A complete understanding of NF-κB dimer DNA-binding specificities and affinities will provide critical insight into mechanisms available for dimer-specific function in the cell. In this study, we examined the DNA-binding preferences of ten NF-κB dimers from mouse and human to a wide-ranging set of 3,285 potential κB site sequences. We anticipate that this large and detailed dataset of κB sites will prove useful for analyses of NF-κB regulatory elements at a genome scale.
Our results have immediate biological and mechanistic implications for each of the three dimer classes (). For the c-Rel and RelA homodimers, one major finding is that c-Rel homodimers bind with substantially higher affinity than RelA homodimers to all kB sites, despite highly correlated binding profiles. This suggests an affinity-dependent mechanism for discriminating these homodimers where c-Rel homodimers out-compete RelA homodimers for κB sites in vivo
on the basis of DNA binding affinity. Under these conditions, RelA homodimers will not preferentially bind to any κB sites. Therefore, to selectively regulate genes in cells that also express c-Rel homodimers (primarily hematopoietic cells), RelA homodimers need to rely on mechanisms other than selective DNA-binding, such as RelA-dependent co-activator interactions34,35
Principles of regulatory specificity for NF-κB dimer classes.
For the heterodimer class, the most important biological and mechanistic implication of our results is that the selective functions of each heterodimer may not be achieved via dimer-specific recognition of κB motifs in target genes. The PBM data for all heterodimers correlated closely, indicating that they recognize the same sequences. It is especially noteworthy that binding data for RelB:p52 correlated closely with those of the other heterodimers. It has been reported that RelB:p52, but not RelB:p50 and RelA:p50, can bind well to the non-traditional murine BLC-kB site16
. More recently, it was reported that RelB:p52 is less discriminatory than RelA:p50 and can bind to a broader set of κB site sequences20
. Binding sites unique to RelB:p52 – the primary dimer activated in response to the alternative NF-κB pathway18,19
– would provide a mechanism for cells to differentiate target genes of the alternative NF-κB pathway from those of the classical pathway activating RelA:p506
However, additional studies report that RelB:p52 and RelA:p50 share highly similar binding specificities, with no clear preference exhibited by RelB:p5221
. We found that RelB:p52 and RelB:p50 do not differ significantly in their DNA-binding preferences (see Supplementary Discussion
). Furthermore, we now extend this to include all NF-κB heterodimers, demonstrating that NF-κB heterodimers exhibit common binding preferences to the ~3,300 κB site sequences examined in this study. Modest binding differences reported for heterodimers20
may be below the resolution of our approach and may prove functionally important in vivo
and will need to be examined in greater depth in the future. However, this work and others21
suggest that the regulation of distinct sets of genes by different heterodimers is likely achieved primarily through alternative mechanisms, such as dimer-specific interactions with co-regulatory proteins34
, dimer-specific synergy with other transcription factors, or dimer-specific conformational differences20,36,37
For the p50 and p52 homodimers, we defined a subset of κB site sequences bound preferentially by these homodimers. Importantly, these sequences include a novel G-rich p50 homodimer recognition motif found upstream of the IFN-inducible Gbp1
gene (5′-GGGGGAAAAA-3′, p50:p50 z-score = 6.2; c-Rel:c-Rel z-score = 4.5) shown to mediate p50 homodimer-dependent repression38
. This suggests that many other non-traditional, G-rich p50:p50-preferred κB sites in our dataset may similarly function as p50:p50-specific target sites in vivo
In addition to the broad principles summarized above, our results highlight additional levels of complexity in NF-κB-DNA interactions. First, we observed that DNA binding site motifs for significantly bound sites exhibited one strong half-site but a degenerate preference for the opposing half-site (). This is in contrast to more symmetric motifs derived from the highest affinity κB sites (). Second, we observed that non-traditional κB sites appear to be shorter (8- to 9-bp long) than traditional κB sites (9- to 11-bp long). These findings suggest a more modest requirement for a κB site: one traditional half-site sequence recognized via a stereotyped pattern of amino acid-base contacts, with a second half-site that can exhibit considerable plasticity. These results are consistent with structural analyses showing considerable plasticity in both the global conformation of the protein-DNA complex and the amino acid-base contacts mediated by the dimer subunits12,28,39
. Analyses of c-Rel and RelA homodimers bound to different κB sequences revealed stereotyped amino acid-base interactions with the consensus 5′-GGAA-3′ half-site common to each structure, but highly variable contacts with the half-site sequences that differed between the structures. We propose that structural plasticity afforded by the ability of NF-κB dimers to bind to many κB sites with only a single strong half-site provides a mechanism to partially disentangle DNA binding from structural conformation. In turn, this may allow increased structural diversity and the potential for allosteric mechanisms in transcriptional control as have been reported in several cases36,37
In addition to highlighting the challenge of understanding how DNA sequence may influence NF-kB conformation and the functional consequences of NF-kB binding, our results emphasize the importance of the relationship between binding specificity, affinity and function. Our dataset reveals NF-κB binding to a remarkably diverse range of sequences, and suggests that many functionally important sequences (e.g., the Il2
CD28 RE) may diverge considerably from the optimal κB site. Furthermore, we observed highly overlapping binding specificity of NF-kB dimers and considerable potential for competitive binding. It is well established that, in reporter assays, high-affinity binding sites for NF-κB and other factors lead to stronger transcription than low-affinity sites40
. However, in a physiological setting within the context of native chromatin, it is not known whether an affinity threshold must be achieved for function, whether a simple relationship exists between affinity and transcriptional output, or what role the effects of co-regulatory proteins and other DNA-bound transcription factors will play. The results reported here provide a further step toward addressing these fundamental questions. Unlike basic consensus sequences and PWMs, which reveal preferences at each position of a recognition motif, data provided by PBMs and other high-throughput methods41,42
reveal preferences throughout the continuum of possible binding sequences. These datasets will be invaluable for detailed analyses of the DNA sequence-dependence of transcriptional regulatory control.