Mechanisms of NBR2, a Long Non-Coding RNA, in Human Ovarian Aging

About This Grant

PROJECT SUMMARY/ABSTRACT This research proposal is in response to the NOT-OD-24-079 “Notice of Special Interest: Women’s Health Research” focused on health conditions that are female-specific. The ovary is the first organ to age in the human body. Ovarian aging negatively influences lifespan and a broad range of health outcomes in cardiovascular, skeletal, metabolic, immune, and neurocognitive systems in women. Despite these broad impacts, ovarian aging has received limited scientific attention. The biological mechanisms that drive ovarian aging, and how they influence broader healthspan in women, remain poorly understood. The objective of this proposal is to investigate the molecular mechanisms by which NBR2, a long non-coding RNA, contributes to the remarkably complex processes of ovarian aging. By performing genome wide association studies (GWAS) of two reproductive aging- related traits, age of natural menopause (ANM) and reproductive lifespan (RL), we found that genetic variants within a gene-rich haplotype block at the BRCA1 locus are associated with both traits. Our integrative post- GWAS analysis, combined with functional genomic studies in human ovarian cell models, identified a causal non-coding regulatory variant (rs2298862 T>C) associated with both later ANM and longer RL. Although previous ANM GWAS studies identified BRCA1 as the causal gene at this locus, our functional genomic studies experimentally validated that NBR2 at the true target gene. The variant downregulated NBR2 expression, suggesting that NBR2 is a likely driver of the reproductive longevity phenotypes. The major goal of this project is to uncover the mechanisms by which NBR2 modulates female reproductive longevity (Aim 1) and elucidate the mechanisms by which the causal regulatory variant regulates NBR2 expression in diverse ovarian cell types (Aim 2). In Aim 1, I will test the hypothesis that reduced NBR2 expression delays ovarian aging by modulating pro-longevity signaling pathways. I will generate CRISPR/Cas9-mediated knockout NBR2 cell models and perform unbiased RNA-immunoprecipitation followed by mass spectrometry to identify NBR2 interactors and downstream targets. In Aim 2, I will test the hypothesis that the rs2298862 (T>C) variant reduces NBR2 expression by altering long-range chromatin interactions, disrupting transcription factor binding, and modulating enhancer activity. I will generate multiple ovarian cell types from CRISPR-engineered human embryonic stem cells carrying the variant and assess its impact on NBR2 expression, chromatin architecture, transcription factor (TF) binding, and aging-related cellular phenotypes. By identifying pathways and regulatory mechanisms by which NBR2 and its downstream regulators influence ovarian aging, this project will provide a molecular framework for understanding human reproductive longevity and may reveal targets for preserving ovarian function and healthspan in women.