Changes of histone H3 lysine 23 acetylation and methylation in porcine somatic cells, oocytes and preimplantation embryos

Highlights

• The acetylation and mono-, di- and tri-methylations of noncanonical histone H3 at lysine 23 (H3K23ac, H3K23me1, H3K23me2 and H3K23me3) during porcine oocyte maturation and pre-implantation development were analyzed.

• H3K23ac and -me3 were detectable through oocyte meiotic resumption.

• H3K23me3 was very weakly detected in the pronuclei of zygotes and the nuclei of blastocysts.

• H3K23ac signals were clearly detected in the nuclei of PA- and IVF-derived blastocysts.

• These results suggest that H3K23 modifications are widely distributed in maturing porcine oocytes and developing preimplantation embryos.

Abstract

Histone modifications play important roles in regulating the expression of developmental genes during preimplantation embryonic development. Here, we analyzed the temporal and spatial distribution of the acetylation and mono-, di- and tri-methylations of noncanonical histone H3 at lysine 23 (H3K23ac, H3K23me1, H3K23me2 and H3K23me3) during porcine oocyte maturation and pre-implantation development, as well as in porcine fetal fibroblasts. H3K23ac, -me1, -me2 and -me3 were enhanced in EdU-positive fetal fibroblasts (S-phase) compared to EdU-negative fetal fibroblasts (G1 and/or G2-phase). More than 91% of the DNA replication foci were well colocalized with H3K23 methylation sites in porcine fetal fibroblasts. H3K23ac and -me3 were detectable through oocyte meiotic resumption. After parthenogenic activation (PA), H3K23me3 was very weakly detected in the pronuclei of zygotes and the nuclei of blastocysts. After in vitro fertilization (IVF), no H3K23me3 signal was observed in the nuclei of IVF-derived embryos, with the exception of the residual polar bodies. In contrast, H3K23ac signals were clearly detected in the nuclei of PA- and IVF-derived blastocysts. The RNA polymerase inhibitor, actinomycin D, reduced the H3K23ac signal in porcine blastocysts. These findings may serve as a valuable reference for further studies of how H3K23 modifications contribute to the regulation of oocyte maturation and early embryonic development in mammals.

Introduction

The basic structural subunit of chromatin in eukaryotic cells is the nucleosome, which consists of 147 base pairs of DNA and an octamer of histone proteins (two each of histones H2A, H2B, H3 and H4) [1]. The unstructured N-termini (tails) of the histones are subject to numerous post-translational modifications, including acetylation, methylation, phosphorylation, ubiquitylation, ADP-ribosylation, sumoylation, etc. [2,3]. Histone acetylation and methylation are largely involved in regulating chromatin structure and gene expression during early mammalian embryonic development [4]. Histone acetylation is generally involved in transcriptional activation. Histone methylation, which includes the monomethylated (me1), dimethylated (me2) and trimethylated (me3) forms [5,6], can be associated with activation or repression of gene expression, depending on which residue is methylated.

Histone H3 is a core component of the nucleosome, and the roles of its acetylation and methylation have been well studied during gametogenesis and in preimplantation embryos. Histone H3 trimethylated at lysine 27 (H3K27me3) [7,8] and histone H3 dimethylated and trimethylated at lysine 9 (H3K9me2 and H3K9me3, respectively) [1] are associated with the repression of gene expression. However, histone H3 trimethylated at lysine 4 (H3K4me3) [9,10] and histone H3 acetylated at lysine 27 (H3K27ac) [11] are exclusively linked to active gene expression. Dimethylation and trimethylation of H3 at lysine 79 (H3K79me2 and -me3) were suggested to be active gene markers [4], while trimethylation of H3 at lysine 36 (H3K36me3) has been associated with transcriptional elongation [1,12].

Growing oocytes actively express genes during meiotic maturation, but thereafter gene expression is relatively silent until oocytes are fully grown [13]. Upon fertilization of the egg by a sperm, the totipotent zygote begins to express genes [4]. Histone modifications play important roles in regulating the expression of developmental genes during preimplantation embryonic development [14]. Generally, histone modifications are highly dynamic and change extensively in oocytes and preimplantation embryos [15]. Most of the studies that have examined histone modifications in mammalian oocytes and embryos to date have focused on evaluating modifications of canonical histone H3 at lysines 4 [9,16], 9 [17,18], 27 [10,19], 36 [1,12], etc. These studies have provided very helpful resources and form the foundation for epigenetic studies in mammals. However, although H3K23ac is reportedly associated with transcriptional regulation in human glioblastoma cells [20], little information is known about the expression pattern and function of H3K23ac in animal somatic cells or embryos. In addition, the expression and function of H3K23 methylation has not been reported in pig somatic cells, oocytes and embryos.

Here, to gain additional insights into the epigenetic changes that occur during oocyte maturation, early embryonic development and cells, we used immunofluorescence staining and confocal microscopy to systematically analyze the localization patterns of the acetylation and mono-, di- and tri-methylations of histone H3 at lysine 23 (H3K23ac, H3K23me1, H3K23me2 and H3K23me3) during porcine oocyte and pre-implantation development, as well as in porcine somatic cells.