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. 2014 Jan;42(2):848-59.
doi: 10.1093/nar/gkt950. Epub 2013 Oct 23.

Characterization of the Interaction Between HMGB1 and H3-a Possible Means of Positioning HMGB1 in Chromatin

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Free PMC article

Characterization of the Interaction Between HMGB1 and H3-a Possible Means of Positioning HMGB1 in Chromatin

Matthew Watson et al. Nucleic Acids Res. .
Free PMC article

Abstract

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.

Figures

Figure 1.
Figure 1.
Dynamic association of domains in HMGB1. Schematic indicating the dynamic equilibrium between closed (auto-inhibited) and open (binding-competent) conformations of full-length HMGB1 (only the fully closed and open structures are shown for simplicity). HMG-box DNA-binding domains in red and blue, basic N-terminal, inter-box- and C-terminal extensions in yellow and acidic part of the C-terminal tail in green. [Adapted from (20)].
Figure 2.
Figure 2.
Interaction of HMGB1 with histone tails in chromatin. Sucrose gradient sedimentation of (linker-histone-depleted) chromatin pre-incubated with HMGB1, analysed in SDS/polyacrylamide gels. (a) Medium-length chromatin; (b) medium-length trypsinized chromatin from which the four core histone tails have been removed (denoted by asterisk). Lanes 3–10 correspond to fraction numbers I–VIII (bottom to top of gradient). Lane 1, molecular mass marker; lane 2, input chromatin.
Figure 3.
Figure 3.
Interaction of HMGB1 and tail-truncated derivatives with chromatin. (a) Schematic of HMGB1. The sequence of the C-terminal intrinsically disordered acidic tail is shown, with the positions of the various truncations. (b) Label-transfer for HMGB1 and its truncated products modified with the biotin label-transfer reagent Sulfo-SBED, either alone or after incubation with linker-histone-depleted chromatin. After cross-linking, and cleavage of the reagent, proteins were resolved by SDS/PAGE and transferred to nitrocellulose membrane. The biotin label was detected by probing sequentially with streptavidin and anti-streptavidin (i), and the identity of the H3 band was confirmed by stripping and re-probing the same blot with anti-H3 (ii).
Figure 4.
Figure 4.
EDC cross-linking of HMGB1 and tail-truncated derivatives to H3 in chromatin. Linker-histone-depleted chromatin alone (lanes 1,2), chromatin with HMG proteins (lanes 3 and 4, 7 and 8, 11 and 12) and HMG proteins alone (lanes 5 and 6, 9 and 10, 13 and 14), untreated or treated with EDC, were resolved by SDS/PAGE and detected by Coomassie staining (a). Western blot of identical gels probed with (b) anti-HMGB1, (c) anti-H3 antibodies, to identify products containing both HMGB1 and H3. Asterisks mark the bands of highest mobility that were detected by both antibodies and probably represent HMG-H3 heterodimers.
Figure 5.
Figure 5.
EDC cross-linking of HMGB1 and tail-truncated derivatives to H3(1–40); the effect of DNA. (a) Schematic of the H3 N-terminal region, including the region removed by trypsin, the region that passes between the two gyres of DNA and the N-terminal helix. The construct used here, H3(1–40), is indicated below, and roughly corresponds to the region of the tail that extends beyond the two gyres of DNA in the nucleosome. (b) (i) EDC cross-linking of HMG proteins and H3(1–40), or H3(1–40) alone; and (ii) the corresponding HMG proteins in the absence of H3. (c), as (b) except in the presence of sonicated calf thymus DNA. Lane 1, molecular mass marker. Asterisks indicate H3(1–40)-HMG heterodimers. Samples were resolved by SDS/PAGE and detected by Coomassie Blue staining.
Figure 6.
Figure 6.
Interaction of H3(1–40) with HMGB1. (a) Overlay of 15N-HSQC spectra of HMGB1, HMGB1 on addition of H3(1–40) to a molar ratio or 1:1 and HMGB1 lacking the acidic tail and therefore representative of the fully ‘tail-free’ state (Δ30; red). (b) Chemical-shift perturbations (Δδ) are displayed for each residue that could be assigned; the noticeable gap spanning most of the acidic tail (187–212) was due to lack of sequence-specific assignments. (c) Representative examples of peaks that shift on addition of H3(1–40) to HMGB1 (compare black and blue). Peaks that shift do so along the same trajectory as those in the tailless protein (Δ30; red), indicated by arrows, suggesting that the addition of H3(1–40) reduces the fraction of the ‘tail-bound’ form. Peaks marked with an asterisk are aliased in the 15N dimension.
Figure 7.
Figure 7.
Interaction of H3(1–40) with the HMGB1 acidic tail peptide. 15N-HSQC spectra of the HMGB1 acidic tail peptide alone and on addition of H3(1–40) to the following H3:acidic tail molar ratios: 0.25:1, 0.5:1, 0.75:1 and 1:1. The progression of three representative peaks during the titration is indicated with arrows.
Figure 8.
Figure 8.
Interaction of HMGB1 and the acidic tail peptide with H3(1–40). (a) Overlay of the 15N-HSQC spectra of H3(1–40) alone and in the presence of HMGB1, or the acidic tail peptide at a 1:1 molar ratio. (b) Chemical-shift perturbations for all non-proline residues in H3(1–40) on interaction with HMGB1 or acidic tail peptide. Helical regions predicted by Jpred are shown above (consensus between algorithms in dark purple; maximum extent in light purple). (Residues <1 remain after thrombin cleavage of the His-tag and are not part of the H3 sequence.) (c) {1H}15N Heteronuclear NOE values for all non-proline residues in the H3(1–40) peptide alone (black circles) and on addition of acidic tail peptide (red circles). (d) Far-UV CD spectra of the individual acidic tail peptide, H3(1–40) and the 1:1 mixture of the two peptides. The sum of the spectra for the two individual peptides normalized for concentration was calculated to determine whether any structural changes occur on interaction.
Figure 9.
Figure 9.
Proposed mechanism for the interaction of HMGB1 and H3 in chromatin. HMGB1 is shown in its closed (auto-inhibited) and open (binding-competent) forms (see Figure 1). HMGB1 binds to the linker DNA at the DNA entry/exit point of the nucleosome, close to where the H3 N-terminal tail exits between the two gyres of DNA, and binding is stabilized by acidic tail/H3 tail interactions. A combination of (i) disruption of H3 tail/DNA interactions, and (ii) HMG-induced untwisting of DNA to produce a positive writhe that promotes unwrapping of DNA, result in destabilization of the nucleosome core.

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