lincRNA-1585在转录因子调控胚胎干细胞多能性维持中的功能和机制

In recent years, the function of Long intergenic non-coding RNA has caused wide attention in the field of life science. We have recently found that lincRNA1585 plays a very important role in somatic cell reprogramming process, reduced the expression of lincRNA1585 not only seriously hinder the formation of induced pluripotent stem cells, and lead to loss the pluripotency of embryonic stem cell. Combined with our long intergenic non-coding RNA and iPS cells (Proceedings of the National Academy of Sciences 2013, Cell Res 2013, Stem Cells 2012) and preliminary experimental data, we hypothesized that, lincRNA1585 mainly through lincRNA1585, transcription factor and the epigenetic complexes play in the maintenance of pluripotency and promote the formation of iPS cells. Therefore, our project intends using crispr-cas9, RNA Immunoprecipitation, RNA pull-down, Chromatin Immunoprecipitation sequence, single-cell sequencing experiment method to study epigenetic mechanism of lincRNA1585 for the acquisition of pluripotency and maintenance in ES cells. The research of this regulatory complex is not only conducive to develop a better understanding of gene regulatory network in the maintenance of pluripotency and provide more molecular cues and theoretical basis for the research of regulatory lincRNA in the iPS generation, but also is beneficial for the study of mechanisms in the somatic cells reprogramming.

近年来长链非编码RNA的功能引起了生命科学领域的广泛关注。我们最近发现lincRNA1585等在体细胞重编程过程中扮演着非常重要的作用，下调其表达不仅严重阻碍iPS细胞的形成，而且导致胚胎干细胞多能性丢失。结合我们非编码RNA和iPS细胞关系的工作（PNAS 2013、Cell Res 2013、 Stem Cells 2012）和预实验研究，我们推测，lincRNA1585主要通过形成lincRNA1585-转录因子-表观遗传分子复合物发挥其维持干细胞多能性和促进iPS细胞形成的功能。为此，本课题拟利用CRISPR-Cas9、RIP、RNA pull-down、ChIP-seq、单细胞测序等实验手段系统研究lincRNA1585调节多能性获得和维持的分子与表观遗传机制。我们的研究不仅有助于理解干细胞多能性维持的分子调控网络，而且为iPS诱导过程中的lincRNA调控机制研究提供参考。

长链非编码RNA能够和核心转录因子共同调控ES细胞多能性网络，但是lincRNA是如何影响染色体结构和基因转录的机制仍然是不清楚。我们的研究发现了一条lincRNA（linc1585）能够受到关键转录因子如Oct4, Sox2, Nanog和c-Myc等的直接结合调节，在干细胞多能性维持和获得过程中都具有非常重要的作用。我们的研究结果显示Linc1585还能够作为Sox2的共调控分子参与ES细胞的多能性维持，并主要介导了Sox2对于发育基因的转录抑制作用。另外，我们研究中发现PRC2的重要组分Ezh2同样能够与linc1585直接结合，来帮助行使lincRNA的转录抑制功能。因此，我们的研究证实了linc1585和Sox2的相互作用能够作为一个招募的平台，并通过引导PRC2复合物到发育基因的调控区域，来介导发育基因的转录沉默，从而维持干细胞的多能性。