干细胞维持及分化中TET复合体招募机制及其功能学的研究

TET family cytosine di-oxygenases are important epigenetic factors regulating DNA methylation. Recent studies have shown that TETs are able to oxidize 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), a process that is believed leading to DNA demethylation. Many studies, including ours, have established the importance of TET activity in maintaining cellular level of 5hmC in embryonic stem cells, demonstrated the requirement of TET activity during reprogramming, and observed an acute reduction of TET1/2 expression upon embryonic stem cell (ESC) differentiation. These findings all pointed out the biological processes governed by TET1/2 are essential in regulating appropriate epigenetic landscape in ESCs. However, it is still not fully understood that the recruitment mechanism regulating TET1/2 to its specific target regions on chromatin in ESCs. Our previous work identified several RNA binding proteins existing in the protein complexes of all three TETs and further confirmed the interactions through co-immunoprecipitation and pull-down assays. We hypothesize that these RNA binding proteins may play important role in TET protein recruitment mediated by yet unknown RNA related pathway. We plan to establish the genome-wide co-localization analyses among TET complex components by ChIP-seq approach, determine the identity of RNA species bound by TET complexes by RNA-seq and their function in TET recruitment, and investigate the potential tumor suppressive function mediated by these components in TET complex. Most of the proposed analyses have already been completed for Nono, and will be further carried out on other candidate proteins. The completion of this study will improve our understanding of how 5hmC is established and maintained in ESC state and regulated during differentiation.

TET家族蛋白重要的表观遗传修饰因子，能催化DNA甲基化至羟、醛、羧甲基化，并介导DNA去甲基化。前期研究已确定TET1/2是维持胚胎干细胞表观遗传信息的关键因子，TET1/2的酶活对细胞重编程过程至关重要，而TET1/2的表达在干细胞分化后被迅速关闭。但TET1/2在胚胎干细胞中是如何被招募至染色质上特定的位置发挥其催化活性的机制尚不明了。本课题组数据显示三类TET复合体中均存在多种染色质结合蛋白尤其是RNA结合蛋白，它们可在体外与TET直接相互作用，且部分候选蛋白的测序分析表明其与TET1在小鼠胚胎干细胞基因组中显著共定位。据此我们推测：TET复合体中的蛋白或RNA组分招募TET到特定染色质区域从而发挥羟甲基化酶作用。本课题已利用TET亲和纯化技术结合RNA-seq检测TET复合体RNA依赖性调节机制，并采用干细胞和小鼠模型验证其复合体中的部分组分在胚胎干细胞分化发育过程的生物学功能。

Nono是一类RNA结合蛋白，是分化细胞核内Paraspeckle（旁粒）的重要组分。然而旁粒结构并不存在与胚胎干细胞中，我们推测在胚胎干细胞中，Nono可能发挥未知的功能。在小鼠胚胎干细胞（mESC）中，本项目通过一系列分子表观遗传手段分析，发现Nono定位与双价染色质上并与Erk信号通路相互作用，调控了mESC的干性维持和分化。具体的，我们发现，Nono敲除的mESC倾向于更强的自我更新，与2i（GSK和Erk抑制物双处理）所诱导的“原始态”（Ground State）在转录组和表观组上有大量相似之处。Erk与Nono相互作用，并为Nono的染色质结合所必须。Nono与Erk共同结合的双价结构域靶基因表现出高水平的RNA聚合酶II丝氨酸五位磷酸化。Nono缺失引起Erk活性下调，影响RNA聚合酶的待活化状态（Poise），并导致分化受阻。总体来说，我们的研究发现了Nono与Erk信号通路协同并调控了双价结构域以及mESC的干性维持。本工作发表在2016年10月的Cell Reports上。..在另一项相关的针对“增强子”的研究中，发现增强子具有“过度活化态”，被表观遗传学信号——组蛋白甲基化H3K4me3和乙酰化H3K27Ac共同控制。该“过度活化态”常被癌细胞利用，引起癌基因过度激活，促进癌细胞转移。本研究还发现了表观遗传因子RACK7和KDM5C可抑制增强子的过度活化，从而防止癌细胞恶化和转移。该机制具有普遍性，为癌症转移机制与治疗手段提供了全新思路，并提出“增强子过度活化态”学术理论，为转化医学与癌症治疗带来新启发。文章发表在2016年4月的Cell上。