Lysine Malonylation and SIRT5 in Epigenetic Regulation

项目摘要 表观遗传因素已经成为代谢紊乱和衰老的关键因素，这两个因素都是 通常与广泛的基因表达变化有关。这项提议的总体目标是 研究一种新的组蛋白修饰，赖氨酸丙二酰化的作用以及NAD+- 依赖蛋白脱酰酶SIRT5作为一种新的组蛋白修饰物参与基因表达的表观遗传调控 对新陈代谢变化的反应。该模型是基于我们最近鉴定的组蛋白H_2B赖氨酸5 (H2BK5)作为受SIRT5调控的丙二酸化位点。我们认为，组蛋白的动态丙二酸化 细胞丙二酰辅酶A介导的H_2B和SIRT5介导的去丙酮化调控染色质结构和基因 抄写。开发了两个特定的目标来在小鼠肝脏中全局测试该模型，然后 在培养细胞中的机械作用。首先，与丙二酸化组蛋白(H_2BK5)和SIRT5结合的基因组区域 将使用来自野生型和SIRT5-/-小鼠的组织使用CHIP-SEQ进行比较。这些研究的目的将是 确定SIRT5介导组蛋白去丙氨酸化的位点。组蛋白的功能后果 丙二酸化将通过比较野生型和野生型之间的基因转录变化来评估。 和SIRT5-/-小鼠。该数据集将与SIRT5与基因组结合的位置进行比较 使组蛋白去丙二醛，以确定SIRT5作用的直接基因组位置。第二，我们将在中国学习 SIRT5和细胞内波动如何动态调节组蛋白丙二酸化的机制 丙二酰辅酶A在小鼠喂食和禁食期间发生。在原代培养的小鼠肝细胞中，我们还将 测试饲喂丙二酸(细胞内转化为丙二酰辅酶A)的效果以及 通过乙酰辅酶A羧基酶控制细胞丙二酰辅酶A的合成或降解 丙二酰辅酶A脱羧酶(降解)。 这项研究计划向理解这种新发现的组蛋白的功能迈出了第一步 修饰，丙二酸化，及其擦除器，SIRT5，在表观遗传调节中。它将对我们的 长期以来，人们一直在努力揭开“组蛋白密码”--它与中间代谢的交集--的面纱， 提高表观遗传因子对基因表达调控的认识。拟议项目的结果 因此，将允许对组蛋白丙二酸化和SIRT5的意义进行机械性后续研究 表观遗传调节以及调节失调如何导致这两种病理状态，如代谢 综合症和糖尿病，以及正常衰老。

PROJECT ABSTRACT Epigenetic factors have emerged as crucial players in metabolic disorders and in aging, both of which are typically associated with a wide range of gene expression changes. The overall objective of this proposal is to investigate the roles of a novel histone modification, lysine malonylation, and its regulation by the NAD+- dependent protein deacylase SIRT5, as a novel histone modifier in epigenetic regulation of gene expression in response to metabolic changes. The model is based on our recent identification of histone H2B lysine 5 (H2BK5) as a site of malonylation regulated by SIRT5. We propose that the dynamic malonylation of histone H2B by cellular malonyl-CoA and demalonylation by SIRT5 regulates chromatin structure and gene transcription. Two specific aims are developed to test this model globally in mouse liver and then mechanistically in cultured cells. First, genomic regions bound with malonylated histones (H2BK5) and SIRT5 will be compared using ChIP-seq using tissues from wild type and Sirt5-/- mice. These studies will aim to identify the sites of SIRT5-mediated histone demalonylation. The functional consequences of histone malonylation will be evaluated by comparing gene transcriptional changes using RNA-seq between wild type and Sirt5-/- mice. This data set will be compared to the sites where SIRT5 binds to the genome and demalonylates histones to identify the direct genomic sites of SIRT5 action. Second, we will study in mechanistic details how histone malonylation is dynamically regulated by SIRT5 and by fluctuations in cellular malonyl-CoA that occur during feeding and fasting in mice. In primary cultured mouse hepatocytes, we will also test the effect of feeding malonate (which is converted into malonyl-CoA intracellularly) and the effect of manipulating the cellular synthesis or degradation of malonyl-CoA via acetyl-CoA carboxylase (synthesis) and malonyl-coA decarboxylase (degradation). This research proposal takes the first step towards understanding the function of this newly discovered histone modification, malonylation, and its eraser, SIRT5, in epigenetic regulation. It will contribute significantly to our longstanding effort of unveiling the “histone code”, its intersection with intermediary metabolism, and to advance our knowledge of gene expression regulation by epigenetic factors. Results from the proposed project will therefore permit mechanistic follow-up studies of the significance of histone malonylation and SIRT5 in epigenetic regulation and how dysregulation may contribute to both pathological conditions, such as metabolic syndrome and diabetes, and to normal aging.