Selection of optimal kappa B/Rel DNA-binding motifs: interaction of both subunits of NF-kappa B with DNA is required for transcriptional activation.
Analysis of the p50 and p65 subunits of the NF-kappa B transcription factor complex has revealed that both proteins can interact with related DNA sequences through either homo- or heterodimer formation. In addition, the product of the proto-oncogene c-rel can bind to similar DNA motifs by itself or as a heterodimer with p50 or p65. However, these studies have used a limited number of known kappa B DNA motifs, and the question of the optimal DNA sequences preferred by each homodimer has not been addressed. Using purified recombinant p50, p65, and c-Rel proteins, optimal DNA-binding motifs were selected from a pool of random oligonucleotides. Alignment of the selected sequences allowed us to predict a consensus sequence for binding of the individual homodimeric Rel-related proteins, and DNA-protein binding analysis of the selected DNA sequences revealed sequence specificity of the proteins. Contrary to previous assumptions, we observed that p65 homodimers can interact with a subset of DNA sequences not recognized by p50 homodimers. Differential binding affinities were also obtained with p50- and c-Rel-selected sequences. Using either a p50- or p65-selected kappa B motif, which displayed differential binding with respect to the other protein, little to no binding was observed with the heterodimeric NF-kappa B complex. Similarly, in transfection experiments in which the selective kappa B binding sites were used to drive the expression of a chloramphenicol acetyltransferase reporter construct, the p65- and p50-selected motifs were activated only in the presence of p65 and p50/65 (a chimeric protein with the p50 DNA binding domain and p65 activation domain) expression vectors, respectively, and neither demonstrated a significant response to stimuli that induce NF-kappa B activity. These findings demonstrate that interaction of both subunits of the heterodimeric NF-kappa B complex with DNA is required for DNA binding and transcriptional activation and suggest that transcriptional activation mediated by the individual rel-related proteins will differ dramatically, depending on the specific kappa B motifs present.
Kunsch
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C
Department of Gene Regulation, Roche Institute of Molecular Biology, Nutley, New Jersey 07110.
Ruben
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SM
Rosen
C A
CA
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020569:Proto-Oncogene Proteins c-rel; D013601:T-Lymphocytes; D014158:Transcription, Genetic
C000715470:MAS1 protein, human; D016328:NF-kappa B; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D004247:DNA