Fig. 2

STAT5 specifically binds to the E3 and E4 regions of c-Myc super-enhancer. a Schematic representation of the mouse STAT5 target genes Cis, Osm and c-Myc and of the qPCR amplicons analysed following chromatin immunoprecipitation. Nucleotide positions are relative to the respective transcription start sites (TSS). Functional STAT binding sites within the individual promoter regions are indicated as grey bars. P1 and P2 designate c-Myc dual promoters, P2 being predominant in normal cells. E1 to E5 symbolise the five domains of c-Myc 3′ super-enhancer. Black boxes underneath the respective genes represent the qPCR amplicons. Primers amplifying regions within Cis and c-Myc open reading frame (ORF) were used as controls. b Chromatin immunoprecipitation (ChIP) was performed with whole-cell lysates from Ba/F3 cells (conventional ChIP protocol), either unstimulated or stimulated 30 min with IL-3, using antibodies specific for STAT5A + STAT5B (STAT5 ChIP). Immunoprecipitated genomic DNA (gDNA) was analysed by qPCR using primers shown in a. One-way ANOVA with Dunnett’s post test was used to evaluate IL-3-induced STAT5 enrichment at the various loci compared to the signal detected at the c-Myc “ORF” region, used as a reference and background control; ***P < 0.001; a P < 0.05 was considered statistically significant. c ChIP was conducted with nuclear lysates (alternative ChIP protocol) from Ba/F3-1*6 cells grown in the absence of IL-3, using antibodies specific for STAT5A, which recognize STAT5A-1*6 mutant. Immunoprecipitated gDNA was analysed by qPCR, as in b. One-way ANOVA with Dunnett’s multiple comparison test was used to evaluate STAT5 enrichment at the various loci vs. c-Myc “ORF” region, used as a reference and background control; *P < 0.05, ***P < 0.001