RNAs associate with chromatin through various ways and carry out diverse functions. One mechanism by which RNAs interact with chromatin is by the complementarity of RNA with DNA, forming a three-stranded nucleic acid structure named R-loop. R-loops have been shown to regulate transcription initiation, RNA modification, and immunoglobulin class switching. However, R-loops accumulated in the genome can be a major source of genome instability, meaning that they must be tightly regulated. This thesis aims to identify R-loop-binding proteins systemically and study their regulation of R-loops.

Using immunoprecipitation of R-loops followed by mass spectrometry, with or without crosslinking, a total of 364 proteins were identified. Among them RNA-interacting proteins were prevalent, including some already known R-loop regulators. I found that a large fraction of the R-loop interactome consists of proteins localized to the nucleolus. By examining several DEAD-box helicases, I showed that they regulate rRNA processing and a shared set of mRNAs. Investigation of an R-loop-interacting protein named CEBPZ revealed its nucleolar localization, its depletion caused down-regulation of R-loops associated with rRNA and mRNA. Characterization of the genomic distribution of CEBPZ revealed its colocalization with insulator-regulator CTCF. When studying if CEBPZ recruits CTCF, I found that instead of regulating CTCF binding, CEBPZ depletion has a major effect on the performance of CUT&RUN, a technique for identifying DNA binding sites of proteins. How CEBPZ affects CUT&RUN is still under investigation, the study of which may help us understand the roles of CEBPZ in regulation of global chromatin structure and genome integrity.