ABSTRACT
PURPOSE: To investigate the effect of epigenetic modification on pattern, time and capacity of transcription activation of POU5F1, the key marker of pluripotency, in cloned bovine embryos. METHODS: Bovine fibroblasts were stably transfected with POU5F1 promoter-driven enhanced green fluorescent protein (EGFP). This provided a visible marker to investigate the effect of post-activation treatment of cloned bovine embryos with trichostatin A (TSA) on time and capacity of POU5F1 expression and its subsequent effect on in vitro development of cloned bovine embryos. RESULTS: Irrespective of TSA treatment, POU5F1 expression was not detected until 8-16 cell stage, but was detected in both inner cell mass and trophectoderm at the blastocyst stage. TSA treatment significantly increased POU5F1 expression, and the yield and quality of cloned embryo development compared to control. CONCLUSION: The POU5F1 expression of cloned embryos is strictly controlled by the stage of embryo development and may not be altered by TSA-mediated changes occur in DNA-methylation and histone-acetylation of the genome.
Subject(s)
Cloning, Organism/methods , Embryo, Mammalian/physiology , Epigenesis, Genetic , Octamer Transcription Factor-3/genetics , Transcriptional Activation , Acetylation , Animals , Blastocyst/metabolism , Cattle , DNA Methylation , Embryo, Mammalian/metabolism , Embryonic Development/genetics , Gene Expression Regulation, Developmental/drug effects , Histones/genetics , Histones/metabolism , Hydroxamic Acids/pharmacologyABSTRACT
5-Aza-2'-deoxycytidine (AzC), trichostatin A (TSA), and its natural mimetic, sodium butyrate (NaB), are antineoplastic drugs that can modify the epigenetic status of donor cells prior to somatic cell nuclear transfer (SCNT). In this study, we used fibroblast cells treated with these drugs to investigate the direct and indirect effects of induced changes in DNA methylation and acetylation of the lysine 9 residue of histone H3 (H3K9). Additionally, we assayed cellular characteristics (cell growth, cell proliferation, cell cycle progression, and apoptosis) and SCNT efficiency in response to these drugs as well as monitoring these effects 24 h after removing the drugs. We observed the following: (1) AzC, TSA, and NaB all showed dose-dependent effects on different cellular characteristics; (2) TSA and NaB induced H3K9 hyperacetylation accompanied by DNA hypermethylation, whereas AzC induced DNA hypomethylation with no effect on H3K9 hyperacetylation; (3) TSA and NaB improved cloning efficiency, whereas AzC reduced it; and (4) unlike AzC, the effects of TSA and NaB on cellular characteristics and SCNT efficiency were reversed following drug removal. Our results indicate that somatic cells treated with TSA and NaB show better survival and recovery rates following the removal of these drugs. Moreover, H3K9 hyperacetylation (induced with TSA and NaB), but not DNA hypomethylation (induced with AzC), favors cloning efficiency.
