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. 2010 Jul 23;329(5990):444-8.
doi: 10.1126/science.1190485.

Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes

Hao Wu  1 , Volkan Coskun, Jifang Tao, Wei Xie, Weihong Ge, Kazuaki Yoshikawa, En Li, Yi Zhang, Yi Eve Sun
Affiliations

Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes

Hao Wu et al. Science. .

Abstract

DNA methylation at proximal promoters facilitates lineage restriction by silencing cell type-specific genes. However, euchromatic DNA methylation frequently occurs in regions outside promoters. The functions of such nonproximal promoter DNA methylation are unclear. Here we show that the de novo DNA methyltransferase Dnmt3a is expressed in postnatal neural stem cells (NSCs) and is required for neurogenesis. Genome-wide analysis of postnatal NSCs indicates that Dnmt3a occupies and methylates intergenic regions and gene bodies flanking proximal promoters of a large cohort of transcriptionally permissive genes, many of which encode regulators of neurogenesis. Surprisingly, Dnmt3a-dependent nonproximal promoter methylation promotes expression of these neurogenic genes by functionally antagonizing Polycomb repression. Thus, nonpromoter DNA methylation by Dnmt3a may be used for maintaining active chromatin states of genes critical for development.

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Figures

Fig. 1
Fig. 1
Essential roles of Dnmt3a in postnatal neurogenesis. (A) Cell nuclei staining of olfactory bulb (OB) in WT and KO mice at postnatal day (P) 24. The schematic on the right outlines the postnatal neurogenesis from SEZ/SVZ NSCs, and the red boxes denote the portion of the rostral migratory stream (RMS) with newborn neurons entering the OB. Note that both the OB size and the number of newborn neurons in KO mice (red box) are reduced. Arrows indicate radially migrating immature neurons in the postnatal OB. Scale bars, 200 μm. (B) Immunohistochemistry and quantification of Dcx+ neuroblasts in WT and KO RMS (P21 to P24). Hpf, high-power field; n, number of fields from three pairs of littermate mice. Error bars indicate SEM (*P < 0.01). Scale bars, 50 μm. (C) Immunocytochemistry of the immature neuronal marker Tuj1 in differentiating WT and KO SEZ/SVZ cultures. Scale bars, 50 μm. (D) Quantification of Tuj1+ neurons in differentiating WT, KO, and rescued NSC cultures. Error bars indicate SEM (*P < 0.01, n = 10 to 15).
Fig. 2
Fig. 2
Dnmt3a occupies and methylates defined regions at both transcriptionally active and inactive genes. (A) Heat map representations of CpG islands (blue/white) and Dnmt3a (red/green) are shown for both Dnmt3a targets and nontargets. Red/blue represents enrichment, whereas green/white represents no enrichment. Genes were rank-ordered by CpG-island length within 4-kb regions flanking TSSs. FDR, false discovery rate; n, number of genes. (B) Distribution of Dnmt3a binding-sites within regions flanking TSSs of H3K4me3-high/CpG-rich and H3K4me3-low/CpG-poor targets. (C) Distribution of Dnmt3a-dependent DNA methylation within regions flanking TSSs of CpG-rich and CpG-poor genes. (D) Dnmt3a occupancy and Dnmt3a-dependent DNA methylation within representative CpG-rich/H3K4me3-high (Gbx2) and CpG-poor/ H3K4me3-low (Gfap) targets. Regions validated by bisulphite sequencing are shown in gray. IP, immunoprecipitation; WCE, whole-cell extract. (E) Box plots of expression levels in WT postnatal NSCs are shown for genes associated with Dnmt3a, H3K4me3, H3K27me3, and proximal promoter methylation (sperm-specific genes). Middle bars, medians; notches, standard errors; boxes, interquartile ranges; and whiskers, 10th and 90th percentiles.
Fig. 3
Fig. 3
Dnmt3a promotes transcription through nonpromoter methylation. (A) Heat map representations of relative changes in expression (from four experiments), DNA methylation, H3K4me3, H3K27me3, and total histone H3 levels (panH3) between KO and WT NSCs are shown for differentially expressed Dnmt3a targets, which are ranked by changes in expression between KO and WT NSCs. n, number of targets. (B) Distributions of DNA demethylation and H3K27me3 increase in KO NSCs are shown for up-regulated targets (red), down-regulated targets (green), targets without expression changes (blue), and nontargets (gray).
Fig. 4
Fig. 4
Dnmt3a promotes transcription by antagonizing Polycomb repression. (A) Ezh2, Suz12, and H3K27me3 occupancy at representative genes in WT and KO NSCs. Regions analyzed by ChIP-qPCR (in fig. S11E) are shaded in gray. (B) Quantitative PCR analysis of Suz12 and H3K27me3 at representative targets in WT, KO, and rescued NSCs. (C) Relative binding of PRC2 subunits on methylated or unmethylated chromatin arrays was quantified by densitometry (*P = 0.027, n = 4 experiments; **P = 0.0013, n = 6). (D) Quantification of Tuj1+ neurons in control and PRC2-deficient differentiating NSCs. Error bars indicate SEM of randomly selected fields from two independent cultures (*P < 0.05; **P < 0.01).
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