Transcriptional regulation is a key mechanism that controls fundamental biological processes such as cell proliferation, differentiation and tumorigenesis. The intent of this investigation is to decipher new transcriptional regulatory mechanisms involving a CtBP co-repressor complex. CtBP (C-terminal Binding Protein) was initially identified as a cellular protein that binds the adenoviral E1A oncoprotein. Disruption of this interaction enhances the ability of E1A to induce transformation, suggesting that CtBP may play crucial roles in cellular transformation. CtBP was subsequently shown to function as a transcriptional co-repressor important for animal development. However, detailed molecular events after recruitment of CtBP to DNA were largely unknown. To address this issue, we isolated a CtBP co-repressor complex, which has an unconventional composition consisting of six potential enzymatic activities. These include histone deacetylases and methylases that coordinate histone modifications for repression, a sumo E3 ligase that regulates CtBP subcellular localization, a putative polyamine oxidase (nPAO), which may repress transcription via a novel mechanism, and a histone H4-specific acetylase (HAT) (CDYL) whose role is unclear. We also showed that CtBP, which shares sequence homology with 2-hydroxy acid dehydrogenases (DH), possesses the predicted enzymatic activity. These exciting findings form the basis of this proposal. Unlike the histone modifying enzymes and the sumo E3 ligase, the function and mechanism of action of the dehydrogenase, the putative PAO and the histone H4-specific HAT are unclear, and are the focus of this application. We will use genetic and molecular approaches to delineate the role of CtBP DH activity in transcription and development in C. elegans. We will carry out biochemical and functional tests to investigate the model that nPAO represses transcription via polyamine modification. Current transcription research is focused mainly on histone modifications. The identification of a novel enzyme that regulates transcription by modifying polyamines, the non-histone component of the chromatin, will break new ground for mechanistic considerations. Since acetylation is correlated with transcriptional activation, the association of a HAT (CDYL) with a co-repressor complex is unusual and provides a unique opportunity to address the role of HAT in co-repressor function. Based on our strong initial results, new paradigms are likely to emerge from the proposed studies that will significantly impact our views of eukaryotic gene regulation.
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