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. 2024 Mar 5;14(1):5438.
doi: 10.1038/s41598-024-56248-2.

Structural insights into the binding mechanism of Clr4 methyltransferase to H3K9 methylated nucleosome

Christopher Saab  1   2 , Joseph Stephan  3 , Elias Akoury  4
Affiliations

Structural insights into the binding mechanism of Clr4 methyltransferase to H3K9 methylated nucleosome

Christopher Saab et al. Sci Rep. .

Abstract

The establishment and maintenance of heterochromatin, a specific chromatin structure essential for genomic stability and regulation, rely on intricate interactions between chromatin-modifying enzymes and nucleosomal histone proteins. However, the precise trigger for these modifications remains unclear, thus highlighting the need for a deeper understanding of how methyltransferases facilitate histone methylation among others. Here, we investigate the molecular mechanisms underlying heterochromatin assembly by studying the interaction between the H3K9 methyltransferase Clr4 and H3K9-methylated nucleosomes. Using a combination of liquid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy, we elucidate the structural basis of Clr4 binding to H3K9-methylated nucleosomes. Our results reveal that Clr4 engages with nucleosomes through its chromodomain and disordered regions to promote de novo methylation. This study provides crucial insights into the molecular mechanisms governing heterochromatin formation by highlighting the significance of chromatin-modifying enzymes in genome regulation and disease pathology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Heterochromatin formation in Schizosaccharomyces pombe. (A) The enzyme Dicer generates siRNAs from dsRNA. (B) siRNAs are subsequently loaded on Argonaute (Ago1). (C) Upon Ago1 interaction with Tas3 and Chp1, (D) the RITS complex associates with chromatin through base-pairing interactions between siRNAs and non-coding transcripts. This prompts the interaction of the Chp1’s chromodomain with H3K9 methylated nucleosomes further recruiting RDRC and Dicer to the centromeric repeats to synthesize new dsRNA and amplify siRNAs. This RNAi cycle promotes (E) RITS-directed recruitment of CLRC methyltransferase and enhances H3K9 methylation. Consequently, efficient silencing and heterochromatin formation require binding of the two HP1 proteins Swi6 and Chp2. Swi6 promotes further association of RITS with the non-coding RNA; and (F) Chp2 associates with the SHREC deacetylase complex to form SHREC2. Upon H3K14 deacetylation, SHREC2 mediates transcriptional gene silencing by limiting RNA polymerase II (Pol II) access to the heterochromatin. Note that SHREC consists of the four proteins Clr1, Clr2, Clr3 and Mit1.
Figure 2
Figure 2
Assembly of FL Clr4-H3KC9me3 Methylated Nucleosome Complex. (A) Coomassie-stained SDS-PAGE of purified N-terminal His-tagged and C-terminal FLAG-tagged protein constructs: Clr4 FL, ΔCD, 192–490, and SET-post. (B) Coomassie-stained 15% SDS-PAGE of purified H3, H2A, H2B and H4 histone proteins from FPLC-purified fractions A11-B7. (C) Native gel showing the assembly of methylated, unmodified, and tailless nucleosomes. (D) In vitro pulldown assay using silver stained SDS-PAGE and western blot analysis of the interaction between Clr4 constructs and H3KC9me3. Clr4 constructs were bound to FLAG resin, incubated with H3KC9me3 nucleosome, eluted from the resin and detected with anti-H3K9me3 antibody, demonstrating the necessity of the Clr4 disordered region for the formation of the Clr4-H3KC9me3 nucleosome complex. The full-length membranes of all gels are reported in Supplementary Fig. 3F.
Figure 3
Figure 3
Overview of The Clr4-Nucleosome Structure. (A) Cryo-EM maps of Clr4 methyltransferase in its (A) unbound (3.8 Å) and (B) bound form to H3KC9me3 nucleosomes (4.5 Å). (C) Both maps are overlaid to confirm the additional densities corresponding to Clr4 interaction with H3KC9me3 nucleosomes. Docking of the Clr4 Chromodomain NMR solution structure (PDB 1G6Z yellow) and the crystal structure of Clr4 Pre-SET-post domains (PDB 1MVH, green) into the cryo-EM Clr4 FL-H3KC9me3 nucleosome complex map. Cryo-EM data collection, structure refinement and validation statistics are reported in Supplementary Table 2.
Figure 4
Figure 4
Deciphering the Interaction of Clr4-H3KC9me3 Methylated Nucleosome Complex by NMR. (A) A schematic diagram representing the domain organization of FL Clr4 protein, CD, ΔCD, 1–191 and 192–490 constructs (N = N-terminal, C = C-terminal, CD = Chromodomain, SET = SET domain, FL = Full length). The numbering reflects the primary sequence of the protein. Two-dimensional 1H-15N HSQC spectra of isotope-labeled (B) Clr4 CD, (C) 1–19140 and (D) 192–490 constructs with backbone amide assignments. For visualization purposes, only selected assignments are shown in (C) and (D) and the full chemical shift assignment is reported in Supplementary Table 3. The shifts in red (spectrum C) belong to the disordered part of Clr4 (residues 70 to 191). Spectra were recorded on a Bruker Avance III 800 MHz spectrometer at 288 K. Intensity ratio plots of E) Clr4 1–191 and (D) Clr4 192–490 in the presence of H3KC9me3 nucleosome (1:0.5 ratio). Intensity ratios represent the backbone amide resonances with the residue patches of the chromodomain, disordered regions, and Set domain showing the most significant decrease in signal intensities in presence of H3KC9me3 nucleosomes (detailed in Supplementary Fig. 7). The secondary structure elements of Clr4 are represented. (G) Based on the intensity ratio plots, the binding sites of H3KC9me3 nucleosome are mapped on the Clr4 CD (PDB 1G6Z, in red), on the disordered regions and on the SET domain (PDB MVH1 in orange). (H) Microscale Thermophoresis assay reporting the binding curves of FL Clr4, ΔCD and CD with the dissociation constants (Kd) of 541 nM, 422 nM and 310 nM, respectively.
Figure 5
Figure 5
Cryo-EM Reconstruction of FL Clr4–H3KC9me3 Nucleosome Complex. (A) Molecular docking of the H3K9 nucleosome crystal structure (PDB 1AOI), the Clr4 CD NMR solution structure (PDB 1G6Z yellow) and the crystal structure of Clr4 Pre-SET-post domains (PDB 1MVH, green) into the cryo-EM Clr4 FL-H3KC9me3 nucleosome complex map. (B) Docking of the corresponding secondary structures reveals an extra density (indicated by red arrow) which explains the interactions of the Clr4 1–191 and Clr4 192–490 constructs with the core of H3KC9me3 methylated nucleosome as accessed by NMR spectroscopy. Cryo-EM data collection, structure refinement and validation statistics are reported in Supplementary Table 2.
Figure 6
Figure 6
Interaction of Clr4 CD and Set Domain with H3KC9me3 Nucleosome. Key residues important for interaction of Clr4 with the H3KC9me3 nucleosome were identified after molecular docking based on the NMR and cryo-EM results. These included patch 62RRLK65 with H4 (H77, K78); patch 190NPSKL194 with H4 (K78, Q94) and H2A (S124); patch 196SYT198 and 201SFY203 with H2A (Y88) and H2B (S124); patch 227VDDE230 with H2A (K76) and H2B (K121); patch 340WGVRSLRF347 with H2B (E94, Q96, K109); patch 405SRFFNH410 with H2B (H83); and patch 431YDLAFF436 with H2B (Y43, K44). Several side chains of the residues are shown in stick representation.
Figure 7
Figure 7
Structural model of The Clr4-H3KC9me3 nucleosome complex. The Cryo-EM/NMR model of Clr4 binding to the H3KC9me3 nucleosome is denoted by space-filling representation. The histone proteins and DNA are color-coded (H2A, H2B, H4 in green, H3 in cyan and DNA in orange) and two nucleosome units are shown: the unmethylated and the H3KC9-methylated, respectively. The domains of full-length Clr4 are indicated. In its free form, the H3KC9-methylated nucleosome is approached by the Clr4 CD (red space-filling representation) to bind the H3 histone protein. In the bound form, the disordered part of Clr4 reinforces this interaction by tethering to both the methylated and unmethylated H3KC9 nucleosomes. This action brings the SET domain closer to the unmethylated H3KC9 nucleosome, for specific methylation of the H3 protein.
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