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. 2016 Aug 2;11(8):553-62.
doi: 10.1080/15592294.2015.1103424. Epub 2015 Oct 19.

Dynamics of histone H3 acetylation in the nucleosome core during mouse pre-implantation development

Céline Ziegler-Birling  1 , Sylvain Daujat  1 , Robert Schneider  1 , Maria-Elena Torres-Padilla  1
Affiliations

Dynamics of histone H3 acetylation in the nucleosome core during mouse pre-implantation development

Céline Ziegler-Birling et al. Epigenetics. .

Abstract

In mammals, the time period that follows fertilization is characterized by extensive chromatin remodeling, which enables epigenetic reprogramming of the gametes. Major changes in chromatin structure persist until the time of implantation, when the embryo develops into a blastocyst, which comprises the inner cell mass and the trophectoderm. Changes in DNA methylation, histone variant incorporation, and covalent modifications of the histones tails have been intensively studied during pre-implantation development. However, modifications within the core of the nucleosomes have not been systematically analyzed. Here, we report the first characterization and temporal analysis of 3 key acetylated residues in the core of the histone H3: H3K64ac, H3K122ac, and H3K56ac, all located at structurally important positions close to the DNA. We found that all 3 acetylations occur during pre-implantation development, but with different temporal kinetics. Globally, H3K64ac and H3K56ac were detected throughout cleavage stages, while H3K122ac was only weakly detectable during this time. Our work contributes to the understanding of the contribution of histone modifications in the core of the nucleosome to the "marking" of the newly established embryonic chromatin and unveils new modification pathways potentially involved in epigenetic reprogramming.

Keywords: Epigenetics; histone acetylation; lateral surface; mammalian embryo; nucleosome core.

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Figures

Figure 1.
Figure 1.
Dynamics of H3K64ac during mouse pre-implantation development. Freshly collected embryos were fixed and stained with an H3K64ac antibody (green). DNA is shown in blue. A. Maximal projections of Z-sections taken every 1 μm on a confocal microscope of oocytes, zygotes at different pronuclear (PN) stages and early 2-cell stage embryos. The arrowheads point to the polar body; male and female pronuclei are indicated. Images were acquired using the same confocal parameters and therefore the fluorescence levels are directly comparable. B. H3K64me3 opposes the temporal kinetics of H3K64ac. Oocytes at the germinal vesicle (GV) or MI stage, zygotes at different PN stages and early 2-cell stage embryos were immunostained with an H3K64me3 antibody (green). The DNA was stained with DAPI (blue). Shown are maximal intensity projections of confocal Z-sections acquired as in A. Male and female pronuclei are indicated, and the polar body is demarcated by an arrow. Note that H3K64me3 is absent from the paternal chromatin throughout. C. H3K64ac levels at different developmental stages as indicated. Top and middle parts show full projections of Z-sections taken every 1 μm (cleavage stages) or 2 μm (blastocyst) on a confocal microscope of DNA (blue) and H3K64ac (green). The ‘Merge’ panels show a merge image of the corresponding middle sections for the blue (DNA) and green (H3K64ac) channels. The same confocal section corresponding to the H3K64ac staining is shown in gray scale at the bottom. From A through C, between 12 and 24 embryos per stage were analyzed in at least 3 independent experiments. Scale bar is 12 μm.
Figure 2.
Figure 2.
H3K122ac is present at low levels in cleavage stage embryos. (A) H3K122ac was analyzed in oocytes and zygotes immediately after fertilization, freshly collected from natural matings. The top 2 panels show maximal projections of confocal Z-sections for H3K122ac (green) or DAPI (blue) of representative embryos of at least 16 analyzed per stage. The bottom panel shows a middle confocal section where the gray and blue channels are merged. When present, the polar body is indicated by an arrowhead. The male and female pronuclei are indicated. Scale bar is 12 μm. (B) Embryos at the indicated stages were freshly collected, fixed, stained with an H3K122ac (green) antibody, and analyzed using confocal microscopy. DNA is shown in blue. Shown are full projections of Z-sections taken every 1 μm (cleavage) or 2 μm (blastocyst). The bottom panel is the same channel for the merge shown on the third row, but showing the H3K122ac channel in gray scale. When present, the polar body is indicated by an arrowhead. The male and female pronuclei are indicated. At least 10 embryos from independent experiments were analyzed per stage. Embryos shown were imaged under identical conditions, thus fluorescence levels are directly comparable. Scale bar is 12 μm.
Figure 3.
Figure 3.
H3K56ac is first detected at the mid-zygote stage after fertilization and persists throughout pre-implantation development. (A) Analysis of H3K56ac immediately after fertilization and until the early 2-cell stage. Freshly collected mouse embryos from natural matings at the indicated stages were stained with an H3K56ac antibody (green) and with DAPI (blue). Full projections of Z-sections acquired every 1 μm (top 2 parts) and middle, merge section (third part) are shown. The gray scale view of the green (H3K56ac) channel of the merge image confocal section is shown at the bottom. Male and female pronuclei are indicated, and the polar body, where visible, is demarcated by an arrow. The PN classification was done as described by Adenot and colleagues. At least 10 embryos were analyzed per stage, performed in 2 independent experiments. Scale bar is 12 μm. (B) Dynamics of H3K56ac throughout cleave stages. Embryos were collected at the indicated stages, fixed, and processed for immunostaining using an H3K56ac. As in A, top and second panels are maximal projections of confocal Z-sections acquired every 1 μm (cleavage stages) or 2 μm (blastocyst) of DAPI (blue) or H3K56ac (green) channels. The merge image shows a middle confocal section of the same embryos and the bottom panel is the gray scale of the green (H3K56ac) channel. At least 14 embryos were analyzed per stage, performed in 2 independent experiments. Scale bar is 12 μm.
Figure 4.
Figure 4.
Relationship between H3K56ac or H3K64ac and S-phase progression. (A) Higher magnification of a representative female pronucleus or nuclei from 2-, 8-, or 16-cell stage embryos stained with the H3K56ac or the H3K64ac antibody. Shown are merge images of single confocal sections. Scale bar is 12 μm. (B) Zygotes were subjected to a BrdU pulse for 30 min during early/mid or late S-phase and processed for immunostaining with an anti-BrdU and an anti-H3K64ac (left) or an anti-H3K56ac (right) antibody. Representative zygotes of 10 analyzed at different stages of S-phase derived from 3 (H3K56ac) or 2 (H3K64ac) independent experiments are shown. Note, that the late replication pattern is characterized by BrdU incorporation at the satellite sequences around the NLBs and remaining BrdU at the peripheral nuclear regions. Shown are single sections of Z-stack sections taken every 0.5 μm where the diameter of the male pronucleus is maximal. Male and female pronuclei are shown. Scale bar is 12 μm. (C) Middle section of an early blastocyst stained with the H3K64ac, the H3K122ac, or the H3K56ac antibody. Shown are the merge images of the green (antibody), blue (DAPI), or red (cortical actin stained with phalloidin) channels. The white line delineates the blastocyst cavity. Scale bar is 20 μm. Representative embryos from 8, 9, or 12 embryos for the H3K64ac, H3K122ac, and H3K56ac antibodies are shown.
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Grants and funding

M.E.T.-P acknowledges funding by EpiGeneSys NoE, ERC-Stg ‘NuclearPotency’, the EMBO YIP and the Fondation Schlumberger pour l'Education et la Recherche. C. Z-B is supported through the Université de Strasbourg. Work in R.S. laboratory is supported by the Fondation pour la Recherche Médicale (FRM), by the Agence Nationale de Recherche (ANR, CoreAc), La Ligue National Contre Le Cancer (Equipe Labellisée) and INSERM Plan Cancer (Epigénétique et Cancer).

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