Genome-Wide Dynamic Profiling of Histone Methylation during Nuclear Transfer-Mediated Porcine Somatic Cell Reprogramming

Abstract

The low full-term developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos is mainly attributed to imperfect epigenetic reprogramming in the early embryos. However, dynamic expression patterns of histone methylation involved in epigenetic reprogramming progression during porcine SCNT embryo early development remain to be unknown. In this study, we characterized and compared the expression patterns of multiple histone methylation markers including transcriptionally repressive (H3K9me2, H3K9me3, H3K27me2, H3K27me3, H4K20me2 and H4K20me3) and active modifications (H3K4me2, H3K4me3, H3K36me2, H3K36me3, H3K79me2 and H3K79me3) in SCNT early embryos from different developmental stages with that from in vitro fertilization (IVF) counterparts. We found that the expression level of H3K9me2, H3K9me3 and H4K20me3 of SCNT embryos from 1-cell to 4-cell stages was significantly higher than that in the IVF embryos. We also detected a symmetric distribution pattern of H3K9me2 between inner cell mass (ICM) and trophectoderm (TE) in SCNT blastocysts. The expression level of H3K9me2 in both lineages from SCNT expanded blastocyst onwards was significantly higher than that in IVF counterparts. The expression level of H4K20me2 was significantly lower in SCNT embryos from morula to blastocyst stage compared with IVF embryos. However, no aberrant dynamic reprogramming of H3K27me2/3 occurred during early developmental stages of SCNT embryos. The expression of H3K4me3 was higher in SCNT embryos at 4-cell stage than that of IVF embryos. H3K4me2 expression in SCNT embryos from 8-cell stage to blastocyst stage was lower than that in the IVF embryos. Dynamic patterns of other active histone methylation markers were similar between SCNT and IVF embryos. Taken together, histone methylation exhibited developmentally stage-specific abnormal expression patterns in porcine SCNT early embryos.

TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos

“…Recently, H3K9me3 and H3K79me3 are identified as two negative reprogramming regulators for generating the induced pluripotent stem cells in mouse [15] and human [16]. Similarly, abnormal histone methylation profiles involving H3K9me2 and H3K9me3 are discovered during porcine early SCNT embryonic development in our previous study [17]. Based on these studies, we hypothesize that abnormal histone methylation leads to the reduction of developmental efficiency of SCNT embryos.…”

Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

“…Cao et al found that ZGA of porcine fertilized embryos occurred at the 4cell stage in porcine fertilized embryos and was delayed in SCNT embryos via genome-wide gene expression analysis [42], but, a comprehensive analysis of global epigenetic modification of porcine preimplantation embryos has not previously been conducted. Previous studies characterized and compared dynamic genomewide profiles of 12 kinds of histone methylation modification by IF staining [43], but no studies have been conducted on dynamic mRNA profiling of global epigenetic modification enzymes. In our study, we conducted an overall analysis of the expression profiles of 84 representative chromatin modification enzymes by means of RT² Profiler PCR arrays.…”

Down-Regulation of H3K4me3 by MM-102 Facilitates Epigenetic Reprogramming of Porcine Somatic Cell Nuclear Transfer Embryos

“…H3K4me3 was confirmed as a barrier for mouse SCNT 4-cell arrest, and the down-regulation of H3K4me3 level by over-expression of the H3K4 demethylase KDM5B could promote mouse 4-cell SCNT embryos development [28]. The ZGA of porcine fertilized embryos occurs at the 4-cell stage and is delayed in SCNT embryos [29], and H3K4me3 levels in porcine 4-cell SCNT embryos were confirmed to be abnormally higher than those in IVF embryos by IF staining [14], but the study on how abnormally high H3K4me3 affects ZGA and the development of porcine SCNT embryos, still has not yet been reported.…”

“…Cao et al characterized and compared global dynamic patterns of 12 different histone methylation modifications during porcine SCNT reprogramming by IF staining [14], but no comprehensive analysis has been performed on dynamic mRNA profiling of global epigenetic modification enzymes during porcine SCNT preimplantation embryo development. A previous study suggested that abnormally high mRNA expression of DNMT1/3A would cause either embryonic apoptosis or incomplete reprogramming in porcine cloned embryos [34], but no analysis of DNMT3B was shown.…”

“…The down-regulation of H3K9me3 and H3K4me3 could improve transcriptional reprogramming of mouse SCNT preimplantation embryos and the production of cloned descendants [13]. H3K9 and H3K4 methylation also show abnormal expression levels in porcine SCNT embryos [14]. The down-regulation of H3K9 methylation by BIX-01294 could improve epigenetic reprogramming of porcine SCNT embryos [15], but there have been very few studies about whether the down-regulation of H3K4 methylation can play a similar role during porcine SCNT embryo development.…”