CBFs activate PU.1 AsPr. (A) Schematic of the human thymic T-lymphoid differentiation hierarchy. (B) Schematic representation of the PU.1 locus with the AsPr containing a consensus RUNX-binding site (black square) and the genomic coordinates in GRCh37/hg19. Gene expression by transcript sequencing (RNA-seq) in thymic progenitors and differentiated T cells (pro-T, CD1a^– pro-T cell; CD1a, CD1a⁺ pro–T cell; DP, double-positive CD4⁺/CD8⁺ T cell; CD4, CD4⁺ T cell; CD8, CD8⁺ T cell) for PU.1 (C) and RUNX1 (D) (n = 2). Data are represented as RPKM normalized to GAPDH housekeeping gene. RUNX1 ChIP-seq in Jurkat (E) and CD34⁺ (F) HSPCs (with immunoglobulin G control). (G) Gel electromobility shift assay with labeled PU.1 AsPr probe oligonucleotide in the presence of RUNX1 (Jurkat cell line). Luciferase reporter assays in HEK293T cells transiently transfected with PU.1 AsPr reporter plasmid and increasing RUNX1 (H), RUNX1-ETO (I), and CBFβ-MYH11 (J) expression plasmids (plasmid concentration [nanogram], n = 4). Data are represented as mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001, Student t test. CLP, common lymphoid progenitor; ETP, early thymic progenitor; HSC-MPP, hematopoietic stem cell and multipotent progenitor; LMPP, lymphoid-primed multipotent progenitor; RUNX compet. Probe, the competition probe with mutated RUNX binding site.