Identification and characterization of nuclear factor kappaB binding sites in the murine bcl-x promoter.
Signal transduction pathways that mediate neuronal commitment to apoptosis involve the nuclear factor kappaB (NF-kappaB) 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-kappaB DNA binding sequences clustered upstream of the brain-specific transcription start site in the upstream promoter region. Recombinant p50/p50 and NF-kappaB proteins from nuclear extracts bound to these sites as determined by electrophoretic mobility shift assay and biotin-oligonucleotide/streptavidin affinity assays. NF-kappaB overexpression, coupled with bcl-x promoter/reporter 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-kappaB. The mutagenic removal of NF-kappaB binding sites individually or in combination revealed altered response patterns to p49/p65 and p50/p65 overexpression. These results support the hypothesis that NF-kappaB can act to enhance Bcl-X(L) expression via highly selective interactions, where NF-kappaB binding and bcl-x promoter activation are dependent on both DNA binding site sequence and NF-kappaB subunit composition. Our data suggest that molecular events associated with NF-kappaB promote regulation of neuronal apoptosis in the developing or injured CNS.
Glasgow
J N
JN
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-0652, USA.
Wood
T
T
Perez-Polo
J R
JR
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D051996:Transcription Factor RelA; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection; D051020:bcl-X Protein
D020121:5' Untranslated Regions; C494811:BCL2L1 protein, human; C494812:Bcl2l1 protein, mouse; C494813:Bcl2l1 protein, rat; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051996:Transcription Factor RelA; D051020:bcl-X Protein; D004247:DNA