The Function And Mechanism Of EZH2 Inhibitor DZNep In Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia(CML)is a malignant hematological disease in which gene mutation or chromosomal translocation blocks the differentiation of hematopoietic stem cells and impairs hematopoietic function.Studies have shown that the histone methyltransferase EZH2,a important subunit of the PRC2(Polycomb Group Protein2,PRC2)protein complex,affects the development of CML by regulating the modification of H3K27me3.EZH2 mediated epigenetic modification is a reversible and dynamically regulated process.Although the current clinical research using EZH2 specific small molecule inhibitors to treat leukemia has made some progress,drug resistance caused by serial use of the drug,as well as the multiple side effects caused by cell non-specificity impeds in the disease treatment.Therefore,this research aims to elucidate the specific regulation mechanism of EZH2.In this study,EZH2 small molecule inhibitor DZNep was used to treat the K562 cell line to explore the effect of EZH2 on CML cells and its molecular mechanism.Firstly,real-time quantitative PCR and Western Blot showed that after DZNep treatment,the expression of EZH2 was significantly reduced in K562 cells;the level of H3K27me3 modification in the whole genome was significantly decreased and the level of H3K9ac modification was significantly increased.Meanwhile,MTS and flow cytometry results indicacted that DZNep significantly inhibited cell proliferation,promoted cell apoptosis and erythroid differentiation,while did not change the cell cycle significantly.In order to explore the regulatory role of EZH2 in the process of erythroid differentiation,we used the histone-modified CHIP-Seq data of K562 and human CD34~+myeloid cells in the Encode database for systematic analysis.H3K9ac modified genes were significantly enriched in the pathways that are related to regulating erythroid differentiation of hematopoietic cells,while H3K27me3 modified genes were not enriched in the pathways of erythroid differentiation.In order to prove the correlation between EZH2and H3K9ac modification,we knocked down the EZH2 gene in K562cells,and the results showed that EHZ2 downregulation significantly promoted the upregulation of histone acetyltransferase P300,indicating that EZH2 downregulate H3K27me3 and promote the expression of P300through regulating H3K27me3 methylation of P300.Next,we knocked down the acetyltransferase P300 in K562 and analyzed the biological behavior of the cells.Knocking down P300 inhibited erythroid differentiation of K562 cell significantly.In order to prove the mechanism of H3K9ac promoted erythroid differentiation,we used real-time quantitative PCR to detect the expression of erythroid differentiation markers TALL,MED1,TRIM58 and GATA1 in P300 knock down K562cells.The results showed that knocking down of P300 significantly down-regulated the mRNA level of TALL,MED1,TRIM58 and GATA1.The above results indicate that DZNep promotes the expression of acetyltransferase P300 by inhibiting the H3K27me3 modification mediated by EZH2,thereby regulating the H3K9ac modification,affecting the expression of key genes related to erythroid differentiation such as GATA1、TAL1,MED1 and TRIM58.This study provides additional insights to the DZNep treatment of CML.Moreover,we discovered that the mutual regulation of different modification sites of histones plays an important role in the production of red blood cells,which might be a valuable perspect for further research in this field.