Binding loci of RelA-containing nuclear factor-kappaB dimers in promoter regions of PHM1-31 myometrial smooth muscle cells

Human parturition is associated with many pro-inflammatory mediators which are regulated by the nuclear factor-kappaB (NF-kappa B) family of transcription factors. In the present study, we employed a ChIP-on-chip approach to define genomic loci within chromatin of PHM1-31 myometrial cells that were occupied by RelA-containing NF-kappa B dimers in response to a TNF stimulation of 1 h. In TNF-stimulated PHM1-31 cells, anti-RelA serum enriched 13 300 chromatin regions; importantly, 11 110 regions were also enriched by anti-RelA antibodies in the absence of TNF. DNA sequences in these regions, from both unstimulated or TNF-stimulated PHM1-31 cultures, were associated with genic regions including I kappa B alpha, COX-2, IL6RN, Jun and KCNMB3. TNF-induced binding events at a consensus kappa B site numbered 1667; these were represented by 112 different instances of the consensus kappa B motif. Of the 1667 consensus kappa B motif occurrences, 770 (46.2%) were identified within intronic regions. In unstimulated PHM1-31 cells, anti-RelA-serum-enriched regions were associated with sequences corresponding to open reading frames of ion channel subunit genes including CACNB3 and KCNB1. Moreover, in unstimulated cells, the consensus kappa B site was identified 2116 times, being defined by 103 different sequence instances of this motif. Of these 2116 consensus kappa B motifs, 1089 (51.5%) were identified within intronic regions. Parallel expression array analyses in PHM1-31 cultures demonstrated that TNF stimulated a >2-fold induction in 51 genes and a fold repression of >1.5 in 18 others. We identified 14 anti-RelA-serum-enriched genomic regions that correlated with 17 TNF-inducible genes, such as COX2, Egr-1, Jun, I kappa B alpha and IL6, as well as five regions associated with TNF-mediated gene repression, including Col1A2.