Transcriptional mechanisms in cardiac hypertrophy

心脏肥大的转录机制

• 批准号: 9893424
• 负责人: Maha Abdellatif
• 金额: $ 63.36万
• 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 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9893424
• 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，需要代谢中间体的激活或失活， 其中包括：用于组蛋白乙酰化的乙酰辅酶A（乙酰-COA），A-酮戊二酸（AKG）作为辅因子作为辅助因子 组蛋白和DNA脱甲基酶，以及用于组蛋白的琥珀酰辅酶A（SUCOA），而不是打折其他 酰基-COA。由于没有COA连接的代谢产物可以从线粒体，核，， 必须获得其乙酰辅酶A，例如，主要是通过在底物期间从线粒体出口的柠檬酸盐 抽象，然后通过ATP柠檬酸裂解酶转化为核中的乙酰辅酶A。另一方面， Nucleus的Akg，Suc-CoA或其他短链酰基辅助核的来源（例如丁酰果，丙酰coa，丙酰磷脂，丙酰基 - COA…等），未完全考虑。另一个未解决的问题是，基因如何选择性地被基因激活 特定的底物，这如何影响器官的体内稳态？ 教条一直是氧化酶和底物氧化专门局限于 线粒体。但是，在最近的公正屏幕中，使用染色质免疫沉淀和质量 用于发现与H2A.Z结合染色质的蛋白质发现的光谱法，我们发现了 TCA循环的线粒体酶，B-氧化和分支链氨基酸分解代谢中的线粒体酶，在细胞核中， 位于基因的转录起始位点（TSS）。重组绿色荧光融合蛋白合并 随着所选酶的推定核定位信号的突变，包括乙酰-COA酰基转移酶 2（ACAA2），氧甲酸酯脱氢酶（OGDH）和异位酸脱氢酶2证实了它们的核 啮齿动物和人类细胞中的定位和染色质结合。更结论性地，染色质 免疫沉淀 - 深度测序（CHIP-SEQ）确认了ACAA2和OGDH的选择性关联 使用H2A.Z占有转录开始站点。最后，在小鼠或人类细胞中敲低或敲除h2a.z 与翻译后Hisstone修饰减少有关的代谢基因的结合减少 包括乙酰化和琥珀酰化。脂肪引起的增加的相关性，例如 结合染色质的ACAA2和基因表达的差异调制，这是由核促进的 定位信号（NLS）突变。在该提案中，我们将专注于研究2种酶的核作用， 表示氧化葡萄糖和脂肪酸的两种途径。包括转换AKG的OGDH 进入Suc-COA和ACAA2，在最后一个反应中，将3-酮酰基-COA转换为乙酰辅酶A和乙酰辅酶A B-氧化螺旋分别。我们假设，1-核包含的线粒体酶 TCA循环和B氧化螺旋螺旋，这些螺旋螺旋在SELECT的TSS处特异性定位于H2A.Z结合的染色质 基因。 2-根据，这使特定基因直接响应葡萄糖或脂肪酸， 乙酰辅酶A，Suc-COA和AKG的生产/消费的局部生产，这是组蛋白所必需的 转录激活或表达所需的修改。 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，在 选择启动子，会影响心脏肥大和失败的发展。那就是我们的目标是：1- 识别ACAA2和OGDH的染色质缔合位点，基础组蛋白标记及其调节 按照正常和肥大的心脏进行饮食。 2-确定核ACAA2和OGDH在调节中的作用 组蛋白的修饰和心肌肥大期间的基因转录。 3-确定核的作用 ACAA2和OGDH在小鼠心脏肥大和衰竭发展中。