Mitochondrial 2-hydroxyglutarate dehydrogenases modulate the cellular epitranscriptome

线粒体 2-羟基戊二酸脱氢酶调节细胞表观转录组

• 批准号: 10541234
• 负责人: Ricardo C Aguiar
• 金额: $ 31万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10541234
• 关键词: Acetyl Coenzyme A; Acetylation; Agreement; B-Lymphocytes; Biological Models; Biological Process; Cell Line; Cells; Chemicals; Complex; Cysteine; DNA; DNA Methylation; Data; Dioxygenases; Distal; Enzymes; Epigenetic Process; Exercise; Gene Expression; Gene Expression Profile; Generations; Genetic Models; Genetic Transcription; Hexosamines; Histones; Homeostasis; Human; Hypermethylation; In Vitro; Knowledge; Link; Location; Mediating; Messenger RNA; Metabolism; Methylation; Methyltransferase; Mitochondria; Mitochondrial RNA; Mixed Function Oxygenases; Modeling; Modification; Mutation; Nuclear; O-GlcNAc transferase; Organelles; Output; Oxidoreductase; Pathology; Pathway interactions; Phenotype; Physiology; Play; Post-Translational Protein Processing; Production; Proliferating; Proteins; RNA; RNA methylation; Reactive Oxygen Species; Reporter; Reporting; Research; Role; S-Adenosylhomocysteine; S-Adenosylmethionine; Signal Transduction; Stem Cell Development; Testing; Therapeutic; Transcriptional Activation; Transgenic Mice; Work; alpha ketoglutarate; chromatin immunoprecipitation; control theory; demethylation; epitranscriptome; field study; histone demethylase; histone methylation; human disease; in vitro Model; in vivo; loss of function; mitochondrial metabolism; mouse model; novel; oxidation

There is increasing recognition of mitochondria as signaling organelles. An important facet of this “adjunct” mitochondrial function is epigenetic modulation, as exemplified by the generation of acetyl-CoA and S- adenosylmethionine used in the acetylation and methylation, respectively, of DNA and histones. In addition, alpha-ketoglutarate (αKG) and 2-hydroxyglutarate (2-HG), metabolites generated almost exclusively in the mitochondria, are found to modulate the activity of αKG-dependent dioxygenases, including TET DNA hydroxylases and histone demethylase (HDM), thus controlling DNA and histone methylation. Notably, our group discovered that αKG activates and 2-HG inhibits FTO and ALKBH5, RNA demethylases that act on N6- methyladenosine (m6A), a reversible chemical modification of mRNA (the epitranscriptome) that influences gene expression. Similar to other epigenetic marks, RNA methylation is dynamically controlled and m6A abundance influence various biological functions, while its misregulation associates with human diseases. Considering that αKG/2-HG are generated mainly by intermediary mitochondrial metabolism, and that the activity of RNA demethylases are modulated by these metabolites, it is reasonable to speculate that mitochondria play an important role in the control of RNA methylation homeostasis. In particular, we postulate that the mitochondrial enzymes D-2- and L-2-hydroxyglutarate dehydrogenase (D2HGDH and L2HGDH), which catalyze the interconversion of 2-HG to αKG, are integral to the interplay between mitochondrial metabolism and the control of RNA methylation. This hypothesis is supported by our earlier discovery that loss of function D2HGDH mutations leads to decreased activity of the αKG-dependent TET and HDM enzymes. We recently expanded on this concept by identifying upstream signals that regulate D2HGDH and L2HGDH expression/activity. Using ChIP assays, inducible cell lines and a transgenic mouse model we discovered that MYC transcriptionally activates D2HGDH and L2HGDH, and that in a D2/L2HGDH/αKG-dependent manner it induces FTO and ALKBH5 function leading to RNA demethylation in vitro and in vivo. Remarkably, we found that the MYC-D2/L2HGDH-αKG axis also promotes the nuclear accumulation of FTO and ALKBH5, in association with enhanced O-GlcNAcylation, a post-translational modification executed by another mitochondrial enzyme, O-GlcNAc transferase (OGT). Here, using multiple genetic models in vitro and in vivo, we will test the hypothesis that a novel mitochondrial signaling axis, which includes MYC at the proximal point, D2/L2HGDH and OGT at the center, and, distally, FTO/ALKBH5 activity, controls the cellular epitranscriptome. Our specific aims are: 1) characterize the contribution of D2HGDH/L2HGDH and of intermediate metabolites to the control of m6A levels, 2) determine the mechanistic basis for the increased O-GlcNAcylation mediated by the MYC-D2/L2HGDH-αKG axis and its role in promoting RNA demethylation, 3) define a mitochondrial metabolism-dependent methylRNA/gene expression signature in human cells.

越来越多的人认识到线粒体作为信号细胞器。这个“附属品”的一个重要方面是 线粒体功能是表观遗传调节，如乙酰辅酶A和S-乙酰辅酶A的产生所例示的。 腺苷甲硫氨酸分别用于DNA和组蛋白的乙酰化和甲基化。此外，本发明还提供了一种方法， α-酮戊二酸（αKG）和2-羟基戊二酸（2-HG），代谢产物几乎仅在 线粒体，被发现调节α KG依赖性双加氧酶的活性，包括泰特DNA 羟化酶和组蛋白脱甲基酶（HDM），从而控制DNA和组蛋白甲基化。值得注意的是，我们 研究小组发现，αKG激活和2-HG抑制FTO和ALKBH 5，RNA去甲基化酶作用于N6- 甲基腺苷（m6 A），一种可逆的mRNA化学修饰（表转录组），影响 基因表达。与其他表观遗传标记类似，RNA甲基化是动态控制的，m6 A 其丰度影响多种生物学功能，而其失调与人类疾病有关。 考虑到αKG/2-HG主要通过线粒体中间代谢产生， RNA去甲基化酶的活性受这些代谢物的调节，可以合理地推测， 线粒体在控制RNA甲基化稳态中起重要作用。特别是，我们假设 线粒体酶D-2-和L-2-羟戊二酸脱氢酶（D2 HGDH和L2 HGDH）， 催化2-HG转化为αKG，是线粒体之间相互作用的组成部分。 代谢和RNA甲基化的控制。这一假设得到了我们早期发现的支持， 功能D2 HGDH突变导致α KG依赖性泰特和HDM酶活性降低。我们 最近通过鉴定调节D2 HGDH和L2 HGDH的上游信号扩展了这一概念 表达/活动。使用ChIP测定、诱导型细胞系和转基因小鼠模型，我们发现， MYC转录激活D2 HGDH和L2 HGDH，并且以D2/L2 HGDH/α KG依赖的方式， 诱导FTO和ALKBH 5功能，导致体外和体内RNA去甲基化。值得注意的是，我们发现 MYC-D2/L2 HGDH-αKG轴也促进FTO和ALKBH 5的核积累， 与增强的O-GlcNAc酰化相关，这是一种由另一种酶执行的翻译后修饰， 线粒体酶，O-GlcNAc转移酶（OGT）。在这里，使用多种体外和体内遗传模型， 我们将检验一种新的线粒体信号传导轴的假设，它包括近端的MYC， 中心的D2/L2 HGDH和OGT以及远端的FTO/ALKBH 5活性控制细胞表位转录组。 我们的具体目标是：1）表征D2 HGDH/L2 HGDH和中间代谢产物对 m6 A水平的控制，2）确定由m6 A介导的O-GlcNAc酰化增加的机制基础。 MYC-D2/L2 HGDH-αKG轴及其在促进RNA去甲基化中的作用，3）定义了线粒体 代谢依赖性甲基RNA/基因表达特征。