Gradual DNA demethylation of the <i>Oct4</i> promoter in cloned mouse embryos

Yamazaki, Yukiko; Fujita, Toko C.; Low, Eleanor W.; Alarcón, Vernadeth B.; Yanagimachi, Ryuzo; Marikawa, Yusuke

doi:10.1002/mrd.20411

Cited by 64 publications

(50 citation statements)

References 63 publications

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“…3B, H3K27me3 could be clearly detected in the normal blastocyst lysates lane, whereas no H3K27me3 could be detected in the somatic cloned blastocysts lane. Similar to a previous report (27,28), Oct4 expression was much weaker in the somatic clones than the normal embryos. ␣-Tubulin was used as a control, and there was no difference in its expression between cloned and normal embryos.…”

Section: Aberrant Reprogramming Of H3k27me3 In Somatic Cellsupporting

confidence: 90%

Defective Chromatin Structure in Somatic Cell Cloned Mouse Embryos

Zhang

Wang

Kou

et al. 2009

Journal of Biological Chemistry

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Epigenetic reprogramming plays a central role in the development of cloned embryos generated by somatic cell nuclear transfer, and it is believed that aberrant reprogramming leads to the abnormal development of most cloned embryos. Recent studies show that trimethylation of H3K27 (H3K27me3) contributes to the maintenance of embryonic stem cell pluripotency because the differentiation genes are always occupied by nucleosomes trimethylated at H3K27, which represses gene expression. Here, we provide evidence that differential H3K27me3 modification exists between normal fertilization-produced blastocysts and somatic cell nuclear transfer cloned blastocysts; H3K27me3 was specifically found in cells of the inner cell mass (ICM) of normal blastocysts, whereas there was no modification of H3K27me3 in the ICM of cloned blastocysts. Subsequently, we demonstrated that the differentiation-related genes, which are marked by H3K27me3 in embryonic stem cells, were expressed at significantly higher levels in cloned embryos than in normal embryos. The polycomb repressive complex 2 (PRC2) component genes (Eed, Ezh2, and Suz12), which are responsible for the generation of H3K27me3, were expressed at lower levels in the cloned embryos. Our results suggest that reduced expression of PRC2 component genes in cloned embryos results in defective modification of H3K27me3 to the differentiation-related genes in pluripotent ICM cells. This results in premature expression of developmental genes and death of somatic cloned embryos shortly after implantation. Taken together, these studies suggest that H3K27me3 might be an important epigenetic marker with which to evaluate the developmental potential of cloned embryos. Embryonic stem (ES)3 cells are derived from the inner cell mass (ICM) of a blastocyst. ES cells are pluripotent and can maintain self-renewal under appropriate culture conditions (1-3). Gene expression profiles of ES cells revealed that there is a core pluripotency regulatory circuitry formed by Oct4, Sox2, and Nanog, which coordinated to maintain the transcriptional program required for the maintenance of ES cell pluripotency (4). Results from recent studies have indicated that ES cells possess a bivalent chromatin structure, which has been suggested to be essential for maintaining their self-renewal and pluripotency characteristics. The bivalent chromatin domains of ES cells consisted of large regions of H3K27 methylation and smaller regions of H3K4 methylation (5). Trimethylation of H3K27 is associated with the activity of the polycomb repressive complex 2 (PRC2); and recent studies have indicated that PRC2 specifically targets developmental genes, which are marked by H3K27me3, to maintain ES cell pluripotency by repressing expression of these genes (6 -12).Somatic cell nuclear transfer (SCNT) represents a remarkable process in which the differentiated somatic cell genome can be converted into a totipotent or at least a pluripotent state (13-16). However, the efficiency of cloning is extremely low, as less than 5% of cloned e...

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Section: Aberrant Reprogramming Of H3k27me3 In Somatic Cellsupporting

confidence: 90%

Defective Chromatin Structure in Somatic Cell Cloned Mouse Embryos

Zhang

Wang

Kou

et al. 2009

Journal of Biological Chemistry

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“…In our previous study, we had analyzed the reprogramming pattern of the PE and found that this locus was completely demethylated by 48 hours postfusion (unpublished data). In mammalian cloning, the onset of Oct4 expression (visualized by Oct4-GFP fluorescence signal) coincides with the four-to eight-cell stage [4], although further demethylation of the Oct4 PP is gradual and is only comparable to that of the naturally produced embryo at the blastocyst stage [19,20]. One of the objectives of the current study was to determine whether the Oct4 PP in OG2 hybrid cells has been completely demethylated by day 2 postfusion, as is the case for the PE, or becomes gradually demethylated from the onset of Oct4-GFP reactivation onward, as is the case for reconstructed embryos.…”

Section: /Rosa26mentioning

confidence: 99%

Pluripotential Reprogramming of the Somatic Genome in Hybrid Cells Occurs with the First Cell Cycle

et al. 2007

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The fusion of pluripotent embryonic cells with somatic cells results in reprogramming of the somatic cell genome. Oct4-green fluorescent protein (GFP) transgenes that do not contain the proximal enhancer (PE) region are widely used to visualize reprogramming of the somatic to the pluripotent cell state. The temporal onset of Oct4-GFP activation has been found to occur 40 -48 hours postfusion. We asked whether activation of the transgene actually reflects activation of the endogenous Oct4 gene. In the current study, we show that activation of an Oct4-GFP transgene that contains the PE region occurs within 22 hours of fusion. In addition, demethylation of the Oct4-GFP transgene and that of the endogenous Oct4 and Nanog genes was found to occur within 24 hours of fusion. As this timing corresponds with the timing of cell cycle completion in embryonic stem cells and fusion hybrids (ϳ22 hours), we postulate that pluripotential reprogramming of the somatic cell genome begins during the first cell cycle after the fusion of a somatic cell with a pluripotent cell and has been completed by day 2 postfusion. STEM CELLS 2008;26:445-454 Disclosure of potential conflicts of interest is found at the end of this article.

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“…Post-translational modifications have been shown to be able to modify the activity of Oct4, such as sumoylation (Wei et al, 2007) and ubiquitination (Xu et al, 2004). During the reprogramming process epigenetic marks are changed such as the removal of methyl groups on DNA (DNA demethylation) of the Oct4 promoter which has been shown during SCNT (Simonsson and Gurdon, 2004) and has also been observed in mouse (Yamazaki et al, 2006). The growth arrest and DNA damage inducible protein Gadd45a and deaminase Aid was shown to promote DNA demethylation of the Oct4 and Nanog promoters (Barreto et al, 2007;Bhutani et al, 2010).…”

Section: Molecular Mechanisms Of Reprogrammingmentioning

confidence: 99%