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. (2) and some of the same lysine side chains can be either methylated or acetylated. These modifications alter chromatin structure, often by providing entry sites for proteins that determine higher-order chromatin organization, leading to the activation or inactivation of specific genes. In addition, methylation and demethylation of p53 and NFκB are carried out by enzymes previously known to modify only histones. For p53, the reactions occur on K370, K372, and K382 (3). For NFκB, K37 is methylated by SET9 (4), and K218 and K221 are methylated by NSD1 and demethylated by FBXL11 (5).STAT3 is phosphorylated on tyrosine and serine residues in response to many different cytokines and growth factors, leading to the formation of dimers through reciprocal phosphotyrosine-SH2 interactions (6). Activated STAT3 dimers bind to and activate the promoters of target genes. In addition to phosphorylation, STAT3 was reported to be acetylated at K685 following cytokine stimulation, and the K685R mutation blocked its activation (7), but these observations have been disputed (8). Ray et al. (9) reported that K49 and K87 of STAT3 are acetylated by p300 and that the K-R mutations resulted in a STAT3 protein that is able to translocate into nuclei, but unable to bind to p300. Here, we show that, in response to IL-6, STAT3 is methylated on K140 by the H3K4 methyl transferase SET9 and demethylated by the H3K4 demethylase LSD1 (lysine-specific demethylase 1, also named BHC110). Prevention of methylation by mutation of K140 greatly enhances the induction of one group of genes in response to IL-6, but has little effect on a second group, and inhibits the activation of a third group. Several lines of evidence indicate that methylation takes place as STAT3 is bound to promoters in the f...