Fig. 1. Myc is a STAT3 target gene in ES cells. (A) LIF-maintained ES cells or embryoid bodies (-LIF) generated from ES cells were harvested and mRNA analysed by RT-PCR analysis using primers designed against Rex1, Oct4, Fgf5, brachyury, Myc and ß-actin. (B) Chromatin immunoprecipitation (ChIP) analysis with a FLAG monoclonal antibody was performed on crosslinked chromatin from a cell line expressing FLAG-tagged STAT3 (STAT3_FLAG). Immunoprecipitated chromatin was then used to determine if STAT3_FLAG bound the endogenous Myc promoter using a specific primer pair that flanks an E2F/E-box in the Myc P2 promoter (left panels). The same chromatin was used as a template in a PCR reaction using primer for the CDK1 promoter that is not implicated as a STAT3 target gene (right panel). Addition of FLAG antibody in ChIPs is as indicated (+). To control for the amount of total chromatin in the ChIP assay, equivalent amounts of total template (`input'), equivalent to 0.03% of total chromatin DNA used in ChIP assays, were used in a parallel PCR reaction using Myc or CDK1 specific primers. Immunoblot analysis: relative expression levels of total STAT3 in the transfected cell line is shown relative to endogenous STAT3 levels (vector alone transfectant). (C) STAT3 trans-activates the Myc gene in ES cells. LIF maintained STAT3-ER cells were deprived of LIF for 36 hours, stimulated with 4OHT (10 nM) or LIF (1x10³ units/ml) and assayed 24 hours later by RT-PCR analysis.