Mitochondrial 2-hydroxyglutarate dehydrogenases modulate the cellular epitranscriptome

人们越来越多地认识到线粒体是信号细胞器。这个“附属品”的一个重要方面 线粒体的功能是表观遗传调节，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。此外, α-酮戊二酸(αKG)和2-羟基戊二酸(2-HG)，代谢产物几乎完全在 线粒体，被发现调节αKG依赖的双加氧酶的活性，包括TetDNA 羟基酶和组蛋白去甲基酶(HDM)，从而控制DNA和组蛋白甲基化。值得注意的是，我们的 研究小组发现，αKG激活而2-HG抑制作用于N6-的FTO和ALKBH5去甲基酶。 甲基腺苷(M6A)，一种可逆的mRNA的化学修饰(表位转录组)，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，m6A 丰度影响各种生物功能，而其失调与人类疾病有关。 考虑到αKG/2-HG主要由线粒体中间代谢产生，而 RNA去甲基酶的活性受这些代谢物的调节，因此有理由推测 线粒体在控制RNA甲基化动态平衡中起着重要作用。特别是，我们假设 线粒体D-2-和L-2-羟基戊二酸脱氢酶(D2HGDH和L2HGDH)， 它们催化2-HG相互转化为αKG，是线粒体之间相互作用所不可或缺的 新陈代谢和RNA甲基化的控制。这一假设得到了我们早先发现的损失的支持 功能D2HGDH突变导致依赖于αKG的TET和HDM酶活性降低。我们 最近通过识别调节D2HGDH和L2HGDH的上游信号对这一概念进行了扩展 表情/活动。利用芯片分析、可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依赖的方式激活 在体外和体内诱导FTO和ALKBH5功能，导致RNA去甲基化。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-GlcN酰化相关，这是由另一个人执行的翻译后修饰 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， 我们将检验这一假设，即一个新的线粒体信号轴，包括近端的MYC， 位于中心的D2/L2HGDH和OGT，以及远端的FTO/ALKBH5活性，控制着细胞表观转录组学。 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)决定了O-GlcN酰化增加的机制基础 MYC-D2/L2HGDH-αKG轴及其在促进RNA去甲基化中的作用：3)确定线粒体 人类细胞代谢依赖的甲基核糖核酸/基因表达特征。

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.