SUMMARYRTK/Ras/MAPK signaling pathways play key functions in metazoan development, but how they control expression of downstream genes is not well understood. In Drosophila, it is generally assumed that most transcriptional responses to RTK signal activation depend on binding of Ets-family proteins to specific cis-acting sites in target enhancers. Here, we show that several Drosophila RTK pathways control expression of downstream genes through common octameric elements that are binding sites for the HMGbox factor Capicua, a transcriptional repressor that is downregulated by RTK signaling in different contexts. We show that Torso RTK-dependent regulation of terminal gap gene expression in the early embryo critically depends on Capicua octameric sites, and that binding of Capicua to these sites is essential for recruitment of the Groucho co-repressor to the huckebein enhancer in vivo. We then show that subsequent activation of the EGFR RTK pathway in the neuroectodermal region of the embryo controls dorsal-ventral gene expression by downregulating the Capicua protein, and that this control also depends on Capicua octameric motifs. Thus, a similar mechanism of RTK regulation operates during subdivision of the anterior-posterior and dorsal-ventral embryonic axes. We also find that identical DNA octamers mediate Capicua-dependent regulation of another EGFR target in the developing wing. Remarkably, a simple combination of activator-binding sites and Capicua motifs is sufficient to establish complex patterns of gene expression in response to both Torso and EGFR activation in different tissues. We conclude that Capicua octamers are general response elements for RTK signaling in Drosophila.

Crosstalk between signaling pathways is crucial for the generation of complex and varied transcriptional networks. Antagonism between the EGF-receptor (EGFR) and Notch pathways in particular is well documented, although the underlying mechanism is poorly understood. The global corepressor Groucho (Gro) and its transducin-like Enhancer-of-split (TLE) mammalian homologs mediate repression by a myriad of repressors, including effectors of the Notch, Wnt (Wg) and TGF-beta (Dpp) signaling cascades. Given that there are genetic interactions between gro and components of the EGFR pathway (ref. 9 and P.H. et al., unpublished results), we tested whether Gro is at a crossroad between this and other pathways. Here we show that phosphorylation of Gro in response to MAPK activation weakens its repressor capacity, attenuating Gro-dependent transcriptional silencing by the Enhancer-of-split proteins, effectors of the Notch cascade. Thus, Gro is a new junction between signaling pathways, enabling EGFR signaling to antagonize transcriptional output by Notch and potentially other Gro-dependent pathways.

Groucho (Gro) is a Drosophila melanogaster transcriptional corepressor that directly interacts with the histone deacetylase Rpd3. Although previous studies suggest that this interaction is required for repression of Gro-responsive reporters in cultured cells, the in vivo significance of this interaction and the mechanism by which it leads to repression remain largely unexplored. In this study, we show that Gro is partially dependent on Rpd3 for repression, supporting the idea that Rpd3-mediated repression is one mode of Gro-mediated repression. We demonstrate that Gro colocalizes with Rpd3 to the chromatin of a target gene and that this is accompanied by the deacetylation of specific lysines within the N-terminal tails of histones H3 and H4. Gro overexpression leads to wing patterning defects and ectopic repression in the wing disc of transcription directed by the vestigial quadrant enhancer. These effects are reversed by the histone deacetylase inhibitors TSA and HC-Toxin and by the reduction of Rpd3 gene dosage. Furthermore, repression of the vestigial quadrant enhancer is accompanied by a Gro-mediated increase in nucleosome density, an effect that is reversed by histone deacetylase inhibitors. We propose a model in which Gro-mediated histone deacetylation results in increased nucleosome density leading to transcriptional repression.

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The Drosophila Groucho (Gro) protein is the founding member of a family of metazoan transcriptional corepressors with diverse roles in development. Gro directs long‐range repression, which contrasts with the short‐range repression directed by the corepressor CtBP. However, the mechanistic difference between these two types of repression is not clear. To determine the structure of domains of Gro‐mediated repression, we examined the genome‐wide distribution of Gro‐binding sites in early embryos and wing discs by ChIP‐chip. Our findings suggest that communication between the sites to which repressors recruit Gro and promoters may involve a combination of linear Gro spreading and DNA looping. This, in turn, leads to an increase in nucleosome density. The increase in nucleosome density appears to require histone deacetylase activity, as the wing discs of flies raised in media containing HDAC inhibitors show dramatically increased levels of histone H3 and histone H4 acetylation coincident with reduced repression and decreased histone density at Gro target genes.