In vivo binding of NF-{kappa}B to the I{kappa}B{beta} promoter is insufficient for transcriptional activation
Résumé
Despite certain structural and biochemical similarities, differences exist in the function of the NF{kappa}B inhibitory proteins I{kappa}B{alpha} and I{kappa}B{beta}. The functional disparity arises in part from variance at the level of gene regulation, and in particular from the substantial induction of I{kappa}B{alpha}, but not I{kappa}B{beta}, gene expression post-NF{kappa}B activation. Here we probe the differential effects of IL-1b on induction of I{kappa}B{alpha} and I{kappa}B{beta}, and more especially perform the first characterisation of the human I{kappa}B{beta} promoter. A consensus NFkB-binding site, capable of binding NF{kappa}B both in vitro and in vivo is found in the I{kappa}B{beta} gene 5‘ flanking region. However, the I{kappa}B{beta} promoter was not substantially activated by pro-inflammatory cytokines, such as IL-1b and TNF{alpha}, that are known to cause strong activation of NF{kappa}B. Furthermore, in contrast to I{kappa}B{alpha}, NF{kappa}B activation did not increase expression of endogenous I{kappa}B{beta} as assessed by analysis of mRNA and protein levels. Unlike kB-responsive promoters, I{kappa}B{beta} promoter-bound p65 inefficiently recruits RNA polymerase II, which stalls at the promoter. We present evidence that this stalling is likely due to the absence of TFIIH engagement, a prerequisite for RNA polymerase II phosphorylation and transcriptional initiation. Differences in the conformation of promoter-bound NF{kappa}B may underlie the variation in the ability to engage the basal transcriptional apparatus at the I{kappa}B{beta} and kB-responsive promoters. This accounts for the differential expression of I{kappa}B family members in response to NF{kappa}B activation and furthers our understanding of the mechanisms involved in transcription factor activity and I{kappa}B{beta} gene regulation.
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