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. 2015 Nov 24;13(8):1552-60.
doi: 10.1016/j.celrep.2015.10.031. Epub 2015 Nov 12.

A DNMT3A2-HDAC2 Complex Is Essential for Genomic Imprinting and Genome Integrity in Mouse Oocytes

Pengpeng Ma  1 , Eric de Waal  2 , Jamie R Weaver  2 , Marisa S Bartolomei  2 , Richard M Schultz  3
Affiliations

A DNMT3A2-HDAC2 Complex Is Essential for Genomic Imprinting and Genome Integrity in Mouse Oocytes

Pengpeng Ma et al. Cell Rep. .

Abstract

Maternal genomic imprints are established during oogenesis. Histone deacetylases (HDACs) 1 and 2 are required for oocyte development in mouse, but their role in genomic imprinting is unknown. We find that Hdac1:Hdac2(-/-) double-mutant growing oocytes exhibit global DNA hypomethylation and fail to establish imprinting marks for Igf2r, Peg3, and Srnpn. Global hypomethylation correlates with increased retrotransposon expression and double-strand DNA breaks. Nuclear-associated DNMT3A2 is reduced in double-mutant oocytes, and injecting these oocytes with Hdac2 partially restores DNMT3A2 nuclear staining. DNMT3A2 co-immunoprecipitates with HDAC2 in mouse embryonic stem cells. Partial loss of nuclear DNMT3A2 and HDAC2 occurs in Sin3a(-/-) oocytes, which exhibit decreased DNA methylation of imprinting control regions for Igf2r and Srnpn, but not Peg3. These results suggest seminal roles of HDAC1/2 in establishing maternal genomic imprints and maintaining genomic integrity in oocytes mediated in part through a SIN3A complex that interacts with DNMT3A2.

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Figures

Figure 1
Figure 1. Deletion of both Hdac1 and Hdac2 results in global decrease of 5-mC without affecting 5-hmC in oocytes
(A) Immunocytochemical detection of 5-mC (green) and HDAC2 (red) in oocytes obtained from wild-type (WT) mice 12-days-of-age and Hdac1:2−/− mice. Shown are representative images and only the nucleus is shown. In this and other figures, the bar corresponds to 10 μm. Quantification of the 5-mC nuclear staining signals shown in lower panel, the nuclear staining intensity of 5-mC in the WT oocytes was set to 1. All data are expressed as mean ± SEM, * P<0.05. (B) Quantification of the 5-mC and 5-hmC nuclear staining signals in panel A. The nuclear staining intensity in the WT oocytes was set to 1 and the data are expressed as mean ± SEM, * P<0.05.
Figure 2
Figure 2. DNA methylation analysis in Hdac1:2−/− growing oocytes
Bisulfite sequencing analysis of DNA methylation at the ICRs of several maternally [Snrpn (A), Igf2r (B), Peg3 (C)] or paternally [H19 (D)] methylated genes and repetitive elements [Line1 [E), Iap (F)] in WT and Hdac1:2−/− growing oocytes obtained from mice 12 days-of-age. Open circles and filled circles represent unmethylated and methylated CpG sites, respectively, and each row represents data from a single DNA molecule.
Figure 3
Figure 3. Increased expression of repetitive elements and incidence of DNA double-strand breaks (DSBs) in Hdac1:2−/− oocytes
(A) Relative abundance of repetitive element mRNA in WT and Hdac1:2−/− growing oocytes obtained from mice 12 days-of-age. Data are expressed relative to that in WT oocytes as mean ± SEM. *, p< 0.05. (B) Immunocytochemical detection of DSBs with anti-γH2AX (green). DNA was detected with propidium iodide (red). Shown are representative images and only the nucleus is shown.
Figure 4
Figure 4. Expression of DNMT1, DNMT3A, DNMT3B and DNMT3L in Hdac1:2−/− oocytes and expressing Hdac2 in Hdac1:2−/− growing oocytes partially restores DNMT3A2 nuclear stainning
(A) Relative amount of DNMT1, DNMT3A, DNMT3B and DNMT3L determined by immunoblot analysis. Extracts for immunoblot analysis were prepared from oocytes obtained from at least four WT or mutant mice 12 days-of-age and total protein extract equivalent to 150 oocytes was loaded per lane. The experiment was conducted 3 times, and similar results were obtained in each case. ACTB was used as a loading control. (B) Immunocytochemical detection of DNMT31, DNMT3A, DNMT3B and DNMT3L in WT and mutant oocytes obtained from mice 12-days-of-age. Shown are representative images and only the nucleus is shown. (C) Immunocytochemical detection of DNMT3A in oocytes obtained from mice 12-days-of-age and lacking different combinations of Hdac1 and Hdac2. Shown are representative images and only the nucleus is shown. DNA was counterstained with propidium iodide. (D) Quantification of the data shown in C in which the nuclear staining intensity of DNMT3A in WT oocytes was set to 1. All data are expressed as mean ± SEM. *, p < 0.05. (E) Mutant oocytes were injected with either a cRNA (0.4 μg/μl) encoding Hdac2 (Hdac1:2−/−-OE) or MiliQ water (Hdac1:2−/−-C) and incubated in CZB medium; controls were wild-type oocytes (WT-C). Oocytes were removed 30 h after injection for immunoctyochemical detection of DNMT3A. Shown are representative images. DNA was counterstained with propidium iodide. (F) Quantification of the data shown in panel A. Staining intensity of DNMT3A2 in WT oocytes was set to 1 and the data are expressed as mean ± SEM. Signal intensities relative to WT in Hdac1:2−/−-C and Hdac1:2−/−-OE are 45±4%, and 63±6% respectively. *, p< 0.02.
Figure 5
Figure 5. Expression of HDAC1, HDAC2 and DNMT3A2 and DNA methylation in Sin3a−/− Oocytes
(A) Relative amount of SIN3A, HDAC1, HDAC2 and DNMT3A2 was determined by immunoblot analysis by using total protein extracts from WT and Hdac1:2−/− growing oocytes obtained from mice 12 days-of-age. Equal numbers (200) of oocytes were loaded per lane and and beta-actin (ACTB) was used as a loading control. The experiment was conducted 2 times, and similar results were obtained in each case. (B) Immunocytochemical detection of SIN3A, HDAC1, HDAC2 and DNMT3A2 in WT and Sin3a−/− oocytes obtained from mice 12-days-of-age. Shown are representative images and only the nucleus is shown. (C) Quantification of the data of HDAC2 and DNMT3A2 imunostaining shown in panel B. Nuclear staining intensity of HDAC2 and DNMT3A2 in WT oocytes was set to 1, and the data are expressed as mean ± SEM. Bisulfite sequencing analysis of DNA methylation at the ICRs of maternally methylated genes, Snrpn (D), Igf2r (E), Peg3 (F) or H19 (G) that is a paternally methylated gene, and repetitive elements, LINE1 (H) and IAP (I) in WT and Sin3a−/− growing oocytes obtained from mice 12 days-of-age. Filled circles represent methylated sites and open circles represent unmethylated sites. Each row represents data from a single DNA molecule. The numbers below each set of DNA strands indicate the percent of methylated CpG sites.
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