Induction of RUNX1 expression in RUNX1^−/− ES cells carrying an inducible RUNX1 allele rapidly induces chromatin unfolding at Pu.1. (A) Experimental strategy. (B) Expression analyses of Runx1, Pu.1, and Fli1 mRNA in the indicated cell types and with/without doxycycline (DOX) induction (concentration 0.1 μg/mL). (C) DNaseI in vivo footprinting assays examining the Pu.1 promoter in the indicated cell types. Signal intensities were measured across the displayed regions and were calculated relative to the rDNA control, with ES cells set as 1. (D) iRUNX1 cells were differentiated into embryoid bodies and induced with 0.1 μg/mL DOX for 4 hours or left uninduced. Flk1⁺ cells were purified by magnetic cell sorting. ChIP assays were performed in duplicate with 10⁷ cells for each assay and were normalized against the signal with an amplicon located on chromosome 2 (Chr2 control) representing a nonexpressed GAPDH pseudogene. Numbers in panels B and D are mean values between 2 measurements.