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Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3 - PubMed

  • ️Mon Jan 01 2001

Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3

S A Jacobs et al. EMBO J. 2001.

Abstract

Recent studies show that heterochromatin-associated protein-1 (HP1) recognizes a 'histone code' involving methylated Lys9 (methyl-K9) in histone H3. Using in situ immunofluorescence, we demonstrate that methyl-K9 H3 and HP1 co-localize to the heterochromatic regions of Drosophila polytene chromosomes. NMR spectra show that methyl-K9 binding of HP1 occurs via its chromo (chromosome organization modifier) domain. This interaction requires methyl-K9 to reside within the proper context of H3 sequence. NMR studies indicate that the methylated H3 tail binds in a groove of HP1 consisting of conserved residues. Using fluorescence anisotropy and isothermal titration calorimetry, we determined that this interaction occurs with a K(D) of approximately 100 microM, with the binding enthalpically driven. A V26M mutation in HP1, which disrupts its gene silencing function, severely destabilizes the H3-binding interface, and abolishes methyl-K9 H3 tail binding. Finally, we note that sequence diversity in chromo domains may lead to diverse functions in eukaryotic gene regulation. For example, the chromo domain of the yeast histone acetyltransferase Esa1 does not interact with methyl- K9 H3, but instead shows preference for unmodified H3 tail.

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Figures

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Fig. 1. A specific antibody for methyl-K9 H3, α-methyl-K9 H3, co-localizes with HP1 in polytene chromosomes. (A) ELISA analysis of α-methyl-K9 H3 showing specificity of the antiserum for methyl-K9 H3 peptide. The α-methyl-K9 H3 antibody was produced by using a methyl-lysine-containing H3 sequence, TARKSTGGC, coupled to keyhole limpet hemocyanin (KLH). (B) Western analysis (12% SDS–PAGE) of recombinant Xenopus H3 (rX.l. H3) (gift from K.Luger), human core histones (hMCF7) and Drosophila core histones (dS2) using α-methyl-K9 H3. The right lane of the two dS2 lanes corresponds to the experiment where antibodies were pre-absorbed with methyl-K9 H3 peptide. (C–E) Third instar polytene chromosomes were analyzed by indirect immunofluoresence using the general DNA stain DAPI (C), α-methyl-K9 H3 (D) and anti-HP1 antibodies, α-HP1 (E). The white arrows point to chromocenters.

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Fig. 2. Two-dimensional [15N–1H]-HSQC NMR spectra demonstrating the specific interaction of the HP1 chromo domain with methyl-K9 H3 peptide. (A) Superposition of the chromo (amino acids 1–84 in green cross-peaks) and chromo shadow (amino acids 132–206 in red cross-peaks) domains spectra on the intact HP1 (amino acids 1–206 in black cross-peaks). In order to improve the NMR spectra of the chromo shadow domain (20 kDa dimer) and intact (50 kDa dimer) proteins, we incorporated 50% perdeuteration of non-labile hydrogens in the samples. This was achieved by growing E.coli in the media containing 50% D2O–50% H2O. (B) Superposition of the chromo domain free (black cross-peaks) and in complex with methyl-K9 H3 peptide (red cross-peaks); stoichiometric binding was achieved at the protein to peptide concentration ratio of 1:4. (C) Superposition of the chromo domain free (black cross-peaks) and in the presence of saturating amount of methyl-K4 H3 peptide (red cross-peaks).

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Fig. 3. Binding of the HP1 chromo domain to fluorescein-labeled H3 tail peptides as measured by fluorescence polarization. Curves represent the best fit to the data as described in Materials and methods. The fraction bound corresponds to the normalized anisotropy. Binding assays were performed for the HP1 chromo domain with unmodified H3 (triangles), methyl-K9 H3 (circles), methyl-K4 H3 (crosses) and methyl-K4/methyl-K9 H3 (diamonds). The binding assay performed for intact HP1 with methyl-K9 H3 is shown with squares.

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Fig. 4. Binding of the HP1 chromo domain to methyl-K9 H3 peptide as measured by isothermal titration calorimetry at 25°C. (A) Raw data for injections of the peptide into the chromo domain as described in Materials and methods. The asterisk corresponds to the first injection, which was excluded from the analysis. (B) Integrated heats of injections, with the solid line corresponding to the best fit of the data using the MicroCal software.

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Fig. 5. NMR-detected differences between the complex (with methyl-K9 H3 peptide) and free chromo domain. (A) Backbone chemical shift differences between the chromo domain bound to methyl-K9 H3 peptide and free chromo domain are tabulated in the form of weighted average chemical shift difference, Δδave, as described in the text; the asterisks correspond to the sites with no available data. Secondary structure elements are shown at the top. (B) 15N relaxation data measured at 60.8 MHz for 1 mM free (triangles) and complex (circles) chromo domain.

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Fig. 6. Map of the binding surface of the chromo domain for methyl-K9 H3 peptide. (A) The amino acid sequence of the Drosophila HP1 (dmHP1) chromo domain (as prepared for this study) is shown in an alignment (using CLUSTALW) with other chromo domains: human HP1 types α, β and γ, S.pombe Swi6p (amino acids 59–145), Clr4 (amino acids 1–72), human MOF (amino acids 370–456) and S.cerevesiae Esa1 (amino acids 11–97). Secondary structure elements present in dmHP1 are marked. Residues that participate in peptide binding are marked by magenta triangles. Residues in red correspond to the sites of phosphorylation in dmHP1. The boxed sequence in HP1β corresponds to the range of the three-dimensional structure of the core of this chromo domain (Ball et al., 1997) that is used to prepare (B)–(D). (B and C) Two surface views of the HP1 chromo domain related by 180° rotation. The magenta surface corresponds to residues demonstrating large perturbations, Δδave ≥0.2 (Figure 5A). (D) The surface electrostatic potential for the methyl-lysine-binding face. The residue numbers corresponding to magenta regions in (C) are labeled. In addition, residues L43, W45 and E56 were labeled in the putative binding pocket. (B)–(D) were prepared using GRASP (Nicholls, 1993).

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Fig. 7. Effect of point mutations in the specificity of the HP1 chromo domain for methyl-K9 H3 peptide. (A) Fluorescence polarization assays are illustrated for wild-type (circles), V26M (squares) and Y24F/A25P (triangles); measurements were performed at 25°C in 50 mM sodium phosphate pH 6 and 25 mM NaCl buffer. (B) The NMR chemical shift perturbations in backbone 1HN and 15N nuclei of the V26M mutant are marked on the structure (Ball et al., 1997) using the same view as in Figure 6C. Regions in magenta correspond to the sites where NMR signals disappear (residues 25–28, 43, 55, 62, 66 and 70) and regions in yellow (residues 54, 56 and 68) correspond to the sites with weighted chemical shift perturbations in the range 0.1 < Δδ < 0.2. Regions shown in white do not show any significant perturbation upon mutation.

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Fig. 8. Fluorescence polarization assays for interaction of the Esa1 chromo domain with fluorescein-labeled unmodified H3 (circles), methyl-K4 H3 (triangles), methyl-K4/methyl-K9 H3 (open squares) and unmodified H4 (diamonds). Measurements were performed at 25°C in 50 mM sodium phosphate pH 8 and 25 mM NaCl buffer. Curves represent the best fit to the data as described in Materials and methods. Fraction bound corresponds to the normalized anisotropy.

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