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：
10322194
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
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/10322194
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.

线粒体作为信号细胞器的识别越来越多。这个“辅助”的重要方面线粒体功能是表观遗传调节的，例如乙酰辅酶A和S-的产生体现 DNA和组蛋白的乙酰化和甲基化分别用于乙酰化和甲基化。此外， α-酮戊二酸（αkg）和2-羟基戊二酸（2-HG），代谢物几乎完全在该代谢中产生线粒体被发现调节αkg依赖性双氧酶的活性，包括TET 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，我们的组发现αkg激活，2-HG抑制了对N6-作用的RNA脱甲基酶的FTO和ALKBH5 甲基腺苷（M6A），一种影响影响的mRNA的可逆化学修饰（表观转录组）基因表达。与其他表观遗传标记相似，RNA甲基化受动态控制和M6A 抽象会影响各种生物学功能，而其与人类疾病的不正当关联。考虑到αkg/2-Hg主要由中间线粒体代谢产生，并且是 RNA脱甲基酶的活性是由这些代谢物调节的，可以合理推测线粒体在控制RNA甲基化稳态中起重要作用。特别是我们假设线粒体酶D-2-2-和L-2-羟基戊二酸脱氢酶（D2HGDH和L2HGDH），催化2-Hg至αkg的相互转换与线粒体之间的相互作用不可或缺代谢和RNA甲基化的控制。我们较早发现的损失支持了这一假设功能D2HGDH突变的功能导致αkg依赖性TET和HDM酶的活性降低。我们最近通过识别调节D2HGDH和L2HGDH的上游信号来扩展此概念表达/活动。使用芯片分析，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，并以D2/L2HGDH/αkg依赖性方式激活IT 诱导FTO和ALKBH5功能，导致体外和体内RNA脱甲基化。值得注意的是，我们发现 MYC-D2/L2HGDH-αkg轴也促进了FTO和ALKBH5的核积累与增强的O-Glcnacylation相关，这是另一种人执行的后翻译后修改线粒体酶，O-GLCNAC转移酶（OGT）。在这里，使用多个遗传模型在体外和体内，我们将检验以下假设：新型线粒体信号轴，该信号轴（包括代理点的MYC）中心的D2/L2HGDH和OGT，分别控制FTO/ALKBH5活性，控制了细胞表演组。我们的具体目的是：1）表征D2HGDH/L2HGDH和中间代谢物对 M6A水平的控制，2）确定由O-Glcnacylation增加的机械基础 MYC-D2/L2HGDH-αkg轴及其在促进RNA脱甲基化中的作用，3）定义线粒体代谢依赖性的甲基na/基因表达在人类细胞中。