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-kappaB 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 kappaB 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 kappaB motif, which displayed differential binding with respect to the other protein, little to no binding was observed with the heterodimeric NF-kappaB complex. Similarly, in transfection experiments in which the selective kappaB 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-kappaB activity. These findings demonstrate that interaction of both subunits of the heterodimeric NF-kappaB 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 kappaB motifs present.