Stable oxidative cytosine modifications accumulate in cardiac mesenchymal cells from Type2 diabetes patients: rescue by alpha-ketoglutarate and TET-TDG functional reactivation [mouse muscle RNA-seq]. Stable oxidative cytosine modifications accumulate in cardiac mesenchymal cells from Type2 diabetes patients: rescue by alpha-ketoglutarate and TET-TDG functional reactivation [mouse muscle RNA-seq]

Background: Here, the role of α-ketoglutarate (αKG) in the epi-metabolic control of DNA demethylation has been investigated in therapeutically relevant cardiac mesenchymal cells (CMSCs) isolated from controls and type 2 diabetes donors. Methods & results: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing and gene specific GC methylation detection revealed an accumulation of 5mC, 5hmC and 5fC in the genomic DNA of human CMSCs isolated from diabetic (D) donors (D-CMSCs). Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high fat diet (HFD), injected with streptozotocin (STZ) or both in combination (STZ-HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in D-CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised thymine DNA glycosylase (TDG) and ten eleven translocation protein 1 (TET1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatched or 5fC. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5fC accumulation thus partially mimicking the diabetic epigenetic landscape in cells of non- diabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG-dehydrogenase, increased the αKG level in D- CMSCs and in the heart of HFD mice eliciting DNA demethylation, glucose uptake and insulin response. Conclusions: In this report we established that diabetes may epigenetically modify and compromise function of therapeutically relevant cardiac mesenchymal cells. Restoring the epi-metabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes. Overall design: RNA was isolated from mouse skeletal muscle samples (3 control, 3 high fat diet, and 3 high fat diet + AA6) and sequenced.

**背景**：本研究针对从健康对照及2型糖尿病供体中分离得到的、具有治疗应用价值的心脏间充质细胞（cardiac mesenchymal cells, CMSCs），探究了α-酮戊二酸（α-ketoglutarate, αKG）在DNA去甲基化的表观代谢调控中的作用。
**方法与结果**：通过定量全基因组分析、甲基化DNA测序、羟甲基化DNA测序及基因特异性GC甲基化检测，研究发现糖尿病供体来源的人心脏间充质细胞（D-CMSCs）的基因组DNA中，5-甲基胞嘧啶（5mC）、5-羟甲基胞嘧啶（5hmC）与5-甲酰基胞嘧啶（5fC）出现累积。对高脂饮食（high fat diet, HFD）、链脲佐菌素（streptozotocin, STZ）单独或联合注射（STZ-HFD）造模小鼠的全心脏基因组DNA分析显示，其胞嘧啶氧化修饰出现持续性累积。在此背景下，非靶向及靶向代谢组学分析表明，D-CMSCs及高脂饮食小鼠的全心脏组织中，细胞内αKG合成水平出现下降。该现象伴随胸腺嘧啶DNA糖苷酶（thymine DNA glycosylase, TDG）与十十一易位蛋白1（ten eleven translocation protein 1, TET1）的相互作用及功能受损，且TET1发生核迁出。分子动力学与突变分析表明，αKG可结合TDG的Arg275位点，从而实现酶的别构激活；在此作用下，TDG清除G/T核苷酸错配及5fC的能力显著提升。因此，外源性补充αKG可通过促进TDG功能、恢复TET1的核定位以及增强TET/TDG相互作用，修复DNA去甲基化循环。通过CRISPR/Cas9基因敲除或TET/TDG小干扰RNA（siRNA）敲低实现TDG失活后，可诱导5fC累积，从而在非糖尿病来源的细胞中部分模拟糖尿病相关的表观遗传特征。本研究鉴定出的新型化合物(S)-2-[(2,6-二氯苯甲酰基)氨基]琥珀酸（AA6）是αKG脱氢酶抑制剂，可提升D-CMSCs及高脂饮食小鼠心脏组织中的αKG水平，进而诱导DNA去甲基化、葡萄糖摄取及胰岛素应答反应。
**结论**：本研究证实，糖尿病可通过表观遗传修饰改变治疗相关心脏间充质细胞的功能。修复DNA去甲基化循环的表观代谢调控，有望为受环境代谢异常影响的细胞提供治疗获益。
**整体实验设计**：从小鼠骨骼肌样本（3例对照组、3例高脂饮食组、3例高脂饮食+AA6给药组）中提取RNA并进行测序。