Active chromatin is maintained after RUNX1 withdrawal. (A) In vivo DMS footprinting assay examining the Pu.1 3′ URE conducted with 3T3 cells, macrophages (Mac), and CD41⁺ cells sorted after 4 days of a blast culture that had been continuously induced with 1 μg/mL doxycycline at day 2 (+DOX) and where the inducer was removed after day 3 (−DOX). Transcription factor binding sites are indicated on the right. Guanines displaying hyperreactivity to DMS-methylation are marked by •; protections from methylation are indicated by white circles; the weak protection over the RUNX site is indicated by a gray circle. Only reproducible changes are marked. Remaining band intensity (with protected bands) or fold enhancement (with hyperreactive bands) is indicated next to the bands with the G reaction set as 1. (B) ChIP assay demonstrating binding of C/EBPβ to its binding sites at the 3′ URE and the Pu.1 promoter in blast cultures from wt cells (■) and induced iRUNX1 cells (iRUNX-DOX) where DOX has been withdrawn as described in panel A. (C) Network diagram illustrating the initiating role of RUNX1 in establishing open chromatin and the expression of hematopoietic master regulators such as PU.1 in pluripotent hematopoietic precursor cells as well as the maintenance of the active state in differentiating myeloid cells. Once Runx1 is activated in pluripotent hematopoietic cells,^60,61  a regulatory circuit is established capable of maintaining open chromatin in the absence of RUNX1 and driving myelopoiesis. The induction of secondary factors driving macrophage-specific genes has been described in Krysinska et al⁹  and Laslo et al.⁶²  The relative importance of the different transcription factors is illustrated by thin (less important) and bold (more important) lines. Numbers in panel B represent mean values of 2 independent experiments analyzed in duplicate.