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， 其中包括：用于组蛋白乙酰化的乙酰辅酶 A (乙酰辅酶 A)、作为辅因子的 α-酮戊二酸 (aKG) 组蛋白和 DNA 去甲基化酶，以及用于组蛋白琥珀酰化的琥珀酰辅酶 A (suc-CoA)，不排除其他酶 酰基辅酶A。由于 CoA 连接的代谢物都不能从线粒体、细胞核中输出， 例如，必须主要通过底物过程中从线粒体输出柠檬酸盐来获得其乙酰辅酶A 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， 细胞核的 aKG、suc-CoA 或其他短链酰基辅酶 A（例如丁酰辅酶 A、丙二酰辅酶 A、丙酰辅酶 A）来源 CoA…等），未完全考虑在内。另一个悬而未决的问题是，基因是如何选择性激活的？ 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特别局限于 线粒体。然而，在最近的无偏筛选中，使用染色质免疫沉淀和质量 我们发现，通过光谱测定法发现与心脏中 H2A.Z 结合染色质相关的蛋白质 细胞核中 TCA 循环、b-氧化和支链氨基酸分解代谢的线粒体酶， 定位于基因的转录起始位点（TSS）。重组绿色花融合蛋白组合 具有选定酶（包括乙酰辅酶A酰基转移酶）的推定核定位信号的突变 2 (ACAA2)、氧化戊二酸脱氢酶 (OGDH) 和异柠檬酸脱氢酶 2 证实其核 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 ACAA2 和 OGDH 的选择性关联 具有 H2A.Z 占据的转录起始位点。最后，在小鼠或人类细胞中敲低或敲除 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。其中的相关性可以通过脂肪诱导的 染色质结合 ACAA2 和基因表达的差异调节，该调节被核 定位信号（NLS）突变。在本提案中，我们将重点研究 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，将 aKG 转换为 转化为 suc-CoA 和 ACAA2，ACAA2 在最后一个反应中将 3-酮酰基-CoA 转化为乙酰基-CoA 和酰基-CoA 分别为b-氧化螺旋。我们假设， 1- 细胞核中含有线粒体酶 TCA 循环和 b 氧化螺旋专门定位于选择的 TSS 处的 H2A.Z 结合染色质 基因。 2- 因此，这使得特定基因通过 组蛋白所需的乙酰辅酶 A、suc-CoA 的局部产生以及 aKG 的产生/消耗 转录激活或抑制所需的修饰。 3- 核的扰动 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，影响心脏肥大和衰竭的发展。因此，我们的目标是：1- 识别 ACAA2 和 OGDH 的染色质关联位点、潜在的组蛋白标记及其调控 在正常和肥厚的心脏中通过饮食。 2-确定核ACAA2和OGDH在调节中的作用 肌细胞肥大期间的组蛋白修饰和基因转录。 3- 确定核的作用 ACAA2 和 OGDH 在小鼠心脏肥大和衰竭发展中的作用。