Fig. 4

BRD2 co-localizes with STAT5 at E3/E4 in Ba/F3 cells transformed by constitutive active STAT5A-1*6. a Ba/F3-1*6 cells were treated for 60 min with 200 nM TSA, 1 μM JQ1 or 0.02 % DMSO (vehicle). Nuclei isolation and ChIP was performed following the alternative protocol, and using BRD2-specific antibodies or the same amount of rabbit IgG as a control. qPCR primers are depicted in Fig. 2a. Only the vehicle-treated IgG control is shown. TSA- and JQ1-treated IgG controls exhibited similar background levels (data not shown), as previously reported [31]. BRD2 binds strongly to the Cis transcription start site (TSS), the c-Myc promoter region (STAT1) and enhancer regions E3 and E4. BRD2 association is lost upon JQ1 treatment and is reduced at Cis TSS and c-Myc E3/E4 upon treatment with the deacetylase inhibitor TSA. Background cut-off (horizontal line) was defined as the mean of the signal generated by the IgG negative controls plus 2x the standard deviation (SD) of the IgG control (mean IgG + 2x SD). BRD2 enrichment at the various loci in the vehicle-treated condition was evaluated using One-way ANOVA with Dunnett’s multiple comparison vs. c-Myc “ORF” region, used as a reference; of note, BRD2 signal intensity at the c-Myc “ORF” region is not reduced upon JQ1 treatment, suggesting that it corresponds to background BRD2 signal; **P < 0.01, ***P < 0.001; a P < 0.05 was considered statistically significant. One-way ANOVA with Dunnett’s post test was used to assess differences between TSA- or JQ1-treated conditions and the vehicle-treated control; ^## P < 0.01, ^### P < 0.001; a P < 0.05 was considered statistically significant. b Ba/F3 cells were stimulated with IL-3 for 30 min and BRD2 ChIP was conducted with whole-cell lysates using BRD2-specific antibodies or rabbit IgG, following the conventional ChIP protocol [12, 31]. Genomic DNA was analysed as in a. Only background signals were detected along the c-Myc gene locus and downstream super-enhancer in Ba/F3 cells, while BRD2 was recruited in response to IL-3 at the transcription start site (TSS) of the Cis gene. Background cut-off (horizontal line) was defined as described in A (mean IgG + 2x SD). Student’s t test was used to monitor IL-3-induced BRD2 recruitment at Cis TSS; **P < 0.01; a P < 0.05 was considered statistically significant