Relatively little is known about the molecular mechanisms involved in transcriptional repression, despite its importance in development and differentiation. Recent evidence suggests that some transcriptional repressors act by way of adaptor molecules known as corepressors. Here, we use in vivo functional assays to test whether different repressor activities are mediated by the Groucho (Gro) corepressor in the Drosophila embryo. Previously, Gro was proposed to mediate repression by the Hairy-related family of basic helix-loop-helix proteins. Our results indicate not only that repression by Hairy requires Gro, but that a repressor domain from the Engrailed (En) homeodomain protein is also Gro dependent. The latter result correlates with an ability of this En domain to bind to Gro in vitro. In contrast, repressor regions from the Even-skipped, Snail, Krü ppel, and Knirps transcription factors are effective in the absence of Gro. These results show that Gro is not generally required for repression, but acts as a specific corepressor for a fraction of negative regulators, including Hairy and En.

Pax5 (BSAP) functions as both a transcriptional activator and repressor during midbrain patterning, B-cell development and lymphomagenesis. Here we demonstrate that Pax5 exerts its repression function by recruiting members of the Groucho corepressor family. In a yeast two-hybrid screen, the grouchorelated gene product Grg4 was identi®ed as a Pax5 partner protein. Both proteins interact cooperatively via two separate domains: the N-terminal Q and central SP regions of Grg4, and the octapeptide motif and C-terminal transactivation domain of Pax5. The phosphorylation state of Grg4 is altered in vivo upon Pax5 binding. Moreover, Grg4 ef®ciently represses the transcriptional activity of Pax5 in an octapeptidedependent manner. Similar protein interactions resulting in transcriptional repression were also observed between distantly related members of both the Pax2/5/8 and Groucho protein families. In agreement with this evolutionary conservation, the octapeptide motif of Pax proteins functions as a Groucho-dependent repression domain in Drosophila embryos. These data indicate that Pax proteins can be converted from transcriptional activators to repressors through interaction with corepressors of the Groucho protein family.

Early Drosophila development requires two receptor tyrosine kinase (RTK) pathways: the Torso and the Epidermal growth factor receptor (EGFR) pathways, which regulate terminal and dorsal-ventral patterning, respectively. Previous studies have shown that these pathways, either directly or indirectly, lead to post-transcriptional downregulation of the Capicua repressor in the early embryo and in the ovary. Here, we show that both regulatory effects are direct and depend on a MAPK docking site in Capicua that physically interacts with the MAPK Rolled. Capicua derivatives lacking this docking site cause dominant phenotypes similar to those resulting from loss of Torso and EGFR activities. Such phenotypes arise from inappropriate repression of genes normally expressed in response to Torso and EGFR signaling. Our results are consistent with a model whereby Capicua is the main nuclear effector of the Torso pathway, but only one of different effectors responding to EGFR signaling. Finally, we describe differences in the modes of Capicua downregulation by Torso and EGFR signaling, raising the possibility that such differences contribute to the tissue specificity of both signals.

SummaryReceptor tyrosine kinase (RTK) signaling pathways control multiple cellular decisions in metazoans, often by regulating the expression of downstream genes. In Drosophila melanogaster and other systems, E-twenty-six (ETS) transcription factors are considered to be the predominant nuclear effectors of RTK pathways. Here, we highlight recent progress in identifying the HMG-box protein Capicua (CIC) as a key sensor of RTK signaling in both Drosophila and mammals. Several studies have shown that CIC functions as a repressor of RTK-responsive genes, keeping them silent in the absence of signaling. Following the activation of RTK signaling, CIC repression is relieved, and this allows the expression of the targeted gene in response to local or ubiquitous activators. This regulatory switch is essential for several RTK responses in Drosophila, from the determination of cell fate to cell proliferation. Furthermore, increasing evidence supports the notion that this mechanism is conserved in mammals, where CIC has been implicated in cancer and neurodegeneration. In addition to summarizing our current knowledge on CIC, we also discuss the implications of these findings for our understanding of RTK signaling specificity in different biological processes. IntroductionReceptor tyrosine kinase (RTK) signaling pathways regulate many biological processes in all metazoans. Their activities elicit diverse cellular responses, such as proliferation, differentiation, metabolism and migration, and abnormal RTK signaling can lead to multiple diseases, most notably cancer. RTK signaling is initiated following the binding of extracellular ligands to cellsurface RTKs, which then typically oligomerize, and either autoor trans-phosphorylate tyrosine residues in their intracellular domains. This, in turn, stimulates an array of intracellular signaling cascades that primarily act through the small GTPase Ras, and a core of three serine/threonine kinases [Raf, mitogenactivated protein kinase kinase (MEK) and mitogen-activated protein kinase (MAPK, also known as ERK)], but also through the phosphatidyl-inositol-3-kinase (PI3K) and phospholipase Cc (PLCc) pathways (Lemmon and Schlessinger, 2010).Because RTK signaling pathways often lead to changes in gene expression, the nuclear factors that are directly phosphorylated by components of these pathways, for example by MAPK, have a key role in the interpretation of RTK responses. In Drosophila melanogaster, where many RTK responses have been studied in detail, the best-characterized RTK-Ras-MAPK effectors belong to the ETS transcription factor superfamily. Thus, two ETS factors, the activator Pointed-P2 and the repressor Yan, mediate multiple RTK-regulated decisions and are direct substrates of MAPK (O'Neill et al., 1994;Brunner et al., 1994;Rebay and Rubin, 1995;Gabay et al., 1996;Tootle and Rebay, 2005). Similarly, ETS proteins in other species, such as Caenorhabditis elegans LIN-1 and mammalian ELK1, are important targets of Ras-MAPK