Selection of Optimal κB/Rel DNA-Binding Motifs: Interaction of Both Subunits of NF-κB with DNA Is Required for Transcriptional Activation

Abstract

Analysis of the p50 and p65 subunits of the NF-κB 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 12260448kB12260448 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 12260448kB12260448 motif, which displayed differential binding with respect to the other protein, little to no binding was observed with the heterodimeric NF-κB complex. Similarly, in transfection experiments in which the selective 12260448kB12260448 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-κB activity. These findings demonstrate that interaction of both subunits of the heterodimeric NF-κB 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 12260448kB12260448 motifs present.