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.

Tet酶介导的DNA胞嘧啶5-羟甲基化（5-hydroxymethylcytosine, 5hmC）在小鼠胚胎干细胞（embryonic stem cells, ESCs）中发挥关键调控作用。近期研究亦证实RNA中存在5-羟甲基胞嘧啶（5hmC），但其生理功能目前仍未得到充分阐释。本研究阐明了该修饰在小鼠ESCs及分化中拟胚体中的贡献与功能。对ESCs开展的全转录组定位分析显示，数百条信使RNA（messenger RNA, mRNA）在具备明确独特共有序列与特定结构特征的位点上带有5hmC修饰。细胞分化过程中，大量转录本（包括诸多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ESCs，我们发现Tet酶可部分负责介导mRNA中的5hmC修饰沉积。全转录组筛选进一步揭示了Tet1与Tet2结合的mRNA靶标，其结合位点的拓扑结构与5hmC位点高度相似。研究证实，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。我们据此提出：Tet介导的RNA胞嘧啶5-羟甲基化是一种转录组灵活性标记，与多能性与谱系定向之间的平衡密不可分。