An efficient approach to isolate STAT regulated enhancers uncovers STAT92E fundamental role in Drosophila tracheal development

Abstract

The ventral veinless (vvl) and trachealess (trh) genes are determinants of the Drosophila trachea. Early in development both genes are independently activated in the tracheal primordia by signals that are ill defined. Mutants blocking JAK/STAT signaling at any level do not form a tracheal tree suggesting that STAT92E may be an upstream transcriptional activator of the early trachea determinants. To test this hypothesis we have searched for STAT92E responsive enhancers activating the expression of vvl and trh in the tracheal primordia. We show that STAT92E regulated enhancers can be rapidly and efficiently isolated by focusing the analysis on genomic regions with clusters of putative STAT binding sites where at least some of them are phylogenetically conserved. Detailed analysis of a vvl early tracheal enhancer shows that non-conserved sites collaborate with conserved sites for enhancer activation. We find that STAT92E regulated enhancers can be located as far 60 kb from the promoters. Our results indicate that vvl and trh are independently activated by STAT92E which is the most important transcription factor required for trachea specification.

JAK/Stat signaling regulates heart precursor diversification inDrosophila

“…The heart tube formation defects in Stat92E Membryos therefore reflect the pleiotropic functions of the JAK/Stat pathway outside the mesoderm. For example, JAK/Stat signaling regulates segmentation (Harrison et al, 1998), ectoderm morphogenesis (Bertet et al, 2009) and the development of ectoderm derivatives, including the tracheal system (Sotillos et al, 2010). Incomplete ectoderm development probably exacerbates the mesoderm patterning defects in Stat92E M-embryos, causing incomplete cardiac morphogenesis.…”

“…The ChIPchip analysis identified additional Stat92E target genes including ventral veins lacking and trachealess (Sotillos et al, 2010) (see Table S1 in the supplementary material), suggesting that the ChIPchip data would be a useful tool to identify Stat92E target genes in the mesoderm. Although a total of 107 loci fall within 10 kb of a reported Stat92E binding region (see Table S1 in the supplementary material), the tin transcriptional start site is over 262 kb from the closest Stat92E binding region (3R:17466843).…”

“…Because the HLHm5 cisregulatory region lacks a conserved SCBS, we predict that Stat92E regulates HLHm5 expression through a non-consensus binding site. Alternatively, Stat92E acts at long distances to regulate gene expression (Sotillos et al, 2010). The SCBSs in E(spl)-C could be 4635 RESEARCH ARTICLE Stat92E restricts tin expression a platform from which Stat92E regulates multiple E(spl)-C genes that, in turn, regulate HLHm5 expression.…”

Trachealess (Trh) regulates all tracheal genes during Drosophila embryogenesis

“…The dorsal limit on trachea formation is set by the dorsal patterning gene decapentaplegic ( dpp ) (Isaac and Andrew, 1996; Wilk et al, 1996). The segment polarity gene wingless ( wg ) limits formation of tracheal primordia to the cells between its stripes of expression within each segment (de Celis et al, 1995; Wilk et al, 1996), whereas STAT92E, the single Drosophila STAT, which also functions as a segment polarity gene in early development, is essential for tracheal formation (Brown et al, 2001; Li et al, 2003; Sotillos et al, 2010). …”

“…Indeed, more recent findings reveal that the initial expression of both trh and vvl is directly and independently activated by STAT92E (Sotillos et al, 2010). vvl has been reported to be required for the maintenance of trh expression, suggesting that although the initial expression of early tracheal transcription factor genes is independent of the others, these genes subsequently become dependent on each other for their continued expression (Zelzer and Shilo, 2000).…”