An Expanded Interplay Network between NF-κB p65 (RelA) and E2F1 Transcription Factors: Roles in Physiology and Pathology

Abstract

Transcription Factors (TFs) are the main regulators of gene expression, controlling among others cell homeostasis, identity, and fate. TFs may either act synergistically or antagonistically on nearby regulatory elements and their interplay may activate or repress gene expression. The family of NF-κB TFs is among the most important TFs in the regulation of inflammation, immunity, and stress-like responses, while they also control cell growth and survival, and are involved in inflammatory diseases and cancer. The family of E2F TFs are major regulators of cell cycle progression in most cell types. Several studies have suggested the interplay between these two TFs in the regulation of numerous genes controlling several biological processes. In the present study, we compared the genomic binding landscape of NF-κB RelA/p65 subunit and E2F1 TFs, based on high throughput ChIP-seq and RNA-seq data in different cell types. We confirmed that RelA/p65 has a binding profile with a high preference for distal enhancers bearing active chromatin marks which is distinct to that of E2F1, which mostly generates promoter-specific binding. Moreover, the RelA/p65 subunit and E2F1 cistromes have limited overlap and tend to bind chromatin that is in an active state even prior to immunogenic stimulation. Finally, we found that a fraction of the E2F1 cistrome is recruited by NF-κΒ near pro-inflammatory genes following LPS stimulation in immune cell types.

Keywords: E2F1; NF-κB; genomics; lung cancer; transcription factor interactions/interplay.

Figure 1

(A) Venn plots depicting the common E2F1-p65, the E2F1-specific, and the p65-specific binding regions (cistromes) in HeLa cancer cells. (B–D) Aggregate plots (up) and heatmaps (down) illustrating the signal distribution for E2F1, p65, H3K27ac, and DNaseI-seq accessibility around the E2F1-specific, the common E2F1-p65, and the p65-specific binding regions, respectively.

Figure 2

(A–C) The figure panels depict the genomic distribution of E2F1-specific, E2F1-p65 common, and p65-specific peaks respectively in HeLa cells. (D,E) The figure panels show the top motif for E2F1-specific and p65-specific peaks, respectively. (F–H) The figure panels illustrate the top biological processes for E2F1-specific, E2F1-p65 common, and p65-specific peaks, respectively.

Figure 3

(A) Shown are E2F1-p65 common (up) and p65-specific peaks categorised according to whether they bear p300 binding prior and upon TNFα treatment in HeLa cancer cells. (B) Shown is the p300 signal around the common p65-E2F1 and p65-only peaks prior and upon TNFα treatment.

Figure 4

The percentage of TNFα-inducible genes in HeLa cells targeted by p65 alone or by E2F1-p65.

Figure 5

Upset plot showing multi-way comparisons between p65 and E2F1 binding sites in HeLa and U2OS cells.

Figure 6

(A) Venn diagram depicting comparisons between E2F1 and p65 peaks prior and upon treatment with LPS for 120 min. (B) Aggregate plots (up) and heatmaps (down) depicting E2F1, p65, and H3K27ac levels around the E2F1 peaks at basal state (left) and 120 min after LPS treatment (right). (C) Aggregate plots (up) and heatmaps (down) showing E2F1, p65, and HK327ac levels around the 78 inducible E2F1 peaks 120 min after LPS treatment. (D) Closest genes to E2F1 inducible peaks 120 min after LPS treatment. (E) The genomic distribution of E2F1 inducible peaks 120 min after LPS treatment.

Figure 7

Model for the proper transcriptional regulation of the E2F1-p65 target genes. (A) Prior to stimulation, intergenic or intragenic active regulatory elements, which have all the necessary “signatures” for active regulatory elements, are looped to the promoter of the target genes, prior to stimulation. (B) Upon stimulation, the loop is preserved, p65 is recruited to the active regulatory elements activating its target genes. At the same time, for the p65-E2F1 target genes, (C) E2F1 could bind first and (D) then p65 is recruited. Alternatively, (B) p65 which is already bound on chromatin, (D) can attract and recruit E2F1 on chromatin. Otherwise, (D) E2F1 and p65 bind chromatin independently of each other (Please see text for more details).