Figure 2. RUNX1, GATA2, SP1, MYC and RNA Pol II co-localize on the SET minimal functional promoter region (-318bp-TSS).

Luciferase assays with the SET promoter constructs in HL-60 cells 48h after transfection. A. Results represent relative Firefly/Renilla luciferase activities considering the empty pGL3basic vector as 1. Data are the means ± SD of three independent experiments. Asterisks denote the statistical significance of the differences between pGL3b and the constructs or between the paired constructs where indicated. *P < 0.05, **P < 0.01, ***P < 0.001, Two-way ANOVA and Bonferroni tests were used. B. Chromatin Immunoprecipitation assay performed in the HL-60 cell line. Two promoter sub-regions were defined, a distal region (-932/-587bp) enriched with RUNX1 and GATA2 predicted binding sites and the minimal functional promoter region (-318/-144bp) with SP1 and MYC E-box putative binding sites. A distal genomic region on the same chromosome 9 was used as a negative control. QRT–PCR results were calculated using the 2-Ct method and they are presented as the fold enrichment of chromatin DNA precipitated by the specific antibody versus chromatin DNA precipitated by a non-related IgG. Values are the mean of three independent experiments. Asterisks denote the statistical significance differences where indicated. *P < 0.05, **P < 0.01, ***P < 0.001, Two-way ANOVA and Bonferroni tests were used. C. ChIP-re-ChIP assay performed in the HL-60 cell line. Technical procedures were carried out as described in Materials and Methods. MYC antibody was used for the first immunoprecipitation, and SP1, RUNX1, and GATA2 antibodies were used for the second immunoprecipitation. Re-ChIP assay values are the mean of two independent experiments.