BIOLOGICAL OVERVIEW

bHLH-PAS proteins represent a class of transcription factors involved in diverse biological activities. Previous experiments have demonstrated that the PAS domain confers target specificity. This suggests an association between the PAS domain and additional DNA-binding proteins, Such an association is essential for the induction of specific target genes. A candidate for interaction with PAS domain protein Trachealess (Trh) is Drifter/Ventral veinless. A dual requirement for Trh and Drifter has been identified for the autoregulation of Trh and Drifter expression. Furthermore, ectopic expression of both Trh and Dfr (but not each one alone) triggers trh autoregulation in several embryonic tissues. A direct interaction between Drifter and Trh proteins, mediated by the PAS domain of Trh and the POU domain of Drifter, has been demonstrated (Zelzer, 2000).

Transcription of the trh gene is autoregulated, thus maintaining its expression throughout tracheal development, after the initial cues that determine the position of the tracheal placodes have disappeared. However, several experimental results suggest that the Trh/ARNT heterodimer is not sufficient for autoregulation of the trh gene. (1) Examination of Trh-Sim chimeras demonstrates that target gene specificity is determined by the PAS domain, possibly through interactions with other proteins. (2) Ubiquitous Trh can induce ectopic trh expression occasionally, at stage 11, but only at the position of tracheal pits in segments that do not normally form tracheal pits, suggesting that additional protein(s) expressed in this pattern need to cooperate with Trh. A candidate protein that may interact with Trh is the POU-domain protein Drifter/Ventral veinless (Dfr). This protein was previously shown to participate in tracheal morphogenesis. Initially, dfr is expressed in the ten tracheal placodes, as well as in the position of placodes in segments that normally do not produce tracheal pits. dfr mutations show a reduced expression of tracheal-specific genes such as breathless (btl), and accordingly exhibit migration defects that are reminiscent of the btl phenotype (Zelzer, 2000 and references).

An important feature of both Trh and Dfr expression is their capacity to be autoregulated. Once the exogenous cues that direct expression of these genes in the tracheal placodes diminish, expression is maintained by autoregulation. Since the trh and dfr genes themselves can be regarded as targets for Trh or Dfr, respectively, a test was performed to see whether autoregulation of each of the two genes requires both Trh and Dfr. Two phases of Trh expression have been defined; at stage 12, expression induced by exogenous cues is diminished and autoregulation ensues. Staining for the Trh protein in dfr mutant embryos has demonstrated that the initial phase of Trh expression in the placodes is normal. However, starting at stage 12 the levels of Trh are reduced, and are almost undetectable by stage 15. Failure of the cells in the tracheal pits of dfr mutant embryos to express Trh is not due to the death of these cells. Previous examination of the tracheal pits of dfr mutant embryos has shown that the cells are viable and capable of secreting tracheal lumen material, regardless of their failure to migrate properly. It can be concluded that Dfr is required for the autoregulation, and hence the maintenance of trh expression (Zelzer, 2000).

In the case of Dfr, a distinct 514 bp fragment has been defined as the dfr-autoregulatory element, which begins to drive Dfr expression at stage 11/12. This fragment also confers expression in the oenocytes. In trh mutant embryos, lacZ expression driven by this fragment in the oenocytes is retained, but completely abolished in the trachea. Again, the absence of expression in the tracheal placodes, which fail to invaginate in the trh mutant background, is not due to death of these cells. Staining of trh mutant embryos with anti-Dfr antibodies or with a probe detecting dfr RNA, has revealed the early, Trh-independent phase of expression up to stage 11. The uninvaginated placode cells in trh mutant embryos are thus intact, but fail to express the dfr autoregulation reporter. These experiments demonstrate that Trh and Dfr are required simultaneously for the autoregulation of Trh and Dfr themselves (Zelzer, 2000).

Rho (Rhomboid) functions as a regulator for processing the EGF receptor ligand Spitz, and is expressed a embryonic stage 9/10 in the midline glial cells, as well as in cells positioned at the center of the tracheal placodes. The parallel expression of rho in the tissues where Sim and Trh are functional, suggests that it may be a transcriptional target of these two bHLH-PAS proteins. In trh mutant embryos, expression of rho in the tracheal placodes is abolished. Similarly, in sim mutant embryos, expression of rho in the midline is eliminated. To determine if rho expression is regulated by direct binding of Sim and Trh, a 762 bp fragment of the rho 50 regulatory region was dissected: this is sufficient for midline and tracheal expression. The sequence of this fragment contains four sites with the Sim/Trh (ST) binding consensus. Similar sites have previously been shown by in vivo and in vitro analysis to represent the binding sites for Sim/ARNT or Trh/ARNT heterodimers. The 762 bp rho regulatory region was further dissected, and the capacity of smaller fragments to induce midline or tracheal expression in embryos was followed. The following conclusions were reached: Sim/Trh binding sites STc and STd are neither sufficient nor necessary for tracheal or midline expression. In contrast, Sim/Trh binding sites STa and STb are essential for midline and tracheal expression. Distinct cis elements appear to be required to promote midline vs. tracheal expression (Zelzer, 2000).

The paradigm that Trh or Dfr alone are not sufficient to induce their target genes or autoregulation, broadens the scope of activities of the two proteins. Trh is required not only for the induction of tracheal fates, but also for patterning the salivary ducts and posterior spiracles. It is possible that in these tissues, Trh associates with other proteins and induces a different set of tissue-specific target genes. Similarly, Dfr is also expressed in the midline cells. Dfr is not necessary for the induction of Sim-target genes, as can be deduced from the normal expression of rhomboid in the midline of dfr-mutant embryos. However, Dfr could be functioning in conjunction with other midline proteins such as the Sox-domain protein Dichaete (Zelzer, 2000).

Genome-wide identification of Grainy head targets in Drosophila reveals regulatory interactions with the POU-domain transcription factor, Vvl

Grainy head (Grh) is a conserved transcription factor (TF) controlling epithelial differentiation and regeneration. To elucidate Grh functions, embryonic Grh targets were identified by ChIP-seq and gene expression analysis. Grh was shown to control hundreds of target genes. Repression or activation correlates with the distance of Grh binding sites to the transcription start sites of its targets. Analysis of 54 Grh-responsive enhancers during development and upon wounding suggests cooperation with distinct TFs in different contexts. In the airways, Grh repressed genes encode key TFs involved in branching and cell differentiation. Reduction of the POU-domain TF, Vvl, (ventral veins lacking) largely ameliorates the airway morphogenesis defects of grh mutants. Vvl and Grh proteins additionally interact with each other and regulate a set of common enhancers during epithelial morphogenesis. It is concluded that Grh and Vvl participate in a regulatory network controlling epithelial maturation (Yao, 2017).

Grh controls epithelial development and regeneration in multiple organisms. ChIP-seq data provide a broad view of Grh-binding to its targets in all Grh-expressing tissues. The analysis of Grh-dependent regulatory sequences indicates that the majority of the 5599 peaks that include the consensus Grh-binding sequence identify true Grh targets. Hitherto, analysis of Grh targets in development focused on proteins involved in epidermal barrier formation, adhesion molecules and junctional proteins. Identification of functional Grh targets in the airways adds large groups of proteins involved in lipid metabolism, cell signaling and TFs. This suggests additional functions of Grh in tubulogenesis that might explain several of its additional roles. For example, the phenotype of grh mutants in the airways includes the selective expansion of the epithelial apical membranes, a phenotype that has not been detected in any of the mutants affecting junctional proteins or the formation and modification of the apical extracellular barrier. Definition of new Grh targets during airway maturation provides a rich resource for future studies addressing how Grh controls epithelial morphogenesis (Yao, 2017).

A prevalent group of Grh targets in the epidermis and airways includes genes involved in innate immune responses ranging from pattern recognition receptors to effectors. Interestingly, several putative GRHL2 targets in human bronchial epithelial cells, such as serpins and chitinase 3-like proteins, have been implicated in immune responses. Analysis of PGRP-LC reveals a direct role of Grh in endowing epithelial cells the ability to combat infections. Although the PGRP-LC reporter expression was not inducible by wounding or bacterial injection, it remains possible that Grh also directly controls the activation of epithelial immune responses upon infection. Indeed, partial inactivation of Grh by RNAi in adult flies resulted in increased morbidity and mortality upon bacterial infection (Yao, 2017).

Analysis of 47 new Grh-activated enhancers in epithelial development suggests the presence of distinct, tissue-specific Grh co-factors in the control of target genes in different epithelial cell types. The activation of some of these reporters upon injury expands the repertoire of Grh-activated enhancers and is in line with previous models proposing wound-induced interactions of Grh with other factors. These interactions could be induced by post-translational modifications of Grh or its co-factors by Rolled and other kinases downstream of Stit receptor kinase signaling and might facilitate the activation of transcription by Grh pre-bound to chromatin (Yao, 2017).

The ChIP-seq and gene expression analysis also reveal a potential role for Grh as a repressor. Such a repressor function of Grh is consistent with previous studies addressing Grh function on individual targets in flies and mammals. A higher correlation of PcG-binding sites and repressive chromatin marks were found around the Grh-binding sites of repressed targets as compared with the binding sites of activated genes. The positioning of Grh-binding sites relative to the TSS of repressed versus activated or unaffected genes also differs: Grh-binding sites are usually further from the TSS in repressed target genes. This observation is supported by the analysis of vvl ds3 and vvl 1.8, the only two identified repressible enhancers, which are located more than 2 kb from the vvl TSS. The difference in the structure of the repressed and activated Grh enhancers suggests that Grh repression might require chromatin looping and involve co-repressors. Further work is needed to elucidate a potential direct function of Grh in transcriptional repression (Yao, 2017).

A characteristic group of Grh targets in the airways includes TFs involved in epithelial cell differentiation. This resembles the complex regulatory functions of Grh during neuronal specification. For instance, in neuroblasts, Grh demarcates the last time window for TF expression by repressing Castor. In intermediate neural progenitors (INPs), Grh is detected in the 'middle-aged' INPs and overlaps with the expression of the TFs Dichaete and Eyeless. The three TFs cross-regulate each other. Similar cross-talk between Grh and its TF targets might specify and maintain epithelial differentiation. Since reduction of vvl in grh mutants largely ameliorates the tube elongation defects, the direct or indirect repression of genes encoding TFs is likely to be a crucial function of Grh in the airways. The shared expression pattern of Vvl and Grh, their binding to a set of common enhancers and their ability to form complexes suggest that they collectively control tube growth during airway maturation. Given their co-expression in other contexts, they might also co-operate during neural cell specification and epithelial immune responses (Yao, 2017).

Earlier descriptions of Vvl biology

In developmental biology it often happens that a gene product can serve multiple functions, and be involved at different developmental stages as well. Because of the long history of research into Drosophila developmental biology, genes may receive multiple names, corresponding to their multiple functions. For example, the genes ventral veins lacking (aka: ventral veinless) and drifter were both cloned in 1995. They turn out to be two names for the same gene, a gene previously called Cf1, identified as a POU-domain gene, one that binds to a neuron-specific regulatory element C, of the dopa decarboxylase gene. (Johnson, 1990). Two other POU-homeodomain transcription factors are found in Drosophila, PDM-1 and PDM-2.

drifter is the name given to vvl based on vvl's tracheal phenotype. drifter mutants display severe tracheal defects and defects in ventral midline glia migration. The glial defects result in defective commissure formation resulting from defects in axon pathfinding. Drifter is expressed in midline glia and in tracheal cells (Anderson, 1995).

Defects in tracheal cell migration are similar to those seen in breathless and pointed mutations. breathless is a homolog of the vertebrate FGF receptor tyrosine kinase; pointed is an ETS domain transcription factor. breathless functions upstream of Ras and Raf. The tracheal and glial cell defects seem attributable to defective cell migration. It is therefore likely that genes affecting migration are regulated by Drifter. It would be reasonable to look at the ras/raf pathway as a candidate for ventral veins lacking regulation.

Slow border cells, a gene implicated in migration of follicle cells is unlikely to regulate breathless expression during embryogenesis because SLBO expression in the tracheal system does not begin until long after breathless expression (Rorth, 1992). Drifter, may enhance btl expression in tracheal cells. Drifter protein is expressed in tracheal cells near the time that btl expression initiates: the dfr mutant phenotype is similar to btl; and dfr expression is not altered in btl mutants (Anderson, 1995). Thus it is possible, even likely, that dfr regulates btl expression. Preliminary experments suggest that dfr is not expressed in the border cells. One interpretation then, is that DFR may regulate btl in the embryo in much the same way that SLBO does in the ovary (Murphy, 1995).

ventral veins lacking mutations evince a variable phenotype consisting of the absence of proximal stretches of specific wing veins. It appears that mutant clones in dorsal veins do not affect ventral veins, and vice versa. vvl is expressed in both dorsal and ventral regions of the presumptive wing blade and wing base coinciding with the sites of future veins. A similar expression profile has been noted for rhomboid, a gene central to the induction of wing veins. Since rhomboid is involved in enhancing responses to the EGF receptor, which triggers the ras/raf pathway, this pathway is again implicated in vvl regulation (de Celis, 1995).

What are the targets of vvl and its cognate drifter? Are they the same or do they have significant differences? To what extent do they overlap? Perhaps dopa decarboylase is a common element in both functions. Knowing the array of targets could result in some generalization as to why the same transcription factor has a role in such different developmental pathways.

Earlier descriptions of Vvl biology

GENE STRUCTURE

The use of alternative polyadenylation sites produces two VVL mRNA transcripts (Anderson, 1995).

Exons - one

Bases in 3' UTR - 1588

PROTEIN STRUCTURE

Structural Domains

The POU domain protein includes both a divergent homeodomain and an additional POU-specific domain that function together as a bipartite DNA-binding domain (Klemm 1994).

The 75 amino acid POU-specific (POUs) domain and a 60 amino acid carboxy-terminal homeo (POUh) domain are joined by a hypervariable linker segment that can vary from 15 to 56 amino acids in length in different POU domain proteins. Thus the POU domain is not a single structural domain; indeed, the POUs and POUh segments form separate structurally independent domains. The POUs and POUh domains are, however, always found together and have therefore coevolved. Both POUs and POUh domains contain helix-turn-helix motifs. The POUs-domain structure is very similar to that of lambda and 434 bacteriophage proteins, but there are significant differences in the length of the first alpha helix, and the "turn" connecting the two HTH alpha helices is also longer. Both POUs and POUh bind DNA, and the length of the linker regulates the efficacy of binding various DNA sequence motifs, especially because POUs and POUh DNA binding sites have different spacings in different promoter elements (Herr, 1995).

date revised: 20 Feb 97 

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