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The disordered N-terminal domain of DNMT3A recognizes H2AK119ub and is required for postnatal development

Nature Genetics volume 54, pages 625–636 (2022)Cite this article

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Abstract

DNA methyltransferase 3a (DNMT3A) plays a crucial role during mammalian development. Two isoforms of DNMT3A are differentially expressed from stem cells to somatic tissues, but their individual functions remain largely uncharacterized. Here we report that the long isoform DNMT3A1, but not the short DNMT3A2, is essential for mouse postnatal development. DNMT3A1 binds to and regulates bivalent neurodevelopmental genes in the brain. Strikingly, Dnmt3a1 knockout perinatal lethality could be partially rescued by DNMT3A1 restoration in the nervous system. We further show that the intrinsically disordered N terminus of DNMT3A1 is required for normal development and DNA methylation at DNMT3A1-enriched regions. Mechanistically, a ubiquitin-interacting motif embedded in a putative α-helix within the N terminus binds to mono-ubiquitinated histone H2AK119, probably mediating recruitment of DNMT3A1 to Polycomb-regulated regions. These data demonstrate an isoform-specific role for DNMT3A1 in mouse postnatal development and reveal the N terminus as a necessary regulatory domain for DNMT3A1 chromatin occupancy and functions in the nervous system.

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Fig. 1: DNMT3A1, but not DNMT3A2, is essential for mouse postnatal development.
Fig. 2: DNMT3A1 binds to and regulates neurodevelopmental genes in the cerebral cortex.
Fig. 3: DNMT3A1 restoration in the nervous system by Nestin-Cre partially rescued Dnmt3a1 KO lethality.
Fig. 4: Deletion of DNMT3A1 N terminus leads to impaired postnatal development.
Fig. 5: The N terminus is required for DNMT3A1-regulated DNA methylation in the cerebral cortex.
Fig. 6: Integrative analyses of DNA methylation and gene expression changes in Dnmt3a1–/– neuron nuclei.
Fig. 7: The N terminus facilitates DNMT3A1 enrichment around bivalent promoters by binding to H2AK119ub.

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Data availability

All new data sets generated for this work have been deposited to GEO under accession number GSE164265. Gene ontology-based mouse gene set collections for GSEA were downloaded from GO2MSIG database (http://www.bioinformatics.org/go2msig/, April 2015 release). The genomic coordinates for mouse transcripts and CpG islands were downloaded from GENCODE (https://www.gencodegenes.org/mouse/) and UCSC genome browser (http://hgdownload.cse.ucsc.edu/) respectively. Source data are provided with this paper.

Code availability

Custom scripts developed for this study are available at https://github.com/DapengHao/DNMT3A1N_terminus.

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Acknowledgements

We thank the Genetically Engineered Rodent Models Core at the Baylor College of Medicine (BCM) for help with generation of mouse models and the Cytometry and Cell Sorting Core Facility at BCM for help with flow cytometry and sorting. We thank the Protein and Monoclonal Antibody Production Core and Y. Zhu at BCM for help with recombinant protein purification. We also thank X. Cheng, X. Zhang and M. T. Bedford (The University of Texas MD Anderson Cancer Center) for helpful discussions. This work was supported by National Institutes of Health grants: R01HL134780 and R01HL146852 (to Y.H.), R01HG007538 and R01CA228140 (to W.L.), NS071153 (to B.D.), R01GM112003 (to Y.Z.), R01CA183252 and R01DK092883 (to M.A.G.) and P30CA125123. This work was also supported by American Cancer Society (RSG-18-043-01-LIB, to Y.H.) and Welch Foundation (BE-1913-20190330, to Y.Z.).

Author information

Author notes

  1. Dapeng Hao

    Present address: School of Basic Medical Sciences, Harbin Medical University, Harbin, China

  2. Teng-Wei Huang

    Present address: Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan

  3. Xueqiu Lin

    Present address: Department of Bioengineering, Stanford University, Stanford, CA, USA

  4. These authors contributed equally: Tianpeng Gu, Dapeng Hao.

Authors and Affiliations

  1. Molecular and Cellular Biology Department, Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX, USA

    Tianpeng Gu, Anna G. Guzman, Ayala Tovy, Carina Rosas, Mira Jeong & Margaret A. Goodell

  2. Division of Biostatistics, Dan L Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA

    Dapeng Hao & Xueqiu Lin

  3. Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA

    Junsung Woo, Teng-Wei Huang & Benjamin Deneen

  4. Department of Translational Medical Science, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, USA

    Lei Guo, Yubin Zhou & Yun Huang

  5. Department of Neurosurgery, Baylor College of Medicine, Houston, TX, USA

    Benjamin Deneen

  6. Division of Computational Biomedicine, Department of Biological Chemistry, School of Medicine, University of California, Irvine, Irvine, CA, USA

    Wei Li

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Contributions

M.A.G. and T.G. conceived the study. T.G. performed most experiments with help from M.J., T.-W.H., A.G.G., A.T. and C.R. D.H. carried out computational analysis of all the high-throughput sequencing data and X.L. carried out initial analysis of DNMT3A ChIP–seq data under supervision of W.L. J.W. performed the LTP experiment under supervision of B.D. L.G. performed the OptoIDR assay under supervision of Y.Z. and Y.H. M.A.G., T.G. and D.H. designed the figures. M.A.G. and T.G. wrote the manuscript with input from the other authors. All authors approved the final version of the manuscript.

Corresponding author

Correspondence to Margaret A. Goodell.

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The authors declare no competing interests.

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Nature Genetics thanks Déborah Bourc’his, Wolf Reik, Aled Parry and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Expression profiles of DNMT3A isoforms in mouse postnatal tissues and unaltered cell populations in the spleen and thymus of Dnmt3a1–/– mice.

a, RT-qPCR expression analysis of Dnmt3a and its transcript variants in WT mouse tissues at P18. n = 2 biological independent mice. Data are presented as mean with individual values. b, A diagram representing the generation of Dnmt3a-FLAG knock-in (KI) mouse model. 3× FLAG tag was added to the C terminus of the endogenous Dnmt3a by CRISPR-Cas9 mediated homologous recombination. Linker amino acid sequences: GGSG. c, Western blots for DNMT3A isoforms within P18 Dnmt3a-FLAG mouse tissues. The left panel shows specific detection of FLAG-tagged DNMT3A isoforms in the KI mouse thymus. The experiment was repeated two times independently with similar results. d, Alignment of the sequence of residual N-terminal fragment potentially left in cells after exon 4 deletion to that of DNMT3A1. The reading frame shifts after Proline 56, and the premature stop codon occurs 11 amino acids afterwards. e, Western blots to verify the loss of DNMT3A1 or DNMT3A2 protein respectively in P18 knockout mouse thymuses and cortices. Samples of each genotype were biological replicates. f, Quantification of DNMT3A1 expression in Western blot analysis of P18 mouse cortices (e, right). n = 2 biological independent samples. Data are shown as mean with individual values. g,h, Smaller spleens in P21 Dnmt3a1–/– mice. Representative images of KO and WT spleens are shown (g). Spleen weight was normalized to the body weight (h). Data are shown as mean ± s.e.m. ** P = 0.0014; ns: not significant, P = 0.9707 (two-sided unpaired t tests). i,j, Smaller thymuses in P21 Dnmt3a1–/– mice. Data are shown as mean ± s.e.m. (j). * P = 0.0142; ns: not significant, P = 0.4033 (two-sided unpaired t tests). k, Flow cytometry analysis of cell populations on viable splenic cells from a WT mouse: Gr1/Mac-1 for myeloid cells, CD4/CD8 for T cells and B220 for B cells. The sequential gating strategy is displayed in Supplementary Fig. 1. l, Quantification of frequencies for cell subsets as shown in k. Data are presented as mean ± s.d. m, A representative flow cytometry plot of CD4 and CD8 on viable thymocytes from a WT mouse. SP: single positive; DP: double positive, DN: double negative. The sequential gating strategy is provided in Supplementary Fig. 2. n, Quantification of frequencies for cell subsets as shown in m. Data are presented as mean ± s.d. The number of mice analyzed in h,l and j,n: WT littermates for Dnmt3a1 KO, n = 4; Dnmt3a1 KO, n = 5; WT littermates for Dnmt3a2 KO, n = 4; Dnmt3a2 KO, n = 5.

Source data

Extended Data Fig. 2 General morphology and cell populations in Dnmt3a1–/– mouse brain.

a–c, Double immunofluorescence (IF) staining for DNMT3A1 and the neuronal marker NEUN (a), astroglial marker SOX9 (b), or oligodendroglial marker OLIG2 (c) on sagittal brain sections of P23 WT mice. White arrows point to cells positive for SOX9 or OLIG2. IF was performed with an N-terminal anti-DNMT3A antibody which is DNMT3A1-specific. Section thickness: 15 μm. Scale bar = 100 μm. d, Quantification of DNMT3A1 signal intensity in DNMT3A1 positive and NEUN, SOX9, or OLIG2 positive cells in the same IF images. Positive cells were detected and measured using QuPath software. e, Dnmt3a1–/– mice had smaller brains, but the ratio of brain to body weight was greater than WT littermates at P21. WT, n = 8; Dnmt3a1 KO, n = 7. All data are presented as mean ± s.d. with individual values. P values for brain weight, *** P = 0.0005; body weight, **** P < 0.0001 and brain/body ratio, **** P < 0.0001 (two-sided unpaired t tests). f, Normal brain morphology of Dnmt3a1–/– mouse revealed by Nissl staining of sagittal brain sections. Scale bar = 1.0 mm. The experiment was repeated two times with independent biological samples. g, IF staining showing deficiency of DNMT3A1 protein and unaltered cell populations of neurons (NEUN+), astrocytes (SOX9+) and oligodendrocytes (OLIG2+) in the cerebral cortex of Dnmt3a1–/– mice at P23. Scale bar = 100 μm. The experiment was repeated three times and cell counts are provided below. h, Schematic representation of a sagittal section of the mouse brain. The black squares indicate regions selected for cell counting. i, Percentages of neurons, astrocytes, and oligodendrocytes in the indicated brain regions (h) of Dnmt3a1–/– (n = 3) and WT (n = 3) mice. Two sections from each mouse brain were stained for each cell marker. All data are presented as mean ± s.d. j, Representative IF staining for NEUN and the motoneuron-specific marker ChAT on coronal sections through the hypoglossal nucleus of P23 mouse brain. Motor neurons are positive for both ChAT and NEUN. Scale bar = 100 μm. The experiment was repeated two times. k, Total numbers of motor neurons in the hypoglossal nucleus of Dnmt3a1–/– (n = 3) and WT (n = 2) mice. Data are presented as mean with individual values.

Source data

Extended Data Fig. 3 Correlation between DNMT3A1 and H3K27me3 across gene bodies and gene expression analysis for Dnmt3a1–/– mouse brain.

a, Heatmaps of H3K4me3, H3K27me3 and DNMT3A1 across genes (left) in P18 Dnmt3a-FLAG cortex. Genes were ranked by H3K4me3 occupancy, and the curves represent smoothing cubic splines (right) fitted by the median signals of H3K27me3 and DNMT3A1 across each gene. b, Unchanged expression levels of DNA methylation regulators in Dnmt3a1–/– cortex. TPM: transcripts per million. WT, n = 3; Dnmt3a1 KO, n = 3 biological replicates. Data are shown as mean ± s.d. *** P = 0.0002 (two-sided unpaired t test). c, RT-qPCR verification of depletion of Dnmt3a1 full-length transcripts and down-regulation of neural development-related genes in P21 Dnmt3a1–/– cerebral cortex. The expression level of each gene was normalized to that of Gapdh. WT, n = 4; Dnmt3a1 KO, n = 5 biological replicates. Data are shown as mean with s.d. P values for Dnmt3a1, **** P < 0.0001; Neurod6, * P = 0.0173; Bdnf, * P = 0.0280; Wnt7a, *** P = 0.0002 (two-sided unpaired t tests). d,e, Heatmaps highlighting the relative expression level of members in gene sets synapse assembly (d) and neuromuscular process (e). f, RT-qPCR verification of expression changes for selected members in gene sets synapse assembly and neuromuscular process in P21 Dnmt3a1–/– cerebral cortex. WT, n = 4; Dnmt3a1 KO, n = 5 biological replicates. Data are shown as mean ± s.d. P values for Wnt5a, ** P = 0.0081; Homer1, * P = 0.0215; Shank1, *** P = 0.0006; Shank2, ** P = 0.0053 (two-sided unpaired t tests). g, GSEA of Dnmt3a1–/– hippocampal transcriptome. Top dysregulated gene sets were listed and ranked by NES. h, Enrichment plots for down-regulated gene set regulation of synaptic transmission in Dnmt3a1–/– hippocampus. i, Heatmap and average density of wildtype DNMT3A1 binding across DEGs (up- or down-DEGs) and non-DEGs (other) of Dnmt3a1–/– cortex. Input was subtracted from the signal of DNMT3A1 ChIP-seq. Genes are ordered by P values, with up-DEGs and down-DEGs on the top and bottom ends, respectively.

Source data

Extended Data Fig. 4 DNMT3A1 N-terminal domain is an intrinsically disordered region.

a, Prediction of intrinsic disorder for DNMT3A1 by PONDR (Predictor of Natural Disordered Regions) online. Amino acid positions are shown on the x axis. The cyan bars designate the N-terminal regions investigated. b, Fluorescence images of living Hela cells expressing Cry2-mCherry or N-terminal IDR fusion proteins (optoN219 and optoN278). All cells were subjected to 488 nm blue light stimulation under identical conditions. The experiment was repeated three times independently with similar results. Representative images after 80-second stimulation are presented. Scale bar = 10 μm. c, Purification of 6×His tagged GFP and N-terminal IDR-GFP fusion proteins for in vitro droplet formation assay. d, Visualization of turbidity of indicated protein solutions (20 µM) in droplet formation buffer in the absence (–) or presence (+) of 8% PEG-8000. e, Representative images of GFP-positive spherical protein droplets formed at concentrations of 5 µM and 20 µM. Proteins were diluted to the final concentrations with droplet formation buffer in the presence of 8% PEG-8000. The experiment was repeated four times with similar results. Scale bar = 20 μm. f, Fusion events between proximal droplets of N219-GFP (top) or N278-GFP (bottom). g, Live-cell images of FRAP analysis on GFP-DNMT3A1 expressed in NIH 3T3 cells. Scale bar = 10 μm. h, Average fluorescence recovery trace in GFP-DNMT3A1 FRAP experiments (n = 12 cells). All data are presented as mean ± s.d. i, Live-cell images of FRAP analysis on GFP-MeCP2. Scale bar = 10 μm. j, Average fluorescence recovery trace in GFP-MeCP2 FRAP experiments (n = 6 cells).

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Extended Data Fig. 5 Dnmt3a1ΔN mice showed a reduced rate of weight gain and impaired behaviors.

a, Body weights of female Dnmt3a1ΔN (n = 5) mice and WT littermates (n = 5) at the age of 2, 4 and 6 months. Data are shown as mean ± s.d. ** P = 0.003166; *** P = 0.000360; **** P < 0.0001 (multiple t tests). b, Representative images of Dnmt3a1ΔN and WT females at 6 months. c, Dnmt3a1ΔN mice exhibited an increase in fecal boli deposits during 30-min open field test. **** P < 0.0001 (two-sided unpaired t tests). d, Dnmt3a1ΔN mice showed a similar level of anxiety with WT littermates in a Light-dark exploration test. ns: not significant (two-sided unpaired t tests), dark duration, P = 0.5814; light duration, P = 0.5810; entries, P = 0.4214. The number of mice analyzed in c,d: Dnmt3a1ΔN, n = 19; WT, n = 16. Data are shown as mean ± s.e.m. with individual values.

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Extended Data Fig. 6 DNA methylation and gene expression analyses for Dnmt3a1–/–, Dnmt3a2–/– and Dnmt3a1ΔN P21 cortices.

a, Average levels, and changes of CpG methylation genome-wide (overall), and at various genomic features in the cerebral cortex of WT, Dnmt3a1–/–, Dnmt3a2–/– and Dnmt3a1ΔN P21 mice. Promoters: 2,000 bp upstream to 500 bp downstream of TSSs. Canyons were defined as long unmethylated regions over 3.5 kb with an average methylation level < 0.1 in WT cortices (n = 855). CGI or Canyon shores: ± 2 kb regions flanking CGI or Canyons. Data are shown as mean with individual values. b, Genome-wide average CpH (non-CpG) methylation levels in WT and KO cerebral cortices (n = 2 biological replicates each genotype). Data are shown as mean with individual values. c, DNA methylation levels across bivalent genes and two other clusters of genes defined in Fig. 2a. d, Scatterplot of DMR methylation levels in KOs and WT. Each dot represents a DMR. Regions with methylation difference less than 0.1 are not included, giving rise to the gaps. e, Smooth curves of the percentage of each gene covered by DMRs in KO cortices. Genes were ordered by wildtype DNMT3A signal (Fig. 5h). f, Venn diagram of overlapping DMRs (FDR < 0.05) between KO cortices. g, Venn diagram representations of overlapping up-regulated DEGs (left, P < 0.01) and down-regulated DEGs (right) between KOs. h, GSEA analysis of KO cortex transcriptomes. GO terms (biological processes) with FDR < 0.05 in any of the differential expression analysis (Dnmt3a1 KO versus WT, Dnmt3a2 KO versus WT, or Dnmt3a1ΔN versus WT) were included (n = 49).

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Extended Data Fig. 7 Neuron nuclei sorting from the cerebral cortex.

a, Flow cytometry analysis of purified cortical cell nuclei by sucrose ultracentrifugation. NEUN-positive single nuclei were sorted and used for DNA/RNA extraction and ChIP experiments. b, Representative IF images of presort and sorted nuclei. The purity of sorted neuron nuclei was above 95%, and it was checked for every sample after sorting. Scale bar = 50 μm.

Extended Data Fig. 8 DNA methylation and gene expression analyses in sorted neuron nuclei.

a, Violin plots for the distribution of average CpG methylation ratios of 5 kb bins (top) and CpH methylation ratio of 50 kb bins (bottom) over the genome in WT, Dnmt3a1–/–, Dnmt3a2–/– and Dnmt3a1ΔN neuron nuclei (n = 2 biological replicates each genotype). The lower and upper hinges of boxplots correspond to the first and third quartiles. The lower or upper whisker extends from the hinge to the smallest or largest value within 1.5× interquartile range of the hinge respectively. b, DNA methylation levels at Canyons (left), CpG islands (right) and flanking regions. c, IGV displays of DNMT3A1 (cortex), H3K4me3, H3K27me3 and H2AK119ub enrichment in Dnmt3a-FLAG neuron nuclei, and CpG methylation in WT and KO neuron nuclei at Bmp7 gene locus. The differentially methylated region is highlighted in light green. d, Heatmaps for relative CpG methylation levels at Dnmt3a1–/– DMRs (FDR < 0.05) in WT, Dnmt3a1–/–, Dnmt3a2–/– and Dnmt3a1ΔN neuron nuclei. Overlapping DMRs in Dnmt3a2–/– or Dnmt3a1ΔN neuron genome are displayed on the right. e, Identification of enhancers (poised and active) by histone modifications H3K4me1, H3K4me3 and H3K27ac. H3K27me3 and DNMT3A1 were plotted accordingly. f, Average DNA methylation levels at poised and active enhancers in in WT, Dnmt3a1–/–, Dnmt3a2–/– and Dnmt3a1ΔN neuron nuclei. g, Volcano plot of the distribution of differentially expressed genes (P < 0.01) in Dnmt3a1–/– neuron nuclei. h, Heatmap for wildtype DNMT3A1 binding across Dnmt3a1–/–-neuron nuclei DEGs (up- or down-DEGs) and non-DEGs (other). Density plot was presented in Fig. 6c. i, Average DNA methylation levels across Dnmt3a1–/–-neuron nuclei DEGs and other genes in WT and Dnmt3a1–/– neuron nuclei. j, Heatmaps of DMR distribution across up- and down-regulated DEGs in Dnmt3a1–/– neuron nuclei. The gene body was scaled to a 10 kb region. Each red line represents a DMR. k, Fraction of genes covered by DMRs in the gene body and ± 5 kb flanking regions in Dnmt3a1–/– neuron nuclei. l, Average DNA methylation levels across Dnmt3a1–/–-cortex DEGs and other genes in WT and Dnmt3a1–/– cortices. m, Changes of CpG methylation levels (Dnmt3a1 KO–WT) across Dnmt3a1–/– cortical DEGs and other genes.

Extended Data Fig. 9 The N-terminus is required for DNMT3A1 enrichment around bivalent promoters via binding to H2AK119ub.

a, Schematic diagram of DNMT3A constructs that were re-expressed in Dnmt3a–/– mouse ESCs. A 3× FLAG tag and a nuclear localization signal (NLS) were added at the N-terminus of each protein. b, Western blots showing re-expression of DNMT3A variants in established stable ESC lines. The experiment was repeated two times with similar results. c, Density plots of H3K4me3, H3K27me3 and H3K36me3 ChIP-seq signals at each group of TSSs indicated in Fig. 7a. d, Heatmaps for DNMT3A1 (WT) and DNMT3A1ΔN binding profiles at regions flanking TSSs in the cerebral cortex. A C-terminus antibody was used in DNMT3A ChIP-seq. The same heatmaps for H3K4me3 and H3K27me3 marks are also shown in Fig. 2a. e, Coomassie blue staining of purified GFP-fused DNMT3A1 N-terminal fragments: N121 and N122–219 (left), N219 and N219 mutants (right). f, DNMT3A1 protein structure predicted by AlphaFold (https://alphafold.ebi.ac.uk). The UIM is a part of an α-helix in the flexible N-terminus of DNMT3A1. Met 220 is the first amino acid of DNMT3A2. g, Coomassie blue staining of purified GFP-fused DNMT3A1 N278 and DNMT3B1 N-terminus (aa 1–222). h, Western blot for DNMT3A1 N278 and DNMT3B1 N-terminus after pulldown assays with the indicated nucleosomes. The experiment was repeated three times independently with similar results. i, Heatmap showing genome-wide Spearman correlation between H3K4me3, H3K27me3, H2AK119ub marks and DNMT3A1 (cortex) binding in neuron nuclei. j, IGV displays of H3K4me3, H3K27me3, H2AK119ub and DNMT3A1 enrichment at a region on chromosome 2 in the cerebral cortex and neuron nuclei.

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Gu, T., Hao, D., Woo, J. et al. The disordered N-terminal domain of DNMT3A recognizes H2AK119ub and is required for postnatal development. Nat Genet 54, 625–636 (2022). https://doi.org/10.1038/s41588-022-01063-6

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