Figure 4.
Figure 4. CEBP factors and OCT-1 induce activity of the proximal PU.1 promoter in HT93A APL cells following ATRA stimulation. (A) A series of PU.1 promoter constructs were ligated to the luciferase gene in the pxp2 vector. HT93A cells were transfected with equal amounts of each of the deletion constructs and induced for 18 hours with 1 μM ATRA. The results are given as fold activation compared with HT93A cells transfected with the empty pxp2 vector only. (B) The sequence between -48 bp and -86 bp of the human PU.1 promoter contains a CEBP and an octamer consensus binding site (with the sites underlined and italicized). The brackets demonstrate the sequences used in the EMSA assays. (C) EMSA studying binding activity to the CEBP consensus site present in the PU.1 promoter with the CEBP probe (-80 to -60 bp). HT93A cells were stimulated with 1 μM ATRA and harvested at the time points indicated. SS indicates supershift using specific antibodies against CEBPA, CEBPB, and CEBPE. Specificity of the antibodies was verified by supershifting extracts from K562 cells transfected with human CEBPA, CEBPB, or CEBPE cDNAs; no cross-reactivity was observed (data not shown). C indicates competition with cold oligo. (D) EMSA of binding to the OCT site in the PU.1 promoter before and after 18 hours of stimulation with 1 μM ATRA (left panel). The probe is depicted in panel B as “OCT-1” extending from -66 to -42. Cos OCT-1 indicates Cos cells transfected with an OCT-1 expression plasmid as a positive control (lanes 2 and 3). Cos OCT-2 indicates Cos cells transfected with an OCT-2 expression plasmid as a positive control (lanes 4 and 5). Supershifts are depicted for OCT-1 (lanes 3, 7, 9, 11) and OCT-2 (lane 5). U937 indicates binding activity in lane 6, and supershift with OCT-1 in lane 7; HT93A, increase of binding activity before and after induction with ATRA (lanes 8 and 10), with OCT-1 supershifts in lanes 9 and 11; and P, free probe alone. Right panel shows EMSA with a probe with a mutation in the OCT site; no binding activity was detectable with this probe. (E) Western blot analysis of HT93A cells treated with 1 μM ATRA using a CEBPA antibody (top), a CEBPB antibody (second), a CEBPE antibody (third), an OCT-1 antibody (fourth), and a β-actin antibody (bottom). (F) H1299 cells were transiently transfected with the -86-bp PU.1 promoter (top 8 bars) and pcDNA3 vector (V) or equal amounts of expression plasmids for CEBPA, CEBPB, CEBPE, and/or OCT-1. OCT-1 was always cotransfected with equimolar amounts of BOB-1. Results are given as fold induction of the -86-bp promoter alone (to P = 1-fold). CEBP mut bars represent transfections with the -86-bp construct with the CEBP site mutated, whereas OCT mut bars are experiments with the -86-bp construct with the OCT site mutated. The bottom 4 bars are transfections with the -86-bp construct with both sites mutated (CEBP+OCT). (G) ChIP assay of the PU.1 promoter in HT93A cells treated with 1 μM ATRA. Depicted are the results of the quantitative RT-PCR for CEBPA, CEBPB, CEBPE, and OCT-1 binding to the PU.1 promoter at days 0, 1, 3, and 5 after ATRA treatment. (A, F-G) Mean values and SD (error bars) are depicted.

CEBP factors and OCT-1 induce activity of the proximal PU.1 promoter in HT93A APL cells following ATRA stimulation. (A) A series of PU.1 promoter constructs were ligated to the luciferase gene in the pxp2 vector. HT93A cells were transfected with equal amounts of each of the deletion constructs and induced for 18 hours with 1 μM ATRA. The results are given as fold activation compared with HT93A cells transfected with the empty pxp2 vector only. (B) The sequence between -48 bp and -86 bp of the human PU.1 promoter contains a CEBP and an octamer consensus binding site (with the sites underlined and italicized). The brackets demonstrate the sequences used in the EMSA assays. (C) EMSA studying binding activity to the CEBP consensus site present in the PU.1 promoter with the CEBP probe (-80 to -60 bp). HT93A cells were stimulated with 1 μM ATRA and harvested at the time points indicated. SS indicates supershift using specific antibodies against CEBPA, CEBPB, and CEBPE. Specificity of the antibodies was verified by supershifting extracts from K562 cells transfected with human CEBPA, CEBPB, or CEBPE cDNAs; no cross-reactivity was observed (data not shown). C indicates competition with cold oligo. (D) EMSA of binding to the OCT site in the PU.1 promoter before and after 18 hours of stimulation with 1 μM ATRA (left panel). The probe is depicted in panel B as “OCT-1” extending from -66 to -42. Cos OCT-1 indicates Cos cells transfected with an OCT-1 expression plasmid as a positive control (lanes 2 and 3). Cos OCT-2 indicates Cos cells transfected with an OCT-2 expression plasmid as a positive control (lanes 4 and 5). Supershifts are depicted for OCT-1 (lanes 3, 7, 9, 11) and OCT-2 (lane 5). U937 indicates binding activity in lane 6, and supershift with OCT-1 in lane 7; HT93A, increase of binding activity before and after induction with ATRA (lanes 8 and 10), with OCT-1 supershifts in lanes 9 and 11; and P, free probe alone. Right panel shows EMSA with a probe with a mutation in the OCT site; no binding activity was detectable with this probe. (E) Western blot analysis of HT93A cells treated with 1 μM ATRA using a CEBPA antibody (top), a CEBPB antibody (second), a CEBPE antibody (third), an OCT-1 antibody (fourth), and a β-actin antibody (bottom). (F) H1299 cells were transiently transfected with the -86-bp PU.1 promoter (top 8 bars) and pcDNA3 vector (V) or equal amounts of expression plasmids for CEBPA, CEBPB, CEBPE, and/or OCT-1. OCT-1 was always cotransfected with equimolar amounts of BOB-1. Results are given as fold induction of the -86-bp promoter alone (to P = 1-fold). CEBP mut bars represent transfections with the -86-bp construct with the CEBP site mutated, whereas OCT mut bars are experiments with the -86-bp construct with the OCT site mutated. The bottom 4 bars are transfections with the -86-bp construct with both sites mutated (CEBP+OCT). (G) ChIP assay of the PU.1 promoter in HT93A cells treated with 1 μM ATRA. Depicted are the results of the quantitative RT-PCR for CEBPA, CEBPB, CEBPE, and OCT-1 binding to the PU.1 promoter at days 0, 1, 3, and 5 after ATRA treatment. (A, F-G) Mean values and SD (error bars) are depicted.

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