Figure 2.

RUNX1 upregulates TRAIL transcription independent of the consensus RUNX1 sequences on the TRAIL promoter. (A) Nucleotide sequence of the human TRAIL promoter element are depicted. RUNX1-binding consensus sequences, ACCACA and TGTGGT, are shown in red boxes (Sites 1 and 2). The transcription start site is indicated by the red arrow. (B and C) RUNX1 increased TRAIL promoter activity. (B) RUNX1 and CBFβ expression plasmids were transfected alongside the TRAIL promoter-luciferase reporter plasmid (pTRAIL) into HeLa cells. At 48 h post-transfection, cell lysates were prepared and luciferase assays were performed. (C) The RUNX1 and/or CBFβ plasmids were transfected alongside the pTRAIL-promoter construct in HeLa cells before luciferase activities were measured. Results were normalized to Renilla luciferase driven by the TK promoter. (D) Site-directed mutagenesis of sites 1 and 2 was used to generate mutant 1 and mutant 2, respectively. (E) pTRAIL/-819 luciferase constructs encoding the TRAIL promoter with the Mt1 or Mt2 mutations were transfected into HeLa cells alongside the RUNX1 and CBFβ expression plasmids, before their activities were measured and compared with those of the pTRAIL construct pTRAIL/-819. pGL2m, an empty reporter plasmid, was used as the control. (F) Electrophoresis mobility shift assay was performed to detect the formation RUNX1 protein-DNA complexes. Recombinant His-tagged RUNX1 and His-tagged CBFβ produced in E. coli were incubated with probes containing sequences shown in (D). CT is a control probe with the RUNX1 consensus sequence. The positions for of the protein-DNA complexes (shift bands) and free probes are indicated on the right side of gel image. ^*P<0.05 and ^**P<0.01. RUNX1, Runt-related transcription factor 1; CBFβ, core-binding factor β; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; WT, wild-type; Mt, mutant.