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. 2016 Apr 19:2:16004.
doi: 10.1038/celldisc.2016.4. eCollection 2016.

The Chp1 chromodomain binds the H3K9me tail and the nucleosome core to assemble heterochromatin

Manuel Zocco  1 , Mirela Marasovic  1 , Paola Pisacane  1 , Silvija Bilokapic  1 , Mario Halic  1
Affiliations

The Chp1 chromodomain binds the H3K9me tail and the nucleosome core to assemble heterochromatin

Manuel Zocco et al. Cell Discov. .

Abstract

To maintain genome stability, cells pack large portions of their genome into silent chromatin or heterochromatin. Histone H3 lysine 9 methylation, a hallmark of heterochromatin, is recognized by conserved readers called chromodomains. But how chromodomains interact with their actual binding partner, the H3K9 methylated nucleosome, remains elusive. We have determined the structure of a nucleosome trimethylated at lysine 9 of histone H3 (H3K9me3 Nucleosome) in a complex with the chromodomain of Chp1, a protein required for RNA interference-dependent heterochromatin formation in fission yeast. The cryo-electron microscopy structure reveals that the chromodomain of Chp1 binds the histone H3 lysine 9 methylated tail and the core of the nucleosome, primarily histones H3 and H2B. Mutations in chromodomain of Chp1 loops, which interact with the nucleosome core, abolished this interaction in vitro. Moreover, fission yeast cells with Chp1 loop mutations have a defect in Chp1 recruitment and heterochromatin formation. This study reveals the structural basis for heterochromatic silencing and suggests that chromodomains could read histone code in the H3 tail and the nucleosome core, which would provide an additional layer of regulation.

Keywords: Chp 1; RNAi; S. pombe; chromodomain; cryo-EM; heterochromatin; nucleosome.

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Figures

Figure 1
Figure 1
Cryo-EM reconstruction of Chp1CD-H3K9me3Nucleosome complex. (a) Cryo-EM map of Chp1CD-H3K9me3Nucleosome complex at 10 Å (FSC 0.143 cutoff of two independently refined data sets). The map was reconstructed from the C15 subclass that had defined electron density in addition to the core of the nucleosome (depicted in red). The nucleosome is shown in light blue. No symmetry was applied. (b) Cryo-EM map of the H3K9me3Nucleosome control at 7.3 Å (FSC 0.143 cutoff of two independently refined data sets). The map shows the core of the nucleosome (light blue). No density is found at the position of Chp1CD density in the Chp1CD-H3K9me3Nucleosome complex cryo-EM map. C2 symmetry was applied. (c) Fourier shell correlation (FSC) curve showing the resolution of cryo-EM maps shown in a and b. The resolution is shown at 0.143 cutoff for both reconstructions. The mask including nucleosome and the Chp1 density was applied for resolution calculation for both density maps. Both data sets were split into two halves that were independently refined.
Figure 2
Figure 2
Model of Chp1CD bound to the nucleosome core. (a) Local resolution for Chp1CD-H3K9me3Nucleosome Cryo-EM map was calculated with the Resolution Map software (ResMap). The local resolution calculation shows that nucleosome core resolution is 9–10 Å, whereas Chp1CD has a slightly lower resolution of 10 Å. (b and c) Fitting of Chp1CD (PDB code 3G7L, red) crystal structure into the Chp1CD-H3K9me3Nucleosome Cryo-EM map (cross-correlation 0.91). Note the separation of Chp1CD density into two features where α-helix and β-sheet were fitted in the cryo-EM map (transparent red). The nucleosome is shown in light blue.
Figure 3
Figure 3
Interaction of Chp1CD with the nucleosome core. (a) Molecular interface of Chp1CD interaction with the H3K9me3Nucleosome. Three contacts can be observed. Chp1CD LOOP1 (31–37aa) interacts with the histone H3 loop (77–81aa), whereas Chp1CD LOOP2 interacts with H4 (55–63aa) and potentially also with H4 tail. Chp1CD α-helix interacts with acidic patch formed by H2B (105–113aa). Chp1CD is shown in red and the nucleosome in blue. The cryo-EM map is shown in transparent light blue. Red dots represent couple of residues present in the protein but absent in the crystal structure. (b) Depiction of the nucleosomal regions that make contacts with Chp1CD (colored yellow). Primary interaction is with histone H3 region 76–81aa. Second observed interaction is with an acidic patch formed by H2A and H2B (H2B, region 105–113aa). At lower contour level we observe an interaction with H4 region 55–63aa and possibly with H4 tail.
Figure 4
Figure 4
Chp1CD interaction with the nucleosome core is required for binding to H3K9me3Nucleosomes. (a) Model of Chp1CD interacting with the H3K9me3Nucleosome. Mutations used in this study are depicted on the model. LOOP1A/B mutations are shown in yellow, LOOP2A in cyan, LOOP2B in blue. (b) Thermophoresis assay showing binding curves of wild-type Chp1CD and LOOP1B/2B Chp1CD mutant to H3K9me3 peptide. Kd is shown below. (c) In vitro pulldown assay showing that Chp1CD interacts with the core of the nucleosome and that LOOP1B/2B mutations abolish this interaction. (d) In vitro pulldown assay showing Chp1CD interaction with H3K9me3Nucleosomes. LOOP1B/2B mutations strongly reduce the interaction with H3K9me3Nucleosomes. (e) Thermophoresis assay showing binding curves of wild-type Chp1CD and LOOP1B/2B Chp1CD mutant to H3K9me3Nucleosomes. Kd is shown below the image. (f) In vitro pulldown assay showing that Chp1CD-H3K9me3Nucleosome complex interacts with RNA. Chp1CD LOOP1B/2B mutations abolished this interaction.
Figure 5
Figure 5
Chp1CD interaction with the core of the nucleosome is required for heterochromatin formation. (a) Silencing assay showing that Chp1CD LOOP1B/2B mutant cells have a defect in heterochromatin formation at centromeric repeats. Tenfold serial dilutions were spotted on YES, YES+FOA and EMMC-Ura plates. (b) Relative expression of centromeric dh transcripts in wt and Chp1CD mutant cells. Yeast cells with Chp1CD LOOP1B/2B mutations show accumulation of pericentromeric dh transcripts to the levels of chp1Δ. Error bars indicate s.e.m. (c) ChIP experiment showing that H3K9me is reduced at centromeric dh repeats in Chp1CD LOOP1B/2B mutant cells to the level of chp1Δ. Error bars indicate s.e.m. (d) ChIP experiment showing that Chp1CD LOOP1B and 2B mutants are less efficiently recruited to centromeric dh repeats. Error bars indicate s.e.m. (e) Silencing assay showing that genomically integrated Chp1CD LOOP1B/2B mutant cells have a defect in heterochromatin formation at centromeric repeats. Tenfold serial dilutions were spotted on YES and YES+FOA plates. (f) Relative expression of centromeric dg transcripts in wt and genomically integrated Chp1CD mutant cells. Yeast cells with Chp1CD LOOP1B/2B mutations show accumulation of pericentromeric dg transcripts to the levels of chp1Δ. Error bars indicate s.e.m.
Figure 6
Figure 6
Chp1CD interaction with the core of the nucleosome is required for heterochromatin formation. Model showing Chp1CD interaction with the H3K9me3Nucleosome. In establishment mode, Chp1CD will be tethered to centromeric repeats by sRNAs and Argonaute. Chp1CD will bind to the core of the nucleosome. This will tether Argonaute to chromatin even in absence of H3K9me. After deposition of initial H3K9me, Chp1 will bind H3K9me histone tail to further stabilize interaction with the chromatin. The interaction with the nucleosome core is required for heterochromatin formation and silencing of centromeric repeats in fission yeast.
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