Selection of optimal kappa B/Rel DNA-binding motifs: interaction of both subunits of NF-kappa B with DNA is required for transcriptional activation.

Abstract

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

Regulation of DNA binding by Rel/NF-κB transcription factors: structural views

“…The di erent Rel/NFkB dimer species each have di erent a nities for these kB sites as revealed by EMSA (Fujita et al, 1992;Kunsch et al, 1992). One reason for the observed varying a nities is the primary sequence di erences between family members.…”

“…Not only are some dimers not observed, but the remainder form with various a nities. For example, the p50/RelA heterodimer forms preferentially over the p50 homodimer, which forms with greater a nity than the RelA homodimer (Ganchi et al, 1993;Kunsch et al, 1992). A current model (Huang et al, 1997;Sengchanthalangsy et al, 1999) proposes that amino acids Asp-254 and Tyr-267 in p50 and their amino acid analogs in the other Rel family members play key roles in determining dimerization a nity.…”

Activators and target genes of Rel/NF-κB transcription factors

“…The various members of the Rel/NF-kB family di er in their preference for speci®c DNA-binding sites (Kunsch et al, 1992). Using random oligonucleotides and a DNA-binding assay, Kunsch et al (1992) selected optimal DNA-binding sites for p50, RelA and c-Rel, as homodimers.…”

“…Using random oligonucleotides and a DNA-binding assay, Kunsch et al (1992) selected optimal DNA-binding sites for p50, RelA and c-Rel, as homodimers. The preferred binding site di ers for each homodimer.…”

Cellular delivery of a double-stranded oligonucleotide

“…Since the NFkB complexes have different DNA-binding specificities (cf. Moerman et al, 57 Kunsch et al, 58 Schmid et al, 59 Toledano et al 60 ) it may be possible to design an NFkB decoy that blocks the effect of NFkB in glial cells but not in neuronal cells.…”