Previously unknown TF combinations are at play in promoter-distal looping interactions. (A) Schematic of promoter-distal looping interaction. TF complex(es) can be seen to be bound to both the promoter (P) and enhancer (E) elements; upon transcriptional activation, these regulatory elements are brought within close proximity allowing the interaction of these TF complexes. (B) Heatmap showing hierarchical clustering of the TFs bound at promoter and distal elements. To control for numbers of binding peaks per experiment, the data were normalized to the average of 32 iterations of a randomized selection of total ChIP-Seq peaks, weighted according to the number of peaks per TF. (C) Co-immunoprecipitation data (Co-IP) showing protein-protein interactions between PU.1/GFI1B, MEIS1/GFI1B, GFI1B/RUNX1, and RUNX1/MEIS1. Expression constructs were transfected into 293T cells and putative protein interactions assayed by Co-IP/western blot (WB) analysis. (i) Following transfection of GFI1B and SPI1/PU.1, lysates were immunoprecipitated (IP) with an anti-PU.1 antibody and immune complexes were then analyzed to detect the presence of GFI1B. (ii) After transfection of GFI1B and flag-tagged MEIS1, lysates were immunoprecipitated with an anti-FLAG antibody (MEIS1), followed by western blot using anti-GFI1B antibody. (iii) MYC-tagged RUNX1 and GFI1B were transfected, and the lysates were immunoprecipitated with an anti-GFI1B antibody, followed by western blot using anti-RUNX1 antibody. (iv) MYC-tagged RUNX1 and MEIS1 were transfected, and the lysates were immunoprecipitated with anti-MYC antibody (RUNX1) and immune complexes were analyzed by western blots using an anti-MEIS1 antibody. IgG, immunoglobulin G.