on downstream effectors such as p70S6K and protein translation, activation of FRAP/TOR, and inhibition of GSK-3beta. Interestingly, heat shock stimulates the activity of GSK-3beta and ERK MAPK. The increase in GSK-3beta activity may occur through its phosphorylation on a tyrosine residue by an unknown tyrosine kinase. Thus, it appears that when activated, HSF-1 reduces the expression of most other genes and must be inactivated in a timely manner for cell proliferation to continue. The cell has developed an elegant mechanism for doing this, since some of the enzymes that control cell proliferation are capable of inactivating HSF-1 (He, 1998 and references).

Heat-shock factor-1 (HSF1) is a major transactivator of stress-inducible genes in response to environmental changes, but it is also implicated in extra-embryonic development and female fertility in mice. Mouse embryos whose mothers lack this protein are unable to develop properly beyond the zygotic stage, although oocytes are ovulated and fertilized normally. Wild-type spermatozoa do not save zygotes from lethality, indicating that the reproductive failure of females deficient in this factor is caused by a 'maternal effect' mutation, and that HSF1 from the mother normally controls early post-fertilization development (Christians, 2000).

Female mice lacking the gene encoding HSF1 (Hsf1^-/- females) have normal ovaries and reproductive tracts, indicating that folliculogenesis and oogenesis do not require HSF1 expression, by contrast with the Drosophila Hsf mutation and others affecting fertility in mammals. Ovulated eggs of Hsf1^-/- females remain properly arrested at metaphase II until fertilization, after which zygotes form with two pronuclei and a second polar body. Although mutant embryos produced by Hsf1 ^-/- females can initiate early development, they do not survive after transplantation into the oviduct of Hsf1 wild-type mice, indicating that the causes of Hsf1^-/- infertility are intrinsic rather than extrinsic (Christians, 2000).

To determine at which stage development breaks down, an analysis was made of the cleavage efficiency of preimplantation embryos produced by Hsf1^-/- females, using Hsf1^+/- females as controls, mated with either wild-type or homozygous males. The percentage of blastocysts at 3.5 days of development was the same and independent of the genotype of the father. In contrast, the embryos of homozygous females mated with either Hsf1 ^+/+ or Hsf1^-/- males are blocked mainly at the 1-cell stage, and development at 3.5 days is poor . Unlike the embryos produced by heterozygous females, none of these mutant embryos reach the blastocyst stage at the appropriate time, and 18% embryos from the Hsf1 ^-/- x Hsf1^-/- intercross reached the 2-cell stage. A significantly higher fraction of those sired by Hsf1^+/+ males progress beyond the 1-cell division, but the difference does not persist beyond this stage. These findings show that the survival of the offspring is determined mainly by the maternal genotype, and that Hsf1^-/- is therefore a maternal-effect mutation (Christians, 2000).

Embryos might die at the 1-2-cell stage because of a failure to initiate the zygotic transcriptional activity required to complete the transition from maternal to embryonic control of development. Since spontaneous expression of Hsp70.1 indicates the onset of transcriptional activity at the late 1-cell stage, Hsp70.1-Luc transgenic lines, in which a luciferase reporter gene is driven by the murine Hsp70.1 promoter, were used to test this idea directly. At 1.5 days of development, embryos from Hsf1 ^-/- and Hsf1^-/- females that had been mated with Hsf1^+/+ transgenic males both showed luciferase activity significantly above the background level. The Hsf1-mutant zygotes therefore apparently express heat-shock proteins, showing that zygotic transcriptional activity can begin without HSF1 (Christians, 2000).

These results do not exclude the possibility that the level of transcriptional activity, or the control of the target genes to be transcribed, might have been altered in mutant embryos. Furthermore, ultrastructural abnormalities affect mainly the nuclei of embryos from null females at the 2-cell stage, indicating that, even with transcriptional activity, reduced survival is in part related to the decrease in structural integrity of the early embryonic nucleus. The key role for control by maternal-origin HSF1 in early development raises the possibility that in defective form it may be a cause of post-fertilization abnormalities associated with infertility in mammals, including humans (Christians, 2000).

Heat shock factor oligomerization and DNA-binding specificity

Heat shock transcription factors (HSFs) are multi-zipper proteins with high-affinity binding to DNA that is regulated by heat shock-induced trimerization. Formation of HSF trimers is dependent on hydrophobic heptad repeats located in the amino-terminal region of the protein. Two subregions at the carboxyl-terminal end of human HSF1 have been identified that maintain the monomeric form of the protein under normal conditions. One of these contains a leucine zipper motif that is conserved between vertebrate and insect HSFs. These results suggest that the carboxyl-terminal zipper may suppress formation of trimers by the amino-terminal HSF zipper elements by means of intramolecular coiled-coil interactions that are sensitive to heat shock (Rabindran, 1994).

In unstressed mammalian cells, HSF1 is present mainly in complexes with an apparent molecular mass of about 200 kDa, unable to bind to DNA. Heat treatment induces a shift in the apparent molecular mass of HSF1 to about 700 kDa, concomitant with the acquisition of DNA-binding ability. Cross-linking experiments suggest that this change in complex size may reflect the trimerization of monomeric HSF1. Human HSF1 expressed in Xenopus oocytes does not bind DNA, but derepression of DNA-binding activity, as well as oligomerization of HSF1, occurs during heat treatment at the same temperature at which hsp gene expression is induced in this organism, suggesting that a conserved Xenopus protein(s) plays a role in this regulation. Inactive HSF1 resides in the cytoplasm of human cells; on activation it rapidly translocates to a soluble nuclear fraction, and shortly thereafter it becomes associated with the nuclear pellet. On heat shock, activatable HSF1, which might already have been posttranslationally modified in the unstressed cell, undergoes further modification (Baler, 1993).

Heat stress regulation of human heat shock genes is mediated by human heat shock transcription factor hHSF1, which contains three 4-3 hydrophobic repeats (LZ1 to LZ3). In unstressed human cells (37 degrees C), hHSF1 appears to be in an inactive, monomeric state that may be maintained through intramolecular interactions stabilized by transient interaction with hsp70. Heat stress (39 to 42 degrees C) disrupts these interactions, and hHSF1 homotrimerizes and acquires heat shock element DNA-binding ability. hHSF1 expressed in Xenopus oocytes also assumes a monomeric, non-DNA-binding state and is converted to a trimeric, DNA-binding form upon exposure of the oocytes to heat shock (35 to 37 degrees C in this organism). Because endogenous HSF DNA-binding activity is low, and anti-hHSF1 antibody does not recognize Xenopus HSF, this system was used for mapping regions in hHSF1 that are required for the maintenance of the monomeric state. The results of mutagenesis analyses strongly suggest that the inactive hHSF1 monomer is stabilized by hydrophobic interactions involving all three leucine zippers that may form a triple-stranded coiled coil. Trimerization may enable the DNA-binding function of hHSF1 by facilitating cooperative binding of monomeric DNA-binding domains to the heat shock element motif. This view is supported by observations that several different LexA DNA-binding domain-hHSF1 chimeras bind to a LexA-binding site in a heat-regulated fashion; that single amino acid replacements disrupting the integrity of hydrophobic repeats render these chimeras both constitutively trimeric and DNA binding, and that in these assays LexA itself binds stably to DNA only as a dimer but not as a monomer (Zuo, 1994).

Comparison of wild-type and mutant hHSF1 expressed in Xenopus oocytes and human HeLa cells suggests that retention of hHSF1 in the monomeric form depends on hydrophobic repeats (LZ1 to LZ3) and a carboxy-terminal sequence element in hHSF1 as well as on the presence of a titratable factor in the cell. Oligomerization of hHSF1 appears to induce DNA-binding activity as well as to uncover an amino-terminally located nuclear localization signal. A mechanism distinct from that controlling oligomerization regulates the transcriptional competence of hHSF1. Components of this mechanism have been mapped to a region, including LZ2 and nearby sequences downstream from LZ2, that is clearly separated from the carboxy-terminally located transcription activation domain(s). A fold-back structure is thought to mask the transcription activation domain in the unstressed cell but it is opened up by modification of hHSF1 and/or binding of a factor facilitating hHSF1 unfolding in the stressed cell. Activation of hHSF1 appears to involve at least two independently regulated structural transitions (Zuo, 1995).

HSF1 exists in unstressed cells in an inactive form, which is converted to the DNA binding from upon exposure of cells to elevated temperature. A protocol has been developed for isolation of the non-DNA binding form of recombinant mouse HSF1, involving expression and affinity purification of HSF1 as a fusion with the glutathione S-transferase protein in Escherichia coli, followed by specific protease cleavage to release pure HSF1 protein. The purified inactive HSF1 can be converted to the DNA binding form by heat treatment in vitro. This conversion is accompanied by oligomerization of HSF1 from a monomeric to a trimeric native structure, similar to that observed for HSF1 in heat-shocked cells. These results indicate that elements residing in the HSF1 polypeptide are sufficient both for maintenance of this factor in the non-DNA binding form and for its heat-induced conversion to the DNA binding form, and support a role for HSF1 as the "molecular thermostat" in eukaryotic cells, which senses adverse environmental conditions and activates the cellular stress response (Goodson, 1995a).

One step in the pathway leading to transcriptional activation of heat shock genes involves heat shock factor 1 (HSF1) trimerization, required for high-affinity binding of this activator protein to heat shock elements (HSEs) in the promoters. Previous studies have shown that in vivo the trimerization is negatively regulated at physiological temperatures by a mechanism that requires multiple hydrophobic heptad repeats (HRs) which may form a coiled coil in the monomer. To investigate the minimal requirements for negative regulation, mouse HSF1 translated in both rabbit reticulocyte lysate or extracted from Escherichia coli were examined after limited expression. Under these conditions HSF1 behaves as a monomer that can be induced by increases in temperature to form active HSE-binding trimers. Mutations of either HR region cause activation in both systems. Temperature elevations and acidic buffers activate purified HSF1, and mild proteolysis excises fragments that form HSE-binding oligomers. These results suggest that oligomerization can be repressed in the monomer and that repression can be relieved in the apparent absence of regulatory proteins. An intramolecular mechanism may be central for the regulation of this transcription factor in mammalian cells, although not necessarily sufficient (Farkas, 1998).

Eukaryotic heat shock transcription factors (HSF) regulate an evolutionarily conserved stress-response pathway essential for survival against a variety of environmental and developmental stresses. Although the highly similar HSF family members have distinct roles in responding to stress and activating target gene expression, the mechanisms that govern these roles are unknown. A loop within the HSF1 DNA-binding domain has been identified that dictates HSF isoform specific DNA binding in vitro and preferential target gene activation by HSF family members in both a yeast transcription assay and in mammalian cells. These characteristics of the HSF1 loop region are transposable to HSF2 and sufficient to confer DNA-binding specificity, heat shock inducible HSP gene expression and protection from heat-induced apoptosis in vivo. In addition, the loop suppresses formation of the HSF1 trimer under basal conditions and is required for heat-inducible trimerization in a purified system in vitro, suggesting that this domain is a critical part of the HSF1 heat-stress-sensing mechanism. It is proposed that this domain defines a signature for HSF1 that constitutes an important determinant for how cells utilize a family of transcription factors to respond to distinct stresses (Ahn, 2001).

In mammals, three nonredundant HSF family members have been identified. The physiological and environmental signals that activate HSF2 and HSF4, as well as the respective roles of all three HSFs to developmental pathways, remain to be elucidated; however, detailed biochemical analyses of these proteins have revealed interesting and significant differences with respect to their activation and DNA binding characteristics. Previous genetic analyses of hsf1^-/- cells have established that HSF1 is the only heat-inducible HSF in mice, and in vitro biochemical studies have shown differences between HSF1 and HSF2 in the degree of cooperative DNA binding on the hsp70 promoter. A multifunctional region of the HSF1 DNA-binding domain, the loop, has been mapped that allows discrimination between different HSE-HSF interactions and acts as a part of the sensing and regulatory mechanism for responding to thermal stress. The HSF1-specific DNA-binding property can be transposed to HSF2 by creating a hybrid of HSF2 with the loop of HSF1, and significantly, the HSF2Loop1 chimera is capable of activating transcription of an HSP70-lacZ reporter in yeast and the endogenous inducible hsp70 gene in mouse cells. Based on previous data that HSF1 binds to HSEs with greater cooperativity than HSF2 does, it is proposed that the HSF1 loop facilitates interactions between HSF1 trimers, thereby promoting its binding to extended HSEs. Such cooperative DNA binding has also been established for lower eukaryotic HSFs from yeast and fly. The HSF2 loop seems much less capable of promoting such cooperativity between trimers, as evidenced by its smaller footprint over the hsp70 HSE and weaker activation of the yeast HSP70-lacZ reporter. Therefore the loop domain may be a critical determinant in allowing HSF1 and HSF2 to activate target genes selectively based on the arrangement of the HSE (Ahn, 2001).

The idea that different arrangements of response elements for a transciption factor may influence the pattern of gene regulation was proposed to explain how the glucocorticoid receptor could function as both a transcriptional activator and repressor depending upon the promoter context. The allosteric control hypothesis posited that distinct DNA recognition sequences influence the activity of a transcriptional activator by dictating the conformation of the DNA-binding protein bound to the site, which in turn specifies the protein surfaces available for interactions with other transcription factors. Similarly, differences in the orientation of the HSE have been reported to promote distinct binding by yeast HSF. An arrangement of two repeats of the tail-tail sequence (nTTCnnGAAn) was found to bind two HSF trimers much more efficiently than two repeats of the head-head orientation (nGAAnnTTCn). Furthermore, a comparison of yeast HSF binding to the gapped CUP1 promoter and a continuous HSE by proteolytic cleavage assays revealed differences in the sensitivity of HSF to the protease. This difference in conformation was suggested to be one factor in determining the temperature threshold of HSF target gene activation. Although these studies show that yeast HSF is capable of adapting to different HSEs, this analysis of HSF1 and HSF2 suggests that the mammalian proteins exhibit more defined preferences for DNA binding and target gene activation. Although both proteins bind to the same core HSE sequences, differential binding and transactivation from shorter versus longer arrays of the pentameric HSE repeats by HSF2 and HSF1, respectively, would provide a basis for differential target gene activation in mammals. Interactions with other cis- and trans-acting factors may influence the protein-DNA interactions at some promoters. Although these data have addressed the molecular basis for discrimination for HSEs by HSF1 and HSF2, there may be overlapping targets for HSF1 and HSF2. For example, hsp70 can be activated in an HSF2-dependent manner during hemin-induced differentiation of K562 cells. However, it was observed that HSF1 and HSF2 differentially occupy the hsp70 HSE and activate the gene to significantly different levels in response to the heat shock and hemin, respectively. Thus, it is proposed that distinctions in the arrangement of the HSEs will provide an additional layer of selectivity among HSF-dependent target gene expression, especially under limiting levels of HSF1 and HSF2 protein concentrations, and may also modulate transcriptional potency. A full characterization of HSF2-preferred HSEs in vivo must await the identification of bona fide HSF2 target genes (Ahn, 2001).

A question that is raised is how might the loop facilitate cooperative interactions. A potential answer may be found from the cocrystal structure of two K. lactis HSF DBDs bound to two inverted HSE repeats. Although disordered in that structural model, the loop was predicted to lie in the interface between the two DNA-bound HSF DBDs. The loop of one DBD was thought to be juxatoposed against the turn of the helix-turn-helix motif of an adjacent DBD. Although not encompassed by the structure, the loop-and-turn interface was also predicted to bridge interactions between adjacent trimers bound to an extended HSE. Thus the loop may comprise part of a critical protein-protein interaction surface that is responsible for stabilizing contacts between individual DBDs within a DNA-bound trimer and between adjacent trimers (Ahn, 2001).

Even more striking, it has been found that the HSF1 loop is sufficient for conferring heat-inducible trimerization to purified HSF2 in vitro when transposed into this isoform, suggesting that the loop is part of the critical heat-sensing mechanism that distinguishes HSF1. A physiological significance for the HSF1 loop in cells is shown because the HSF2Loop1 protein, like HSF1 but not HSF2, is capable of rescuing transfected hsf1^-/- MEFs from heat-induced apoptosis. Therefore, it is proposed that the HSF1 loop modulates the monomer-to-trimer transition in response to heat stress in vitro and in cells in addition to facilitating cooperative binding on extended HSEs. A potential model is that the loop undergoes a conformational change upon heat stress, which relieves intramolecular repression of the monomer and exposes the trimerization domain (Ahn, 2001).

Heat shock factor alternative splicing

Mammalian cells express two distinct HSF1 mRNA isoforms that arise via alternative splicing of the HSF1 pre-mRNA. The two HSF1 mRNA isoforms differ by a single 66 bp exon of the HSF1 gene, which is spliced into the HSF1-alpha mRNA isoform but skipped in the HSF1-beta mRNA isoform. This 66 bp exon encodes a 22 amino acid sequence, whose molecular weight (2.3 kDa) matches the difference in size between the HSF1-beta and HSF1-alpha protein isoforms (69 and 71 kDa). Further analysis reveals that this extra 22 amino acid sequence, whose insertion site in the HSF1-alpha isoform is located immediately adjacent to a C-terminal leucine zipper motif (leucine zipper 4) previously shown to be involved in maintenance of HSF1 in the non-DNA-binding control form, contains an additional, previously unidentified leucine zipper motif (leucine zipper 5). The levels of the two HSF1 isoforms are regulated in a tissue dependent manner, with testis expressing higher levels of the HSF1-beta isoform while heart and brain express higher levels of the HSF1-alpha isoform. These results demonstrate a new mechanism by which HSF1 expression is regulated in mammalian cells and suggest a potential role for the HSF1 isoforms in mediating tissue-dependent regulation of the cellular stress response (Goodson, 1995b).

Heat shock factor interaction with chaperones

Heat shock and other proteotoxic stresses cause accumulation of nonnative proteins that trigger activation of heat shock protein (Hsp) genes. A chaperone/Hsp functioning as a repressor of heat shock transcription factor (HSF) could make activation of Hsp genes dependent on protein unfolding. In a novel in vitro system, in which human HSF1 can be activated by nonnative protein, heat, and geldanamycin, the addition of Hsp90 inhibits activation. Reduction of the level of Hsp90 but not of Hsp/c70, Hop, Hip, p23, CyP40, or Hsp40 dramatically activates HSF1. In vivo, geldanamycin activates HSF1 under conditions in which it is an Hsp90-specific reagent. Hsp90-containing HSF1 complex is present in the unstressed cell and dissociates during stress. It is concluded that Hsp90, by itself and/or associated with multichaperone complexes, is a major repressor of HSF1 (Zou, 1998).

Transcriptional activation of heat shock genes is a reversible and multistep process involving conversion of inactive heat shock factor 1 (HSF1) monomers into heat shock element (HSE)-binding homotrimers as well ashyperphosphorylation, and further modifications that induce full transcriptional competence. HSF1 is controlled by multiple regulatory mechanisms, including suppression by additional cellular factors, physical interactions with HSP70, and integration into different cellular signaling cascades. However, the signaling mechanisms by which cells respond to stress and control the HSF1 activation-deactivation pathway are not known. HSP90, a cellular chaperone known to regulate several signal transduction molecules and transcription factors, functions in the regulation of HSF1. The existence of HSF1-HSP90 heterocomplexes was demonstrated by coimmunoprecipitation of HSP90 with HSF1 from unshocked and heat-shocked nuclear extracts: the recognition of HSF1-HSE complexes in vitro was shown by using HSP90 antibodies (Abs), and the recognition of HSF1 in vivo by HSP90 Abs microinjected directly into oocyte nuclei. The functional impact of HSP90-HSF1 interactions was analyzed using two strategies: direct nuclear injection of HSP90 Abs and treatment of cells with geldanamycin (GA), an agent that specifically blocks the chaperoning activity of HSP90. Both HSP90 Abs and GA delay the disassembly of HSF1 trimers during recovery from heat shock and specifically inhibit heat-induced transcription from a chloramphenicol acetyltransferase reporter construct under control of the hsp70 promoter. HSP90 Abs activate HSE binding in the absence of heat shock, an effect that can be reversed by subsequent injection of purified HSP90. GA does not activate HSE binding under nonshock conditions but increases the quantity of HSE binding induced by heat shock. On the basis of these findings and the known properties of HSP90, a new regulatory model is proposed in which HSP90 participates in modulating HSF1 at different points along the activation-deactivation pathway, influencing the interconversion between monomeric and trimeric conformations as well as transcriptional activation. HSP90 is proposed to link HSF1 to cellular signaling molecules coordinating the stress response (Ali, 1998).

Heat shock factor phosphorylation

HSF1 is the mediator of stress-induced heat shock gene transcription in humans. HSF1 displays stress-induced DNA-binding activity, oligomerization, and nuclear localization, while HSF2 does not. HSF1 undergoes phosphorylation in cells exposed to heat or cadmium sulfate but not in cells treated with the amino acid analog L-azetidine-2-carboxylic acid, indicating that phosphorylation of HSF1 is not essential for its activation. Interestingly, HSF1 and HSF2 overexpressed in transfected 3T3 cells both display constitutive DNA-binding activity, oligomerization, and transcriptional activity. These results demonstrate that HSF1 can be activated in the absence of physiological stress and also provide support for a model of regulation of HSF1 and HSF2 activity by a titratable negative regulatory factor (Sarge, 1993).

Heat shock transcription factor 1 (HSF1) is constitutively expressed in mammalian cells and negatively regulated for DNA binding and transcriptional activity. At serine residues distal to the transcriptional activation domain, constitutive phosphorylation of HSF1 functions to repress HSF1's transactivation function. A region of constitutive phosphorylation encompasses serine residues 303-307. Whereas HSF1 is constitutively repressed for transactivation and derepressed by heat shock, mutations of serines 303 and 307 to alanine result in derepression to a high level of constitutive activity. The relationship between MAP kinase phosphorylation of HSF1 and the constitutive activation of HSF1 reported in this paper is unclear (Kline, 1997).

Human heat shock transcription factor 1 (HSF1) is responsible for stress-induced transcription of heat shock protein genes. The activity of the HSF1 transcriptional activation domains is modulated by a separate regulatory domain, which confers repression at control temperature and heat inducibility. A high level of phosphorylation at the low (control) temperature seems to result in HSF1 inactivation. Two specific proline-directed serine motifs are important for function of the regulatory domain in the following ways: mutation of these serines to alanine results in derepressed HSF1 activity at control temperature, and mutation to glutamic acid, mimicking a phosphorylated serine, results in normal repression at control temperature, with normal heat shock inducibility. Tryptic mapping shows that these serines are the major phosphorylation sites of HSF1 at control temperature in vivo. Stimulation of the Raf/ERK pathway in vivo results in an increased level of phosphorylation of these major sites. The regulatory domain is an excellent substrate in vitro for the mitogen-activated MAPK/ERK. Phosphorylation of the regulatory domain of HSF1 appears to decrease the activity of HSF1 at control temperature, and suggests a mechanism for modification of HSF1 activity by growth control signals (Knauf, 1996).

Heat shock factor sumoylation

Covalent modification of proteins by the small ubiquitin-related modifier SUMO regulates diverse biological functions (see Drosophila SUMO). Sumoylation usually requires a consensus tetrapeptide, through which the binding of the SUMO-conjugating enzyme Ubc9 to the target protein is directed. However, additional specificity determinants are in many cases required. To gain insights into SUMO substrate selection, the differential sumoylation of highly similar loop structures within the DNA-binding domains of heat shock transcription factor 1 (HSF1) and HSF2 were used. Site-specific mutagenesis in combination with molecular modeling revealed that the sumoylation specificity is determined by several amino acids near the consensus site, which are likely to present the SUMO consensus motif to Ubc9. Importantly, sumoylation of the HSF2 loop impedes HSF2 DNA-binding activity, without affecting its oligomerization. Hence, SUMO modification of the HSF2 loop contributes to HSF-specific regulation of DNA binding and broadens the concept of sumoylation in the negative regulation of gene expression (Anckar, 2006)

Heat shock factor interaction with target sites

The interaction of recombinant mouse heat shock transcription factors 1 and 2 (mHSF1 and mHSF2) with their binding site, the heat shock element (HSE) has been characterized using human HSP70 HSE. It consists of three perfect 5'-nGAAn-3' sites (1, 3, and 4) and two imperfect sites (2 and 5) arranged as tandem inverted repeats. Recombinant mHSF1 and mHSF2, which exist as trimers in solution, both bind specifically to this HSE and stimulate transcription of a human HSP70-CAT construct in vitro. Footprinting analyses reveal differential binding of mHSF1 and mHSF2 to the HSP70 HSE. Specifically, mHSF1 binds all five pentameric sites, whereas mHSF2 fails to interact with the first site of the HSE but binds to sites 2 to 5. Missing-nucleoside analysis demonstrates that the third and fourth nGAAn sites are essential for mHSF1 and mHSF2 binding. The binding of the initial mHSF1 trimer to the HSE exhibits preference for sites 3, 4, and 5; then binding of a second trimer occurs at sites 1 and 2. These results suggest that HSF may recognize its binding site through the dyad symmetry of sites 3 and 4 but requires an adjacent site for stable interaction. Thus mHSF1 and mHSF2 bind specifically to the HSE through major groove interactions. Footprinting reveals structural differences in the first and third repeats of the HSE, suggesting that the DNA is distorted in this region. The possibility that the HSE region is naturally distorted may assist in understanding how a trimer of HSF can bind to what is essentially an inverted repeat binding site (Kroeger, 1993).

Heat shock transcription factor 1 (HSF1) functions as the master regulator of the heat shock response in eukaryotes. In addition to its role as a transcription factor, HSF1 stimulates the activity of the DNA-dependent protein kinase (DNA-PK). DNA-PK is composed of two components: a 460-kDa catalytic subunit and a 70- and 86-kDa heterodimeric regulatory component, also known as the Ku protein. HSF1 binds specifically to each of the two components of DNA-PK. Binding occurs in the absence of DNA. The complex with the Ku protein is stable and forms at a stoichiometry close to unity between the Ku protein heterodimer and the active HSF1 trimer. The binding is blocked by antibodies against HSF1. These results show that HSF1 also binds directly, but more weakly, to the catalytic subunit of DNA-PK. Both interactions are dependent on a specific region within the HSF1 regulatory domain. This sequence is necessary but not sufficient for HSF1 stimulation of DNA-PK activity. The ability of HSF1 to interact with both components of DNA-PK provides a potential mechanism for the activation of DNA-PK in response to heat and other forms of stress (Huang, 1997).

In response to stress, heat shock factor 1 (HSF1) acquires rapid DNA binding and transient transcriptional activity while undergoing conformational transition from an inert non-DNA-binding monomer to active functional trimers. Attenuation of the inducible transcriptional response occurs during heat shock or upon recovery at non-stress conditions and involves dissociation of the HSF1 trimer and loss of activity. The hydrophobic repeats of the HSF1 trimerization domain were used in the yeast two-hybrid protein interaction assay to identify heat shock factor binding protein 1 (HSBP1), a novel, conserved, 76-amino-acid protein that contains two extended arrays of hydrophobic repeats that interact with the HSF1 heptad repeats. HSBP1 is nuclear-localized and interacts in vivo with the active trimeric state of HSF1 that appears during heat shock. During attenuation of HSF1 to the inert monomer, HSBP1 associates with Hsp70. HSBP1 negatively affects HSF1 DNA-binding activity, and overexpression of HSBP1 in mammalian cells represses the transactivation activity of HSF1. To establish a biological role for HSBP1, the homologous Caenorhabditis elegans protein was overexpressed in body wall muscle cells and was shown to block activation of the heat shock response from a heat shock promoter-reporter construct. Alteration in the level of HSBP1 expression in C. elegans has severe effects on survival of the animals after thermal and chemical stress, consistent with a role for HSBP1 as a negative regulator of the heat shock response (Satyal, 1998).

Developmently regulated heat shock factors

The spontaneous expression of heat shock genes during development is well documented in many animal species, but the mechanisms responsible for this developmental regulation are only poorly understood. In vertebrates, additional heat shock transcription factors, distinct from heat shock factor 1 (HSF1) involved in the stress response, are thought to be involved in this developmental control. In particular, the mouse HSF2 has been found to be active in testis and during preimplantation development. In mice, HSF2 is regulated during the postimplantation phase of development. It is expressed at least until 15.5 days of embryogenesis. It becomes restricted to the central nervous system during the second half of gestation. It is expressed in the ventricular layer of the neural tube, which contains mitotically active cells, but not in postmitotic neurons. Parallel results are obtained for mRNA, protein, and activity levels, demonstrating that the main level of control is transcriptional. The detailed analysis of the activity of a luciferase reporter gene under the control of the hsp70.1 promoter, as well as the description of the protein expression patterns of the major heat shock proteins in the central nervous system, show that HSF2 and heat shock protein expression domains do not coincide. This result suggests that HFS2 might be involved in other regulatory developmental pathways and paves the way to new functional approaches (Rallu, 1997).

The inducible DNA-binding activities of HSF1 and HSF2 are regulated through distinct pathways. HSF1 is activated by heat shock and other forms of stress, whereas HSF2 is activated during hemin-induced differentiation of human K562 erythroleukemia cells, suggesting a role for HSF2 in regulating heat shock gene expression under nonstress conditions, such as differentiation and development. The inactive, non-DNA-binding forms of HSF2 and HSF1 exist primarily in the cytoplasm of untreated K562 cells as a dimer and monomer, respectively. This difference in the control oligomeric states suggests that distinct mechanisms are used to control the DNA-binding activities of HSF2 and HSF1. Upon activation, both factors acquire DNA-binding activity, oligomerize to a trimeric state, and translocate into the nucleus. Simultaneous activation of both HSF2 and HSF1 in K562 cells subjected to hemin treatment followed by heat shock results in the synergistic induction of hsp70 gene transcription, suggesting a novel level of complex regulation of heat shock gene expression (Sistonen, 1994).

Acquisition of heat shock factor 2 (HSF2) DNA binding activity is accompanied by induced transcription of heat shock genes in hemin-treated K562 cells undergoing erythroid differentiation. HSF2 consists of two alternatively spliced isoforms, HSF2-alpha and HSF2-beta, whose relative abundance is developmentally regulated and varies between different tissues. To investigate whether the molar ratio of HSF2-alpha and HSF2-beta isoforms is crucial for the activation of HSF2 and whether the HSF2 isoforms play functionally distinct roles during the hemin-mediated erythroid differentiation, cell clones were generated expressing different levels of HSF2-alpha and HSF2-beta. In parental K562 cells, the HSF2-alpha isoform is predominantly expressed and HSF2 can be activated upon hemin treatment. In contrast, when HSF2-beta is expressed at levels exceeding those of endogenous HSF2-alpha, the hemin-induced DNA binding activity and transcription of heat shock genes are repressed, whereas overexpression of HSF2-alpha results in an enhanced hemin response. Furthermore, the hemin-induced accumulation of globin, known as a marker of erythroid differentiation, is decreased in cells overexpressing HSF2-beta. It is suggested that HSF2-beta acts as a negative regulator of HSF2 activity during hemin-mediated erythroid differentiation of K562 cells (Leppa, 1997).

HSF2 alternative splicing is developmentally regulated; a switch in expression occurs during testis postnatal development from the HSF2-beta mRNA isoform to the HSF2-alpha isoform. Transfection analysis shows that the HSF2-alpha protein, the predominant isoform expressed in testis cells, is a more potent transcriptional activator than the HSF2-beta isoform. These results reveal a new mechanism for the control of HSF2 function in mammalian cells, in which regulated alternative splicing is used to modulate HSF2 transcriptional activity in a tissue-dependent manner (Goodson, 1995c).

Heat shock transcription factors (HSFs) are generally known as regulators of cellular stress response. The mammalian HSF1 functions as a classical stress factor, whereas HSF2 is active during certain developmental processes, including embryogenesis and spermatogenesis. In the present study, HSF2 expression was examined at specific stages of the rat seminiferous epithelial cycle. Expression of the alternatively spliced HSF2-alpha and HSF2-beta isoforms is developmentally regulated in a stage-specific manner. Studies on cellular localization demonstrate that HSF2 is present in the nuclei of early pachytene spermatocytes at stages I to IV and in the nuclei of round spermatids at stages V to VIIab. In contrast, a strong HSF2 immunoreactivity is detected in small distinct cytoplasmic regions from zygotene spermatocytes to maturation phase spermatids. Immunoelectron microscopic analysis reveals that these structures are mainly cytoplasmic bridges between germ cells. These results on cellular localization of HSF2 and stage-specific expression of the HSF2 isoforms indicate that HSF2, in addition to its function as a nuclear transcription factor, may be involved in other cellular processes during spermatogenesis, possibly in the sharing process of gene products between the germ cells (Alastalo, 1998).

Mammalian cells coexpress a family of heat shock factors (HSFs) whose activities are regulated by diverse stress conditions to coordinate the inducible expression of heat shock genes. Distinct from HSF1, which is expressed ubiquitously and activated by heat shock and other stresses that result in the appearance of nonnative proteins, the stress signal for HSF2 has not yet been identified. HSF2 activity has been associated with development and differentiation, and the activation properties of HSF2 have been characterized in hemin-treated human K562 erythroleukemia cells. A stress signal for HSF2 activation occurs when the ubiquitin-proteasome pathway is inhibited. HSF2 DNA-binding activity is induced upon exposure of mammalian cells to the proteasome inhibitors hemin, MG132, and lactacystin, and in the mouse ts85 cell line, which carries a temperature sensitivity mutation in the ubiquitin-activating enzyme (E1) when shifted to the nonpermissive temperature. HSF2 is labile, and its activation requires both continued protein synthesis and reduced degradation. The downstream effect of HSF2 activation by proteasome inhibitors is the induction of the same set of heat shock genes that are induced during heat shock by HSF1, thus revealing that HSF2 affords the cell with a novel heat shock gene-regulatory mechanism to respond to changes in the protein-degradative machinery (Mathew, 1998).

In vertebrates, the presence of multiple heat shock transcription factors (HSFs) indicates that these factors may be regulated by distinct stress signals. HSF3 is specifically activated in unstressed proliferating cells by direct binding to the c-myb proto-oncogene product (c-Myb). These factors formed a complex through their DNA binding domains that stimulates the nuclear entry and formation of the transcriptionally active trimer of HSF3. Because c-Myb participates in cellular proliferation, this regulatory pathway may provide a link between cellular proliferation and the stress response (Kanei-Ishii, 1997).

Human HSF4 (hHSF4) is a novel member of the hHSF family that shares properties with other members of the HSF family yet appears to be functionally distinct. hHSF4 lacks the carboxyl-terminal hydrophobic repeat that is shared among all vertebrate HSFs and is thought to be involved in the negative regulation of DNA binding activity. hHSF4 is preferentially expressed in the human heart, brain, skeletal muscle, and pancreas. Transient transfection of hHSF4 in HeLa cells, which do not express hHSF4, results in a constitutively active DNA binding trimer that (unlike other members of the HSF family) lacks the properties of a transcriptional activator. Constitutive overexpression of hHSF4 in HeLa cells results in reduced expression of the endogenous hsp70, hsp90, and hsp27 genes. hHSF4 represents a novel hHSF that exhibits tissue-specific expression and functions to repress the expression of genes encoding heat shock proteins and molecular chaperones (Nakai, 1997).

E2F coregulates an essential HSF developmental program that is distinct from the heat-shock response

Heat-shock factor (HSF; see Drosophila Hsf) is the master transcriptional regulator of the heat-shock response (HSR) and is essential for stress resilience. HSF is also required for metazoan development; however, its function and regulation in this process are poorly understood. This study characterize the genomic distribution and transcriptional activity of Caenorhabditis elegans HSF-1 during larval development and showed that the developmental HSF-1 transcriptional program is distinct from the HSR. HSF-1 developmental activation requires binding of E2F/DP (see Drosophila E2f) to a GC-rich motif that facilitates HSF-1 binding to a heat-shock element (HSE) that is degenerate from the consensus HSE sequence and adjacent to the E2F-binding site at promoters. In contrast, induction of the HSR is independent of these promoter elements or E2F/DP and instead requires a distinct set of tandem canonical HSEs. Together, E2F and HSF-1 directly regulate a gene network, including a specific subset of chaperones, to promote protein biogenesis and anabolic metabolism, which are essential in development (Li , 2016).

Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis

Heat shock factor 1 (HSF1) is the master regulator of the heat shock response in eukaryotes, a very highly conserved protective mechanism. HSF1 function increases survival under a great many pathophysiological conditions. How it might be involved in malignancy remains largely unexplored. This study reports that eliminating HSF1 protects mice from tumors induced by mutations of the RAS oncogene or a hot spot mutation in the tumor suppressor p53. In cell culture, HSF1 supports malignant transformation by orchestrating a network of core cellular functions including proliferation, survival, protein synthesis, and glucose metabolism. The striking effects of HSF1 on oncogenic transformation are not limited to mouse systems or tumor initiation; human cancer lines of diverse origins show much greater dependence on HSF1 function to maintain proliferation and survival than their nontransformed counterparts. While it enhances organismal survival and longevity under most circumstances, HSF1 has the opposite effect in supporting the lethal phenomenon of cancer (Dai, 2007).

REFERENCES

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Achary, B. G., Campbell, K. M., Co, I. S. and Gilmour, D. S. (2014). RNAi screen in Drosophila larvae identifies histone deacetylase 3 as a positive regulator of the hsp70 heat shock gene expression during heat shock. Biochim Biophys Acta 1839(5): 355-363. PubMed ID: 24607507

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Alastalo, T. P., et al. (1998). Stage-specific expression and cellular localization of the heat shock factor 2 isoforms in the rat seminiferous epithelium. Exp. Cell Res. 240(1): 16-27. PubMed Citation: 9570917

Ali, A., et al. (1998). HSP90 interacts with and regulates the activity of heat shock factor 1 in Xenopus oocytes. Mol. Cell. Biol. 18(9): 4949-60. PubMed Citation: 9710578

Amin, J., et al., (1994). Cooperative binding of heat shock transcription factor to the Hsp70 promoter in vivo and in vitro. J. Biol. Chem. 269: 4804-11. PubMed Citation: 8106450

Anckar, J., et al. (2006). Inhibition of DNA binding by differential sumoylation of heat shock factors. Mol. Cell. Biol. 26(3): 955-64. 16428449

Baler, R., Dahl, G. and Voellmy, R. (1993). Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1. Mol. Cell. Biol. 13: 2486-96. PubMed Citation: 8455624

Boehm, A. K., Saunders, A., Werner, J. and Lis, J. T. (2003). Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock. Mol Cell Biol. 23(21): 7628-37. 14560008

Brown, S. A. and Kingston, R. E. (1997). Disruption of downstream chromatin directed by a transcriptional activator. Genes Dev. 11(23): 3116-3121. PubMed Citation: 9389644

Chen, C., et al. (1997). Heat shock factor 1 represses Ras-induced transcriptional activation of the c-fos gene. J. Biol. Chem. 272(43): 26803-26806. PubMed Citation: 9341107

Chen, Y. C., et al. (2009). The Torso signaling pathway modulates a dual transcriptional switch to regulate tailless expression. Nucleic Acids Res. 37(4): 1061-72. PubMed Citation: 19129218

Christians, E., et al. (2000). Embryonic development: Maternal effect of Hsf1 on reproductive success. Nature 407: 693-694. PubMed Citation: 11048707

Clos, J., et al. (1990). Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation. Cell 63: 1085-97. PubMed Citation: 2257625

Clos, J., et al. (1993). Induction temperature of human heat shock factor is reprogrammed in a Drosophila cell environment. Nature 364: 252-5. PubMed Citation: 8321322

Dai, C., et al. (2007). Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell 130: 1005-1018. Medline abstract: 17889646

Farkas, T., Kutskova, Y. A. and Zimarino, V. (1998). Intramolecular repression of mouse heat shock factor 1. Mol. Cell. Biol. 18(2): 906-918

Fernandes, M., Xiao, H. and Lis, J.T. (1993). Fine structure analyses of the Drosophila and Saccharomyces heat shock factor--heat shock element interactions. Nucleic Acids Res. 22: 167-73

Fernandes, M., Xiao, H. and Lis, J. T. (1995). Binding of heat shock factor to and transcriptional activation of heat shock genes in Drosophila. Nucleic Acids Res. 23: 4799-4804

Giardina, C. and Lis, J. T. (1995a). Dynamic protein-DNA architecture of a yeast heat shock promoter. Mol. Cell. Biol. 15: 2737-2744

Giardina, C. and Lis, J. T. (1995b). Sodium salicylate and yeast heat shock gene transcription. J. Biol. Chem. 270: 10369-10372

Goodson, M. L. and Sarge, K. D. (1995a). Heat-inducible DNA binding of purified heat shock transcription factor 1. J. Biol. Chem. 270: 2447-2450

Goodson, M. L. and Sarge, K. D. (1995b). Regulated expression of heat shock factor 1 isoforms with distinct leucine zipper arrays via tissue-dependent alternative splicing. Biochem. Biophys. Res. Commun. 211: 943-949

Goodson, M. L., Park-Sarge, O. K. and Sarge, K. D. (1995c). Tissue-dependent expression of heat shock factor 2 isoforms with distinct transcriptional activities. Mol. Cell. Biol. 15: 5288-5293

Gordon, S., et al. (1997). Distinct stress-inducible and developmentally regulated heat shock transcription factors in Xenopus oocytes. Dev. Biol. 181: 47-63

Ghosh, S. K., Missra, A., Gilmour, D. S. (2011). Negative elongation factor accelerates the rate at which heat shock genes are shut off by facilitating dissociation of heat shock factor. Mol. Cell Biol. 31(20): 4232-43. PubMed Citation: 21859888

Green, M., et al. (1995). A heat shock-responsive domain of human HSF1 that regulates transcription activation domain function. Mol. Cell. Biol. 15: 3354-62

Guertin, M. J. and Lis, J. T. (2010). Chromatin landscape dictates HSF binding to target DNA elements. PLoS Genet. 6(9): e1001114. PubMed Citation: 20844575

Guertin, M. J., Martins, A. L., Siepel, A. and Lis, J. T. (2012). Accurate prediction of inducible transcription factor binding intensities in vivo. PLoS Genet 8: e1002610. Pubmed: 22479205

Hajdu-Cronin, Y. M., Chen, W. J. and Sternberg, P. W. (2004). The L-type cyclin CYL-1 and the heat-shock-factor HSF-1 are required for heat-shock-induced protein expression in Caenorhabditis elegans. Genetics 168(4): 1937-49. 15611166

He, B., Meng, Y. H. and Mivechi, N. F. (1998). Glycogen synthase kinase 3beta and extracellular signal-regulated kinase inactivate heat shock transcription factor 1 by facilitating the disappearance of transcriptionally active granules after heat shock. Mol. Cell. Biol. 18(11): 6624-33

Huang, J., et al., (1997). Heat shock transcription factor 1 binds selectively in vitro to Ku protein and the catalytic subunit of the DNA-dependent protein kinase. J. Biol. Chem. 272(41): 26009-26016

Jedlicka, P., Morton, M. A. and Wu, C. (1997). Multiple functions of Drosophila heat shock transcription factor in vivo. EMBO J. 16: 2452-62

Jurivich, D. A., et al. (1994). Arachidonate is a potent modulator of human heat shock gene transcription. Proc. Natl. Acad. Sci. 91: 2280-4

Kanei-Ishii, C., et al. (1997). Activation of heat shock transcription factor 3 by c-Myb in the absence of cellular stress. Science 277(5323): 246-248

Karunanithi, S., et al. (1999). Neuroprotection at Drosophila synapses conferred by prior heat shock. J. Neurosci. 19(11): 4360-9. PubMed ID: 10341239

Kline, M. P. and Morimoto, R. I. https://pubmed.ncbi.nlm.nih.gov/tor 1 transcriptional acivation domain is modulated by constitutive phosphorylation. Mol. Cell. Biol. 17: 2107-15

Knauf, U., et al. (1996). Repression of human heat shock factor 1 activity at control temperature by phosphorylation. Genes Dev. 10:2782-2793

Kroeger, P. E., Sarge, K. D. and Morimoto, R. I. (1993). Mouse heat shock transcription factors 1 and 2 prefer a trimeric binding site but interact differently with the HSP70 heat shock element. Mol. Cell. Biol. 13: 3370-83

Kumsta, C., Ching, T. T., Nishimura, M., Davis, A. E., Gelino, S., Catan, H. H., Yu, X., Chu, C. C., Ong, B., Panowski, S. H., Baird, N., Bodmer, R., Hsu, A. L. and Hansen, M. (2013). Integrin-linked kinase modulates longevity and thermotolerance in C. elegans through neuronal control of HSF-1. Aging Cell [Epub ahead of print]. PubMed ID: 24314125

Kust, N., Rybalkina, E., Mertsalov, I., Savchenko, E., Revishchin, A. and Pavlova, G. (2014). Functional analysis of Drosophila HSP70 promoter with different HSE numbers in human cells. PLoS One 9: e101994. PubMed ID: 25101947

Li, J., Chauve, L., Phelps, G., Brielmann, R. M. and Morimoto, R. I. (2016). E2F coregulates an essential HSF developmental program that is distinct from the heat-shock response. Genes Dev 30: 2062-2075. PubMed ID: 27688402

Lee, S., et al. (2000). The yeast heat shock transcription factor changes conformation in response to superoxide and temperature. Mol. Biol. Cell 11: 1753-1764. PubMed ID: 10793149

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Levy-Holtzman, R., Clos, J. and Schechter, I. (1995). Differences in DNA sequence recognition by the heat-shock factors of Drosophila melanogaster and the parasitic helminth Schistosoma mansoni. Biochim. Biophys. Acta 1264: 134-140

Lis, J. T., et al. (2000). P-TEFb kinase recruitment and function at heat shock loci. Genes Dev. 14: 792-803. 10766736

Marchler, G. and Wu, C. (2001). Modulation of Drosophila heat shock transcription factor activity by the molecular chaperone DROJ1. EMBO J. 20: 499-509. 11157756

Mason, P. B. and Lis, J. T. (1997). Cooperative and competitive protein interactions at the hsp70 promoter. J. Biol. Chem. 272(52): 33227-33233

Mathew, A., Mathur, S. K. and Morimoto, R. I.. (1998). Heat shock response and protein degradation: regulation of HSF2 by the ubiquitin-proteasome pathway. Mol. Cell. Biol. 18(9): 5091-8

Mercier, P. A., et al. (1997). Xenopus heat shock factor 1 is a nuclear protein before heat stress. J. Biol. Chem. 272 (22): 14147-14151

Michaud, S., et al. (1997). Cell-specific expression and heat-shock induction of Hsps during spermatogenesis in Drosophila melanogaster. J. Cell Sci. 110: 1989-1997

Morimoto, R. I., A. Tissieres and C. Georgopoulos, editors (1994). The biology of heat shock proteins and molecular chaperones. Cold Spring Harbor, NY: Cold Spring Laboratory Press.

Nakai, A., et al. (1997). HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator. Mol. Cell. Biol. 17: 469-48

Newton, E. M., et al. (1996). The regulatory domain of human heat shock factor 1 is sufficient to sense heat stress. Mol. Cell. Biol. 16: 939-846

Nightingale, K. P., et al. (1998). Histone acetylation facilitates RNA polymerase II transcription of the Drosophila hsp26 gene in chromatin. EMBO J. 17: 2865-2876

Nowak, S. J. and Corces, V. G. (2000). Phosphorylation of histone H3 correlates with transcriptionally active loci. Genes Dev. 14: 3003-3013. 11114889

O'Brien, T. and Lis, J. T. (1993). Rapid changes in Drosophila transcription after an instantaneous heat shock. Mol. Cell. Biol. 13: 3456-63

Orosz, A., Wisniewski, J. and Wu, C. (1996). Regulation of Drosophila heat shock factor trimerization: Global sequence requirements and independence of nuclear localization. Mol. Cell. Biol. 16: 7016-30

Park, J. M., et al. (2001). Drosophila mediator complex is broadly utilized by diverse gene-specific transcription factors at different types of core promoters. Mol. Cell. Bio. 21: 2312-2323. 11259581

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Rabindran, S. K., et al. (1994). Interaction between heat shock factor and hsp70 is insufficient to suppress induction of DNA-binding activity in vivo. Mol. Cell. Biol. 14: 6552-60

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Sandaltzopoulos, R., et al. (1995). Dual regulation of the Drosophila hsp26 promoter in vitro. Nucleic Acids Res 23: 2479-2487

Sandaltzopoulos, R. and Becker, P. B. (1998). Heat shock factor increases the reinitiation rate from potentiated chromatin templates. Mol. Cell. Biol. 18(1): 361-367

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Satyal, S. H., et al. (1998). Negative regulation of the heat shock transcriptional response by HSBP1. Genes Dev. 12(13): 1962-1974

Shopland, L. S. (1995). HSF access to heat shock elements in vivo depends critically on promoter architecture defined by GAGA factor, TFIID, and RNA polymerase II binding sites. Genes Dev. 9: 2756-2769

Sistonen, L., Sarge, K. D. and Morimoto, R. I. (1994). Human heat shock factors 1 and 2 are differentially activated and can synergistically induce hsp70 gene transcription. Mol. Cell. Biol. 14: 2087-99

Tsukiyama, T., Becker, P. B. and Wu, C. (1994). ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor. Nature 367: 525-32

Tsukiyama, T. and Wu, C. (1995a). Purification and properties of an ATP-dependent nucleosome remodeling factor. Cell 83: 1011-1020

Tsukiyama, T., et al. (1995b). ISWI, a member of the SWI2/SNF2 ATPase family, encodes the 140 kDa subunit of the nucleosome remodeling factor. Cell 83: 1021-1026

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Westwood, J. T. and Wu, C. (1993). Activation of Drosophila heat shock factor: conformational change associated with a monomer-to-trimer transition. Mol. Cell. Biol. 13: 3481-6

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