Doctor of Philosophy (Ph.D.)
Degree Granting Department
Biology (Cell Biology, Microbiology, Molecular Biology)
Younghoon Kee, Ph.D.
Kristina Schmidt, Ph.D.
Meera Nanjundan, Ph.D.
Gary Daughdrill, Ph.D.
DNA Damage, Genomic Stability, Replication, Transcription
The coordination of transcription, replication, and DNA damage response (DDR) is vital for maintaining normal cellular homeostasis. All of these processes take place on the chromatin and thus, the temporal and spatial separation of the factors responsible are necessary for each to be correctly completed. Here we detail several novel processes contributing to this network.
BMI1 is a component of the Polycomb Repressive Complex 1 (PRC1) which plays a key role in maintaining epigenetic silencing programs during development. Recently, BMI1 and other members of PRC1 like RNF2 have been implicated gene silencing during the DDR; however, the mechanism through which BMI1 and RNF2 impose this transcriptional repression is still under investigation. We have identified a novel relationship between the E3 ubiquitin ligase UBR5 and these PRC1 components during the DDR to repress transcription at these sites. We show here that UBR5 can work downstream of the PRC1 complex to suppress RNAPII elongation by negatively regulating the FACT histone chaperone complex. We show that UBR5 is recruited to sites of DNA damage through interactions with BMI1 and acts to prevent FACT from erroneously promoting transcription in that area.
We have found that this process is especially critical for preserving the integrity of common fragile sites (CFSs), these genomic loci are prone to breaks and rearrangements upon acute replication stress; importantly, these sites often encode for cancer associated genes and are closely linked to the progression of several genetic diseases. Previous work has determined that CFSs can become more unstable when they are being transcribed, however, the epigenetic regulators of CFS transcription an area of active inquiry. Given that we identified BMI1 and RNF2 as regulators of transcription at DNA damage sites we went on to characterize their contribution to the preservation of CFS stability. We found that cells lacking BMI1 and RNF2 show hallmarks of replication associated damage at CFS and present with decreased replication fork progression. Transcriptional elongation at CFSs is also increased in the absence of BMI1 and RNF2. We show that this creates an environment prone to transcription-replication conflicts (TRC) at CFSs. Previous work has shown that TRCs can result in the formation of R-loops, these RNA-DNA hybrid structures pose a significant barrier to replication and can result in double strand break formation. The cellular response to R-loops is an area of growing interest as these structures can have very diverse functions and outcomes in the cell. Here we show that R-loops are increased at CFS in the absence of BMI1 or RNF2, this is most likely the result of increased TRC. We investigated factors known to respond to R-loops and found the FA family proteins FANCD2 and FANCI as responders to R-loops generated by BMI1 and RNF2 deficiency. In the absence of BMI1 or RNF2 these factors become essential for resolving the increased R-loops, loss of FANCD2 or FANCI in RNF2 KO cells is highly genotoxic.
Taken together, we have identified a system for the preservation of genomic integrity at CFS, this is essential for preventing aberrations and the progression of disease. This entails the recruitment of UBR5 to damage sites by BMI1 or RNF2 followed by repression of FACT activity by UBR5. This can work to prevent aberrant transcription and TRCs resulting from that activity. When this regulation is not in place the cells experience increased genomic instability resulting from R-loop formation, these R-loops can be responded to by FA family proteins.
Scholar Commons Citation
Sanchez, Anthony Richard IV, "Genome Maintenance Roles of Polycomb Transcriptional Repressors BMI1 and RNF2" (2020). Graduate Theses and Dissertations.