Identification by in vivo genomic footprinting of a transcriptional switch containing NF-kappaB and Sp1 that regulates the IkappaBalpha promoter.

Abstract

In unstimulated cells, NF-kappaB transcription factors are retained in the cytoplasm by inhibitory IkappaB proteins. Upon stimulation by multiple inducers including cytokines or viruses, IkappaBalpha is rapidly phosphorylated and degraded, resulting in the release of NF-kappaB and the subsequent increase in NF-kappaB-regulated gene expression. IkappaBalpha gene expression is also regulated by an NF-kappaB autoregulatory mechanism, via NF-kappaB binding sites in the IkappaBalpha promoter. In previous studies, tetracycline-inducible expression of transdominant repressors of IkappaBalpha (TD-IkappaBalpha) progressively decreased endogenous IkappaBalpha protein levels. In the present study, we demonstrate that expression of TD-IkappaBalpha blocked phorbol myristate acetate-phytohemagglutinin or tumor necrosis factor alpha-induced IkappaBalpha gene transcription and abolished NF-kappaB DNA binding activity, due to the continued cytoplasmic sequestration of RelA(p65) by TD-IkappaBalpha. In vivo genomic footprinting revealed stimulus-responsive protein-DNA binding not only to the -63 to -53 kappaB1 site but also to the adjacent -44 to -36 Sp1 site of the IkappaBalpha promoter. In vivo protection of both sites was inhibited by tetracycline-inducible TD-IkappaBalpha expression. Prolonged NF-kappaB binding and a temporal switch in the composition of NF-kappaB complexes bound to the -63 to -53 kappaB1 site of the IkappaBalpha 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 kappaB1 site. Mutation of either the kappaB1 site or the Sp1 site abolished transcription factor binding to the respective sites and the inducibility of the IkappaBalpha promoter in transient transfection studies. These observations provide the first in vivo characterization of a promoter proximal transcriptional switch involving NF-kappaB and Sp1 that is essential for autoregulation of the IkappaBalpha promoter.