RUNX1 interaction is essential for E2A-PBX1–enhanced gene activation. (A) Schematic of PBX1 domains (left). GST pull-down assays (right) showing direct RUNX1 binding by the IH-HD domain of C-terminal region of PBX1 (PBX1C). (B) Co-IP assay showing RUNX1 binding to WT but not the IH-HD domain-deleted (mt) E2A-PBX1. (C) ChIP-qPCR with anti-HA at enhancers and (D) RT-qPCR of indicated genes in 697 cells transduced by lentivirus expressing WT or IH-HD–deleted (dIH-HD) E2A-PBX1. The NCAPD2 locus was used as a negative control for E2A-PBX1 binding. Data from 3 independent experiments are presented as mean ± SD. Statistics by Student t test. *P < .05; **P < .01; ***P < .001. (E) Model for the coactivator function of E2A-PBX1 in regulating the expression of RUNX1 and RUNX1 programs in pre–B-ALL. Based on previous and current results, the following working model for E2A-PBX1–mediated gene activation is proposed: (i) the E2A-PBX1 fusion protein is expressed under the control of the E2A promoter; (ii) E2A-PBX1, by interaction with RUNX1, is recruited to the RUNX1 locus and sustains high-level expression of RUNX1; (iii) RUNX1 mediates recruitment of E2A-PBX1, which in turn recruits p300 through E2A AD1/2-p300 KIX domain interactions and TFIID through E2A-AD3-TFIID TAF4 interactions; and (iv) these interactions, and potentially other cofactor interactions of E2A-PBX1 and/or RUNX1, enhance expression of a subset of RUNX1 targets that lead to leukemogenesis. DM, dimerization domain; GTFs, general transcription factors; HCM, Hox cooperative motif; HD, homeodomain; IH, inhibitory helix.