Investigation of an unconventional co-repressor complex

描述（由申请人提供）：转录调控是控制细胞增殖、分化和肿瘤发生等基本生物过程的关键机制。这项研究的目的是破译新的转录调控机制涉及CtBP共阻遏复合物。CtBP （c端结合蛋白）最初被鉴定为一种结合腺病毒E1A癌蛋白的细胞蛋白。这种相互作用的破坏增强了E1A诱导转化的能力，表明CtBP可能在细胞转化中发挥关键作用。CtBP随后被证明是一种对动物发育很重要的转录共抑制因子。然而，CtBP被招募到DNA后的详细分子事件在很大程度上是未知的。为了解决这个问题，我们分离了一个CtBP共抑制物复合物，它具有由六种潜在酶活性组成的非常规成分。其中包括协调组蛋白修饰抑制的组蛋白去乙酰化酶和甲基化酶，调节CtBP亚细胞定位的sumo E3连接酶，可能通过新机制抑制转录的推测的多胺氧化酶（nPAO），以及作用尚不清楚的组蛋白h4特异性乙酰化酶（HAT） （CDYL）。我们还发现CtBP与2-羟基酸脱氢酶（DH）具有序列同源性，具有预测的酶活性。这些令人兴奋的发现构成了这一建议的基础。

DESCRIPTION (provided by applicant): 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.