The Biological Significance Of H2A.Z Ubiquitination In Cancer Cells

Part 1 Expression of H2 A.Z in hepatocellular carcinoma and its clinical significanceObjective: To investigate the expression of H2 A.Z in hepatocellular carcinoma and its potentialclinical significance.Methods: The expression level of H2 A.Z in hepatocellular carcinoma and its relationship with pathological stage were comprehensively evaluated based on the meta-analysis of data from the GEO and TCGA.The diagnostic prognostic value of H2 A.Z in HCC,the relationship between H2 A.Z expression and various clinical parameters were evaluated based on TCGA.In addition,we detected H2 A.Z expression by immunohistochemistry(IHC)staining.Results: In our study,a total of 56 datasets were included,including 2,824 cases and 2,181 controls.Meta-analysis revealed that H2 AFZ was significantly up-regulated in hepatocellular carcinoma(SMD=1.23,95% CI:1.05,1.42)and advanced hepatocellular carcinoma(SMD=0.25,95%CI: 0.08,0.42).The expression level of H2 AFV was significantly up-regulated in hepatocellular carcinoma(SMD=0.74,95% CI: 1.05,1.42).The area under the ROC curve for H2 AFZ and H2 AFV diagnosis of hepatocellular carcinoma was 0.96 and 0.93,respectively.In addition,the expression level of H2 AFZ was significantlyassociated with alpha fetoprotein level,histological grade,pathological stage and T stage(P<0.05),and H2 AFV levels was remarkably correlated with sex,alpha fetoprotein level,histological grade,pathological stage and T The stage(P<0.05).The results of immunohistochemistry showed that the H2 A.Z protein level was significantly correlated with Edmondson grade and Arginase-1 level(P<0.05).High expression of H2 AFZ and H2 AFV was significantly associated with their poor prognosis.Conclusions: H2 AFZ and H2 AFV were highly expressed in hepatocellular carcinoma.H2 AFZ was significantly associated with pathological stage and grade of HCC.H2 AFV is notably associated with pathological grade of HCC.H2 A.Z.1 and H2 A.Z.2 serve as potential biomarkers for diagnosis and prognosis of hepatocellular carcinoma,which may play an important role in the occurrence and development of HCC.Part 2 Biological significance of H2 A.Z ubiquitination in cancer cellsChapter 1 Establishment of H2 A.Z wt and H2 A.Z mut-transfected cell linesObjective: Our previous studies showed that H2 A.Z may play an important role in the occurrence and development of cancer.As we now know,the function of histones is usually achieved by histone modifications.Ubiquitination is an important form of histone modification.The purpose of this study is to explore the biological significance of H2 A.Z ubiquitination.Therefore,this study mainly constructs the H2 A.Z wt and H2 A.Z mut overexpression vectors,and then establishes stable transfectant cell lines,which will lay the foundation for the subsequent research on the function of H2 A.Z.Methods: Firstly,the FLAG-H2 A.Z wt overexpression vector was constructed by molecular biology techniques.Secondly,site-directed mutagenesis was used to site-direct mutation of the ubiquitination sites of H2 A.Z on the flag-H2 A.Z wt vector.Finally,we detected the overexpression vectors by DNA sequencing,and confirmed the stable transfectant cell lines by immunofluorescence staining and western blot.Results: DNA sequencing of FLAG-H2 A.Z wt overexpression vector showed that we successfully inserted the target DNA fragment.DNA sequencing of the FLAG-H2 A.Z mut overexpression vector showed that the codons encoding the amino acids 120,121 and 125 of H2 A.Z(wt)on the vector were AAG to AGG,AAA to AGA and AAG to AGG,respectively.Immunofluorescence staining showed that the positive rate of the cells was 100% after screening by IL-2Rα-beads,and the exogenous H2 A.Z located inthe nucleus.Westem blot validated that the FLAG-H2 A.Z(wt)-transfected cell line had an unubiquitinated Flag-H2 A.Z(wt)and an ubiquitinated FlagH2 A.Z(wt).However,only the Flag-H2 A.Z(mut),which could not be ubiquitinated,was detected in the FLAG-H2 A.Z(mut)-transfected cell line.These results indicated that we had successfully constructed the FLAGH2 A.Z(wt)and FLAG-H2 A.Z(mut)overexpression vectors and established the corresponding stable transfectant cell lines.Conclusions: Lysines at positions 120,121 and 125 of H2 A.Z were its ubiquitination sites.Chapter 2 Effect of H2 A.Z ubiquitination on the stability of H2A-H2 A.Z heterotypic nucleosomesObjective: To investigate whether H2A-H2 A.Z heterotypic nucleosomes exist in eukaryotic cells and to study the mechanism of their stability.Methods: Mononucleosomes were isolated from the chromatin by MNase and were purified on a 5%-30% sucrose gradient centrifugation.Then,we detected H2A-H2 A.Z heterotypic nucleosomes by Co-Immunoprecipitation and western blot,and examined the effect of ubiquitination and high salt conditions on its stability.Results: The mononucleosomes separation liquid of H2 A.Z wt and H2 A.Z mut cell lines contained Flag-H2 A.Z(wt)and Flag-H2 A.Z(mut)mononucleosomes,respectively,both contained H2A-H2 A.Z heterotypic nucleosome core particles.And the relative content of H2A-H2 A.Z heterotypic nucleosomes of the former is higher than the latter.H2 A.Z ubiquitination was down-regulated by RING2-shRNA vector,and the relative content of H2AH2 A.Z heterotypic nucleosomes was found to be different from the two stable transfectant cell lines.The sequence from high to low was as follows: H2 A.Z(wt)cell line > RING2-shRNA and FLAG-H2 A.Z wt co-transfected cell line > H2 A.Z(mut)cell line.The relative content of H2A-H2 A.Z heterotypic nucleosomes in M132 treatment group was lower than the control,and was inversely associated with the treatment time.We compared the relative amounts of H2A-H2 A.Z heterotypic nucleosomes between the NEM group and the control,and found that the former was higher than the latter.In addition,when they were treated with high-salt solution(100 mM)at the same time,the relativecontents of H2A-H2 A.Z heterotypic nucleosomes had decreased,but the NEM group was still higher than the control.These results indicated that the stability of H2A-H2 A.Z heterotypic nucleosomes depended on the monoubiquitylation of H2 A.Z.On the contrary,deubiquitination and high salt conditions could significantly reduce its stability.Conclusions: For the first time,we confirmed the presence of H2AH2 A.Z heterotypic nucleosomes in eukaryotic cells,and successfully isolated intact nucleosome core particles of H2A-H2 A.Z heterotypic nucleosomes.At the same time,we revealed the mechanism of their stability,which depended on H2 A.Z ubiquitination.Chapter 3 Effect of H2 A.Z ubiquitination on the binding of transcription factors with H2 A.ZObjective: To explore the effect of H2 A.Z ubiquitination on the binding of transcription factors with H2 A.Z,to study the mechanism of ubiquitination regulating H2 A.Z-mediated cellular biological processes.Methods: Mononucleosomes were Co-Immunoprecipitation with M2,and the Co-Immunoprecipitation products were eluted competitively with Flag peptide.Then,we detected proteins with protein silver staining.Finally,they were identified by protein mass spectrometry.Results: Protein silver staining showed that the type and quantity of interaction proteins,which binding to H2 A.Z or ubH2 A.Z,were not exactly the same.There were about 19 different protein bands.Furthermore,their interaction proteins were identified by protein mass spectrometry.The proteins that interact with H2 A.Z were EEF1 D,ZRANB2,H2 ai,CBF,SRSF1,PSIP1,HMGA1,H2 B,H4,H1.3,NSBP1,HMGN3,CBX1,EEF1B2,CSE1 L,YBX1,ANP32 E,FUS,HSPA5,hnRNP D,HMGN4,RBM3,SFRS2 B,RcNSEP1,DEK,HMGN1,MIER1,RPS28.And the proteins that bind to ubH2 A.Z were polyUB,H2 AFV,NCL,NSBP1,H2 B,SERBP1,HMGN1,EIF4 B,HMGN3,CSDA,FUS,PSIP1,HMGA1,ZRANB2,UQCRH,H4,H3,RBM3,MRPL12,HMGN4,MRPL1,HnRNP A2/B1,YBX2,CANX,H1 FX.We observed that they were not exactly the same.This suggested that they may be involved in different cellular biological processes,and ubiquitination regulates H2 A.Z-mediated gene expression regulation.Conclusions: The transcription factors that interact with H2 A.Z and ubH2 A.Z were not exactly the same,suggesting that ubiquitination regulatesH2A.Z-mediated cellular biological processes.The main mechanism may be to promote the recruitment of different transcription factors through H2 A.Z ubiquitination and deubiquitination,and thus generate corresponding transcription states.Chapter 4 Effect of H2 A.Z ubiquitination on regulation of gene expressionObjective: To explore the effect of H2 A.Z ubiquitination on regulation of gene expression based on whole genome level by ChIP-Seq.Methods: In this study,we mainly used chromatin immunoprecipitation sequencing technology(ChIP-Seq).Results: We analyzed the binding sites of the H2 A.Z(wt)and H2 A.Z(mut)in the genome.The binding sites of H2 A.Z(wt)were distributed in the intergenic region of the gene,about 50.9%,upstream 23.1%,introns 22.2%,downstream 2.4%,and exons 1.4%.The distribution of H2 A.Z(mut)binding sites in the genome was similar to that of H2 A.Z(wt),intergenic region 50.2%,upstream 24.1%,introns 22%,downstream 2.2%,and exons 1.5%.It indicated that both H2 A.Z(wt)and H2 A.Z(mut)regulate the target genes by binding to their intergenic,upstream,and introns regions.Gene Ontology(GO)analysis revealed that these target genes were mainly involved in biological processes such as cellular processes,metabolic processes,single-organism processes,biological regulation,etc.The cellular components mediated by these genes included cell and cell part,organelles,cell membranes,etc.Their molecular functions were primarily related to binding and catalytic activity,etc.Their differential genes were CALD1,DPP6,NFIB,PROX1,PTPN7,PTPRN2,RORB,RGS5,SEMA5 A,MUC12,FBXL7,MCF2 L,TMEM98,UNC93 A,HHIP,TTLL2,MAML2,PLXNA4,PHACTR3,LOC284395,VSTM2 B,HHIPAS1,LOC654342.The binding sites of H2 A.Z(wt)or H2 A.Z(mut)to these genes were mainly distributed in the intergenic 66.2%,introns 26.89%,upstream 5.6%,and downstream 1.4%.It suggested that they were regulated by H2 A.Z(wt)or H2 A.Z(mut)binding to their intergenic and introns regions.They mainlyinvolved in biological processes such as cellular processes,metabolic processes,multicellular organismal processes,developmental processes,signaling,etc.The cellular components mediated by these genes included cell and cell part,organelles,etc.Their molecular functions were mainly related to binding and catalytic activity,etc.The Pathway analysis revealed that they were primarily involved in IL-17 signaling pathway,Hedgehog signaling pathway,Basal cell carcinoma,Th1 and Th2 cell differentiation,Type I diabetes mellitus,MAPK signaling pathway,c AMP signaling pathway,Notch signaling pathway,etc.Motif of H2 A.Z(wt)had a conservative sequence,i.e.CCTCBGCCTCCC.And motif of H2 A.Z(mut)also included a conserved sequence,i.e.TTTTTTTTTTTT.Conclusions: These results indicated that H2 A.Z regulates gene transcription,both activation and inhibition.H2 A.Z ubiquitination resulted in a change in the site of H2 A.Z binding to the target gene,indicating that H2 A.Z ubiquitination may alter its spatial conformation and thus regulate gene transcription.