Changes of histone H3 lysine 23 acetylation and methylation in porcine somatic cells, oocytes and preimplantation embryos
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.
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Chemicals and animal ethics statement
All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise indicated. Animal experiments were approved by the Institutional Animal Care and Use Committee of Chung Nam National University, Republic of Korea.
Culturing of porcine fetal fibroblasts
Porcine fetal fibroblast cells were thawed and cultured in 6-well dishes containing 2 mL DMEM plus 10% fetal bovine serum (FBS) per well. Cells were grown to confluence at 38.5 °C in a humidified atmosphere under 5% CO2. Porcine fetal fibroblasts were passaged
Subnuclear localization of H3K23 acetylation and methylations in porcine somatic cells
As the localization of H3K23 acetylation and methylation had not previously been reported in porcine somatic cells, we examined the subnuclear localization of H3K23ac, -me1, -me2 and -me3 in porcine fetal fibroblasts. Immunofluorescence analysis using their specific antibodies revealed that the localization patterns for H3K23ac, -me1, -me2 and -me3 were similar in porcine fetal fibroblasts cells. As shown in Fig. S1, the fluorescence signals of H3K23ac, -me1, -me2 and -me3 in porcine fetal
Discussion
To gain new insights into the epigenetic changes that occur during oocyte meiotic resumption and early embryonic development, we examined the dynamic changes of H3K23 acetylation and methylation in porcine oocytes, preimplantation embryos and fetal fibroblasts. The signals corresponding to H3K23ac, -me1, -me2 and -me3 were widely distributed in porcine fetal fibroblasts and exhibited differences in oocytes and embryos, suggesting that they may play discrete functions in regulating cell cycle,
Ethics approval and consent to participate
Animal experiments were approved by the Institutional Animal Care and Use Committee of Chungnam National University, Republic of Korea.
Author’s contributions
Conceived and designed the experiments: DIJ, TL. Performed the experiments: TL, LS, JEL. Analyzed the data: TL, JBL, SYK. Wrote the paper: TL, DIJ.
CRediT authorship contribution statement
Tao Lin: Conceptualization, Formal analysis, Investigation, Writing - original draft. Ling Sun: Formal analysis. Jae Eun Lee: Formal analysis. Joo Bin Lee: Investigation. So Yeon Kim: Investigation. Dong Il Jin: Conceptualization, Writing - original draft.
Declaration of competing interest
The authors declare that there is no conflict of interest.
Acknowledgments
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Ministry of Education, Science and Technology (MEST) (NRF-2019R1I1A3A01061877).
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These authors contributed equally to this study.