Scientific Abstract Genome integrity is constantly threatened by various forms of genotoxic stresses, including radiation or endogenous metabolic products. Damaged DNA lesions in actively proliferating cells cause stalling of DNA replication polymerases as well as RNA polymerases, which can lead to replication fork arrest or transcriptional arrest, respectively. Understanding the cellular response to monitor and execute the polymerase stalling will give us better insight on how stressed cells, such as cancer cells, deal with the challenges. The BMI1-containing Polycomb complex (PRC1; Polycomb Repressive Complex 1) is an important regulator of epigenetic silencing programs during development, in part by inducing H2AK119 monoubiquitination (H2AK119-Ub) associated with repressed RNA Polymerase II (Pol II). Recently, the role of BMI1 in DNA damage response has gained much attention, but in what aspect and mechanism it participates in the process is unclear. In our preliminary studies, we identified UBR5 as a downstream factor whose chromatin recruitment is regulated by the BMI1-containing PRC1 complex. While transcription is repressed at UV-induced damaged sites on chromatin, depletion of the PRC1 members or UBR5 alone de-repressed transcription elongation at the damaged sites, suggesting that UBR5 functions in a linear pathway with PRC1 in inducing gene silencing at the lesions. We found that UBR5 interacts with BMI1 as well as histone chaperone complex FACT, and that UBR5 co-localizes with FACT component SPT16 to the UV-induced lesions in a BMI1-dependent manner. We further provide evidence that UBR5 directly ubiquitinates SPT16. Our evidence suggests a model that the BMI1-UBR5 axis antagonizes the FACT-dependent Pol II elongation upon UV insults. Based on these data, we propose a hypothesis that UBR5 is a key downstream effector of the PRC1- mediated gene silencing.
The specific aims are to determine: 1) role of PRC1 and histone modification in UBR5 recruitment, 2) role of BMI1 and UBR5 in regulating transcriptional repression under DNA damaged or unperturbed conditions, 3) role of OTUD5 deubiquitinating enzyme in the UBR5 and PRC1-induced transcriptional repression at the lesions. The successful outcome of these studies will establish UBR5 as a key downstream mediator of the transcriptional regulation mediated by the PRC1 complex. The proposed work will fill the gap of important yet largely unknown function of the Polycomb complex in gene silencing, and will have an impact that advances the broader area of transcription, DNA damage response, and cancer development.
Proper regulation of RNA synthesis upon genotoxic stress is crucial for genome and proteome integrity. Completion of our study will lead to better understanding of cellular mechanisms that responds to genotoxic stresses that organisms encounter.
|de Vivo, Angelo; Sanchez, Anthony; Yegres, Jose et al. (2018) The OTUD5-UBR5 complex regulates FACT-mediated transcription at damaged chromatin. Nucleic Acids Res :|