Abstract
DNA methylation deregulation at partially methylated domains (PMDs) represents an epigenetic signature of aging and cancer, yet the underlying molecular basis and resulting biological consequences remain unresolved. We report herein a mechanistic link between disrupted DNA methylation at PMDs and the spatial relocalization of H3K9me3-marked heterochromatin in aged hematopoietic stem and progenitor cells (HSPCs) or those with impaired DNA methylation. We uncover that TET2 modulates the spatial redistribution of H3K9me3-marked heterochromatin to mediate the upregulation of endogenous retroviruses (ERVs) and interferon-stimulated genes (ISGs), hence contributing to functional decline of aged HSPCs. TET2-deficient HSPCs retain perinuclear distribution of heterochromatin and exhibit age-related clonal expansion. Reverse transcriptase inhibitors suppress ERVs and ISGs expression, thereby restoring age-related defects in aged HSPCs. Collectively, our findings deepen the understanding of the functional interplay between DNA methylation and histone modifications, which is vital for maintaining heterochromatin function and safeguarding genome stability in stem cells.
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Data availability
The sequencing datasets were deposited into the NCBI BioProject under the accession number GSE183675. The majority of data are available in the source data file. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.
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Acknowledgements
We are grateful for the Epigenetic core at the Institute of Biosciences and Technology at the Texas A&M University. This work was supported by grants from Cancer Prevention and Research Institute of Texas (RP210070 to Y.Z.), National Institute of Health grants (R35HL166557, R01DK132286 and R01CA240258 to Y.H.; R01CA232017 and R21CA277257 to Y.Z.; P01AG036695, DK092883, CA183252, P30CA125123 and P01CA265748 to M.A.G.), Leukemia & Lymphoma Society (LLS-TRP 6680-24 to Y.Z.) and the Welch Foundation (BE-1913-20220331 to Y.Z.).
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Y.H. and Y.Z. directed and supervised the project. T.H. performed most animal-related work, molecular characterization and sequencing library preparation. J.L. performed all the bioinformatics analysis on high-throughput sequencing data. L.G., T.W. and S.F. supported sequencing library preparation. Y.D. performed cell sorting. A.D. and M.C. performed genotyping and supported molecular cloning. A.G., K.W., C.R. and C.K. supported animal-related work. Y.Y., C.C.Y., S.L. and M.J.Y. provided human bone marrow samples. M.A.G. and X.C. provided essential resources and key intellectual inputs to support this study. Y.H. and Y.Z. wrote the paper. All the authors participated in the discussion, data interpretation and paper editing or discussion.
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Nature Aging thanks Gerald de Haan, Francesco Neri, and Karl Lenhard Rudolph for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Tet2 depletion mitigates age-related decline in HSPC self-renewal and repopulation capability (Related to Fig. 1).
a, Immunoblot analysis of Tet2 expression in cKit+ cells from bone marrow of WT or Tet2KO mice (6 ~ 8-week-old, n = 3 male and 2 female mice). b, The total cell numbers (Top) and the fraction of LSK cells (Bottom) in the indicated replating times. CFU assay was used to assess HSPCs function. Data are shown as mean ± S.D; n = 3 biological replicates. (Two-way ANOVA test; Supplementary Table 1). c, The reconstitution efficiency of lethally irradiated CD45.1 mice transferred with Lin─cKit+Sca1+ (LSK) cells (n = 3 male and 2 female donor mice / group). The reconstitution efficiency was measured biweekly from the peripheral blood till 16 weeks post-BMT (n = 5-7 recipient mice with equal number of male and female mice / group, each dot represents an individual recipient mouse). (Two-way ANOVA test; Supplementary Table 1). d, Lineage analysis in the peripheral blood collected from lethally irradiated CD45.1 mice transferred with LSK cells (n = 5-7 recipient mice /group, each dot represents an individual recipient mouse). Data are shown as mean ± S.D. (Two-way ANOVA test; Supplementary Table 1). e, The gating strategies. Lin─, lineage-negative; LSK, Lin─cKit+Sca1+; LT-HSC, long-term HSC; MPP, multipotential progenitor; ST-HSC, short-term HSC. f, Lineage analysis in the peripheral blood collected from recipient mice undergoing a second bone marrow transplantation using donor cells from the recipient mice transferred with young and old Tet2KO HSPCs listed in Extended Data Fig. 1c, d. Data are shown as mean ± S.D. (n = 3 male donor mice and 3-5 male recipient mice / group). g, Representative Hematoxylin and Eosin (H&E) stained sections of heart, liver, lung, and spleen tissues collected from the CD45.1 recipient mice with secondary BMT (18 weeks post-BMT). Donor cells were total bone marrow cells isolated from mice transplanted Tet2KO LSKs (young or old) as shown in Extended Data Fig. 1c, d (n = 3 donor mice). Scale bar, 200 μm. h, Lineage analysis in the peripheral blood collected from the recipient mice transferred with mixed HSPCs as shown in Fig. 1e. Data are shown as mean ± S.D. (Two-way ANOVA test; Supplementary Table 1).
Extended Data Fig. 2 Distinct transcriptional changes during WT and Tet2KO HSPCs aging (Related to Fig. 2).
a, Heatmap showing the expression of the marker genes used to annotate cell populations. b, Pseudotime inference reveals the differentiation process projected in UMAP-based embedding (shown in Fig. 1a) in all the analyzed cells. Dark blue represents cells in earlier differentiation stages; while dark red represents cells in later differentiation stages. c, The cell type ratio correlation analysis of scRNA-seq data collected in this study (WT-Young and Tet2KO-young) with the previously published dataset (GSE124822). d, Lineage analysis in the bone marrow collected from gender- and aged-matched congenic WT-young, WT-old, Tet2KO-young, and Tet2KO-old mice. Young, 6-8 weeks; Old, 18–24 months. Data are shown as mean ± S.D; n = 4-13 (equal number of male and female mice) / group, each dot represents an individual mouse. (Two-tailed Student’s t-test). e, (Left) Erythroid lineage analysis on bone marrow cells collected from gender- and aged-matched congenic WT-young, WT-old, Tet2KO-young, and Tet2KO-old mice. Data are shown as mean ± S.D; n = 4-12 (equal number of male and female mice) / group, each dot represents an individual recipient mouse (Two-tailed Student’s t-test). (Right) The gating strategies of erythroid progenitors. f, Heatmap showing the expression of marker genes used to annotate cell cycles. g, Heatmap representing genes associated with the pseudotime trajectory for HSC as shown in Fig. 2e.
Extended Data Fig. 3 Measurements of Tet expression and 5hmC in young and aged HSPC (Related to Fig. 3).
a, RT-qPCR analysis of Tet1 and Tet3 expression in LSK cells from WT-young, WT-old, Tet2KO-young, and Tet2KO-old mice. Data are shown as mean ± S.D (n = 3 independent experiments). b, Summary of Pearson’s correlation coefficients of 5hmC (left) and H3K9me3 (right) in the indicated groups. c, The percentage of age-related DHMRs in various genomic regions (distal, intergenic, genebody, 3’UTR, or promoter regions) for the indicated comparison groups (left, WT-Young vs WT-Old; right, Tet2KO-Young vs Tet2KO-Old). d, The top 5 enriched biological functions of age-related DHMRs identified from WT or Tet2KO HSPCs (young vs old) using the GREAT analysis. e, The genome browser views of 5hmC (blue, CMSIP) (scale: 0 to 5) in LSK cells purified from WT-young and WT-old mice. H3K9me3 track (scale: −0.5 to 0.5) in LSK cells purified from the WT-young group is used as the reference to discriminate between heterochromatin and euchromatin.
Extended Data Fig. 4 Tet2 loss prevents the spatial relocalization of H3K9me3-marked heterochromatin from the nuclear periphery to the nucleoplasm during HSPC aging (Related to Fig. 3).
a, Additional representative images of immunofluorescence (IF) staining of H3K9me3 in LSK cells purified from WT-young (6-8 weeks old) and WT-old (18 months old) mice. DAPI was used to stain the whole nuclei. Scale bar, 5 µm. b, Immunoblot analysis (left) and quantification (right) of the indicated histone marks in LK cells purified from WT-young, WT-old, Tet2KO-young, and Tet2KO-old mice. Data are shown as mean ± S.D; n = 3 independent biological replicates. c, Immunofluorescence analysis (top) and quantification (bottom) of H3K9me3 in CD34+ cells purified from human bone marrow at the indicated ages. Data are shown as mean ± S.D; n = 35-40 cells, Scale bar, 5 µm. d, (Left) Immunofluorescence staining of H3K4me3, H3K27ac, and lamin B1 in LSK cells. Scale bar, 5 µm. (Right) The quantification of fluorescence intensities of H3K4me3 and H3K27ac. Data are shown as mean ± S.D; n = 35-45 cells (n = 3-10 mice with equal number of male and female mice).
Extended Data Fig. 5 DNA methylation deregulation contributes to the spatial relocalization of H3K9me3 in HSPCs. (Related to Fig. 4).
a, Average DNA methylation levels within the coding regions in LSK cells purified WT-young (6-8 weeks), WT-old (18 months), Tet2KO-young (6-8 weeks), and Tet2KO-old (18 months) mice. b, Violin plot of DNA methylation levels in the indicated groups. c, Scatterplot of age-related DMRs (young vs old) identified from WGBS data in this study and the published WGBS data (GSE47819). The Pearson’s correlation coefficients were listed to compare the DNA methylation levels within age-related DMRs for the indicated groups. d, Genomic distribution of age-related DMRs (young vs old) identified from WT and Tet2KO groups. e, Genome browser views illustrating H3K9me3, WGBS, 5mC changes (∆5mC), the standard deviation of 5mC changes (5mC S.D.), and 5hmC in WT-young and WT-old HSPCs. f, PCR-based genotyping results using genomic DNA isolated from the tails collected from MxCre and MxCre-Dnmt3af/f mice treated with pIpC. Genotyping was performed 4 weeks after pIpC treatment. g, Average DNA methylation levels within the coding regions in LSK cells purified from MxCre (blue) and MxCre-Dnmt3af/f mice (red) treated with pIpC and analyzed 4 weeks after pIpC treatment. h, RT-qPCR analysis of Dnmt1 expression in LSK cells purified from WT-young, WT-old, Tet2KO-young and Tet2KO-old mice. Data are shown as mean ± S.D (n = 3). (Two-tailed Student’s t-test). i, Summary of the Pearson’s correlation coefficients of H3K9me3 ChIP-seq data for LSK cells purified from MxCre (control) or MxCre-Dnmt3af/f (Dnmt3aKO) mice treated with pIpC. j, The scatterplot analysis to identify genomic regions exhibiting significant differential H3K9me3 enrichment between MxCre (control) and MxCre-Dnmt3af/f (Dnmt3aKO) HSPCs. k, Genome browser view of H3K9me3 in LSK cells purified from MxCre (control) and MxCre-Dnmt3af/f mice treated with pIpC. l, Schematic illustrating the impact of aging, Tet2 and Dnmt3a in regulating the spatial localization of H3K9me3-marked heterochromatin.
Extended Data Fig. 6 Upregulation of repetitive elements in aged HSPCs contributes to their functional decline (Related to Fig. 5).
a, Single-cell expression profiles of repetitive elements from each cell cluster in all the analyzed Lin─ cells (n = 8,352). b, UMAP dimensionality reduction showing the cell distribution, taking into account the expression of repetitive elements as listed in Extended Data Fig. 6a, of all analyzed Lin─ cells with high coverage (more than 300 genes and 30000 UMIs) (n = 8,352) purified from WT-young, WT-old, Tet2KO-young and Tet2KO-old mice. Young mice: 6-8 weeks old; Old mice: 18–24 months old. HSC, hematopoietic stem cells; Mono, monocyte progenitor; Ery, erythroid progenitor; MkP, megakaryocyte progenitor; Bcell-P: B cell progenitors. c, (Left) UMAP dimensionality reduction (n = 929 cells) in HSCs identified from the analysis performed in panel A. Four distinct clusters were identified based on the gene expression signatures including repetitive elements listed in Extended Data Fig. 6b. (Right) UMAP plot showing the distribution of HSCs in the indicated groups. d, Venn diagram to illustrate the overlapping genomic regions with age-related compartment changes in WT and Tet2KO groups. e, Violin plot of 5hmC levels in H3K9me3-marked genomic regions with age-related compartment changes in the indicated groups. f, Normalized expression of SINE and LINE elements within H3K9me3-marked genomic regions with age-related compartment changes (B-to-A or A-to-B) in the indicated groups.
Supplementary information
Supplementary Table 1
ANOVA test.
Supplementary Table 2
Marker genes in scRNA-seq analysis.
Supplementary Table 3
Age-related DEGs.
Supplementary Table 4
Age-related DHMRs.
Supplementary Table 5
Age-related DMR analysis.
Supplementary Table 6
List of qPCR primers.
Source data
Source Data Fig. 1
Unprocessed western blots.
Source Data Fig. 4
Unprocessed western blots.
Source Data Fig. 5
Unprocessed western blots.
Statistical Source Data
Source data for statistical analysis.
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Hong, T., Li, J., Guo, L. et al. TET2 modulates spatial relocalization of heterochromatin in aged hematopoietic stem and progenitor cells. Nat Aging 3, 1387–1400 (2023). https://doi.org/10.1038/s43587-023-00505-y
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DOI: https://doi.org/10.1038/s43587-023-00505-y
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