The PU.1 SubTAD is dynamic during monocytic differentiation. (A) Real-time PCR showing PU.1 mRNA expression in undifferentiated (monocytic) and 72-hour PMA + VD3–differentiated (macrophage-like) THP-1 cells, along with primary human monocytes for comparison. All values are relative to those of GAPDH. *P < .05; **P < .01 (significant different variances calculated by 1-way analysis of variance). Mean and standard error of the mean are presented. (B) Near-cis 4C-seq profile visualization with the PU.1 promoter VP (gray dashed line) of undifferentiated (top) and 72-hour PMA + VD3–differentiated (bottom) THP-1 cells. Raw fragment data are visible as gray dots and running medians as blue dots; the trend line is LOESS smoothed, and the gray shadowing represents quantiles. Red dashed lines mark the boundaries of the PU.1 SubTAD. The UCSC gene track is shown at the top. The plots are representative of 2 biological repeats each. (C) Quantitative 3C assay demonstrating increased crosslinking frequency of the URE with the PU.1 promoter in mouse bone marrow–derived macrophages (Macs) as compared with Lineage-Sca1⁺-ckit⁺ bone marrow stem and progenitor cells (LSK). We also measured the interaction of the PU.1 promoter with a control region outside of the SubTAD (+71 kb), revealing no increase in crosslinking frequency. The highest value was set to 1. Error bars represent SD of 2 independent experiments. (D) ChIP-seq profiles of H3K4me1 (top) and H3K27ac (bottom) in undifferentiated (THP-1) and 72-hour PMA + VD3–differentiated THP-1 cells (THP-1 PMAVD3), displayed as UCSC browser tracks. Green arrowheads represent the position of the PU.1 promoter (PP) and the URE.