Dissect the Molecular Mechanisms of HERV-H Mediated Shaping of Chromatin 3D Structure in Human Pluripotent Stem Cells

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 year, 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 lncRNAs no doubt unravels pathogenic pathways of the associated diseases, and provide potential solutions (e.g. drug targets) to effective therapies.