Investigate Novel Epigenetic Silencing Mechanisms in Mouse ESCs

 描述（由申请人提供）：我们拟议研究的长期目标是确定调节重要生物过程的新表观遗传机制。研究表观遗传沉默复合物在控制小鼠胚胎干细胞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Rif 1基因的新作用， 作为2-细胞胚胎基因和滋养外胚层基因ESC的表观遗传沉默因子。这种抑制对于ESC中的细胞命运维持至关重要。先前的研究表明，一个罕见的内源性反转录转座子家族（<0.05%）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，滋养外胚层基因需要沉默，以保持胚胎干细胞的承诺，胚胎组织，但分子基础仍然难以捉摸。除了这些有趣的问题，在该领域的一个长期的兴趣是开发简单而可靠的方法来研究表观遗传因素在一个特定的基因座的方式。 我们最近的初步研究已经确定了一种新的表观遗传沉默复合物在胚胎干细胞，Rif 1表观遗传沉默复合物（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（位于染色体内位点的端粒样序列），这是强烈富集在MERV-L的侧翼区域，在RESC介导的2-细胞胚胎基因沉默中起着关键作用。其次，Rif 1与多能性转录因子的相互作用对于抑制滋养外胚层基因是至关重要的，这些基因没有MERV-L作为启动子。基于这些发现，我们假设RESC利用不同的机制靶向不同的基因。此外，我们一直在开发基于修饰的CRISPR/Cas9系统的新方法，以基因座特异性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拴系（CRISPR拴系）或通过生物化学手段从特定基因座拉出因子（CRISPR拉出）。在这项提案中，我们将充分研究RESC介导的沉默的机制，并充分开发基于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，成果 将揭示招募表观遗传因子以及ESC中细胞命运决定的新机制。其次，这项工作将增加一个有趣的扭曲经典的位置杂色效应（端粒位置效应），即在早期胚胎基因的表观遗传沉默的作用，这是从来没有在哺乳动物细胞中报道的间质端粒序列。第三，正在开发的新方法将提供有用的工具，研究转录调控和表观遗传学的基因座特异性的方式。

 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.