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, 141 (6), 1313-23

Ontogeny, Conservation and Functional Significance of Maternally Inherited DNA Methylation at Two Classes of Non-Imprinted Genes

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Ontogeny, Conservation and Functional Significance of Maternally Inherited DNA Methylation at Two Classes of Non-Imprinted Genes

Charlotte E Rutledge et al. Development.

Abstract

A functional role for DNA methylation has been well-established at imprinted loci, which inherit methylation uniparentally, most commonly from the mother via the oocyte. Many CpG islands not associated with imprinting also inherit methylation from the oocyte, although the functional significance of this, and the common features of the genes affected, are unclear. We identify two major subclasses of genes associated with these gametic differentially methylated regions (gDMRs), namely those important for brain and for testis function. The gDMRs at these genes retain the methylation acquired in the oocyte through preimplantation development, but become fully methylated postimplantation by de novo methylation of the paternal allele. Each gene class displays unique features, with the gDMR located at the promoter of the testis genes but intragenically for the brain genes. Significantly, demethylation using knockout, knockdown or pharmacological approaches in mouse stem cells and fibroblasts resulted in transcriptional derepression of the testis genes, indicating that they may be affected by environmental exposures, in either mother or offspring, that cause demethylation. Features of the brain gene group suggest that they might represent a pool from which many imprinted genes have evolved. The locations of the gDMRs, as well as methylation levels and repression effects, were also conserved in human cells.

Keywords: DNA methylation; Epigenetics; Imprinting.

Figures

Fig. 1.
Fig. 1.
Two novel gene classes with gametic differentially methylated regions (gDMRs). (A) Numbers of genes (n) showing methylation (Meth) in oocyte, blastocyst or both from reanalysis of published genome-wide data (Smallwood et al., 2011). DNMT3L-dependent: genes with >50% loss of methylation in DNMT3L-deficient oocytes. (B) Top-ranking classes of genes by ontology analysis of data from: (i) reduced representation bisulfite sequencing (RRBS) in meiosis II (MII) oocytes (as in A); (ii) whole bisulfitome amplified DNA sequencing on germinal vesicle (GV) oocytes [WBA-seq (Kobayashi et al., 2012)]; and (iii) methylated DNA immunoprecipitation (meDIP) on preimplantation embryos (Borgel et al., 2010). P-value and false discovery rate (FDR) are indicated. (C) Top-scoring class from B(i) compared with novel imprinted genes (De Veale et al., 2012) and those identified by meDIP [B(iii)]. (D) Methylation ontogeny of representative groups of genes from each gDMR class; genes methylated postimplantation are also shown as a control. 3L KO, oocytes from Dnmt3l-/- females; Blast, blastocyst. Error bars represent s.e.m. (E) Structure of prototypical brain (Crocc) and testis (Rhox13) gDMR genes. Black boxes: CpG islands (CGIs), with ID number, identified by CFP1 binding (Illingworth et al., 2010). Green boxes: CGI identified by conventional means, with number of CpGs indicated. Pyro, region covered by pyrosequencing assay. (F) Proportions of brain gDMRs at known (RefSeq) transcriptional start sites (TSSs), those showing TSS-associated chromatin marks in ESCs or brain cells, and those with no evidence of being TSSs.
Fig. 2.
Fig. 2.
Validation of methylation at novel gDMRs in gametes and effects of DNMT3L loss. (A) COBRA of methylation in wild-type (WT) and DNMT3L-deficient (3L) mouse oocytes. un, uncut; u, unmethylated; asterisk, limit digest band. (B) Clonal analysis, with methylation as a percentage of all sites indicated. Arrowheads indicate sites also analysed by COBRA. (C) As for A. Post-impl, genes known to be methylated only after implantation. (D) In situ hybridisation of testis tissue. Arrow indicates spermatogonial stem cells; arrowhead indicates spermatozoa. (E) Methylation of gDMRs as detected by pyrosequencing in sperm and oocytes. (F) RT-qPCR showing transcript levels in WT and 3L KO ovaries for the indicated genes. Error bars indicate s.e.m. **P<0.01, ***P<0.001 by t-test.
Fig. 3.
Fig. 3.
Methylation and repression at novel gDMRs in adult mouse tissues. (A) COBRA on the adult mouse tissues and cell lines indicated at the top. Fibro, fibroblasts without (-) or with (+aza) 5-aza-2′-deoxycytidine. (B) Methylation analysis by pyroassay. (C) All analysed genes showed significant demethylation (P<0.05, ANOVA) by pyroassay following Aza treatment. (D) RT-PCR for the indicated genes in treated fibroblasts. Testis is a positive control (Pos ctrl), except for Adcy6 and Grin3b, where brain was used. IAP and Sycp3 are known to be repressed by methylation. Asterisked genes have low-density CGIs. Hprt is a loading control. (E) RT-qPCR of samples from D. UT, untreated. Error bars indicate s.e.m.
Fig. 4.
Fig. 4.
Conservation of methylation and repression in human. (A) Methylation levels as determined by pyrosequencing in hTERT-1604 normal human fibroblasts. (B) RT-PCR showing derepression by Aza treatment (lane +) is comparable between testis gDMR genes (left) and genes methylated postimplantation (right). Adult testis is a positive control; ACTB is for loading. (C) Significant differences in methylation were seen at all analysed genes in cells exposed to Aza or depleted of DNMT1 by siRNA (P<0.05, except for FKBP6 with Aza which was not significant). (D) RT-qPCR for the same samples. All upregulation >5-fold was significant at P<0.05. Error bars indicate s.e.m.
Fig. 5.
Fig. 5.
Enzyme dependence for gDMR classes in mouse stem cells. (A) Methylation levels in parental J1 ESCs (WT) and DNMT1-deficient derivatives (1KO) assessed by COBRA. un, uncut control. (B) Clonal analysis of a gDMR from an imprinted, testis or brain gene (Snrpn, Fkbp6 and Grin3b, respectively). (C) COBRA showing methylation levels in clonally derived ESCs (16aabb, 7aabb) carrying double knockouts (DKO) in both Dnmt3a and Dnmt3b. (D) Pyrosequencing for the indicated gDMRs. Blastocyst methylation levels are derived from the analysis shown in Fig. 1D. Differences between DKO and WT were significant (P<0.05) for all analysed genes except Crocc. (E) Fold reactivation in mutant versus WT cells as assessed by RT-qPCR. 3ab, DKO cells (as above); TKO, triple KO cells lacking DNMT1, DNMT3A and DNMT3B, data from Karimi et al. (Karimi et al., 2011b); brain and imprint gDMR unavailable. Error bars indicate s.e.m.
Fig. 6.
Fig. 6.
Reprogramming ability of the different gDMR classes. (A) Methylation by COBRA in ESCs with normal (WT), absent (1KO) and rescued (1KO+1) DNMT1 status. (B) Pyroassay indicates significant (P<0.05) gain in methylation in 1KO+1 cells for all analysed genes except Peg1 and Adcy6. (C) Overall gain in methylation in 1KO+1 cells assayed by LUMA. (D) Clonal analysis for one gene from each class of gDMR. (E) Analysis of clones indicates significant (P<0.05) gain in methylation in 1KO+1 cells for all non-imprinted genes analysed. (F) Timecourse of Dnmt1 knockdown and recovery by RT-qPCR following transient transfection of adult mouse fibroblasts with siRNA. Scr, scrambled control; d, days. (G) Pyroassay showing methylation loss and re-establishment for samples shown in F; untreated set to 100%. All genes were significantly demethylated (P<0.05) at 4 days, but were no longer significantly different from untreated (UT) at 18 days, except for Fkbp6. (H) RT-qPCR of same samples showing re-establishment of repression. Day 4 values were set to 100%. KD, knockdown. Error bars indicate s.e.m.
Fig. 7.
Fig. 7.
Methylation dynamics and methyltransferase dependency of genes inheriting methylation from the mother. (Top) Methylation ontogeny of brain, testis and imprinted gDMRs, as well as of postimplantation genes. The average methylation of each gene class (see key, bottom right) is shown: filled bar, 100% methylation; empty bar, 0% methylation. Methyltransferase requirements at different stages, where established, are shown within the central blue panel. (Bottom) The effect of loss and restoration of methyltransferase activity in different experimental systems. The dish on the right represents ESCs.

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