Three NF-kappa B sites in the I kappa B-alpha promoter are required for induction of gene expression by TNF alpha

Nucleic Acids Res. 1994 Sep 11;22(18):3787-92. doi: 10.1093/nar/22.18.3787.

Abstract

NF-kappa B was first identified as a postive regulator which bound to a 10 bp sequence in the first intron of the Ig kappa light chain gene. Further characterization of this transcription factor has revealed that NF-kappa B is kept from binding to its consensus sequence by its inhibitor, IkB-alpha, which retains NF-kappa B in the cytoplasm. Upon receiving various extra- and intracellular signals, I kappa B-alpha is rapidly degraded and NF-kappa B is induced to translocate into the nucleus. This process precedes the rapid induction of I kappa B-alpha mRNA and protein. To understand how I kappa B-alpha is replenished, we have cloned and sequenced the 5' flanking region of the I kappa B-alpha gene and have identified the transcription start site and three NF-kappa B sites in this region. Further characterization of these NF-kappa 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 I kappa B-alpha promoter-CAT reporter construct and all three NF-kappa B sites are required for full activation of the I kappa B-alpha gene following stimulation with TNF-alpha. Our data confirm a transcriptional autoregulatory loop involved in maintaining appropriate NF-kappa B and I kappa B-alpha levels in the cell.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Binding Sites
  • Cloning, Molecular
  • DNA / metabolism
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Gene Expression Regulation / drug effects
  • Gene Expression Regulation / physiology*
  • HeLa Cells
  • Humans
  • I-kappa B Proteins*
  • Molecular Sequence Data
  • Mutation / physiology
  • NF-KappaB Inhibitor alpha
  • NF-kappa B / antagonists & inhibitors*
  • NF-kappa B / metabolism*
  • Promoter Regions, Genetic / genetics*
  • RNA, Messenger / biosynthesis
  • Sequence Analysis, DNA
  • Transcription, Genetic
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • DNA-Binding Proteins
  • I-kappa B Proteins
  • NF-kappa B
  • NFKBIA protein, human
  • RNA, Messenger
  • Tumor Necrosis Factor-alpha
  • NF-KappaB Inhibitor alpha
  • DNA

Associated data

  • GENBANK/U08468