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.
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