PU.1-dependent LDB1 recruitment to the PU.1 locus upon differentiation. (A) RNA-seq with undifferentiated (left column, n = 3) and 72-hour PMA-differentiated THP-1 cells (middle column, n = 3) as well as with primary human monocytes (right column, n = 4). Individual data points as well as means ± SD are indicated *P < .1; **P < .05 (false discovery rate–adjusted P values. (B) LDB1 protein levels in undifferentiated and 72-hour PMA + VD3–differentiated THP-1 cells as detected by western blotting. VCP was used as loading control. The molecular protein mass is indicated. (C) LDB1 (top) and IgG (bottom) ChIP-seq tracks of 12-hour PMA + VD3–differentiated THP-1 cells 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 positions of the PU.1 promoter (PP) and the URE. (D) LDB1 binding to the PU.1 locus increases with differentiation. ChIP-qPCR with primers specific for the PU.1 URE (left and middle) or an adjacent negative control region (right), which was chosen based on lack of a LDB1 binding peak in the LDB1 ChIP-seq data shown in panel C. Chromatin was from undifferentiated (left) or 12-hour PMA + VD3–differentiated THP-1 cells (middle and right) that was precipitated with an antibody against LDB1 (red bars) or an IgG control antibody (black bars). (E) LDB1 chromatin binding requires PU.1. ChIP-qPCR with primers for the URE and chromatin template of 72-hour Dox-treated THP-1 cells expressing a negative control shRNA (left) or shRNAs against PU.1 (middle and right). The cells were also treated with PMA + VD3 12 hours before harvest to induce LDB1 binding. Error bars in panels D and E represent SD of the mean. n.s., not significant; **P < .01; ***P < .001 (significantly different means calculated by the Student t test).