Identification and characterization of nuclear factor kappa B binding sites in the murine bcl-x promoter

Signal transduction pathways that mediate neuronal commitment to apoptosis involve the nuclear factor kappa B (NF-kappa B) transcription factor. Bcl-X -L is a potent regulator of apoptosis in the CNS and is highly expressed in the developing and adult brain. We identified three putative NF-kappa B DN A binding sequences clustered upstream of the brain-specific transcription start site in the upstream promoter region. Recombinant p50/p50 and NF-kapp a B proteins from nuclear extracts bound to these sites as determined by el ectrophoretic mobility shift assay and biotin-oligonucleotide/streptavidin affinity assays. NF-kappa B overexpression, coupled with bcl-x promoter/rep orter assays using a series of murine bcl-x promoter and deletion mutants, has identified the downstream 1.1 kb of the bcl-x promoter as necessary for basal promoter activity and induction by NF-kappa B. The mutagenic removal of NF-kappa B binding sites individually or in combination revealed altere d response patterns to p49/p65 and p50/p65 overexpression. These results su pport the hypothesis that NF-kappa B can act to enhance Bcl-X-L expression via highly selective interactions, where NF-kappa B binding and bcl-x promo ter activation are dependent on both DNA binding site sequence and NF-kappa B subunit composition. Our data suggest that molecular events associated w ith NF-kappa B promote regulation of neuronal apoptosis in the developing o r injured CNS.