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The effects of 5-aza-2'- deoxycytidine and trichostatin A on gene expression and DNA methylation status in cloned bovine blastocysts.

ABSTRACT: We previously found that treatment of both donor cells and early cloned embryos with combination of 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A (TSA) significantly improve the in vitro and full-term development of nuclear transfer (NT) bovine embryos. To investigate how this treatment improved the epigenetic reprogramming of somatic cell nuclei, we compared the expression levels of DNA methylation-, chromatin structure-, and development-related genes in in vitro fertilized (IVF group), NT (C-NT group), and 5-aza-dC and TSA-treated NT (T-NT group) single blastocyst using quantitative real-time PCR. We also compared the DNA methylation status of satellite I among three groups using bisulfite sequencing analysis and combined bisulfite restriction analysis (COBRA). There were significantly lower levels of DNMT1, DNMT3b, HDAC2, and IGF2 transcripts in T-NT blastocysts than in C-NT blastocysts, whereas the relative abundance of OCT4 and SOX2 mRNA was significantly increased in T-NT blastocysts compared to C-NT blastocysts. In addition, the treatment also reduced the DNA methylation levels of NT blastocysts on satellite I sequence. It is likely that TSA may act synergistically with 5-aza-dC to exert such modifications in gene expression and DNA methylation, subsequently enhancing developmental potential (in vitro and full-term) of treated cloned embryos.

SUBMITTER: Wang Y

PROVIDER: S-EPMC3146745 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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The effects of 5-aza-2'- deoxycytidine and trichostatin A on gene expression and DNA methylation status in cloned bovine blastocysts.

Wang Yongsheng Y Su Jianmin J Wang Lijun L Xu Wenbing W Quan Fusheng F Liu Jun J Zhang Yong Y

Cellular reprogramming 20110412 4

We previously found that treatment of both donor cells and early cloned embryos with combination of 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A (TSA) significantly improve the in vitro and full-term development of nuclear transfer (NT) bovine embryos. To investigate how this treatment improved the epigenetic reprogramming of somatic cell nuclei, we compared the expression levels of DNA methylation-, chromatin structure-, and development-related genes in in vitro fertilized (IVF group), ...[more]

PMID: 21486115

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Project description:The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.

Project description:BACKGROUND: DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes. METHOD: Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC). Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR. RESULTS: Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS). In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated. CONCLUSION: These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically silenced gene is associated with the long term reversion of gene expression level brought about by alterations in the epigenetic status following 5-aza-dC treatment.

Dataset Information

The effects of 5-aza-2'- deoxycytidine and trichostatin A on gene expression and DNA methylation status in cloned bovine blastocysts.

Publications

The effects of 5-aza-2'- deoxycytidine and trichostatin A on gene expression and DNA methylation status in cloned bovine blastocysts.

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