LOUP cooperates with RUNX1 to facilitate URE-PrPr interaction. (A) Gene track view of the ∼26-kb region encompassing the URE and the PrPr. Top panel: RUNX1 ChIP-seq tracks derived from CD34⁺ cells from healthy donors (GSM1097884), and a patient with FLT3-ITD AML (GSM1581788). Bottom panel: a schematic showing the corresponding genomic locations of LOUP and the 5′ region of PU.1. (B) DNA pulldown assay showing binding of RUNX1 to the RUNX1-binding motifs at the URE and the PrPr. Proteins captured by biotinylated DNA oligos (wild-type oligo containing RUNX1-binding motif [wt]; oligo with mutated RUNX1-binding motif [mt]) in U937 nuclear lysate were detected by immunoblot. (C) ChIP-qPCR analysis of RUNX1 occupancy at the URE and the PrPr. LOUP-depleted U937 (sgLOUP, L2a) and control (sgControl, N1) clones were used. PCR amplicons include the URE (contains known RUNX1-binding motif at the URE), PrPr (contains putative RUNX1-binding motif in the PrPr), and GENE DESERT (a genome region that is devoid of protein-coding genes). Error bars indicate SD (n = 3). **P < .01; ****P < .0001. (D) RNA pulldown analysis of the RUNX1-LOUP interaction. Top panel: schematic diagram of LOUP showing the relative position of the repetitive region (RR). Arrows underneath the diagram illustrate direction and relative lengths of in vitro–transcribed and biotin-labeled LOUP fragments (AS, full-length antisense control; Bead, no RNA control; EGFP, EGFP mRNA control; RR; S, full-length sense). Bottom panel: LOUP fragments were incubated with U937 nuclear lysate. Retrieved proteins were identified by immunoblot. (E) Schematic diagram of the repetitive region (RR) showing predicted binding regions R1 and R2. (F-G) RNAP-binding analysis of R1 and R2 with recombinant full-length and Runt domain of RUNX1. In vitro-transcribed and biotin-labeled RNAs include R1-AS (R1 antisense control); R1-S (R1 sense); and R2-S (R2 sense). The vertical line demarcates where an unrelated lane was removed from the figure. See also supplemental Figure 5.