5hmC RNA role in mouse Embryonic stem cell differentiation to Embryonic bodies [RNA-seq]

Tet-enzyme-mediated 5-hydroxymethylation of cytosine in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs revealed hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), showed decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we found Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further revealed mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation was found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment. Overall design: Transcriptome of wild type mouse embryonic stem cells (3 samples), amanitin-treated 0h (2 samples), amanitin-treated 4h (2 samples) and Tet1,2,3 triple knock-out mouse embryonic stell cell amanitin-treated 0h (2 samples) and amanitin-treated 4h (2 samples)

Tet酶（Tet-enzyme）介导的DNA胞嘧啶5-羟甲基化（5-hydroxymethylation）在小鼠胚胎干细胞（embryonic stem cells，ESCs）中发挥关键作用。近年来，RNA中也被证实存在5-羟甲基胞嘧啶（5-hydroxymethylcytosine，5hmC），但其生理作用仍大多未被阐明。本研究揭示了该修饰标记在小鼠胚胎干细胞及分化中的拟胚体（embryoid bodies）中的作用与功能。对小鼠胚胎干细胞进行全转录组定位分析后，发现数百条信使RNA（messenger RNA，mRNA）在具有明确独特共有序列及特定特征的位点上带有5hmC修饰。细胞分化过程中，大量转录本（包括多个编码关键多能性相关因子如Eed、Jarid2的转录本）的胞嘧啶羟甲基化水平出现下降。利用Tet基因敲除（Tet-knockout）的小鼠胚胎干细胞，我们证实Tet酶是介导mRNA上5hmC修饰沉积的部分因子。进一步的全转录组筛选发现，Tet1与Tet2结合的mRNA靶位点，其拓扑结构与5hmC修饰位点高度相似。研究表明，Tet介导的RNA羟甲基化会降低关键多能性促进转录本的稳定性。我们提出，Tet酶介导的RNA胞嘧啶5-羟甲基化是转录组可塑性（transcriptome flexibility）的一种标记，与多能性与细胞谱系定型（lineage commitment）之间的平衡密不可分。实验设计概述：野生型小鼠胚胎干细胞（3个样本）、鹅膏毒肽（amanitin）处理0小时组（2个样本）、鹅膏毒肽处理4小时组（2个样本），以及Tet1、Tet2、Tet3三基因敲除小鼠胚胎干细胞经鹅膏毒肽处理0小时组（2个样本）与处理4小时组（2个样本）的转录组分析