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YAP induces an oncogenic transcriptional program through TET1-mediated epigenetic remodeling in liver growth and tumorigenesis

Abstract

Epigenetic remodeling is essential for oncogene-induced cellular transformation and malignancy. In contrast to histone post-translational modifications, how DNA methylation is remodeled by oncogenic signaling remains poorly understood. The oncoprotein YAP, a coactivator of the TEAD transcription factors mediating Hippo signaling, is widely activated in human cancers. Here, we identify the 5-methylcytosine dioxygenase TET1 as a direct YAP target and a master regulator that coordinates the genome-wide epigenetic and transcriptional reprogramming of YAP target genes in the liver. YAP activation induces the expression of TET1, which physically interacts with TEAD to cause regional DNA demethylation, histone H3K27 acetylation and chromatin opening in YAP target genes to facilitate transcriptional activation. Loss of TET1 not only reverses YAP-induced epigenetic and transcriptional changes but also suppresses YAP-induced hepatomegaly and tumorigenesis. These findings exemplify how oncogenic signaling regulates the site specificity of DNA demethylation to promote tumorigenesis and implicate TET1 as a potential target for modulating YAP signaling in physiology and disease.

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Fig. 1: Tet1 is a direct target of the YAP–TEAD complex.
Fig. 2: YAP activation causes regional DNA demethylation in enhancers.
Fig. 3: TET1 interacts with transcription factor TEAD4 to cause regional histone acetylation and chromatin opening.
Fig. 4: Loss of Tet1 suppresses YAP-induced hepatomegaly and transcriptional activation.
Fig. 5: Tet1 deletion suppresses YAP-induced hepatomegaly and tumorigenesis in diverse genetic models of YAP activation.
Fig. 6: YAP activation drives TEAD4 expression through TET1-dependent epigenetic remodeling.
Fig. 7: Drug-induced DNA demethylation partially compensates for TET1 function in YAPTg Tet1 -/- livers.

Data availability

RNA-seq, MBD–seq and ChIP–seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE178227. Source data are provided with this paper. All other data and reagents supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank the McDermott Sequencing Core, the Next Generation Sequencing Core, the Bioinformatics Lab, the Animal Resource Center and the Transgenic Core Facility at University of Texas Southwestern Medical Center for assistance with this work. This work was supported in part by grants from the National Institutes of Health (EY015708 to D.P. and R35GM136316 to E.H.C.) and Department of Defense (PR190360 to D.P.). D.P. is an investigator of the Howard Hughes Medical Institute.

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B.W. and D.P. conceived the study. B.W. performed experiments. B.W., S.M. and E.H.C. contributed to the bioinformatic data analysis. B.W., Y.Z. and D.P. wrote the paper.

Corresponding author

Correspondence to Duojia Pan.

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Nature Genetics thanks Yi Zhang, Georg Halder 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 YAP activation induces TET1 expression.

a, RNA-seq analysis showing the expression of canonical YAP target genes, YAP upstream tumor suppressor genes and DNA methylation-related genes in induced YAPTg compared to control livers. Mice were treated with 50 mg/l Dox for 10 days starting at 1 month of age. b, Representative immunostaining of livers from induced YAPTg and Mst1/2 mutant mice at 1 month of age (n = 4), showing the induction of the hepatoblast marker AFP, but not the bile duct marker CK19, in YAP-activated hepatocytes. Also note the normal expression of the hepatocyte markers ALB and HNF4A in the proliferating hepatocytes (PH3). White scale bar, 100 μm. c, Heatmap of representative gene expression, derived from the published microarray data from YAPS127A transgenic livers after 7 days Dox induction. Note the induction of Tet1 and hepatoblast marker (Afp), but not bile duct markers (Krt7 and Krt19), in YAPS127A transgenic livers. d-e, RT-qPCR of Tet1 mRNA level in Yap mutant livers at neonatal P0.5 (n = 6) (d) and active mutant TAZ4SA transgenic (TAZ4SATg) livers (CTL n = 3; TAZ4SATg n = 4) which were treated with 50 mg/l Dox for 3 days starting at 1 month of age (e). Tet1 mRNA level was normalized to control livers. f, Western blot of TET1 protein in Mst1/2 mutant livers. Arrowhead marks the specific TET1 protein band. Images are representative of three independent experiments. g, Genomic tracks displaying MBD-seq, TET1 ChIP-seq, TEAD4 ChIP-seq and H3K27ac ChIP-seq reads at TET1 promoter. h, Genomic tracks displaying TEAD4 ChIP-seq reads at TET1 promoter in human embryonic stem cells (H1-hESC, green) and in mouse trophoblast stem cells (mTS, red). i, ChIP-qPCR at Tet1 promoter in Mst1/2 mutant livers (n = 3). Values represent mean ± s.e.m. (d, e, i). P values are calculated with unpaired two-tailed Student’s t-test (d, e, i).

Source data

Extended Data Fig. 2 YAP and TET1 are dispensable for TCP-induced hepatomegaly.

a, Representative gross image of livers (top panel) and quantification of liver-to-body weight ratio (bottom panel) from vehicle-treated (n = 8) and TCP-treated (n = 7) mice. 1-month-old mice were injected intraperitoneally with a single dose of 3 mg/kg TCP. Mice were sacrificed 7 days post injection. b, c, RT-qPCR showing mRNA levels of the YAP downstream target genes (b) or upstream suppressor genes (c) in TCP-treated livers. mRNA levels were normalized to vehicle-treated control livers (n = 4). d, Representative gross image of livers (top panel) and quantification of liver-to-body weight ratio (bottom panel) from 1-month-old Yap mutant mice (CTL n = 12; Yap/ n = 13) subjected to TCP-induced hepatomegaly. e, MBD-qPCR of methylation levels at the indicated rDMRs in TCP-treated mouse livers. Methylation levels were normalized to vehicle-treated control livers (n = 3). f, Representative gross image of livers (top panel) and quantification of liver-to-body weight ratio (bottom panel) from 1-month-old Tet1 mutant mice (CTL n = 10; Tet1/ n = 15) subjected to TCP-induced hepatomegaly. Values represent mean ± s.e.m. (a, b, c, d, e, f). P values are calculated with unpaired two-tailed Student’s t-test (a, b, c, d, e, f).

Source data

Extended Data Fig. 3 Elevated YAP/TAZ and TET1 abundance in foregut cancers shows a significant and positive correlation.

a, Elevated expression of YAP and TAZ in five human cancers (LIHC, CHOL, ESCA, PAAD and STAD) obtained from the TCGA database. The center lines show the medians, the box limits mark the 25th to the 75th percentiles and the whiskers indicate 1.5× the interquartile range. b, c, Spearman’s correlation coefficient analysis between the expression of YAP/TAZ and TET1 in LIHC, CHOL, ESCA, PAAD, STAD (b), LGG and GBM (c). d, Elevated expression of TEAD1 and TEAD4 in human foregut cancers (LIHC, CHOL, ESCA, PAAD and STAD) obtained from the TCGA database. e, Spearman’s correlation coefficient analysis between the expression of YAP/TAZ and TEAD1 (left panel) or TEAD4 (right panel) in human foregut cancers (LIHC, CHOL, ESCA, PAAD and STAD) obtained from the TCGA database. f, Kaplan-Meier analysis of overall survival in patients with human foregut cancers (LIHC, CHOL, ESCA, PAAD and STAD) according to high or low mRNA expression of YAP/TAZ, TET1, TEAD1 or TEAD4. P values are calculated with two-tailed Spearman’s correlation test (b, c, e) or two-sided log-rank test (f).

Extended Data Fig. 4 YAP activation causes regional DNA demethylation in canonical YAP target genes.

a, DNA dot blot of genomic 5mC and 5hmC levels in 1-month-old Mst1/2 mutant livers (top panels). The blot was then stained with methylene blue as loading control (bottom panels). Images are representative of two independent experiments. b, 5mC and 5hmC validation at the indicated rDMRs in Mst1/2 mutant livers. Methylation (top panel) and hydroxymethylation (bottom panel) levels were determined by MBD-qPCR and hMeDIP-qPCR, respectively (n = 4). Values represent mean ± s.e.m. P values are calculated with unpaired two-tailed Student’s t-test. c, Genomic tracks displaying MBD-seq and H3K4me1 ChIP-seq reads at the rDMR loci of the indicated YAP target genes. Grey columns represent YAP-induced rDMRs. The cloning-based locus-specific bisulfite sequencing sites are also marked. Note the robust enhancer mark H3K4me1 occupancy at the YAP-induced rDMRs. d, Cloning-based traditional bisulfite sequencing (left panels) and TAB-seq (right panels) of CpG sites at the rDMR loci of YAP target genes. All sequencing results include 10 independent clones.

Source data

Extended Data Fig. 5 YAP activation causes regional DNA demethylation in enhancers.

a, Pie charts showing the distribution of methylation peaks and YAP-induced rDMRs to mm10 mouse genomic features. b, Average peak profile of methylation peaks and YAP-induced rDMRs mapped to nearest TSS. Note the disassociation of YAP-induced rDMRs from TSS. c, The de novo motif analysis of YAP-induced rDMRs. Both RUNX and TEAD recognition motifs were identified in the top 10 enriched motifs. d, e, Enrichment profiles representing the TEAD motif centered at YAP-induced rDMRs (d) and TET1 peaks (e). f, Enrichment profiles representing the TET1, TEAD4 and H3K27ac ChIP-seq reads centered at YAP-induced rDMRs.

Extended Data Fig. 6 TEAD4 interacts with TET1 through its YAP binding domain (YBD).

a, Co-IP assay showing physical interactions between TET1 and RUNX1 or TEAD1/TEAD4. The indicated constructs were transfected into HEK293T cells, lysed and subjected to immunoprecipitation assays. Normal IgG immunoprecipitates serve as negative control. b, Co-IP assay showing no interactions between TET1 and YAP. Co-IP assay was conducted under same condition as in (a). c, Co-IP assay showing physical interactions between TET1 and RUNX1 or TEAD1/TEAD4 in the presence or absence of DNA/RNA nuclease. The indicated constructs were transfected into HEK293T cells, lysed with or without benzonase treatment before immunoprecipitation assays. Note the co-IP between TET1 and RUNX1 or TEAD1/TEAD4, regardless of nuclease treatment. d, Co-IP assay showing physical interactions between TET1 and TEAD1/TEAD4. Note that the TET1-TEAD1/TEAD4 interaction were abolished by deletion of the CXXC domain in TET1 protein. e, Co-IP assay showing physical interactions between TEAD4 and TET1. The schematic diagram indicates the TEAD4 mutants used: TEAD4ΔΤΕΑ is defective in DNA-binding and TEAD4-Y429H is defective in YAP-binding. Both TEAD4 mutants interacted with TET1. f, Co-IP assay showing physical interactions between TEAD4 and TET1. Note that TEAD4 interacts with TET1 through its YAP binding domain (YBD). g, Co-IP assay showing physical interactions between TEAD4 and TET1/2/3. Note the co-IP between TEAD4 and TET1, not TET2 or TET3. h, Co-IP assay showing physical interactions between endogenous TET1 and TEAD1 in YAPTg livers. i, Genome track showing Tet1 RNA-seq in YAPTg livers. j, TET1 exon usage profile in LIHC samples from the TCGA database. Exon usage profile was visualized with TSVdb web tool. Patients are represents in x-axis and exon usage of each exon are showed in y-axis. Images are representative of two (a, b, c, g) or three (d, e, f, h) independent experiments.

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Extended Data Fig. 7 TET1 interacts with TEAD4 transcription factor to cause regional histone acetylation and chromatin opening at the rDMRs of canonical YAP target genes.

a, Validation of TEAD4 and TET1 antibody specificity. Western blot of TEAD4 or TET1 protein in HEK293T cells with non-targeting control (siCon) or siRNA targeting TEAD4 (siTEAD4) or siRNA targeting TET1 (siTET1) treatment. Images are representative of two independent experiments. b, Enrichment profiles representing the TET1 ChIP-seq reads from control and YAPTg livers centered at TET1 peaks (left panel) and the TEAD4 ChIP-seq reads from control and YAPTg livers centered at TEAD4 peaks (right panel). c, Spearman’s correlation coefficient analysis using the signals of peaks between H3K27ac and TET1 or TEAD4 from the identified 8,022 TET1/TEAD4/H3K27ac overlapped peaks. d, Genomic tracks displaying MBD-seq and ChIP-seq reads at the rDMR loci of Cyr61 and Ctgf. Grey columns represent YAP-induced rDMR regions. e, ChIP-qPCR at rDMRs in YAPTg livers (n = 3). f, g, ChIP-qPCR (n = 3) (f) and FAIRE-qPCR (n = 4) (g) at rDMRs in Mst1/2 mutant livers. h, Luciferase reporter assay showing enhancer activity of unmethylated and methylated Myc and Cyr61 rDMRs of three independent experiments. Reporter construct containing respective rDMRs with or without in vitro methylation was co-transfected with YAP-expressing vector into HEK293T cells. Luciferase activity was normalized to unmethylated rDMR. i, Luciferase reporter assay showing enhancer activity of methylated Myc or Cyr61 rDMR with non-targeting control (siCon) or siRNA targeting TET1 (siTET1) of three independent experiments. Reporter construct containing methylated rDMR was co-transfected with YAP-expressing vector together with either non-targeting control or siRNA targeting TET1 into HEK293T cells. Luciferase activity was normalized to non-targeting control. Values represent mean ± s.e.m. (e, f, g, h, i). P values are calculated with unpaired two-tailed Student’s t-test (e, f, g, h, i) or two-tailed Spearman’s correlation test (c).

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Extended Data Fig. 8 Loss of Tet1 suppresses YAP-induced hepatomegaly and tumorigenesis.

a, Normal liver function in Tet1 mutant mice, as indicated by similar serum levels of ALB, AST and ALT compared to control mice (n = 5). b, c, Representative H&E and Ki-67 staining of liver sections (b) and quantification of Ki-67 positive cells (c) from the indicated mice (n = 4) treated with Dox for 10 days starting at 1 month of age. d, Quantification of Ki-67 positive cells in YAPTg livers treated with Tet1 shRNA lentivirus (n = 4). e, Principal components analysis (PCA) for transcriptome profiles from the indicated mice (n = 3). f, A volcano plot showing differential expression of 2,963 YAP-upregulated genes in YAPTg Tet1/ compared to YAPTg livers. Red dots represent remarkable (fold change ≥ 2) and significant (P < 0.00001) genes. g, i, Representative gross image and H&E staining of livers (g) and quantification of liver-to-body weight ratio (i) from CTL (n = 7), Tet1−/− (n = 9), YAPTg (n = 13) and YAPTg Tet1−/− (n = 18) mice treated with Dox starting at birth for ~2 months. h, Kaplan-Meier survival curve showing the survival of the indicated mice treated with Dox starting at birth, monitored for 120 days. j, Quantification of LIHC incidence under same condition as in (g). k, l, Representative gross image and H&E staining of livers (k) and quantification of liver-to-body weight ratio (l) from CTL (n = 6), Tet1−/− (n = 6), YAPTg (n = 22) and YAPTg Tet1−/− (n = 14) mice at ~14 months of age without Dox treatment. m, Quantification of LIHC incidence under same condition as in (k). Values represent mean ± s.e.m. (a, c, d, i, l). P values are calculated with unpaired two-tailed Student’s t-test (a) or one-way ANOVA with Tukey’s test (c, d, i, l).

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Extended Data Fig. 9 TET1-mediated DNA methylation is required for transcriptional induction of TEAD1/4.

a, Clustered heatmaps of MBD-seq data (top panel) and RNA-seq data (bottom panel) displaying sample-to-sample distance between the indicated genetic background. b, Methylation profile representing the MBD-seq signals from the indicated mutant livers centered at TET1 peaks. c, Absolute distance of methylation peaks, YAP-induced rDMRs and TET1-dependent rDMRs relative to nearest TSS. d, Genomic distribution of methylation peaks, YAP-induced rDMRs and TET1-dependent rDMRs. e, Average peak profile of methylation peaks, YAP-induced rDMRs and TET1-dependent rDMRs mapped to nearest TSS. f, Genomic tracks displaying MBD-seq, TET1 ChIP-seq, TEAD4 ChIP-seq, H3K4me1 ChIP-seq and H3K27ac ChIP-seq reads at the Tead1 rDMR locus. Grey column represents a YAP-induced rDMR region. g, Cloning-based traditional bisulfite sequencing (left panels) and TAB-seq (right panels) of 16 CpG sites at the Tead1 rDMR locus. All sequencing results include 10 independent clones. h, 5mC and 5hmC validation at the Tead1 and Tead4 rDMRs in the indicated mutant livers. Methylation (top panel) and hydroxymethylation (bottom panel) levels were determined by MBD-qPCR and hMeDIP-qPCR, respectively (n = 4). i, RT-qPCR of Tead1 and Tead4 in the indicated mutant livers. mRNA level was normalized to control livers (n = 6). j, RT-qPCR of Tead1 and Tead4 in the indicated 1-month-old mutant livers (CTL n = 5, 5, 6 and KO n = 7, 6, 6 from left to right). mRNA level was normalized to control livers. k, Western blot of TEAD1 and TEAD4 protein in Mst1/2 mutant livers. Images are representative of three independent experiments. l, Genomic tracks displaying TEAD4 ChIP-seq reads at TEAD1 and TEAD4 genes in human embryonic stem cells (H1-hESC).Values represent mean ± s.e.m. (h, i, j). P values are calculated with unpaired two-tailed Student’s t-test (j) or one-way ANOVA with Tukey’s test (h, i).

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Extended Data Fig. 10 YAP directly induces the transcription of Tead1 and Tead4 in a TET1-dependent manner.

a, b, ChIP-qPCR (n = 3) (a) and FAIRE-qPCR (n = 3) (b) at rDMRs in the indicated mutant livers (also see Fig. 3l and Extended Data Fig. 7e). c, d, ChIP-qPCR (n = 3) (c) and FAIRE-qPCR (n =4) (d) at Tead1 and Tead4 rDMRs in Mst1/2 mutant livers. e, A volcano plot for differential DNA methylation of 1,376 rDMRs sites (associated with 1,003 TET1-dependent YAP target genes) in DAC-treated compared to untreated YAPTg Tet1/ livers. Red dots represent remarkable (fold change ≥ 2) and significant (P < 0.1) genes. Note that 27.4% of rDMRs (377 out of 1,376) were DNA demethylation by DAC treatment. f, Heatmaps of RNA-seq (left panel) and MBD-seq (right panel) data displaying changes of 1,003 TET1-dependent YAP target genes between the indicated mice, ranked based on P values (between YAPTg Tet1/ and YAPTg Tet1/ /DAC) with the most significant on top. g, Schematic model showing a YAP-TET1-TEAD positive feedforward loop sustains YAP signaling. In control livers, the YAP-TEAD complex has limited access to the promoter and enhancer of YAP target genes due to closed chromatin. In YAP-activated livers, YAP-induced expression of TET1 protein causes regional DNA demethylation and local chromatin opening to facilitate YAP-TEAD binding. As a transcriptional coactivator, YAP then recruits histone-modifying enzymes such as NCOA6 and P300, as well as chromatin remodelers such as SWI/SNF complex, to further increase chromatin opening. This further facilitates the association of the YAP-TEAD complex to YAP target genes to activate their transcription. Induction of Tead1 and Tead4 by the YAP-TEAD complex constitutes a feedforward loop to amplify YAP signaling. Values represent mean ± s.e.m. (a, b, c, d). P values are calculated with unpaired two-tailed Student’s t-test (c, d) or one-way ANOVA with Tukey’s test (a, b).

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Wu, BK., Mei, SC., Chen, E.H. et al. YAP induces an oncogenic transcriptional program through TET1-mediated epigenetic remodeling in liver growth and tumorigenesis. Nat Genet 54, 1202–1213 (2022). https://doi.org/10.1038/s41588-022-01119-7

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