Basal expression of IkappaBalpha is controlled by the mammalian transcriptional repressor RBP-J (CBF1) and its activator Notch1.
By using the hepatic stellate cell (HSC) as a paradigm for cells that undergo long term re-programming of NF-kappaB-dependent transcription, we have determined a novel mechanism by which mammalian cells establish their basal NF-kappaB activity. Elevation of NF-kappaB activity during HSC activation is accompanied by induction of CBF1 expression and DNA binding activity. We show that the transcriptional repressor CBF1 interacts with a dual NF-kappaB/CBF1-binding site (kappaB2) in the IkappaBalpha promoter. Nucleotide substitutions that disrupt CBF1 binding to the kappaB2 site result in an elevation of IkappaBalpha promoter activity and loss of responsiveness of the promoter to a transfected CBF1 reporter vector. Overexpression of CBF1 in COS1 cells was associated with markedly reduced IkappaBalpha protein expression and elevated NF-kappaB DNA binding activity. CBF1-induced repression of IkappaBalpha promoter activity was reversed in HSC transfected with the Notch1 intracellular domain (NICD). The ability of NICD to enhance IkappaBalpha gene transcription was confirmed in COS1 cells and was found to be dependent on an intact RAM domain of NICD that has been shown previously to help mediate the interaction of NICD with CBF1. One of the mechanisms by which NICD is thought to convert CBF1 into an activator of transcription is via the recruitment of transcriptional co-activators/histone acetylases to gene promoters. Co-transfection of HSC with NICD and p53 caused a diminution of IkappaBalpha promoter activity, by contrast overexpression of p300 enhanced IkappaBalpha promoter function. Taken together, these data suggest that basal IkappaBalpha expression (and as a consequence NF-kappaB activity) is under the control of the various components of the CBF1/Notch signal transduction pathway.
Oakley
Fiona
F
Liver Group, Division of Infection, Inflammation and Repair, University of Southampton, United Kingdom.
Mann
Jelena
J
Ruddell
Richard G
RG
Pickford
Jessica
J
Weinmaster
Gerry
G
Mann
Derek A
DA
D000818:Animals; D019556:COS Cells; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D020672:I-kappa B Proteins / Q000096:biosynthesis* / Q000235:genetics; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D008565:Membrane Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; C497763:Notch1 protein, rat; D009687:Nuclear Proteins; C497572:Rbpjl2 protein, rat; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D014157:Transcription Factors; D000072000:NF-KappaB Inhibitor alpha