Abstract A06: Cisplatin-induced DNA damage modifies the chromatin landscape of histone H2B monoubiquitination in a p53-dependent manner

Abstract

Abstract Post-translational modifications (PTMs) of histone tails can function as epigenomic regulators of the cellular response to DNA damage. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a central histone PTM involved in the DNA damage response. H2Bub1 localizes to double-strand breaks where it functions to decondense chromatin, making it accessible to DNA repair factors. H2Bub1 is also known to be enriched at coding sequences of highly expressed genes, demonstrating the fundamental importance of H2Bub1 for an open chromatin structure that facilitates transcription. H2Bub1 is a dynamic process, written predominantly by the E3 ubiquitin ligase complex of the ring finger proteins RNF20 and RNF40 and erased by multiple deubiquitinases, including those from the ubiquitin-specific peptidase subfamily USP7 and USP44. RNF20 is reported to interact with p53. Platinum drugs are used as standard-of-care for the treatment of high-grade serous ovarian cancer (HGSOC) based on their capacity to damage DNA. We sought to determine the response of ubiquitinated histone tails to a platinum drug with the goal of improving the efficacy of current best practice therapy for HGSOC. Following treatment of a wild-type p53 cell line with an IC75 dose of cisplatin, we combined H2Bub1 chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to determine genomic enrichment of H2Bub1 in response to DNA damage. Further, we coupled this with RNA-seq (RNA sequencing) to enable correlation of genomic enrichment of H2Bub1 with levels of gene expression. KEGG pathway and gene ontology analyses demonstrated greatest H2Bub1 enrichment in response to DNA damage in p53 signalling pathway genes, including CDKN1A, BBC3, MDM2 and BAX. This correlated with increased expression of p53 target genes in response to DNA damage. Wild-type TP53 itself did not show H2Bub1 enrichment upon treatment with cisplatin. H2Bub1 ChIP experiments were repeated in HGSOC mutant p53 cell line models, specifically OVCAR-3 with the known gain-of-function TP53 mutation R248Q and Kuramochi with the missense TP53 mutation D281Y. H2Bub1 was not enriched in response to DNA damage at wild-type p53 gene targets in these cell lines, nor were these genes highly expressed. We therefore propose that DNA damage induced H2Bub1-enrichment in mutant p53 cell lines will have an entirely distinct genomic profile compared to wild-type p53 cell lines, with important roles in enabling the expression of mutant p53 target genes in HGSOC. Experiments are currently under way to directly address this hypothesis. Lastly, we have used clonogenic cell survival assays to demonstrate that downregulation of RNF20, either transiently using short interfering (si) RNA or stably,using short hairpin (sh) RNA, decreased the ability of HGSOC cells to survive treatment with cisplatin. Down-regulation of H2Bub1 by downregulation of its E3 ubiquitin ligase RNF20 may represent a novel treatment strategy to increase the efficacy of DNA damage-based therapy for HGSOC. Citation Format: Alexander J. Cole, Kristie-Ann Dickson, Christopher Liddle, Clare Stirzaker, Roderick Clifton-Bligh, Deborah J. Marsh. Cisplatin-induced DNA damage modifies the chromatin landscape of histone H2B monoubiquitination in a p53-dependent manner. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A06.