HOXB4 transcriptionally activates and binds to the promoter of Hemgn bothex vivoand in vivo. (A) Schematic illustration of the multiple putative HOXB4-binding sites on the promoter region of Hemgn (sites 1, 2, 3, and 4). (B) HOXB4-dependent increase in promoter activity of Hemgn-luciferase in MEL cell cotransfection experiments. *P < .05. (C) HOXB4-specific binding caused band shift of probes containing the putative HOXB4-binding site (site 1). Lanes 1 and 2 indicate biotin-labeled WT probe (−1562 bp to −1540 bp of Hemgn promoter); lanes 3 and 4, labeled WT probe: competitive unlabeled probe = 1:200; lanes 5 and 6, probe with “TAAT” core sequence mutated to “AGCA”; lanes 7 and 8, labeled WT probe plus anti HOXB4 antibody (I12); solid arrow indicates band shift by HOXB4; and open arrow, supershift by I12. #Degraded GST-HOXB4 caused band shift. (D) Semiquantitative PCR detected enrichment of the promoter fragments of Hemgn by HOXB4 specific binding after ChIP assay in 5-FU BM cells. (E) Quantitative real-time PCR detected relative enrichment of individual regions in Hemgn promoter by HOXB4 after ChIP assay in 5-FU BM cells. All values were normalized to the corresponding input control sample.