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. 2012 Sep 28;287(40):33756-65.
doi: 10.1074/jbc.M112.390849. Epub 2012 Jul 19.

Methylation of lysine 9 in histone H3 directs alternative modes of highly dynamic interaction of heterochromatin protein hHP1β with the nucleosome

Francesca Munari  1 , Szabolcs Soeroes, Hans Michael Zenn, Adrian Schomburg, Nils Kost, Sabrina Schröder, Rebecca Klingberg, Nasrollah Rezaei-Ghaleh, Alexandra Stützer, Kathy Ann Gelato, Peter Jomo Walla, Stefan Becker, Dirk Schwarzer, Bastian Zimmermann, Wolfgang Fischle, Markus Zweckstetter
Affiliations

Methylation of lysine 9 in histone H3 directs alternative modes of highly dynamic interaction of heterochromatin protein hHP1β with the nucleosome

Francesca Munari et al. J Biol Chem. .

Abstract

Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) mark is a hallmark of establishment and maintenance of heterochromatin. Although genetic and cell biological aspects have been elucidated, the molecular details of HP1 binding to H3K9me3 nucleosomes are unknown. Using a combination of NMR spectroscopy and biophysical measurements on fully defined recombinant experimental systems, we demonstrate that H3K9me3 works as an on/off switch regulating distinct binding modes of hHP1β to the nucleosome. The methyl-mark determines a highly flexible and very dynamic interaction of the chromodomain of hHP1β with the H3-tail. There are no other constraints of interaction or additional multimerization interfaces. In contrast, in the absence of methylation, the hinge region and the N-terminal tail form weak nucleosome contacts mainly with DNA. In agreement with the high flexibility within the hHP1β-H3K9me3 nucleosome complex, the chromoshadow domain does not provide a direct binding interface. Our results report the first detailed structural analysis of a dynamic protein-nucleosome complex directed by a histone modification and provide a conceptual framework for understanding similar interactions in the context of chromatin.

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Figures

FIGURE 1.
FIGURE 1.
hHP1β specifically interacts with H3K9me3 nucleosomes. A and B, pulldown experiments using synthetic nucleosomes uniformly containing the indicated histone H3 modification status immobilized via biotinylated DNA on streptavidin-coated magnetic beads and soluble recombinant hHP1β. Recovered material was analyzed by Western blotting using the indicated antibodies and by Ponceau staining. Note that streptavidin stripped from the beads runs with H4 on the SDS-polyacrylamide gel. C, SPR analysis of titration series of soluble hHP1β WT interacting with unmodified (immobilization of 940 response units (RU)) and H3K9me3 (immobilization of 950 RU) nucleosomes immobilized via biotinylated DNA on a sensor chip. D, pulldown experiments using symmetrically or asymmetrically H3KC9me3-modified nucleosomes. E, comparative steady state evaluation of hHP1β binding to symmetric or asymmetric H3KC9me3 nucleosomes. SPR binding responses shortly before the end of the association phase were plotted against the analyte concentration and fitted by nonlinear regression assuming a 1:1 interaction model. Data of two independent measurements were normalized to Rmax = 100% and averaged.
FIGURE 2.
FIGURE 2.
Molecular determinants of hHP1β binding to H3KC9me3 nucleosome defined by NMR spectroscopy. A, two-dimensional TROSY-1H,15N HSQC spectra of hHP1β alone (blue) and with H3KC9me3 nucleosome at a molar ratio of 1:2 (red). Residues experiencing large changes are annotated. B, intensity loss (I/I0) and weighted chemical shift difference (ΔδNH) values from the spectra in A are plotted as a function of hHP1β primary sequence. A cluster of signals (Val-22 to Val-23, Lys-43 to Gly-44, and Glu-55 to Asp-62) disappeared already at low molar ratio. Other missing values correspond to proline residues or residues with severe signal overlap. The domain organization of hHP1β is schematically shown at the top. C, nucleosome-to-hHP1β cross-saturation transfer experiments: intensity ratio of hHP1β signals recorded with (Isat) or without (Iref) selective saturation of nucleosome aliphatic protons. Error bars were calculated on the basis of the signal-to-noise-ratios in the two spectra. Missing signals are due to severe overlap, and some residues (i.e. 164–173) broadened beyond detection below 290 K (supplemental Fig. 2). D, intensity ratio values representing the success of cross-saturation transfer via direct binding are mapped onto the three-dimensional structure of the chromodomain (Protein Data Bank code 1AP0 (6)). Residues without experimental values (proline or overlapped) are in gray.
FIGURE 3.
FIGURE 3.
Effect of H3KC9me3 nucleosome and H3K9me3/H3KC9me3 peptide binding on the structure of hHP1β CD. A, selected regions from TROSY 1H,15N HSQC spectra of hHP1β alone (blue), with unlabeled H3KC9me3 nucleosome at a 1:2 molar ratio (red), or with H3K9me3 peptide at a 1:2 molar ratio (green). Black arrows highlight some of the CD peaks shifting in the same direction in the two binding events. B, chemical shift differences of methyl resonances (ΔδCH) in U-2H,12C, selective methyl Val and Leu 13CH3,12CD3-labeled hHP1β, upon binding to H3KC9me3 nucleosome (black; hHP1β/nucleosome molar ratio of 1:2) and to H3K9me3 peptide (green; hHP1β/peptide molar ratio of 1:2) relative to the free state. C, 1H,13C chemical shift changes of methyl groups of hHP1β upon binding to H3KC9me3 nucleosome are mapped onto the three-dimensional structures of CD (Protein Data Bank code 1AP0, top panel) and CSD (Protein Data Bank code 1DZ1 (13), bottom panel). Methyl groups are shown as spheres. D, comparison of 15N (upper panel) and 1H (lower panel) chemical shift changes in the CD upon binding of hHP1β to H3KC9me3 peptide (gray) and H3KC9me3 nucleosome (red). E, correlation plot of relative changes of 15N and 1H chemical shifts (i.e. 0.2ΔδN/ΔδH) between hHP1β bound to H3KC9me3 peptide (x axis) and H3KC9me3 nucleosome (y axis) with respect to the free form. Only CD residues with chemical exchange that is fast on the NMR time scale were considered.
FIGURE 4.
FIGURE 4.
Dynamics of the hHP1β-nucleosome complex. A, 15N cross-correlation rates (ηxy) measured for U-2H,15N- labeled hHP1β in the free state (blue) or with H3KC9me3 nucleosome at a molar ratio of 1:2 (red). The dashed line at 110 s−1 marks the expected ηxy value for the CD if it would tumble together with the core of the nucleosome particle as a single rigid body. In the calculation (see supplemental Methods), the population-weighted average of the rates of free and bound forms was considered. B, “tail-transplantation” pulldown experiments of nucleosomes uniformly containing unmodified H3, H3K9me3, or a truncated version of H3(Δ1–20) together with a H2B hybrid where the first 20 amino acids of H3 containing K9me3 were fused to the N terminus (see schematic representation on the left). Recovered material was analyzed by Western blotting using the indicated antibodies and by Ponceau staining.
FIGURE 5.
FIGURE 5.
Interaction interfaces of hHP1β with unmodified and H3KC9me3 nucleosome are different. A, selected regions of the TROSY 1H,15N HSQC spectrum of free hHP1β (black) and of hHP1β in the presence of unmodified nucleosome (orange), free 601-DNA (red), or unmodified H3-tail peptide (light blue). In all three cases, the hHP1β/ligand molar ratio was 1:1. Arrows highlight the direction of peak shifts for some residues in the CD. B, hHP1β-1H,15N resonance intensity loss upon binding to unmodified nucleosome (orange) and H3KC9me3 nucleosome (black) as a function of residue number. The hHP1β/ligand molar ratio was 1:2 in both cases. C, chemical shift changes (ΔδNH) upon hHP1β binding to unmodified nucleosome. D, comparison of chemical shift changes (ΔδNH) in WT hHP1β (orange) and hHP1βΔNΔC (green) in the presence of unmodified nucleosome, both at 1:1 molar ratio. E, lysine patches in the hHP1β N-terminal and hinge region (see also supplemental Fig. 5F). F, comparison of chemical shift changes induced in WT hHP1β upon binding to free 601-DNA (red) and unmodified nucleosome (black), both at 1:1 molar ratio. G, 1H,15N chemical shift changes in WT hHP1β (red) and hHP1βΔNΔC (green) in the presence of 601-DNA at molar ratios of 1:1.
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