Nucleic Acids Research, 1994, Vol. 22, No. 18 3787-3792
© 1994
MOLECULAR BIOLOGY |
Three NF-
B sites in the IB-
promoter are required for induction of gene expression by TNF
1Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA 2Lineberger Comprehensive Cancer Research Center and Department of Biology, University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA
*To whom correspondence should be addressed
Received April 13, 1994. Revised August 3, 1994. Accepted August 3, 1994.
NF-
B was first identified as a postive regulator which bound to a 10 bp sequence in the first intron of the Ig light chain gene. Further characterization of this transcription factor has revealed that NF-B is kept from binding to its consensus sequence by its inhibitor, IkB-
, which retains NF-B in the cytoplasm. Upon receiving various extra- and intracellular signals, IB- is rapidly degraded and NF-B is induced to translocate into the nucleus. This process precedes the rapid induction of I;B- mRNA and protein. To understand how IB- is replenished, we have cloned and sequenced the 5' flanking region of the IB- gene and have identified the transcription start site and three NF-B sites in this region. Further characterization of these NF-B sites show that they have different affinities for three specific protein complexes which we identify here to consist of various members of the Rel family. In transient assays, cotransfection with a p65 expression vector is able to activate an IB- promoter- CAT reporter construct and all three NF-B sites are required for full activation of the IB- gene following stimulation with TNF-. Our data confirm a transcriptional autoregulatory loop involved in maintaining appropriate NF-B and IB- levels in the cell.
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