Identification by In Vivo Genomic Footprinting of a Transcriptional Switch Containing NF-κB and Sp1 That Regulates the IκBα Promoter

Abstract

In unstimulated cells, NF-κB transcription factors are retained in the cytoplasm by inhibitory IκB proteins. Upon stimulation by multiple inducers including cytokines or viruses, IκBα is rapidly phosphorylated and degraded, resulting in the release of NF-κB and the subsequent increase in NF-κB-regulated gene expression. IκBα gene expression is also regulated by an NF-κB autoregulatory mechanism, via NF-κB binding sites in the IκBα promoter. In previous studies, tetracycline-inducible expression of transdominant repressors of IκBα (TD-IκBα) progressively decreased endogenous IκBα protein levels. In the present study, we demonstrate that expression of TD-IκBα blocked phorbol myristate acetate-phytohemagglutinin or tumor necrosis factor alpha-induced IκBα gene transcription and abolished NF-κB DNA binding activity, due to the continued cytoplasmic sequestration of RelA(p65) by TD-IκBα. In vivo genomic footprinting revealed stimulus-responsive protein-DNA binding not only to the −63 to −53 κB1 site but also to the adjacent −44 to −36 Sp1 site of the IκBα promoter. In vivo protection of both sites was inhibited by tetracycline-inducible TD-IκBα expression. Prolonged NF-κB binding and a temporal switch in the composition of NF-κB complexes bound to the −63 to −53 κB1 site of the IκBα 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 κB1 site. Mutation of either the κB1 site or the Sp1 site abolished transcription factor binding to the respective sites and the inducibility of the IκBα promoter in transient transfection studies. These observations provide the first in vivo characterization of a promoter proximal transcriptional switch involving NF-κB and Sp1 that is essential for autoregulation of the IκBα promoter.

ACKNOWLEDGMENTS

M.A., H.J.K., and P.G. contributed equally to this work.

We thank Alain Israël and Ron Hay for reagents used in this study. We also thank members of the Molecular Oncology Group, Lady Davis Institute for helpful discussions.

This research was supported by grants from the Medical Research Council of Canada and CANFAR. M.A. and P.G. were supported by FRSQ postdoctoral fellowships, H.K. was supported by an FCAR studentship and J.H. was supported by an MRC Senior Scientist award.