Figure 2. miR-155 precursor BIC is a transcriptional target of STAT5. (A) Analysis of the BIC promoter region yielded one STAT binding site (highlighted in blue). Primers flanking the binding site that were used for PCR analysis of ChIP samples are indicated in red. The beginning of the BIC transcript is highlighted in bold script. (B) ChIP-seq reads from the BIC (MIR155HG) gene promoter region in malignant MyLa2059 cells. Reads (76 bases) obtained from immunoprecipitation of STAT5, STAT3, RelA and a negative control (rabbit IgG, bottom). The chromosomal positions of MIR155HG refer to hg19. Forward reads are indicated in green and reverse reads in red. (C) PCR analysis of ChIP samples using the primer set indicated in (B). The 190 bp amplicon was only detected in the STAT5-precipitated samples and the positive control (histone H3). (D) Similar results were obtained by qPCR: STAT5-precipitated samples displayed a 10-fold enrichment of the amplified sequence in relation to a negative control, whereas detection in STAT3 precipitated samples ranged at background level. U6 rRNA was used for normalization. (E) Luciferase assay for BIC promoter activity. CHO-K1 cells were transiently transfected with pCMV-LacZ, empty pGL3 or pGL3-BIC-WT, together with STAT5A, STAT5A-CA, STAT5B or STAT5B-CA (CA, constitutively active). Transfection with the constitutively active STAT5A-CA and STAT5B-CA resulted in a 28.6- (p < 0.001) and 29-fold (p = 0.002) induction of BIC promoter activity, respectively. Data were obtained from three independent experiments.