PU.1 initiates spatial chromatin activity in the PU.1 SubTAD. (A) PU.1 ChIP-seq tracks of undifferentiated THP-1 (green, top) and 72-hour PMA + VD3–treated THP-1 cells (blue, bottom) around the PU.1 gene locus (220-kb range). The dashed red lines indicate the boundaries of the PU.1 SubTAD (white region). The UCSC browser gene track is shown above. Green arrowheads represent position of the PU.1 promoter (PP) and the URE. (B) western blot confirming PU.1 knockdown in transgenic THP-1 cells carrying an inducible shRNA against PU.1 (shPU.1 kd), in comparison with control transgenic THP-1 cells carrying an inducible SC4 shRNA. The cells were first treated with Dox for 24 hours and then with PMA + VD3 for another 12 hours. VCP is shown as loading control. The molecular protein mass is indicated in kilodaltons using a prestained protein ladder (#26617, Thermo Scientific). (C-D) 4C-seq profiles from 2 independent experiments displaying impaired spatial contacts of the PU.1 promoter (PP VP; C) or the reciprocal URE (URE VP; D) VP within the PU.1 SubTAD after PU.1 knockdown early in differentiation. The THP-1 cells carrying PU.1 shRNA or SC4 control shRNA constructs were Dox and PMA + VD3 treated as in panel B. Green arrows at the top indicate the direction of VP interaction with the URE or the PU.1 promoter, respectively. Green arrowheads at the bottom represent position of the PU.1 promoter (PP) and the URE. Dashed blue lines represent heights of the URE or promoter interacting with the used VP.