InteractiveFly: GeneBrief

tango : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The cloning of tango also known as Drosophila Arnt, a homolog of the vertebrate Ah receptor nuclear translocator (Arnt) was reported simultaneously by three different laboratories (Zelzer, 1997, Ohshiro, 1997 and Sonnenfeld, 1997). Tango heterodimerizes with two transcription factors, Trachealess (Trh) and Single minded (Sim), to regulate transcription in the trachea and central midline, respectively. Tango, like Trh and Sim, is a bHLH-PAS transcription factor; all are able to form heterodimers via interaction through their PAS domains (Zelzer, 1997).

Using several criteria, biochemical studies provide evidence that Tango associates with Sim and Trh. (1)Tgo protein is expressed in all cells in the embryo and thus overlaps in expression with Sim in the CNS midline cells and Trh in tracheal cells. (2) Studies using the yeast two-hybrid assay indicate that Tango can form dimers with Sim, Trh and Sima (a Drosophila gene related to mammalian hypoxia-indicible factor 1alpha [Nambu, 1996]). (3) Co-immunoprecipitation experiments with caculoviral-expressing proteins indicate that Tango forms dimers with Sim and Trh. (4) Expression studies in cultured cells suggest two pairwise interactions: Sim-Tgo and Trh-Tgo, with either of the two pair capable of interaction in order to bind DNA and activate transcription. (5) Mutations in tango result in CNS midline and tracheal defects, defects similar to those observed with mutations in sim and trh, respectively. (6) Gene dosage experiments suggest that the two pairs (sim-tgo and trh-tgo) interact to control CNS midline and tracheal cell transformation and development, respectively (Sonnenfeld, 1997).

The development of Drosophila trachea is under the control of spatially and/or quantitatively regulated activity involving the FGF receptor known as Breathless, which is also essential for midline glial migration. Examination of the proximal promoter region of the breathless promoter reveals three conserved elements (central midline elements or CMEs) resembling previously identified putative binding sites for Sim/Arnt heterodimers (Swanson, 1995) within a 150 base pair region, from -606 to -447 bases, relative to the P2 transcriptional initiation site. These three sites account for breathless expression in midline precursor cells (Ohshiro, 1997).

breathless expression in developing trachea is regulated by direct interactions between Trachealess/Tango heterodimers and three identical central midline elements (TACGTGs) situated in the minimum enhancer region. To test whether the heterodimer of Tango and Trh is capable of binding to CMEs, Trh and Tango fusion proteins were subjected to electrophoretic mobility shift assays of a CME1-containing oligonucleotide. In the absence of either Tango or Trh or both, there is little or nor protein-DNA interaction. In contrast, a retardation band can be seen when the oligonucleotide is incubated with a reaction mixture containing both Tango and Trh, indicating that Trh and Tango are capable of forming a heterodimer which exhibits DNA-binding activity (Ohshiro, 1997).

Beside regulation of central midline and tracheal development, there are likely to be additional roles for Tango. Tango interacts biochemically with the Drosophila Sima protein. The function of Sima is currently unknown, but its ubiuquitous expression pattern and primary sequence suggests it may be related functionally to mammalian HIF-1alpha; it may control the Drosophila response to hypoxia. Drosophila cell culture experiments have revealed the existence of a CME-binding factor that is induced under hypoxic conditions (Nagao, 1996). Therefore, Tango could be involved in controlling the hypoxia response.

The Drosophila spineless (ss) gene encodes a basic-helix-loop-helix-PAS transcription factor that is required for proper specification of distal antennal identity, establishment of the tarsal regions of the legs, and normal bristle growth. ss is the closest known homolog of the mammalian aryl hydrocarbon receptor (Ahr), also known as the dioxin receptor. Dioxin and other aryl hydrocarbons bind to the PAS domain of Ahr, causing Ahr to translocate to the nucleus, where it dimerizes with another bHLH-PAS protein, the aryl hydrocarbon receptor nuclear translocator (Arnt). Ahr:Arnt heterodimers then activate transcription of target genes that encode enzymes involved in metabolizing aryl hydrocarbons. Ss functions as a heterodimer with the Drosophila ortholog of Arnt, Tango. The ss and tgo genes have a close functional relationship: loss-of-function alleles of tgo were recovered as dominant enhancers of a ss mutation, and tgo-mutant somatic clones show antennal, leg, and bristle defects almost identical to those caused by ss minus mutations. The results of yeast two-hybrid assays indicate that the Ss and Tgo proteins interact directly, presumably by forming heterodimers. Coexpression of Ss and Tgo in Drosophila SL2 cells causes transcriptional activation of reporters containing mammalian Ahr:Arnt response elements, indicating that Ss:Tgo heterodimers are very similar to Ahr:Arnt heterodimers in DNA-binding specificity and transcriptional activation ability. During embryogenesis, Tgo is localized to the nucleus at sites of ss expression. This localization is lost in a ss null mutant, suggesting that Tgo requires heterodimerization for translocation to the nucleus (Emmons, 1999).

Ectopic expression of ss causes coincident ectopic nuclear localization of Tgo, independent of cell type or developmental stage. In the embryo, ss is expressed in the antennal segment, the gnathal segments, the leg anlage, and the peripheral nervous system. Strong nuclear accumulation of Tgo is seen in the antennal segment, which expresses the highest level of ss. Nuclear accumulation of Tgo is also observed in the gnathal segments (mandibular, maxillary, and labial), but the intensity of staining is relatively weak compared to the antennal segment. This correlates with the relatively weak expression of ss in the gnathal segments, when compared to the antennal segment. Nuclear localization of Tgo in the antennal and gnathal segments is dependent on ss, since it is not seen in a ss null mutant. The expression of ss in the appendage primordia and the peripheral nervous system also correlates with Tgo nuclear accumulation. Sensory cells that express ss are in close proximity to the tracheal cells that express trh. To distinguish these, embryos were labeled with anti-Trh and anti-Tgo. Non-tracheal cells that show nuclear Tgo are observed in the location of ss-expressing sensory cells. This non-tracheal Tgo nuclear accumulation is absent in ss mutant embryos. These results indicate that Tgo accumulates in the nuclei of ss-expressing antennal, gnathal and sensory cells, consistent with the formation and nuclear accumulation of Ss:Tgo heterodimers in vivo. Surprisingly, no significant Tgo nuclear accumulation is seen in the limb primordia, even though ss is expressed in these cells. This may reflect regulatory events idiosyncratic to the limb primordia, or a lack of sensitivity of the immunostaining, since the limb primordia express ss at considerably lower levels than the antennal segment. Tgo nuclear accumulation is also observed in the cells of the dorsal vessel. Since, sim, ss and trh are not expressed in the dorsal vessel, an additional bHLH-PAS protein may function in combination with Tgo in controlling the development or physiology of these cells, which comprise the Drosophila circulatory system. When ectopic expression of a UAS-ss transgene is driven by en-Gal4, Tgo is found to accumulate in nuclei in circumferential ectodermal en stripes. Similarly, expression of ss in mesodermal cells (driven by twi-GAL4) causes nuclear accumulation of Tgo in the mesoderm. These experiments support the conclusion that Ss and Tgo interact in vivo, and suggest that their interaction and nuclear accumulation does not depend on additional, spatially-restricted, factors. Despite the very different biological roles of Ahr and Arnt in insects and mammals, the molecular mechanisms by which these proteins function appear to be largely conserved (Emmons, 1999).

How did Ss and Ahr come to have such different functions in vertebrates and arthropods? One possibility is that Ahr functioned as some type of chemosensory protein in an ancestral organism. In vertebrates, this function became utilized by all cells to sense aryl hydrocarbon toxins, whereas in arthropods it became intimately associated with the specification of a major chemosensory organ, the antenna. It is hoped that studies of organisms from other lineages will shed light on how Ss and Ahr came to adopt such different roles (Emmons, 1999).

A comparison of midline and tracheal gene regulation during Drosophila development

Within the Drosophila embryo, two related bHLH-PAS proteins, Single-minded and Trachealess, control development of the central nervous system midline and the trachea, respectively. These two proteins are bHLH-PAS transcription factors and independently form heterodimers with another bHLH-PAS protein, Tango. During early embryogenesis, expression of Single-minded is restricted to the midline and Trachealess to the trachea and salivary glands, whereas Tango is ubiquitously expressed. Both Single-minded/Tango and Trachealess/Tango heterodimers bind to the same DNA sequence, called the CNS midline element (CME) within cis-regulatory sequences of downstream target genes. While Single-minded/Tango and Trachealess/Tango activate some of the same genes in their respective tissues during embryogenesis, they also activate a number of different genes restricted to only certain tissues. The goal of this research is to understand how these two related heterodimers bind different enhancers to activate different genes, thereby regulating the development of functionally diverse tissues. Existing data indicates that Single-minded and Trachealess may bind to different co-factors restricted to various tissues, causing them to interact with the CME only within certain sequence contexts. This would lead to the activation of different target genes in different cell types. To understand how the context surrounding the CME is recognized by different bHLH-PAS heterodimers and their co-factors, novel enhancers were identified and analyzed that drive midline and/or tracheal expression, and they were compared to previously characterized enhancers. In addition, expression was tested of synthetic reporter genes containing the CME flanked by different sequences. Taken together, these experiments identify elements overrepresented within midline and tracheal enhancers and suggest that sequences immediately surrounding a CME help dictate whether a gene is expressed in the midline or trachea (Long, 2014).

Several families of transcription factors contain members that bind related, but slightly different DNA recognition sequences. Examples include members of the nuclear receptor family and bHLH proteins. Nuclear receptor homodimers and heterodimers bind DNA response elements consisting of two inverted repeats separated by a trinucleotide spacer. Specificity is determined by interactions between protein loops on the second zinc finger of a particular steroid receptor DNA binding domain and the trinucleotide spacer within the DNA recognition site. Similarly, the recognition sequence of bHLH transcription factors is called the E box and consists of the sequence CANNTG. Specific bHLH heterodimers preferentially bind E boxes containing various internal dinucleotides (represented by the NN within the E box). The bHLH-PAS proteins investigated in this study are a subfamily within the bHLH superfamily of transcription factors. The PAS domain helps stabilize protein-protein interactions with other PAS proteins, as well as with additional co-factors, some of which mediate interactions with the environment. The evolutionary relationship of bHLH and bHLH-PAS proteins is also reflected in the similarity of their DNA recognition sequences. The CME is related to the E box and historically has been considered to consist of a five rather than six base pair consensus. Previous results indicated bHLH-PAS heterodimers strongly prefer the internal two nucleotides of the binding site to be 'CG', while the nucleotide immediately 5' to this core helps to discriminate which heterodimer binds the site. The first crystal structure of a bHLH-PAS heterodimer bound to DNA reveals that the recognition sequence of the human Clock/Bmal bHLH-PAS heterodimer actually consists of seven base pairs, rather than five. This is consistent with results reported in this study that suggest Sim/Tgo and Trh/Tgo heterodimers preferentially bind highly related, but slightly different seven base pair sequences. In addition, experiments with fly Sim and human Tgo, called Aryl hydrocarbon receptor nuclear translocator protein (Arnt), using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach, identify the sequence DDRCGTG (D = A, C or T and R = either purine) as the Sim/Tgo binding site. The current results agree with this, although the consensus sequence this study identified by examining known enhancers, is shifted by one nucleotide (DACGTG). In the midline and tracheal enhancers, sixty-six copies of the CME, ACGTG, were found and forty-eight copies of the related sequence, GCGTG, also identified in the SELEX experiments. Half of these GCGTG sites fit the seven bp consensus TGCGTGR and future experiments are needed to determine their importance within the various enhancers. The results indicated that the CME context favored within midline and tracheal enhancers as well as enhancers active in both tissues, was very similar, yet clearly distinct from binding sites of other bHLH and bHLH-PAS heterodimers. Based on the expression pattern of certain reporter genes examined in this study, the same CME may be bound by Sim/Tgo in the midline and Trh/Tgo in the trachea within certain enhancers. Within other contexts, the CME appears to be discriminated by these different heterodimers, because some enhancers drive expression in only one tissue or the other (Long, 2014).

Results from both endogenous enhancers and the synthetic reporter genes confirm the importance of the proximal sequences in limiting expression to either the midline or trachea. While the proximal context of the CME plays a role, additional sequences clearly combine with the CME to ultimately determine if an enhancer is functional in the midline or trachea. Taken together, these results indicate that proximal motifs combine with additional sequences not only to determine whether or not a gene is expressed in the midline or trachea, but also to determine which cellular subtypes express the gene and when it is activated within a tissue. Future experiments will reveal if 1) changing the sequence, AACGTGC, to TACGTGC within a midline enhancer will cause the enhancer to drive expression in trachea as well and 2) if changing the sequence, TACGTGC, to AACGTGC within an enhancer that drives expression in both the midline and trachea, will restrict expression to only the trachea. Sequences proximal to the CME likely affect the affinity of either Sim/Tgo and/or Trh/Tgo heterodimers to the DNA, but binding sites for additional factors that interact cooperatively to stabilize an entire transcription complex are needed for high levels of expression within a particular cell. Moreover, recent experiments indicate that enhancers containing multiple CMEs are activated earlier in the embryonic midline than enhancers containing only one CME. It has been suggested that Sim/Tgo binding sites may be sufficient for activation in the early embryo, but that binding sites for additional transcription factors must combine with the CME to drive expression within the later, more complex embryo (Long, 2014).

Damage-responsive neuro-glial clusters coordinate the recruitment of dormant neural stem cells in Drosophila

Recruitment of stem cells is crucial for tissue repair. Although stem cell niches can provide important signals, little is known about mechanisms that coordinate the engagement of disseminated stem cells across an injured tissue. In Drosophila, adult brain lesions trigger local recruitment of scattered dormant neural stem cells suggesting a mechanism for creating a transient stem cell activation zone. This study found that injury triggers a coordinated response in neuro-glial clusters that promotes the spread of a neuron-derived stem cell factor via glial secretion of the lipocalin-like transporter Swim. Strikingly, swim is induced in a Hif1-α-dependent manner in response to brain hypoxia. Mammalian Swim (Lcn7) is also upregulated in glia of the mouse hippocampus upon brain injury. These results identify a central role of neuro-glial clusters in promoting neural stem cell activation at a distance, suggesting a conserved function of the HIF1-α/Swim/Wnt module in connecting injury-sensing and regenerative outcomes (Simoes, 2022).

Injury is known to stimulate diverse forms of plasticity, which serve to restore organ function. Many tissues harbor a small number of undifferentiated adult stem cells that are engaged in tissue turnover or become activated following injury to replace damaged cells. Some tissues, such as muscle or brain, contain mainly dormant stem cells that are not dividing and reside in a reversible state of quiescence. Niche cells in intimate contact with quiescent stem cells have been found to provide activating cues upon tissue damage. However, little is known how the activation of multiple dispersed stem cell units is coordinated to establish an adequate stem cell response zone across an injured tissue (Simoes, 2022).

Quiescent progenitor cells in muscle and the brain respond to injury in mammals, but also in fruit flies (Drosophila). This allows to harness the extensive genetic tools available in Drosophila to dissect injury-dependent stem cell activation. Although still unclear, the presence of dormant stem cells in short-lived insects indicates that these cells may play a beneficial role for tissue plasticity or repair upon predator attacks or inter-species aggressions (Simoes, 2022).

In the adult fly brain, experimental stab lesions to the optic lobes (OLs) or the central brain trigger a proliferative response resulting in local neurogenesis several days after injury (AI), which has been linked to activation of normally quiescent neural progenitor cells (qNPs). qNPs have also been found to promote adult brain plasticity in contexts unrelated to injury. On the other hand, stab lesions can also trigger glial divisions shortly after injury (Simoes, 2022).

Despite extensive knowledge on neural stem cell proliferation during fly development, the signals governing qNP activation in response to injury are unknown. A ubiquitous pulse of Drosophila Myc (dMyc) overexpression has been previously shown to promote qNP division, but the signals detected by qNPs remained enigmatic (Simoes, 2022).

In mammals, a wide variety of signals are known to regulate quiescent neural stem cells (qNSCs) in homeostatic conditions, whereas their response to tissue damage is less well understood. qNSCs are located in two main niches, the subventricular zone and the dentate gyrus of the hippocampus, buried within the brain. Upon brain injury, qNSCs only partially enter an activated state, and neuroblast recruitment to infarcted brain regions and local neurogenesis is limited (Simoes, 2022).

Strikingly, the initial consequences triggered by brain injury, which include neural cell death, upregulation of antioxidant defense, and c-Jun N-terminal kinase (JNK) stress signaling, are very conserved in flies and mice suggesting that injury sensing of qNSCs/qNPs may rely on common principles. In this work, injury-induced changes were studied in the adult fly brain leading to recruitment of isolated qNPs near the injury site. A crucial role was identified of damage-responsive neuro-glial clusters (DNGCs), which enable proliferation of distant qNPs by promoting an enlarged stem cell activation zone. Evidence is provided that these multicellular units orchestrate the spatial and temporal availability of an essential, but localized stem cell factor for qNPs via injury-stimulated secretion of the transport protein Swim. As Swim production is dependent on the injury-sensitive transcription factor HIF1-α, the identified mechanism may serve to spatially and temporary adjust the stem cell activation zone to the extent of damage suffered in a given tissue area, resulting in locally calibrated pulses of stem cell activity (Simoes, 2022).

How tissue damage is sensed and how the recruitment of multiple stem cell units is coordinated in response to local, heterogeneous tissue damage represents a fundamental question. By investigating how dispersed qNPs are locally recruited to injury, we have identified a mechanism that creates a defined zone of stem cell activation in the adult fly brain. The process is dependent on DNGCs, which depending on their size and possibly composition may regulate the extent by which a localized stem cell factor such as Wg/Wnt can travel to rare qNPs in the vicinity. Whereas the neuronal cells provide Wg/Wnt, the glial component supplies the carrier protein Swim, thereby promoting the dispersion of the signal. This cooperative interaction of two different cell types to gain long range function of Wg/Wnt is rather unique (Simoes, 2022).

At the cellular level, a model is proposed whereby injury-sensitive HIF1-α directs Swim synthesis in glial cells. Swim transporters diffuse and facilitate the spread of localized neural-derived Wg ligands, probably by binding to and shielding the lipid-residues of Wg/Wnt in the aqueous extracellular space. Mobile Wg-Swim complexes are consequently able to reach and activate qNPs in the injured brain domain. Wg/Wnt signal transduction and downstream upregulation of dmyc is shown to be crucial for the proliferation of this novel cell type. Overall, it is proposed that the described mechanism provides a means to match recruited stem cell activity to the spatial and temporal persistence of damage in the injured brain. Activation of dormant neural progenitors by high levels of Wg/Wnt Wg/Wnt signaling is probably one of the most universal pathways driving stem cell proliferation. Nevertheless, an understanding of Wg/Wnt signals for dormant stem cells has only recently emerged. Dormant muscle stem cells, for example, maintain quiescence by raising their threshold for Wnt transduction via cytoplasmic sequestration of &betal-catenin, and qNSC in the hippocampus do not rely on Wnt signaling under homeostasis but display a high capability to respond to Wnt in a graded manner when exposed. Similarly, the results demonstrate that qNPs start proliferating when high Wg/Wnt levels are provided in an autocrine fashion (Simoes, 2022).

Overall, the results suggest that activation of qNPs in the adult fly brain is mainly prevented by the low availability of Wg/Wnt ligands under homeostatic conditions. Although Wnt signaling normally occurs between adjacent cells, this study provides evidence that Wg functions at a tissue scale in the injured fly brain (Simoes, 2022).

This study describes the property of Swim to extend the signaling range of Wg/Wnt. Further research will be required to determine whether other stem cell-relevant factors can be transported by Swim. In zebrafish, reduced levels of Swim/Lcn7 produce craniofacial defects due to compromised Wnt3 signaling, highlighting a different context of Wnt/Swim interaction. A Wg/Swim interaction has previously been proposed in developing epithelia in flies, although the effect was not observed in a more recent study (Simoes, 2022).

Swim::mCherry is strongly expressed in the adult ovary germline of flies, in agreement with data from the recently published Fly Cell Atlas. Remarkably, swim KO flies showed reduced fertility, a phenotype which has also been reported for lcn7/tinagl1 KO mice (Takahashi, 2016). Interestingly, Swim expression in the germarium strongly overlapped with Wg::GFP, in line with previous findings describing a requirement of extensive Wg travel from the niche to distant follicular stem cells (Simoes, 2022).

Finally, this study elucidated how the Swim/Wg stem cell-activating signal is connected to damage sensing in the injured brain. Both in flies and mice, swim/lcn7 induction occurs in glial cells in response to brain injury. Remarkably, stroke-induced lcn7 induction is not observed in mouse brains, in which Hif1-α has been deleted from mature neurons and glial cells. This suggests that HIF1-α-dependent swim regulation is conserved in mammals (Simoes, 2022).

According to the current model, the damage responsiveness of stem cells is strongly gated by the availability of stable HIF1-α during acute hypoxia. Such a limited activation pulse would effectively restrict the mitotic effect of Swim/Wg complexes to the acute phase of repair, acting as a safeguard mechanism against overgrowth. Moreover, the hypoxia-dependent secretion of Swim would allow to temporally and locally fine-tune the realm of the stem cell activation zone to injury. Local oxygen concentrations modulate the activity of adult stem cells in different niches. In the fly larval OL, Dpn-expressing neural progenitors proliferate in a pronounced hypoxic environment, which bears parallels to the situation following brain injury (Simoes, 2022).

In the mammalian brain, injury-induced Wnt ligands may not efficiently reach qNSCs in distant neurogenic niches, resulting in poor stem cell activation. As such, Wnt pathway stimulating approaches hold promise as possible treatment for brain injury as they are known to support regeneration at several levels including qNSC activation, neurogenesis, and axon outgrowth. Increasing the mobility or stability of Wg/Wnts by Swim-like transporters may therefore represent a successful strategy to engage endogenous progenitors into regeneration. Given the fact that Wg/Wnts can support tissue renewal and regeneration in numerous tissues, the properties of Swim to transform a restricted tissue area into a temporary stem cell-activating zone, uncovered in this study may have important applications in regenerative medicine (Simoes, 2022).

Although the current experiments have revealed an impaired distribution of Wg in the injured brain in the absence of Swim transporters, it cannot be completely rule out that Swim may alter Wg function by other means than physical binding and direct transport. Ideally, the injury-induced formation of Wg-Swim complexes should be observable in the extracellular space. Although colocalization of Swim and Wg signals was detected, it was not possible to image Wg-Swim complexes at high resolution due to elevated background of Wg and mCherry antibodies when performing extracellular stainings. Overcoming these current limitations with overexpression systems or optimized immunodetection should allow to capture the dynamics of Wg-Swim interactions in injured brain tissue in the future (Simoes, 2022).

REGULATION

Targets of Activity

The development of the Drosophila trachea is under the control of spatially and/or quantitatively regulated activity involving the FGF receptor known as Breathless, which is also essential for midline glial migration. This study has identified the minimum enhancer region of breathless. btl is expressed in trachea, midline precursors (MLPs), midgut precursors and salivary duct glands. Three central midline elements (CME), consisting of binding sites for Single minded/Arnt (Ah receptor nuclear translocator) heterodimers, are identified within a 150 base pair region, from -606 to -447 bases, relative to the P2 transcriptional initiation site. These three sites account for breathless expression in MLPs (Ohshiro, 1997).

breathless expression in developing trachea is regulated by direct interaction between Trachealess/Tango heterodimers and three identical central midline elements (TACGTGs) situated in the minimum enhancer region. These results also show that Single-minded/Tango heterodimers, which are essential for breathless expression in midline precursor cells, share DNA targets in common with Trachealess/Tango, indicating that two different basic helix-loop-helix-PAS protein complexes act through the same target sites in vivo. It is also thought that additional nucleotide sequences flanking CMEs may serve additional cis-regulatory elements for tracheal expression. Late breathless expression might be considered to be under the control of the ligand Branchless, which activates genes expressed at late stages, including pointed and blistered/pruned/DSRF (Ohshiro, 1997).

When Sim and Tango are cotransfected into cultured cells with a LacZ reporter carrying six central midline elements (CME), LacZ transcription is induced to high levels. A transgene carrying four CME is expressed in developing and mature trachea, posterior spiracles, and salivary ducts. This expression pattern resembles the combined sim and trh expression patterns, and suggests that the CME is an in vivo target element of Sim, Trh and Tgo. All CNS midline LacZ expression from a four CME transgene is abolished in sim mutants. Similarly, all tracheal LacZ expression is abolished in trh mutant backgounds, while CNS midline expression is unaffected. In tango mutant embryos, both CNS midline and tracheal expression is reduced, although not as severely as that observed in sim and trh mutant embryos, perhaps due to residual maternal tango expression in tango mutants (Sonnenfield, 1997).

Spatially and temporally regulated activity of Branchless/Breathless signaling is essential for trachea development in Drosophila. Early ubiquitous breathless (btl) expression is controlled by binding of Trachealess/Tango heterodimers to the btl minimum enhancer. Branchless/Breathless signaling includes a Sprouty-dependent negative feedback loop. Late btl expression is a target of Branchless/Breathless signaling and hence, Branchless/Breathless signaling contains a positive feedback loop, which may guarantee a continuous supply of fresh receptors to membranes of growing tracheal branch cells. Branchless/Breathless signaling activates MAP-kinase, which in turn, activates late btl expression and destabilizes Anterior-open (Yan), a repressor for late btl expression. Biochemical and genetic analysis has indicated that the minimum btl enhancer includes binding sites of Anterior-open (Ohshiro, 2002).

The minimum btl enhancer consists of B2 and B3 regions, the latter, a late enhancer. lacZ expression driven by B3 enhancer mimics btl late expression. The B3 enhancer possesses two of three CMEs sites for binding of Trh/Tgo complexes. The disruption of three CMEs in the btl enhancer brings about the complete loss of btl expression in tracheal cells at later stages. Thus, Trh/Tgo may also be required for late btl expression. A POU-Homeobox containing protein, Ventral veinless (Vvl)/Drifter is required for maintenance of btl expression in developing trachea. Pnt, Trh/Tgo, and Ventral veinless/Drifter thus quite likely synergistically activate btl expression in DB, VB, and LTa/p whereas Aop and/or Sal activity represses btl to prevent its expression in TC and/or DT (Ohshiro, 2002).

Many organisms respond to toxic compounds in their environment by inducing regulatory networks controlling the expression and activity of cytochrome P450 monooxygenase (P450s) detoxificative enzymes. In particular, black swallowtail (Papilio polyxenes) caterpillars respond to xanthotoxin, a toxic phytochemical in their hostplants, by activating transcription of the CYP6B1 promoter via several regions located within 150 nt of the transcription initiation site. One such element is the xenobiotic response element to xanthotoxin (XRE-Xan) that lies upstream of consensus XRE-AhR (xenobiotic response element to the aryl hydrocarbon receptor) and OCT-1 (octamer-1 binding site) element known to be utilized in mammalian aryl hydrocarbon response cascades. Two-plasmid transfections conducted in Sf9 cells have indicated that XRE-Xan, XRE-AhR and a number of other proximal elements, but not OCT-1, are critical for basal as well as xanthotoxin- and benzo[alpha]pyrene-induced transcription of the CYP6B1 promoter. Four-plasmid transfections with vectors co-expressing the Spineless (Ss) and Tango (Tgo) proteins, the Drosophila melanogaster homologues of mammalian AhR and ARNT, have indicated that these proteins enhance basal expression of the CYP6B1 promoter but not the magnitude of its xanthotoxin and benzo[alpha]pyrene induction. Based on these results, it is proposed that these Drosophila transcription factors modulate basal expression of this promoter in a ligand-independent manner and attenuate its subsequent responses to planar aryl hydrocarbons (benzo[alpha]pyrene) and allelochemicals (xanthotoxin) (Brown, 2005).

Common motifs shared by conserved enhancers of Drosophila midline glial genes

Coding sequences are usually the most highly conserved sectors of DNA, but genomic regions controlling the expression pattern of certain genes can also be conserved across diverse species. In this study, five enhancers were identified capable of activating transcription in the midline glia of Drosophila melanogaster and each contains sequences conserved across at least 11 Drosophila species. In addition, the conserved sequences contain reiterated motifs for binding sites of the known midline transcriptional activators, Single-minded, Tango, Dichaete, and Pointed. To understand the molecular basis for the highly conserved genomic subregions within enhancers of the midline genes, the ability of various motifs to affect midline expression, both individually and in combination, were tested within synthetic reporter constructs. Multiple copies of the binding site for the midline regulators Single-minded and Tango can drive expression in midline cells; however, small changes to the sequences flanking this transcription factor binding site can inactivate expression in midline cells and activate expression in tracheal cells instead. For the midline genes described in this study, the highly conserved sequences appear to juxtapose positive and negative regulatory factors in a configuration that activates genes specifically in the midline glia, while maintaining them inactive in other tissues, including midline neurons and tracheal cells (Fulkerson, 2010).

The results described in this study indicate that the four genes expressed in the midline glia contain enhancers with subregions conserved in 11 or 12 of the sequenced Drosophila genomes. These conserved subregions contain one or more of the four motifs previously identified in the wrapper regulatory region, are highly A/T rich, and needed for robust expression in the midline. These results confirm the importance of several transcription factor-binding sites for midline glial activation. One of these sites, the CME, binds both Sim/Tgo and Trh/Tgo heterodimers and, when multimerized, can drive reporter gene expression in both midline and tracheal cells. Two lines of evidence indicate that the context of the CME determines whether or not it can be utilized to drive expression in these two tissues. (1) The sequences flanking the CMEs are highly conserved in the four genes discussed in this study, Glec, oatp26f, liprinγ and wrapper, suggesting that the location and sequence of other transcription factor-binding sites are constrained. (2) Changing the sequences flanking the CME in the synthetic multimers can eliminate expression in the midline, trachea, or both tissues (Fulkerson, 2010).

A multimerized CME in the context of the 4Toll:GFP reporter was expressed in both the midline and trachea and quite sensitive to slight modifications in flanking sequences. Changing 5-7 nucleotides on either side of the CME within this multimerized construct either substantially elevated expression in the trachea and eliminated it in the midline (T rich:GFP) or eliminated expression in both tissues (Sox:GFP). Additional combinations between the CME and one of the other midline glial motifs restricted expression to the midline (Pnt:GFP) or the trachea (POU:GFP). These results indicate that testing binding sites for two different factors next to one another can disrupt the endogenous ordering and spacing of the sites within the enhancers. Significantly, the Toll:GFP and Pnt:GFP reporters, unlike the intact enhancers described in this study, drive GFP expression in both midline neurons and glia. This midline expression pattern suggests that the synthetic multimers may lack repressor-binding sites that restrict expression to midline glia. Taken together, these results demonstrate the sensitivity of CME function to flanking sequences within the midline enhancers (Fulkerson, 2010).

Existing experimental evidence suggests that unlike most transcription factors, Sim/Tgo heterodimers (as well as Trh/Tgo heterodimers) preferentially binds one sequence over all others: ACGTG, the CME. Within the enhancers described in this study, sites flanking the CME have remained unchanged over evolutionary time due, in part, to similarities between binding sites for Sim and Trh and the molecular consequences of changing nucleotides adjacent to the CME. This conservation may ensure transcription is restricted to the midline glia and repressed in tracheal cells. In addition to the midline enhancers reported in this study, regions conserved among Drosophila species were found within the known midline enhancers. For instance, a 1.0-kb enhancer present in the first intron of slit drives expression in the midline glia and it contains a single CME and a 32-bp sequence conserved in 11 Drosophila species. It is important to note that the number of midline enhancers described in this study is limited and not all the midline glial enhancers are likely to exhibit such a high degree of conservation. For instance, a midline enhancer of the ectoderm3 gene, was identified that exhibits much less conservation among Drosophila species and presently, the basis for the observed variation among enhancers is unknown (Fulkerson, 2010).

The Ets transcriptional activator, pnt, a downstream effector of EGFR signaling, and Drifter, a POU domain protein, are expressed in both embryonic midline glia and tracheal cells. Previous studies have shown that deleting a POU domain-binding site within an enhancer of rhomboid eliminated expression in tracheal cells, but did not affect its midline glial expression. The results described in this study confirm and extend these results and suggest that the location of a POU domain-binding site relative to the CME can play a role in determining if a gene is expressed in the midline glia, the trachea or both. Moreover, swapping the PAS domains between Sim and Trh proteins indicated that additional, midline or tracheal specific cofactors bind to the PAS domains of the individual proteins and likely to determine which genes are expressed in the two different cell types. This may be the reason sequences adjacent to the CME play such a critical and sensitive role in determining which tissues express the various reporter genes described here. To activate the midline genes, Sim may interact with Drifter and Pnt and bind to sequences flanked by different binding sites compared with sequences bound by Trh, Drifter, and Pnt needed to activate tracheal genes. The simplicity of the multimers studied in this paper raise the possibility that different PAS heterodimers may specifically interact with other factors, such as Drifter and Pnt, in a manner that depends on the relative location and/or distance between each binding site, as has been described for nuclear hormone receptor complexes (Fulkerson, 2010).

The results confirm those of Swanson (2010), who found binding sites can be juxtaposed in different ways within enhancers to favor particular short-range interactions, and, in this way, various combinations of transcription factor binding sites (inputs) can result in more than one output. Similarly, the motifs described in this study can be combined in different ways that result in either midline or tracheal expression. The results indicate the proximity of the CME to activators, one another and/or to repressors could contribute to the level of expression observed in the trachea and midline. This study focused on activator sites, but repressor sites are also likely present and restrict expression to certain cell types. Previous studies in Drosophila embryos have revealed the complexity of the transcriptional regulatory 'grammar' and have shown that the transcriptional output from various genes can be determined by the stoichiometry, affinity, spacing, arrangement, and distance between activator and repressor sites (Fulkerson, 2010).

The high degree of conservation within the midline enhancer subregions examined in this study here belies known properties of transcription factors and their recognition sequences, as well as observations made for many early developmental regulators of Drosophila development. Most transcription factors can vary considerably in the sequences they recognize and tend to bind to related sites with different affinities. This property would suggest that enhancers need not be strictly conserved to function, in contrast to what is reported here. The pattern of conserved sequences within these identified enhancers suggests that the transcription factors that bind these regions do so in a conserved order and spacing pattern. These results suggest that Sim and Trh may interact with other proteins to form an 'enhanceosome'-like complex, similar to that observed in the regulation of the interferon-β gene, in which activators and HMG proteins interact to form a specific multiprotein complex, with a defined structure. This model contrasts with the 'information display/billboard' model of enhancer function. In that model, enhancers are bound by a group of independent factors or group of factors that work together to promote or repress transcription in particular cell types. An important distinction between the two models is the arrangement of binding sites within an enhancer. Within an enhanceosome, the arrangement of binding sites relative to each other is constrained, whereas within a billboard enhancer, the relative arrangement of binding sites is rather flexible as long as a sufficient number of binding sites work together, in many possible configurations, to recruit factors for transcriptional activation (Fulkerson, 2010).

Results obtained with the midline glial genes examined in this study suggest that midline enhancers may consist of a nucleating enhanceosome-like region that combines with an 'information display/billboard' constellation of additional binding sites. This is supported by results obtained with the 70-bp conserved region of wrapper. When tested alone, it only marginally drives midline expression, whereas in the context of the 166-bp enhancer, it works quite well. Moreover, the 166-bp region of virilis cannot function on its own, but drives high levels of expression in the midline glia of melanogaster in the context of the larger, 476 bp region. That the 166-bp region from virilis cannot work efficiently in the midline suggests the transcription complex that binds to this region may be slightly different in virilis compared with melanogaster. For each enhancer described in this study, the presence of the conserved region is required to obtain expression in the midline glia (Fulkerson, 2010).

After comparing vertebrate genomes and generating reporter constructs with highly conserved noncoding sequences, Bailey (2006) noticed that many of these direct expression to regions of the CNS. It is possible that enhancers of CNS genes are more conserved compared with other gene sets, such as early developmental regulators of Drosophila that have been studied in detail. This may be due to the highly conserved nature of the transcription factors that regulate gene expression in this tissue, many of which have analogous functions in flies and mammals). Sox-binding sites are present throughout conserved regions of CNS genes and one of the similarities between these conserved CNS genes, the extensively characterized interferon-β enhanceosome and midline glial genes is the importance of HMG proteins. These proteins may bend the DNA, facilitating binding to highly structured, multiprotein complexes. The enhancers described here likely bind PAS and Sox proteins together with other conserved CNS regulators and it may be this combination of transcription factors that contributes to the similarly conserved arrangement of binding sites (Fulkerson, 2010).

Numerous combinations of transcription factor binding sites can be used to drive expression in many tissue types. Despite the conservation found in this study, binding sites for transcription factors do vary considerably, making it, at times, difficult to identify enhancers based on sequence conservation. In certain cases, changes within enhancers can generate diverse phenotypes between Drosophila populations. The continuing challenge is to understand both the forces constraining the enhancer sequences between Drosophila species, as well as how changes in these regions lead to significant modifications in the expression pattern of a gene, which over the long term, leads to variation among Drosophila populations and eventually, Drosophila species. For the midline genes described in this study, selection has stabilized the constellation of binding sites found within enhancers, resulting in their conservation among Drosophila species over approximately 40 million years of evolution (Fulkerson, 2010).

The Drosophila jing gene is a downstream target in the Trachealess/Tango tracheal pathway

Primary branching in the Drosophila trachea is regulated by the Trachealess (Trh) and Tango (Tgo) basic helix-loop-helix-PAS (bHLH-PAS) heterodimers, the POU protein Drifter (Dfr)/Ventral Veinless (Vvl), and the Pointed (Pnt) ETS transcription factor. The jing gene encodes a zinc finger protein also required for tracheal development. Three Trh/Tgo DNA-binding sites, known as CNS midline elements, in 1.5 kb of jing 5'cis-regulatory sequence (jing1.5) previously suggested a downstream role for jing in the pathway. This study shows that jing is a direct downstream target of Trh/Tgo and that Vvl and Pnt are also involved in jing tracheal activation. In vivo lacZ enhancer detection assays were used to identify cis-regulatory elements mediating embryonic expression patterns of jing. A 2.8-kb jing enhancer (jing2.8) drove lacZ expression in all tracheal cell lineages, the CNS midline and Engrailed-positive segmental stripes, mimicking endogenous jing expression. A 1.3-kb element within jing2.8 drove expression that was restricted to Engrailed-positive CNS midline cells and segmental ectodermal stripes. Surprisingly, jing1.5-lacZ expression was restricted to tracheal fusion cells despite the presence of consensus DNA-binding sites for bHLH-PAS, ETS, and POU domain transcription factors. Given the absence of Trh/Tgo DNA-binding sites in the jing1.3 enhancer, these results are consistent with previous observations suggesting a combinatorial basis to Trh-/Tgo-mediated transcriptional regulation in the trachea (Morozova, 2010).

In the developing Drosophila trachea, transcriptional regulation must be precisely coordinated with growth factor signaling to induce the appropriate cellular response. Studies of downstream transcriptional response elements in the transforming growth factor β (TGF-β) signaling pathway show the importance of discrete sequence changes differentiating an activation versus repressive response. Furthermore, such an activating enhancer element in the knirps gene in this pathway requires a cooperative effect with Trh and Tgo to possibly direct tissue specificity in the trachea. Tracheal gene expression is also controlled combinatorially by Trh/Tgo and Dfr/Vvl or either alone. Similarly, this study shows that Trh/Tgo response elements in the jing gene require additional elements to specify embryonic tracheal expression (Morozova, 2010).

Jing is implicated in transcriptional regulation in numerous biological processes, but its exact role is not known. This study extend previous observations of a role for jing in the trachea by establishing it as a direct downstream target of Trh/Tgo heterodimers. By analyzing jing 5' cis-regulatory regions, this study shows combinatorial basis to Trh/Tgo-mediated jing activation. A 2.8-kb jing enhancer recapitulates endogenous jing expression in the embryonic trachea, ectodermal stripes, and CNS midline. jing2.8 includes a distal 1.5-kb of genomic DNA that has three CMEs which are known for their involvement in combinatorial transcriptional regulation. The best evidence that Trh/Tgo complexes are able to directly activate the jing1.5 enhancer was gathered from Drosophila S2 cells by Luciferase reporter and ChIP assays. The CMEs in jing1.5-luc were required for activation by Trh/Tgo suggesting a protein-DNA interaction. Furthermore, Trh/Tgo heterodimers associated with and activated the jing1.5 enhancer. However, the combination of DNA-binding sites for bHLH-PAS, POU, and ETS transcription factors in jing1.5 is not capable of driving tracheal β-Gal expression in a pattern similar to that of endogenous jing. The jing1.3 enhancer cannot drive tracheal expression. Evidence is shown, in vitro and in vivo, that trh, pnt, and dfr/vvl regulate jing mRNA and even jing1.5-lacZ fusion cell expression. Given these results, along with the absence of additional CMEs and consensus POU domain-binding sites in jing1.3, it is proposed that trh and dfr/vvl regulate jing tracheal expression in combination with additional elements in jing1.3 (Morozova, 2010).

jing1.5 specifies a fusion cell component of jing expression that may instead be regulated by the bHLH-PAS transcription factors, Dys/Tgo. This is consistent with the presence of preferred and less preferred Dys/Tgo DNA-binding sites in the jing 1.5-lacZ enhancer. Prior to embryonic stage 12, trh is required for dys expression and then Dys and Archipelago downregulate trh specifically in fusion cells during stage 12. Therefore, Trh cannot activate jing1.5-lacZ in fusion cells from stage 12 which is consistent with the presence of fusion cell lacZ expression in embryos carrying CME deletions in jing1.5. The reductions in jing1.5-lacZ expression in the fusion cells of trh mutants may therefore result from subsequent reductions in dys expression (Morozova, 2010).

This study also characterized jing cis-regulatory elements controlling different aspects of jing expression in CNS glia and Engrailed-expressing midline neurons and segmental ectodermal cells. The midline expression of jing enhancers provided an opportunity to compare jing transcriptional regulation in two tissues. The data show that jing1.5 is sufficient to drive expression in MG and neurons where Jing is normally expressed. The CNS midline identity of jing1.5-lacZ-expressing cells was shown in several ways. First, jing1.5-lacZ expression was absent in a homozygous sim mutant background. Second, the jing1.5-lacZ expression domain was expanded by activating the Spitz Egfr ligand thereby forcing midline glial survival. Lastly, MG characteristics, such as oblong shape and dorsal positions, are shown by some jing1.5-lacZ-expressing midline cells. Therefore, this enhancer is differentially activated in the CNS midline and trachea suggesting that there may be differences in the mechanism by which Sim/Tgo and Trh/Tgo heterodimers activate transcription. This is consistent with the differential abilities of Sim/Tgo and Trh/Tgo to associate with Dfr/Vvl in vitro and the inability of trh to induce ectopic CNS midline gene expression (Morozova, 2010).

Strong CNS midline expression was also driven by the jing1.3 enhancer despite the absence of Sim/Tgo or Dfr/Vvl consensus DNA-binding sites. However, upon further characterization, the jing1.3-lacZ-expressing midline cells were found to express the segment polarity gene, engrailed (en). En-expressing CNS midline cells take up the posterior-most position within each VNC segment. Another En-positive midline cell lineage includes four to six MGP which are present at stage 13 but not at stage 17. The round shape of En-positive jing1.3-lacZ-expressing midline cells suggests that they belong to the MNB lineage and its progeny and do not belong to the MGP lineage. The mechanism of midline activation of jing1.3 is not known, but the ability of Jing to function as a repressor suggests that it may function combinatorially with En in segmental patterning. Further studies will be aimed at determining whether jing plays a role in segmental ectodermal patterning and its associated gene expression programs (Morozova, 2010).

Protein Interactions

The Drosophila single-minded and trachealess bHLH-PAS genes control transcription and development of the CNS midline cell lineage and tracheal tubules, respectively. Single-minded and Trachealess activate transcription by forming dimers with the Drosophila Tango protein that is an ortholog of the mammalian Arnt protein. Tgo interacts strongly with murine AhR. Heterodimers of Tango/Arnt are observed with Single minded, Sima and Trachealess, but no heterodimers are formed between pairwise combinations of Sim, Sima, Trh and Ahr (Sonnenfeld, 1997).

The basic-helix-loop-helix-PAS protein heterodimer formed by Drosophila Single-minded (Sim) and Tango (Tgo) controls transcription and embryonic development of the CNS midline cells, while another heterodimer formed by Trachealess (Trk) and Tango controls tracheal cell and salivary duct transcription and development. Expression of both single-minded and trachealess are highly restricted to their respective cell lineages, however tango is broadly expressed. The developmental control of subcellular localization of these proteins was investigated because of Tango's similarity to the mammalian basic-helix-loop-helix-PAS Aromatic hydrocarbon receptor, whose nuclear localization is dependent on ligand binding. Confocal imaging of Single-minded and Trachealess protein localization indicates that these proteins accumulate in cell nuclei when initially synthesized in their respective cell lineages and remain nuclear throughout embryogenesis. Ectopic expression experiments show that Single-minded and Trachealess are localized to nuclei in cells throughout the ectoderm and mesoderm, indicating that nuclear accumulation is not regulated in a cell-specific fashion and unlikely to be ligand dependent. In contrast, nuclear localization of Tango is developmentally regulated; it is localized to the cytoplasm in most cells except the CNS midline, salivary duct, and tracheal cells; in these tissues it accumulates in nuclei. Genetic and ectopic expression experiments indicate that Tango nuclear localization is dependent on the presence of a basic-helix-loop-helix-PAS protein such as Single-minded or Trachealess. Drosophila cell culture experiments show that Single-minded and Trachealess nuclear localization is dependent on Tango since, in the absence of Tango, these proteins are cytoplasmic. These results suggest a model in which Single-minded and Trachealess dimerize with Tango in the cytoplasm of the CNS midline cells and trachea, respectively, and the dimeric complex accumulates in nuclei in a ligand-independent mode and regulates lineage-specific transcription (Ward, 1998).

Once activated transcriptionally in their respective cell lineages, SIM and TRH mRNAs are translated; the Sim and Trh proteins dimerize with Tgo, and the complex translocates to the nucleus. Sim and Trh do not act as receptors for developmentally relevant molecules that trigger translocation to nuclei upon binding; instead their presence in cells is the developmental signal itself. The bHLH-PAS developmental regulatory proteins described here are controlled by transcriptional activation and not ligand-binding; it will be interesting to see if this correlation is a general feature as other bHLH-PAS proteins of developmental significance are analyzed (Ward, 1998).

The Drosophila single-minded gene controls CNS midline cell development by both activating midline gene expression and repressing lateral CNS gene expression in the midline cells. The mechanism by which Single-minded represses transcription was examined using the ventral nervous system defective gene as a target gene. Transgenic-lacZ analysis of constructs containing fragments of the ventral nervous system defective regulatory region have identified sequences required for lateral CNS transcription and midline repression. Elimination of Single-minded:Tango binding sites within the ventral nervous system defective gene does not affect midline repression. Mutants of Single-minded that remove the DNA binding and transcriptional activation regions abolish ventral nervous system defective repression, as well as transcriptional activation of other genes. The replacement of the Single-minded transcriptional activation region with a heterologous VP16 transcriptional activation region restores the ability of Single-minded to both activate and repress transcription. These results indicate that Single-minded indirectly represses transcription by activating the expression of repressive factors. Single-minded provides a model system for how regulatory proteins that act only as transcriptional activators can control lineage-specific transcription in both positive and negative modes (Estes, 2001).

Three general models of Sim-mediated repression were tested: (1) Sim directly represses target genes by binding their DNA and repressing transcription in association with a corepressor(s); (2) Sim does not bind DNA of target genes but interacts with positively acting factors preventing their action, and (3) Sim represses indirectly by activating transcription of genes encoding repressive factors. Several complementary experiments demonstrate that midline repression requires activation of repressive gene expression by Sim (Model 3). Ectopic expression experiments utilizing mutant forms of Sim demonstrate that the basic region, PAS domain, and C-terminal regions are all required for both transcriptional activation and repression. Removal of the PAS domain also abolished the ability of Sim to form dimers with Tgo, suggesting that Tgo is necessary for repression. More informative is Db-Sim. This mutant protein was able to dimerize with Tgo and the protein complex accumulates in the nucleus. However, neither midline transcription nor repression occurs, presumably due to the inability of the Sim:Tgo dimer to bind DNA. This argues against a model in which Sim interacts with an activator protein in a non-DNA-binding mode (Model 2) and instead suggests that DNA binding is required for Sim repression (Model 1 or Model 3). However, analysis of the vnd gene using lacZ transgenes indicates that Sim:Tgo binding sites are not required for midline repression (Model 1); mutation of the single CNS midline element (CME; ACGTG) in fragment 2.5RB or mutation of three CMEs in 5.3RS does not affect lacZ expression. Transient transfection experiments have shown that CMEs are relevant targets of Sim:Tgo binding, and in vivo analyses of five different genes have shown that the CME functions in vivo as a Sim:Tgo binding site. However, it remains possible that Sim:Tgo could bind a variant sequence within the vnd gene. Arguing against this are the results indicating that Sim represses indirectly by activating transcription (Estes, 2001).

The C-terminal region of Sim that follows the PAS domain contains multiple transcriptional activation domains. Removal of the C-terminal 211 aa eliminates those activation domains and additional residues. The DeltaC-Sim protein is unable to activate midline transcription or repress vnd expression, even though it dimerized with Tgo and the complex accumulates in nuclei. This is consistent with Sim repressing vnd expression by activating the transcription of repressive factors. However, it is also possible that there is a domain within the C-terminal region that could directly mediate repression. Fusing the VP16 activation domain onto DeltaC-Sim and functionally assaying the fusion protein in vivo tested this. The results show that addition of the VP16 activation domain restores the ability of DeltaC-Sim to activate transcription and repress vnd. These experiments demonstrate that vnd repression correlates with the ability of Sim to activate transcription (Model 3). Another construct removed the Sim AAQ repeat region (a repeating stretch of 10 Ala-Ala-Gln repeats followed by several imperfect repeats). Its deletion does not affect the ability of Sim to dimerize with Tgo, accumulate in nuclei, activate transcription, or repress vnd. Although striking in sequence, its function remains a mystery. The combination of the vnd-lacZ and ectopic Sim-mutant experiments demonstrate that Sim does not directly repress or inhibit vnd gene expression but, instead, activates transcription of genes that encode repressive factors consistent with the third model of repression. This model is also consistent with the delayed timing of vnd repression seen in early embryonic development (Estes, 2001).

In mammalian systems, the heterodimeric basic helix-loop-helix (bHLH)-PAS transcription hypoxia-inducible factor (HIF) has emerged as the key regulator of responses to decreased oxygen concentrations . A homologous system is present in Drosophila, and its activity has been characterized in vivo during development. By using transcriptional reporters in developing transgenic flies, it has been shown that hypoxia-inducible activity rises to a peak in late embryogenesis and is most pronounced in tracheal cells. The bHLH-PAS proteins Similar (Sima) and Tango function as HIF-alpha and HIF-ß homologs, respectively; a conserved mode of regulation for Sima by oxygen has been demonstrated. Sima protein, but not its mRNA, is upregulated in hypoxia. Time course experiments following pulsed ectopic expression demonstrate that Sima is stabilized in hypoxia and that degradation relies on a central domain encompassing amino acids 692 to 863. Continuous ectopic expression overrode Sima degradation, which remains cytoplasmic in normoxia, and translocates to the nucleus only in hypoxia, revealing a second oxygen-regulated activation step. Abrogation of the Drosophila Egl-9 prolyl hydroxylase homolog, CG1114, causes both stabilization and nuclear localization of Sima, indicating a central involvement in both processes. Tight conservation of the HIF/prolyl hydroxylase system in Drosophila provides a new focus for understanding oxygen homeostasis in intact multicellular organisms (Lavista-Llanos, 2002).

To test this a proposed role for Tgo in the hypoxic response, embryos that were homozygous for a strong tgo mutant allele (tgo⁵) were examined. These embryos failed to induce the reporter in hypoxia, strongly supporting the role of Tgo as the HIF-ß subunit and indicating that, as in mammalian cells, this protein is absolutely required for the hypoxia response (Lavista-Llanos, 2002).

The development of the mature insect trachea requires a complex series of cellular events, including tracheal cell specification, cell migration, tubule branching, and tubule fusion. The Drosophila dysfusion gene encodes a basic helix-loop-helix (bHLH)-PAS protein conserved between Caenorhabditis elegans, insects, and humans; dysfusion controls tracheal fusion events. The Dysfusion protein functions as a heterodimer with the Tango bHLH-PAS protein in vivo to form a putative DNA-binding complex. The dysfusion gene is expressed in a variety of embryonic cell types, including tracheal-fusion, leading-edge, foregut atrium cells, nervous system, hindgut, and anal pad cells. RNAi experiments indicate that dysfusion is required for dorsal branch, lateral trunk, and ganglionic branch fusion but not for fusion of the dorsal trunk. The escargot gene, which is also expressed in fusion cells and is required for tracheal fusion, precedes dysfusion expression. Analysis of escargot mutants indicates a complex pattern of dysfusion regulation, such that dysfusion expression is dependent on escargot in the dorsal and ganglionic branches but not the dorsal trunk. Early in tracheal development, the Trachealess bHLH-PAS protein is present at uniformly high levels in all tracheal cells, but when the levels of Dysfusion rise in wild-type fusion cells, the levels of Trachealess in fusion cells decline. The downregulation of Trachealess is dependent on dysfusion function. These results suggest the possibility that competitive interactions between basic helix-loop-helix-PAS proteins (Dysfusion, Trachealess, and possibly Similar) may be important for the proper development of the trachea (Jiang, 2003).

The bHLH-PAS transcription factor Dysfusion regulates tarsal joint formation in response to Notch activity during Drosophila leg development

A characteristic of all arthropods is the presence of flexible structures called joints that connect all leg segments. Drosophila legs include two types of joints: the proximal or 'true' joints that are motile due to the presence of muscle attachment and the distal joints that lack musculature. These joints are not only morphologically, functionally and evolutionarily different, but also the morphogenetic program that forms them is distinct. Development of both proximal and distal joints requires Notch activity; however, it is still unknown how this pathway can control the development of such homologous although distinct structures. This study shows that the bHLH-PAS transcription factor encoded by the gene dysfusion (dys), is expressed and absolutely required for tarsal joint development while it is dispensable for proximal joints. In the presumptive tarsal joints, Dys regulates the expression of the pro-apoptotic genes reaper and head involution defective and the expression of the RhoGTPases modulators, RhoGEf2 and RhoGap71E, thus directing key morphogenetic events required for tarsal joint development. When ectopically expressed, dys is able to induce some aspects of the morphogenetic program necessary for distal joint development such as fold formation and programmed cell death. This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes. A dedicated dys cis-regulatory module was identified that regulates dys expression in the tarsal presumptive leg joints through direct Su(H) binding. All these data place dys as a key player downstream of Notch, directing distal versus proximal joint morphogenesis (Cordoba, 2014: PubMed).

DEVELOPMENTAL BIOLOGY

Embryonic

Tango protein is found either in cell nuclei or cytoplasm, depending on the cell type and time of development. In the precellular blastoderm, Tango protein is uniformly distributed, presumable due to maternal contribution. Staining is more intense in the cytoplasm than in the nucleus. During the extended germband stage, Tgo protein is detected in all three germ layers. As tracheal pits form, the cells surrounding the pits show enhanced levels of Tgo protein and RNA, as compared to surrounding cells. Amounts of Tgo protein above ubiquitous levels continue to be observed in the tracheal cells, including the posterior spiracles, from stage 11 to the end of embryogenesis at stage 17. As the CNS forms, uniformly high levels of Tgo protein are found in the brain and ventral nerve cord (Sonnenfeld, 1997).

EFFECTS OF MUTATION

Isolation and analysis of tango mutants reveal CNS midline and tracheal defects, and gene dosage studies demonstrate in vivo interactions between single-minded::tango and trachealess::tango. Defects in CNS midline neurons and glia were examined using enhancer trap reporters. In wild-type embryos, the AA142 enhancer trap is expressed in an average of 3.5 midline glia per segment by stage 14 of embryogenesis. In tango mutant embryos, there is a reduction in the number of stained midline glia to approximately one cell per segment. The X55 enhancer trap gene stains the ventral unpaired median neurons (VUMs) and the median neuroblast (MNB) and its progeny in the ventral region of the CNS. In tango mutant embryos, the number of VUM neurons and MNB progeny are reduced in number (60% of wild-type) and do not migrate into the ventral regions of the ventral cord. The role of tango in tracheal development was examined by staining tango mutant embryos with monoclonal antibody 2A12, which stains the lumen of the tracheal tubes. tango mutant embryos are shown to have a variety of tracheal defects. Experiments with heterozygotes show that tango interacts genetically with both trachealess and single minded (Sonnenfeld, 1997).

EVOLUTIONARY HOMOLOGS

Invertebrate Arnt homologs

Juvenile hormone analog (JHA) insecticides are relatively nontoxic to vertebrates and offer effective control of certain insect pests. Recent reports of resistance in whiteflies and mosquitoes demonstrate the need to identify and understand genes for resistance to this class of insect growth regulators. Mutants of the Methoprene-tolerant (Met) gene in Drosophila melanogaster show resistance to both JHAs and JH, and previous biochemical studies have demonstrated a mechanism of resistance involving an intracellular JH binding-protein that has reduced ligand affinity in Met flies. Met flies are resistant to the toxic and morphogenetic effects of JH and several JHAs, but not to other classes of insecticide. Biochemical studies reveal a target-site resistance mechanism, that of reduced JH binding in cytosolic extracts from either of two JH target tissues in Met flies. This property of reduced JH binding was cytogenetically localized to the Met region on the X chromosome and can account for the resistance. Possible identities for this binding protein include either an accessory JH-binding protein in the cytoplasm, similar to the cellular retinoic acid-binding protein in vertebrates, or a JH receptor protein involved in the action of JH (Ashok, 1998).

The Met+ gene has been cloned by transposable P-element tagging and reduced transcript level has been found in several mutant alleles, showing that underproduction of the normal gene product can lead to insecticide resistance. Transformation of Met flies with a Met+ cDNA results in susceptibility to methoprene, indicating that the cDNA encodes a functional Met+ protein. Met shows homology to the basic helix-loop-helix (bHLH)-PAS family of transcriptional regulators, implicating Met in the action of JH at the gene level in insects. This family also includes the vertebrate dioxin receptor, a transcriptional regulator known to bind a variety of environmental toxicants. Met shows three regions of homology to members of a family of transcriptional activators known as bHLH-PAS proteins. Met generally has higher homology to the vertebrate bHLH-PAS proteins than to those identified in D. melanogaster. A D. melanogaster ARNT-like gene has recently been cloned, and DARNT has higher homology to vertebrate ARNT than does Met, suggesting that DARNT, not Met, may function like ARNT in flies. Met homology to these proteins includes the bHLH region that is involved in DNA binding (30-38% identity), the PAS-A region (28-40%), and the PAS-B region (22-35%). The arrangement of these domains in the Met gene is the same as for other bHLH-PAS genes (Ashok, 1998).

Hypoxia-inducible factor, a heterodimeric transcription complex, regulates cellular and systemic responses to low oxygen levels (hypoxia) during normal mammalian development or tumor progression. Evidence is presented that a similar complex mediates response to hypoxia in C. elegans. This complex consists of HIF-1 and AHA-1, which are encoded by C. elegans homologs of the hypoxia-inducible factor (HIF) alpha and ß subunits, respectively. hif-1 mutants exhibit no severe defects under standard laboratory conditions, but they are unable to adapt to hypoxia. Although wild-type animals can survive and reproduce in 1% oxygen, the majority of hif-1-defective animals die in these conditions. The expression of an HIF-1:green fluorescent protein fusion protein is induced by hypoxia and is subsequently reduced upon reoxygenation. Both hif-1 and aha-1 are expressed in most cell types, and the gene products can be coimmunoprecipitated. It is concluded that the mechanisms of hypoxia signaling are likely conserved among metazoans. Additionally, it is found that nuclear localization of AHA-1 is disrupted in an hif-1 mutant. This finding suggests that heterodimerization may be a prerequisite for efficient nuclear translocation of AHA-1 (Jiang, 2001).

Although mammalian HIF-1alpha has an essential role in embryonic development, C. elegans hif-1 mutants are viable and fertile when cultured in standard laboratory conditions. This reflects the relatively simple physiology of C. elegans. A mammalian embryo relies on hypoxia-induced angiogenesis to oxygenate tissues. Hif-1alpha -/- mice die by E9.0 with severe vascular defects. In contrast, an adult C. elegans hermaphrodite has no apparent need for specialized respiratory structures or a complex circulatory system. Any cell in the organism is only a few cell widths from the outer surface of the worm or the intestinal lumen. Oxygen sensing and hif-1 function is likely to be very important in the soil environment inhabited naturally by C. elegans. In the laboratory, C. elegans are cultured on top of an agar-based medium, and the ambient oxygen concentrations are relatively high. However, high concentrations of bacteria, the C. elegans food source, can deplete oxygen in a soil microenvironment. The nematodes must be able to sense and adapt to these hypoxic conditions (Jiang, 2001).

AHA-1 translocation to the nuclei of intestinal cells is inefficient in hif-1 mutants. This result was not predicted by the prevalent models for hypoxia signaling. In the mammalian cell lines commonly used to study hypoxia-inducible factor or aryl hydrocarbon receptor (AHR) signaling, ARNT is localized to the nucleus constitutively. After HIF-1alpha or activated AHR translocates to the nucleus, it forms a dimer with ARNT. However, in the Drosophila embryo, Drosophila ARNT (encoded by the tango gene) apparently remains cytoplasmic until a bHLH-PAS dimerization partner is expressed. It is concluded that the role of AHA-1 in the formation and nuclear localization of an active transcriptional complex may depend on cell type-specific factors, such as the expression of other bHLH-PAS proteins. This hypothesis will be explored by examining the expression of other bHLH-PAS genes in those cells that localize AHA-1 to the nucleus in the absence of hif-1 (Jiang, 2001).

Mammalian Arnts

cDNAs encoding two distinct basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) proteins with similarity to the mammalian aryl hydrocarbon nuclear translocator (Arnt) protein were isolated from RTG-2 rainbow trout gonad cells. The deduced proteins, termed rtARNTa and rtARNTb, are identical over the first 533 amino acids and contain a basic helix-loop-helix domain that is 100% identical to human Arnt. rtARNTa and rtARNTb differ in their COOH-terminal domains due to the presence of an additional 373 base pairs of sequence that manifest the characteristics of an alternatively spliced exon. The presence of the 373-base pair region causes a shift in the reading frame. rtARNTa lacks the sequence and has a COOH-terminal domain of 104 residues rich in proline, serine, and threonine. rtARNTb contains the sequence and has a COOH-terminal domain of 190 residues rich in glutamine and asparagine. mRNAs for both rtARNT splice variants are detected in RTG-2 gonad cells, trout liver, and gonad tissue. rtARNTa and rtARNTb protein were identified in cell lysates from RTG-2 cells. Transfection of rtARNT expression vectors into murine Hepa-1 cells that are defective in Arnt function (type II) results in rtARNT protein expression localized to the nucleus. Treatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a 20-fold greater induction of endogenous P4501A1 protein in cells expressing rtARNTb when compared with rtARNTa, even though both proteins effectively dimerize with the aryl hydrocarbon receptor. The decreased function of rtARNTa appears to be due to inefficient binding of rtARNTa.AhR complexes to DNA. The presence of rtARNTa can reduce the aryl hydrocarbon receptor-dependent function of rtARNTb in vivo and in vitro. It is concluded that rtARNTa is a dominant negative activity in the bHLH/PAS family (Pollenz, 1995).

Arnt2, a new member of the basic-helix-loop-helix transcription factor family, was cloned from rat brain cDNAs. Its deduced 712 amino acid sequence displays 63% identity with that of the aryl hydrocarbon receptor nuclear translocator (Arnt1). Whereas Arnt2 gene expression occurs selectively in brain and kidney, the expression of Arnt1 is ubiquitous, suggesting that the two proteins play distinct roles, presumably via dimerization and DNA binding with different partners (Drutel, 1996).

In an effort to better understand the toxicity mechanism of 2,3,7, 8-tetrachlorodibenzo-p-dioxin, an iterative search of sequence tags expressed in humans was employed to identify novel basic-helix-loop-helix-PAS (bHLH-PAS) proteins that interact with either the Ah receptor (AhR) or the Ah receptor nuclear translocator (Arnt). Five new "members of the PAS superfamily" (MOPs 1-5), similar in size and structural organization to the AhR and Arnt, have been characterized. MOPs 1-4 have N-terminal bHLH and PAS domains and C-terminal variable regions. MOP5 contain the characteristic PAS domain and a variable C terminus; it is possible that the cDNA contains a bHLH domain, but the entire open reading frame has yet to be completed. Coimmunoprecipitation studies, yeast two-hybrid analysis, and transient transfection experiments all demonstrate that MOP1 and MOP2 dimerize with Arnt and that these complexes are transcriptionally active at defined DNA enhancer sequences in vivo. MOP3 is found to associate with the AhR in vitro but not in vivo. This observation, coupled with the fact that MOP3 forms tighter associations with the 90-kDa heat shock protein than the human AhR, suggests that MOP3 may be a conditionally active bHLH-PAS protein that requires activation by an unknown ligand. The expression profiles of the AhR, MOP1, and MOP2 mRNAs, coupled with the observation that they all share Arnt as a common dimeric partner, suggest that the cellular pathways mediated by MOP1 and MOP2 may influence or respond to the dioxin signaling pathway (Hogenesch, 1997).

Expression of Arnt, a mammalian Tango homolog

Dioxins are environmental pollutants, whose detrimental effects on health are the cause of wide public concern due to their accumulation in the food chain and resistance to metabolism. The most well known dioxin is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dioxins exert their effects through a ligand activated transcription factor termed the dioxin or aryl hydrocarbon receptor (AhR), which acts in concert with another structurally related protein: the aryl hydrocarbon nuclear translocator (Arnt). In situ hybridization was used to study the localization of the mRNAs for these two proteins in the rat brain. mRNAs for both AhR and Arnt are found predominantly in the same neuronal populations: the olfactory bulb, the hippocampus, and the cerebral and cerebellar cortices. Arnt, however, has a more widespread expression than AhR in the brain. The present results demonstrate that dioxins may act directly in the brain and that the effects of dioxin may occur in discrete neuronal populations. However, in some parts of the brain, e.g. the hypothalamus, that are thought to be targets of the toxic effects of dioxins, detectable levels of AhR mRNA are not observed. Furthermore, it appears that Arnt may have additional functions in the brain, apart from being the heterodimerization partner of AhR, possibly through heterodimerizing with other transcription factors (Kainu, 1995).

The AhR is a ligand-activated partner of the Arnt protein. Both proteins are required to transcriptionally regulate gene expression. Arnt must be complexed to AhR to permit binding to the regulatory DNA sequence. The AhR-ligand complex is known to mediate a range of biological responses, such as developmental toxicity, induction of cleft palate, and hydronephrosis. AhR and Arnt are expressed in human embryonic palatal cells and AhR has a specific developmental pattern of expression in the mouse embryo. In the present study, expression of Arnt is characterized in C57Bl/6N mouse embryos from gestation day 10-16. The day 10-11 embryos show highest levels of Arnt in neuroepithelial cells of the neural tube, visceral arches, otic and optic placodes, and preganglionic complexes. The heart also has significant expression of Arnt, with strong nuclear localization. After day 11, expression in heart and brain declines. In day 12-13 embryos expression is highest in the liver, where expression increases from day 12 to 16. At days 15-16 the highest levels of Arnt occurs in adrenal gland and liver, although Arnt is also detected in the submandibular gland, the ectoderm, the tongue, and in bone and muscle. In all of these tissues Arnt is cytoplasmic as well as nuclear, except in some of the cortical adrenal cells, in which Arnt is strongly cytoplasmic with little or no nuclear localization. These specific patterns of Arnt expression, which differ in certain tissues from the expression of AhR, suggest that Arnt may have additional roles in normal embryonic development (Abbott, 1995).

Mutation of Arnt

The arylhydrocarbon-receptor nuclear translocator (Arnt) is a member of the basic-helix-loop-helix-PAS family of heterodimeric transcription factors, which includes the arylhydrocarbon receptor (AhR), hypoxia-inducible factor-1alpha (HIF-1alpha) and the Drosophila Single-minded protein (Sim). Arnt forms heterodimeric complexes with the aryl hydrocarbon receptor, HIF-1alpha, Sim and the PAS protein Per. In response to environmental pollutants, AhR-Arnt heterodimers regulate genes involved in the metabolism of xenobiotics, whereas Arnt-HIF-1alpha heterodimers probably regulate those involved in the response to oxygen deprivation. By generating a targeted disruption of the Arnt locus in the mouse, it is shown that Arnt-/- embryonic stem cells fail to activate genes that normally respond to low oxygen tension. Arnt-/- ES cells also failed to respond to a decrease in glucose concentration, indicating that Arnt is crucial in the response to hypoxia and to hypoglycemia. Arnt-/- embryos are not viable past embryonic day 10.5 and show defective angiogenesis of the yolk sac and branchial arches, stunted development and embryo wasting. The defect in blood vessel formation in Arnt-/- yolk sacs is similar to the angiogenic abnormalities reported for mice deficient in vascular endothelial growth factor or tissue factor. On the basis of these findings, a model is proposed in which increasing tissue mass during organogenesis leads to the formation of hypoxic/nutrient-deprived cells, the subsequent activation of Arnt, and a concomitant increase in the expression of genes (including the gene encoding vascular endothelial growth factor) that promote vascularization of the developing yolk sac and solid tissues (Maltepe, 1997).

Homologous recombination was used in embryonic stem cells to generate mice heterozygous for an aryl hydrocarbon nuclear translocator (Arnt) null mutation. These mice were intercrossed, but no live homozygous Arnt-/- knockout mice were produced among 64 newborns. Homozygotes die in utero between 9.5 and 10.5 days of gestation. Abnormalities include neural tube closure defects, forebrain hypoplasia, delayed rotation of the embryo, placental hemorrhaging, and visceral arch abnormalities. However, the primary cause of lethality appears to be failure of the embryonic component of the placenta to vascularize and form the labyrinthine spongiotrophoblast. This may be related to Arnt's known role in hypoxic induction of angiogenesis. No defects were found in yolk sac circulation (Kozak, 1997).

Domain structure of Arnt

Gene regulation by dioxins is mediated by the dioxin receptor-Arnt heterodimer, a ligand generated complex of two basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) transcription factors. By using dioxin receptor chimeras where the dimerization and DNA binding bHLH motif has been replaced by a heterologous DNA binding domain, the ability of Arnt to interact with the dioxin receptor via the PAS domain has been detected in a mammalian 'hybrid interaction' system. By coimmunoprecipitation assays, the ability of PAS domains of the dioxin receptor and Arnt to mediate independent heterodimerization has been confirmed in vitro. Selectivity for PAS dimerization is noted in the hybrid interaction system, because neither the dioxin receptor nor Arnt PAS-mediated homodimers are detected. Surprisingly, however, the PAS domain of Drosophila Period can dimerize with both the dioxin receptor and Arnt subunits in vitro, and disrupt the ability of these subunits to form a DNA binding heterodimer. Ectopic expression of Per blocks dioxin signaling in mammalian cells. The PAS domains of the dioxin receptor and Arnt are therefore novel dimerizing regions critical to the formation of a functional dioxin receptor-Arnt complex, while the PerPAS domain is a potential negative regulator of bHLH/PAS factor function (Lindebro, 1995).

The aryl hydrocarbon (or dioxin) receptor (AhR) is a ligand-activated basic helix-loop-helix (bHLH) protein that heterodimerizes with the bHLH protein AhR nuclear translocator (Arnt) to form a complex that binds to xenobiotic regulatory elements in the enhancers of target genes. A series of fusion proteins, with a heterologous DNA-binding domain, was used to independently study the trans-activating function of the human AhR and Arnt proteins in yeast. The results confirm that both the human AhR and Arnt contain carboxyl-terminal trans-activation domains. The AhR has a complex trans-activation domain that is composed of multiple segments, which function independently and exhibit varying levels of activation. These regions within the AhR cooperate when linked together, resulting in a synergistic activation of transcription. Fusion proteins of the AhR and Arnt trans-activation domains with the LexA DNA-binding domain, expressed in bacteria and purified to near-homogeneity, stimulate transcription of a minimal promoter in vitro in yeast nuclear extracts. Using this in vitro transcription assay, it is also possible to demonstrate that the AhR and Arnt trans-activation domains, in the absence of a DNA-binding domain, inhibit activated and basal transcription. In vitro, the receptor binds selectively to the basal transcription factors, the TATA-binding protein and TFIIF, whereas Arnt binds preferentially to TFIIF. Taken together, these results suggest that AhR and Arnt activate target gene expression, at least in part, through direct interactions with basal transcription factors (Rowlands, 1996).

Binding of Arnt to DNA

The Ah receptor (AhR) and its DNA binding partner, the Ah receptor nuclear translocator (Arnt), are basic helix-loop-helix proteins distinguished by their Per, AhR, Arnt, and Sim(PAS) homology regions. To identify the amino acids of the AhR.Arnt heterodimer that contact the TNGCGTG recognition sequence, deletion mapping and amino acid substitutions have been performed within the N termini of both the AhR and Arnt. The ability of the variant AhR and Arnt proteins to bind DNA and activate gene transcription was determined by gel shift analysis and transient transfection assays. The amino acids of Arnt that contact DNA are similar to those of other basic/helix-loop-helix proteins and include glutamic acid residue 83 and arginine residues 86 and 87. Although initial experiments indicate that DNA binding of the AhR may involve two regions (bordered by amino acids 9-17 and amino acids 34-42), further analysis demonstrates that only amino acids 34-39 are critical for the AhR.TNGC interaction. These experiments indicate that while the structural features of the Arnt.GTG complex may closely resemble that deduced for proteins such as Max, E47, and USF, the AhR.TNGC complex may represent a unique DNA binding form of basic/helix-loop-helix proteins (Swanson, 1996).

The Ah receptor (AhR), the Ah receptor nuclear translocator protein (Arnt), and single-minded protein (SIM) are members of the basic helix-loop-helix-PAS (bHLH-PAS) family of regulatory proteins. The DNA half-site recognition and pairing rules for these proteins were examined using oligonucleotide selection-amplification and coprecipitation protocols. Oligonucleotide selection-amplification reveals that a variety of bHLH-PAS protein combinations can interact, each one generating a unique DNA binding specificity. To validate the selection-amplification protocol, the preference of the AhR.Arnt complex was demonstrated for the sequence commonly found in dioxin-responsive enhancers in vivo (TNGCGTG). The Arnt protein is capable of forming a homodimer with a binding preference for the palindromic E-box sequence, CACGTG. Further examination indicates that Arnt may have a relaxed partner specificity, since it is also capable of forming a heterodimer with SIM and recognizing the sequence GT(G/A)CGTG. Coprecipitation experiments using various PAS proteins and Arnt are consistent with the idea that the Arnt protein is capable of a broad range of interactions among the bHLH-PAS proteins, while the other members appear more restricted in their interactions. Comparison of this in vitro data with sites known to be bound in vivo suggests that the high affinity half-site recognition sequences for the AhR, SIM, and Arnt are T(C/T)GC, GT(G/A)C (5'-half-sites), and GTG (3'-half-sites), respectively (Swanson, 1995).

Arnt is a nuclear basic helix-loop-helix (bHLH) transcription factor that, contiguous with the bHLH motif, contains a region of homology (PAS) with the Drosophila factors Per and Sim. Arnt dimerizes in a ligand-dependent manner with the bHLH dioxin receptor, a process that enables the dioxin-(2,3,7,8-tetrachlorodibenzo-p-dioxin)-activated Arnt-dioxin receptor complex to recognize dioxin response elements of target promoters. In the absence of dioxin, Arnt does not bind to this target sequence motif. Arnt constitutively binds the E box motif CACGTG, also recognized by a number of distinct bHLH factors, including USF and Max. Amino acids that have been identified to be critical for E box recognition by Max and USF are conserved in Arnt. Consistent with these observations, full-length Arnt, but not an Arnt deletion mutant lacking its potent C-terminal transactivation domain, constitutively activates CACGTG E box-driven reporter genes in vivo. These results indicate a role for Arnt in the regulation of a network of target genes that is distinct from the regulatory role played by the Arnt-dioxin receptor complex in dioxin-stimulated cells (Antonsson, 1995).

The aryl hydrocarbon receptor (AhR) and the aryl hydrocarbon receptor nuclear translocator (Arnt) belong to a novel subclass of basic helix-loop-helix transcription factors. The AhR.Arnt heterodimer binds to the xenobiotic responsive element (XRE). Substitution of each of four amino acids in the basic region of Arnt with alanine severely diminishes or abolishes XRE binding, intimating that these amino acids contact DNA bases. Three of these amino acids are conserved among basic helix-loop-helix proteins, and the corresponding amino acids of Max and USF are known to contact DNA bases. Alanine scanning mutagenesis of the basic domain of AhR and substitution with conservative amino acids at particular positions in this domain and in a more amino-proximal AhR segment previously shown to be required for XRE binding demonstrate that the most carboxyl-proximal amino acid position of the basic domain and a position within the amino-proximal segment are intolerant to amino acid substitution with regard to XRE binding, suggesting that these two amino acids make base contacts. Amino acid positions in these AhR regions and in the Arnt basic region are less adversely affected by substitution are also identified. The amino acids at these positions may contact the phosphodiester backbone. The apparent bipartite nature of the DNA binding region of AhR and the identity of those of its amino acids that apparently make DNA contacts impute a novel protein-DNA binding behavior for AhR (Bacsi, 1996).

Expression of CYP1A1 gene is regulated in a substrate-inducible manner through at least two kinds of regulatory DNA elements, in addition to the TATA sequence, XRE (xenobiotic responsive element), and BTE (basic transcription element), a GC box sequence. The trans-acting factor on the XRE is a heterodimer consisting of the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt), while Sp1 acts as a regulatory factor on the BTE. An investigation was carried out of how these factors interact with one another to induce expression of the CYP1A1 gene. Both in vivo transfection assays using Drosophila Schneider line 2 (SL2) cells, which is devoid of endogenous Sp1, AhR, and Arnt, and in vitro transcription assays using baculovirus-expressed AhR, Arnt, and Sp1 proteins, reveal that these factors synergistically enhance expression of the reporter genes driven by a model CYP1A1 promoter, consisting of four repeated XRE sequences and a BTE sequence. Both AhR and Arnt interact with the zinc finger domain of Sp1 via their basic HLH/PAS domains. When either the AhR.Arnt heterodimer of Sp1 is bound to its cognate DNA element, DNA binding of the second factor is facilitated. A survey of DNA sequences in the promoter region shows that the XRE and GC box elements are commonly found in the genes whose expressions are induced by polycyclic aromatic hydrocarbons, suggesting that the two regulatory DNA elements and their cognate trans-acting factors constitute a common mechanism for induction of a group of drug-metabolizing enzymes (Kobayashi, 1996).

CBP/p300, an Arnt coactivator

A heterodimer of AhR (aryl hydrocarbon receptor) and Arnt (AhR nuclear translocator) conveys a transactivation signal of aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene to the genes for a group of drug-metabolizing enzymes. This inducible expression of the genes is inhibited by adenovirus E1A, suggesting that CBP/p300 is somehow involved in the transactivation of the genes by the AhR and Arnt heterodimer. Yeast and mammalian two hybrid systems revealed that CBP/p300 interacts with the transactivation domain of Arnt, but not with that of AhR, via the CREB-binding domain. A pull down assay using GST-Arnt hybrid protein confirms the interaction between Arnt and CBP/p300. Considering these results and that Arnt or Arnt2 functions as a common partner in the formation of transcriptional regulators with other bHLH/PAS proteins (such as AhR, HLF, and HIF-1alpha), the possibility arises that CBP/p300 is extensively involved as a coactivator in the transactivation process by bHLH/PAS heterodimer transcription factors through the interaction with Arnt or Arnt2 (Kobayashi, 1997).

Ah receptor/Arnt heterodimers

The human aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator protein (Arnt) were coexpressed in the yeast Saccharomyces cerevisiae to create a system for the study of the Ahr/Arnt heterodimeric transcription factor. Specific transcriptional activation mediated by AhR/Arnt heterodimer (which is a functional indicator of receptor expression) was assessed by beta-galactosidase activity produced from a reporter plasmid. Yeast expressing AhR and Arnt display constitutive transcriptional activity that is not augmented by the addition of AhR agonists in strains that required exogenous tryptophan for viability. In contrast, strains with an intact pathway for tryptophan biosynthesis do respond to AhR agonists and have lower levels of background beta-galactosidase activity. In the yeast system, hexachlorobenzene, benzo(a)pyrene, and beta-naphthoflavone are effective AhR agonists for beta-galactosidase activity induction. Tryptophan, indole, indole acetic acid, and tryptamine activate transcription in yeast coexpressing AhR and Arnt. Indole-3-carbinol is an exceptionally potent AhR agonist in yeast. This yeast system is useful for the study of AhR/Arnt protein complexes, and may prove to be generally applicable to the investigation of other multiprotein complexes (Miller, 1997).

In mouse hepatoma cells, the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, or dioxin) induces Cyp1A1 gene transcription, a process that requires two basic helix-loop-helix regulatory proteins: the aromatic hydrocarbon receptor (AhR) and the aromatic hydrocarbon receptor nuclear translocator (Arnt). Ligation-mediated PCR technique was used to analyze dioxin-induced changes in protein-DNA interactions and chromatin structure of the Cyp1A1 enhancer-promoter in its native chromosomal setting. Dioxin-induced binding of the AhR/Arnt heterodimer to enhancer chromatin is associated with a localized (about 200 bp) alteration in chromatin structure that is manifested by increased accessibility of the DNA; these changes probably reflect direct disruption of a nucleosome by AhR/Arnt. Dioxin induces analogous AhR/Arnt-dependent changes in chromatin structure and accessibility at the Cyp1A1 promoter. However, the changes at the promoter must occur by a different, more indirect mechanism, because they are induced from a distance and do not reflect a local effect of AhR/Arnt binding. Dose-response experiments indicate that the changes in chromatin structure at the enhancer and promoter are graded, mirroring the graded induction of Cyp1A1 transcription by dioxin (Okino, 1995).

Function of Ah receptor/Arnt heterodimers: a role for phosphorylation

The Ah receptor binds aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with high affinity. After binding aryl hydrocarbons, the receptor releases the 90-kDa heat shock protein and forms a heterodimer with the Arnt protein capable of binding at xenobiotic-responsive elements (XREs) and stimulating the transcription of genes involved in the metabolism of aryl hydrocarbons. The activity of the Ah receptor/Arnt dimer can be decreased by treatments that cause the down-regulation of protein kinase C and decrease the nuclear accumulation of the receptor. Incubation with acid phosphatase or with alkaline phosphatase has been reported to block XRE binding. Thus the literature suggests that phosphorylation regulates Ah receptor activity by affecting DNA binding and/or nuclear transport. A reporter plasmid containing two XREs was used to investigate the effects of phosphatase inhibitors on TCDD-dependent transcription carried out by the Hepa-1 mouse liver cell line. The inhibitors calyculin A and okadaic acid cause two- to threefold increases in TCDD-dependent transcription, at concentrations capable of selectively inhibiting protein phosphatase 1 and protein phosphatase 2A. The inhibitor cyclosporin A doubles TCDD-dependent transcription at a concentration capable of selectively inhibiting protein phosphatase 2B. All three of the phosphatase inhibitors increase TCDD-dependent transcription without affecting transcription in the absence of TCDD. Nuclear extracts were prepared from cells treated with concentrations of either okadaic acid or cyclosporin A, both of which substantially stimulate TCDD-dependent transcription. Neither of the inhibitors significantly increase the level of TCDD-dependent XRE binding in the extracts. GAL4-Arnt fusion proteins were used to further investigate whether the phosphatase inhibitors affected a step other than DNA binding. Okadaic acid treatment specifically increases the ability of a GAL4 fusion protein containing the Arnt PAS and transactivation domains to stimulate transcription. These results suggest that serine/threonine-specific protein phosphatases can act at a level subsequent to XRE binding to inhibit the ability of the Ah receptor/Arnt dimer to stimulate transcription (Li, 1997).

Interaction of Ahr with HSP90

Functional domains of the mouse aryl hydrocarbon receptor (Ahr) were investigated by deletion analysis. Ligand binding is localized to a region encompassing the PAS B repeat. The ligand-mediated dissociation of Ahr from the 90-kDa heat shock protein (HSP90) does not require the aryl hydrocarbon receptor nuclear translocator (Arnt), but it is slightly enhanced by this protein. One HSP90 molecule appears to bind within the PAS region. The other molecule of HSP90 appears to require interaction at two sites: one over the basic helix-loop-helix region, and the other located within the PAS region. Each mutant was analyzed for dimerization with full-length mouse Arnt and subsequent binding of the dimer to the xenobiotic responsive element (XRE). In order to minimize any artificial steric hindrances to dimerization and XRE binding, each Ahr mutant was also tested with an equivalently deleted Arnt mutant. The basic region of Ahr is required for XRE binding but not for dimerization. Both the first and second helices of the basic helix-loop-helix motif and the PAS region are required for dimerization. These last results are analogous to those previously obtained for Arnt, compatible with the notion that equivalent regions of Ahr and Arnt associate with each other. Deletion of the carboxyl-terminal half of Ahr does not affect dimerization or XRE binding but, in contrast to an equivalent deletion of Arnt, eliminates biological activity, as assessed by an in vivo transcriptional activation assay, suggesting that this region of Ahr plays a more prominent role in transcriptional activation of the cyp1a1 gene than does the corresponding region of Arnt (Fukunaga, 1995).

Expression of a series of Ah receptor (AhR) deletion mutants in an in vitro translation system has been previously used to map several functional domains of the murine AhR. In this report, quantitative immunoprecipitation of 90-kDa heat shock protein (hsp90) from reticulocyte lysate allowed a measurement of expression levels for the AhR and AhR deletion mutants, complexed with hsp90. After translation of a series of deletion mutants it was determined that there are two distinct domains important in forming a stable AhR/hsp90 complex, corresponding to amino acid sequences 1-166 and 289-347 of the AhR. Neither Arnt, nor Per are able to stably interact with hsp90. Thus, the AhR appears to be a unique member of the PAS domain family of proteins that binds a known ligand and stably interacts with hsp90 (Perdew, 1996).

CYP1A1, a target of Ah receptor/Arnt heterodimers

Introduction of a retroviral expression vector for the aryl hydrocarbon receptor (AhR) restores CYP1A1 inducibility to a mutant derivative of the Hepa-1 cell line that is defective in induction of CYP1A1 by ligands for the receptor. An AhR protein with normal ligand binding activity is expressed in the mutant but ligand treatment of mutant cell extract fails to induce binding of the AhR/Arnt dimer to the xenobiotic responsive element (XRE). AhR cDNAs derived from the mutant encode a protein that is unimpaired in ligand-dependent dimerization with Arnt, but the AhR.Arnt dimer so formed is severely impaired for XRE binding activity. The mutant cDNAs contain a C to G mutation at base 648, causing a cysteine to tryptophan alteration at amino acid 216, located between the Per-Arnt-Sim (PAS) homology region A and PAS B repeats. Introduction of the same mutation in the wild-type AhR sequence by site-directed mutagenesis similarity impairs XRE binding activity. Substitution with the conservative amino acid, serine, has no effect on XRE binding. The tryptophan mutation, but not the wild-type allele, is detectable in the genomic DNA of the mutant. The implication that an amino acid within the PAS region may be involved in DNA binding indicates that the DNA binding behavior of AhR may be more anomalous than previously suspected (Sun, 1997).

The ligand-activated aromatic hydrocarbon receptor (AhR) dimerizes with the AhR nuclear translocator (Arnt) to form a functional complex that transactivates expression of the cytochrome P-450 CYP1A1 gene and other genes in the dioxin-inducible [Ah] gene battery. The activity of the CYP1A1 enzyme negatively regulates this process. To study the relationship between CYP1A1 activity and Ah receptor activation CYP1A1-deficient mouse hepatoma c37 cells and CYP1A1- and AhR-deficient African green monkey kidney CV-1 cells were used. c37 cells that have not been exposed to exogenous Ah receptor ligands already contain transcriptionally active AhR-Arnt complexes, a finding that is also observed in wild-type Hepa-1 cells treated with Ellipticine, a CYP1A1 inhibitor. In CV-1 cells, transient expression of AhR and Arnt leads to high levels of AhR-Arnt-dependent luciferase gene expression, even in the absence of an agonist. Elevated reporter gene expression correlates with constitutive nuclear localization of the AhR. Transcriptional activation of the luciferase reporter gene observed in CV-1 cells is significantly decreased by (1) expression of a functional CYP1A1 enzyme, (2) competition with chimeric or truncated AhR proteins containing the AhR ligand-binding domain, and (3) treatment with the AhR antagonist alpha-naphthoflavone. These results suggest that a CYP1A1 substrate, which accumulates in cells lacking CYP1A1 enzymatic activity, is an AhR ligand responsible for endogenous activation of the Ah receptor (Chang, 1998).

A xenobiotic-responsive element (XRE)-binding factor(s) other than the AhR.Arnt complex inhibits the transcription of CYP1A1 gene in the liver from adult rabbits, known to be nonresponsive to CYP1A1 inducers. The constitutive factor(s) in liver nuclear extracts binds to the core sequence of XRE. The binding is eliminated by the presence of an excess amount of the AhR.Arnt complex synthesized in vitro. To identify the constitutive factor(s), a sequence similar to rabbit XRE was sought. The sequence of rabbit XRE overlaps with that of the upstream stimulatory factor 1 (USF1)-binding site in the mouse metallothionein I promoter. In fact, a super shift assay using a specific antibody against human USF1 indicates that USF1 is capable of binding to rabbit XRE. The AhR.Arnt-mediated activation of XRE-TK/Luc reporter gene in RK13 cells is blocked by the transfection with a USF1 expression vector with the amounts of the expression vector transfected. These results indicate that the XRE of the rabbit CYP1A1 gene is recognized by the basic helix-loop-helix proteins as a regulator of the expression of CYP1A1 in both an agonistic (AhR.Arnt) and an antagonistic (USF1) manner (Takahishi, 1997).

Transcriptional activation of the human CYP1A1 gene by halogenated and polycyclic aromatic hydrocarbons is mediated by the aryl hydrocarbon receptor (AhR) complex, a ligand-dependent transcription factor. A competent AhR comprises at least two components following nuclear translocation and DNA binding, the AhR and the AhR nuclear translocator (Arnt) protein, whose combined action on human CYP1A1 gene transcription is shown to be dependent upon functional protein kinase C (PKC). In the present study, the effects of phorbol 12-myristate 13-acetate, a potent PKC activator, were examined on the ligand-induced transcriptional activation of the CYP1A1 gene and cellular function of the AhR in human HepG2 101L cells. The 101L cells carry a stable transgene consisting of 1800 bases of 5'-flanking DNA and the promoter of the human CYP1A1 gene linked to the firefly luciferase structural gene. Pretreatment of cells with 12-myristate 13-acetate enhances ligand-induced CYP1A1 gene expression two- to three-fold. Inhibition of PKC activity blocks directly the transcriptional activation and the transactivation of the CYP1A1 gene, indicating a role for PKC in the AhR-mediated transcriptional activation process. However, the DNA binding activities of the in vitro activated and the induced nuclear AhR as measured by electrophoretic mobility shift analysis are not affected when CYP1A1 transcription is inhibited, indicating the actions of PKC to be a nuclear event that works in concert with or precedes AhR binding to the gene. These results illustrate that PKC is absolutely essential for the cellular and molecular events that control induction of CYP1A1 gene transcription (Chen, 1996).

Hypoxia-inducible factor 1 (HIF-1), and Arnt-containing, heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes

In response to hypoxia, the hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of a network of genes encoding erythropoietin, vascular endothelial growth factor, and several glycolytic enzymes. HIF-1 consists of a heterodimer of two basic helix-loop-helix PAS (Per/Arnt/Sim) proteins, HIF-1alpha (Drosophila homolog: Similar) and Arnt. HIF-1alpha and Arnt mRNAs are constitutively expressed and are not altered upon exposure of HeLa or HepG2 cells to hypoxia, suggesting that the activity of the HIF-1alpha-Arnt complex may be regulated by some as yet unknown posttranscriptional mechanism. In support of this model, it has been demonstrated that Arnt protein levels are not increased under conditions that induce an hypoxic response in HeLa and HepG2 cells. However, under identical conditions, HIF-1alpha protein levels are rapidly and dramatically up-regulated, as assessed by immunoblot analysis. In addition, HIF-1alpha acquires a new conformational state upon dimerization with Arnt, rendering HIF-1alpha more resistant to proteolytic digestion in vitro. Dimerization as such is not sufficient to elicit the conformational change in HIF-1alpha, since truncated forms of Arnt that are capable of dimerizing with HIF-1alpha do not induce this effect. The high affinity DNA binding form of the HIF-1alpha-Arnt complex is only generated by forms of Arnt capable of eliciting the allosteric change in conformation. In conclusion, the combination of enhanced protein levels and allosteric change by dimerization defines a novel mechanism for modulation of transcription factor activity (Kallio, 1997).

Hypoxia-inducible factor-1 (HIF-1), a DNA-binding complex implicated in the regulation of gene expression by oxygen, has been shown to consist of a heterodimer of two basic helix-loop-helix PAS proteins, HIF-1alpha, and HIF-1beta. One partner, HIF-1beta, is the aryl hydrocarbon receptor nuclear translocator (Arnt), an essential component of the xenobiotic response. In the present work, Arnt-deficient mutant cells, originally derived from the mouse hepatoma line Hepa1c1c7, have been used to analyze the role of Arnt/HIF-1beta in oxygen-regulated gene expression. Two stimuli were examined: hypoxia itself and desferrioxamine, an iron-chelating agent that also activates HIF-1. Induction of the DNA binding and transcriptional activity of HIF-1 is absent in the mutant cells, indicating an essential role for Arnt/HIF-1beta. Analysis of deleted Arnt/HIF-1beta genes indicates that the basic, helix-loop-helix, and PAS domains, but not the amino or carboxyl termini, are necessary for function in the response to hypoxia. Comparison of gene expression in wild type and mutant cells demonstrates the critical importance of Arnt/HIF-1beta in the hypoxic induction of a wide variety of genes. Nevertheless, for some genes a reduced response to hypoxia and desferrioxamine persists in these mutant cells, clearly distinguishing Arnt/HIF-1beta-dependent and Arnt/HIF-1beta-independent mechanisms of gene activation by both these stimuli (Wood, 1996).

Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding heterodimeric protein complex originally described in the transcriptional activation of the erythropoietin gene by hypoxia. This protein complex is composed of two subunits, HIF-1alpha and HIF-1beta (synonymous with aryl hydrocarbon receptor nuclear translocator, Arnt). In this study, Arnt-deficient cells, derived from the mouse hepatoma cell line Hepa1c1c7, were used to further characterize HIF-1 complex formation and its relationship with gene activation by hypoxia and desferrioxamine (Df). Gel shift assays reveal that Arnt is absolutely required for the formation of the HIF-1 DNA-binding complex. Results from RNase protection assays and Northern blots show that the lack of functional HIF-1 complex completely abrogates the response to hypoxia of three genes, vascular endothelial growth factor (VEGF), the glycolytic enzymes aldolase A (ALDA), and phosphoglycerate kinase 1 (PGK-1), each of which is known to be upregulated by low oxygen tension. Desferrioxamine induction of VEGF and PGK-1 genes is reduced in the Arnt-deficient cells, but unlike the response to hypoxia, the induction is not completely suppressed. These results suggest that Df is able to activate gene transcription through HIF-1-independent mechanisms. Exposure to either hypoxia or Df does not induce any changes in HIF-1alpha and -1beta mRNA levels, suggesting that posttranscriptional mechanisms are involved in HIF-1 complex activation (Salceda, 1996).

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes, including the human erythropoietin (EPO) gene. Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin gene in hypoxic cells. HIF-1 is involved in the activation of VEGF transcription. VEGF 5'-flanking sequences mediate transcriptional activation of reporter gene expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF transcription initiation site mediates hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element, which is otherwise minimally responsive to hypoxia. When reporters containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40 promoter, are cotransfected with expression vectors encoding HIF-1alpha and Arnt, reporter gene transcription is much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site is present in the 47-bp hypoxia response element; a 3-bp substitution eliminates the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF-1alpha inhibits the activation of reporter transcription in hypoxic cells in a dose-dependent manner. VEGF mRNA is not induced by hypoxia in mutant cells that do not express the Arnt subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic cells (Forsythe, 1996).

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia and dioxin signaling, respectively. Both proteins are conditionally regulated basic helix-loop-helix (bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) region of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt. HIF-1 alpha requires Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and PAS motifs of Arnt are critical for dimerization with HIF-1 alpha. Strikingly, HIF-1 alpha exhibits very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha inhibits ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated signaling pathways are not mutually exclusive, since activation of dioxin receptor function does not impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs are expressed constitutively in a large number of human tissues and cell lines, and these steady-state expression levels are not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1 alpha function. HIF-1 alpha is associated with the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism, possibly by binding to an as yet unknown class of ligands (Gradin, 1996).

bHLH PAS transcriptional regulators control critical developmental and metabolic processes, including transcriptional responses to stimuli such as hypoxia and environmental pollutants, mediated respectively by hypoxia inducible factors (HIF-alpha) and the dioxin (aryl hydrocarbon) receptor (DR). The bHLH proteins contain a basic DNA binding sequence adjacent to a helix-loop-helix dimerization domain. Dimerization among bHLH.PAS proteins is additionally regulated by the PAS region, which controls the specificity of partner choice such that HIF-alpha and DR must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes. Purified bacterially expressed proteins encompassing the N-terminal bHLH and bHLH.PAS regions of Arnt, DR, and HIF-1alpha have been analyzed and the contribution of the PAS domains to DNA binding in vitro was examined. Recovery of functional DNA binding proteins from bacteria was dramatically enhanced by coexpression of the bHLH.PAS regions of DR or HIF-1alpha with the corresponding region of Arnt. Formation of stable protein-DNA complexes by DR/Arnt and HIF-1alpha/Arnt heterodimers with their cognate DNA sequences requires the PAS A domains and exhibits KD values of 0.4 nM and approximately 50 nM, respectively. In contrast, the presence of the PAS domains of Arnt has little effect on DNA binding by Arnt homodimers, and these bind DNA with a KD of 45 nM. In the case of the DR, both high affinity DNA binding and dimer stability are specific to the Arnt native PAS domain, since a chimera in which the PAS A domain was substituted with the equivalent domain of Arnt generates a destabilized protein that binds DNA poorly (Chapman-Smith, 2004).

The structure and interactions of the C-terminal PAS domain of human HIF-2alpha has been studied by NMR spectroscopy. HIF-2alpha PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2alpha central beta-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending these findings to other PAS domains, this beta-sheet interface is found to be widely used for both intra- and inter-molecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins (Erbel, 2003).

Hypoxia-inducible factor-dependent histone deacetylase activity determines stem cell fate in the placenta

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of HIFalpha and the arylhydrocarbon receptor nuclear translocator (ARNT/HIF1ß). ARNT function is required for murine placental development. Cultured trophoblast stem (TS) cells were used to investigate the molecular basis of this requirement. In vitro, wild-type TS cell differentiation is largely restricted to spongiotrophoblasts and giant cells. Interestingly, Arnt-null TS cells differentiate into chorionic trophoblasts and syncytiotrophoblasts, as demonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and the HIV receptor CXCR4. During this process, a region of the differentiating Arnt-null TS cells undergo granzyme B-mediated apoptosis, suggesting a role for this pathway in murine syncytiotrophoblast turnover. Surprisingly, HIF1alpha and HIF2alpha are induced during TS cell differentiation in 20% O₂; additionally, pVHL levels are modulated during the same time period. These results suggest that oxygen-independent HIF functions are crucial to this differentiation process. Since histone deacetylase (HDAC) activity has been linked to HIF-dependent gene expression, whether ARNT deficiency affects this epigenetic regulator was investigated. Interestingly, Arnt-null TS cells have reduced HDAC activity, increased global histone acetylation, and altered class II HDAC subcellular localization. In wild-type TS cells, inhibition of HDAC activity recapitulates the Arnt-null phenotype, suggesting that crosstalk between the HIFs and the HDACs is required for normal trophoblast differentiation. Thus, the HIFs play important roles in modulating the developmental plasticity of stem cells by integrating physiological, transcriptional and epigenetic inputs (Maltepe, 2005).

Characterization of murine ARNT interacting protein (AINT)

Basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) proteins form dimeric transcription factors to mediate diverse biological functions including xenobiotic metabolism, hypoxic response, circadian rhythm and central nervous system midline development. The Ah receptor nuclear translocator protein (ARNT) plays a central role as a common heterodimerization partner. A novel, embryonically expressed, ARNT interacting protein (AINT) is described that may be a member of a larger coiled-coil PAS interacting protein family. The AINT C-terminus mediates interaction with the PAS domain of ARNT in yeast and interacts in vitro with ARNT and ARNT2 specifically. AINT localizes to the cytoplasm and overexpression leads to non-nuclear localization of ARNT. A dynamic pattern of AINT mRNA expression during embryogenesis and cerebellum ontogeny supports a role for AINT in development (Sadek, 2000).

The AINT C-terminus is homologous to two human proteins, TACC1 and TACC2. TACC1 (accession no. AF049910) is embryonically expressed and constitutive expression results in transformation and anchorage independent growth of mouse fibroblasts. The TACC2 (accession no. AF095791) gene is located on chromosome 10 and has yet to be described further. Three additional TACC-related proteins have been described. The coiled-coil region of Xenopus maskin is most closely related to AINT and TACC3 and interacts with CPEB and eIF-4E to restrict polyadenylation-induced translation during oocyte matura- tion. Drosophila TACC (D-TACC) has been shown to interact with and stabilize centrosomal microtubules in embryo extracts via the C-terminal region (Gergely, 2000). Finally, AZU-1, a variant of TACC2, is implicated in tumor suppression and tissue morphogenesis of epithelial cells. A C. elegans sequence cloned from chromosome III (accession no. Q9XWJ0) also appears to encode the conserved coiled-coil domain of TACC proteins although functionally this has not been characterized. To compare this growing protein family and determine important conserved sequences in the coiled-coil domains, the C-terminal ends of each member were aligned and a phylogenetic tree was constucted based on this conserved region. The existing TACC proteins can be aligned into two distinct clades, one consisting of TACC1 and TACC2 proteins and a second containing TACC3 proteins, to which AINT belongs. With the sudden growth in TACC family members it will be interesting to see if future members will resolve the first clade into two separate groups for TACC1 and TACC2 proteins (Sadek, 2000).

AINT is not a bHLH-PAS protein. Many recent studies in the field of bHLH-PAS proteins have served to identify novel family members and most mechanisms described thus far are examples of heterodimerization of two bHLH-PAS proteins via the PAS domain. However, it has become increasingly apparent that PAS proteins also interact heterotypically with non-PAS proteins such as AINT. Examples of this type of interaction may be seen at different stages of the signal transduction pathway. Early in the transduction pathway the AhR interacts with HSP90 and AIP/ARA9/XAP in its non-liganded state, perhaps to maintain a conformation that recognizes ligand. The Drosophila PAS protein Period (Per) forms heterodimers with the circadian clock protein Timeless (Tim), which does not contain a PAS domain. This heterodimerization is important for the regulation of circadian rhythms and is mediated by the PAS domain of Per. In the later stages of signal transduction, HIF-1alpha actively recruits CBP to the transcriptional complex. In addition, SRC-1, SRC-2, and SRC-3 have not been widely studied in the context of PAS interactions and, instead, have been shown to bridge interactions between nuclear receptors and basal transcription machinery via conserved LXXLL motifs termed nuclear receptor boxes. From these many examples, it might not be unusual to have isolated a non-bHLH-PAS protein in the screening for ARNT interacting proteins. Of the heterotypic interactions mentioned above, the Per-Tim interaction may be most relevant in the context of AINT. In the same way that Tim interacts with the PAS domain of Per, evidence is presented that AINT requires an intact PAS domain of ARNT for interaction. Because the PAS domain provides the dimerization surface between many proteins, this could be a clue to the function of AINT in either enhancing or disrupting homotypic interactions between ARNT and other PAS proteins (Sadek, 2000).

Hypoxia requires notch signaling to maintain the undifferentiated cell state

In addition to controlling a switch to glycolytic metabolism and induction of erythropoiesis and angiogenesis, hypoxia promotes the undifferentiated cell state in various stem and precursor cell populations. The latter process requires Notch signaling. Hypoxia blocks neuronal and myogenic differentiation in a Notch-dependent manner. Hypoxia activates Notch-responsive promoters and increases expression of Notch direct downstream genes. The Notch intracellular domain (ICD) interacts with HIF-1alpha, a global regulator of oxygen homeostasis, and HIF-1alpha is recruited to Notch-responsive promoters upon Notch activation under hypoxic conditions. Taken together, these data provide molecular insights into how reduced oxygen levels control the cellular differentiation status and demonstrate a role for Notch in this process (Gustafsson, 2005).

The data presented here indicate that Notch ICD and HIF-1α are important at the convergence point between the two signaling mechanisms. The importance of Notch ICD is underlined by the ability of γ-secretase inhibitors, which block the S3 cleavage of the Notch receptor and thus liberation of Notch ICD, to strongly reduce the hypoxic response on Notch downstream genes and promoters. Furthermore, the signaling output from an exogenously introduced Notch 1 ICD was modified by hypoxia, leading to increased activation of 12XCSL-luc and Hes-luc in a Notch 1 ICD-dependent manner. The importance of HIF-1α in this process receives support from the observed direct physical interaction between HIF-1α and Notch 1 ICD, the lack of an hypoxia-induced effect on Notch signaling in fibroblasts devoid of HIF-1α, and that both the amount and activity status of HIF-1α correlate with the level of Notch activation. The latter notion is based on the observations that: (1) transfected HIF-1α elevates the Notch downstream response; (2) a transactivation-inactive form of HIF-1α leaves the Notch response unchanged, and (3) the response is augmented in cells lacking VHL. Finally, HIF-1α is recruited to the Hey-2 promoter in C2C12 cells in a Notch- and hypoxia-dependent manner. This suggests a mechanism involving an effect of direct transcriptional activation of a Notch-responsive promoter by HIF-1α, probably as part of a Notch ICD/CSL transcriptional complex. This model is consistent with the observation that a transcriptionally inactive form of HIF-1α, which was capable of interacting with Notch 1 ICD, does not augment the Notch downstream response. It is therefore reasoned that hypoxia-dependent stabilization of Notch 1CD is not sufficient for activation of the Notch response but may require the recruitment of a form of HIF-1α containing the C-terminal transactivation domain to the Notch ICD/CSL regulatory complex, possibly potentiating the interaction with transcriptional coactivators. This hypothesis is based on the finding that HIF-1α is recruited to promoters of Notch downstream genes and the observation that a mutated form of Notch ICD, unable to interact with CBP/p300, is transcriptionally active at hypoxia. In this context, it will be interesting to learn whether HIF-1β also participates in such a regulatory complex (Gustafsson, 2005).

The link between hypoxia and Notch described here may have ramifications for other aspects of hypoxia, such as tumor development, in which deregulation of both HIF-1α- and Notch-mediated signaling events have been implicated. Since many tumors show elevated expression of HIF-1α, caused by hypoxia inherent to growing tumors and/or genetic loss of VHL, it will be interesting to investigate whether the elevated levels of HIF-1α are paralleled by increased Notch signaling, and whether the ensuing Notch induction contributes to tumor development (Gustafsson, 2005).

In conclusion, the data presented here demonstrate a link between hypoxia and Notch signaling and provide insights into how hypoxia maintains the undifferentiated cell state, by using the Notch signaling mechanism. The data also point to an important role for HIF-1α in this process and to the fact that it can interact with the Notch intracellular domain to link hypoxic information to a Notch response. These data advance the understanding of how Notch crosstalks with other signaling mechanisms and may open up possibilities to control various aspects of the hypoxic response by experimentally manipulating Notch signaling (Gustafsson, 2005).

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