Study On The Mechanism Of Transcriptional Factor TWIST1 Involved In DNA Methylation In Myelodysplastic Syndrome

Myelodysplastic syndromes(MDS)are clonal disorders of hematopoietic stem/precursor cells characterized by an inexorable but variably paced trend of progressive cytopenias and to acute myelocytic leukemia(AML)in approximately 30% of cases transformation.Aberrant DNA methylation plays an important role in the pathophysiology of myelodysplastic syndromes(MDS).Silencing of suppressor genes caused by abnormal methylation of the CpG islands near the transcription start site often leads to development of the disease.Hypomethylating agent which decitabine used for treatment of high-risk MDS patients has achieved significant clinical efficacy can improve the survival rate,but some patients did not respond to this drug.At present,the causes of abnormal DNA methylation in MDS and the mechanism of action of hypomethylating agent are not clear.The transcription factor TWIST1 is a highly conserved structure with a basic helix-loop-helix(bHLH)structure that regulates the expression of the target genes in conjunction with the E-box(NCANNTGN)sequence of the target gene.In our previous work,the expression of TWIST1 in the cloned cells of high risk MDS was abnormally increased.And in the preliminary data,it is shown that over expressing TWIST1 in hematopoietic cells KG1 a leads to higher level of methylation in whole genome.We speculated that the expression of TWIST1 in MDS is closely related to DNA methylation machinery.We indicated that the over-expression TWIST1 in KG1 a cells significantly promoted cell proliferation compared to control groups with or without the treatment of DAC.Meanwhile,cell cylce of KG1 a cells showed G0/G1 phase accummulation compared to over-expressed TWIST1 cells.No significant differencens of cell apoptosis were found in over-expressed TWIST1 vs KG1 a cells with or without the treatment of DAC.Western Blot results showed that the expression of methyltransferase DNMT was positively correlated with the expression of TWIST1 in KG1 a cells that were over-expressed TWIST1.Affinity purification coupled to mass spectrometry indicated that TWIST1 is interacting with DNMT3 a,the direct binding of these two proteins was then verified by co-immunoprecipitation and GST-pull down,and Epitope Mapping predicted the binding sites of TWIST1 and DNMT3 a.The difference of methylation levels in KG1 a cells that were over-expressed TWIST1 vs KG1 a cells was detected by genome-wide methylation sequencing.It was found that the methylation level of differentially methylated regions(DMRs)in TWIST1 over-expression group is higher than in control group.Elavated methylation level in promotor area and decreased mRNA levels of tumor suppressor genes CDKN2 B and MYOD1,which were the target genes of TWIST1 and reported to abbrently expressed in MDS,were found by methylation-specific PCR in our study.In this study,we investigated cell proliferation and cell apoptosis as well as cell cycle in hematopoietic cell KG1 a that were over expressed TWIST1,explored the direct binding affinity of TWIST1 and DNMT3 a,and indicated the role of TWIST1-DNMT3 a complex in regulating methylation in the promotor area of TWIST1 target genes.Our study showed transcriptional factor TWIST1 is involved in DNA methylation machinery in hematopoietic cells,and laid the foundation for understanding the molecular mechanism of disease responses in demethylated treated patients with MDS.