tango

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).

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).

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