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High mobility group protein B1 (HMGB1) binds to the internucleosomal linker DNA in chromatin and abuts the nucleosome. Bending and untwisting of the linker DNA results in transmission of strain to the nucleosome core, disrupting histone/DNA contacts. An interaction between H3 and HMGB1 has been reported. Here we confirm and characterize the interaction of HMGB1 with H3, which lies close to the DNA entry/exit points around the nucleosome dyad, and may be responsible for positioning of HMGB1 on the linker DNA. We show that the interaction is between the N-terminal unstructured tail of H3 and the C-terminal unstructured acidic tail of HMGB1, which are presumably displaced from DNA and the HMG boxes, respectively, in the HMGB1-nucleosome complex. We have characterized the interaction by nuclear magnetic resonance spectroscopy and show that it is extensive for both peptides, and appears not to result in the acquisition of significant secondary structure by either partner.

Facilitated binding of p53 to DNA by high mobility group B1 (HMGB1) may involve interaction between the N-terminal region of p53 and the high mobility group (HMG) boxes, as well as HMG-induced bending of the DNA. Intramolecular shielding of the boxes by the HMGB1 acidic tail results in an unstable complex with p53 until the tail is truncated to half its length, at which point the A box, proposed to be the preferred binding site for p53(1-93), is exposed, leaving the B box to bind and bend DNA. The A box interacts with residues 38-61 (TAD2) of the p53 transactivation domain. Residues 19-26 (TAD1) bind weakly, but only in the context of p53(1-93) and not as a free TAD1 peptide. We have solved the structure of the A-box/p53(1-93) complex by nuclear magnetic resonance spectroscopy. The incipient amphipathic helix in TAD2 recognizes the concave DNA-binding face of the A box and may be acting as a single-stranded DNA mimic., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Histone H1 and HMGB1 (high-mobility group protein B1) are the most abundant chromosomal proteins apart from the core histones (on average, one copy per nucleosome and per ten nucleosomes respectively). They are both highly mobile in the cell nucleus, with high on/off rates for binding. In vivo and in vitro evidence shows that both are able to organize chromatin structure, with H1 binding resulting in a more stable structure and HMGB1 binding in a less stable structure. The binding sites for H1 and HMGB1 in chromatin are partially overlapping, and replacement of H1 by HMGB1 through the highly dynamic nature of their binding, possibly facilitated by interaction between them, could result in switching of chromatin states. Binding of HMGB1 to DNA or chromatin is regulated by its long and highly acidic tail, which is also involved in H1 binding. The present article focuses mainly on HMGB1 and its interaction with chromatin and H1, as well as its chaperone role in the binding of certain transcription factors (e.g. p53) to their cognate DNA.

The architectural DNA-binding protein HMGB1 consists of two tandem HMG-box domains joined by a basic linker to a C-terminal acidic tail, which negatively regulates HMGB1-DNA interactions by binding intramolecularly to the DNA-binding faces of both basic HMG boxes. Here we demonstrate, using NMR chemical-shift mapping at different salt concentrations, that the tail has a higher affinity for the B box and that A box-tail interactions are preferentially disrupted. Previously, we proposed a model in which the boxes are brought together in a collapsed, tail-mediated assembly, which is in dynamic equilibrium with a more extended form. Small-angle X-ray scattering data are consistent with such a dynamic equilibrium between collapsed and extended structures and are best represented by an ensemble. The ensembles contain a significantly higher proportion of collapsed structures when the tail is present. (15)N NMR relaxation measurements show that full-length HMGB1 has a significantly lower rate of rotational diffusion than the tail-less protein, consistent with the loss of independent domain motions in an assembled complex. Mapping studies using the paramagnetic spin label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidin-3-yl)methyl methanethiosulfonate] placed at three locations in the tail confirm our previous findings that the tail binds to both boxes with some degree of specificity. The end of the tail lies further from the body of the protein and is therefore potentially free to interact with other proteins. MTSL labelling at a single site in the A domain (C44) causes detectable relaxation enhancements of B domain residues, suggesting the existence of a "sandwich"-like collapsed structure in which the tail enables the close approach of the basic domains. These intramolecular interactions are presumably important for the dynamic association of HMGB1 with chromatin and provide a mechanism by which protein-protein interactions or posttranslational modifications might regulate the function of the protein at particular sites, or at particular stages in the cell cycle., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

H1 and HMGB1 bind to linker DNA in chromatin, in the vicinity of the nucleosome dyad. They appear to have opposing effects on the nucleosome, H1 stabilising it by "sealing" two turns of DNA around the octamer, and HMGB1 destabilising it, probably by bending the adjacent DNA. Their presence in chromatin might be mutually exclusive. Displacement/replacement of one by the other as a result of their highly dynamic binding in vivo might, in principle, involve interactions between them. Chemical cross-linking and gel-filtration show that a 1:1 linker histone/HMGB1 complex is formed, which persists at physiological ionic strength, and that complex formation requires the acidic tail of HMGB1. NMR spectroscopy shows that the linker histone binds, predominantly through its basic C-terminal domain, to the acidic tail of HMGB1, thereby disrupting the interaction of the tail with the DNA-binding faces of the HMG boxes. A potential consequence of this interaction is enhanced DNA binding by HMGB1, and concomitantly lowered affinity of H1 for DNA. In a chromatin context, this might facilitate displacement of H1 by HMGB1.

The structure-specific DNA-binding protein HMGB1 (high-mobility group protein B1) which comprises two tandem HMG boxes (A and B) and an acidic C-terminal tail, is acetylated in vivo at Lys(2) and Lys(11) in the A box. Mutation to alanine of both residues in the isolated A domain, which has a strong preference for pre-bent DNA, abolishes binding to four-way junctions and 88 bp DNA minicircles. The same mutations in full-length HMGB1 also abolish its binding to four-way junctions, and binding to minicircles is substantially impaired. In contrast, when the acidic tail is absent (AB di-domain) there is little effect of the double mutation on four-way junction binding, although binding to minicircles is reduced approximately 15-fold. Therefore it appears that in AB the B domain is able to substitute for the non-functional A domain, whereas in full-length HMGB1 the B domain is masked by the acidic tail. In no case does single substitution of Lys(2) or Lys(11) abolish DNA binding. The double mutation does not significantly perturb the structure of the A domain. We conclude that Lys(2) and Lys(11) are critical for binding of the isolated A domain and HMGB1 to distorted DNA substrates.

The mechanism underlying negative regulation of HMGB1-DNA interaction by the acidic C-terminal tail is ill defined. To address this issue, we have devised a novel NMR chemical-shift perturbation mapping strategy to elucidate interactions between the tail, which consists solely of aspartic acid and glutamic acid residues, and the two well characterized HMG-box DNA-binding domains. A series of HMGB1 tail-truncation mutants differing from each other by five residues was generated. Chemical-shift perturbation mapping using these mutants shows that tails of different lengths bind with different affinities. Nevertheless, the truncated tails bind along the same path on the HMG boxes as the full-length tail, differences in length being manifested in differences in the "reach". The tail makes extensive contacts with the DNA-binding surfaces of both HMG boxes, thus explaining the basis of negative regulation of HMGB1-DNA interaction by the tail.