The Regulatory R-loops in Pluripotency, Early Development, and Tissue Homeostasis

PROJECT SUMMARY R-loops are three-stranded nucleic acid structures composed of an RNA-DNA hybrid and a free single-stranded DNA. The opened DNA strands may lead to DNA damage; thus, R-loops are a risk factor for genome integrity. Improper R-loop accumulation contributes to abnormal human development and diseases. In contrast to the detrimental effects of R-loops, growing evidence suggests that they also regulate gene transcription, mitosis, and homologous recombination, thus contributing to many fundamental physiological processes. The formation and resolution of R-loops must be tightly regulated. Therefore, the goal for the next five years of my research is to investigate the mechanisms by which R-loops epigenetically regulate gene transcription in pluripotency, early development, and tissue homeostasis. My unpublished work in mouse ESCs (mESCs) strongly suggests that Zfp281 (human: ZNF281) is an R-loop-dependent transcription factor and recruits Tet1 and Brca2 for DNA demethylation and DNA damage repair, respectively. We will explore the functions of R-loops as a DNA epigenetic regulator in the mouse naive-formative-primed pluripotent state transition through pathways that include Zfp281/Tet1-mediated R-loop formation for gene activation and Zfp281/Brca2-mediated R-loop resolution for genome stability. In human development, naive human ESCs (hESCs) have a prolonged developmental plasticity and can differentiate into extraembryonic lineages. My unpublished work showed that ZNF281 is highly enriched in naive hESCs, with a widespread occupation of Polycomb repressive complex 2 (PRC2) and the repressive histone H3K27me3 mark at the extraembryonic master gene loci (e.g., GATA4, CDX2). Therefore, we will investigate the effects of R-loops and ZNF281 on PRC2-repressed low-transcription genes in naive hESCs and their differentiation into the human extraembryonic endoderm and trophectoderm lineages. Moreover, we will investigate the relationship between DNA damage and genome-wide gains of ZNF281, PRC2, and H3K27me3 in naive hESCs. And last, we will develop a research program to explore R- loop functions in tissue homeostasis and in disease settings. We will focus on B-cell homeostasis, as evidence shows that aberrant accumulation of R-loops accelerates the progression of diffuse large B-cell lymphoma (DLBCL), a disease originating from the malignant transformation of B-cells in the germinal center. My unpublished work showed that B-cell-specific Zfp281 conditional knockout mice had accumulated pre-/pro-B cells in the bone marrow and had increased IgG1-expressing activated B-cells in the spleen upon immunization. Using the DLBCL lines and the Zfp281 deficiency mouse model, we will investigate the effects of abnormal R- loop accumulation induced by Zfp281 deficiency on B-cell development and the progression of B-cell lymphoma. In summary, we aim to establish a coherent view of regulatory R-loops as DNA and histone epigenetic modifiers, as well as a potential risk factor if they are not properly resolved, in different cellular systems.