Histone Demethylase KDM5B Is A Key Regulator Of Genome Stability

Objectives:Maintenance of genomic stability is essential for normal organismal development and is vital to prevent diseases such as cancer.As genetic information is packaged into chromatin,it has become increasingly clear that the chromatin environment plays an important role in DNA damage response.However,how DNA repair is controlled by epigenetic mechanisms is not fully understood.Here we intend to identify the function of lysine-specific histone demethylase 5B(KDM5B)in multiple aspects of DNA double-strand break(DSB)response.Methods and Results:1)Nuclear protein were extracted and fractioned.We found that KDM5 B was preferentially accumulated in the chromatin fraction after DNA damage.2)Using a cell-based system in which a recognition site for endonuclease I-Sce1 or AsiSI is stably incorporated into genome and a DSB could be generated by transient transfection with I-Sce1 or AsiSI,quantitative ChIP showed that KDM5 B was recruited to DSB site upon DNA damage.3)Immunoprecipitation demonstrated that KDM5 B was coimmunoprecipitated with PARP1 and the genotoxic insult could enhance the interaction between PARP1 and KDM5 B.Moreover,KDM5 B was highly PARylated when cells were exposed to genotoxic insult.4)Nuclear protein were extracted and fractioned.We found that loss of PARP1 led to a diminished chromatin binding of KDM5 B.Analogously,reduced level of KDM5 B binding to chromatin was detected when cells were treated with PARP1 enzymatic inhibitor PJ-34.Quantitative ChIP showed that the PARP1 enzymatic activity as well as the physical association between KDM5 B and PARP1 could influence the redistribution of KDM5 B to site-specific damaged chromatin.5)Using the NHEJ and HR-DRGFP system followed by FACS,the results demonstrated that KDM5 B depletion impaired HR and NHEJ efficiency.6)Transfecting the demethylase activity defective KDM5 B mutation and using the NHEJ or HR-DRGFP system followed by FACS demonstrated that demethylase activity of KDM5 B was required for its role in DSB repair.Moreover,Quantitative ChIP showed that KDM5 B could change the level of H3K4me3 around the break region.7)Applying the peptide pull-down and immunofluorescence assay,the results showed that KDM5 B was responsible for the recruitment of BRCA1 to the break site and influenced the BRCA1 foci formation through its catalytic activity.But KDM5 B knockdown did not affect the formation of gH2AX IRIF or that of MDC1 IRIF.Also,we used the laser microirradiation and the immunofluorescence and found that KDM5 B was responsible for the recruitment of Ku70 via its catalytic activity.8)Exposing cells to IR or NCS to generate DSB,we found that KDM5 B depletion increased IR-induced DNA damage checkpoint proteins phosphorylation.9)Knock down KDM5B/ATM and expose cells to IR or NCS.The results indicated that KDM5 B depletion sensitized cells to genotoxic insults.10)Exposing cells to IR or transfecting KDM5 B siRNA followed by FACS to identify the cell cycle,the results indicated that KDM5 B could affect the cell cycle progression by delaying the G1/S transition and irradiation could exacerbate the situation.11)Knock down KDM5 B in p53+/+ cells or p53-/-cells and analyse the expression level by Western blotting.The results showed that the absence of KDM5 B in p53+/+ cells,but not in p53-/-cells,could elevate the expression level of the relative proteins in p53 signaling pathway.Using FACS we found that KDM5 B depletion induced delay of G1/S transition was only observed in p53+/+cells.The effect was dependent on p53 activation.Conclusion:KDM5B becomes enriched in DNA-damage sites after ironizing radiation and endonuclease treatment in a poly(ADP ribose)polymerase 1 dependent manner.KDM5 B is required for efficient DSB repair and for the recruitment of Ku70 and BRCA1,the essential component of nonhomologous end-joining and homologous recombination,respectively.Significantly,KDM5 B deficiency activates p53 signaling and sensitizes cells to genotoxic insults.Our results suggest that KDM5 B is a bona fide DNA damage response protein and indicate that KDM5 B is an important genome caretaker and a critical regulator of genome stability,adding to the understanding of the roles of epigenetics in the maintenance of genetic fidelity.