Purification And Identification Of A Novel Protein Component With H2A.Z-exchange Activity From HeLa Nuclear Extract

In eukaryotic cells,genetic information stores in DNAs,which packaged into nhcleosome as a basic unit of the chromatin.H2A.Z is a variant of the canonical histone H2A conserved in yeast and humans.The histone elements of a nucleosome are considered highly related to transcriptional regulation,DNA double strand breaks(DSB)repair and maintenance of genome stability.Histone substitutions in chromosome is essential to chromatin remodeling.Papers have been published to unveil remodeling enzymes which have the H2A.Z exchange actixvity.For instance,Swr1 in yeast and its two homologs in human,SRCAP and P400/TIP60,are well-established chromatin remodeling enzymes.Recent studies have shown that TIP60 complex can swap H2A.Z into the DSB foci independently of its acetyltransferase activity.H2A.Z deficiency leads to recruit:ment failure of KU70/KU80 complex to the DSB foci,so as to prevent the non-homologous end joining(NHEJ)pathways in the DSB repair process.Whether H2A.Z is involved in transcriptional activation or inhibition is not clear,but the presence of H2A.Z in promoter region can change DNA accessibility to RNA polymerases,as far as to affect gene transcription.In addition,H2A.Z also plays an important role in DSB repair.In order to screen out novel protein components that may have H2A.Z exchange activities,we performed multiple column chromatography with 100 litters of HeLa nuclear extract,followed by in vitro histone varnant exchange assay to track the H2AZ exchange activity.We found protein components which can exchange H2A.Z into the nucleosomes,and further confirmed their activities together with other properties using in vitro and in vivo assays.The mass spectrometry showed that positive components were rich of DNA-PKcs and all subunits of condensing ?complex.Followed hydrophilic-hydrophobic chromatography separated the overlapping peaks.H2A.Z replacement activity was mainly present in the component containing DNA-PKcs.Later we revealed that condensing II complex which purified from stable cell line did not have H2A.Z exchange activity,whereas the DNA-PKcs which purified from over-expressing cells can exchange H2A.Z into nucleosome in a dose dependent manner.Since H2A.Z accumulation is dynamic changed on DSB foci,,we knocked down DNA-PKcs in two DSB models to observe the effect on H2A.Z recruitment.In X-ray radiation model,H2A.Z was normally recruited to damage foci at around one hour,whereas knocking down(KD)DNA-PKcs leaded to a decrease and delay in H2A.Z recruitment.In 4OHT-induced DSB model,KD DNA-PKcs resulted in a signimicantly delay of H2A.Z recruitment by two hours at the DSB foci.Further,we examine the regulation mechanism of DNA-PKcs on H2A.Z exchange.Results indicated that DNA-PKcs may have changed the chromatin structure by phosphorylating H2A,and then facilitated H2A.Z exchange into nucleosones.This activity can be inhibited by DNA-PKcs kinase inhibitor NU7026.However,whether DNA-PKcs phosphorylates other histones during this process,and the phosphorylated sites on H2A is remain to be investigated.In summary,we established DNA-PKcs as a H2A.Z exchange enzyme,of which the activity is related to H2A phosphorylation.But the specific mechanism remains to be further elucidated.