The Effect Of Trichostatin A And5-AZA-2’-Deoxycytidine On The Epigenetic Modificationsand Related Genes Expression Of Bovine Fibroblasts

With the creation of somatic cell nuclear transfer technology, different types of cells have been successfully used as donor cells in a variety of species. However, the somatic cell nuclear transfer efficiency is still low. Studies have shown that epigenetic reprogramming of somatic cell effects cloned embryo development, and inadequate reprogramming of somatic cells is the leading cause for to early embryonic death and birth abnormalities. DNA methylation and histone acetylation are two common epigenetic modifications associated with chromatin structure and gene expression regulation. Studies have shown that treatment of donor cells with histone deacetylase inhibitor trichostatin A (Trichostatin A, TSA) and DNA methylation inhibitor5-aza-2-deoxycytidine (5-Aza-2’-deoxycytidine) can change its epigenetic state to improve the cloning efficiency. However, the effects of these drugs on the donor cell epigenetic modifications the mechanisms by which they improve the efficiency of nuclear transfer is not well understood. In this study, different concentrations of TSA and5-Aza-dC were used to treat bovine fetal fibroblast cells. After treatment, the changes on cell growth, histone acetylation, pluripotency gene expression, and pluripotency gene promoter DNA methylation were measured.1. Effect of TSA on cell growth, morphology and the level of histone acetylation of bovine fibroblasts(1) Bovine fibroblasts were treated with0ng/mL,10ng/mL,25ng/mL,50ng/mL, and75ng/mL TSA for24h. The cell growth curve showed S type.10ng/mL and25ng/mL VPA inhibited cell growth, while high concentrations of50ng/mL and75ng/ml VPA caused an sever cell death.(2) Immunofluorescence showed that the histone H3K9> H3K14acetylation levels increased as the increase of TSA concentration after treatment.2. Effect of TSA on expression and promoter DNA methylation of pluripotency gene in bovine fibroblasts(1) Realtime PCR was used to detect the expression of pluripotent genes in bovine fibroblasts treated with0ng/mL,10ng/mL,25ng/mL,50ng/mL, and75ng/mL TSA for24h. The results showed that VPA upregulated the expression of OCT4and NANOG in a concentration-dependent manner. When cells were treated with25ng/mL TSA for6h,12h,24h,36h,48h and60h, the highest expression of OCT4and NANOG emerged at24h and12h respectively.(2) The changes in DNA methylation at the promoters of pluripotency genes OCT4, NANOG and SOX2was measured bisulfate sequencing. The results showed that the DNA methylation level of OCT4promoter was significantly reduced by TSA treatment, but the DNA methylation levels of NANOG、SOX2promoter were not changed by the treatment.3. Effect of5-aza-dC treatment and combined treatment with5-aza-dC and TSA on pluripotency gene and epigenetic gene expression in bovine fibroblasts(1) Bovine fetal fibroblasts were treated with0μM,0.01μM,0.1μM and1μM DNA methyl-transferase inhibitor5-Aza-dC for72h. Cell growth curve showed S type. Compared with the control group, treated group showed a certain degree of inhibition of cell growth.(2) Realtime PCR was used to detect epigenetic modification gene and pluripotency gene expression. The results showed that5-aza-dC treatment had no effect on OCT4, NANOG and HDAC1expression, but down-regulated DNMT1expression.(3) Combination treatment of bovine fetal fibroblasts with TSA and5-aza-dC up-regulated the expression of OCT4and had no effect of NANOG expression.Conclusion:Treatment of bovine fibroblasts with TSA improves the levels of H3K9ac, H3K14ac and H3K9me3, and up-regulates the expression of OCT4and NANOG. Treatment of bovine fibroblasts with5-Aza-dC down-regulates the expression of DNMT1. These changes facilitate to convert fibroblasts to a more suitable state for using as donor cells in somatic cell nuclear transfer.