Functional analysis of RUNX1-PTD in vitro. (A) Western blot analysis of RUNX1-PTD expression. The analyzed cells included KPAM1 expressing RUNX1C-PTD under the control of a Dox-inducible promoter (lanes 1, 2, 3, and 4), ML-DS (number 21) without RUNX1-PTD (lane 5), and ML-DS (number 28) with RUNX1-PTD (lane 6). Dox treatment concentrations are indicated. (B) Protein domains of RUNX1C, RUNX1C-PTD (Ex3-6), and RUNX1C-PTD (Ex3-7). The structures of RUNX1C and RUNX1C-PTDs comprise runt-homology domains (RHD), a transactivation domain (TAD), and a VWRPY motif. (C) Luciferase reporters from the GP1BA promoter were cotransfected with the indicated constructs in QT6 cells. Reporter, mock vector; Wild, WT RUNX1C; Ex3–6, RUNX1C-PTD with duplication of Ex3-6; Ex3-7, RUNX1C-PTD with duplication of exons 3 to 7; ∗∗P < .01 by Student t test. Data are presented as mean ± SD. (D) Overlap of ChIP-seq peaks of WT RUNX1C and RUNX1-PTDs. KPAM1 cells were transfected with Xpress-tagged RUNX1C, RUNX1C-PTD (Ex3-6), and RUNX1C-PTD (Ex3-7). ChIP-seq was performed using anti-Xpress antibody. The significance of the overlap was assessed using the peakPermTest function of the ChIPpeakAnno package; ∗∗P = .001 by peakPermTest. (E) Relative distribution of the RUNX1C and RUNX1-PTD ChIP-seq peak regions in the genome. (F) Results of de novo motif analysis of ChIP-seq data. The 2 most significant motifs were found by the MEME algorithms in RUNX1C (top), RUNX1C-PTD (Ex3-6) (middle), and RUNX1C-PTD (Ex3-7) (bottom) ChIP-seq data. RUNX1 binding motifs were the most significantly enriched (>97%). The second most frequently occurring motif, which was found in ∼25% of the peaks, was GATA1. (G) Subcellular localization of RUNX1C and RUNX1C-PTD proteins in HEK293 cells transfected with Xpress-tagged RUNX1C-PTD or WT RUNX1C.