The Effect Of Trichostatin A On DNA Methylation Of OCT4 Promoter Of Human Embryonic Fibroblasts And On Development Of Human Somatic Nuclear Transfer Embryos

Objective:To investigate the effect of trichostatin A(TSA)on DNA methylation of OCT4 promoter of human embryonic fibroblasts(hEFs) and development of human somatic nuclear tansfer(hSCNT)embryos.Methods:hEFs(3×105cells/90-mm dish)were allowed to attach for at least 48 hours and then treated without or with TSA for 24,48 and 72 hours with final concentrations of 50,100,200,300,400,500,600,700 and 800nM.After the incubation,the FACS,immunohistochemistry were evaluted.The FACS analysis of apoptosis was carried out to evaluate cytotoxicity of TSA.Immunohistochemistry was used to detect the expression of ES specic transcriptional factor oct4 and acethl-Histone H3. Expression pattern of OCT4 mRNA in hEFs before and after treatment with TSA was analyzed by reverse-transcription PCR(RT-PCR).And DNA methyltransferasel(DNMT1)mRNA also was detected by RT-PCR. We analyzed DNA methylation of CpG sites located in the OCT4 promoter by the bisulfate sequencing PCR(BSP)method to evaluate the effect of TSA on DNA methylation of CpG sites located in the OCT4 promoter of hEFs.Then,we treated human cloned embryos with 50nM TSA for 4hr to evauted the effect of TSA on the development of human cloned embryos.Result:The FACS analysis revealed that TSA may cause an increase in apoptosis in a concentration-dependent manner and in a time-independent manner after incubation of hEFs. Immunohistochemistry showed us the hEFs incubating with TSA was negative for the oct4.RT-PCR detection for the OCT4 transcript consistented with the result mentioned above.We observed that treating hEFs with 100 nM TSA for 48h might increase the level of acethl-Histone H3 significantly compared with the control.And we also found expression level of DNMT1 mRNA was down-regulated by TSA in a concentration-dependent manner after 48h of hEFs treatment.BSP showed the DNA methylation level of OCT4 promoter of 100nM TSA-8h-treated hEFs was approximately similar to those of non-treated cells.It remained hypermethylation compared with hES cells.With treatment of 50nM TSA for 4 hr after activation,we found that the≥8cell rate of TSA-treated cloned embryos(36.8%)was more than two times higher than that of untreated embryos(17.6%);And we also found there are double blastocysts of 19 TSA-treated cloned embryos,but none of 17 untreated embryos.Our findings indicated that TSA could induce histone acetylation and down-regulated DNMTI expression,but failed to demethylate OCT4 promoter of hEFs and to reactivate the expression of OCT4.Also,our results suggested that TSA-treatment improve developmental competence of hSCNT embryos and the effect of TSA on histone acetylation and down-regulating DNMT1 mRNA may accounted for it.