NFIX and PU.1 cobind regions of open chromatin, especially super-enhancers. (A) The ability of NFIX and PU.1 to physically interact was tested by co-IP following transient transfection in 293T cells. Proteins were either unexpressed (lane 1), expressed singly (lanes 2 and 3) or coexpressed (lane 4) as indicated at top, and expression was verified (inputs). IP of MYC-NFIX was selectively able to co-IP FLAG-PU.1 (middle panel, lower blot, lane 4) and reciprocally IP of FLAG-PU.1 was able to co-IP MYC-NFIX (bottom, upper blot, lane 4). (B) Transcription factor footprint plot for NFIX and PU.1 using HPC5 ATAC-seq data. Top: average Tn5 cutting frequency. Bottom: heatmap of Tn5 cutting frequency. (C) Genomic annotation pie chart for NFIX-PU.1 peaks. (D) Signal plots of ATAC-seq and H3K27ac for NFIX-PU.1 cobound peaks and NFIX background peaks in HPC5 cells. Background represents NFIX peaks not overlapping with PU.1. Top: average of normalized signal. Bottom: heatmap of epigenetic signals on NFIX-PU.1 peaks and background peaks. (E) Venn diagram showing overlaps between super-enhancers and NFIX-PU.1 peaks. (F) Representative tracks showing NFIX and PU.1 peak overlap near target genes, Pdcd1, Socs3, and Meis1. An asterisk (∗) is included to indicate differential PU.1 peak signals in Nfix−/− HPC5 ChIP-seq samples (∗P value <1 × 10−5). co-IP, coimmunoprecipitation.
