H3K36me3 and PSIP1/LEDGF associate with several DNA repair proteins, suggesting their role in efficient DNA repair at actively transcribing loci

Abstract

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses an H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology-directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry (qMS) to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). We also performed stable isotope labelling with amino acids in cell culture (SILAC) followed by qMS for a longer isoform of PSIP1 (PSIP/p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts ( MEFs). Furthermore, immunoprecipitation followed by western blotting was performed to validate the qMS data. DNA damage in PSIP1 knockout MEFs was assayed by a comet assay. Results: Proteomic analysis shows the association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP /p75. We further validated the association of PSIP/p75 with PARP1, hnRNPU and gamma H2A.X  and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP/p75 in promoting homology-directed repair (HDR), our data support a wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting proteins involved in DNA damage response pathways to the actively transcribed loci.