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).

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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