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...

Hepatocarcinogenesis is a multistage process in which precursor lesions progress into early hepatocellular carcinomas (eHCC) by sequential accumulation of multiple genetic and epigenetic alterations. To decode the molecular events during early stages of liver carcinogenesis, we performed gene expression profiling on cirrhotic (regenerative) and dysplastic nodules (DN), as well as eHCC. Although considerable heterogeneity was observed at the regenerative and dysplastic stages, overall, 460 differentially expressed genes were detected between DN and eHCC. Functional analysis of the significant gene set identified the MYC oncogene as a plausible driver gene for malignant conversion of the DNs. In addition, gene set enrichment analysis revealed global activation of the MYC up-regulated gene set in eHCC versus dysplasia. Presence of the MYC signature significantly correlated with increased expression of CSN5, as well as with higher overall transcription rate of genes located in the 8q chromosome region. Furthermore, a classifier constructed from MYC target genes could robustly discriminate eHCC from high-grade and lowgrade DNs. In conclusion, our study identified unique expression patterns associated with the transition of highgrade DNs into eHCC and showed that activation of the MYC transcription signature is strongly associated with the malignant conversion of preneoplastic liver lesions. [Cancer Res 2009;69(7):2775-82]

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Interferon regulatory factor 3 (IRF-3) plays a central role in inducing the expression of cellular antiviral genes, including the interferon-␤ gene, in response to Pattern Recognition Receptors. IRF-3 is targeted for proteasome-mediated degradation, which modulates the strength and duration of the innate immune responses that depend on it. We have found that caspase-8, which is activated by cytosolic RIG-I-dependent signaling, catalyzes an essential intermediate step in the ubiquitination and proteasome-mediated degradation of IRF-3. Mutation of a consensus cleavage site within IRF-3 generates a form that is not cleaved by caspase-8 and that is protected from ubiquitination and degradation. An in vitro assay confirms the direct action of caspase-8 cleavage on IRF-3. We also show that caspase-8-mediated cleavage of IRF-3 helps to modulate dsRNA-dependent gene induction.

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PurposeTo develop a method to measure the rate of glaucomatous visual field (VF) deterioration and to identify fast progressors.MethodsRetrospective, longitudinal, observational study of 8486 eyes of 4610 glaucomatous patients with ≥6 VFs and ≥3 years of follow-up. A Glaucoma Rate Index (GRI) was calculated. VF locations were partitioned into exponential decay or exponential improvement models. A pointwise rate of change (PRC) was estimated with an exponential fit and expressed as the percent/year change of the age- and location-matched normal perimetric range, presented as a spatially conserved VF map. PRCs were summed and normalized with boundary rates set by simulated decaying and improving VF series on a scale of −100 to +100, respectively.ResultsA total of 89,704 VF examinations with 425,039 test location series was used. Median follow-up and number of VFs/eye were 9.7 years and 9 VFs, respectively. Initial and final mean deviations (±SD) were −4.2 (±5.2) and −5.7 (±6.4) dB. The proportions of test locations designated as decayed, improved, and unchanged were 13%, 4%, and 83%, respectively. Mean PRCs for decay, improvement, and no change were −3.7 (±4.7)%/y, 2.5 (±2.6)%/y, and −0.5 (±2.1)%/y, respectively. The number of eyes with negative and positive GRIs was 5802 (68%) and 2390 eyes (28%), respectively. The proportion of eyes defined as fast progressors was 6.8%.ConclusionsGRI provides a robust measure of glaucomatous VF change, operates without discontinuity over the entire perimetric range, and can be used to identify fast progressors.Translational RelevanceThis study describes a novel method that can help the clinician to determine VF progression.

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