The virus-induced human IFN-β enhancer is arguably the most thoroughly characterized transcriptional regulatory element in any higher eukaryotic genome. The structure reported here allows the assembly of a model of the full enhancer bound to the DNA-binding domains of all the relevant transcription factors. It shows in molecular detail the interactions of ATF-2/c-Jun, IRF-3, IRF-7 and NFκB with the enhancer DNA.
The nucleotide sequence of the IFN-® enhancer is nearly invariant over roughly 100 million years of evolution, unlike the sequence of the gene (Figure S6
). Thus, the precise organization of the assembled transcription factors has had strong and continuing selective advantage. Moreover, mutational analyses have shown that virtually every nucleotide in the enhancer DNA sequence matters for some aspect of the response to viral infection (Du and Maniatis, 1992
; Goodbourn and Maniatis, 1988
; Thanos and Maniatis, 1992
). The enhanceosome structure accounts for this conservation by showing that the transcription factors form a composite surface for recognition of the entire sequence and that adjacent trascription-factor binding sites overlap ( and ). For example, IRF-3 and IRF-7 specify additional bases around the core IRF binding site 5′ AANNGAAA 3′ through non-conserved amino-acid residues such as Leu42, Arg78 and Arg86 in IRF-3 and Thr93 in IRF-7. These additional DNA contacts explain a number of observed sequence preferences (Lin et al., 2000a
; Morin et al., 2002
). They also account for the requirement of IRF-7 in the early IFN-β response to viral infection (Honda et al., 2005
), by showing how loop L2 of IRF-7 at site D avoids interference with NFκB and how DNA sequence just outside the cores of sites B and D leads to a preference for IRF-7 over other family members.
A hallmark of combinatorial transcriptional control is synergy, mediated largely in this case by enhanceosome formation (Merika and Thanos, 2001
; Struhl, 2001
). Synergy implies strong cooperativity at some level of assembly, such as direct interactions between adjacently bound transcription factors. When c-Fos, c-Jun, and NFAT bind the ARRE2 site of the IL-2 enhancer, contacts between adjacent proteins do impart both cooperativity and specificity: an extended network of polar interactions, which includes all three proteins and the DNA backbone, establishes a preferred orientation for the Fos:Jun heterodimer on its binding site and a particular conformation for the two-domain RHR of NFAT (Chen et al., 1998b
). Extended contacts between transcription factors are noticeably absent in the IFN-β enhanceosome, however. Despite the density with which the eight bound proteins are packed along the essentially straight segment of enhancer DNA, the structure and the binding measurements reported here (Figure S2
) and in our previous paper (Panne et al., 2004
) show that the relatively tenuous local protein interfaces between abutting DNA-binding domains impart very little cooperativity. For example, the L2 loop of IRF-7 has an insertion of 9 amino-acid residues (with respect to IRF-3) between helicesα2 and α3 (Figure S3
). Although this loop extends towards p50, IRF-7 does not bind cooperatively with NFκB to the enhancer (Figure S2
), and the structure shows that the glycine- and proline-rich L2 loop is largely disordered and moves out of the way to accommodate loop L1 of p50 without making extensive contacts (Figure S4
What interactions can give rise to cooperativity of transcription factor association with the IFN-β enhancer in the absence of strong contacts between adjacent proteins? In principle, cooperative binding can arise through nucleotide sequence dependent structural changes in the DNA that allow formation of complementary DNA conformations for adjacently bound transcription factors (Escalante et al., 2002a
; Klemm and Pabo, 1996
; Panne et al., 2004
). This conformational complementarity appears to be the case for ATF-2/c-Jun and all four IRFs (but not for NFκB, which has a site that does not overlap that of its neighbor). We have shown that cooperative binding of ATF-2/c-Jun and IRF-3 depends on the inherent asymmetry of the ATF-2/c-Jun binding site and that modifying it into a consensus AP-1 recognition element eliminates the cooperativity (Panne et al., 2004
). That is, the ATF-2/c-Jun site is actually a composite element that accommodates not just ATF-2/c-Jun but also part of the adjacent IRF-3. Similarly, all four IRF binding sites are composite elements, and the structures show a remarkably precise sequence organization to accommodate a specific array of IRFs.
Local complementarity of DNA conformation at overlapping sites cannot, however, account for the strong in vivo
synergy of IFN-β gene regulation, as binding analyzed by the EMSA experiments in Figure S2
would than have shown a more striking cooperative character. Previous work has shown that interactions beyond the DNA-binding domains provide additional driving force for cooperative assembly. Have we failed to visualize important pairwise interactions between the transcription factors? Except for the dimerization domains of IRF-3 and IRF-7, which not only hold the dimers together but also bind the co-activator CBP/p300 (Qin et al., 2005
), essentially all of the regions of the various DNA-bound proteins known to have well-defined, folded structure are included in the structures and in our binding measurements. That is, the remaining parts of ATF-2, c-Jun, and NFκB are probably flexibly extended, and various segments are known to interact with specific co-activators or co-repressors or to serve as signals for nuclear localization or for degradation. These extended regions are unlikely to form specific contacts with each other or with the IRFs. The absence of pairwise interactions in vitro,
using purified full-length activators further supports this contention (D.Panne; unpublished observation).
The high mobility group protein HMGA1a has also been implicated in cooperative enhanceosome assembly (Thanos et al, 1993
). Unlike the stably folded, “architectural” HMG proteins such as LEF-1, HMG-1, and SRY, which alter DNA conformation and create a platform for association of transcriptional activators, HMGA1a merely requires an accessible, A:T-rich minor groove. The enhanceosome structure shows that the mapped HMGA1a binding sites are not accessible and that HMGA1a is unlikely to be part of the final assembly. Enhanceosome assembly is asynchronous (Munshi et al., 2001
). HMGA1a could therefore act as a molecular chaperone during different stages of the assembly process and then dissociate from the final complex – a mode of action also proposed for HMG-1 in certain cases (Thomas, 2001
Multivalent interactions of the co-activators, CBP and p300, with all the assembled transcription factors participate in activating transcription directed by the IFN-β enhancer (Merika et al., 1998
; Wathelet et al., 1998
). CBP and p300 are large, extended, flexible molecules, with a series of domains, some widely spaced, that bind segments of the activation regions of various transcription factors. The IRF-binding domain (IBiD) near the C-terminus of CBP interacts with IRF-3 (Lin et al., 2001
; Qin et al., 2005
); the KIX domain, near the N-terminus, with RelA and c-Jun (Bannister et al., 1995
); the CH2 domain, between KIX and IBiD, with ATF-2 (Kawasaki et al., 1998
In transient transfection experiments with IFN-β reporter genes, insertion of an integral DNA turn (10 base pairs) between the PRDI-PRDII and the PRDIV-PRDIII domains of the IFN-β enhancer does not compromise activation; insertion of a half-integral turn of the helix (5 bp) between the sites essentially disables the enhancer (Thanos and Maniatis, 1995
). These experiments reveal the importance of the position of transcription factors on the face of the DNA helix in the assembly of the preinitiation complex, and they illustrate the adaptability of CBP and p300 in spanning variable intervals between DNA-bound transcription factors. They do not, however, reflect all the biological specificity that has led to the evolutionary invariance of the enhancer sequence. In particular, the transfection experiments are unlikely to reflect the subtleties of enhanceosome assembly and enhancer function of the endogenous gene in the context of chromatin. For example, the level of induction of the endogenous gene is orders of magnitude higher than observed with the transfected reporter (T.M. upublished data), and thus the effects of insertions on cooperative binding might not be observed at all in the transfection experiments.
The IFN-β enhanceosome is a precise and specific assembly of “generic” transcription factors that participate in many other regulatory complexes as well. Faithful coincidence detection requires that a functional response should occur only when the right set of transcription factors is on the enhancer and only when all those factors are indeed present. The structure shows that this combinatorial specificity is encoded not just in the various binding sites but also in their overlap and in their positions with respect to each other. That is, precision of the assembly contributes directly to its specificity (e.g., to the requirement for IRF-3 and IRF-7), even in the absence of extended protein-protein interfaces. The strict evolutionary conservation of the IFN-β enhancer sequence correlates with its organizational precision, and we suggest that other strictly conserved enhancer sequences – for example, the 300 bp IL-2 enhancer/promoter – may have similar structural characteristics. These characteristics also imply that non-consensus binding-site sequences can have critical functional importance, a property that will need to be included in computional algorithms for detecting transcription-factor sites in genome sequences.
The IFN-β enhanceosome structure further shows that cooperativity of assembly probably resides at a level of interaction not represented by contacts between neighboring DNA-binding domains but probably at the level of co-activators. The flexibility of CBP/p300 allows it to serve as a signal integrator not only for enhanceosomes of tightly defined geometry, but also for “modular” enhancers with more variably spaced binding sites. One of the best-studied modular elements, the even-skipped stripe-2 enhancer of Drosophila ( Small et al., 1991
), shows local evolutionary conservation over segments longer than a single transcription-factor binding site, even when the larger-scale organization of the enhancer is clearly variable. Thus, conserved sub-elements may have a precise, enhanceosome-like molecular architecture within a generally more flexible complete enhancer (Ludwig et al., 2000
; Ludwig et al., 2005
). A generic adaptor (CBP/p300) would then pass on to the Pol II machinery a summary of tightly regulated signals from several specifically arrayed sets of generic activators.