Research ArticleGENETICS

TET2 coactivates gene expression through demethylation of enhancers

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Science Advances  07 Nov 2018:
Vol. 4, no. 11, eaau6986
DOI: 10.1126/sciadv.aau6986
  • Fig. 1 TET2 is a coactivator of ERα.

    (A) TET2 and ERα protein levels were determined by Western blotting in TET2-WT and TET2-KO CAL51 cells (ERα) and MCF7 cells (ERα+). HSP90 was used as an internal control (n = 3). (B) Cells (2 × 104) of each cell line were seeded in six-well plates, and the cell growth ability of TET2-WT and TET2-KO cells was determined by cell counting at the indicated days. Data are means ± SD; n = 3 independent experiments. **P < 0.01, two-tailed unpaired Student’s t test. (C) Representative images of the cell morphology of MCF7-TET2-WT and MCF7-TET2-KO clones (n = 3). (D) MCF7-TET2-WT and MCF7-TET2-KO cells were maintained in phenol red–free medium containing 5% charcoal-stripped fetal bovine serum (FBS) for 4 days. Subsequently, 5 × 104 cells were seeded in six-well plates in the presence of dimethyl sulfoxide (DMSO) or E2 at 10 nM, and cell growth was determined by cell counting at the indicated days. Data are means ± SD; n = 3 independent experiments. **P < 0.01, two-tailed unpaired Student’s t test. (E) Schematic of the RNA-seq experimental workflow using MCF7-TET2-WT or MCF7-TET2-KO cells treated with either DMSO or E2 (10 nM) for 4 hours (n = 2). (F and G) Venn diagrams (F) and heat maps (G) showing the overlap of genes induced by E2 between TET2-WT and TET2-KO cells. Log2FC, log2 fold change. (H) Representative RNA-seq tracks of genes differentially induced by E2 in TET2-WT and TET2-KO cells (n = 2).

  • Fig. 2 Enhancer TET2 mediates proper recruitment of ERα.

    (A) TET2-WT and TET2-KO MCF7 cells were maintained in phenol red–free medium containing 5% charcoal-stripped FBS for 4 days, followed by treatment with either DMSO or E2 at 100 nM for 45 min. Heat maps generated from ChIP-seq data showing the occupancy of ERα in DMSO- and E2-treated cells. All rows are centered on ERα peaks and further divided into TSS and non-TSS regions. TSS and non-TSS regions were further divided into three clusters each by k-means (n = 2). (B) Left: Log2 fold change heat map shows the comparison of ERα occupancy between TET2-WT and TET2-KO cells treated with DMSO versus E2. Right: Log2 (fold change) of nearby gene expression in Tet2-WT or Tet2-KO cells treated with either DMSO or E2 (n = 2). (C) Heat maps generated from ChIP-seq data showing the occupancy of TET2, which is centered on the ERα peaks, and rows ordered as in (A) (n = 2). (D) Representative genome browser tracks of TET2 and ERα occupancy at enhancers. (E) Venn diagram showing common peaks between total TET2 peaks and ERα cluster 1 peaks. (F) Box plot quantifying changes on H3K4me3, H3K4me1, H3K27me3, and H3K27Ac occupancy at ERα alone and ERα/TET2 co-occupied cluster 1 peaks. (G to J) Well-observed CpG methylation around ERα-binding sites overlapping TSS sites (G) and non-TSS regions (H to J) separated by clusters identified in (A). Average CpG methylation values ± SEM for two biological replicates per cell type are plotted for the center of ERα-binding sites ± 2500 base pairs (bp). NTD, N-terminal domain.

  • Fig. 3 Positive feedback between TET2 and ERα at enhancers.

    (A) Parental MCF7 cells were maintained in phenol red–free medium containing 5% charcoal-stripped FBS for 4 days, followed by treatment with either DMSO or E2 at 10 nM for 4 hours. Heat maps generated from RNA-seq data showing the expression changes of selected epigenetic factors including histone and DNA modifiers (n = 2). (B) Real-time PCR was performed to determine the expression of TET1 to TET3 after E2 treatment. Data are means ± SD; n = 3 independent experiments. **P < 0.01, two-tailed unpaired Student’s t test. (C) Parental MCF7 cells were maintained in phenol red–free medium containing 5% charcoal-stripped FBS for 4 days, followed by E2 treatment for the indicated times. TET2 protein levels were determined by Western blotting. HSP90 was used as an internal control (n = 3). (D) Parental MCF7 cells were maintained in phenol red–free medium containing 5% charcoal-stripped FBS for 4 days, followed by treatment with either DMSO or E2 (100 nM) for 45 min. Representative genome browser tracks showing the occupancy of ERα, H3K4me3, H3K27ac, and H3K4me1 levels at TET2 (n = 2). MCF7 cells were treated with tamoxifen (1 μM) for 72 hours. (E) Protein levels of TET2 were determined by Western blotting. HSP90 was used as an internal control (n = 3). (F) Representative RNA-seq tracks showing TET2 expression changes in response to tamoxifen treatment (n = 2). (G and H) Representative ChIP-seq tracks (G) and average plot (H) showing loss of TET2 chromatin occupancy in response to tamoxifen treatment.

  • Fig. 4 Loss of MLL3, but not MLL4, COMPASS disrupts the TET2-ERα axis.

    (A) Cartoon of MLL3 and MLL4 COMPASS regulation of gene expression from enhancers. (B) Level of expression of MLL3, MLL4, and TET2, as assessed by real-time PCR in MCF7 cells treated with shNONT, shMLL3, and shMLL4. Data are means ± SD; n = 3 independent experiments. **P < 0.01, two-tailed unpaired Student’s t test. (C) Protein levels of MLL3, MLL4, and TET2 as determined by Western blotting in MCF7 cells treated with shNONT, shMLL3, and shMLL4 (n = 3). (D) Heat maps of MLL3, H3K4me1, and H3K27ac log2 fold changes in response to E2 treatment. Rows are centered on the non-TSS ERα peaks and ordered as in Fig. 2A (n = 2). (E) Representative genome browser tracks showing the occupancy of MLL3 at TET2 enhancers (n = 2). (F) Representative tracks showing the recruitment of MLL3 and increased occupancy of H3K4me1 at TET2 enhancers induced by E2 treatment (n = 2). (G) Representative RNA-seq tracks showing the expression of TET2 induced by E2 in shNONT and shMLL3 cells (n = 2).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/11/eaau6986/DC1

    Fig. S1. TET2 is a coactivator of ERα.

    Fig. S2. Enhancer-bound TET2 mediates proper recruitment of ERα.

    Fig. S3. MLL3 COMPASS is responsible for proper estrogen induction of gene expression.

    Fig. S4. Cell type–specific enhancer specificity of MLL3 COMPASS.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. TET2 is a coactivator of ERα.
    • Fig. S2. Enhancer-bound TET2 mediates proper recruitment of ERα.
    • Fig. S3. MLL3 COMPASS is responsible for proper estrogen induction of gene expression.
    • Fig. S4. Cell type–specific enhancer specificity of MLL3 COMPASS.

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