The effect of NaAsO2 on the binding of methyl CpG binding protein 2, DNA methyltransferase 1 and histone deacetylase 1 to the promoter of MGMT gene in HaCaT cells / 中国地方病学杂志

ABSTRACT

Objective To investigate the effect of NaAsO2 on the binding levels of methyl CpG binding protein 2(MeCP2),DNA methyltransferase 1 (DNMT1) and histone deacetylase 1(HDAC1) to the hypermethylation promoter region of MGMT gene in HaCaT cells,in order to provide a basis to deepen the interpretation of the role of arsenic poisoning mechanism.Methods HaCaT cells were treated repeatedly and interval with different concentrations of NaAsO2(3.13,6.25,12.50,25.00 μnol/L,respectively) for 72 h.Untreated HaCaT was used as blank control group and human epidermal squamous carcinoma cell line(A431 cells) as positive control group.The binding levels to the two transcription regulatory regions(ChIP1,ChIP2) and to the coding region(ChIP3) of MGMT 8ene were detected by chromatin immuno-precipitation combined with quantitative PCR.Results The differences of binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each group were significant (F=7.387,84.634,78.442 and 19.263,69.649,26.546,all P ＜ 0.05).The binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each NaAsO2 exposed group[3.13 μmol/L NaAsO2 exposed group(136.00 ±16.97)％,(145.00 ± 2.83)％,(88.50 ± 19.09)％ and (106.50 ± 37.48)％,(112.34 ± 8.73)％,(59.71 ± 8.49)％;6.25 μmol/L NaAsO2 exposed group(130.00 ± 42.43)％,(154.50 ± 4.95)％,(101.00 ± 1.27)％ and (88.50 ±3.54)％,(134.32 ± 2.82)％,(102.75 ± 19.91)％ ; 12.50 μmol/L NaAsO2 exposed group(141.50 ± 23.33)％,(161.50 ± 7.78)％,(125.00 ± 11.31)％ and (119.50 ± 24.75)％,(171.59 ± 3.54)％,(167.61 ± 10.61)％; 25.00μmol/L NaAsO2 exposed group(134.50 ± 43.13)％,(472.50+ 50.20)％,(383.50 ± 30.41)％ and (180.09 ±12.73)％,(348.50 ± 27.58)％,(158.45 ± 12.02)％] were higher than that in the blank control group[(51.50 ±9.19)％,(82.00 ± 12.73)％,(25.03 ± 2.91)％ and (37.02 ± 4.24)％,(91.56 ± 26.16)％,(19.09 ± 2.90)％,all P ＜ 0.05].The differences of binding levels of MeCP2 to ChIP3 in each group were not significant(F =1.670,P ＞0.05),but the differences of binding levels of DNMT1 and HDAC1 to ChIP3 were significant (F =4.404,9.863,all P ＜ 0.05),and only the binding levels in the 25.00 μmol/L NaAsO2 exposed group [(615.85 ± 29.63)％,(306.09 ± 59.40)％] were higher than that in the blank control group[(99.70 ± 12.02)％,(92.45 ± 48.79)％,all P ＜ 0.05].Conclusions MeCP2 can bind to the methylated MGMT gene transcriptional regulatory regions which are induced by arsenic and leads to histone deacetylation by the recruitment of DNMT1 and HDAC1 and,meanwhile,DNMT1 can bind to the coding region of MGMT gene to recruit HDAC1 in a methyl DNA binding protein(MBD) independence manner and media MGMT gene silencing through the chromatin remodeling way,which might be the early molecular events of arsenic poisoning.