Coordinated binding of NF-kappaB family members in the response of human cells to lipopolysaccharide

Abstract

The NF-kappaB family of transcription factors plays a critical role in numerous cellular processes, particularly the immune response. Our understanding of how the different NF-kappaB subunits act coordinately to regulate gene expression is based on a limited set of genes. We used genome-scale location analysis to identify targets of all five NF-kappaB proteins before and after stimulation of monocytic cells with bacterial lipopolysaccharide (LPS). In unstimulated cells, p50 and p52 bound to a large number of gene promoters that were also occupied by RNA polymerase II. After LPS stimulation, additional NF-kappaB subunits bound to these genes and to other genes. Genes that became bound by multiple NF-kappaB subunits were the most likely to show increases in RNA polymerase II occupancy and gene expression. This study identifies NF-kappaB target genes, reveals how the different NF-kappaB proteins coordinate their activity, and provides an initial map of the transcriptional regulatory network that underlies the host response to infection.

Fig. 1.

The overlap between genes bound before and after LPS stimulation by each NF-κB subunit and all NF-κB subunits together, displayed as Venn diagrams. The number of genes occupied by each subunit exclusively in unstimulated cells, exclusively in LPS-stimulated cells, or occupied in both are indicated. All binding events are significant at a P value threshold of 0.002.

Fig. 2.

Co-occupation of promoters by NF-κB and RNA polymerase II in unstimulated cells. (A) DNA binding of RNA polymerase II and NF-κB subunits in unstimulated cells. The genes are sorted by RNA polymerase II binding ratio (marked on the left). Genes occupied by each NF-κB subunit are marked by a blue dash. (B) Binding and expression data for 135 genes that are bound by at least one NF-κB subunit in unstimulated cells and annotated in the expression data. Expression levels are relative to the average expression of genes across 79 different human tissue or cell types. Red, higher than average expression; green, lower than average, according to the scale shown to the right. Selected overexpressed and underexpressed genes are named on the right.

Fig. 3.

After LPS stimulation, NF-κB subunits bind to promoters that were previously bound by p50. Fraction of bound genes in activated cells that were prebound by p50 before stimulation at two significance levels, 0.002 (dark blue) and 0.01 (light blue). All NF-κB subunits bound to significant numbers of p50 target genes. In contrast, the binding pattern of E2F4 was not related to p50 binding and followed the pattern expected by chance (randomized data).

Fig. 4.

Recruitment of multiple NF-κB family members leads to increased RNA polymerase II occupancy and gene expression. (A) NF-κB binding at genes to which RNA polymerase II is recruited and to genes where it is lost (top 5% of genes for both categories). The change in RNA polymerase binding is represented as a ratio, with red indicating an increase and green indicating a decrease, according to the scale on the left. Only genes bound by RNA polymerase II in either unstimulated or activated cells were used. Gray, binding of NF-κB subunits at P values < 0.01; blue, binding at P values < 0.002. (B) NF-κB binding at genes that are up-regulated or down-regulated over time in response to LPS in U937 cells. Genes were either up-regulated by 2-fold or down-regulated by 2-fold at two consecutive time points. Red, increase in expression; green, decrease in expression, according to the scale on the left. (C) The percentage of genes to which 1, 2, 3, or 4 or more different NF-κB family members are recruited that show an increase in RNA polymerase II occupancy (blue bars) or an increase in RNA abundance (gray bars) after LPS stimulation. (D) The percentage of genes whose promoters contain 1, 2, 3, or 4 or more NF-κB binding motifs that show an increase in RNA polymerase II occupancy (blue bars) or an increase in RNA abundance (gray bars) after LPS stimulation. The binding motif was defined as GGGRNNYYCC and was counted if present within or 200 bp either side of the promoter element represented on the array.

Fig. 5.

NF-κB target genes in the host response to pathogens. Changes in the expression of 61 NF-κB target genes during the response of U937 cells to LPS and the response of macrophages to LPS and different bacterial species. The genes have previously been identified as regulated in response to pathogens (10, 11). Genes named in red have not previously been identified as NF-κB targets. Expression changes are shown over time (0–24 h) and are colored according to the scale shown to the left. EC, Escherichia coli; EHEC, Enterohemorrhagic Escherichia coli; ST, Salmonella typhi; STm, Salmonella typhimurium; SA, Staphylococcus aureus; LM, Listeria monocytogenes; MTB, Mycobacterium tuberculosis; MB, Mycobacterium bovis.