Fig. 4: Selective binding of Rel and RelA in mouse BMDMs examined by ChIP–seq.

a, ChIP–seq was performed with RelA and Rel antibodies in lipid-A-stimulated (1 h) BMDMs (five and three biological replicates, respectively). The 6,700 strongest peaks for each antibody were merged, yielding 8,134 peaks. Rel/RelA RPKM ratios are plotted on the y axis, with the 8,134 peaks along the x axis (displayed as the percentage of total peaks). On the right, numbers of peaks with different ranges of RPKM ratios are shown. b, The most enriched motifs from de novo motif analyses performed with 300 peaks representing the largest or smallest ratios and 300 peaks with intermediate ratios. c, The 8,134 peaks were divided into 27 equal bins based on Rel/RelA RPKM ratios. The enrichment of three consensus NF-κB motifs within HOMER, and the consensus NF-κB motif from b, was then examined. d, Among the 8,134 peaks, 1,050 were annotated to promoter regions (−1,500 bp to +500 bp relative to the transcription start site). The Rel/RelA RPKM ratios of these peaks were plotted and used to rank the peaks in the promoter regions of Rel-dependent genes (Fig. 1b). The table lists the Rel-dependent genes, the peak distance from the transcription start site, the RPKM rank of each peak, the peak rank percentile, the Rel/RelA RPKM ratio and the annotated peak number. e, IGV tracks are shown for two Rel-dependent genes (Il12b and Tnfsf9) and for one promoter peak with strong RelA binding in comparison with Rel (Camp). Tracks are for Rel, RelA and p50 from lipid-A-stimulated cells (1 h). f, Rel/RelA RPKM ratios calculated for 65 previously defined promoter peaks for lipid-A-inducible genes that coincided with recognizable NF-κB motifs (Supplementary Table 4). These peaks were separated into two groups based on PBM z scores for Rel homodimer binding: z > 9 (n = 40) and z < 9 (n = 25). The Rel/RelA RPKM ratios for each peak within each group are shown in the box plot. The lower and upper edges of the box represent the first and third quartiles. The upper whisker extends from the third quartile to the maxima within 1.5 times the interquartile range. The lower whisker extends from the first quartile to the minima within 1.5 times the interquartile range. Any values that fell beyond the whiskers were considered to be outliers. The statistical significance of the difference between the two groups (P value determined by Welch’s two sample t-test (two sided) is shown at the top.