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

基本信息

批准号：
10117575
负责人：
Ricardo C Aguiar
金额：
$ 30.87万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
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/10117575
关键词：
Acetyl Coenzyme A Acetylation Agreement B-Lymphocytes Biological Models Biological Process Cell Line Cells Chemicals Cysteine DNA DNA Methylation Data Dioxygenases Distal Enzymes Epigenetic Process Exercise Gene Expression Gene Expression Profile Generations Genes 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 Physiology Play Post-Translational Protein Processing Production Proteins RNA RNA methylation Reactive Oxygen Species Reporter Research Role S-Adenosylmethionine Signal Transduction Stem Cell Development Testing Therapeutic Transcriptional Activation Transgenic Mice Work alpha ketoglutarate chromatin immunoprecipitation demethylation epitranscriptome field study histone demethylase histone methylation human disease in vitro Model in vivo loss of function mitochondrial metabolism mouse model novel oxidation

项目摘要

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.

人们越来越认识到线粒体作为信号细胞器的一个重要方面。线粒体功能是表观遗传调节，例如乙酰辅酶A和S-的产生腺苷甲硫氨酸分别用于 DNA 和组蛋白的乙酰化和甲基化。 α-酮戊二酸 (αKG) 和 2-羟基戊二酸 (2-HG)，代谢物几乎全部在线粒体，被发现可以调节 αKG 依赖性双加氧酶的活性，包括 TET DNA 羟化酶和组蛋白去甲基化酶 (HDM)，从而控制 DNA 和组蛋白甲基化。研究小组发现，αKG 激活而 2-HG 抑制 FTO 和 ALKBH5，这些 RNA 去甲基酶作用于 N6- 甲基腺苷 (m6A)，一种 mRNA（表观转录组）的可逆化学修饰，可影响与其他表观遗传标记类似，RNA 甲基化是动态控制的并且 m6A 丰度影响多种生物功能，而其失调则与人类疾病相关。考虑到αKG/2-HG主要由线粒体中间代谢产生，并且 RNA 去甲基化酶的活性受到这些代谢物的调节，因此可以合理地推测：我们假设线粒体在 RNA 甲基化稳态的控制中发挥着重要作用。线粒体酶 D-2- 和 L-2- 羟基戊二酸脱氢酶（D2HGDH 和 L2HGDH），催化 2-HG 向 αKG 的相互转化，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。 D2HGDH 突变导致 αKG 依赖性 TET 和 HDM 酶活性降低。最近通过识别调节 D2HGDH 和 L2HGDH 的上游信号扩展了这一概念使用 ChIP 测定、诱导细胞系和转基因小鼠模型，我们发现 MYC 转录激活 D2HGDH 和 L2HGDH，并且以 D2/L2HGDH/αKG 依赖性方式激活值得注意的是，我们发现 FTO 和 ALKBH5 功能导致 RNA 去甲基化。 MYC-D2/L2HGDH-αKG 轴还促进 FTO 和 ALKBH5 的核积累，与增强的 O-GlcNAcylation 相关，O-GlcNAcyllation 是由另一个执行的翻译后修饰线粒体酶，O-GlcNAc 转移酶（OGT）在这里，使用体外和体内的多种遗传模型，我们将测试一个新的线粒体信号轴的假设，其中包括近端的 MYC，中心的 D2/L2HGDH 和 OGT 以及远端的 FTO/ALKBH5 活性控制着细胞表观转录组。我们的具体目标是：1）表征 D2HGDH/L2HGDH 和中间代谢物对 m6A 水平的控制，2) 确定 O-GlcNAcNAc 介导的增加的机制基础 MYC-D2/L2HGDH-αKG 轴及其在促进 RNA 去甲基化中的作用，3) 定义线粒体人类细胞中代谢依赖性甲基RNA/基因表达特征。