Effects of RUNX1-ETO on regulatory function of LOUP. (A) qRT-PCR analysis of samples from patients with AML. Normal karyotype (NK), n = 14; t(8;21) karyotype, n = 7. Mann-Whitney U test: **P < .01; *P < .05. (B) qRT-PCR expression analysis of LOUP RNA (left panel) and PU.1 mRNA (right panel) in U937 cells with inducible expression of RUNX1-ETO (+/−, with/without induction). Cells were transfected with empty vector (EV) and LOUP cDNA. Error bars indicate SD (n = 3). *P < .05; **P < .01. (C) Gene track view at the LOUP locus including the URE where LOUP transcription initiation is located. From top to bottom are RUNX1-ETO ChIP-seq tracks, H3K9Ac ChIP-seq tracks, and DNase-seq tracks of Kasumi-1 cells upon depletion of RUNX1-ETO. Cells were transfected with either nontargeting siRNA (siControl) or RUNX1-ETO–targerting siRNA. Data were processed from a published data set (GEO GSE29222) and integrated into the UCSC genome browser. (D) DNA pulldown assay showing binding of RUNX1-ETO to the RUNX1-binding motifs at the URE. Proteins captured by biotinylated DNA oligos (wild-type oligo containing RUNX1-binding motif [wt]; oligo with mutated RUNX1-binding motif [mt]) in Kasumi-1 nuclear lysate were detected by immunoblot (cytosol fraction [cyt]; nuclear fraction [nuc]). (E) A model of how LOUP coordinates with RUNX1 to modulate chromatin looping resulting in PU.1 induction, myeloid differentiation, and cell growth, and how RUNX1-ETO interferes with LOUP-mediated molecular functions.