Human INO80 Chromatin Remodeler Contributes To Activating Doxorubicin-induced P21 Expression Through Removal Of H2A.Z From P21 Promoter

Epigenetic regulation is mainly divided into four ways: DNA methylation,non-coding RNA action,chromatin remodeling and histone modification.As a class of chromatin remodeling enzymes,INO80 can alter the chromatin structure using the energy produced by hydrolysis of ATP,including causing the sliding of DNA on nucleosomes and the replacement between classical histones and histone variants.H2A.Z is a variant of the classical histone H2A.Comparing to H2A,its structural changes lead to differences in its ability to bind to DNA.It is widely distributed in specific sites on chromatin to perform corresponding specific functions.As a class of histone acetylase,MOF can acetylate the N-terminal tail of histone H4.At present,whether human INO80 can replace H2A.Z with H2A,and what kind of functional role after replacement does it perform,how does acetylation of histone H4 by MOF participate in chromatin activity,it is all unclear.We purified the activated INO80 complex from Hela cells and confirmed that INO80 can remove H2A.Z from recombinant nucleosomes in vitro histone exchange assay.The INO80 mutant(INO80E653Q),which lost DNA-sliding catalytic activity in vitro,also lost its ability to exchange H2A.Z with H2A in our experiments,further confirmed that INO80 has a replacement activity for H2A.Z,in addition to DNA sliding activity.Interestingly,the MOF complex,also purified from Hela cells,acetylated histone H4 of recombinant nucleosomes in vitro,and significantly enhanced the ability of INO80 to remove H2A.Z from nucleosomes.This fully reflects the close relationship between histone modification and chromatin remodeling in chromatin activity.At the genome-wide level,we found the overall distribution of H2A.Z within 3kb upstream and downstream of transcription start sites(TSSs)increased after knocking down INO80.The distribution of acetylated H2A.Z(H2A.Zac)has also accordingly increased.To further validate this conclusion,we extracted nuclear chromatin after knocking down INO80,and found the proportion of H2A.Z in the chromatin increased compared to the control group.Therefore,we demonstrate that INO80 is involved in the regulation of H2A.Z distribution at the genomic level and is primarily involved in the process of removing H2A.Z from chromatin.In order to study the specific function of INO80 on the replacement activity of H2A.Z,we focused our attention on the transcriptional regulation of p21 gene.It was confirmed that INO80 and H2A.Z were co-localized at the p53 binding site of the p21 promoter.This indicates that INO80 is likely to regulate the distribution of H2A.Z on the p21 promoter.When Doxo induces p21 transcriptional activation,H2A.Z is rapidly displaced from the p53 binding site on the p21 promoter,thereby facilitating the recruitment of p53 and activating transcription.After knocking down INO80,the process of H2A.Z rapidly removing from nucleosomes was delayed,resulting in the corresponding inhibition of transcriptional activation of p21.It was demonstrated that the transcriptional activation of p21 relies on the removement of H2A.Z by INO80.At the same time,after knocking down MOF,it was not only directly decrease the level of H4K16 ac at the p53 binding site of the p21 promoter,but also delays the rapid removement of H2A.Z from nucleosomes.Predictably,p21 transcriptional activation was also inhibited.This indicates that the p21 transcriptional activation,which relies on replaceing of H2A.Z by INO80,is also affected by MOF-mediated histone acetylation.Based on current studies and in vitro results,we hypothesized that MOF maintains relatively high level of H4K16 ac at the p53 binding site on the p21 promoter,thereby recreating the nucleosome active(more accessible).Thus,it is easier for INO80 to displace H2A.Z from this site and promote p21 transcriptional activation.In summary,we confirmed that INO80 can remove H2A.Z from recombinant nucleosomes in vitro;INO80 regulates Doxo-induced p21 transcriptional activation through its replacement activity.In addition,MOF-mediated histone acetylation promotes the transcriptional activation of p21 though assisting replacement of H2A.Z by INO80.