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Cited by 113Acute Myeloid Leukemia ResearchEpigenetics and DNA MethylationGenomics and Chromatin Dynamics

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TET2 mutations are associated with hypermethylation at key regulatory enhancers in normal and malignant hematopoiesis

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Tulstrup, Morten, Soerensen, Mette, Hansen, Jakob Werner, Gillberg, Linn, Needhamsen, Maria, Kaastrup, Katja, Helin, Kristian, Christensen, Kaare, Weischenfeldt, Joachim, Grønbæk, Kirsten·2021·Nature Communications
DOI 10.1038/s41467-021-26093-2PMC PMC8523747
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Abstract

Mutations in the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). Here, we investigate associations between TET2 mutations and DNA methylation in whole blood in 305 elderly twins, 15 patients with CCUS and 18 healthy controls. We find that TET2 mutations are associated with DNA hypermethylation at enhancer sites in whole blood in CHIP and in both granulocytes and mononuclear cells in CCUS. These hypermethylated sites are associated with leukocyte function and immune response and ETS-related and C/EBP-related transcription factor motifs. While the majority of TET2- associated hypermethylation sites are shared between CHIP and in AML, we find a set of AML-specific hypermethylated loci at active enhancer elements in hematopoietic stem cells. In summary, we show that TET2 mutations is associated with hypermethylated enhancers involved in myeloid differentiation in both CHIP, CCUS and AML patients.

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Fig. 1 Epigenome-wide association studies of DNMT3A and TET2 mutations. a QQ plot the P value distribution in differential methylation analyses by DNMT3A mutation status (55 DNMT3A-mutated inviduals compared to 189 individuals without CHIP). b Same as A, but comparing 44 TET2-mutated individuals to 189 without CHIP. c Volcano plot of results from TET2-specific analyses. Horizontal dashed line indicates P = 1.4 × 10−5. Numbers in corners indicate the number of CpG sites in each quadrant separated by dashed lines. Red and blue dots represent hyper- and hypomethylated CpG sites, respectively, below the significance threshold. d Selection of P value cutoff used in C. Red line displays the increasing proportion of hypermethylated sites with decreasing P value cutoff. Horizontal dashed line indicates 95% of sites are hypermethylated. Vertical dashed line indicates selected P cutoff of 1.4 × 10−5. e Manhattan plot with the 2741 CpG sites showing TET2 mutation-associated hypermethylation highlighted in red. P values and effect size estimates in all panels derived using a linear mixed effects regression with twin pair as random intercept. All P values are two-sided and not adjusted for multiple comparisons.
Fig. 1 Epigenome-wide association studies of DNMT3A and TET2 mutations. a QQ plot the P value distribution in differential methylation analyses by DNMT3A mutation status (55 DNMT3A-mutated inviduals compared to 189 individuals without CHIP). b Same as A, but comparing 44 TET2-mutated individuals to 189 without CHIP. c Volcano plot of results from TET2-specific analyses. Horizontal dashed line indicates P = 1.4 × 10−5. Numbers in corners indicate the number of CpG sites in each quadrant separated by dashed lines. Red and blue dots represent hyper- and hypomethylated CpG sites, respectively, below the significance threshold. d Selection of P value cutoff used in C. Red line displays the increasing proportion of hypermethylated sites with decreasing P value cutoff. Horizontal dashed line indicates 95% of sites are hypermethylated. Vertical dashed line indicates selected P cutoff of 1.4 × 10−5. e Manhattan plot with the 2741 CpG sites showing TET2 mutation-associated hypermethylation highlighted in red. P values and effect size estimates in all panels derived using a linear mixed effects regression with twin pair as random intercept. All P values are two-sided and not adjusted for multiple comparisons.CC BY · PubMed Central
Fig. 2 Characteristics of the 2741 CpG sites hypermethylated in TET2-mutated CHIP. a Enrichment of Gene Ontology terms for 2741 hypermethylated sites. P values derived using the region-based binomial test in GREAT and are not adjusted for multiple comparisons. b Monocyte chromatin states at hypermethylated and non-hypermethylated sites. c CpG island relations for hypermethylated and non-hypermethylated sites.
Fig. 2 Characteristics of the 2741 CpG sites hypermethylated in TET2-mutated CHIP. a Enrichment of Gene Ontology terms for 2741 hypermethylated sites. P values derived using the region-based binomial test in GREAT and are not adjusted for multiple comparisons. b Monocyte chromatin states at hypermethylated and non-hypermethylated sites. c CpG island relations for hypermethylated and non-hypermethylated sites.CC BY · PubMed Central
Fig. 3 Results of epigenome-wide analyses of TET2 mutations in granulocyte DNA from five CCUS patients and eight healthy controls. a Volcano plot of CCUS results. Red dots indicate CpG sites from the set of 2741 hypermethylated sites in CHIP (Fig. 1c). Margins display distributions of the 2741 sites versus the rest of the EPIC array. Numbers in corners indicate the number of dots (both colors) in each quadrant separated by dashed lines. P values and effect size estimates derived using a linear model in Limma. P values are two-sided and not adjusted for multiple comparisons. b Volcano plot of CHIP results (same as 1C), yellow dots indicate CpG sites from the set of 12,096 significantly hypermethylated sites in CCUS granulocytes (upper right quadrant in panel a). Only sites analyzed in both CHIP and CCUS are shown. P values and effect size estimates derived using a linear model in Limma. P values are two-sided and not adjusted for multiple comparisons. c Enrichment of Gene Ontology terms for 12,096 hypermethylated sites in CCUS. P values derived using the region-based binomial test in GREAT and are not adjusted for multiple comparisong. d Monocyte chromatin states for hypermethylated and non-hypermethylated sites. e CpG island relations for hypermethylated and non-hypermethylated sites.
Fig. 3 Results of epigenome-wide analyses of TET2 mutations in granulocyte DNA from five CCUS patients and eight healthy controls. a Volcano plot of CCUS results. Red dots indicate CpG sites from the set of 2741 hypermethylated sites in CHIP (Fig. 1c). Margins display distributions of the 2741 sites versus the rest of the EPIC array. Numbers in corners indicate the number of dots (both colors) in each quadrant separated by dashed lines. P values and effect size estimates derived using a linear model in Limma. P values are two-sided and not adjusted for multiple comparisons. b Volcano plot of CHIP results (same as 1C), yellow dots indicate CpG sites from the set of 12,096 significantly hypermethylated sites in CCUS granulocytes (upper right quadrant in panel a). Only sites analyzed in both CHIP and CCUS are shown. P values and effect size estimates derived using a linear model in Limma. P values are two-sided and not adjusted for multiple comparisons. c Enrichment of Gene Ontology terms for 12,096 hypermethylated sites in CCUS. P values derived using the region-based binomial test in GREAT and are not adjusted for multiple comparisong. d Monocyte chromatin states for hypermethylated and non-hypermethylated sites. e CpG island relations for hypermethylated and non-hypermethylated sites.CC BY · PubMed Central
Fig. 4 Selected enriched transcription factor binding motifs at hypermethylated monocyte enhancer regions in CHIP and CCUS. Hypermethylated regions defined as 200 bp flanking regions around CpG sites located in an enhancer region. Enrichment (E) values derived using the Fischer’s exact test method in Analysis of Motif Enrichment. E values are adjusted P values with correction for the number of tested transcription factor binding motifs from the HOCOMOCO v11 full dataset (N = 769).
Fig. 4 Selected enriched transcription factor binding motifs at hypermethylated monocyte enhancer regions in CHIP and CCUS. Hypermethylated regions defined as 200 bp flanking regions around CpG sites located in an enhancer region. Enrichment (E) values derived using the Fischer’s exact test method in Analysis of Motif Enrichment. E values are adjusted P values with correction for the number of tested transcription factor binding motifs from the HOCOMOCO v11 full dataset (N = 769).CC BY · PubMed Central
Fig. 5 TET2 mutation-associated hypermethylation in TCGA LAML data. a Results of epigenome-wide association studies in four and 86 AML patients with and without TET2 mutations, respectively. Red dots indicate CpG sites from the set of 2741 significantly hypermethylated sites in CHIP (Fig. 1c). Horizontal dashed line indicates P = 0.01. Margins display distributions of the 2741 sites versus the rest of the sites. Numbers in corners indicate the number of dots (both colors) in each quadrant separated by dashed lines. P values and effect size estimates derived using a linear model in Limma. P values are two-sided and not adjusted for multiple comparisons. b Similar to a. Orange dots indicate CpG sites from the set of 12,096 significantly hypermethylated sites in CCUS granulocytes. Only overlapping sites between the two sets are shown. P values and effect size estimates derived using a linear model in Limma. P values are two-sided and not adjusted for multiple comparisons. c Volcano plot of CCUS granulocyte results (same as in Fig. 3a) but with the 2000 significantly hypermethylated sites in AML highlighted (purple dots). P values and effect size estimates derived using a linear model in Limma. P values are two-sided and not adjusted for multiple comparisons. d Proportion of hypermethylated and non-hypermethylated sites in AML located in hematopoietic stem cell (HSC)-specific (N = 14,398) and monocyte-specific enhancers (N = 14,017). e Mean scaled effect size estimates of TET2 mutations at all CpG sites located in HSC-specific (N = 47,748) and monocyte-specific enhancers (N = 37,518). Error bars indicate 95% confidence intervals. See Supplementary Fig. 13 for plots of data distributions.
Fig. 5 TET2 mutation-associated hypermethylation in TCGA LAML data. a Results of epigenome-wide association studies in four and 86 AML patients with and without TET2 mutations, respectively. Red dots indicate CpG sites from the set of 2741 significantly hypermethylated sites in CHIP (Fig. 1c). Horizontal dashed line indicates P = 0.01. Margins display distributions of the 2741 sites versus the rest of the sites. Numbers in corners indicate the number of dots (both colors) in each quadrant separated by dashed lines. P values and effect size estimates derived using a linear model in Limma. P values are two-sided and not adjusted for multiple comparisons. b Similar to a. Orange dots indicate CpG sites from the set of 12,096 significantly hypermethylated sites in CCUS granulocytes. Only overlapping sites between the two sets are shown. P values and effect size estimates derived using a linear model in Limma. P values are two-sided and not adjusted for multiple comparisons. c Volcano plot of CCUS granulocyte results (same as in Fig. 3a) but with the 2000 significantly hypermethylated sites in AML highlighted (purple dots). P values and effect size estimates derived using a linear model in Limma. P values are two-sided and not adjusted for multiple comparisons. d Proportion of hypermethylated and non-hypermethylated sites in AML located in hematopoietic stem cell (HSC)-specific (N = 14,398) and monocyte-specific enhancers (N = 14,017). e Mean scaled effect size estimates of TET2 mutations at all CpG sites located in HSC-specific (N = 47,748) and monocyte-specific enhancers (N = 37,518). Error bars indicate 95% confidence intervals. See Supplementary Fig. 13 for plots of data distributions.CC BY · PubMed Central

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Authors
Tulstrup, M., Soerensen, M., Hansen, J. W., Gillberg, L., Needhamsen, M., Kaastrup, K., Helin, K., Christensen, K., Weischenfeldt, J., & Grønbæk, K.
Affiliations
  • University of Copenhagen (DK)
  • University of Southern Denmark (DK)
  • Karolinska Institutet (SE)
Corresponding author
  • Weischenfeldt, Joachim
  • Grønbæk, Kirsten
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2021-10-18
Volume
12
Issue
1
Pages
6061-6061
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-021-26093-2
PMC
PMC8523747
OpenAlex
W3205085619
Cited by
108
References
50
Citations by year
2026: 19 · 2025: 19 · 2024: 17 · 2023: 30 · 2022: 23
FWCI
12.25
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
  • University of Southern Denmark Research Portal (University of Southern Denmark) (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Novo Nordisk Fonden (NNF17CC0027852)
Topics
  • Health Sciences › Medicine › Hematology › Acute Myeloid Leukemia Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
TET2 mutation, DNA hypermethylation, clonal hematopoiesis of indeterminate potential, clonal cytopenia of undetermined significance, acute myeloid leukemia, hematopoietic stem cells, myeloid differentiation, leukocyte function, immune response, transcription factor binding motifs, epigenetic regulators
MeSH terms
DNA Methyltransferase 3A, DNA (Cytosine-5-)-Methyltransferases, DNA-Binding Proteins, Enhancer Elements, Genetic, Hematopoiesis, Hematopoietic Stem Cells, Humans, Mutation, Myeloproliferative Disorders, Transcription Factors, DNA Methylation, Dioxygenases

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