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

> **NIH NIH R35** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2024 · $411,250

## Abstract

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 My unpublished work in mouse ESCs (mESCs) strongly suggests that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 My unpublished work showed that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 potent...

## Key facts

- **NIH application ID:** 10939680
- **Project number:** 1R35GM154906-01
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Xin Huang
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $411,250
- **Award type:** 1
- **Project period:** 2024-07-05 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10939680

## Citation

> US National Institutes of Health, RePORTER application 10939680, The Regulatory R-loops in Pluripotency, Early Development, and Tissue Homeostasis (1R35GM154906-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10939680. Licensed CC0.

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