Identification by in vivo genomic footprinting of a transcriptional switchcontaining NF-kappa B and Sp1 that regulates the I kappa B alpha promoter

In unstimulated cells, NF-kappa B transcription factors are retained in the cytoplasm by inhibitory I kappa B proteins. Upon stimulation by multiple i nducers including cytokines or viruses, I kappa B alpha is rapidly phosphor ylated and degraded, resulting in the release of NP-kappa B and the subsequ ent increase in NF-kappa B-regulated gene expression. I kappa B alpha gene expression is also regulated by an NF-kappa B autoregulatory mechanism, via NF-kappa B binding sites in the I kappa B alpha promoter. In previous stud ies, tetracycline-inducible expression of transdominant repressors of I kap pa B alpha (TD-I kappa B alpha) progressively decreased endogenous I kappa B alpha protein levels. In the present study, we demonstrate that expressio n of TD-I kappa B alpha blocked phorbol myristate acetate-phytohemagglutini n or tumor necrosis factor alpha-induced I kappa B alpha gene transcription and abolished NF-kappa B DNA binding activity, due to the continued cytopl asmic sequestration of RelA(p65) by TD-I kappa B alpha. In vivo genomic foo tprinting 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 th e I kappa B alpha promoter. In vivo protection of both sites was inhibited by tetracycline-inducible TD-I kappa B alpha expression. Prolonged NF-kappa B binding and a temporal snitch in the composition of NF-kappa B complexes bound to the -63 to -53 kappa B1 site of the I kappa B alpha promoter were also observed; with time after induction, decreased levels of transcriptio nally 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 abo lished transcription factor binding to the respective sites and the inducib ility of the I kappa B alpha promoter in transient transfection studies. Th ese observations provide the first in vive characterization of a promoter p roximal transcriptional snitch involving NF-kappa B and Sp1 that is essenti al for autoregulation of the I kappa B alpha promoter.