Identification and analysis of non-coding loci critical for cell viability and chromatin organization

项目摘要： 鉴定功能性非编码序列的努力主要集中在调控元件上，如 增强子和非编码RNA。然而，尽管约70%的人类基因组是表观遗传学上静止的，即， 没有任何表观遗传修饰的标志，表明功能不活动，大多数体细胞突变 与疾病相关的基因发生在非编码区。一个新出现的问题是为什么以及如何体细胞化 表观遗传学静止基因座的突变会影响细胞功能并导致疾病形成。一 可能的机制是这些基因座的突变可能改变邻近的染色质组织， 通过3D基因组传播变化以产生对表型的深刻影响。虽然这 假设是诱人的，但在本领域中，这种非编码基因座尚未被广泛鉴定、表征和分析。 人类基因组在拟议的工作中，我们的目标是整合计算预测和CRISPR库 筛选以系统地揭示不存在任何功能元件并且甚至不存在任何功能元件的非编码基因座。 以组蛋白修饰、蛋白结合或开放染色质为标志，但对细胞存活很重要（特异性 目标1和2）。我们将研究这些位点的缺失如何导致细胞功能障碍的机制 使用Hi-C、单细胞RNA-seq和单ATAC-seq分析（Specific Aim 3）。一旦完成， 拟议的工作将提供一个新的方面来理解表观遗传学上的静止非编码的功能， 基因座，并促进开发靶向这些基因座的基于基因组编辑的疗法。

Project Summary: The effort to identify functional non-coding sequences has largely focused on the regulatory elements such as enhancers and non-coding RNAs. However, while ~70% of the human genome is epigenetically quiescent, i.e. not marked by any epigenetic modification indicating functional inactivity, the majority of somatic mutations associated with diseases occur in the non-coding regions. An emerging question is why and how somatic mutations in epigenetically quiescent loci would affect cellular functions and cause disease formation. A possible mechanism is that the mutations in these loci may alter the neighbor chromatin organization and such a change is propagated through the 3D genome to generate a profound impact on phenotype. Although this hypothesis is tempting, such non-coding loci have not been widely identified, characterized and analyzed in the human genome. In the proposed work, we aim to integrate computational prediction and CRISPR library screen to systematically uncover non-coding loci that do not host any functional element and are not even marked by histone modification, protein binding or open chromatin but are important for cell survival (Specific Aim 1 and 2). We will investigate the mechanisms of how the deletion of these loci lead to cell dysfunction using Hi-C, single cell RNA-seq and single ATAC-seq analyses (Specific Aim 3). Once completed, the proposed work will provide a new aspect of understanding the functions of epigenetically quiescent non-coding loci and facilitate developing genome-editing based therapeutics targeting these loci.