PU.1 and PML-RARA are direct regulators of the HK3 promoter. (A) Schematic representation of a 6.6-kb human HK3 promoter fragment. Lanes A, B, and C are 3 putative PU.1 binding sites (circles) found in the HK3 promoter as determined by MatInspector. (B) In vivo binding of PU.1 to the 3 binding sites was shown by ChIP in NB4 cells. ChIP was performed with antibodies against PU.1, PML, and RARA. Antibodies against acetyl-histone H3 and IgG served as positive and negative controls, respectively. As a negative control for the different pull-downs, absence of GAPDH amplification is shown. (C) HK3 promoter transactivation assays. H1299 cells were transiently transfected with 40 ng HK3 promoter reporter construct and PU.1 or PML-RARA expression vectors as indicated. Total amount of transfected DNA was adjusted with pcDNA3.1 empty vector as indicated. pRL-TK Renilla luciferase vector (40 ng) was cotransfected in each experiment as an internal control for transfection efficiency. After 24 hours, luciferase activity was measured. The promoter activity is shown as relative light units (RLU). Results are the mean ± SD of 5 independent experiments. ***P < .0001; **P < .001 (Mann-Whitney U tests).