Investigation of an unconventional co-repressor complex

描述（由申请人提供）：修饰组蛋白尾部的酶在转录、细胞增殖、分化、衰老和肿瘤发生中起关键作用。我的实验室的主要兴趣是了解CtBP （c -末端结合蛋白）超复合物的分子机制和生物学，它包含组蛋白去乙酰化酶（hdac），甲基化酶（G9a/EuHMT1），去甲基化酶（LSD1）和核蛋白（CDYL），它们与脂质代谢酶具有序列同源性。在过去的资助期内，我们发现了第一个组蛋白去甲基化酶LSD1，并在了解CtBP和CDYL的生物学作用方面取得了突破。Ce-CtBP功能的缺失导致秀丽隐杆线虫的寿命延长，这取决于叉头转录因子DAF-16/FOXO，该因子是调节寿命的多种信号通路的交汇点。微阵列分析鉴定出约200个推测的Ce-CtBP靶基因，这些基因在代谢、应激反应、转录和翻译等方面具有功能。我们的模型是Ce-CtBP通过与这些信号通路交叉和拮抗DAF-16来调节寿命。在这个应用中，我将通过探索Ce-CtBP与调节寿命的信号通路之间的遗传和生化关系来研究潜在的分子机制。由于DAF-16在体外与Ce-CtBP相互作用，我将研究这种相互作用是否由DAF-16中假定的ctbp结合基序PIDLE介导，并确定物理相互作用对Ce-CtBP拮抗daf16介导的转录和寿命调节是否重要。我将通过ChIP和ChIP- ChIP鉴定Ce-CtBP和DAF-16共同调控的靶基因，并通过RNAi和过表达实验研究它们在寿命中的作用。拟议的研究将确定参与ce - ctbp介导的寿命调节的参与者和途径。CDYL相互作用蛋白的纯化使我们有了令人兴奋的发现，CDYL连接了神经元基因主调控因子REST和组蛋白甲基化酶G9a的相互作用，以抑制基因转录。REST也是一种新发现的肿瘤抑制因子。我们发现CDYL和G9a，而不是CoREST（另一种REST协同抑制因子）可以介导REST肿瘤抑制，从而确定CDYL和G9a是新的候选肿瘤抑制因子。我们将研究CDYL在临床肿瘤中的抑瘤作用，探索CDYL调控肿瘤抑制的分子机制。鉴于其与脂质代谢酶的同源性以及与CoA的结合能力，我们还将探讨CoA及其脂质衍生物的结合是否调节CDYL的转录和肿瘤抑制功能。我们提出的对CtBP和CDYL的研究提供了一个独特的机会，可以获得衰老和癌症等关键生物过程中染色质调节的新见解，并有可能揭示染色质代谢调节的新方面。本申请拟研究两种分别调节寿命、代谢和肿瘤发生的蛋白。研究结果将为控制这些过程的机制提供新的见解，并可能有助于未来开发调节代谢、衰老和肿瘤发生的小分子。

DESCRIPTION (provided by applicant): Enzymes that modify histone tails play critical roles in transcription, cell proliferation, differentiation, aging and tumorigenesis. A main interest of my lab is to understand the molecular mechanism and biology of the CtBP (C-terminal binding protein) super- complex, which contains histone deacetylases (HDACs), methylases (G9a/EuHMT1), a demethylase (LSD1), and a nuclear protein (CDYL) that shares sequence homology with lipid metabolizing enzymes. In the past funding period, we discovered the first histone demethylase LSD1 and made breakthroughs in understanding the biological roles of CtBP and CDYL. Loss of Ce-CtBP function results in C. elegans life span extension, dependent on the forkhead transcription factor DAF-16/FOXO, which is the point of convergence of multiple signaling pathways that regulate life span. Microarray analysis identified ~200 putative Ce-CtBP target genes with functions in metabolism, stress response, transcription and translation. Our model is that Ce-CtBP regulates life span by intersecting with these signaling pathways and by antagonizing DAF-16. In this application, I will investigate the underlying molecular mechanisms by exploring the genetic and biochemical relationship between Ce-CtBP and the signaling pathways that regulate life span. Since DAF-16 interacts with Ce-CtBP in vitro, I will investigate whether this interaction is mediated by the putative CtBP-binding motif PIDLE located within DAF-16, and determine if physical interaction is important for Ce-CtBP to antagonize DAF16-mediated transcription and life span regulation. I will identify common target genes regulated by Ce-CtBP and DAF-16 by ChIP and ChIP-chip and investigate their roles in life span by RNAi and over-expression experiments. The proposed studies will identify players and pathways involved in Ce-CtBP-mediated life span regulation. Purification of CDYL-interacting proteins led us to the exciting discovery that CDYL bridges the interaction of the neuronal gene master regulator, REST, and the histone methylase G9a to repress gene transcription. REST is also a newly identified tumor suppressor. We found that CDYL and G9a, but not CoREST (another REST co- repressor) may mediate REST tumor suppression, identifying CDYL and G9a as novel candidate tumor suppressors. We will investigate CDYL tumor suppression in clinical cancers and explore the molecular mechanisms by which CDYL regulates tumor suppression. Given its homology to lipid metabolizing enzymes and ability to bind CoA, we will also explore whether binding of CoA and its lipid derivatives regulate CDYL transcription and tumor suppression functions. Our proposed investigations of CtBP and CDYL offer a unique opportunity to gain novel insights into chromatin regulation in critical biological processes such as ageing and cancer, and have the potential to uncover a new facet of metabolic regulation of chromatin-based processes. PUBLIC HEALTH RELEVANCE This application proposes to study two proteins that regulate life span, metabolism and tumorigenensis, respectively. Findings will provide new insights into mechanisms that control these processes and may help future development of small molecules that modulate metabolism, aging and oncogenesis.