Mechanism of κB DNA binding by Rel/NF-κB dimers

Abstract

The DNA binding of three different NF-B dimers, the p50 and p65 homodimers and the p50/p65 heterodimer, has been examined using a combination of gel mobility shift and fluorescence anisotropy assays. The NF-B p50/p65 heterodimer is shown here to bind the B DNA target site of the immunoglobulin enhancer (Ig-B) with an affinity of approximately 10 nM. The p50 and p65 homodimers bind to the same site with roughly 5-and 15-fold lower affinity, respectively. The nature of the binding isotherms indicates a cooperative mode of binding for all three dimers to the DNA targets. We have further characterized the role of pH, salt, and temperature on the formation of the p50/p65 heterodimer-Ig-B complex. The heterodimer binds to the Ig-B DNA target in a pH-dependent manner, with the highest affinity between pH 7.0 and 7.5. A strong salt-dependent interaction between Ig-B and the p50/p65 heterodimer is observed, with optimum binding occurring at monovalent salt concentrations below 75 mM, with binding becoming virtually nonspecific at a salt concentration of 200 mM. Binding of the heterodimer to DNA was unchanged across a temperature range between 4°C and 42°C. The sensitivity to ionic environment and insensitivity to temperature indicate that NF-B p50/p65 heterodimers form complexes with specific DNA in an entropically driven manner.The Rel/NF-B transcription factors constitute one of the most important families of regulatory transcription factors. Members of the Rel/NF-B family are essential for diverse biological functions such as the regulation of innate and adaptive immunity, development, and apoptosis in a wide array of eukaryotes from Drosophila to man (1-4). Like most transcription factors, dimers of NF-B proteins modulate transcription by directly binding to enhancer sequences located in the regulatory regions of numerous genes. These DNA sequences are collectively known as B DNA sequences. In mammals, the Rel/NF-B dimers arise from five polypeptides, p50, p52, p65, c-Rel, and RelB. The most abundant of these dimers are the p50/p65 heterodimer and the p50 homodimer. The existences of some, but not all, of the other possible dimers have been shown to exist in cells.The NF-B family can be divided into two subgroups based on the presence or absence of an activation domain. p50 and p52 do not contain a distinct activation domain and belong to class I. The other three members constitute the class II subfamily. It is generally believed that the homodimers of p50 and p52 and the p50/p52 heterodimer function as transcriptional repressors. The remaining combinations of dimeric NF-B proteins, containing at least one monomer of p65, c-Rel, or RelB, act as activators. Rel/NF-B proteins share a region that shows over 45% sequence similarity across the entire family. This region, known as the Rel homology region (RHR), 1 is responsible for DNA binding and subunit dimerization. High resolution x-ray crystal structures of RHRs are known for four homodimers, p50, p52, p65, and c-Rel in their DNA-bound conformations (5-8). These struc...

Thermodynamic and structural insights into CSL‐DNA complexes

“…In contrast to CSL, p50 binding to DNA was very sensitive to temperature with >30-fold higher affinity observed at 30 C than binding experiments performed at 10 C; nonetheless, over the temperature range tested, p50 in general, has a similarly modest affinity for DNA, suggesting that both CSL, and p50 binding to DNA may be modulated by interactions with neighboring transcription factors. Two other points should also be mentioned, first, our experimentally determined values for the affinity of p50 for DNA by ITC are very similar to the values previously reported using gelshift and fluorescence anisotropy binding assays 30 ; and second, the moderate affinity of CSL for DNA we determined here is not an artifact of our experimental setup; as shown in Table S3, a number of transcription factors with low nanomolar dissociation constants for DNA have been characterized using ITC. Taken together, the modest affinity of CSL for DNA suggests that not all CSL binding sites in vivo are occupied at all times and that CSL-coregulator complexes are likely forming/exchanging in the nucleoplasm and on DNA, which in our mind, places much more emphasis on cooperative mechanisms that recruit CSL to sites on the DNA than previously appreciated.…”

“…4). Although the molecular basis for this nonlinearity is outside the scope of this study, it should be mentioned that a similar nonlinear dependence of binding has been observed for the structurally related transcription factor nuclear factor (NF)-kappaB, 30 and for other DNA-binding proteins 31 . A closer examination of the enthalpic and entropic contributions to binding at 150 and 200 mM salt reveals a modest change in the enthalpy of binding (less than 1.0 kcal/mol), but a large entropic penalty of $3.0 kcal/mol for both reactions.…”

Copine-I represses NF-κB transcription by endoproteolysis of p65

“…Consistent with the loss of p65 DNA-binding activity, the p65(51-551) protein was unable to bind to DNA (Figure 6b). Although p65:p65 homodimers are not believed to display the same high affinity for DNA as the p65:p50 heterodimer, both homo-and heterodimers readily form DNA-protein complexes Phelps et al, 2000). Consistent with this observation, we detected p65:p65 homodimer and p65:p50 heterodimers complexes, which were confirmed by super-shift analysis using a-Flag and a-p50 antibodies (Figure 6b; data not shown).…”

Transcriptional regulation via the NF-κB signaling module

“…More recent solution-based in vitro binding measurements revealed the following: p50:RelA binds to 10 or 11 bp consensus sequences with similar affinities (B10 nM) (Phelps et al, 2000b). RelA and c-Rel homodimers bind the same sequences with at least 10-fold lower affinity.…”

“…For example, on the interferon-b promoter, both p50:RelA and RelA:RelA dimers can activate transcription even though these two dimers bind the b-interferon kB site with significantly different affinities (Phelps et al, 2000b). Considering the large assembly of activators and co-activators, a single activator bound to a single response element is rarely sufficient to induce transcription.…”