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Bioinformatics analysis of transcriptional control is guided by knowledge of the characteristics of cis-regulatory regions or enhancers. Features such as clustering of binding sites and co-occurrence of binding sites have aided enhancer identification, but quantitative predictions of enhancer function are not yet generally feasible. To facilitate the analysis of regulatory sequences in Drosophila melanogaster, we identified quantitative parameters that affect the activity of short-range transcriptional repressors, proteins that play key roles in development. In addition to the previously noted distance dependence, repression is strongly influenced by the stoichiometry, affinity, spacing, and arrangement of activator binding sites. Repression is insensitive to the type of activation domain, suggesting that short-range repression may primarily affect activators at the level of DNA binding. The activity of several short-range, but not long-range, repressors is circumscribed by the same quantitative parameters. This cis-regulatory ‘grammar’ may aid the identification of enhancers regulated by short-range repressors and facilitate bioinformatic prediction of the functional output of transcriptional regulatory sequences. [ABSTRACT FROM AUTHOR]

Transcriptional repressors often employ multiple activities, but the molecular mechanisms and physiological relevance of this functional diversity remain obscure. The Drosophila melanogaster Knirps repressor uses CtBP corepressor-dependent and -independent pathways. To separately analyze the components of Knirps repression activity, we elucidated the specific repression properties of CtBP and of Knirps subdomains. Like Knirps, CtBP represses adjacent transcriptional activators; but unlike Knirps, CtBP is unable to repress basal promoter elements. We determined that the ability of CtBP to recapitulate only a subset of Knirps activities is due to a quantitative, rather than qualitative, deficiency in repression activity. The CtBP-dependent portion of Knirps synergizes with the CtBP-independent repression activity to potently repress promoter elements from enhancer- or promoter-proximal positions. This result indicates that multiple repression activities are combined to exceed critical thresholds on target genes. CtBP mutant proteins unable to bind NAD fail to interact with DNA-bound factors. We show that DNA-binding Gal4-CtBP fusion proteins also require NAD binding for activity, indicating that NAD plays a role in repression at a step subsequent to CtBP recruitment to the promoter. [ABSTRACT FROM AUTHOR]

Transcriptional corepressors play complex roles in developmental gene regulation. These proteins control transcription by recruiting diverse chromatin-modifying enzymes, but it is not known whether corepressor activities are finely regulated in different developmental settings or whether their basic activities are identical in most contexts. The evolutionarily conserved C-terminal binding protein (CtBP) is recruited by a variety of transcription factors that play crucial roles in development and disease. CtBP contains a central NAD(H) binding core domain that is homologous to D2 hydroxy acid dehydrogenase enzymes, as well as an unstructured C-terminal domain. NAD(H) binding is important for CtBP function, but the significance of its intrinsic dehydrogenase activity, as well as that of the unstructured C terminus, is poorly understood. To clarify the biological relevance of these features, we established genetic rescue assays to determine how different forms of CtBP function in the context of Drosophila melanogaster development. The mutant phenotypes and specific gene regulatory effects indicate that both the catalytic site of CtBP and the C-terminal extension play important, if nonessential roles in development. Our results indicate that the structural and enzymatic features of CtBP, previously thought to be dispensable for overall transcriptional control, are critical for modulating this protein's activity in diverse developmental settings. [ABSTRACT FROM AUTHOR]

Transcriptional repressor proteins play key roles in the control of gene expression in development. For the Drosophila embryo, the following two functional classes of repressors have been described: short-range repressors such as Knirps that locally inhibit the activity of enhancers and long-range repressors such as Hairy that can dominantly inhibit distal elements. Several long-range repressors interact with Groucho, a conserved corepressor that is homologous to mammalian TLE proteins. Groucho interacts with histone deacetylases and histone proteins, suggesting that it may effect repression by means of chromatin modification; however, it is not known how long-range effects are mediated. Using embryo chromatin immunoprecipitation, we have analyzed a Hairy-repressible gene in the embryo during activation and repression. When inactivated, repressors, activators, and coactivators cooccupy the promoter, suggesting that repression is not accomplished by the displacement of activators or coactivators. Strikingly, the Groucho corepressor is found to be recruited to the transcribed region of the gene, contacting a region of several kilobases, concomitant with a loss of histone H3 and H4 acetylation. Groucho has been shown to form higher-order complexes in vitro; thus, our observations suggest that long-range effects may be mediated by a "spreading" mechanism, modifying chromatin over extensive regions to inhibit transcription. [ABSTRACT FROM AUTHOR]