Investigate Novel Epigenetic Silencing Mechanisms in Mouse ESCs

 描述(由申请人提供)：我们提议的研究的长期目标是确定调节重要生物过程的新的表观遗传学机制。研究表观遗传沉默复合体在控制小鼠胚胎干细胞(ESCs)命运中的作用为我们的研究提供了一个理想的平台。我们最近的工作已经确定了Rif1基因的新角色 作为2-细胞胚胎基因和滋养外胚层基因的表观遗传沉默因子。这种抑制对于维持胚胎干细胞的细胞命运至关重要。以往的研究表明，一个罕见的内源性反转录转座子家族(MERV-L)作为2-细胞胚胎基因表达的启动子，但对其调控机制知之甚少。此外，为了维持胚胎干细胞对胚胎组织的承诺，需要沉默滋养外胚层基因，但分子基础仍然难以捉摸。除了这些耐人寻味的问题外，该领域的一个长期兴趣是开发简单而可靠的方法，以特定于基因座的方式研究表观遗传因素。我们最新的初步研究在ESCs中发现了一个新的表观遗传沉默复合体，Rif1表观遗传沉默复合体(RESC)。我们的研究还导致了RESC介导的沉默的意想不到的机制。首先，我们证明了间质端粒序列(位于染色体内染色体内的类似端粒的序列)在resc介导的2-细胞胚胎基因沉默中起着关键作用。端粒序列强烈富含在Merv-L的侧翼区域。其次，Rif1‘S与多能转录因子的相互作用是抑制滋养外胚层基因的关键，而滋养外胚层基因没有Merv-L作为启动子。基于这些发现，我们假设RESC利用不同的机制来靶向不同的基因。此外，我们一直在开发基于改进的CRISPR/Cas9系统的新方法，以座位特异性的方式研究表观遗传沉默。这些方法可以分别将表观遗传因子与靶基因系系(CRISPR系留)或通过生物化学方法(CRISPR Pull-out)将表观遗传因子从特定的基因座中拉出。在这项提案中，我们将充分研究RESC介导的沉默的潜在机制，并充分发展基于CRISPR的方法来研究表观遗传沉默。本研究的意义和创新之处在于几个方面。第一，结果 将揭示在ESC中招募表观遗传因子以及细胞命运决定的新机制。其次，这项工作将为经典的位置差异效应(o端粒位置效应)增加一个有趣的转折，即间质端粒序列在早期胚胎基因表观遗传沉默中的作用，这在哺乳动物细胞中从未报道过。第三，正在开发的新方法将为以特定于基因座的方式研究转录调控和表观遗传学提供有用的工具。

 DESCRIPTION (provided by applicant): The long-term goal of our proposed study is to identify novel epigenetic mechanisms for regulating important biological process. Investigating epigenetic silencing complexes in controlling mouse embryonic stem cells (ESCs) fate provides an ideal platform for our investigation. Our recent work has identified the novel role of Rif1 gene as an epigenetic silencing factor for 2-Cell embryo genes and trophectoderm genes ESCs. This repression is critical for cell fate maintenance in ESCs. Previous studies showed that a rare family of endogenous retrotransposon (<0.05%), MERV-L, serves as promoters for 2-Cell embryo gene expression but little is known about the regulatory mechanisms. In addition, trophectoderm genes need to be silenced in order to maintain the commitment of ESCs to embryonic tissues but the molecular underpinning remains elusive. Besides these intriguing issues, a longstanding interest in the field is to develop simple and reliable approaches to study epigenetic factors in a locus-specific manner. Our most recent preliminary studies have identified a novel epigenetic silencing complex in ESCs, Rif1 epigenetic silencing complex (RESC). Our studies also led to unexpected mechanisms for RESC-mediated silencing. First, we demonstrated that interstitial telomeric sequences (telomere-like sequences located in intrachromosomal sites), which are strongly enriched at flanking regions of MERV-L, play a critical role in RESC-mediated silencing of 2-Cell embryo genes. Second, Rif1's interaction with pluripotency transcription factors is critical for repressing trophectoderm genes, which don't have MERV-L as promoters. Based on these findings, we hypothesize that RESC utilizes distinct mechanisms for targeting different genes. Furthermore, we have been developing new approaches that are based on the modified CRISPR/Cas9 systems to study epigenetic silencing in a locus specific manner. These approaches can tether epigenetic factors to target genes (CRISPR tethering) or pull out factors from specific loci by biochemistry means (CRISPR pull-out), respectively. In this proposal, we will fully investigate the mechanisms underlying RESC- mediated silencing and fully develop CRISPR-based approaches for studying epigenetic silencing. The significance and innovation of this study lie in several fronts. First, the outcomes will reveal novel mechanisms for recruiting epigenetic factors as well as cell fate decisions in ESC. Second, this work will add an interesting twist to the classic position variegation effects (o telomere position effect), i.e. the role of interstitial telomeric sequences in the epigenetic silencing of early embryonic genes, which has never been reported in mammalian cells. Third, the new methodologies under development will provide useful tools for studying transcriptional regulation and epigenetics in a locus specific manner.