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).

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).

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).

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

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