Methylation patterns in 5' terminal regions of pluripotency-related genes in bovine in vitro fertilized and cloned embryos

In order to investigate DNA methylation profiles of five pluripotency-related genes (Oct4, Sox2, Nanog, Rexl and Fgf4) during bovine maternal to zygotic transition (MZT) in both in vitro fertilized (IVF) and nuclear transfer (NT) embryos, sodium bisulfite sequencing method was used to detect DNA methylation levels, accompanied by the statistical analysis of embryo developmental rates. The results showed that Oct4, Nanog, Rexl and Fgf4 were respectively demethylated by 25.22% (P < 0.01), 3.84% (P > 0.05), 31.82% (P < 0.01) and 10% (P > 0.05) while Sox2 retained unmethylation during MZT in IVF embryos. By contrast, Oct4 and Rexl respectively underwent demethylation by 23.04% (P < 0.01) and 6.02% (P > 0.05), and, reversely, Sox2, Nanog and Fgf4 respectively experienced remethylation by 0.84% (P > 0.05), 5.39% (P > 0.05) and 5.46% (P > 0.05) during MZT in NT embryos. Interestingly, the CpG 14 site of Sox2 was specifically methylated in both 8-cell and morula NT embryos. In addition, the development of blastocysts between IVF and NT embryos showed no significant difference. DNA methylation analysis showed that only Oct4 and Sox2 underwent the correct methylation reprogramming process, which may be responsible for the development of blastocysts of NT embryos to a certain extent. In conclusion,the five genes respectively experienced demethylation to different extents and incomplete DNA methylation reprogramming during bo-vine MZT in both IVF and NT embryos, suggesting that they may be used as indicators for bovine embryo developmental competence.     

DNA methylation as an epigenetic regulator of neural 5-lipoxygenase expression: evidence in human NT2 and NT2-N cells

Increased expression of 5-lipoxygenase is associated with various neuropathologies and may be related to epigenetic gene regulation. DNA methylation in promoter regions is typically associated with gene silencing. We found that human NT2 cells, which differentiate into neuron-like NT2-N cells, express 5-lipoxygenase and we investigated the relationship between 5-lipoxygenase expression and the methylation state of the 5-lipoxygenase core promoter. We used the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor valproate to alter DNA methylation and to induce histone modifications. 5-Lipoxygenase expression and DNA methylation were assayed with RT-PCR and bisulfite genomic sequencing, respectively. Neuronal differentiation of proliferating NT2 precursors decreased 5-lipoxygenase expression. 5-Aza-2'-deoxycytidine increased 5-lipoxygenase mRNA levels only in proliferating cells, whereas valproate increased 5-lipoxygenase mRNA levels in a cell cycle-independent manner. In both precursors and differentiated cells, CpG dinucleotides of the promoter were poorly methylated. In precursors, both 5-aza-2'-deoxycytidine and valproate further reduced the number of methylated CpGs. Moreover, we found evidence for cytosine methylation in CpWpG (W=adenine or thymine) and other asymmetrical sequences; CpWpG methylation was reduced by valproate in NT2-N but not in NT2 cells. This is the first report demonstrating that the dynamics of DNA methylation relates to neural 5-lipoxygenase gene expression.     

Promoter CpG methylation contributes to ES cell gene regulation in parallel with Oct4/Nanog, PcG complex, and histone H3 K4/K27 trimethylation

We report here genome-wide mapping of DNA methylation patterns at proximal promoter regions in mouse embryonic stem (mES) cells. Most methylated genes are differentiation associated and repressed in mES cells. By contrast, the unmethylated gene set includes many housekeeping and pluripotency genes. By crossreferencing methylation patterns to genome-wide mapping of histone H3 lysine (K) 4/27 trimethylation and binding of Oct4, Nanog, and Polycomb proteins on gene promoters, we found that promoter DNA methylation is the only marker of this group present on approximately 30% of genes, many of which are silenced in mES cells. In demethylated mutant mES cells, we saw upregulation of a subset of X-linked genes and developmental genes that are methylated in wild-type mES cells, but lack either H3K4 and H3K27 trimethylation or association with Polycomb, Oct4, or Nanog. Our data suggest that in mES cells promoter methylation represents a unique epigenetic program that complements other regulatory mechanisms to ensure appropriate gene expression.     

DNA methylation in embryonic stem cells

Embryonic stem cells (ESCs) are pluripotent, self‐renewing cells. These cells can be used in applications such as cell therapy, drug development, disease modeling, and the study of cellular differentiation. Investigating the interplay of epigenetics, genetics, and gene expression in control of pluripotence and differentiation could give important insights on how these cells function. One of the best known epigenetic factors is DNA methylation, which is a major mechanism for regulation of gene expression. This phenomenon is mostly seen in imprinted genes and X‐chromosome inactivation where DNA methylation of promoter regions leads to repression of gene expression. Differential DNA methylation of pluripotence‐associated genes such as Nanog and Oct4/Pou5f1 has been observed between pluripotent and differentiated cells. It is clear that tight regulation of DNA methylation is necessary for normal development. As more associations between aberrant DNA methylation and disease are reported, the demand for high‐throughput approaches for DNA methylation analysis has increased. In this article, we highlight these methods and discuss recent DNA methylation studies on ESCs. J. Cell. Biochem. 109: 1–6, 2010. © 2009 Wiley‐Liss, Inc.     

Methylation patterns in 5' terminal regions of pluripotency-related genes in bovine in vitro fertilized and cloned embryos

In order to investigate DNA methylation profiles of five pluripotency-related genes(Oct4,Sox2,Nanog,Rex1 and Fgf4)during bovine maternal to zygotic transition(MZT)in both in vitro fertilized(IVF)and nuclear transfer(NT)embryos,sodium bisulfite sequencing method was used to detect DNA methylation levels,accompanied by the statistical analysis of embryo developmental rates.The results showed that Oct4,Nanog,Rex1 and Fgf4 were respectively demethylated by 25.22%(P < 0.01),3.84%(P > 0.05),31.82%(P < 0.01)and 10...     

Epigenetic marks in somatic chromatin are remodelled to resemble pluripotent nuclei by amphibian oocyte extracts

Reprogramming pluripotency after nuclear transplantation shows that molecules in oocytes can remodel somatic chromatin to a stem cell state. Here we report on an ex-ovo system using axolotl oocyte extracts to remodel epigenetic marks of somatic chromatin. Molecules present in axolotl oocyte extracts induce the reduction of the overall levels of H3K9me3, HP1α, and DNA methylation of somatic cells, and they increase the levels of H3K9ac. The levels of signal intensity detected in treated differentiated cells resemble those detected in embryonic stem cells, which are, in contrast, unaffected by these extracts. Analysis of specific genome sequences shows that somatic cells exposed to oocyte extracts undergo demethylation of LINE-1 repeats but Major Satellite repeats and the imprinted gene H19 remain unchanged. In addition, they induce demethylation of the Oct-4 promoter. Finally, the kinetics of activation of Oct-4 and Nanog expression from MEF nuclei treated in extracts suggests that these genes are subject to different levels of epigenetic control. The results demonstrate that axolotl oocyte extracts are a useful tool for studying epigenetic remodelling of somatic cells to a stem cell configuration, and for elucidating oocyte specific mechanisms of nuclear reprogramming.     

Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells

During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Recently, we reported that zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one] acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. Here we show that continuous application of zebularine to T24 cells induces and maintains p16 gene expression and sustains demethylation of the 5' region for over 40 days, preventing remethylation. In addition, continuous zebularine treatment effectively and globally demethylated various hypermethylated regions, especially CpG-poor regions. The drug caused a complete depletion of extractable DNA methyltransferase 1 (DNMT1) and partial depletion of DNMT3a and DNMT3b3. Last, sequential treatment with 5-aza-2'-deoxycytidine followed by zebularine hindered the remethylation of the p16 5' region and gene resilencing, suggesting the possible combination use of both drugs as a potential anticancer regimen.     

Methylation patterns in 5＇ terminal regions of pluripotency-related genes in bovine in vitro fertilized and cloned embryos

In order to investigate DNA methylation profiles of five pluripotency-related genes (Oct4, Sox2, Nanog, Rex1 and Fgf4) during bovine maternal to zygotic transition (MZT) in both in vitro fertilized (IVF) and nuclear transfer (NT) embryos, sodium bisulfite sequencing method was used to detect DNA methylation levels, accompanied by the statistical analysis of embryo developmental rates. The results showed that Oct4, Nanog, Rex1 and Fgf4 were respectively demethylated by 25.22% (P < 0.01), 3.84% (P > 0.05), 31.82% (P < 0.01) and 10% (P > 0.05) while Sox2 retained unmethylation during MZT in IVF embryos. By contrast, Oct4 and Rex1 respectively underwent demethylation by 23.04% (P < 0.01) and 6.02% (P > 0.05), and, reversely, Sox2, Nanog and Fgf4 respectively experienced remethylation by 0.84% (P > 0.05), 5.39% (P > 0.05) and 5.46% (P > 0.05) during MZT in NT embryos. Interestingly, the CpG 14 site of Sox2 was specifically methylated in both 8-cell and morula NT embryos. In addition, the development of blastocysts between IVF and NT embryos showed no significant difference. DNA methylation analysis showed that only Oct4 and Sox2 underwent the correct methylation reprogramming process, which may be responsible for the development of blastocysts of NT embryos to a certain extent. In conclusion, the five genes respectively experienced demethylation to different extents and incomplete DNA methylation reprogramming during bovine MZT in both IVF and NT embryos, suggesting that they may be used as indicators for bovine embryo developmental competence.