DNA羟化酶Tet家族蛋白Tet1和Tet2主要通过其转录抑制活性，调控胚胎干细胞多能性相关基因的表达。本研究采取亲和纯化方法结合SILAC技术筛选并验证了新的Tet1互作蛋白Pspc1。Pspc1表达敲低的表型与Tet1和EED在胚胎干细胞中缺失的表型相似；Pspc1与多梳蛋白Polycomb皆可与lncRNA Neat1结合。这些前期数据提示Pspc1可能是Tet和Polycomb间的桥梁分子，三者通过Neat1形成复合体发挥调控功能。在本项研究中，我们将确认Pspc1与Polycomb和Tet是否共同调控下游基因，Neat1或其他RNA是否介导了三者复合体的形成，以及复合体调控的相关RNA靶分子。本研究的成果，将揭示Tet1-Pspc1 蛋白复合物在RNA调控机制和转录控制中的新功能，并为将来精确定义Pspc1功能及Tet家族在干细胞维持、分化和肿瘤形成中的转录调控奠定坚实的基础。

DNA hydroxylases Tet family (Tet1 and Tet2) play important roles in the maintenance of stem cell pluripotency by functioning mainly as transcription repressor and regulating pluripotency associated genes expression, including Nanog and Oct4. In our studies, we performed affinity purification combined with SILAC-mass spectrometry technology and found a novel Tet1-interacting protein paraspeckle protein 1 (Pspc1). Pspc1 depletion led to defects in EB differentiation of the ESCs, which is similar with the defects found in Tet1 and EED depleted ESCs. We also found that the Pspc1 and Polycomb proteins bind to lncRNA Neat1. These preliminary data suggest that probably Pspc1 is the bridging factor for Tet and Ploycomb and contributes to the Tet-Ploycomb complex formation through Neat1. The current application aims to extend our current Tet interactome studies and investigate functional contribution of a close partner of Tet1, Pspc1 to Tet1's transcriptional regulation of stem cell pluripotency. The identification of transcriptional target genes of Pspc1 shared by Tet1 in ESCs as well as the direct binding noncoding and/or coding RNA targets of Pspc1 will uncover novel functions of the Tet1-Pspc1 complex in RNA regulatory machinery/transcriptional control, and lay a solid function for future more detailed mechanistic studies to define the functional contribution of Pspc1, and paraspeckle in general, to Tet function in transcriptional regulation of stem cell control, differentiation, and cancer.

DNA羟化酶家族蛋白主要通过将5mC氧化为5hmC，调控DNA甲基化以及胚胎干细胞多能性相关基因的表达。在小鼠胚胎干细胞中，Tet1和Tet2是两种最主要的5hmC调节蛋白。Tet1主要负责启动子和转录起始位点的5hmC水平，而Tet2主要调节高表达基因gene body区和外显子边界的5hmC水平。研究表明Tet2在调节早期胚胎发育和免疫反应的转录水平中起重要作用，但是Tet2识别染色质靶点的分子机制尚不清楚。因此我们在胚胎干细胞中对Tet2结合染色质的机理做了深入研究。第一，我们在3xflag-Tet2敲入的ESC中进行SILAC和免疫沉淀偶联质谱，发现新的与Tet2互作的蛋白Pspc1。co-IP证明两者的相互作用高度特异。第二，在敲低Pspc1的ESC中，染色质上Tet2水平下降了30%，表明Pspc1和Tet2的相互作用可能介导了Tet2被招募到染色质的过程。第三，我们利用CRISPR-Cas9建立了Pspc1敲除的ESC细胞系，之后分别表达Pspc1WT和RNA结合区含有4个突变的Pspc1Mut。结果证明Tet2和Pspc1在染色体上的结合都受到Pspc1的RNA结合能力的影响，提示Pspc1与RNA的相互作用可能参与了Tet2被招募的过程。另外利用转录抑制剂α-amanitin处理ESC可以减少Tet2在染色体上的结合。第四，体外RNA免疫共沉淀证明Tet2蛋白复合体与RNA的结合依赖于Pspc1。第五，结合Pspc1的交联与串联免疫沉淀及高通量测序（CLIP-seq）和已有的Tet2染色质免疫共沉淀（ChIP-seq）数据，Pspc1在基因组上的RNA结合峰与Tet2在染色体上的被招募位置具有高相关性，说明Tet2被招募到基因组的位置有可能由Pspc1与RNA的结合决定。总之，我们的发现对揭示Tet2在染色体上结合的分子机理提供了坚实的理论基础。