title: In vivo binding of NF-kappaB to the IkappaBbeta promoter is insufficient for transcriptional activation.
abstract: Despite certain structural and biochemical similarities, differences exist in the function of the NF-kappaB (nuclear factor kappaB) inhibitory proteins IkappaBalpha (inhibitory kappaBalpha) and IkappaBbeta. The functional disparity arises in part from variance at the level of gene regulation, and in particular from the substantial induction of IkappaBalpha, but not IkappaBbeta, gene expression post-NF-kappaB activation. In the present study, we probe the differential effects of IL (interleukin)-1beta on induction of IkappaBalpha and perform the first characterization of the human IkappaBbeta promoter. A consensus NF-kappaB-binding site, capable of binding NF-kappaB both in vitro and in vivo, is found in the IkappaBbeta gene 5' flanking region. However, the IkappaBbeta promoter was not substantially activated by pro-inflammatory cytokines, such as IL-1beta and tumour necrosis factor alpha, that are known to cause strong activation of NF-kappaB. Furthermore, in contrast with IkappaBalpha, NF-kappaB activation did not increase expression of endogenous IkappaBbeta as assessed by analysis of mRNA and protein levels. Unlike kappaB-responsive promoters, IkappaBbeta 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 transcription factor IIH engagement, a prerequisite for RNA polymerase II phosphorylation and transcriptional initiation. Differences in the conformation of promoter-bound NF-kappaB may underlie the variation in the ability to engage the basal transcriptional apparatus at the IkappaBbeta and kappaB-responsive promoters. This accounts for the differential expression of IkappaB family members in response to NF-kappaB activation and furthers our understanding of the mechanisms involved in transcription factor activity and IkappaBbeta gene regulation.
authors.affiliation: UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Griffin
authors.forename: Paul N
authors.initials: PN
authors.lastname: Moynagh
mesh_terms: D024506:5' Flanking Region / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics*; D053583:Interleukin-1beta / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D012319:RNA Polymerase II / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism; D051758:Transcription Factor TFIIH / Q000378:metabolism; D024363:Transcription Initiation Site; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics*; D014162:Transfection
chemical_list: C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D053583:Interleukin-1beta; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA; D051758:Transcription Factor TFIIH; D008156:Luciferases; D012319:RNA Polymerase II