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Molecular and Cellular Biology, September 1999, p. 6140-6153, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Identification by In Vivo Genomic Footprinting of a Transcriptional Switch Containing NF-kappa B and Sp1 That Regulates the Ikappa Balpha Promoter

Michèle Algarté, Hakju Kwon, Pierre Génin, and John Hiscott*

Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, and Departments of Microbiology & Immunology, Medicine, and Oncology, McGill University, Montreal, Canada H3T 1E2

Received 8 April 1999/Returned for modification 10 May 1999/Accepted 9 June 1999

In unstimulated cells, NF-kappa B transcription factors are retained in the cytoplasm by inhibitory Ikappa B proteins. Upon stimulation by multiple inducers including cytokines or viruses, Ikappa Balpha is rapidly phosphorylated and degraded, resulting in the release of NF-kappa B and the subsequent increase in NF-kappa B-regulated gene expression. Ikappa Balpha gene expression is also regulated by an NF-kappa B autoregulatory mechanism, via NF-kappa B binding sites in the Ikappa Balpha promoter. In previous studies, tetracycline-inducible expression of transdominant repressors of Ikappa Balpha (TD-Ikappa Balpha ) progressively decreased endogenous Ikappa Balpha protein levels. In the present study, we demonstrate that expression of TD-Ikappa Balpha blocked phorbol myristate acetate-phytohemagglutinin or tumor necrosis factor alpha-induced Ikappa Balpha gene transcription and abolished NF-kappa B DNA binding activity, due to the continued cytoplasmic sequestration of RelA(p65) by TD-Ikappa Balpha . In vivo genomic footprinting revealed stimulus-responsive protein-DNA binding not only to the -63 to -53 kappa B1 site but also to the adjacent -44 to -36 Sp1 site of the Ikappa Balpha promoter. In vivo protection of both sites was inhibited by tetracycline-inducible TD-Ikappa Balpha expression. Prolonged NF-kappa B binding and a temporal switch in the composition of NF-kappa B complexes bound to the -63 to -53 kappa B1 site of the Ikappa Balpha promoter were also observed; with time after induction, decreased levels of transcriptionally active p50-p65 and increased p50-c-Rel heterodimers were detected at the kappa B1 site. Mutation of either the kappa B1 site or the Sp1 site abolished transcription factor binding to the respective sites and the inducibility of the Ikappa Balpha promoter in transient transfection studies. These observations provide the first in vivo characterization of a promoter proximal transcriptional switch involving NF-kappa B and Sp1 that is essential for autoregulation of the Ikappa Balpha promoter.

* Corresponding author. Mailing address: Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec, Canada H3T1E2. Phone: (514) 340-8222, ext. 5265. Fax: (514) 340-7576. E-mail: mijh{at}musica.mcgill.ca.

Molecular and Cellular Biology, September 1999, p. 6140-6153, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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