Defective Chromatin Structure in Somatic Cell Cloned Mouse Embryos

Abstract

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...

The Polycomb Protein Ezh2 Impacts on Induced Pluripotent Stem Cell Generation

“…Additionally, Ezh2 was required for efficient somatic cell reprogramming by cell fusion and nuclear transfer [9,10]. These findings are in line with the results reported here.…”

“…This is related to the (i) target cells, (ii) reprogramming factors and vector systems used for reprogramming, and (iii) further so far largely unknown factors [50]. In ES cells, many genes are marked with H3K27me3, which is critical for their differentiation potential [10]. In addition, ES cells with disrupted PcG protein expression and consequently altered H3K27me3 show impaired differentiation potential [5,51].…”

“…ES cells lacking the PRC2 components, Ezh2, Eed, and Suz12, were deficient in cell fusion-induced reprogramming of somatic cells toward pluripotency [9]. In somatic cell nuclear transfer (SCNT) experiments, the inner cell mass of cloned embryos showed low H3K27me3 modification compared to fertilized embryos and thus differentiation-related genes were expressed [10]. Furthermore, the low levels of H3K27me3 in SCNT embryos correlate with low Ezh2 expression in such cloned embryos.…”

The Expression Pattern of Polycomb Group Protein Ezh2 During Mouse Embryogenesis

“…The mRNA expression of Ezh2 has been identified in whole mouse embryos at day E9.5 and in the fetal liver, brain and skeletal muscles at day E13 or day E17 (Laible et al, 1997). Studies also suggested that Ezh2-induced H3K27me3 might be an important epigenetic marker for evaluating the developmental potential of cloned embryos (Zhang et al, 2009).…”

Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency

“…Compared to fertilized embryos, cloned embryos display higher levels of DNA methylation and incomplete chromatin remodeling (Zhang et al 2009, Mason et al 2012, Rodriguez-Osorio et al 2012. To improve NT efficiency, several approaches, including the use of chemical inhibitors such as HDAC inhibitors (HDACi) and 5-aza-C, have been devised to facilitate the reprogramming of somatic nuclei and establish embryonic epigenetic patterns (Ogura et al 2013).…”