UC San Diego

The spatial interactions between chromatin, RNA, and proteins inside the cell nucleus underpin the exquisite execution of cellular functions. RNA, one of the most versatile molecules, plays crucial roles in forming membrane less nuclear bodies, modulating chromatin architecture, and regulating gene expression. Despite a few well-studied ncRNAs, the roles of many other RNAs in gene regulation and chromatin organization remain unclear. This thesis aims to provide a systematic approach to delineate functionally important chromatin-associated RNAs (caRNAs).Chapter 1 establishes a baseline model of caRNA localization relative to chromatin structure and the influence of caRNAs on chromatin organization. By jointly comparing chromatin structure with genome-wide RNA-chromatin interactions in human embryonic stem cells, I found that caRNA localization is largely confined and shaped by local chromatin structures such as compartments, topologically associated domains (TADs), and chromatin loops. Chromatin loop anchors are hotspots for caRNA interactions, and depletion of loop anchor caRNAs leads to the formation of new chromatin loops. Chapter 2 addresses the challenge of identifying functional caRNA-chromatin interactions, where nascent transcripts significantly contribute to RNA-chromatin interactions, complicating the identification of functional caRNAs. I proposed a modified version of the iMARGI technique that introduces RNase A treatment before crosslinking. This method effectively removes non-protected caRNAs, such as nascent transcripts, leaving only stringent and protected interactions. We found that RNase-inaccessible caRNAs exhibit higher sequence conservation and are enriched at transcription factor binding sites, histone modifications, and other functional genomic regions. Additionally, we identified specific RNA species associated with transcription factor and histone modification sites. Chapter 3 presents MUSIC, a novel technology I co-developed for simultaneous profiling of DNA-DNA, RNA-DNA, and RNA-RNA interactions along with the transcriptome at single-cell resolution. Applying MUSIC to brain tissue from control and Alzheimer's disease (AD) patients, I find that transcriptomically aged cells have fewer local chromatin contacts and are more prevalent in AD patients. The research also demonstrates that the cell type exhibiting chromatin contacts between cis eQTL and a promoter tends to be that in which these cis eQTL specifically affect the expression of their target gene. Furthermore, I reveal that female cortical cells exhibit highly heterogeneous interactions between XIST non-coding RNA and chromosome X, with decreased XIST-chrX enrichment linked to increased chrX gene expression. In conclusion, this thesis provides valuable tools and insights into the functional roles of caRNAs in nuclear organization and gene regulation, offering new technologies for studying the RNA-DNA interplay in both normal and disease cellular states.