Abstract
The ten-eleven translocation (TET) family of dioxygenases maintain stable local DNA demethylation during cell division and lineage specification. As the major catalytic product of TET enzymes, 5-hydroxymethylcytosine is selectively enriched at specific genomic regions, such as enhancers, in a tissue-dependent manner. However, the mechanisms underlying this selectivity remain unresolved. Here we unveil a low-complexity insert domain within TET2 that facilitates its biomolecular condensation with epigenetic modulators, such as UTX and MLL4. This co-condensation fosters a permissive chromatin environment for precise DNA demethylation. Disrupting low-complexity insert-mediated condensation alters the genomic binding of TET2 to cause promiscuous DNA demethylation and genome reorganization. These changes influence the expression of key genes implicated in leukaemogenesis to curtail leukaemia cell proliferation. Collectively, this study establishes the pivotal role of TET2 condensation in orchestrating precise DNA demethylation and gene transcription to support tumour cell growth.
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Data availability
All of the sequencing data generated in this study, including CMS-IP-seq, WGBS and Micro-C data that support its findings, have been deposited in the Gene Expression Omnibus under the accession code GSE183934. The human AML data were derived from the TCGA research network: http://cancergenome.nih.gov/. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.
Code availability
The script code used to produce Fig. 7i and Extended Data Fig. 9 is available via GitHub at https://github.com/yangjunhuicq/prognostic_signature.
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Acknowledgements
This work was supported by The Welch Foundation (BE-1913-20220331 to Y.Z.), Leukemia & Lymphoma Society (to Y.Z.) and American Cancer Society (RSG-18-043-01-LIB to Y.H.).
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Y.H. and Y.Z. directed and oversaw the project. L.G. performed most of the droplet forming analysis, animal-related work and molecular characterization. Y.-T.L., X.H., G.P., S.K. and X.C. supported the droplet forming and biochemical analyses and functional characterization. L.G. and T.H. supported the sequencing library construction and analysis. J. Li, L.R., R.Z., J.Y. and L.H. performed the bioinformatics analysis on high-throughput sequencing data. Y.D. performed the cell sorting. R.W., J. Liang and Y.Y. supported animal colony maintenance, performed genotyping and histology analysis. W.L., B.D.S. and Y.Z. provided essential resources and key intellectual inputs to support this study. Y.H., Y.Z. and L.G. wrote the manuscript. All authors participated in the initial discussion, data interpretation and manuscript editing.
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Extended data
Extended Data Fig. 1 TET2 forms condensates via its LCI domain.
(a) Schematic for generating a mouse model with GFP knockin (KI) at the C-terminus of endogenous Tet2. 3XFLAG-tag was introduced at the C-terminus of Tet2-ΔLCI. (b-c) Sanger sequencing (B) and genotyping (C) results to confirm the knockin of GFP at the C-terminus of Tet2. n = 3 independent biological replicates. (d) Representative images of mCh-CRY2 fused to six TET2 IDRs predicted by PONDR as shown in Fig. 1e. HEK293T cells were stimulated by blue light at the indicated time points. Scale bar, 10 µm. (e) Quantification of droplet numbers per cell (n = 20 cells from 3 biological replicates, one-way ANOVA with Tukey’s post-hoc test). Data were shown as mean ± S.D. (f) Reversible droplet formation for both human (top) and mouse (bottom) TET2 LCI domain when expressed in HEK293T cells upon repeated blue light stimulation. Scale bar, 5 µm. (g) Representative images (top) and FRAP recovery curves (bottom) of the TET2 LCI domain using purified recombinant protein fused with mCherry. n = 3 droplets. Scale bar, 5 µm. (h-i) Representative images (top) of droplet formation and quantification (bottom) of the puncta size of purified recombinant TET2 LCI fused with mCherry at the indicated protein (h) and NaCl (i) concentrations (n = 40 droplets). The data represent means ± SD (one-way ANOVA with Tukey’s post-hoc test). Scale bar, 10 µm. Related to Fig. 1.
Extended Data Fig. 2 Molecular grammar of TET2 LCI.
(a) Schematic illustrating the amino acid composition and positions in the TET2 LCI domain. (b) Representative images (left) and quantification (right) of droplet numbers per cell in HEK293T cells expressing the indicated TET2 variants fused to mCh-CRY2. (n = 20 cells from 3 independent biological replicates, one-way ANOVA with Tukey’s post-hoc test). Data were shown as mean ± SD. Scale bar, 10 µm. (c) Representative images (left) and quantification (right) of droplet numbers per cell in HEK293T cells expressing the indicated GFP fused TET2 variants. (n = 6 cells, one-way ANOVA with Tukey’s post-hoc test). Data were shown as mean ± SD. Scale bar, 10 µm. Related to Fig. 2.
Extended Data Fig. 3 TET2 co-condensates with enhancer regulators via LCI.
(a) Schematic depicting the experimental setup and analytical procedures of proximity labelling with TET2CD-TurboID and TET2CDΔLCI-TurboID. (b) Immunoblotting analysis to confirm the proximity labelling with TET2CD and TET2CDΔLCI fused to TurboID in HEK293T cells. n = 3 independent biological replicates. (c) Heatmap of ChIP-seq signals showing genomic co-localization of TET2, MLL4 and UTX at H3K27ac-enriched genomic regions in mESCs. TET2 ChIP-seq data were obtained from the GSE115972 dataset (collected from mESCs with 3XFlag knockin at the endogenous loci of Tet2). MLL4 and UTX ChIPseq data were obtained from GSE97703. (d-e) Representative images (d) and quantitative recruitment coefficient analysis (e) on HEK293T cells co-transfected with mCherry-CRY2 fused to the indicated TET2 LCI variants and MLL4-GFP before and after blue light stimulation (n = 6, one-way ANOVA with Tukey’s post-hoc test). Data were shown as mean ± SD. Scale bar, 10 µm. Related to Fig. 3.
Extended Data Fig. 4 LCI-mediated condensation determines precise DNA demethylation of TET2.
(a) Dotblot analysis (left) and statistical quantification (right) of 5hmC levels in HEK293T cells transduced with doxycycline (Dox) inducible TET2CD variants. Western blotting with antibody against FLAG was applied to verify protein expression levels of TET2CD variants. Methylene blue staining and GAPDH blotting were used as the total DNA or protein loading control, respectively. Data were shown as mean ± SD (n = 3 biological replicates). *** p < 0.001 (two-sided unpaired Student’s t-test). (b) Venn diagram showing overlapping status of Dam signals identified from mESCs expressing TET2CD-Dam and TET2CDΔLCI-Dam from two independent biological replicates. (c) Summary of Pearson correlation coefficients among TET2CD-Dam signals, H3K27ac ChIP-seq data (ENCFF163HEV), FLAG ChIP-seq data of FLAG-Tet2 (GSM3196082), and H3K9me3 signals (GSM3517479) in mESCs. (d) Scatter plot of the Dam signals in 5-kb bins identified from mESCs expressing TET2CD-Dam and TET2CDΔLCI-Dam. (e) Immunoblotting of FLAG-tagged TET2CD or TET2CDΔLCI transduced in Tet1/2/3 triple knockout (Tet-TKO) mESCs. GAPDH was used as the loading control. n = 3 independent biological replicates. (f) Scatter plot of the average DNA methylation levels for CpGs covered by at least 10 reads within mESCs expressing TET2CD and TET2CDΔLCI. (g) The average DNA methylation level of mESCs expressing TET2CD or TET2CDΔLCI. (h) Dotblot analysis (left) and quantification (right) of 5hmC levels using an in vitro on beads enzymatic assay. FLAG tagged TET2CD or TET2CDΔLCI were immobilized on anti-FLAG beads and incubated with a 5mC-modified DNA oligonucleotide in vitro. Western blotting (bottom) illustrated the amounts of proteins used for the on beads reaction using an antibody against FLAG. Data were shown as mean ± SD (n = 3 biological replicates; two-sided unpaired Student’s t-test). (i) Venn diagram showing overlapping hyperDMRs identified from Tet-TKO vs TET2CD groups (blue) and Tet-TKO vs TET2CDΔLCI groups (red). Numbers listed in the circle represent the DMR numbers in the corresponding category. Numbers listed below are the percentage of peaks in the corresponding category. Related to Fig. 4.
Extended Data Fig. 5 Disrupting TET2 condensation suppresses leukemia growth.
(a) Immunoblot analysis on the expression of the indicated TET2 chimeras before and after doxycycline treatment (the same concentration as in Fig. 5a) for 24 hours in MOLM13 and THP-1 cells. n = 3 independent biological replicates. (b) Representative images (left) and statistical analysis (right) of colony formation of MOLM13 cells transduced with lentivirus encoding TET2CD or TET2CDΔLCI with or without doxycycline (Dox) treatment (1 µg/ml and 0.2 µg/ml for TET2CD and TET2CDΔLCI respectively). (n = 3 independent experiments, two-sided unpaired Student’s t-test). Data were shown as mean ± SD. Scale bar, 200 µm. (c) Growth curves of MOLM13 and THP-1 cell lines transduced with indicated TET2 chimeras without doxycycline (Dox) induction for 5 days. (d) Growth curves of MOLM13 and HEK293T cells transduced with WT TET2 and TET2-∆LCI. Data were shown as mean ± S.D. (n = 8 independent replicates; two-sided unpaired Student’s t-test). (e) Immunoblot analysis of the apoptosis-related marker, cleaved caspase 3 (C/Caspase3), in MOLM13 and HEK293T cells expressing TET2CDΔLCI from day 0 to day 5. GAPDH was used as the loading control. n = 3 independent biological replicates. (f) Representative images (left) and statistical analysis (right) of dot-blot assay results using an antibody against 5mC in MOLM13 and HEK293T transduced with TET2-∆LCI at the indicated days. Methylene blue (MEB) is used as loading control. Data were shown as mean ± SD. (n = 4 independent replicates; two-sided unpaired Student’s t-test). (g-h) Dotblot analysis (g) and ELISA assay (h) showing 5hmC levels in HEK293T cells transduced with doxycycline (Dox) inducible TET2 CD variants as indicated. Data were shown as mean ± S.D., n = 3 independent replicates. (one-way ANOVA with Tukey’s post-hoc test). (i) Dotblot analysis showing 5hmC levels in MOLM13 cells transduced with catalytically dead versions of the indicated TET2 CD variants. Cells transduced with WT TET2CD were used as positive control. (j) Growth curves of MOLM13 cell lines transduced with the indicated catalytically dead TET2 chimeras after doxycycline (Dox) induction for 5 days. (k) Western blot analysis of chromatin associated (left) and whole cell lysate (WCL) (right) TET2CD variants as indicated. Histone 3 (H3) was used as the loading control. n = 3 independent biological replicates. Related to Fig. 5.
Extended Data Fig. 6 Chemically disrupting TET2 condensation inhibits leukemia growth.
(a) Immunoblot analysis of total lysates from MOLM13 cells transduced with the corresponding constructs with or without rapamycin (Rapa) treatment (2 µM). n = 3 independent biological replicates. (b) Dot-blot analysis of the total 5hmC levels in MOLM13 and THP-1 cells transduced with the indicated constructs with or without rapamycin (Rapa) treatment (2 µM) for 24 hrs. (c) Proliferation analysis of THP-1 cells transduced with the indicated constructs with or without rapamycin (Rapa) treatment (2 µM). Data were shown as mean ± SD. (n = 8 independent biological replicates; one-way ANOVA with Tukey’s post-hoc test). (d) Experimental procedures for monitoring tumor growth in vivo. (e) Representative images of spleens (left) and statistical analysis of spleen weights (right) collected from the recipient mice under the indicated conditions. Data are shown as mean ± SD. (n = 4 mice / group; one-way ANOVA with Tukey’s post-hoc test). Related to Fig. 5.
Extended Data Fig. 7 Generation of a TET2 LCI domain deletion mouse model.
(a) Schematic showing the targeting strategy with CRISPR/Cas9 to generate Tet2 LCI domain (T1389-Y1760) deletion (Tet2-ΔLCI) mice. A FLAG tag was inserted into the C terminus of Tet2. (b) Genotyping primers and use of PCR to identify WT (+/+), Tet2-ΔLCI heterozygotes (+/Del) and homozygotes (Del/Del) mice. n = 3 independent biological replicates. (c) Real-time qPCR analysis of Tet2 mRNA expression with primers targeting LCI and non-LCI regions. Data were shown as mean ± SD; n = 3 independent replicates (two-sided unpaired Student’s t-test). (d) Lineage analysis on peripheral blood (PB) and bone marrow (BM) samples from WT and Tet2-ΔLCI mice at the indicated ages (Top: 6–8 weeks old; bottom: 10–12 months old). Data are shown as mean ± SD (n = 5–7 biologically independent mice; Two-tailed Student’s t-test). (e) Hematopoietic stem and progenitor cell analysis in bone marrow samples isolated from recipient mice transplanted with WT (CD45.1+) and the indicated HSPC groups (at a 1:1 ratio; black, WT; blue, Tet2-KO; red, Tet2-∆LCI). The percentage of CD45.2+ cells in each population was analyzed. MPP: Multipotent progenitors; LT-HSC: long-term hematopoietic stem cells; ST-HSC: short-term hematopoietic stem cells. Data are shown as mean ± SD. (n = 9-10 biologically independent mice; Two-tailed Student’s t-test). Related to Fig. 6.
Extended Data Fig. 8 TET2 loss-of-condensation causes chromatin topological changes in leukemia cells.
(a) Heatmap representation of 5hmC enrichment around ± 2 kb of transcribed regions or H3K27ac (GSM2136938) enriched peak regions in MOLM13 cells (mock) and MOLM13 cells expressing TET2CD or TET2CDΔLCI for 24 or 48 h. n = 2 biological replicates. (b) Violin plot of overall DNA methylation levels in MOLM13 cells (MOCK) (grey) and MOLM13 cells expressing TET2CD (blue) or TET2CDΔLCI (red) for 24 or 48 hours. DNA methylation were determined by WGBS analysis. n = 2 biological replicates, two-sided Kolmogorov-Smirnov tests. (c) The average DNA methylation levels within the coding regions in MOLM13 cells expressing TET2CD (blue and green) or TET2CDΔLCI (red and orange) for 24 or 48 h. (d) The representative genome browser tracks of Micro-C data in MOLM-13 cells expressing TET2CD or TET2CDΔLCI. (e) Scatter plot of Eigenvector (PCA) in 50-kb in MOLM13 cells expressing TET2CD and TET2CDΔLCI for 48 h. (f) Scatter plot of Eigenvector (PCA) and DNA methylation difference at 50-kb between MOLM13 cells expressing TET2CD and TET2CDΔLCI for 48 h. (g) (Left) Representative genome browser views of Micro-C data using Juciebox60 at chromosome 7. (Right) The zoom-in snapshots of genomic regions in the highlighted boxes. The top tracks represent the difference of Eigenvector (PCA) or DNA methylation levels in MOLM13 cells expressing TET2CD and TET2CDΔLCI for 48 h. (h) Volcano plot of differentially expressed genes (DEGs) identified from MOLM13 cells expressing TET2CD and TET2CDΔLCI for 48 h. The DEGs were defined by |Log2fold change | ≥ 1, p-value < 0.05. Related to Fig. 7.
Extended Data Fig. 9 Chromatin topological changes induced by TET2 LCI loss of condensation are linked to tumor suppressive transcriptional program in AML.
(a) Flowchart showing the bioinformatic methods applied to identify 15-gene signatures that were associated with TET2 loss-of-condensation and AML patient outcomes (see details in the Methods). (b) Differential expression of the 15 signature genes between high-risk and low-risk AML cases (see details in the Methods). Two-sided Kolmogorov-Smirnov tests were applied for statistical analysis. Related to Fig. 7.
Supplementary information
Supplementary Table 1
mESC DamID peaks.
Supplementary Table 2
mESC DMRs.
Supplementary Table 3
MOLM-13 5hmC peaks.
Supplementary Table 4
MOLM-13 DMRs.
Supplementary Table 5
MOLM-13 DEGs.
Supplementary Table 6
Antibody list.
Source data
Source Data Figs. 4–6 and Extended Data Figs. 1, 3 and 4–7
Unprocessed gels.
Source Data Figs. 1–6 and Extended Data Figs. 1–7
Statistical source data.
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Guo, L., Hong, T., Lee, YT. et al. Perturbing TET2 condensation promotes aberrant genome-wide DNA methylation and curtails leukaemia cell growth. Nat Cell Biol (2024). https://doi.org/10.1038/s41556-024-01496-7
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DOI: https://doi.org/10.1038/s41556-024-01496-7
