Reversible methylation of promoter-bound STAT3 by histone-modifying enzymes

Abstract

Following its tyrosine phosphorylation, STAT3 is methylated on K140 by the histone methyl transferase SET9 and demethylated by LSD1 when it is bound to a subset of the promoters that it activates. Methylation of K140 is a negative regulatory event, because its blockade greatly increases the steady-state amount of activated STAT3 and the expression of many (i.e., SOCS3) but not all (i.e., CD14) STAT3 target genes. Biological relevance is shown by the observation that overexpression of SOCS3 when K140 cannot be methylated blocks the ability of cells to activate STAT3 in response to IL-6. K140 methylation does not occur with mutants of STAT3 that do not enter nuclei or bind to DNA. Following treatment with IL-6, events at the SOCS3 promoter occur in an ordered sequence, as shown by chromatin immunoprecipitations. Y705-phosphoryl-STAT3 binds first and S727 is then phosphorylated, followed by the coincident binding of SET9 and dimethylation of K140, and lastly by the binding of LSD1. We conclude that the lysine methylation of promoter-bound STAT3 leads to biologically important down-regulation of the dependent responses and that SET9, which is known to help provide an activating methylation mark to H3K4, is recruited to the newly activated SOCS3 promoter by STAT3.

Fig. 1.

K140A or K140R mutation of STAT3 increases its tyrosine phosphorylation and transcriptional activity in response to IL-6. (A) Western analyses for STAT3 and Y705-phosphoryl-STAT3. STAT3-null A4 cells were infected with retroviral constructs and stable pools of cells were selected with G418. DLD1, parental colon cancer cells; A4, STAT3-null cells; WT, A4 cells expressing a normal level of wild-type STAT3; K140A, A4 cells expressing a normal level of K140A STAT3; K140R, A4 cells expressing a normal level of K140R STAT3. The cells were treated with IL-6 for 4 h and total cell lysates were analyzed by Western blot. (B) Northern analysis of gene expression. Cells were treated with IL-6 for 4 h and total RNA was analyzed for GAPDH and SOCS3 mRNAs. (C) EMSAs. Whole-cell extracts were made from DLD1 cells, A4 cells, or A4 cells expressing wild-type, Y705F, K140A, or K140R STAT3, treated with IL-6 for 4 h. A GAS (STAT3 binding) element derived from the SOCS3 promoter was used as the probe. (D) SOCS3 reporter gene induction. Luciferase constructs containing ∼1 kb of the human SOCS3 promoter (from −1 to about −1,000) were cotransfected with a pCH110 control plasmid. Cells stimulated with IL-6 for 4 h or untreated cells were washed with serum-free medium and cultured in complete medium for 12 h more. Cell lysates were analyzed for luciferase activity, which was normalized to the level of β-galactosidase activity from pCH110 control cells in the same extract. Values are means of triplicate determinations and the bars show one SEM. (E) A4 cells expressing wild-type or K140R STAT3 were treated with IL-6 for 4 h. The cells were washed with serum-free medium twice and cultured in complete medium without IL-6. Whole-cell lysates were analyzed by Western blot for total STAT3 and Y705-phosphoryl-STAT3. (F) A4 cells expressing wild-type or K140R STAT3 were treated with IL-6 for 4 h. The cells were washed with serum-free medium twice and cultured in complete medium without IL-6. After 32 h, the above cells were retreated with IL-6 for 4 h, and then washed with serum-free medium twice and cultured in complete medium, again without IL-6. Whole-cell lysates were analyzed by Western blot for total STAT3 and Y705-phosphoryl-STAT3. (G) A4 cells expressing wild-type or K140R-STAT3 were treated with IL-6 for 4 h. The cells were washed with serum-free medium twice and cultured in complete medium without IL-6. Total RNA was analyzed for GAPDH and SOCS3 mRNAs. (H) A4 cells expressing wild-type or K140R STAT3 were treated with IL-6 for 4 h. The cells were washed with serum-free medium twice and cultured in complete medium without IL-6. After 32 h, the cells were retreated with IL-6 for 4 h, washed with serum-free medium twice and cultured in complete medium without IL-6. Total RNA was analyzed for GAPDH and SOCS3 mRNAs.

Fig. 2.

Nuclear import and DNA binding are required for IL-6–induced S727 phosphorylation and K140 dimethylation of STAT3. (A) Western analyses for STAT3, S727-phosphoryl-STAT3, and STAT3-K140me2. STAT3-null A4 cells were infected with retroviral constructs and stable pools were selected with G418. A4, STAT3-null cells; WT, YF, S727A, K140A, K140R, R214/215A and R414/417A, A4 cells expressing normal levels of wild-type or mutant STAT3 proteins. Total lysates prepared from cells treated with IL-6 for 4 h were analyzed by Western blot. (B) A4 cells expressing wild-type STAT3 were treated with IL-6. Cytoplasmic and nuclear fractions representing equal numbers of cells were separated by electrophoresis and analyzed by Western blot. (C) A4 cells expressing wild-type STAT3 were grown on coverslips to 20 to 30% confluence, then treated with IL-6 for 4 h, followed by staining with primary antibodies directed against STAT3, S727-phosphoryl-STAT3 and STAT3-K140me2. Following staining with DAPI (blue nuclear stain) and fluorescent secondary antibodies against STAT3 (green), S727-phosphoryl-STAT3 (red) or STAT3-K140me2 (red), the cells were examined by confocal microscopy. The yellow/pink pixels in the composite image demonstrate the close association of the two proteins. (D) Northern analysis of gene expression. Cells were treated with IL-6 as above for 4 h and total RNAs were analyzed for GAPDH and SOCS3.

Fig. 3.

STAT3 is methylated by SET9 and demethylated by LSD1. (A) Cells were transfected transiently with pcDNA3.1-SET9. After 48 h, the cells were treated with IL-6 for 4 h and total cell lysates were analyzed by Western blot. A4, STAT3-null cells; WT, K140A and K140R, A4 cells expressing normal levels of STAT3 proteins. (B) Cells were transfected transiently with pcDNA3.1-SET9. After 48 h, the cells were treated with IL-6 for 4 h and total RNAs were analyzed by Northern blot. (C) Cells were transfected transiently with pcDNA3.1-LSD1. After 48 h, the cells were treated with IL-6 for 4 h and total cell lysates were analyzed by Western blot. A4, STAT3-nullcells; WT, K140A and K140R, A4 cells expressing normal levels of STAT3 proteins. (D) Cells were transfected transiently with pcDNA3.1-LSD1. After 48 h, the cells were treated with IL-6 as above for 4 h and total RNAs were analyzed by Northern blot. (E) Western analyses for SET9 and LSD1. A4 cells were infected with retroviral constructs and stable pools of cells were selected with G418. DLD1, parental cells; A4, STAT3-null cells; WT, K140A, and K140R, A4 cells expressing normal levels of STAT3 proteins. The cells were treated with IL-6 for 4 h and total cell lysates were analyzed by Western blot. (F) STAT3 was immunoprecipitated from whole-cell extracts of the above cells by using anti-Flag M2 beads. Western analyses were performed to detect SET9 and LSD1. (G) A4 cells expressing wild-type STAT3 were transfected transiently with siRNAs directed against SET9 or LSD1 and, 24 h later, the cells were transfected again with the same siRNAs. After an additional 24 h, some of the cells were transfected with siRNA-resistant SET9 or LSD1 cDNAs. After another 24 h, some of the cells were treated with IL-6 for 4 h more and analyzed for SOCS3 expression by Northern blot and some were left untreated and analyzed for SET9 and LSD1 expression by Western blot.

Fig. 4.

Comparison of genes induced by IL-6 in wild-type cells or in A4 cells expressing K140R STAT3. (A) Cells were treated with IL-6 for 4 or 24 h or were untreated. Total RNAs were analyzed by using an Illumina gene array. Messenger RNAs showing a change in expression of twofold or more, compared with their expression in untreated cells, were scored. Fold-change was calculated from the ratio (K140R-STAT3 treated for 4 h/untreated) ÷ (WT-STAT3 treated for 4 h/untreated). (B) Northern analysis of gene expression. Total RNAs were used and GAPDH was analyzed on the same transfers. (C) SET9 siRNA was used as described in Fig. 3G. After 48 h more, the cells were treated with IL-6 for 4 or 24 h. Total RNA was extracted for Northern analysis. (D) LSD1 siRNA was used as described in Fig. 3G. After an additional 48 h, the cells were treated with IL-6 for 4 or 24 h. Total RNA was extracted for Northern analysis.

Fig. 5.

STAT3-K140me2 binds to the SOCS3 promoter selectively. A ChIP assay was performed with chromatin from IL-6–treated or untreated A4 cells expressing wild-type STAT3, using antibodies against total STAT3, S727-phosphoryl-STAT3, or STAT3-K140me2. The immunoprecipitated DNA was amplified by qPCR, using primers specific for SOCS3, CDCA1, and CD14. Values (percent of the inputs) are the mean ± SD of triplicates performed on three separate chromatin preparations. Data were analyzed using origin75 software and compared by using the unpaired Student's t test. All of the values obtained with each antibody were corrected for background by subtracting the value for untreated cells, which was about 0.1% of the input in each case.

Fig. 6.

The sequence of events that follows the binding of STAT3 homodimers to the SOCS3 promoter. (A) CHIP assays. Quantitative ChIP assays were performed with chromatin from IL-6–treated or untreated A4 cells expressing wild-type STAT3, using antibodies against total STAT3, Y705-phosphoryl-STAT3, S727-phosphoryl-STAT3, STAT3-K140me2, SET9, and LSD1. The immunoprecipitated DNA was amplified by qPCR, using primers specific for SOCS3. Values (percent of the inputs) are the mean ± SD of triplicates performed on three separate chromatin preparations. Data were analyzed using origin75 software and compared by using the unpaired Student's t test. All of the values obtained with each antibody were corrected for background by subtracting the value for untreated cells, which was about 0.1% of the input in each case. Note that the data shown are averages, representing mixed populations of cells. We expect that each individual promoter will not be able to bind both the methyltransferase and the demethylase simultaneously. (B) A model for the sequence of events that follows the binding of STAT3 homodimers to the SOCS3 promoter. Events following the binding of LSD1 and demethylation of STAT3 are not represented because they are not well understood at present.