ETV6-RUNX1 binds to the EPOR gene promoter region and activates its transcription. (A) In silico analysis of mouse and human upstream genomic regions of the EPOR gene revealed the existence of a highly conserved putative RUNX1 binding site located ∼ 200 bp upstream of transcription initiation. (B) ChIP assays at the putative RUNX1 binding site located in the EPOR promoter in mouse 1/27 BaF3 cells induced to express ETV6-RUNX1 (+ MIF) or mock-induced control cells (clone 1 + MIF, n = 6; P = .0048). α-V5 indicates V5 epitope antibody used for ETV6-RUNX1 immunoprecipitation in BaF3 1/27 cells and IgG is isotype control; and negative, an EPOR genomic region negative for ETV6-RUNX1 binding sites (see also supplemental Figure 2). (C) The first 200 bp of the human EPOR promoter was cloned upstream of a pGL4 luciferase vector and transfected into 293T cells with or without expression vectors for GATA1 (positive control) or ETV6-RUNX1 (ER). Both GATA1 (as expected) and ETV6-RUNX1 were able to enhance basal luciferase activity of the promoter (P = .0076). This effect was dependent on an intact RUNX1 binding site as the mutation (mut) of the putative site abolished the increase in luciferase activity observed with ETV6-RUNX1. RLU indicates relative luciferase units, normalized to EPOR promoter vector (EPOR-luc). Data are from 4 independent experiments.