Characterization of R-Loop-Interacting Proteins in Embryonic Stem Cells
Authors
Wu, TongFaculty Advisor
Thomas FazzioAcademic Program
Interdisciplinary Graduate ProgramUMass Chan Affiliations
Department of Molecular, Cell and Cancer BiologyDocument Type
Doctoral DissertationPublication Date
2021-10-30Keywords
R-loopRNA
ribosomal RNA
embryonic stem cells
chromatin
epigenetics
DEAD-box proteins
CEBPZ
CTCF
Bioinformatics
Biology
Biotechnology
Molecular Genetics
Metadata
Show full item recordAbstract
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.DOI
10.13028/aysv-5285Permanent Link to this Item
http://hdl.handle.net/20.500.14038/31393Rights
Copyright is held by the author, with all rights reserved.ae974a485f413a2113503eed53cd6c53
10.13028/aysv-5285