猪体细胞到iPS细胞重编程过程中DNA甲基化的动态变化

DNA methylation and chromatin modi?cations serve as important epigenetic control layers on top of the genome sequence. They determine the chromatin structure and regulate gene expression. Recently, the discovery of a sixth base, 5-hydroxymethylcytosine (5hmC), hinted that the mechanisms responsible for the oxidation of 5-methylcytosine (5mC) into 5hmC might offer a clue to understanding processes of epigenetic control. Indeed, the three 2-oxogluterate (2OG)- and Fe(II)-dependent dioxygenases, Tet1, Tet2, and Tet3, were found to be able to mediate the transition from 5mC to 5hmC, and a substantial amount of all methylcytosine appeared to be converted by this enzymes in mice. Therefor, the role and regulation of Tet genes are associated with and important for the pluripotent state. Our results indicate that Tet3-mediated conversion between 5mC to 5hmC is involved in the active demethylation of paternal genome and methylated plasmid when active demethylation occurs. The generation of induced pluripotent stem cells (iPS) from somatic cells provides a new method to investigate cell reprogramming and treat diseases. At present, however, in the process of iPS induction, dynamic changes of epigenetics are not clear. Based on our previous works, this study aims to determine the methylation level of exogenous genes and endogenous genes changes during the induction of iPS, and the roles of Tet genes in iPS induction and pluripotency maintenance.

DNA甲基化与染色质修饰在基因组层面对表观遗传控制发挥重要作用，它们决定着染色质结构与基因表达。最近发现的5-羟甲基胞嘧啶(5hmC)可能有助于理解表观遗传控制的过程。小鼠中三种双加氧酶Tet1, Tet2和Tet3都能介导5-甲基胞嘧啶（5mC）转化为5hmC。因此Tet家族基因可能与多能性状态相关，并且对于多能性的维持起重要的作用。我们的研究结果显示Tet3介导的5mC到5hmC的转变过程参与小鼠雄原核和甲基化质粒的主动去甲基化。诱导多能干细胞(iPS)的出现，为研究体细胞重编程和疾病治疗提供了一个崭新的方法。目前iPS诱导过程中表观遗传学的动态变化过程还不清楚。本项目在我们前期研究工作的基础上，通过分析iPS诱导过程中DNA甲基化的动态变化，旨在确定iPS细胞诱导过程中外源基因和内源基因甲基化的动态变化过程，并对Tet家族基因在iPS细胞诱导以及iPS多能性维持中的作用进行深入研究。

诱导多能干细胞 (iPS) 开创了再生医学研究的新领域，对生物医学研究和畜牧业生产具有重要意义，但诱导效率低、细胞重编程机制不清仍是制约其应用的瓶颈。本项目利用诱导多能干细胞技术以及RNA干扰技术，旨在研究Tet1基因在猪iPS特性维持中的作用。主要研究结果如下：（1）将猪胚胎成纤维细胞（PEF）重编程为piPS。piPS克隆表达OCT4、SOX2、NANOG、REX1和SSEA4等干细胞标记。piPS在体外能形成表达三胚层标记基因Afp、Bmp4、Gfap的拟胚体。piPS在裸鼠皮下形成畸胎瘤，拟胚体包括外胚层起源的神经上皮，中胚层起源的血管、横纹肌，内胚层起源的滤泡样结构；（2）筛选出了2种能有效干扰Tet1表达的siRNA。Tet1表达下调后，piPS失去自我更新状态，开始分化并影响基因表达。其中谱系分化相关基因Pitx2、Hand1、Gata6和Lef1表达上升，多能性相关基因Lefty2、Klf2、Sox2表达下降；（3）Tet1表达下调后，影响piPS中DNA甲基化状态，其中Oct4、c-Myc启动子区域5mC含量明显上升，Sox2、Nanog启动子区域5mC含量没有变化，Klf4启动子区域5mC含量轻度上升；全基因组水平的5mC含量上升，而5hmC含量下降。本项目通过研究siRNA瞬时干扰piPS中Tet1基因表达后DNA甲基化及基因表达的状态，证实了Tet1基因在piPS多能性维持中发挥重要作用，为阐明猪iPS多能性网络调控和维持机制提供了重要的理论和实验依据。