STAT3-driven transcription depends upon the dimethylation of K49 by EZH2 - PubMed (original) (raw)

STAT3-driven transcription depends upon the dimethylation of K49 by EZH2

Maupali Dasgupta et al. Proc Natl Acad Sci U S A. 2015.

Abstract

Several transcription factors, including p53, NF-κB, and STAT3, are modified by the same enzymes that also modify histones, with important functional consequences. We have identified a previously unrecognized dimethylation of K49 of STAT3 that is crucial for the expression of many IL-6-dependent genes, catalyzed by the histone-modifying enzyme enhancer of zeste homolog 2 (EZH2). Loss of EZH2 is protumorigenic in leukemias, but its overexpression is protumorigenic in solid cancers. Connecting EZH2 to a functionally important methylation of STAT3, which is constitutively activated in many tumors, may help reveal the basis of the opposing roles of EZH2 in liquid and solid tumors and also may identify novel therapeutic opportunities.

Keywords: gene expression; histone methyltransferase; posttranslational modification.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

STAT3 is dimethylated on K49. (A) STAT3-null A4 cells were infected with a retroviral construct expressing WT-STAT3, and stable pools of cells were selected with G418. The level of STAT3 expression, analyzed by the Western method, corresponds to that in the DLD1 parental cells. (B) A4-WT-STAT3 cells were treated with IL-6 (50 ng/mL) and soluble IL-6 receptor (sIL-6R) (62.5 ng/mL) for 1 h, and total cell lysates were analyzed for Y705 phosphorylation. (C) MS analysis of tryptic peptides of STAT3 indicates that K49 is dimethylated. Flag-tagged WT-STAT3 was immunoprecipitated from untreated or IL-6–treated A4-WT-STAT3 cells. The tryptic K49 peptide QFLAPWIESQDWAYAASK was included in the targeted analysis. A peptide consistent with a 28-Da mass modification of K49 was identified. (D) The relative abundance of the modified form of the K49 peptide was greater in the IL-6–treated samples. The levels of the modified forms of the peptides containing Y705, K49, and S727 were analyzed.

Fig. 2.

Fig. 2.

K49 mutation of STAT3 impairs its transcriptional activity in response to IL-6. (A) A4 cells were infected with retroviral constructs expressing WT-STAT3 or the STAT3 mutants K49R, K140R, K685R, or S727A, at levels comparable to the level of WT-STAT3 in DLD1 parental cells. Stable pools of cells were selected with G418. Western analyses of total cell lysates are shown. (B) A4-vector control (A4-V), A4-WT, A4-K49R, A4-K140R, A4-K685R, and A4-S727A cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 4 h, and total cell lysates were analyzed for Y705 phosphorylation. (C) A microarray analysis was performed to identify genes induced in WT-STAT3 and the mutant STAT3 cells upon IL-6 treatment. The heat map shows the induced expression of 59 genes (the full list is given in Table S1). We include only signals that changed by twofold or more, with differential P ≤ 0.05, in IL-6–treated WT-STAT3 cells compared with IL-6–treated vector control cells and untreated WT-STAT3 cells and with average signals >30 in WT cells treated with IL-6. (D) WT-STAT3 cells and A4 cells carrying each of the mutant proteins were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 4 h, and the relative expression of the BCL3, SERPINA1, GADD45G, and SOCS3 genes, compared with the untreated cells, was determined by quantitative PCR (qRCR). Values are shown as means, with SDs, from triplicate experiments.

Fig. 3.

Fig. 3.

STAT3 Y705 phosphorylation is required for K49 dimethylation of STAT3. (A) WT-STAT3 cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 1 or 4 h, respectively, and total cell lysates were analyzed for K49 dimethylation by using an antibody that recognizes this specific modification. (B) A4 cells expressing WT or K49R STAT3 were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 1 h, and total cell lysates were analyzed for K49 dimethylation. (C) A4 cells expressing WT or Y705F STAT3 were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 1 h, and total cell lysates were analyzed for K49 dimethylation and Y705 phosphorylation. (D) hTERT-HME1 cells were treated with OSM (100 ng/mL) or IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 1 h, and total cell lysates were analyzed for K49 dimethylation, Y705 phosphorylation, and total STAT3.

Fig. 4.

Fig. 4.

STAT3 is dimethylated on K49 by EZH2. (A) The sequence of STAT3 surrounding K49 is aligned against the EZH2 target sequence surrounding H3 K27 and the SET9 target sequence surrounding H3 K4. Knockdown of EZH2 in A4 cells expressing WT-STAT3 was confirmed by Western analysis. To determine whether EZH2 knockdown impairs STAT3 Y705 phosphorylation, cells with or without EZH2 knockdown were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 1 h and were analyzed for Y705 phosphorylation. (B) EZH2-knockdown and control WT-STAT3 cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 4 h and were analyzed for K49 dimethylation, Y705 phosphorylation, and total STAT3. (C) WT-STAT3 and K49R-STAT3 cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL), and Y705 phosphorylation of STAT3 was determined. (D) WT-STAT3 cells, with or without EZH2 knockdown, were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 4 h, and the relative expression of the SERPINA1, SOCS3, and GADD45G genes was determined by qPCR. Values are shown as the means, with SDs, from triplicate experiments. (E) EZH2-knockdown and control hTERT-HME1 cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 4 h and were analyzed for EZH2 knockdown, K49 dimethylation, Y705 phosphorylation, and total STAT3 expression. (F) hTERT-HME1 cells, with or without EZH2 knockdown, were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 4 h, and the relative expression of the SOCS3 and BCL3 genes was determined by qPCR. Values are shown as the means, with SDs, from triplicate experiments.

Fig. 5.

Fig. 5.

EZH2 knockdown and K49 mutation of STAT3 in A4 cells have similar effects on IL-6–dependent, STAT3-mediated gene expression. (A) A4 cells expressing WT-STAT3 or the mutants K49R, K49A, or K49Q were analyzed for STAT3 expression. WT-STAT3 cells with or without EZH2 knockdown also were included in the analysis. (B) A4-vector control (V), WT, K49R, K49A, K49Q, WT-shControl, or WT-shEZH2 cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 4 h, and total cell lysates were analyzed for Y705 phosphorylation. (C) Genes induced by IL-6 in WT-STAT3 cells, in A4 cells carrying the K49R, A, or Q mutants, and in WT-shEZH2 cells were compared by microarray analysis. The heat map shows the induced expression of 97 genes (the full list in given in Table S3). We include only signals that changed by twofold or more, with differential P ≤ 0.05, in IL-6–treated WT-STAT3 cells compared with IL-6–treated vector control cells and untreated WT-STAT3 cells and with average signals >30 in IL-6–treated WT-STAT3 cells.

Fig. 6.

Fig. 6.

EZH2 binding to STAT3 is enhanced by IL-6 treatment in a time-dependent manner. (A) WT-STAT3 cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 1 h, followed by immunoprecipitation of STAT3 from whole-cell extracts with anti-FLAG M2 beads. Western analyses were performed to detect EZH2. (B) WT-STAT3 cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for the indicated times, followed by immunoprecipitation of STAT3 from whole-cell extracts with anti-FLAG M2 beads. Western analyses were performed to detect the time dependence of EZH2 binding. (C) A4 cells expressing WT-STAT3 or one of the three K49 mutants were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 1 h, followed by immunoprecipitation of STAT3 from whole-cell extracts with anti-FLAG M2 beads. Western analyses were performed to detect EZH2 binding.

Fig. 7.

Fig. 7.

Knockdown of EZH2 has little effect on the binding of STAT3 to the SOCS3 promoter. A4-WT-STAT3/shControl (NTshRNA) and A4-WT-STAT3/shEZH2 (shEXH2) cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 30 min or were untreated. A ChIP assay then was performed, using an antibody against total STAT3. The immunoprecipitated DNA was amplified by qPCR, using primers specific for STAT3-binding sites on the SOCS3 promoter. Values represent means ± SD of fold enrichments of the IL-6–treated versus untreated samples following analysis by qPCR.

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