InteractiveFly: GeneBrief

Hsp70Aa, Hsp70Ab, Hsp70Ba, Hsp70Bb, Hsp70Bbb and Hsp70Bc: Biological Overview | References

BIOLOGICAL OVERVIEW

Unravelling the molecular mechanisms that govern cell migration is of great importance towards understanding both normal embryogenesis and physiological and pathological processes occurring in the adult. Migration of border cells (BCs) during Drosophila oogenesis provides a simple and attractive model in which to address this problem. This study shows that the molecular chaperone Hsp70 is required for BC migration. Thus, BCs lacking all Hsp70 genes present in the fly genome fail to reorganize their actin cytoskeleton, resulting in migration defects. Similar defects are found when the Hsp70 co-chaperone DnaJ-1, the Drosophila homolog of the human Hsp40, is overexpressed specifically in BCs. In addition, biochemical and genetic evidence is provided for an interaction between DnaJ-1 and PDGF/VEGF receptor (PVR), which is also required for actin-mediated BC migration. Furthermore, the results showing that PVR also interacts genetically with Hsp70 suggest that a mechanism by which the DnaJ-1/Hsp70 chaperone complex regulates BC migration is by modulating PVR function (Cobreros, 2008).

The migration of the border cells (BCs) in the Drosophila egg chamber represents an excellent model system to dissect genetically the mechanisms regulating cell migration in vivo. BCs originate as a group of 6-10 somatic cells located at the anterior terminal pole of the ovarian follicular epithelium. BCs comprise 6-8 outer border cells and two anterior polar cells. At stage 9, BCs undergo a partial epithelial-mesenchymal transition (EMT), change their shape, delaminate from the follicular epithelium (FE) and become migratory as a cluster. By extending F-actin-based protrusions in between the nurse cells (NCs), outer BCs migrate posteriorly until they contact the anterior membrane of the oocyte at stage 10, carrying the non-motile polar cells with them . Once it reaches the oocyte, the BC cluster migrates dorsally to reside eventually at the anterior dorsal corner of the oocyte. Concomitant to BC migration, the main FE moves towards the posterior pole of the egg chamber (Cobreros, 2008).

A variety of molecules that regulate BC migration have been identified. The Drosophila homolog of the C/EBP transcription factor slow border cells (Slbo) is a key regulator of BC migration. It is expressed in BCs before and during their migration. Slbo is essential for BC motility; mutations in Slbo result in a failure in BC migration. However, Slbo is not sufficient to induce BC migration since earlier expression of Slbo in BCs does not cause a precocious migration. Slbo controls the expression of many genes required for BC migration including the Drosophila homolog of E-Cadherin (DE-Cad), which is crucial in both BCs and NCs for BC migration (Cobreros, 2008).

In addition, genetic screens have identified four signalling pathways that control spatially and temporally BC migration. Signalling through the JAK-STAT pathway is necessary and sufficient to convert stationary follicle epithelial cells into migratory and invasive cells. Thus, ectopic expression of the pathway in non-motile cells makes them migratory and mutations in different components of the pathway result in a failure to initiate migration. In addition, the JAK-STAT pathway is required for the recruitment of outer BCs by the anterior polar cells as inhibition of the JAK/STAT pathway results in a smaller number of BCs recruited to the cluster. Mutations in the Ecdysone hormone co-receptor taiman (tai) result in defects in BC migration even though Slbo expression is not altered. Furthermore, the current model proposes that the ecdysone hormone receptor pathway controls BC migration by regulating the turnover of DE-Cad-containing adhesive complexes. Finally, two Receptor Tyrosine Kinase (RTK) pathways, the Platelet-Derived Growth Factor/Vascular Endothelial Growth Factor Receptor (PVR) and the Epithelial Growth Factor Receptor (EGFR) pathways, have also been shown to control BC migration. In this case, as the expression of dominant negative forms of both receptors in BCs results in a delayed and misrouted migration, it has been proposed that these pathways act redundantly to guide BC migration (Cobreros, 2008 and references therein).

Despite the various genetic screens that have been conducted to isolate and characterize genes involved in BC migration, none of these studies have reached saturation and, as such, it is anticipated that a proportion of genes still remain to be identified. This is supported by recent studies that compared the transcriptome of migratory follicle cells with that of all other ovarian cells. The results of these studies revealed that the number of genes whose expression is significantly higher in migratory cells reaches several hundreds. These genes encode for ECM components and regulators as well as proteins involved in transcription, the regulation of the cytoskeleton, cell adhesion or signalling pathways. The myriad of functions performed by the identified genes reflects both the complexity of cell migration and the fact that there is still much to discover (Cobreros, 2008).

In order to isolate new elements required for the developmental regulation of BC migration, a gain-of-function screen was performed using an inducible system that allowed the overexpression of genes specifically in the adult thus avoiding deleterious gene expression during embryonic and larval stages. One of the genes which overexpression resulted in BC migration defects was DnaJ-1, the Drosophila homolog of the human co-chaperone Hsp40. DnaJ1/Hsp40 proteins have been conserved throughout evolution and are important for protein folding, translocation and degradation. They perform these roles primarily by regulating the activity of chaperone proteins, such as the heat-shock protein Hsp70, through stimulation of their ATPase activity (Qiu, 2006). This work has investigated whether Hsp70 proteins are required for BC migration; removal of all Hsp70 genes present in the fly causes a delay in BC migration. In addition, biochemical and genetic interactions are described between DnaJ-1 and the PDGF- and VEGF-related receptor PVR, as well as a genetic interaction between PVR and Hsp70, suggesting that one mechanism by which DnaJ-1/Hsp70 could regulate BC migration is by modulating PVR function (Cobreros, 2008).

Hsp70 proteins are conserved molecular chaperones, found in the cytosol and in other compartments of the cell, that play an essential role in the life cycle of many proteins under both normal and stressful conditions. The house-keeping functions of Hsp70s include degradation of unstable and misfolded proteins, prevention and dissolution of protein aggregates, transport of proteins between cellular compartments, folding and refolding of proteins, uncoating of clathrin-coated vesicles and control of regulatory proteins. In a conventional model for Hsp70's mechanism of action, the 'client' protein with exposed hydrophobic residues is first recognized and bound by the co-chaperone Hsp40, which delivers it to ATP-Hsp70. The J-domain of Hsp40 then triggers ATP hydrolysis and the locking of Hsp70 on the 'client', thus promoting its folding (Fan, 2003; Qiu, 2006). 'Client' proteins whose activity is controlled through transient association with Hsp70 include regulatory proteins such as nuclear receptors (steroid hormone receptor), kinases (Raf, eIF2ξ-Kinase and CyclinB1) and transcription factors (HSF, c-Myc and pRb). Through these interactions, Hsp70 chaperones regulate important physiological processes such as cell cycle, cell differentiation or programmed cell death, as well as pathological processes such as oncogenesis, neurodegenerative diseases, viral infections and aging (Mayer, 2005; Cobreros, 2008).

The work reported in this study reveals a novel role for the molecular chaperone Hsp70 in development: the regulation of border cell migration during Drosophila oogenesis. Interfering with Hsp70 function causes a delay in the migration of BCs. One of the initial steps in BC migration is an epithelial to mesenchymal transition, during which BCs reorganize their actin cytoskeleton, delaminate from the epithelium and begin to migrate between the nurse cells towards the oocyte. This study shows that removal of Hsp70 function results in a failure of F-actin redistribution at the leading front of motile cells. A role for Hsp70 and other chaperones, such as Hsp90, in F-actin reorganization has already been proposed from experiments in cell culture. In fact, incubation of Schwann cells with antibodies against Hsp90 leads to a rearrangement of the actin filaments present in the lamellipodia and to a reduction in the migrating ability of these cells (Sidera, 2004). In addition, Hsp90 has also been involved in the actin-mediated motility of endothelial cells in vitro. Furthermore, both Hsp90 and the Hsp70 biding protein BAG-1 have been found to co-localize with F-actin during cell migration (Naishiro, 1999; Sidera, 2004]). Hsp70 could also act at other levels on the regulation of the actin cytoskeleton. Indeed, chaperones have been shown to perform many different and cooperative roles in the regulation of the cytoskeleton function (Liang, 1997). For instance, Hsp70 and Hsp90 have been shown to have an actin-binding activity that stabilizes the actin filaments by cross-linking. In addition, Hsp70 has also been shown to contribute to actin dynamics by assisting the chaperonin TriC on the folding of newly synthesized actin chains (Siegers, 2003). Finally, the possibility cannot be ruled out that the effects of Hsp70 on actin reorganization are an indirect consequence of the role of Hsp70 on microtubules polymerization and assembly (Liang, 1997). However, preliminary results showing no effects on α-tubulin localization in Hsp70 mutant BCs do not support this possibility. Having shown that Hsp70 regulates BC migration, the use of a new technique that allows live imaging of actin dynamics during this process will help elucidate the role of these chaperones on actin cytoskeleton during cell migration in vivo (Cobreros, 2008).

The experiments reported in this study show that overexpression of DnaJ-1 results in BC migration defects. A shared feature of the DnaJ/Hsp40 family of proteins is that they all contain the J domain, which is responsible for the regulation of Hsp70 ATPase activity. In addition to this domain, many of the DnaJ/Hsp40 family members contain other conserved regions such as a Gly/Phe-rich region and/or cystein repeats. Depending on the presence of these other regions DnaJ proteins can be classified in three groups: Type I proteins harbour all three domains; Type II members possess the Gly/Phe-rich sequence but lack the cysteine repeats; finally, Type III proteins do not contain either of these two conserved regions. Besides their role in assisting Hsp70, type I and type II proteins, but not type III, can bind non-native substrates. However, in some cellular processes, such as the suppression of protein aggregation, while type I members can function independently of Hsp70, type II proteins must function in conjunction with Hsp70 to suppress aggregation (for review see Fan, 2003). DnaJ-1 belongs to the Type II group, which suggests that it normally acts as a co-chaperone for Hsp70 proteins. In this context, overexpression of DnaJ-1 might result in over activation of Hsp70 that in turn can affect BC migration. This is supported by data showing that overexpression of Hsp70 also results in BC migration defects. However, the possibility cannot be discarded that during BC migration DnaJ-1 could also act on its own. In addition, DnaJ/Hsp40 has also been shown to regulate other chaperones, such as the Hsp90 proteins. The isolation of mutants in the DnaJ-1 gene should facilitate further investigation into the interrelationship between DnaJ-1 and Hsp70 in their novel role in BC cell migration (Cobreros, 2008).

The results also reveal a molecular and genetic interaction between DnaJ-1 and the PDGF/VEGF receptor, which is also required for actin cytoskeleton reorganization and cell morphology during border cell migration. This finding opens the possibility for the Hsp40-Hsp70 proteins to exert their function on BC migration partly through the regulation of the activity of PVR, which could in turn trigger intracellular signals leading to motility. In fact, it is becoming increasingly accepted that chaperone interactions serve a variety of functions that go beyond folding insufficiency, as some target proteins do not appear to be generally misfolded. Furthermore, a role for the chaperones Hsp70 and Hsp90 in the maturation of receptors of the steroid and estrogen families has already been shown (for review see Cheung, 2000; Kimmis, 2000]). The current model is that several molecules of the chaperone machinery, including Hsp90, Hsp70 and Hsp40, associate with steroid receptors to form heterocomplexes. These interactions are, on one hand, required for the transition of the receptor into a high affinity state and, on the other, they appear to account for the repression of receptor function that is relieved upon hormone binding (Cheung, 2000; Nair, 1996). In addition, chaperones can also regulate responses downstream of receptors by controlling their recycling or degradation. Thus, overexpression of Hsp70 has been shown to inhibit responses downstream of plasma membrane receptors, such as the insulin receptor, by preventing insulin receptor recycling, a mechanism that has been proposed to operate during heat stress to protect the receptor from thermal damage. In this scenario, one way it can be envisioned why overexpression or loss of function of components of the chaperone complex result in similar phenotypes during BC migration is the following. On one hand, loss of any of the components of the chaperone complex, such as Hsp70, would prevent full activation of PVR, leading to a failure in BC migration. On the other hand, overexpression of components of the chaperone complex, such as Hsp40 or Hsp70, could either sequester the receptor in the heterocomplex interfering with its release by ligand binding, or inhibit its recycling or promote its degradation. Alternatively, the effects of Hsp40 or Hsp70 overexpression in BC migration could also be independent of their interactions with the PVR pathway, and instead be related, for instance, to the role of Hsp proteins in actin stabilization mentioned above. Although at present these possibilities cannot be distinguish, the results show that Hsp70 and Hsp40 levels must be tightly regulated to allow proper BC migration (Cobreros, 2008).

Although there is increasing evidence in support of the role of molecular chaperones on cytoskeleton dynamics during stress, little is known about the involvement of this function in unstressed cells. This study has shown that the Hsp70 and DnaJ-1 are indeed required for the reorganization of the actin cytoskeleton in cell migration events occurring during development. Thus, the potent molecular and genetic tools available in Drosophila can now be used to decipher at the cellular and molecular level the mechanisms by which chaperones regulate cell migration in vivo. Furthermore, there is accumulating new evidence supporting a role for cell-surface chaperones on cancer metastasis (Tsutsumi, 2007). For instance, the expression of Hsp70 in human breast cancer cells has been correlated with metastasis and poor prognosis (Ciocca, 1993). Similarly, over expression of the Hsp70 binding protein BAG-1 accelerates cell motility of human gastric cancer cells (Naishiro, 1999). Thus, furthering understanding of the role of chaperones on cell migration will also help gain better understanding of the molecular mechanisms leading to metastatic spread, and to the identification of therapeutic methods to treat metastasis, one of primary cause of mortality associated with cancer (Cobreros, 2008).

Loss of Hsp70 in Drosophila is pleiotropic, with effects on thermotolerance, recovery from heat shock and neurodegeneration

The heat-shock response is a programmed change in gene expression carried out by cells in response to environmental stress, such as heat. This response is universal and is characterized by the synthesis of a small group of conserved protein chaperones. In Drosophila melanogaster the Hsp70 chaperone dominates the profile of protein synthesis during the heat-shock response. Precise deletion alleles of the Hsp70 genes of D. melanogaster have been generated and those alleles were used to characterize the phenotypes of Hsp70-deficient flies. Flies with Hsp70 deletions have reduced thermotolerance. It was found that Hsp70 is essential to survive a severe heat shock, but is not required to survive a milder heat shock, indicating that a significant degree of thermotolerance remains in the absence of Hsp70. However, flies without Hsp70 have a lengthened heat-shock response and an extended developmental delay after a non-lethal heat shock, indicating Hsp70 has an important role in recovery from stress, even at lower temperatures. Lack of Hsp70 also confers enhanced sensitivity to a temperature-sensitive lethal mutation and to the neurodegenerative effects produced by expression of a human polyglutamine disease protein (Gong, 2006).

The heat-shock response, found in all living organisms, provides an effective defense against exposure to adverse environments. The distinctive feature of the heat-shock response is the synthesis of a set of conserved heat-shock proteins (Hsp's). Hsp's can protect against a number of cellular stresses, including high temperatures, oxidative stress, and a variety of cytotoxins. Convincing evidence for the protective function of Hsp's is derived from the induced thermotolerance test. The ability to survive severe heat stress is increased if an organism is first exposed to a mild heat treatment, but not if protein synthesis is blocked. Such mild heat treatments offer protection against a variety of stresses. The generally accepted interpretation for this phenomenon is that the synthesis of Hsp's that is triggered by the mild heat shock aids subsequent survival under more severe or alternative stresses (Gong, 2006).

The Hsp's and their constitutively synthesized relatives (termed heat-shock cognates, or Hsc proteins) form a diverse group of protein chaperones that can disaggregate proteins from large aggregates or assemblies, prevent aggregation of denatured proteins, aid the renaturation or folding of proteins to reach their proper conformation, direct proteins to degradative pathways, and bind proteins to restrain their function, making them available for ligand binding or allowing them to translocate across membranes. Although some of the classes of Hsp's clearly have distinct activities, they also exhibit overlapping functions, and may share proteins that act as cofactors, known as cochaperones (Gong, 2006).

Among Hsp's, Hsp70 is ubiquitous, with unusually high conservation in protein sequence and functional features (Boorstein, 1994). The chaperone functions of the Hsp70 family of proteins are well established (Skowyra, 1990; Flynn, 1991; Schroder, 1993; Hartl, 1996; Hartl, 2002; Mayer, 2005). Hsp70 and its relatives have several other roles as well. Although Hsp70 is not itself a protease, it is now known that cochaperones can control its activity to direct substrate proteins either to refold or to be degraded. CHIP (carboxyl-terminus of Hsc70 interacting protein) is a ubiquitin ligase that associates with Hsp70 and Hsp90 and their non-stress-induced cognates to direct substrate proteins to the proteasome. CHIP is also involved in regulation of the heat-shock response. Other activities of Hsp70 family proteins are the regulation of apoptosis and eliciting innate and adaptive immunity. Interest in the Hsp70 class of chaperones is growing because of the large variety of cellular processes in which they are involved, as well as their possible participation in aging, cancer, and several neurodegenerative genetic disorders (Gong, 2006 and references therein).

The heat-shock response was first discovered in Drosophila, as a change in the puffing pattern of salivary gland polytene chromosomes in response to heat. In Drosophila this response is particularly dramatic: following a shift to high temperature nearly all transcription and translation is devoted solely to expression of Hsp genes, while other genes are turned off. Hsp70 is the major protein synthesized during this period. Although the synthesis of Hsp70 is nearly undetectable in Drosophila cells at the normal growth temperature of 25°, its expression is rapidly induced at least 1000-fold by raising the temperature to 37° (Velazquez, 1983). The prominent expression of Hsp70 suggests that it may play a large role in thermotolerance, and experimental evidence confirms this supposition. Solomon (1991) found that Drosophila cells with extra copies of Hsp70 genes had increased survival after heat shock, but that interference with Hsp70 reduced their survival. Additionally, when cells carrying a metallothionein-controlled Hsp70 gene were treated with copper at normal temperature, and then shifted directly to severe temperature, a dramatic increase in survival was observed. Additional transgenic copies of Hsp70 provide Drosophila with enhanced thermotolerance (Welte, 1993; Feder, 1996; Krebs, 1998). However, long-term survival is reduced by extra copies of Hsp70 (Krebs, 1997). Thus, the role of Hsp70 in Drosophila thermotolerance is still not fully understood. As a counterpoint to the Drosophila results implicating Hsp70 in thermotolerance, both Saccharomyces cerevisiae and Escherichia coli rely mainly on the Hsp100 family to survive severe temperatures; however, Hsp100 has not been found in animals (Gong, 2006 and references therein).

In addition to its role in the Drosophila heat-shock response, Hsp70 and its cognates are clearly involved in non-heat-shock processes. A recent significant discovery is the finding that Hsp70 can modulate the effects of expressing polyglutamine disease genes. Polyglutamine (polyQ) diseases are a group of dominant inherited neurodegenerative disorders of humans, with the disease alleles characterized by expanded segments of CAG repeats encoding polyglutamine. The pathogenic polyQ proteins are thought to self-associate to form insoluble aggregates inside cells, termed intracellular inclusions. When a segment of the human Machado-Joseph disease gene, which included a segment of CAG repeats, was expressed in the fly eye, developmental defects such as rough eyes and loss of pigment cells and photoreceptor neurons were observed (Warrick, 1998). It was further shown that Hsp70 was located in the aggregates, that overexpression of human Hsp70 suppressed the disease, and expression of a dominant negative mutant of a constitutively expressed Hsp70-cognate gene (Hsc4.k71) enhanced the degeneration (Warrick, 1999). Drosophila appears to be a very useful model organism to study human polyQ diseases and other human neurodegenerative diseases, and to uncover the role of Hsp70 in those pathologies (Feany, 2000; Bonini; 2003; Gong, 2006 and references therein).

This study made use of Drosophila Hsp70 deletion mutants to study the role of Hsp70 in thermotolerance and the regulation of the heat-shock response, protein folding, and neurodegeneration. Hsp70 mutants were found affect all these processes. Flies carry six copies of Hsp70 per haploid genome, situated at two closely linked loci on chromosome 3 (see Drosophila chr3R:7,779,643-7,786,798, chr3R:8,327,180-8,337,693 and chr3R:8,291,026-8,293,500). Deletion mutants were generated by homologous recombination. The deletion of the two Hsp70 genes at the 87A locus is called Df(3R)Hsp70A, the single gene Hsp70Ba deletion allele is called Hsp70Ba³⁰⁴, and the four-gene deletion of all Hsp70B genes is called Df(3R)Hsp70B. By combining Df(3R)Hsp70A with Hsp70Ba³⁰⁴ a chromosome was obtained lacking three of the six copies normally found on that chromosome; combining Df(3R)Hsp70A with Df(3R)Hsp70B produced a chromosome completely lacking Hsp70. All the mutant combinations were viable and fertile as homozygotes, including the Hsp70A Hsp70B recombinant that completely eliminates Hsp70, and all had similar developmental times (Gong, 2004; Gong, 2006).

Flies with a reduction in Hsp70 copy number are viable, including flies with no copies of Hsp70. Similarly, mice that lack all Hsp70 genes are also viable (Hunt, 2004). However, mutations in several of the constitutively expressed homologues of Hsp70 in Drosophila do cause lethality (Elefant, 1999; Burmester, 2000), indicating that the Hsc70 family of proteins carries out critical functions at normal temperatures. Furthermore, this study found that Hsp70-null females, though fertile, have a significant reduction in fertility, indicating that the heat-inducible genes also have some role at normal temperature. Nonetheless, the fact that the Drosophila Hsp70 deletion strains are viable and fertile as homozygotes provided substantial versatility in characterizing the effects of Hsp70 dosage, and also allowed examination of the phenotypes of Hsp70-null flies (Gong, 2006).

When multiple copies of a gene are present, the possibility that those genes have divergent functions must be considered. For instance, in yeast, the SSA1 and SSA2 genes, though encoding nearly identical Hsp70 proteins, differ in their interaction with the [URE3] prion. Evidence has been presented that the two Hsp70 clusters of D. melanogaster are differentially regulated. However, Bettencourt (2002) concluded that the Hsp70 gene copies have not diverged, and likely all provide the same function. The proteins encoded by these six genes have between 98.6% and 100% identity, and 99.5% or greater similarity. The present study assumed that the deletions produced are functionally distinguished only by copy number. Experiments on thermotolerance and the effect of polyQ protein expression are easily interpreted strictly in terms of Hsp70 copy number. For instance, in the adult thermotolerance experiment, the two different six-copy genotypes were indistinguishable. Furthermore, when transgenic copies of Hsp70 were added back to the Df(3R)Hsp70A Hsp70Ba³⁰⁴ genotype, they complemented the larval thermotolerance defect, even though the Hsp70 transgenes represent genes that were not deleted in that genotype (Gong, 2006).

The two Hsp70 clusters differ by intergenic segments. Portions of the S-element transposon are found at both 87A and 87C, and the examination of sequence diversity suggests that these elements are maintained by selection (Maside, 2002). However, there are several other S-elements, including many complete elements, found throughout the genome, so it seems unlikely that these particular copies provide any function that is not also encoded elsewhere. The large intergenic region at 87C hosts the largest euchromatic cluster of transposons found in the genome, and includes heat-shock-transcribed repetitive DNA, the αβ and αγ repeats (Lis, 1978). It is not known whether these transcripts serve any function. Their heat-induced transcription at this location may simply be an accident of proximity to the massively induced Hsp70 genes. Copies of the same sequences found in centric heterochromatin are not induced by heat shock (Lis, 1981). Although it is thought unlikely, the possibility that deletion of these repetitive sequences plays a role in some of the phenotypes described cannot be ruled out. To fully address such questions it may be necessary to introduce point mutations into individual genes, or to delete specific repetitive elements (Gong, 2006).

Not surprisingly, it was found that Hsp70 makes an important contribution to thermotolerance in Drosophila subjected to a severe 39° heat shock. Adult flies with reduced Hsp70 copy number succumb more quickly to lethal high temperatures than do flies with their full complement of Hsp70 genes, larvae are killed at a higher rate, and larvae lag in development in response to non-lethal heat shocks. Many previous studies that examined the effects of Hsp70 overexpression in cell lines or in whole animals, or underexpression in cell lines, also led to the conclusion that Hsp70 is an important component of thermotolerance (Solomon, 1991; Feder, 1996; Feder, 1997; Krebs, 1998; Roberts, 2003). However, none of these prior studies were able to examine the effects of heat on flies that completely lacked Hsp70. Furthermore, some studies show that overexpression of Hsp70 is not always beneficial. Larvae carrying extra transgenic copies of Hsp70 have reduced survival following some heat-shock regimens (Krebs, 1997). Females with extra copies of Hsp70 also show a reduction in fertility following heat shock (Silbermann, 2000). Such studies have led to the idea that the existing copy number of Hsp70 in D. melanogaster was produced by a balance between selection for its chaperone function under stress conditions and against its deleterious effects on growth, viability, and fecundity (Feder, 1999). Thus, it was important to examine the phenotypes of Hsp70-null flies to test whether copy number reduction would also have a deleterious effect on thermotolerance. The results with Hsp70 mutants confirm that Hsp70 plays a major role in thermotolerance in Drosophila and supports the hypothesis that Hsp70 copy number represents a balance arrived at by competition between positive and negative selection (Gong, 2006).

It was surprising to find that Hsp70 is not required to survive a slightly milder 37° 60-min heat shock, even though the temperature was only 2° less than a lethal heat shock. This phenotype of Hsp70 mutants in Drosophila is reminiscent of the phenotype of hsp104 mutants in S. cerevisiae. Hsp104 is required to tolerate extreme heat shocks, but a small degree of thermotolerance remains in hsp104 mutants, and at 37°, a temperature that induces the heat-shock response, the mutants grow as well as cells with a functional HSP104 gene (Sanchez, 1990). The thermotolerance that remains in hsp104 mutants is attributable to Hsp70 (Sanchez, 1993). The Drosophila strains characterized in this study still carry the closely related Hsp68 gene. It is quite possible that the function of Hsp70 is partly provided by Hsp68, much as overexpression of the Hsp70-encoding SSA1 gene can partially compensate for loss of Hsp104 in yeast (Sanchez, 1993). Indeed, by examining protein synthesis in embryos homozygous for large deficiencies that removed the Hsp70 genes (and many other genes as well), Ish-Horowicz, (1979) found that Hsp68 expression increased in the absence of Hsp70. When Hsp68 mutants become available it will be informative to combine them with the Hsp70 mutants to assess phenotypes in the complete absence of this class of heat-induced chaperones. The constitutively expressed forms of Hsp70 may also function at high temperatures to provide a substantial baseline level of thermotolerance. The amount of Hsp70 produced after heat shock is always less than the constitutively synthesized level of Hsc70 proteins (Palter, 1986), allowing for the possibility that the Hsc70 proteins contribute to thermotolerance, though their expression is not induced by heat shock. A role for Hsc70 genes in thermotolerance is suggested by results in several organisms (Gong, 2006 and references therin).

The heat sensitivity of Hsp70 mutant flies likely results from the loss of Hsp70 chaperone function and the consequent reduction in the capacity of cells to refold proteins that were denatured by heat shock (Pelham 1986). Evidence for this mechanism is provided by the finding that shi¹ temperature-sensitive paralytic flies, which normally recover rapidly upon return to normal temperature, recover quite poorly if they lack Hsp70. Feder (1997) showed that Hsp70 overexpression helped to restore alcohol dehydrogenase activity to D. melanogaster larvae after heat shock. Using a luciferase reactivation assay, it has been shown that the Hsp70 homologues from other organisms are also involved in rescuing proteins after thermal denaturation. Hsp104 performs a similar function in yeast in cooperation with Hsp70, and the participation of the Hsp104 homolog in refolding denatured proteins is vital to produce thermotolerance in E. coli (Gong, 2006 and references therein).

In the absence of Hsp70, proteins that have been unfolded by heat must be refolded by alternate pathways, either spontaneously or with the involvement of other chaperones. The loss of the Hsp70, the most highly expressed Hsp in Drosophila, may overload the remaining chaperone systems and delay recovery. Following a severe heat shock the remaining Hsp's are relatively ineffective, as shown by the only minimal increase in survival that is produced by a low-temperature pretreatment in Hsp70-null larvae. The shi¹ experiment shows that the function of some proteins cannot be fully restored without Hsp70. It is likely that they must be synthesized anew to restore function, resulting in developmental delays or lethality. In some cases the sensitivity of genetic screens for temperature-sensitive mutants might be greatly improved by incorporating Hsp70 deficiencies (Gong, 2006).

It is conceivable that the temperature-sensitive dynamin encoded by shi¹ may be exceptionally responsive to Hsp70 chaperone activity. The Hsp70 cognate, Hsc70-4, binds to clathrin and dynamin and participates in the assembly and disassembly of clathrin cages, with mutants showing defects in endocytosis and exocytosis. In Hsp70⁺ flies, perhaps Hsp70 takes the part of Hsc70-4 and associates with clathrin and/or dynamin at high temperature. In shi¹ animals these associations could maintain ts-dynamin in a configuration that allows it to resume its function when the temperature is lowered. In the absence of Hsp70, this association does not occur and a change in the conformation of the temperature-sensitive shi¹ protein may be irreversible. In support of the idea that Hsp70 may substitute for Hsc70-4, Hsp70 is abnormally induced in Hsc70-4 mutant flies when it would otherwise be silent (Elefant. 1999; Bronk, 2001). However, if this hypothesis were true it seems that Hsp70-null flies should exhibit paralysis under the same conditions that inactivate shi¹, and they do not (Gong, 2006).

Hsp70 has been implicated as having a critical role in regulation of the heat-shock response in prokaryotes and in eukaryotes. Originally, a tight correlation between repression of Hsp70 mRNA translation and resumption of non-Hsp mRNA translation was observed (DiDomenico, 1982). Subsequently, interference with Hsp70 expression was seen to cause a delay in repression of Hsp mRNA translation and resumption of normal translation (Solomon, 1991). In S. cerevisiae, the Hsp70-encoding SSA1 gene similarly carries out self-regulation. Under nonstress conditions, Hsp70 participates in repressing the activity of HSF, the positive transcription factor for Hsp genes. It is believed that Hsp70, in cooperation with other Hsp's, sequesters HSF and restrains its activity. Under stress conditions the Hsp's are diverted to chaperoning other denatured proteins, freeing HSF to activate transcription of the Hsp genes. Examination of the duration of heat-shock puffing in Hsp70 wild type and mutant flies confirms that Hsp70 is needed for normal regulation of heat-shock transcription because Hsp repression is delayed in Hsp70-null flies (Gong, 2006).

The functional analysis of Hsp's is certain to be facilitated by the availability of Hsp70 deficiencies. The absence of Hsp70 is likely to help reveal roles of the remaining Hsp's that were obscured in the presence of Hsp70. Phenotypic assessment of mutant combinations has been quite useful for revealing the roles of specific Hsp-encoding genes in yeast. Combining Hsp70 mutations with mutations in other Hsp genes is likely to be equally informative in Drosophila. Hsp70-deficiencies also sensitize flies to at least one type of neurodegeneration, that resulting from the expression of a human polyQ disease gene. The use of Hsp70 mutants may facilitate the identification and analysis of other components that either prevent or contribute to such degeneration (Gong, 2006).

Inducible and constitutive heat shock gene expression responds to modification of Hsp70 copy number in Drosophila melanogaster but does not compensate for loss of thermotolerance in Hsp70 null flies

The heat shock protein Hsp70 promotes inducible thermotolerance in nearly every organism examined to date. Hsp70 interacts with a network of other stress-response proteins, and dissecting the relative roles of these interactions in causing thermotolerance remains difficult. This study examined the effect of Hsp70 gene copy number modification on thermotolerance and the expression of multiple stress-response genes in Drosophila melanogaster, to determine which genes may represent mechanisms of stress tolerance independent of Hsp70. Hsp70 copy number in four strains is positively associated with Hsp70 expression and inducible thermotolerance of severe heat shock. When assayed at carefully chosen temperatures, Hsp70 null flies are almost entirely deficient in thermotolerance. In contrast to expectations, increasing Hsp70 expression levels induced by thermal pretreatment are associated with increasing levels of seven other inducible Hsps across strains. In addition, complete Hsp70 loss causes upregulation of the inducible Hsps and six constitutive stress-response genes following severe heat shocks. It is concluded that modification of Hsp70 copy number quantitatively and qualitatively affects the expression of multiple other stress-response genes. A positive association between absolute expression levels of Hsp70 and other Hsps after thermal pretreatment suggests novel regulatory mechanisms. Severe heat shocks induce both novel gene expression patterns and almost total mortality in the Hsp70 null strain: alteration of gene expression in this strain does not compensate for Hsp70 loss but suggests candidates for overexpression studies (Bettencourt, 2008).

Hsp22, Hsp23, Hsp26, Hsp27, Hsp40, Hsp68 and Hsp83 display coordinate expression patterns within strains, but divergent patterns between Hsp70^- and the other strains. In the Hsp70^A-Ba-, Hsp70⁺ and Hsp70^traIII strains, expression is upregulated at both zero and one hour after PT, with average fold induction relative to C ranging from 17.3- to 51.5-fold. Expression following HS and pretreatment + heatshock (PT+HS) treatments returns to C levels or below, with the exception of Hsp22 and Hsp26, which are slightly upregulated after PT+HS39 in the Hsp70⁺ and Hsp70^traIII strains. Average fold expression relative to C for all seven genes at PT+HS39, at both timepoints, ranges from a decrease of 142.2-fold to an increase of 2-fold. The above patterns are concordant with patterns of Hsp70 expression (Bettencourt, 2008).

The Hsp70^- strain displays a qualitatively different pattern of expression for the inducible genes in comparison with the other strains. Expression of all seven genes is upregulated after PT to a level indistinguishable from the other strains; however, PT+HS treatments also cause upregulation. Expression is especially pronounced 1 h after PT+HS39 (e.g. 1227.4-fold. In contrast, the other strains show net downregulation after the PT+HS treatments (Bettencourt, 2008).

Hsc70-2, Hsc70-3, Hsc70-4, Hsc70-5, Hsp60 and GstE1 also display coordinate expression patterns in the Hsp70^A-Ba-, Hsp70⁺ and Hsp70^traIII strains, and divergence in the Hsp70^- strain. Expression levels during control conditions vary among genes, but within strains, each gene's expression is generally not upregulated by PT (at either timepoint). In the Hsp70^A-Ba-, Hsp70⁺ and Hsp70^traIII strains, HS causes downregulation of the constitutive genes, with the effect increasing with time: by 1 h post-treatment, average expression levels decrease 34.4- to 159.4-fold relative to C. In the Hsp70^- strain, this effect is absent at HS39 and reversed at HS39.5, where average expression increases 26.6- and 12.1-fold . PT+HS treatments cause further downregulation in the Hsp70^A-Ba-, Hsp70⁺ and Hsp70^traIII strains at both timepoints, with the decrease ranging from 14.9- to 1047.2-fold. In contrast, the Hsp70^-strain shows net upregulation after PT+HS treatments. This effect is largely due to Hsc70-3 and Hsc70-4, which have lower C and PT expression levels in the Hsp70^- strain than in the other strains, and rise after PT+HS instead of falling (Bettencourt, 2008).

These results indicate that Hsp70 loss causes alterations of both inducible and constitutive stress gene expression that are ultimately insufficient for inducible tolerance of severe heat shock. In addition, it was found that both increasing Hsp70 copy number and Hsp70 expression are associated with increases in the expression of multiple inducible Hsp genes (Bettencourt, 2008).

These results are consistent with previous research, but highlight the extreme sensitivity of thermotolerance traits to assay temperature. Furthermore, the strong differences between basal and inducible thermotolerance among the strains reflect the potent effect of thermal pretreatment and heat shock protein induction. Previous research reported the maintenance of some degree of thermotolerance in the Hsp70^- strain, when assayed at lower heat shock temperatures and different heat shock durations that may not maximize the input of Hsp70 (e.g., 37°C (Gong, 2006). In this study thermotolerance assays were designed to maximize differences in survival among strains varying in Hsp70 copy number and expression, and it was found the Hsp70^- strain almost completely deficient in basal and inducible tolerance of severe heat shock. Gene expression was measured in animals undergoing the same severe thermal treatments and interpreted differences among the strains accordingly (Bettencourt, 2008).

When measuring gene expression via qrtPCR, researchers typically express candidate gene levels relative to a control gene, often a 'housekeeping' gene that is ubiquitously expressed independently of experimental treatments. This adjustment serves to control for variation in RNA/cDNA extractions. Rather than express measurements in candidate gene:control gene ratios, this study estimated each gene's own expression level according to an ANOVA model which includes expression of the RpL32 control gene as a covariate. The model also includes effects imparted by both experimental error (extraction, PCR replicate) and the biology that was explored (strain, treatment, timepoint). RpL32, strain and treatment all have a significant effect on candidate gene expression, indicating that these factors, rather than error, explain much of the variation in gene expression (Bettencourt, 2008).

Does the fact that RpL32 expression is itself affected by the treatments employed have an impact on the analysis? Specifically, RpL32 expression decreases with HS and PT+HS treatments across strains. Notwithstanding a sensible biological interpretation of this observation (severe stress downregulates housekeeping genes and upregulates heat shock genes), it would be predicted that if RpL32 was a more important factor in candidate gene expression than strain or treatment, the model would perform poorly during HS and PT+HS and, thus, generate least square mean estimates that do not match raw expression values (Bettencourt, 2008).

The expression curves were recalculated using raw data (CTs) rather than least square means and the opposite was found to be true. The curves are strikingly concordant with the model outputs. The effects of treatment and strain on Hsp gene expression levels are evident whether considering modeled or raw data. This indicates that the biological signals (e.g. upregulation of inducible Hsps following thermal pretreatment, differential regulation in the Hsp70^- strain) are stronger than the 'noise' imparted by variation in control gene expression and/or experimental error (Bettencourt, 2008).

The expression levels of seven inducible Hsp genes after pretreatment (PT), relative to control (C) levels, are high in all of the strains examined but are not strongly associated with Hsp70 copy number. However, when considered in isolation, C and PT expression levels appear to increase with Hsp70 copy number. Furthermore, Hsp70 expression clearly increases with Hsp70 copy number when expressed either in absolute or PT-relative-to-C terms. Finally, the housekeeping RpL32 gene levels remain a highly significant covariate in every inducible gene/timepoint combination but two. This indicates that the distribution of error associated with variation in RNA/cDNA manufacture is not unevenly associated with any strain, gene or timepoint, and that direct comparison of absolute inducible gene expression levels (CTs) is possible. Therefore whether this study explored the highest absolute inducible Hsp expression levels in each strain, those produced by PT, could be predicted by corresponding PT Hsp70 expression levels. Since the expression levels of Hsp70 and the other Hsps each have associated error, simple linear regression is inappropriate for exploring this relationship. As such, reduced major axes regression was conducted. Raw zero- and one-hour PT expression data (reciprocal CT values) was pooled within each strain and the other Hsps was regressed on Hsp70 (Bettencourt, 2008).

It was found that after PT, Hsp70 expression is a strong predictor of other inducible Hsp expression (R² = 73%). The four strains cluster on both axes, indicating that increasing Hsp70 copy number is strongly associated with both measures of gene expression. That multiple Hsp genes would show coordinate upregulation following thermal pretreatment is not surprising: the inducible Hsp genes were first noticed as a suite of heat-induced transcriptional puffs and their coexpression is well established (Ritossa, 1962; Morimoto, 1997). It thus might be expected that strains or species that vary in the amount or activity of a known global stress-response regulator, such as HSF, could show coordinate alteration of Hsp gene expression levels. These strains, however, differ only in Hsp70 copy number and Hsp70 expression. This indicates that the Hsp70 gene or Hsp70 protein has coordinate effects on other Hsp expression, and that increasing Hsp70 levels in turn upregulate the other Hsps. A broad effect of Hsp70 on the regulation of other Hsps is consistent with previous research, but the positive direction of the effect is unexpected (Bettencourt, 2008).

In many systems, free Hsp70 protein can bind to HSF and prevent transactivation that stimulates Hsp transcription, thereby negatively regulating Hsp levels in a classic feedback loop. Consistent with this model, overexpression of Hsp70 protein in the absence of stress can repress inducible transcription of Hsp genes. However, the current results indicate that increasing Hsp70 gene expression levels are associated with increases in the expression of other Hsp genes, at least over brief timescales. This unexpected finding hints at additional mechanisms of Hsp transcriptional regulation independent of Hsp70/HSF protein interaction, and requires further research. An intriguing possibility involves a candidate 'cost' of Hsp70 expression in the absence of severe heat shock. Hsp70 is a generalist chaperone that, in the absence of thermally denatured protein substrates to bind, could instead bind diverse proteins and pull them from their native conformations. These newly non-native proteins could in turn induce a further stress response, including the upregulation of additional Hsp genes. This misregulation would be especially deleterious when Hsp70 is overexpressed, such as in the Hsp70^traIIIstrain, and when thermal conditions strongly induce Hsp70 but do not precede a severe heat shock, such as the PT treatment employed in this study. Clearly, existing and future studies of stress induced gene expression in Drosophila, especially those examining global transcriptional responses in mutant or selected strains that may vary in Hsp70 expression, should be examined for coordinate upregulation of other Hsps (Bettencourt, 2008).

In contrast to the association between levels of Hsp70 and the other inducible Hsp genes after PT, the inducible Hsps are upregulated after PT+HS in the Hsp70^- line. This result is clearly not explained by increasing Hsp70 copy number. It is, however, consistent with increased thermal damage in the Hsp70^- strain and/or improper repression of the heat shock response. Thermally denatured proteins are a primary stimulus of the heat shock response. Since Hsp70^- larvae fail to survive PT+HS, perhaps the increase in Hsp expression simply reflects that the severity of stress combined with lack of Hsp70 causes extensive Hsp-inducing protein damage (from which the animals are ultimately unable to recover). Alternatively, the lack of Hsp70 could cause failure of proper Hsp transcriptional attenuation post-stress. This hypothesis was favored by Gong and Golic (2006), who applied a brief, mild heat shock to the Hsp70^-strain and observed transcriptional Hsp 'puffing' on polytene chromosomes that persisted longer than that observed in a wild-type strain. Upregulation of the inducible Hsps after HS in the Hsp70^- strain was not observed in this study, even though survival of HS and PT+HS is equally low. This may indicate that over the short timespan in which gene expression was examined, PT+HS is more damaging than HS (in the absence of Hsp70). To explore whether HS eventually upregulates the inducible Hsps in the Hsp70^- strain, future research will examine patterns of gene expression, thermal tissue damage and cellular damage indicators such as protein aggregates and ubiquitin conjugates at additional, extended timepoints. Whether the Hsp upregulation in the Hsp70^- strain is a result of damage and/or a lack of transcriptional repression is an open question. Regardless of the mechanism of Hsp upregulation after PT+HS in the Hsp70^- strain, the response is clearly not sufficient to generate increased inducible thermotolerance (Bettencourt, 2008).

As was seen with the inducible Hsps, the constitutive genes display coordinate regulation. The direction of regulation, however, is different: the constitutive genes are expressed at low levels after C and PT, and rise with HS39.5 and both PT+HS treatments in the Hsp70^- strain, as opposed to falling from higher C and PT levels to low HS and PT+HS levels in the other strains. Furthermore, Hsp70 levels do not predict levels of the constitutive genes to the degree they did for the inducible genes. These results indicate that Hsp70 may be more uncoupled from the regulation of the constitutive genes. Neal (2006) reported upregulation of constitutive stress gene expression in HSF mutant Drosophila and suggested that such upregulation could compensate for the loss of inducible Hsp expression. The current results suggest that disrupting Hsp70 specifically, instead of the entire inducible Hsp response through HSF, induces similar upregulation of constitutive genes while also influencing the expression of other inducible Hsps. Again, however, the modification of gene expression caused by Hsp70 loss does not provide compensatory thermotolerance in these assays (Bettencourt, 2008).

This study identified genes whose expression is altered by thermal stress in Hsp70 mutant backgrounds: genes that did not provide compensatory thermotolerance in these assays. Future experiments will determine whether inducing any of these genes prior to stress application will promote thermotolerance in the Hsp70^- strain. Given the strong effect of Hsp70 modification on inducible and constitutive Hsp expression, determining whether and how any of these candidate stress protective genes can operate independently of Hsp70 and its associated costs remains a challenge (Bettencourt, 2008).

Hsp70- and Hsp90-mediated proteasomal degradation underlies TPI^sugarkill pathogenesis in Drosophila

Triosephosphate isomerase (TPI) deficiency is a severe glycolytic enzymopathy that causes progressive locomotor impairment and neurodegeneration, susceptibility to infection, and premature death. The recessive missense TPI^sugarkill mutation in Drosophila melanogaster exhibits phenotypes analogous to human TPI deficiency such as progressive locomotor impairment, neurodegeneration, and reduced life span. This study shows that the TPI^sugarkill protein is an active stable dimer; however, the mutant protein is turned over by the proteasome reducing cellular levels of this glycolytic enzyme. As proteasome function is often coupled with molecular chaperone activity, it is hypothesized that TPI^sugarkill is recognized by molecular chaperones that mediate the proteasomal degradation of the mutant protein. Coimmunoprecipitation data and analyses of TPI^sugarkill turnover in animals with reduced or enhanced molecular chaperone activity indicate that both Hsp90 and Hsp70 are important for targeting TPI^sugarkill for degradation. Furthermore, molecular chaperone and proteasome activity modified by pharmacological or genetic manipulations resulted in improved TPI^sugarkill protein levels and rescue some but not all of the disease phenotypes suggesting that TPI deficiency pathology is complex. Overall, these data demonstrate a surprising role for Hsp70 and Hsp90 in the progression of neural dysfunction associated with TPI deficiency (Hrizo, 2010).

Studies of TPI deficiency disease pathogenesis have previously focused on dimer stability and catalytic activity of the mutant enzyme. Several labs have shown that some, but not all of the human disease causing TPI alleles, have reduced dimerization and isomerase function using in vitro assays. However, previously published data suggests that TPI^sugarkill dimer stability is not compromised. As the TPI^sugarkill protein does not appear to aggregate and increased levels of the TPI^sugarkill protein rescues the mutant phenotypes, these data cumulatively suggest that the mutant protein can conform to a functional shape and likely retains significant activity (Hrizo, 2010 and references therein).

Hsp70 and Hsp90 are molecular chaperones that have been implicated in the progression and amelioration of other neurodegenerative diseases. However, TPI^sugarkill is a unique cytosolic model protein for the study of these chaperones and their role in mutant protein turnover and disease pathogenesis as it is a soluble protein that is not aggregation prone. Other neurodegenerative models can be rescued with increased activity of the molecular chaperones as they help target the misfolded protein for degradation before toxic cellular protein aggregates can form. The role of Hsp90 and Hsp70 in TPI^sugarkill pathogenesis is distinct from the previously mentioned examples, as decreased chaperone activity reduces pathogenesis for some mutant phenotypes and TPI^sugarkill protein does not appear to cause the toxic cellular aggregates observed in other neurodegenerative diseases. In the case of TPI^sugarkill the molecular chaperones are targeting a protein for degradation before it can function within the cell, thus reducing the function of the enzyme and contributing to the disease states. TPI is folded in the cytoplasm and previous labs have studied TPI protein folding rates in vitro and have found them to be rather rapid. The mechanism of chaperone identification and targeting of TPI^sugarkill for degradation is not known. For example, the cytosolic TPI^sugarkill protein may be recognized by molecular chaperones and targeted for degradation due to a slower folding rate than the wildtype protein, as is the case with the ER secretory CFTRΔF508 mutant protein. Overall, while other studies have shown that up-regulation of molecular chaperones may prove beneficial for reducing neurodegeneration caused by formation of toxic aggregates, the current study corresponds with previously published work that also suggests that excessive up-regulation or unregulated chaperone and proteasome activity may lead to undesirable side-effects and that a balance of chaperone and proteasome activity may be required for neuronal function and health (Hrizo, 2010).

This study observed that modulation of molecular chaperone activity alone is not sufficient to restore TPI^sugarkill protein to wildtype levels. The results are in line with what is expected for modest hypomorphic or increased expression conditions examined. Transgenic overexpression of mutant TPI^sugarkill protein also rescued TPI^sugarkill phenotypes, consistent with the interpretation that the mutant protein retains function. Thus far any means examined of altering steady state TPI^sugarkill protein, either by increasing rate of synthesis or decreasing proteasomal-dependent degradation, results in the predicted affect on TPI^sugarkill phenotypes. These data demonstrate that TPI^sugarkill degradation is not a protective mechanism, as has been seen with aggregation prone protein models, but rather leads to a loss of functional protein that underlies pathogenesis (Hrizo, 2010).

While the proteasome and molecular chaperones have been shown to be involved in TPI^sugarkill turnover, it is not clear how TPI^sugarkill is recognized and targeted for degradation. The majority but not all proteasomal-targeted proteins are polyubiquitinated prior to recognition and subsequent degradation by the proteasome. Further studies will be needed to clarify the recognition and targeting mechanisms (Hrizo, 2010).

Overall, these experiments suggest that TPI^sugarkill retains activity and that by modulating TPI turnover disease pathogenesis can be altered. In addition, a new mutant substrate that interacts with molecular chaperones and is degraded by the proteasome has been identified for study. Previous studies of cytosolic protein targeting for protein degradation have focused on ER proteins and exogenous mutant proteins such as VHL tumor suppressor. TPI^sugarkill is a unique protein quality control substrate, the further study of which will yield important insight into the mechanisms of identification and targeting of cytosolic proteasomal substrates (Hrizo, 2010).

Heat shock protein-70 (Hsp-70) suppresses paraquat-induced neurodegeneration by inhibiting JNK and Caspase-3 activation in Drosophila model of Parkinson's disease

Parkinson's disease (PD) is one of the most common neurodegenerative disorders with limited clinical interventions. A number of epidemiological as well as case-control studies have revealed an association between pesticide exposure, especially of paraquat (PQ) and occurrence of PD. Hsp70, a molecular chaperone by function, has been shown as one of the modulators of neurological disorders. However, paucity of information regarding the protective role of Hsp70 on PQ-induced PD like symptoms led to a hypothesis that modulation of hsp70 expression in the dopaminergic neurons would improve the health of these cells. Advantage was taken of Drosophila, which is a well-established model for neurological research and also possesses genetic tools for easy manipulation of gene expression with limited ethical concern. Over-expression of hsp70 was found to reduce PQ-induced oxidative stress along with JNK and caspase-3 mediated dopaminergic neuronal cell death in the exposed organism. Further, anti-apoptotic effect of hsp70 was shown to confer better homeostasis in the dopaminergic neurons of the PQ-exposed organism, as evidenced by their improved locomotor performance and survival. The study has merit in the context of human concern since protection of dopaminergic neurons in PQ-exposed organism was observed by over-expressing a human homologue of hsp70, HSPA1L, in these cells. The effect was parallel to that observed with Drosophila hsp70. These findings reflect the potential therapeutic applicability of hsp70 against PQ-induced PD like symptoms in an organism (Shukla, 2014).

Drosophila Spag is the homolog of RNA Polymerase II Associated Protein 3 (RPAP3), and recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the assembly of cellular machineries

The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins: Pih1D1, RPAP3 and the AAA+ ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, snoRNP (small nucleolar RiboNucleoParticles) and PIKK (Phosphatidyl-Inositol 3-Kinase-related Kinases). This study characterized the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, it was shown that spaghetti is necessary for (1) the stabilization of snoRNP core proteins, (2) TOR (Target Of Rapamycin) activity, and likely, the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients, and further shows that Spag, unlike S. cerevisae Tah1, performs essential functions in Metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90, to facilitate their assembly into macro-molecular complexes (Benbahouche, 2014).

Molecular chaperones protect against JNK- and Nmnat-regulated axon degeneration in Drosophila

Axon degeneration is observed at the early stages of many neurodegenerative conditions and this often leads to subsequent neuronal loss. Previous studies have shown that inactivating the c-Jun N-terminal kinase (JNK) pathway leads to axon degeneration in Drosophila mushroom body (MB) neurons. To understand this process, candidate suppressor genes were screened and Wallerian degeneration slow (Wld^S) fusion protein was found to block JNK axonal degeneration. Although the nicotinamide mononucleotide adenylyltransferase (Nmnat1: see Drosophila Nmnat) portion of Wld^S is required, its nicotinamide adenine dinucleotide (NAD⁺) enzyme activity and the Wld^S/ N-terminus (N70) are dispensable, unlike axotomy models of neurodegeneration. It is suggested that Wld^S-Nmnat protects against axonal degeneration through chaperone activity. Furthermore, ectopically expressed heat shock proteins (Hsp26 and Hsp70) also protected against JNK and Nmnat degeneration phenotypes. These results suggest that molecular chaperones are key in JNK- and Nmnat-regulated axonal protective functions (Rallis, 2013).

Wld^S was discovered from the molecular cloning of spontaneously generated slow Wallerian degeneration (Wld^S) mutant mice that showed a strong capacity to promote axonal survival following acute physical lesion. The Wld^S protein has neuroprotective effects across different species and in different neurodegeneration models. The Wld^S gene product results from the fusion of first 70 residues of the UBE4B gene (N70), that is involved in polyubiquitination, with the entire nicotinamide mononucleotide adenylyltransferase protein sequence (Nmnat1) that is involved in nicotinamide adenine dinucleotide (NAD⁺) biosynthesis. Different portions of Wld^S can confer neuroprotective function (Coleman, 2010). However, Wld^S function remains unclear. For example, despite its predominant nuclear localisation, it is axonal localisation that appears to be key to neuroprotection, even though Wld^S and different Nmnat isoforms have subtle and distinct subcellular locations. Also, while in many neurodegenerative paradigms the Nmnat enzyme activity is essential, it is unclear how the NAD⁺ pathway contributes to axonal protection. Furthermore, some studies suggest Nmnat neuroprotective functions are enzyme-independent. To date, the relationship between Wld^S function(s) and axon-neuronal damage and repair also remains unclear, although recent data suggest Wld^S-Nmnat regulation of mitochondrial motility and calcium buffering functions may underlie key neuroprotective responses to physical injury in Drosophila and mouse axons (Avery, 2012). A further report suggests Drosophila Nmnat (dNmnat or nmnat) also controls axonal mitochondria levels and their availability is key to neuroprotection following acute injury (Fang, 2012). Previous data suggest Wld^S-Nmnat localisation within mitochondria may also be the underlying basis of axonal neuroprotection (Rallis, 2013).

When tested ectopically, many Nmnat isoforms and homologs show axonal-protective effects even though some appear to be weaker, possibly due to labile effects. However, apart from Drosophila Nmnat, currently only mouse Nmnat2 has an endogenous role in promoting axonal stability. It is important to note, beyond their neuronal roles, Nmnats also have obligate roles in NAD⁺ metabolism and multiple cellular processes across species. Very recent reports show Nmnat1 mutations cause Leber congenital amaurosis (LCA), highlighting its importance in retinal degenerative diseases in humans (Rallis, 2013).

This study shows that the Wld^S protein protects against axon degeneration triggered by JNK inactivation. Contrary to previous models, while the Nmnat1 region is sufficient, this study found that its enzyme activity is dispensable for Wld^S neuroprotection. The results suggest that Nmnat and JNK axonal-protective functions occur through molecular chaperones (Rallis, 2013).

One previous report showed that Drosophila Nmnat has a non-enzyme function that involves molecular chaperone activity (Zhai, 2008). Drosophila Nmnat was recruited together with the molecular chaperone, Heat shock protein (Hsp) Hsp70 to polyglutamine expanded spinocerebellar ataxin-1 (SCA-1) containing aggregates. Non-enzyme Nmnat functions were involved in regulating protein folding and blocking SCA-1 neurotoxicity. Very recent results show non-enzyme Nmnat also functions to clear tau oligomers in vivo (Ali, 2012). This study tested the effect of Heat shock proteins (Hsps) on the bsk phenotypes in two ways. In bsk-null neuroblast clones, it was found that, like Wld^S and Nmnats1 and 3, ectopic Hsp70 or Hsp26 also blocked the bsk axon degeneration (Rallis, 2013).

Compared to wild-type axons, bsk axons showed more abnormal protrusions and swellings along the axons and terminals. When Hsp70, Wld^S, Nmnat and Nmnat enzyme-inactive forms were expressed in these clones, these were reduced suggesting that this phenotype is also linked to Hsps and non-enzyme Nmnat activities (Rallis, 2013).

To further test the neuroprotective activity of Hsps, Nmnat RNAi assays was used. When Nmnat RNAi was expressed in MB neurons, this resulted in a β-axon loss phenotype similar to nmnat¹ loss-of-function clones above. Some neuronal loss was visible in newly eclosed adults (1-day-old adults). However, almost all neurons were lost in 7-day-old adults, suggesting that Nmnat is an obligate maintenance factor, consistent with previous reports. The Nmnat RNAi axon and neuronal cell loss was rescued by enzyme-inactive forms of mNmnat1 (H24A) and Wld^S-dead. Furthermore, Hsp26 and Hsp70 expression also partially suppressed the Nmnat RNAi phenotype. Together, these results suggest non-enzyme Nmnat and chaperone activities are linked to JNK axonal functions (Rallis, 2013).

Using the GAL80ts system to control JNK temporal expression, it has been shown that JNK activity is required throughout development, even though the axon degeneration phenotype occurs mainly at adult stages. To determine Nmnat's temporal requirements, Nmnat RNAi was coupled to GAL80ts control, and the loss-of-function phenotype was induced at various stages of development. It was found that RNAi throughout the development and adult phase caused the strongest neuronal loss phenotype. RNAi induction at pupal or adult stages also caused neuronal loss, albeit at a weaker levels. These results suggest Nmnat is required throughout development as well as adult stages. Even though the Nmnat RNAi phenotype is more severe in adults, as in bsk mutants, unlike bsk, Nmnat's genetic requirements extend beyond the developmental stages and are essential at adult stages. This suggests Drosophila Nmnat may have additional roles at adult stages that may be independent of JNK activity (Rallis, 2013).

Combined pharmacological induction of hsp70 suppresses prion protein neurotoxicity in Drosophila

Prion diseases are rare and aggressive neurodegenerative disorders caused by the accumulation of misfolded, toxic conformations of the prion protein (PrP). Therapeutic strategies directed at reducing the levels of PrP offer the best chance of delaying or halting disease progression. The challenge, though, is to define pharmacologic targets that result in reduced PrP levels. Previous studies have reported that expression of wild type hamster PrP in flies induces progressive locomotor dysfunction and accumulation of pathogenic PrP conformations, while co-expression of human Hsp70 delayed these changes. To validate the therapeutic potential of Hsp70, flies were treated with drugs known to induce Hsp70 expression, including the Hsp90 inhibitor 17-DMAG and the glucocorticoid dexamethasone. Although the individual treatment with these compounds produced no significant benefits, their combination significantly increased the level of inducible Hsp70, decreased the level of total PrP, reduced the accumulation of pathogenic PrP conformers, and improved locomotor activity. Thus, the combined action of two pharmacological activators of Hsp70 with distinct targets results in sustained high levels of inducible Hsp70 with improved behavioral output. These findings can have important therapeutic applications for the devastating prion diseases and other related proteinopathies (Zhang, 2014).

Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci

To efficiently transcribe genes, RNA Polymerase II (Pol II) must overcome barriers imposed by nucleosomes and higher-order chromatin structure. Many genes, including Drosophila Hsp70, undergo changes in chromatin structure upon activation. To characterize these changes, the nucleosome landscape of Hsp70 after an instantaneous heat shock was mapped at high spatial and temporal resolution. Surprisingly, an initial disruption of nucleosomes was found across the entire gene within 30 s after activation, faster than the rate of Pol II transcription, followed by a second further disruption within 2 min. This initial change occurs independently of Pol II transcription. Furthermore, the rapid loss of nucleosomes extends beyond Hsp70 and halts at the scs and scs' insulating elements. An RNAi screen of 28 transcription and chromatin-related factors reveals that depletion of heat shock factor, GAGA Factor, or Poly(ADP)-Ribose polymerase or its activity abolishes the loss of nucleosomes upon Hsp70 activation (Petesch, 2008).

Using a high resolution in vivo approach to map changes in the chromatin structure of the rapidly induced Hsp70 gene, a broad disruption of nucleosome structure was observed that occurred at a rate faster than transcribing Pol II and broader than a single transcription unit, ceasing at the natural insulating elements. Furthermore, it was found that the initial changes in chromatin architecture at Hsp70 can be decoupled from transcription of the gene, whereas the second disruption by 2 minutes is transcription-dependent. A selective RNAi screen identified HSF, GAF, and PARP as each being necessary for the changes in chromatin landscape at Hsp70 (Petesch, 2008).

Before HS, the Hsp70 gene contains a chromatin landscape that has many general, as well as some distinct features. Like many other TATA containing genes, a highly positioned nucleosome exists downstream of the promoter region and the adjacent nucleosomes on the body of Hsp70 gradually lose their positioning. Likewise, as seen with many genome-wide studies, the promoter, and a region at the 3’ end of the gene, is relatively nucleosome free. It is yet to be determined why 3’ ends of genes are hypersensitive to nucleases. However, while many genes in yeast contain a positioned nucleosome starting within the first 100 bp of the transcription unit, Hsp70 contains a nucleosome free region that extends further, with the first nucleosome centered 330 bp following the TSS. This extended nucleosome-free region may be a more general feature of genes containing a paused polymerase (Petesch, 2008).

The HS time course shows that within 2 minutes following HS, the chromatin landscape of Hsp70 drastically changes. Following 2 minutes of HS, there no longer exists appreciable protection of a contiguous 100 bp piece of DNA that would normally be provided from a histone octamer. However, there are still detectable levels of histone H3 on the body of the gene, albeit three-fold less than NHS levels. Although these results differ from early observations that histone levels on Hsp70 do not change following HS, the 3-fold decrease measured by qPCR agrees with more recent quantifications of histone levels following HS (Adelman, 2006) and may have gone undetected in the qualitative analysis of these early experiments. Early electron microscopy spreads of native transcribing Pol II complexes with a growing RNA chain from D. melanogaster indicate that the bulk of transcribing Pol II in vivo appears to have nucleosomes flanking its path. The current results, however, suggest that at least for the rapidly induced Hsp70 gene, the nucleosomal structure present before HS no longer exists following activation of the gene (Petesch, 2008).

It was also found that changes in chromatin upon Hsp70 induction extend well beyond the transcription unit of Hsp70 and halt at the scs and scs’ insulating elements. Previous studies of scs and scs’ have shown that these insulators are capable of blocking enhancer functions and establishing chromatin domains that are resistant to position effects. However, the scs and scs’ regions have been located by DNA FISH on squashed polytene chromosomes to be within a HS puff at the endogenous 87A HS locus (Kuhn, 2004). This indicates that the scs and scs’ regions by themselves are not absolute boundaries to changes in chromosome architecture, and supports the observation that puffing is maximal at a time well after nucleosome disruption and therefore denotes additional structural alterations beyond those observed here. Although transcription of CG31211, CG3281, and Aurora did not change following HS, and no factor targeted for RNAi permitted the disruption of nucleosomes beyond scs or scs’, both of these regions include a TSS with detectable amounts of Pol II. It is therefore possible that the promoter architecture with Pol II present at these genes may be responsible for establishing a barrier at these sites. Overall, these results show that scs and scs’ provide a primary barrier to the spread of chromatin decondensation, at least at the nucleosomal level, and add to the limited knowledge of the chromatin architecture of a puff (Petesch, 2008).

Previous results, combined with these, indicate that transcription-independent chromatin decondensation may prove more general. Changes in chromatin structure independent of transcription have been implicated at Hsp70 in humans and also at developmentally regulated puffs in Drosophila. Furthermore, the results indicate that the changes in chromatin at D. melanogaster Hsp70 do not depend on many different transcription factors. In Saccharomyces cerevisiae, many HS genes also lose histone density within the body of the gene by 2 minutes of HS, and as in the current study, these changes are independent of SWI/SNF, Gcn5, and Paf1. Overall, transcription-independent chromatin decondensation might allow cells to rapidly activate genes by clearing the obstacles in the path of Pol II prior to its movement, together with its entourage of elongation factors, through the gene (Petesch, 2008).

The results show that in addition to HSF and GAF, which have previously been implicated in the decondensation at Hsp70 loci, PARP is also necessary for rapid changes in the nucleosome architecture of Hsp70. This is consistent with the finding that reduction of PARP expression results in decreased HS puff sizes. The results go further in demonstrating that PARP aids the rapid removal of nucleosomes within 2 minutes of HS. Poly(ADP-)Ribose (PAR) polymers are the enzymatic product of PARP and have similar chemical and structural features as a nucleic acid. Upon activation, PARP polyribosylates itself, which results in PARP’s release from chromatin. The result of this could be two fold. First, since PARP binds to nucleosomes in a similarly repressive manner as linker histone H1, the activation of PARP could result in its release from chromatin to reverse any repressive effects on the chromatin structure at Hsp70. Second, the ADP-ribosylation of histones may destabilize the nucleosome, and the creation of these PAR polymers could act locally as a nucleic acid that attracts and removes histones from the body of the Hsp70 gene. Alternatively, PARP could covalently modify another protein to activate its role in removal of nucleosomes (Petesch, 2008).

In addition to histones, PAR could also attract transcription factors that bind nucleic acids. This could explain the rapid recruitment of Pol II and other important transcription factors to the site of active HS transcription. Likewise, PAR could also provide a means through which transcription factors recruited to the gene are then retained locally. The activation of PARP could thus provide a rapid, transcription-independent method to deplete histones and promote transcription of the Hsp70 gene (Petesch, 2008).

Activator-induced spread of poly(ADP-ribose) polymerase promotes nucleosome loss at Hsp70

Eukaryotic cells possess many transcriptionally regulated mechanisms to alleviate the nucleosome barrier. Dramatic changes to the chromatin structure of Drosophila melanogaster Hsp70 gene loci are dependent on the transcriptional activator, heat shock factor (HSF), and poly(ADP-ribose) polymerase (PARP). This study found that PARP is associated with the 5' end of Hsp70, and its enzymatic activity is rapidly induced by heat shock. This activation causes PARP to redistribute throughout Hsp70 loci and Poly(ADP-ribose) to concurrently accumulate in the wake of PARP's spread. HSF is necessary for both the activation of PARP's enzymatic activity and its redistribution. Upon heat shock, HSF triggers these PARP activities mechanistically by directing Tip60 acetylation of histone H2A lysine 5 at the 5′ end of Hsp70, where inactive PARP resides before heat shock. This acetylation is critical for the activation and spread of PARP as well as for the rapid nucleosome loss over the Hsp70 loci (Petesch, 2012).

This study establishes an ordered mechanism by which a transcription activator binding to a gene's regulatory region leads to rapid removal of nucleosomes throughout the gene locus. Specifically, the transcriptional activator, HSF, stimulates dTip60 acetylation of H2AK5 that in turn activates PARP, causing its redistribution along Hsp70 and reduced nucleosome occupancy over the locus. Moreover, all of these steps can be accomplished independently of transcription. This activation of PARP and its rapid spread throughout the Hsp70 HS loci demonstrate an interesting mechanism by which the nucleosome barrier can be alleviated to facilitate efficient transcription by Pol II (Petesch, 2012).

HSF and many other transcriptional activators have been classically studied for their ability to recruit or release Pol II into transcriptional elongation. The results speak to another function of HSF as an activator to direct changes in chromatin structure upon HS. HSF is able to achieve this function through physically interacting with the dTip60 complex and facilitating its recruitment to Hsp70 following HS (personal communication by Thomas Kusch to Petesch, 2012). Just as the presence of paused Pol II in non-heat-shock (NHS) conditions primes the Hsp70 gene for rapid transcriptional induction, inactive PARP bound in NHS conditions primes Hsp70 for rapid changes in chromatin structure. Interestingly, trimerization and binding of HSF to the promoter of Hsp70 precipitates the activation of both Pol II and PARP through distinct pathways that ultimately synergize to facilitate rapid and robust transcriptional activation. In vitro studies have shown that the DNA-binding and catalytic domains of PARP comprise the minimal structure sufficient for inactive PARP to bind and locally compact nucleosomes and, upon activation, release PARP from chromatin and decompact chromatin structure. Activation of PARP is known to result in the formation of linear and branched anionic polymers with upwards of 200 units of ADP-ribose. Electron micrograph structures of branched PAR make it easy to visualize how creation of these voluminous, dendritic structures causes automodified PARP to expand 3-dimensionally throughout the Hsp70 loci following HS. The results also indicate that PARP is crosslinked to Hsp70 after HS through a PAR linkage to chromatin. Although PARylation of another target, such as histones, cannot be ruled out, the results fit the simplest model where PARP is its own target. In agreement with the aforementioned in vitro studies, PARP automodification would result in its release from nucleosomes bound prior to HS, and the PAR created from this automodification could create a bridging interaction between PARP and chromatin formed during crosslinking. This also is consistent with in vivo studies showing the major target of PARylation is PARP itself. Antibodies specifically recognizing ADP-ribosylated target proteins, such as PARP or histones, are needed to identify the target of PARP following HS at Hsp70 (Petesch, 2012).

The accumulation of PAR throughout the Hsp70 locus provides additional functional insight into how activation of PARP upon HS can affect chromatin structure and transcriptional activation. PAR has remarkable chemical similarity to other nucleic acids, such as DNA and RNA, but it has twice the charge per nucleic acid residue and the potential to form nonlinear, branched structures. As such, in vitro reconstitution assays have shown that PAR has the ability to locally compete with DNA to bind histones and potentially disrupt native chromatin structure. The transient formation of PAR to alter chromatin structure followed by catabolism of PAR to return histones to its DNA template has been referred to as histone shuttling. While initially investigated to explain PARP's role in DNA damage repair, this phenomenon can be equally extended to PARP's role in facilitating transcription. Indeed, the formation of PAR at Hsp70 loci after HS results in formation of a localized compartment that aids in the local retention of transcription factors, including Pol II, to sustain continued transcription activation of Hsp70 (Zobeck, 2010). It is yet to be determined if PAR also aids in the local retention of histones that were previously measured to be lost from Hsp70 after HS (Petesch, 2012).

The activation of PARP through the acetylation of H2AK5 also ascribes a unique function to dTip60. Like PARP, Tip60 has been studied for both its roles in DNA repair and also transcriptional activation (Sapountzi, 2006). In Drosophila, dTip60 is part of a complex containing Domino, an ATPase homologous to the mammalian p400 and SRCAP proteins, which, like Swr1p in S. cerevisiae, catalyzes the exchange of histone variant H2A.Z into H2A-containing nucleosomes. Drosophila contains only one H2A variant, which has properties of both H2A.Z and the C-terminal extension of H2A.X, and, when phosphorylated, marks sites of DNA damage. Before HS, it is known that Hsp70 contains nucleosomes harboring H2Av near the 5' end of the gene that is lost upon HS. Recently, the phosphorylation of H2AvS137 was shown to globally regulate PARP activation and is necessary for full transcriptional activation of Hsp70. dTip60 acetylates K5 on H2Av that is already phosphorylated on its C-terminal domain at S137 (Kusch, 2004). This acetylation stimulates the dTip60 complex to exchange out the modified H2Av. Additionally, in vitro studies show that the ability of H4 to activate PARP is squelched in the context of a nucleosome due to H2A. Collectively, these studies suggest a model in which the phosphorylation of H2AvS137 stimulates dTip60 to acetylate H2AvK5 following its recruitment upon HS. These modifications are sufficient to stimulate the dTip60 complex to remove the modified H2Av and expose PARP to H4 and activate its enzymatic activity. The importance of H2A variant exchange has also been documented in Arabidopsis, where the Swr1 complex is also necessary for changes in chromatin structure at HS genes following HS (Petesch, 2012).

This proposed model for the order of events that lead to the activation of PARP upon HS raises many questions for future exploration. First, is the H2Av that is present before HS already phosphorylated, and what is the kinase responsible for phosphorylation? Second, is phosphorylation of H2Av necessary for dTip60 acetylation of H2AvK5 upon HS? Third, is H2AvK5Ac by itself or in combination with S137 phosphorylation sufficient for PARP activation in vitro? Fourth, is the ATPase activity of the dTip60 complex to exchange H2Av following HS necessary or sufficient for PARP activation? Finally, is the activity of PARP regulated on a genomic scale at sites with H2Av nucleosomes that are both acetylated at K5 and phosphorylated at S137 (Petesch, 2012)?

The fact that transcription-independent nucleosome loss following HS at Hsp70 is reliant on factors that respond to DNA damage provokes the question if changes in chromatin at Hsp70 are the result of a response to DNA repair. Indeed, transcriptional activation can occur in response to PARP activation from a topoisomerase II break in DNA. However, in contrast to that study, this study found that PARP is already present at Hsp70 before HS and is not recruited upon HS. Although topoisomerase II mediated breaks have been mapped to sites near the TSS of Hsp70 before HS, these breaks are not sufficient to detect active PARP at Hsp70 before HS and might be more important for the initial deposition of PARP before HS. An alternative mechanism is proposed for PARP activation whereby a transcriptional activator hijacks DNA repair proteins to aid transcriptional activation. The fact that PARP is bound near the majority of human TSSs containing Pol II as at Drosophila Hsp70, also hints at the generality for a mechanism whereby activation of prebound PARP leads to changes in chromatin structure and ultimately contributes to gene expression (Petesch, 2012).

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).

Regulation of the transcriptional activity of poised RNA polymerase II by the elongation factor ELL

Many developmentally regulated genes contain a poised RNA polymerase II (Pol II) at their promoters under conditions where full-length transcripts are undetectable. It has been proposed that the transcriptional activity of such promoters is regulated at the elongation stage of Pol II transcription. In Drosophila, the heat-shock loci expressing the Hsp70 genes have been used as a model for the regulation of the transcriptional activity of poised Pol II. Drosophila ELL [dELL; Su(Tpl)] is a Pol II elongation factor capable of stimulating the rate of transcription both in vivo and in vitro. Although ELL and the elongation factor Elongin A have indistinguishable effects on RNA polymerase in vitro, the loss-of-function studies indicate that these proteins are not redundant in vivo. This study used RNAi to investigate the physiological properties of dELL and a dELL-associated factor (dEaf) in a living organism. Both ELL and Eaf are essential for fly development. dELL is recruited to heat shock loci upon induction, and its presence with Pol II at such loci is required for proper heat-shock gene expression. Consistent with a role in elongation, dELL knockdown reduces the levels of phosphorylated Pol II at heat-shock loci. This study implicates dELL in the expression of loci regulated by Pol II elongation (Smith, 2008).

Efficient transcription by RNA polymerase II (Pol II) is an intricate process that requires multiple contacts with the DNA template and nascent RNA that inevitably leads to frequent stalling during the transcription of a gene. The average rate of transcription by Pol II in vivo is an order of magnitude higher than that obtained in vitro despite additional impediments, such as traversing through nucleosomes. Using biochemical approaches, two Pol II elongation factors, Eleven nineteen lysine-rich leukemia (ELL) and Elongin A, were isolated from cell extracts as factors capable of stimulating Pol II activity by suppressing transient pausing. Despite similar in vitro activities, the Drosophila orthologs of ELL and Elongin A are each essential for development. This observation indicates that their in vivo activity is not redundant (Smith, 2008).

Recent genome-wide studies have found a large number of developmentally regulated genes that contain a paused Pol II at their promoters. Therefore, it has been proposed that the transcriptional activity of such poised Pol IIs is regulated at the level of transcription elongation. The classic model for studying genes regulated by promoter-proximal paused polymerase is Hsp70 gene induction in Drosophila. Previous studies have shown that several Pol II elongation factors are rapidly recruited to the Hsp70 genes after heat shock. Although much work has been done on the role of these factors in gene regulation in cultured cells, less is known about the role of these factors in the regulation of heat-shock gene expression in the whole organism. Although there are several mutants in the gene encoding Drosophila ELL (dELL), all of these alleles are embryonic lethal (Eissenberg, 2002). Therefore, it was possible to use these alleles to further characterize the role of the elongation factor ELL in the regulation of the transcriptional activity of poised Pol II and Hsp70 loci. To test the role for dELL in gene expression, RNAi was used to reduce expression levels of both dELL and dELL-associated factor (dEaf) expression levels during development, and the in vivo effect of their reduction on transcription and development was examined. It was found that knockdown of dELL and dEaf results in lethality. Furthermore, knockdown of these elongation factors results in reduced Hsp70 transcript accumulation after heat shock. Immunolocalization of phosphorylated Pol II in heat-shocked dELL knockdown salivary glands demonstrates reduced levels of the elongating form of Pol II at the Hsp70 loci in the absence of dELL. These studies demonstrate that dELL is essential for full induction of heat-shock gene expression and are consistent with a role for dELL in Pol II elongation. These findings provide a role for an RNA Pol II elongation factor in the transcriptional regulation of poised Pol II (Smith, 2008).

dELL has been shown to be essential; homozygous mutant clones do not survive in the eye and homozygotes for loss-of-function alleles die at the end of embryogenesis or in early first instar. To investigate the role of dELL in transcription in flies, dELL was knocked down by RNAi, which typically reduces, but does not eliminate, the targeted gene products. A 600-bp portion of the dELL coding region was inserted into a P-element vector that drives the expression of dsRNA through two convergent Gal4 UAS promoters that flank the insert. Several transgenic lines were generated and tested for effects on viability by crossing to an Actin5C-Gal4 driver line that expresses yeast Gal4 under the cytoplasmic actin promoter. All eight dELL RNAi lines show significant loss of viability when expressed under this driver. When adult escapers were obtained, very few males were observed, indicating that males are more susceptible to loss of dELL. Greater numbers of females than males were observed at the third instar larval stage, indicating that males are dying earlier than females. A significant genome-wide reduction of dELL protein is observed by immunofluorescence analysis of dELL RNAi larval polytene chromosomes (Smith, 2008).

Through two-hybrid analysis, two interacting partners of ELL have been characterized in humans, Eaf1 and Eaf2. Eaf1 and Eaf2 are highly related and can stimulate the elongation activity of ELL in vitro. Recently, the association of Eaf with ELL was shown to be evolutionarily conserved, with the finding that Schizosaccharomyces pombe homologs SpEaf and SpELL directly interact with each other. Additionally, SpEaf enhances the stimulation by SpELL of Pol II transcription in vitro. Because Drosophila also has a single Eaf homolog, RNAi was used to knock down dEaf levels and assessed the viability of dEaf-knockdown flies in six different transgenic RNAi lines. In all lines, significant reductions were observed in the number of adult progeny of RNAi-expressing flies compared with control siblings. In addition, a consistent reduction in the male-female sex ratio was observed for dEaf RNAi, suggesting that the male-enhanced lethal phenotype (not observed for other elongation factors) is due to loss of a dELL-dEaf complex (Smith, 2008).

To test for the effectiveness of the RNAi knockdowns, dELL and dEaf mRNA levels were measured in knockdown larvae and their control siblings. Significant reductions in dELL transcripts are observed in the dELL RNAi larvae. dELL transcripts, as measured by RT-PCR, are not reduced by RNAi to the same level as dELL protein, as assessed by immunofluorescence on polytene chromosomes. Previously, it was observed that knockdown of dRTF1 by RNAi was more effective at the protein than the RNA levels presumably because the long dsRNAs produced are processed as miRNAs and interfere with translation. Because dELL is nested in an intron of the gene encoding the chromatin remodeling enzyme dMi-2, transcript levels for this gene were measured and no reduction was found of dMi-2 RNA in dELL RNAi larvae. Additionally, it was found that dEaf RNA levels are reduced in dEaf RNAi larvae. Interestingly, a significant increase in dELL levels is observed in dEaf RNAi larvae, possibly compensating for the lower dEaf levels (Smith, 2008).

dELL was previously shown to be recruited to heat-shock genes upon heat shock. To determine whether dELL is required for heat-shock gene expression, the levels of Hsp70 transcripts after heat shock were compared in dELL knockdown larvae and their control siblings. By immunofluorescence analysis, little or no dELL is seen at the Hsp70 gene after heat shock in dELL knockdown larvae, whereas the control siblings without the Gal4 driver showed the expected recruitment of dELL to the Hsp70 gene. Northern blot analysis showed reduced levels of Hsp70 mRNA levels in the dELL RNAi larvae. A similar analysis was done with dEaf RNAi larvae, and reduced Hsp70 mRNA also occurs after heat shock, although the deficit was less than observed for the dELL RNAi larvae. Similar results were observed when Hsp70 levels were measured by RT-PCR, showing greater reductions in Hsp70 RNA levels in dELL RNAi than dEaf RNAi larvae (Smith, 2008).

Chromosomal levels of dELL are markedly reduced in the absence of Cdk9, the catalytic subunit of the Pol II C-terminal domain (CTD) kinase PTEF-B. To determine whether dELL knockdown affects the recruitment of Pol II to the Hsp70 genes, dELL knockdown and control polytene chromosomes were probed with antibodies to the Ser-2-phosphorylated, elongating form of Pol II. Lower levels of Ser-2-phosphorylated Pol II were consistently observed at the Hsp70 heat-shock loci in dELL-knockdown larvae, suggesting a close link between dELL function and phosphorylation of the Pol II CTD (Smith, 2008).

ELL belongs to a class of transcription elongation factors that have been shown to stimulate the K_m and/or V_max of RNA Pol II in vitro by alleviating pausing on a purified DNA template. Another member of this class is Elongin A and its Drosophila ortholog dEloA. From the present and previous studies, it is clear that both dELL and dEloA localize to the Hsp70 gene upon heat shock, and each is required for full levels of heat-shock gene expression, suggesting that the in vivo roles of these elongation factors in Hsp70 gene transcription are not redundant (Gerber, 2005). Similarly, it was observed that the knockdown phenotypes of these two proteins can be unique, such as the enhanced male lethality in dELL RNAi larvae. How could both elongation factors be redundant in vitro, yet nonredundant in vivo? The in vitro studies were performed on naked DNA templates, whereas the chromatin environment of RNA Pol II-transcribed genes can provide additional challenges to the polymerase. Each of these elongation factors has its own interaction partners and may be recruited to distinct states of the polymerase, such as initiating, elongating, or stalled polymerase. Consistent with this view, knockdown of dELL, but not dEloA, results in decreased levels of Ser-2-phosphorylated Pol II at the Hsp70 and other loci. Interestingly, the chromosomal targeting of dELL, but not dEloA, is dramatically reduced by the knockdown of CDK9, the Pol II CTD kinase, suggesting that dELL and dEloA are recruited to genes by distinct mechanisms. Fine mapping of dELL and dEloA on the well characterized Hsp70 gene at different time points after activation could clarify the distinct roles for these enzymes (Smith, 2008).

The lesser effect of dEaf knockdown on Hsp70 gene induction could be indicative of a requirement of dEaf for optimal function of dELL, whereas dELL can partially function without dEaf. Indeed, in vitro transcription studies have demonstrated that human Eaf proteins, in combination with ELL, stimulate transcription elongation by Pol II above the levels obtained with ELL alone. In dEaf RNAi larvae, it was observed that dELL levels are increased, conceivably as a cellular response to increased pausing resulting from lower dEaf levels (Smith, 2008).

Previous work on the function of dELL made use of alleles of the Su(Tpl) locus, which encodes dELL (Eissenberg, 2002). All known Su(Tpl) alleles are embryonic lethal. In contrast, RNAi of dELL allows survival to the larval or adult stages depending on the insertion line of the dsRNA construct. Interestingly, the few 'escaper' dELL RNAi adults are overwhelmingly female. As seen with the heat-shock defect, the difference in male and female viability is less in dEaf RNAi flies than in dELL RNAi flies, consistent with dEaf enhancing, but not being absolutely required for, dELL function. A previous study showed that males express much higher levels of a dELL transcript than females, although the functional significance of this difference has not been investigated (Gerber, 2001). One hypothesis is that dELL is needed in males as part of the process of X chromosome dosage compensation; Drosophila dosage compensation factors are thought to enhance transcription elongation of X-linked genes in males, and loss of any of these factors leads to male-specific lethality. In addition, reduced levels of several global chromatin regulators, including the supercoiling factor, Jil-1 H3 kinase, heterochromatin protein HP1, and the chromatin remodeler ISWI, have been reported to differentially affect the survival of males and/or the morphology of the X chromosome. However, in dELL knockdowns, MSL localization and the male polytene X chromosome morphology appears similar in dELL knockdown male larvae and their control brothers. Whether there are specific defects in dosage compensation of X-linked genes may be an interesting avenue for future investigations. Alternative explanations for a male-enhanced lethality also should be considered. For example, Drosophila males differ from females not just in having one less X chromosome, but also in carrying a Y chromosome, which comprises ~12% of the male genome. A number of genes are male-lethal due to the presence of the mostly heterochromatic Y chromosome, including modulators of position effect variegation, such as the Su(var)3-3 gene that encodes the histone demethylase LSD1, the uncharacterized Su(var)2-1, as well as the HP1-interacting protein Bonus (dTIF1), an enhancer and suppressor of position-effect variegation. For Su(var)2-1 and Bonus, the Y-lethal effect is not Y-specific but can be phenocopied by other sources of heterochromatin. A role for dELL in the regulation of heterochromatin is unknown but could conceivably be required for the expression of heterochromatin components (Smith, 2008).

Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation

The kinetics with which promoter-proximal paused RNA polymerase II (Pol II) undergoes premature termination versus productive elongation is central to understanding underlying mechanisms of metazoan transcription regulation. To assess the fate of Pol II quantitatively, photoactivatable GFP-tagged Pol II at uninduced Hsp70 was tracked on polytene chromosomes, and it was shown that Pol II is stably paused with a half-life of 5 min. Biochemical analysis of short nascent RNA from Hsp70 reveals that this half-life is determined by two comparable rates of productive elongation and premature termination of paused Pol II. Importantly, heat shock dramatically increases elongating Pol II without decreasing termination, indicating that regulation acts at the step of paused Pol II entry to productive elongation (Buckley, 2014).

Cdk9 is an essential kinase in Drosophila that is required for heat shock gene expression, histone methylation and elongation factor recruitment

Phosphorylation of the large RNA Polymerase II subunit C-terminal domain (CTD) is believed to be important in promoter clearance and for recruiting protein factors that function in messenger RNA synthesis and processing. P-TEFb is a protein kinase that targets the (CTD). The goal of this study was to identify chromatin modifications and associations that require P-TEFb activity in vivo. The catalytic subunit of P-TEFb, Cdk9, was knocked down in Drosophila using RNA interference. Cdk9 knockdown flies die during metamorphosis. Phosphorylation at serine 2 and serine 5 of the CTD heptad repeat were both dramatically reduced in knockdown larvae. Hsp 70 mRNA induction by heat shock was attenuated in Cdk9 knockdown larvae. Both mono- and trimethylation of histone H3 at lysine 4 were dramatically reduced, suggesting a link between CTD phosphorylation and histone methylation in transcribed chromatin in vivo. Levels of the chromo helicase protein CHD1 were reduced in Cdk9 knockdown chromosomes, suggesting that CHD1 is targeted to chromosomes through P-TEFb-dependent histone methylation. Dimethylation of histone H3 at lysine 36 was significantly reduced in knockdown larvae, implicating CTD phosphorylation in the regulation of this chromatin modification. Binding of the RNA Polymerase II elongation factor ELL was reduced in knockdown chromosomes, suggesting that ELL is recruited to active polymerase via CTD phosphorylation (Eissenberg, 2007).

Cdk9, the catalytic subunit of P-TEFb, is highly conserved among eukaryotes. The yeast kinases Ctk1 and Bur1 are both homologs of Cdk9, and both are CTD kinases in Drosophila, although loss of Bur1 has no effect on CTD phosphorylation yeast. Bur1 is essential but Ctk1 is not (Eissenberg, 2007).

RNAi knockdown of Cdk9 in transgenic flies results in lethality at the pupal stage. This is considerably later than the embryonic lethality reported for C. elegans RNAi knockdown of Cdk9. While this difference could reflect differences in the requirements for Cdk9 in these organisms, it is more likely that differences in timing or efficiency of RNAi, Cdk9 protein turnover and/or maternal Cdk9 loading accounts for the much later lethality in knockdown flies. Nevertheless, these results confirm and extend the finding that P-TEFb is essential in metazoan development (Eissenberg, 2007).

In contrast, Cdk9 homologs in fission yeast and Neurospora are not essential. Since CTD phosphorylation has been linked to promoter clearance, pre-mRNA processing and chromatin modification, it is not possible to say what aspect of P-TEFb activity is essential in metazoa. RNAi knockdown of the Drosophila Cdk9 in cultured cells causes arrest of the cell cycle at the G1-S transition, implicating this kinase in cell cycle control. It is unlikely that cell cycle arrest is causing the lethality in knockdown flies, since cell cycle mutations in Drosophila generally are associated with reduced or missing imaginal discs, and the discs in Cdk9 knockdown larvae appear overtly normal. The finding that Hsp70 transcripts are reduced in Cdk9 knockdown larvae is consistent with the reduced Hsp70 transcription previously reported in Cdk9 RNAi cultured cells. Hsp 70 is not essential in Drosophila, but the effects on Hsp70 suggest that defects in gene expression could underlie the essential requirement for Cdk9 in Drosophila development (Eissenberg, 2007).

Cdk9 knock-down flies show dramatic reductions in both serine 2 and serine 5 phosphorylation. In contrast, flavopiridol treatment of cultured cells has been found to selectively reduce serine 2 phosphorylation. The significance of this difference is unclear, but could reflect differences in experimental protocol. For example, flavopiridol treatments were limited to 15-20 min, while RNAi knockdown third instar larvae are subject to knockdown conditions for several days before assay. Longer periods of Cdk9 inactivation may be required for reduction in serine 5 phosphorylation. Alternatively, it is possible that knockdown of Cdk9 protein levels results in inhibition of TFIIH, the other known CTD kinase. Regardless of the mechanism, the RNAi knockdown clearly results in reduced phosphorylation of the CTD, enabling a test of the consequences of loss of CTD phosphorylation on chromatin modification and recruitment of RNA Polymerase II-associated factors (Eissenberg, 2007).

Loss of CTD phosphorylation in Cdk9 knockdown larvae is associated with reduced binding of the RNA Polymerase II elongation factor ELL genome-wide. ELL is broadly co-localized with phosphorylated RNA Polymerase II on polytene chromosomes, and is rapidly recruited to heat shock loci after a brief heat shock. These results suggest that the efficient recruitment of ELL to transcribed loci requires CTD phosphorylation. Whether this reflects a direct interaction of ELL with the CTD is unknown (Eissenberg, 2007).

Despite the fact that Elongin A affects the same kinetic parameter in RNA Polymerase II catalysis as ELL, Elongin A binding is not reduced by loss of CTD phosphorylation. As with ELL, the nature of Elongin A binding to RNA Polymerase II is unknown, but these observations suggest their binding can be distinguished by sensitivity to the phosphorylation state of the CTD. Since no increase of Elongin A was observed under conditions of reduced ELL binding, it seems unlikely that ELL and Elongin A compete for RNA Polymerase II binding (Eissenberg, 2007).

Spt4 and Spt5 are subunits of DSIF, which is implicated in the regulation of RNA Polymerase II elongation. Previous work suggested that reduced serine 2 phosphorylation of the RNA Polymerase II CTD has no effect on Spt5 recruitment to a heat shock gene in cultured cells. In Cdk9 knockdown flies, in which both serine 2 and 5 phosphorylation are reduced, the chromosomal distribution of Spt5 is unchanged genome-wide. This is consistent with previous reports that Spt5 interacts with both phosphorylated and unphosphorylated RNA Polymerase II (Eissenberg, 2007 and references therein).

The chromo domain motif is a binding site for methylated histone tails. The role of the CHD1 chromo domain in methylated histone binding is controversial. However, recent structural data determined that the double chromo domain of mammalian CHD1 binds methylated H3K4 in vitro. This study shows that Cdk9 knockdown leads to a loss of chromosomal CHD1. This observation is most easily interpreted as the result of loss of H3K4 methylation that also occurs in Cdk9 knockdown chromosomes. Thus, the finding reported in this study lends support to the in vitro binding data and strongly suggests that the chromo domain-methylated histone interaction plays a dominant role in targeting CHD1 to active chromatin in vivo (Eissenberg, 2007).

The observation that both H3K4 and H3K36 methylation are significantly reduced in Cdk9 knockdown chromosomes suggests a linkage between phosphorylation of the CTD and histone methylation at transcribed genes. In this respect, Cdk9 subsumes activities found in yeast Bur1/Bur2 and yeast Ctk1. Since no significant difference was observed in ASH1 protein levels on Cdk9 knockdown chromosomes, a model is favored in which Cdk9-dependent RNA Polymerase II elongation plays a mechanistic role in H3 tail methylation. In this model, RNA Polymerase II passage destabilizes histone-DNA contacts, making the histones better substrates for efficient methylation. Reduced CTD phosphorylation would lead to reduced rates of RNA Polymerase II transcription genome-wide, resulting in reduced efficiency of histone tail methylation. While the mechanism connecting CTD phosphorylation to RNA Polymerase II elongation rate is likely to be complex in vivo, the observation that reduced CTD phosphorylation is associated with reduced dELL binding suggests that loss of dELL association could be a contributing factor (Eissenberg, 2007).

Mutation in Ash1 in Drosophila results in loss of all detectable H3K4 methylation, but has no effect on H3K36 methylation. This is consistent with independent mechanisms for these two chromatin modifications. A Polymerase II passage model provides a simple mechanism to account for similar effects on both modifications based on substrate availability (Eissenberg, 2007).

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).

Stress-induced PARP activation mediates recruitment of Drosophila Mi-2 to promote heat shock gene expression

Eukaryotic cells respond to genomic and environmental stresses, such as DNA damage and heat shock (HS), with the synthesis of poly-[ADP-ribose] (PAR) at specific chromatin regions, such as DNA breaks or HS genes, by PAR polymerases (PARP). Little is known about the role of this modification during cellular stress responses. This study shows that the nucleosome remodeler dMi-2 is recruited to active HS genes in a PARP-dependent manner. dMi-2 binds PAR suggesting that this physical interaction is important for recruitment. Indeed, a dMi-2 mutant unable to bind PAR does not localise to active HS loci in vivo. Several dMi-2 regions have been identified that bind PAR independently in vitro, including the chromodomains and regions near the N-terminus containing motifs rich in K and R residues. Moreover, upon HS gene activation, dMi-2 associates with nascent HS gene transcripts, and its catalytic activity is required for efficient transcription and co-transcriptional RNA processing. RNA and PAR compete for dMi-2 binding in vitro, suggesting a two step process for dMi-2 association with active HS genes: initial recruitment to the locus via PAR interaction, followed by binding to nascent RNA transcripts. It is suggested that stress-induced chromatin PARylation serves to rapidly attract factors that are required for an efficient and timely transcriptional response (Murawaska, 2011).

Mi-2 is strongly linked to transcriptional repression in both vertebrate and invertebrate organisms. Within NuRD and dMec complexes it contributes to the repression of cell type-specific genes. Therefore, the widespread colocalisation of dMi-2 with active Pol II and elongation factors at many chromosomal sites is surprising and suggests that dMi-2 might play an unappreciated role during active transcription, at least (or specifically) during environmental stresses such as HS. Indeed, dMi-2 is recruited to HS genes within minutes of HS. This property is not shared by other chromatin remodelers: Brahma (BRM) is not enriched at HS puffs and HS gene activation is independent of BRM function. Moreover, although imitation switch (ISWI) containing complexes are important for HS gene transcription, ISWI does not accumulate to high levels at active HS loci. Recruitment to HS puffs has previously been reported for Drosophila CHD1. Thus, accumulation at active HS genes is shared by at least two members of the CHD family of nucleosome remodelers but not by SWI/SNF and ISWI proteins (Murawaska, 2011).

Depletion of dMi-2 or a reduction of dMi-2 recruitment does not significantly perturb hsp70 transcription in Kc cells and, therefore, dMi-2 is dispensable for HS gene activation in this system. By contrast, depletion of dMi-2 in larvae strongly decreases hsp70, hsp26 and hsp83 activation. It is possible, that the RNAi-mediated depletion of dMi-2 is more efficient in transgenic flies compared to cell lines. In addition, it is believed that several factors contributing to HS gene activation are highly abundant or redundant in Kc cells but more limiting in other contexts. Accordingly, FACT and Spt6 are required for a HS gene activation in flies but are not essential in Kc cells (Murawaska, 2011).

The strong decrease of HS gene activation in dMi-2 RNAi larvae indicates a positive contribution of dMi-2 to transcription in vivo. Overexpression of inactive dMi-2 also results in reduced HS gene transcription implying that its enzymatic activity is critical. It is presently unclear whether this reflects a requirement for dMi-2 catalysed nucleosome remodeling or whether its activity is directed towards different substrates (Murawaska, 2011).

While dMi-2 could indirectly influence transcription by remodeling nucleosomes within the transcribed part of hsp70, its physical association with nascent HS gene transcripts argues for a more direct effect. Indeed, dMi-2 is not only required for high HS gene mRNA levels, but also affects the efficiency of co-transcriptional 3' end formation and splicing. A role of chromatin remodelers in splicing has been suggested before: Both CHD1 and BRG1 bind components of the splicing apparatus. CHD1 associates with Pol II and binds nucleosomes containing H3K4me3, which are enriched near the 5' end of active genes . BRG1 is present at the coding region of genes and influences splice site choice. It has been proposed that CHD1 and BRG1 physically recruit splicing factors but it is unclear if their ATPase activities play a role. Indeed, inactive BRG1 retains the ability to affect exon choice. Inefficient processing of the hsp70 and hsp83 transcripts is not only observed in larvae expressing reduced levels of dMi-2. Importantly, even stronger processing defects are generated by overexpression of inactive dMi-2. This strongly suggests, for the first time, that the catalytic activity of a chromatin remodeler is required for correct co-transcriptional RNA processing. It remains to be determined whether dMi-2 nucleosome remodeling activity influences RNA processing indirectly, e.g. by altering Pol II elongation rates, or whether it has a more direct role (Murawaska, 2011).

A series of complementary results support the hypothesis that dMi-2 interacts with PAR polymers that are rapidly synthesized at activated HS loci. First, the broad distribution of dMi-2 over the entire transcribed region correlates with the distribution of PAR polymer. Second, pharmacological inhibition of PARP greatly decreases dMi-2 binding to activated hsp70. Third, dMi-2 directly binds PAR polymers in vitro. Fourth, a dMi-2 mutant unable to bind PAR also fails to localise to active HS loci. dMi-2 physically associates with nascent HS gene transcripts and binds RNA in vitro. While this interaction is potentially important for the efficiency of transcription and processing, it likely plays a minor role in dMi-2 targeting. Accordingly, inhibition of transcriptional elongation has no significant effect on dMi-2 recruitment (Murawaska, 2011).

It is important to note, that while the results argue for an important role of PAR binding in the recruitment of dMi-2 to HS loci, it cannot be excluded that protein-protein interactions with histone or non-histone proteins also play a role (Murawaska, 2011).

This analysis indicates that dMi-2 harbours several PAR binding motifs in its N-terminal region. It has been demonstrated that human CHD4 is recruited to double stranded DNA breaks in a PARP-dependent manner (Polo, 2010). That study demonstrated PAR binding activity to the region N-terminal of the ATPase domain of CHD4. This agrees well with the current data and suggests that the PAR binding function of CHD4/dMi-2 has been conserved in evolution (Murawaska, 2011).

Two structural protein modules directly interact with PAR, the macrodomain and the PBZ domain; however, these domains are not present in dMi-2. In addition, several shorter PAR binding motifs have been identified. These motifs bear little sequence similarity but share the presence of several K/R residues which are interspersed by hydrophobic residues. The current results have uncovered three K/R-rich regions with PAR binding activity near the N-terminus of dMi-2. Two of these three K/R-rich regions (K/R III and K/R IV) consist of interspersed basic and hydrophobic residues and are therefore reminiscent of the previously described PAR binding motifs, and the third (K/R I) lacks hydrophobic residues completely. None of the three K/R regions matches the consensus PAR binding motifs. It is possible that a consensus motif should generally be chosen less stringently and that a high content of K and R-residues in these regions is sufficient to provide PAR binding activity in vitro. Further characterisation of these regions will be required to resolve this issue. In addition to the K/R regions, the tandem chromodomains of dMi-2 bind PAR in vitro. Previous studies have shown that the chromodomains are required for interacting with nucleosomal DNA in vitro. The new data suggests that these domains can interact with different nucleic acids (Murawaska, 2011).

Several potential molecular functions of PARylation at HS genes have been suggested. First, PARP activity is required for the rapid loss of nucleosomes at hsp70 within the first two minutes after HS (Petesch, 2008). It has been suggested that PARylation of histones aids rapid nucleosome disassembly (Petesch, 2008). Second, at later stages of the HS response (20-60 minutes after HS), PARP activity is required to establish a compartment which restricts the diffusion of factors such as Pol II and Spt6 and promotes efficient factor recycling. The current results suggest that PARylation carries out a third task, namely, to recruit factors via their direct interaction with PAR. The earliest time point when dMi-2 binding to hsp70 can be detected is between 2 and 5 minutes after HS. This places dMi-2 recruitment between the early PARP-dependent nucleosome removal (0-2 minutes after HS) and effects of the transcription compartment (20-60 minutes after HS) (Murawaska, 2011).

The ability of dMi-2 to bind both PAR and RNA and the finding that RNA can compete for PAR binding to dMi-2 is consistent with the hypothesis that dMi-2 association with active HS genes is a two step process. It is proposed that dMi-2 is initially recruited via interaction with PAR polymers. Synthesis of these starts prior to the onset of hsp70 transcription (Petesch, 2008). This results in a rapid local increase of the dMi-2 concentration. In the second step, when hsp70 transcripts are produced by elongating RNA polymerase II at high rates, dMi-2 can switch from binding PAR to interacting with nascent transcripts (Murawaska, 2011).

Severe cellular stresses, such as DNA strand breaks and acute HS, must be dealt with quickly and efficiently. In both cases, a multitude of factors are rapidly recruited to orchestrate the repair of DNA and the massive transcriptional activation of HS genes, respectively. It is postulated that rapid synthesis of PAR polymers at both DNA damage sites and HS genes affords an efficient mechanism to recruit chromatin remodelers and other factors. It has recently been shown that PARylation of DNA breaks is instrumental in recruiting chromatin remodelers, including mammalian dMi-2 homologs, to damaged sites. This study shows that dMi-2?s recruitment to activated HS genes requires PARP activity and that dMi-2 binds PAR directly. The high local concentration of PAR polymers at DNA breaks and HS genes might exploit the general affinity of dMi-2 for nucleic acids. Indeed, dMi-2 binds both DNA and RNA as well as PAR in vitro. In this manner, PAR polymers might act as a scaffold to redirect dMi-2 to chromatin regions where high levels of dMi-2 activity are required, thus acting as a stress-dependent, transient affinity site for chromatin remodeling and possibly RNA processing activities. The results highlight a signaling and scaffolding function for PARP activity during transient environmental stresses other than DNA damage, suggesting that PARylation carries out important modulatory functions in the stress-dependent reprogramming of nuclear activities (Murawaska, 2011).

Recruitment of the ATP-dependent chromatin remodeler dMi-2 to the transcribed region of active heat shock genes

The ATP-dependent chromatin remodeler dMi-2 can play both positive and negative roles in gene transcription. dMi-2 is recruited to the hsp70 gene in a heat shock-dependent manner and is required to achieve high transcript levels. This study used chromatin immunoprecipitation sequencing (ChIP-Seq) to identify other chromatin regions displaying increased dMi-2 binding upon heat shock and to characterize the distribution of dMi-2 over heat shock genes. dMi-2 is shown to be recruited to the body of at least seven heat shock genes. Interestingly, dMi-2 binding extends several hundred base pairs beyond the polyadenylation site into the region where transcriptional termination occurs. dMi-2 does not associate with the entire nucleosome-depleted hsp70 locus 87A. Rather, dMi-2 binding is restricted to transcribed regions. These results suggest that dMi-2 distribution over active heat shock genes are determined by transcriptional activity (Mathieu, 2012).

hsp70 heat shock genes has been used as a model system to study by what parameters chromatin association of dMi-2 is governed. dMi-2 is recruited to heat shock-activated hsp70 genes, and is required for their full activation in flies. dMi-2 appears to occupy several regions within the body of the hsp70 gene. However, it is not known if dMi-2 covers the hsp70 gene completely, if it is evenly distributed or displays preferences for the 5'- or 3'-ends (Mathieu, 2012).

Actively transcribed hsp70 loci are extensively poly-ADP-ribosylated. Binding of dMi-2 to hsp70 in S2 cells is reduced in the presence of a small molecule poly ADP ribose polymerase (PARP) inhibitor. In addition, dMi-2 binds to PAR in vitro and possesses several PAR-binding motifs suggesting that dMi-2 recruitment to hsp70 involves a direct interaction with the PAR polymer (Murawska, 2011). Moreover, dMi-2 binds nascent hsp70 transcripts and can interact both with DNA and RNA in vitro. Based on these results, it is proposed that dMi-2 is initially recruited to the hsp70 locus when this becomes PARylated shortly after heat shock (HS). Once transcription has been activated, dMi-2 engages with nascent transcripts. However, the relative contributions of PAR, DNA and RNA binding to dMi-2 chromatin association and distribution across genes are not well defined (Mathieu, 2012).

Histone PARylation within the hsp70 locus is believed to contribute to the rapid nucleosome loss that occurs within the first 2min of heat shock. Interestingly, nucleosome loss at hsp70 loci is not restricted to the hsp70 transcription units but extends several kilobases up- and down-stream. It is limited on either side by silencer elements (scs and scs'). Nucleosome depletion across the hsp70 locus increases the access of RNAP II and transcription factors for DNA and their concerted action results in the production of thousands of hsp70 RNA molecules per nucleus. It is currently not known if dMi-2 binding is elevated across the entire PARylated hsp70 locus or if dMi-2 binding is restricted to those regions that are actively transcribed (Mathieu, 2012).

In addition to hsp70, the expression of two other HS genes (hsp26 and hsp83) is affected in transgenic flies expressing reduced levels of dMi-2. This raises the possibility that all HS genes require dMi-2 for full activation and that dMi-2 physically associates with other HS genes during the HS response (Mathieu, 2012).

This study extends an analysis of HS-regulated dMi-2-chromatin interaction by addressing several key questions. First, chromatin immunoprecipitation sequencing (ChIP-Seq) has been used to obtain a high resolution, genome-wide dMi-2 binding profile in both untreated and heat-shocked S2 cells. Through this global approach, seven regions were identified which exhibit strong, HS-induced enrichment of dMi-2 binding. In addition to hsp70 genes, these regions harbour six additional HS genes. Inspection of ChIP-Seq profiles revealed that dMi-2 associates with the body of these HS genes. A more detailed analysis of dMi-2 distribution showed that dMi-2 binding closely follows nascent RNA production. Importantly, dMi-2 binding extends several hundred base pairs beyond polyadenylation sites into regions where transcriptional termination occurs. dMi-2 binding within the PARylated hsp70 locus 87A was analyzed and it was found that dMi-2 recruitment is restricted to actively transcribed regions. These results suggest that RNA synthesis, rather than a general increase in DNA accessibility by PARylation and nucleosome depletion, determines the distribution of dMi-2 at active HS loci. However, ChIP-Seq and RT-qPCR analysis of dMi-2 binding to genes that are constitutively transcribed at high levels, and are induced by other forms of stress indicates that strong transcriptional activity is not sufficient to accumulate dMi-2. Together, these results allow identification of transcription as the key parameter that determines the distribution of dMi-2 over active HS genes (Mathieu, 2012).

Transcription by RNAP II continues past the polyadenylation site until transcription is terminated at one of multiple positions downstream. Termination sites for the hsp26 gene have been mapped by KMnO₄ hypersensitive site mapping. The furthest detectable hypersensitive site was located at a distance of 526bp from the polyadenylation site. Increased dMi-2 binding to the active hsp26 gene can be detected ~300-bp downstream of the polyadenylation site. Thus, the dMi-2 bound region lies within the region that is transcribed by RNAP II (Mathieu, 2012).

dMi-2 binds nascent hsp70 and hsp83 transcripts in vivo. It is hypothesized that this interaction of dMi-2 with nascent transcripts governs the distribution of dMi-2 over active heat shock genes. This hypothesis predicts that dMi-2 levels should be lower within the 5' halves of HS genes, where RNA transcripts are still short, higher within the 3' halves of HS genes, where transcripts reach their maximum length, and decline again past the polyadenylation signal, where the message has been cleaved off and only short transcripts are produced prior to their termination. Indeed, the relative enrichment of dMi-2 binding that was observed across the six heat shock genes analysed supports this hypothesis. In all cases, dMi-2 enrichment is higher in the 3' half compared to the 5' half of genes and declined again in the region beyond the polyadenylation site (Mathieu, 2012).

Several HS genes exhibit a 'dip' in dMi-2 binding around the polyadenylation site. This is also consistent with the hypothesis that dMi-2 binding is mediated by an interaction with nascent RNA. It is proposed that the decline in dMi-2 binding near the polyadenylation site is a consequence of RNA cleavage there. The downstream dMi-2 peak might reflect dMi-2 interacting with the RNA produced by terminating RNA polymerase II (Mathieu, 2012).

While the interaction of dMi-2 with nascent RNA appears to contribute to its association with chromatin, it is not sufficient for recruiting dMi-2 to active gene loci. This view is supported by several findings. First, two genes that have been shown to be activated more than 10-fold upon heat shock in a HSF-dependent manner do not display a significant increase in dMi-2 binding. Secondly, genes that are strongly transcribed in a constitutive fashion, such as the genes encoding ribosomal protein subunits, do not bind more dMi-2 than neighbouring, untranscribed regions. Thirdly, strong activation of metallothionein A by Cd treatment does not result in increased association of dMi-2 with the promoter or the transcribed part of the gene. Fourthly, dMi-2 is not recruited to a reporter gene under control of the hsp70 promoter that is upregulated 200-fold following heat shock. Fifthly, inhibition of transcriptional elongation does not affect the recruitment of dMi-2 to several regions within the activated hsp70 gene. These findings suggest that the initial recruitment of dMi-2 to heat shock genes requires additional signals. In case of the hsp70 gene in Kc cells, one signal appears to be provided by poly-ADP-ribosylation of the locus. No consistent effects of treatment with the PARP inhibitor PJ34 on dMi-2 recruitment was observed in the S2 cells used for this study. The relative contribution of poly-ADP ribosylation to dMi-2 recruitment in different biological contexts is therefore unclear. It is also not known, whether poly-ADP-ribosylation does also occur during the activation of other HS genes (Mathieu, 2012).

A rapid loss of nucleosomes from the 87A locus after HS has been described. Interestingly, nucleosome loss is not restricted to the two hsp70 genes residing within 87A. Instead, it includes the entire region flanked by the insulator elements scs and scs'. This property of the 87A locus has allowed addressing the question if dMi-2 chromatin association correlates with nucleosome depletion. Interrogation of the ChIP-Seq data revealed that dMi-2 recruitment was restricted to the transcribed part of the hsp70 genes even within the larger nucleosome-depleted locus. This underscores the importance of transcription for governing dMi-2 chromatin distribution (Mathieu, 2012).

Taken together, these results support a two-step recruitment model of dMi-2. Initial recruitment does not depend on RNA synthesis. Rather, it is likely to be facilitated by other signals that are specific for HS gene activation, one of which might be poly-ADP-ribosylation in certain contexts. Other potential recruitment signals might include binding to PARP itself, which is located near the 5'-end of the hsp70 transcription unit and migrates across the gene following heat shock, the interaction with histone variants deposited at hsp70 or particular histone modifications that are generated during the heat shock response (Mathieu, 2012).

Once dMi-2 is brought to activate HS genes by one or more of these mechanisms, it interacts with nascent RNA and by doing so associates with the transcribed body of the gene. It is tempting to speculate that this association with nascent RNA influences transcription and co-transcriptional processes. Indeed, quantitative changes are detected in levels and processing of hsp70 gene transcripts in transgenic flies with compromised dMi-2 activity. The ChIP-Seq study suggests that dMi-2 associates with and regulates an entire suite of heat shock genes and provides the basis for a more systematic analysis of dMi-2's role in the heat-shock response (Mathieu, 2012).

Degradation of hsp70 and other mRNAs in Drosophila via the 5' 3' pathway and its regulation by heat shock

Two general pathways of mRNA decay have been characterized in yeast. Both start with deadenylation. The major pathway then proceeds via cap hydrolysis and 5'-exonucleolytic degradation whereas the minor pathway consists of 3'-exonucleolytic decay followed by hydrolysis of the remaining cap structure. In higher eukaryotes, these pathways of mRNA decay are believed to be conserved but have not been well characterized. This study investigated the decay of the hsp70 mRNA in Drosophila Schneider cells. As shown by the use of reporter constructs, rapid deadenylation of this mRNA is directed by its 3'-untranslated region. The main deadenylase is the CCR4.NOT complex; the PAN nuclease makes a lesser contribution. Heat shock prevents deadenylation not only of the hsp70 but also of bulk mRNA. A completely deadenylated capped hsp70 mRNA decay intermediate accumulates transiently and is degraded via cap hydrolysis and 5'-decay. Thus, decapping is a slow step in the degradation pathway. Cap hydrolysis is also inhibited during heat shock. Degradation of reporter RNAs from the 3'-end became detectable only upon inhibition of 5'-decay and thus represents a minor decay pathway. Because two reporter RNAs and at least two endogenous mRNAs were degraded primarily from the 5'-end with cap hydrolysis as a slow step, this pathway appears to be of general importance for mRNA decay in Drosophila (Bönisch, 2007).

A characteristic feature of mRNA is its rapid turnover, permitting a continuous qualitative and quantitative adjustment of protein synthesis according to physiological needs. The pathways of eukaryotic mRNA degradation have been characterized mostly in Saccharomyces cerevisiae. The decay of all mRNAs examined in these cells starts with deadenylation, i.e. exonucleolytic shortening of the poly(A) tail. The second step of decay does not occur until the poly(A) tail has been shortened to about a dozen nucleotides. In the major pathway, this second step is hydrolysis of the 5'-cap. Cap hydrolysis results in free m⁷GDP and a 5'-monophosphate left on the mRNA and is catalyzed by the Dcp2p subunit of the Dcp1p·Dcp2p heterodimer. In vivo, the activity of the decapping enzyme is enhanced by several other proteins. The decapped RNA is degraded by the 5'-exonuclease Xrn1p. In a minor pathway, the deadenylated RNA is degraded from the 3'-end by the exosome before the remaining oligonucleotide is decapped by the enzyme Dcs1p, liberating m⁷GMP (Bönisch, 2007).

The deadenylation-dependent decapping pathway of mRNA decay is in a fundamental competition with translation. Two proteins required for cap hydrolysis in vivo, Pat1p and the RNA helicase Dhh1p, act by shifting the balance in favor of cap hydrolysis at the expense of translation. Because the poly(A) tail is involved in translation initiation via the poly(A)-binding protein, deadenylation can be seen as promoting decapping and decay by removing the mRNA from the translated pool. In fact, the inhibitor of decapping is the poly(A)-binding protein, not the poly(A) tail per se (Bönisch, 2007).

In animal cells, the decay of most mRNAs analyzed is also initiated by deadenylation. Subsequent decay of the deadenylated message is thought to proceed via the two pathways analyzed in yeast, mostly because the relevant proteins are conserved, but experimental data on the later steps of mRNA decay are relatively scarce. Based mainly on in vitro evidence, several groups have argued that the exosomal 3'-decay pathway may be the predominant mode of decay of unstable mammalian mRNAs. In vivo studies have supported a role of the 3'-5' pathway, but evidence for the existence of the 5'-3' pathway, sometimes acting on the same RNAs, has also been provided. Conservation of the 5'-3' decay pathway is also supported by the co-localization of the responsible proteins in distinct cytoplasmic structures, P bodies or GW bodies, in both yeast and mammalian cells. Furthermore, microRNA (miRNA)-induced mRNA destabilization in Drosophila Schneider cells requires Dcp1·Dcp2-dependent decapping. However, the relative contributions of the two decay pathways to overall mRNA decay remain unknown, and decay intermediates have been analyzed only to a limited extent (Bönisch, 2007).

Deadenylation is the rate-limiting step in the decay of most mRNAs. Several arguments support this view. First, deadenylation occurs by continuous shortening of the poly(A) tail throughout the lifetime of the RNA; the process can be easily followed in vivo even for unstable RNAs. However, once deadenylation has proceeded beyond a critical limit, the RNA usually disappears rapidly without detectable intermediates, showing that all subsequent steps are fast. Second, unstable RNAs are deadenylated faster than stable RNAs, and sequence manipulations affecting the overall rate of decay change the rate of deadenylation in a similar manner. Third, when a normally unstable RNA is stabilized because of a physiological stimulus, the rate of its deadenylation is reduced. Fourth, kinetic modeling suggests that changes in the rate of deadenylation have the greatest influence on the overall stability of the RNA. For some RNAs cap hydrolysis is a slow step, leading to the accumulation of a fully deadenylated, capped decay intermediate. Slow decapping is often associated with slow deadenylation (Bönisch, 2007).

The main enzyme responsible for deadenylation in yeast as well as in mammals and in Drosophila is the CCR4·NOT complex. A second universally conserved poly(A)-degrading 3'-exonuclease is the PAN2·PAN3 heterodimer, PAN for short. In yeast, this enzyme catalyzes the residual deadenylation observed upon inactivation of the CCR4·NOT complex. Under normal conditions, PAN is thought to act before the CCR4·NOT complex, catalyzing a rapid initial shortening of the poly(A) tail. A third poly(A)-degrading enzyme, the homodimeric PARN, is conserved in most eukaryotes, but not in yeast and in Drosophila. PARN has been implicated in the decay of several unstable RNAs, but its general role in mRNA turnover remains poorly defined (Bönisch, 2007). RNA elements promoting rapid deadenylation and decay are often located in the 3'-UTR, and the so-called AU-rich elements (AREs) are the best-studied class among them. Destabilizing sequences are bound by specific proteins, which can directly recruit deadenylases and other decay enzymes. Recently, miRNAs have also been found to be able to induce mRNA deadenylation (Bönisch, 2007).

The hsp70 mRNA of Drosophila is very unstable at normal growth temperatures with a functional half-life of 15-30 min suggested by indirect measurements. Rapid degradation of the hsp70 message requires its 3'-UTR. Upon heat shock, the RNA is stabilized at least 10-fold. This contributes to the rapid and massive (more than 1000-fold induction of HSP70 protein synthesis. During recovery from heat shock, the normal instability of the message is restored, and production of HSP70 protein ceases. The first step in hsp70 mRNA degradation during recovery is its deadenylation catalyzed by the CCR4·NOT complex. An unusual, transient but pronounced accumulation of a completely deadenylated RNA species is also observed both during heat shock and recovery. Two observations suggest that deadenylation is inhibited by heat shock: First, during heat shock, the fraction of polyadenylated hsp70 RNA increases with increasing temperature. Second, deadenylation is faster after a mild heat shock than after a severe heat shock, and deadenylation during recovery from a severe heat shock is faster if the cells have been preconditioned by a mild heat shock. However, the temperature dependence of deadenylation and its dependence on 3'-UTR sequences have not been tested directly (Bönisch, 2007).

This study has examined the decay pathway of the hsp70 mRNA in Drosophila Schneider cells. This RNA is found to be degraded via rapid deadenylation controlled by its 3'-UTR, followed by slow cap hydrolysis and 5'-decay. Exosome-dependent 3'-decay plays a minor role in the degradation of the hsp70 mRNA. Both deadenylation and decapping are inhibited during heat shock. The 5'-decay pathway is also important for the degradation of several other mRNAs examined in Schneider cells (Bönisch, 2007).

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).

Menin is a regulator of the stress response in Drosophila melanogaster

Menin, the product of the multiple endocrine neoplasia type I gene, has been implicated in several biological processes, including the control of gene expression and apoptosis, the modulation of mitogen-activated protein kinase pathways, and DNA damage sensing or repair. This study investigated the function of menin in Drosophila. Drosophila lines overexpressing menin or an RNA interference for this gene develop normally but are impaired in their response to several stresses, including heat shock, hypoxia, hyperosmolarity and oxidative stress. In the embryo subjected to heat shock, this impairment is characterized by a high degree of developmental arrest and lethality. The overexpression of menin enhances the expression of HSP70 in embryos and interfers with its down-regulation during recovery at the normal temperature. In contrast, the inhibition of menin with RNA interference reduced the induction of HSP70 and blocked the activation of HSP23 upon heat shock, Menin was recruited to the Hsp70 promoter upon heat shock and menin overexpression stimulated the activity of this promoter in embryos. A 70-kDa inducible form of menin was expressed in response to heat shock, indicating that menin is also regulated in conditions of stress. The induction of HSP70 and HSP23 was markedly reduced or absent in mutant embryos harboring a deletion of the menin gene. These embryos, which did not express the heat shock-inducible form of menin, were also hypersensitive to various conditions of stress. These results suggest a novel role for menin in the control of the stress response and in processes associated with the maintenance of protein integrity (Papaconstantinou, 2005).

The misexpression of menin impaired the response of Drosophila embryos, larvae, and flies to heat shock, hypoxia, hyperosmolarity, or oxidative stress. In embryos subjected to heat shock, this impairment resulted in a high degree of developmental arrest and lethality. The overexpression of menin enhanced the expression of HSP70 in response to heat shock and interfered with its repression during recovery at the normal temperature. In contrast, the induction of HSP70 and HSP23 was markedly reduced or absent when menin was down-regulated by RNAi or when embryos of two Mnn1 mutant lines were subjected to heat shock. These results indicate that menin is a positive regulator of heat shock protein expression in Drosophila melanogaster (Papaconstantinou, 2005).

Using Drosophila lines harboring genomic deletions of Hsp70 genes, it was asked whether a 50% reduction in Hsp70 gene copy number reduced the lethality of heat-shocked flies overexpressing menin, but no changes were observed in the rate of lethality. These results suggest that Hsp70 genes are not the main target of menin or that menin is a more global regulator of the stress response, controlling the expression of several heat shock response genes, including Hsp70. The observation that HSP23 expression is also altered in conditions of menin misexpression supports the latter possibility. It is also possible that the remaining copies of the Hsp70 genes were sufficient to provide levels of HSP70 above the threshold level for lethality caused by menin overexpression in conditions of stress. Therefore, whether or not the lethality observed in conditions of menin misexpression reflects predominantly the aberrant induction of HSP70 remains to be determined (Papaconstantinou, 2005).

Menin was recruited to the Hsp70 promoter upon heat shock. Since antibodies recognized both the 83- and 70-kDa forms of menin, the identity of the menin species recruited on the Hsp70 promoter is presently unknown. The results of immunoprecipitation assays indicated that menin does not interact directly with the heat shock factor. Therefore, menin may be part of a multiprotein complex recruited to the Hsp70 promoter in response to stress. Other proteins, including DAXX, Spt5, Spt6, and elongin A, cooperate with heat shock factor in the regulation of heat shock proteins and, like menin, may play an important role in the survival of the organism facing adverse conditions. Since mammalian HSP70 is also part of a negative feedback loop controlling its own expression, it was asked whether menin interacts with HSP70 in Drosophila embryos, but no interactions were observed using immunoprecipitation and Western blotting analyses (Papaconstantinou, 2005).

A 70-kDa form of menin accumulated in response to heat shock, indicating that menin expression is itself regulated in conditions of stress. In contrast, the 83-kDa form of menin did not accumulate in control embryos subjected to heat shock (parental strains nos-GAL4 and Hsp70-GAL4) or in UAS-Mnn1 strains harboring the nos-GAL4 driver. Since the Mnn1 RNAi blocked the expression of the 70-kDa menin species and Mnn1 mutant embryos did not express this protein, full induction of HSP70, HSP23, and possibly other heat shock proteins may depend on this heat shock-inducible form of menin. Testing of this hypothesis will depend on the molecular characterization of the 70-kDa menin and awaits the development of reagents and Drosophila strains specific for single menin protein species (Papaconstantinou, 2005).

Two alternative forms of menin, differing at the C terminus and encoding proteins 763 and 530 amino acids in length, are predicted by the sequence of existing cDNA clones for Mnn1. Since the 70-kDa menin is recognized by antibodies generated against the central domain of the protein or the predicted C terminus of the 83-kDa form of menin (763 amino acids in length), it is not the 530-amino-acid menin protein encoded by the Mnn1-RB transcript. The expression of the 70-kDa menin is presently under investigation (Papaconstantinou, 2005).

This study did not investigate the expression or mechanism of action of menin in the response to hypoxia, hyperosmolarity, or oxidative stress. Since the expression of heat shock proteins is induced in response to several conditions of stress, it is probable that menin exerts a similar effect on the expression of HSP70 and HSP23 in response to these stresses. A recent study concluded that Drosophila larvae and embryos with a mutation of the menin gene are characterized by genome instability and are more susceptible to a variety of chemical mutagens (Busygina, 2004). Interestingly, the effect of several of these mutagens was only observed at 29°C. Whether or not these observations reflect the impairment of the stress response caused by the absence of menin remains to be investigated (Papaconstantinou, 2005).

The survival of organisms subjected to heat shock depends on the induction of stress-responsive genes. Interfering with the expression or activity of heat shock proteins results in increased lethality in organisms subjected to high temperatures. However, precise regulation of these genes is also required for survival since forced expression of heat shock proteins, such as HSP70, is toxic. This demonstrates that, in Drosophila, proper expression of heat shock proteins depends on menin gene function and that menin is itself regulated by heat shock. Consistent with these findings are recent reports describing a role for the BRCA1 tumor suppressor in the expression of HSP27 in mammals. In addition, BRCA1 was processed and inactivated at high temperatures, indicating that it is also regulated in response to heat stress. Similarly, Tid1, the mammalian homolog of the Drosophila tumor suppressor lethal (2) tumorous imaginal disks, is a cochaperone of HSP70 and therefore a candidate regulator of the stress response (Papaconstantinou, 2005).

The current experiments did not address whether this novel function of menin is relevant for its role as a tumor suppressor in humans. However, they are consistent with the idea that processes involved in the maintenance of protein integrity may also be important for ensuring the integrity of the genome. These processes may depend on a number of common factors, such as menin, BRCA1, and possibly Tid1 (Papaconstantinou, 2005).

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).

Modulation of Drosophila heat shock transcription factor activity by the molecular chaperone DROJ1

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).

Search PubMed for articles about Drosophila Hsp70

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

Adelman, K., et al. (2006). Drosophila Paf1 modulates chromatin structure at actively transcribed genes. Mol. Cell. Biol. 26: 250-260. PubMed ID: 16354696

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date revised: 10 July 2014

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