Variation in Genome-Wide NF-κB RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile

Abstract

NF-κB transcription factors are master regulators of the innate immune response. Activated downstream of pathogen recognition receptors, they regulate the expression of genes to help fight infections as well as recruit the adaptive immune system. NF-κB responds to a wide variety of signals, but the processes by which stimulus specificity is attained remain unclear. In this article, we characterized the response of one NF-κB member, RELA, to four stimuli mimicking infection in human nasopharyngeal epithelial cells. Comparing genome-wide RELA binding, we observed stimulus-specific sites, although most sites overlapped across stimuli. Specifically, the response to poly I:C (mimicking viral dsRNA and signaling through TLR3) induced a distinct RELA profile, binding in the vicinity of antiviral genes and correlating with corresponding gene expression. This group of binding sites was also enriched in IFN regulatory factor motifs and showed overlapping with IFN regulatory factor binding sites. A novel NF-κB target, , was further validated and showed TLR3-specific activation. This work showed that some RELA DNA binding sites varied in activation response following different stimulations and that interaction with more specialized factors could help achieve this stimulus-specific activity. Our data provide a genomic view of regulated host response to different pathogen stimuli.

Changes in H3K27ac following lipopolysaccharide stimulation of nasopharyngeal epithelial cells

“…Next, we examined the effect of the changes in H3K27ac on gene expression, using data from RNA-seq experiment generated previously [14]. First, we noticed that the LPS-increased enhancers identified were located closer to genes’ transcription start sites (TSS) as compared to non LPS-increased H3K27ac peaks (Fig.…”

“…We then used the RELA ChIP-seq data we generated previously [14] to investigate overlap between RELA binding and LPS-increased H3K27ac regions. Interestingly, 82.3% (515 out of 626) of the LPS-increased peaks overlapped with RELA binding sites in the same conditions (Fig.…”

“…Cells were treated with LPS or with fresh medium (Control - no treatment) for 80 min in Detroit 562 cells. ChIP-seq was performed according to the library-on-beads protocol from Wallerman et al [32] using the NEBnext DNA library prep kit (New England Biolabs) as described previously [14] with the following modifications. 5 μg of H3K27ac antibody (Active motif, reference 39,138) was mixed with 50 uL of Dynabeads Protein G before immunoprecipitation.…”

“…The Gene expression data used in this study consists of RNA-seq experiments performed in control and LPS (treatment for 100 min with 1μg/mL) condition in the same Detroit 562 cells [14] and available under the accession number GSE91019 in the NCBI’s Gene Expression Omnibus (GEO) depository.…”

“…We have recently investigated the response of one of the master regulator of innate immunity, Nuclear Factor kappa B (NF-kB) member RELA, to the same stimulus and in the same cell line [14]. Thus, we were able to examine LPS-induced H3K27ac regions identified with regards to binding of RELA, one of the NF-kB member activated downstream of TLR4.…”