开发新方法研究内源逆转录病毒HERV-H在胚胎发育中调控染色质高级结构的分子机理

哺乳动物细胞核内染色质高级结构对于转录调控至关重要。然而，除了知道一些结构蛋白（如CTCF）是形成和维持染色质相互作用的重要调控蛋白外，我们对于远距离染色质相互作用的调控机制了解甚少。最近科学家发现，灵长类特异的内源性H型逆转录病毒（HERV-H）及其转录本参与多能干细胞染色质高级结构的形成和维持，但其具体分子作用途径尚未明晰。. 在这个项目中，我们计划开发一种可以同时检测活细胞中非编码RNA结合蛋白和基因组结合位点的新方法。将这种方法应用于人类胚胎干细胞及其衍生的心肌细胞中的HERV-H RNA，以便为我们提供HERV-H RNA结合蛋白和基因组结合位点在细胞分化过程中动态信息。本项目的研究结果将增加我们对多能细胞和细胞分化过程中3D基因组调控机制的认识。这项新的技术还可以广泛应用于揭示各类非编码RNA的分子功能。

The three-meter long chromatin fibers are folded into a high-order structure so that they can be packaged into a few micron-sized nuclei in mammalian cells. The high-order structure also allows distant DNA fragments to contact and exchange information between each other, which is fundamental for transcriptional regulation. For example, distal enhancers, a type of cis-regulatory element for gene expression, can activate the target gene promoter through chromatin long-range interaction that brings enhancers and promoters together in 3D space. However, very little has been known about how the long-range chromatin interactions can be regulated. In recent years, a few protein factors have been identified playing roles in maintaining and organizing the high-order structure of chromatins. For example, structural proteins such as CTCF and Cohesin, are required for the formation and maintenance of the self-interacting genomic segments, termed topologically associating domains (TADs). More recently, another class of macromolecule non-coding RNA is also discovered potentially with a regulatory role of organizing TAD structure. Scientists found that transcription of a primate specific endogenous retrovirus Type-H (HERV-H) was indispensable for formation and maintenance of TADs in human pluripotent cells. Unfortunately, the molecular pathway of how hHERV-H RNA contributes to creating TADs is not clearly revealed either...Delineating the proteins and chromatin loci associated with HERV-H RNA is fundamental to understanding the molecular mechanisms of HERV-H in various biological events. In this project, we propose to develop a novel method that can simultaneously detect the binding proteins and HERV-H genomic binding sites in living cells. Applying this method to HERV-H RNA in the human embryonic stem cell as well as its derived cardiac progenitor cells and cardiomyocytes provides us a dynamic map of HERV-H binding protein partners and genomic binding sites. This will reveal how HERV-H is engaged in regulating the chromatin high-order structure. We believe that the outcome of this project would improve our knowledge of how 3D genome is regulated in pluripotent cells and during cellular differentiation. The new technology can also be widely applied to uncover the function of non-coding RNAs in a variety of biological systems. In a long run, the discovery could benefit clinical research. For example, many lncRNAs are already used as biomarkers in diagnosis of multiple severe diseases, including cancers. Understanding the molecular function of these ncRNAs undoubtedly unravels pathogenic pathways of the associated diseases, and provide potential solutions (e.g. drug targets) to effective therapies.