Transcriptional mechanisms in cardiac hypertrophy

心脏肥大的转录机制

• 批准号: 10335218
• 负责人: Maha Abdellatif
• 金额: $ 63.77万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-15 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10335218
• 关键词: ATP Citrate (pro-S)-Lyase; Acetyl Coenzyme A; Acetylation; Acyl Coenzyme A; Acyltransferase; Belief; Binding; Branched-Chain Amino Acids; Cardiac development; Catabolism; Cell Nucleus; Cells; Chimeric Proteins; Chromatin; Citrates; Citric Acid Cycle; Coenzyme A; Consumption; DNA; DNA Modification Process; Diet; Disease; Enzymes; Fatty Acids; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Glucose; Goals; Health; Heart; Heart Hypertrophy; Heart failure; Histone Acetylation; Histones; Homeostasis; Human; Hypertrophy; Isocitrate Dehydrogenase; Ketoglutarate Dehydrogenase Complex; Knock-out; Link; Malonyl Coenzyme A; Mass Spectrum Analysis; Metabolic; Metabolism; Mitochondria; Mus; Muscle Cells; Mutation; Nuclear; Nuclear Localization Signal; Organ; Oxides; Palmitates; Pathway interactions; Phenotype; Process; Production; Proteins; Reaction; Recombinants; Regulation; Rodent; Role; Site; Source; Time; Transcription Initiation Site; Transcriptional Activation; alpha ketoglutarate; chromatin immunoprecipitation; cofactor; deep sequencing; enzyme substrate; gene repression; histone modification; knock-down; oxidation; promoter; propionyl-coenzyme A; succinyl-coenzyme A

Our goal is to understand the mechanisms that govern transcription in the heart during health and disease. Transcription is a highly dynamic process that requires metabolic intermediates for its activation or deactivation, these include: acetyl-Coenzyme A (acetyl-CoA) for histone acetylation, a-ketoglutarate (aKG) as a cofactor for histone and DNA demethylases, and succinyl-CoA (suc-CoA) for histone succinylation, not discounting other acyl-CoAs. Since none of the CoA-linked metabolites could be exported out of the mitochondria, the nucleus, must acquire its acetyl-CoA, for example, mainly via export of citrate from the mitochondria during substrate abundance, which is then converted to acetyl-CoA in the nucleus via ATP citrate lyase. On the other hand, the nucleus’s source of aKG, suc-CoA, or other short-chain acyl-CoAs (e.g. butyryl-CoA, malonyl-CoA, propionyl- CoA…etc.), is not fully accounted for. The other unanswered question, is how are genes selectively activated by specific substrates, and how does this influence an organ’s homeostasis? The dogma has always been that oxidative enzymes and substrate oxidation are specifically confined to the mitochondria. However, in a recent unbiased screen, using chromatin immunoprecipitation and mass spectrometry for discovery of proteins that associate with H2A.Z-bound chromatin in the heart, we uncovered mitochondrial enzymes of the TCA cycle, b-oxidation, and branched-chain amino acid catabolism, in the nucleus, localized to the transcription start sites (TSS) of genes. Recombinant green florescence fusion proteins combined with mutations of putative nuclear localization signals of select enzymes, including acetyl-CoA acyltransferase 2 (ACAA2), oxoglutarate dehydrogenase (OGDH), and isocitrate dehydrogenase 2 confirmed their nuclear localization and chromatin binding in both rodent and human cells. More conclusively, chromatin immunoprecipitation-deep sequencing (ChIP-Seq), confirmed the selective association of ACAA2 and OGDH with H2A.Z-occupied transcription start sites. Finally, knockdown or knockout of H2A.Z in mouse or human cells reduced binding of metabolic genes that was associated with reduced posttranslational histone modifications including acetylation and succinylation. The relevance, of which, is exemplified by fatty-induced increase in chromatin-bound ACAA2 and differential modulation of gene expression, which is abrogated by a nuclear localization signal (NLS) mutation. In this proposal, we will focus on investigating the nuclear role of 2 enzymes, representatives of the two pathways that oxidize glucose and fatty acids; including, OGDH, which converts aKG into suc-CoA, and ACAA2, which converts 3-ketoacyl-CoA into acetyl-CoA and acyl-CoA in the last reaction of the b-oxidation spiral, respectively. We hypothesize that, 1- The nucleus harbors mitochondrial enzymes of the TCA cycle and b-oxidation spiral that are specifically localized to H2A.Z-bound chromatin at the TSS of select genes. 2- In accordance, this renders specific genes directly responsive to either glucose or fatty acids, via the local production of acetyl-CoA, suc-CoA, and the production/consumption of aKG, which are required for histone modifications necessary for transcriptional activation or repression. 3- Perturbations of the nuclear concentrations of these genes results in substrate-dependent modulation of histone marks and transcription, at select promoters, which influences the development of cardiac hypertrophy and failure. Thus, our aims are to:1- Identify the chromatin-association sites of ACAA2 and OGDH, the underlying histone marks, and their regulation by diet in the normal and hypertrophied hearts. 2- Determine the roles of nuclear ACAA2 and OGDH in regulating histone modifications and gene transcription during myocyte hypertrophy. 3- Determine the roles of nuclear ACAA2 and OGDH in the development of cardiac hypertrophy and failure in mice.

我们的目标是了解在健康和疾病期间控制心脏转录的机制。 转录是一个高度动态的过程，需要代谢中间体来激活或失活， 这些包括：用于组蛋白乙酰化的乙酰辅酶A（acetyl-CoA），作为组蛋白乙酰化的辅因子的α-酮戊二酸（aKG）， 组蛋白和DNA脱甲基酶，以及用于组蛋白琥珀酰化的琥珀酰辅酶A（suc-CoA），不排除其他 酰基辅酶A由于没有一种CoA连接的代谢物可以从线粒体、细胞核、 必须获得其乙酰辅酶A，例如，主要通过出口柠檬酸盐从线粒体在底物 丰度，然后通过ATP柠檬酸裂解酶在细胞核中转化为乙酰辅酶A。另一方面 - aKG、suc-CoA或其他短链酰基-CoA（例如丁酰-CoA、丙二酰-CoA、丙酰-CoA）的核来源。 CoA等），并没有完全解释清楚。另一个未解的问题是， 特定的底物，这如何影响器官的稳态？ 一直以来的教条是氧化酶和底物氧化是特别局限于 线粒体然而，在最近的一次无偏筛选中，使用染色质免疫沉淀和质量 光谱法发现与心脏中H2A.Z结合染色质相关的蛋白质，我们发现 细胞核中的TCA循环、β-氧化和支链氨基酸催化剂的线粒体酶， 定位于基因的转录起始位点（TSS）。重组绿色荧光融合蛋白组合物 选择酶的推定核定位信号突变，包括乙酰辅酶A酰基转移酶 2（ACAA 2），酮戊二酸脱氢酶（OGDH）和异柠檬酸脱氢酶2证实了它们的核 在啮齿动物和人类细胞中的定位和染色质结合。更确切地说，染色质 免疫沉淀-深度测序（ChIP-Seq）证实了ACAA 2和OGDH的选择性结合 Z-占据转录起始位点。最后，在小鼠或人细胞中敲低或敲除H2A.Z 与翻译后组蛋白修饰减少相关的代谢基因结合减少 包括乙酰化和琥珀酰化。相关性，其中，例证是脂肪诱导的增加， 染色质结合的ACAA 2和基因表达的差异调节，这是废除了核 定位信号（NLS）突变。在这个提议中，我们将集中研究2种酶的核作用， 氧化葡萄糖和脂肪酸的两种途径的代表;包括OGDH，其转化aKG 转化为suc-CoA和ACAA 2，ACAA 2将3-酮脂酰CoA转化为乙酰CoA和酰基CoA， B-氧化螺旋。我们假设，1-细胞核内含有线粒体酶， TCA循环和b-氧化螺旋特异性定位于H2A.Z结合的染色质，在选择的TSS 基因. 2-相应地，这使得特定基因直接响应于葡萄糖或脂肪酸，通过 组蛋白所需的乙酰辅酶A、suc-CoA的本地生产以及aKG的生产/消耗 转录激活或抑制所必需的修饰。3-核扰动 这些基因的浓度导致组蛋白标记和转录的底物依赖性调节， 选择启动子，其影响心脏肥大和衰竭的发展。因此，我们的目标是： 确定ACAA 2和OGDH的染色质结合位点，潜在的组蛋白标记及其调控 在正常和肥大的心脏中。2-确定核ACAA 2和OGDH在调节细胞凋亡中的作用。 肌细胞肥大过程中组蛋白修饰和基因转录。3-确定核武器的作用 ACAA 2和OGDH在小鼠心肌肥厚和心力衰竭中的作用