1. Structural Basis of Heterochromatin Formation by Human HP1
Shinichi Machida, Yoshimasa Takizawa, Masakazu Ishimaru, Yukihiko Sugita, Satoshi Sekine, Jun-ichi Nakayama, Matthias Wolf, Hitoshi Kurumizaka 
Molecular Cell 
Volume 69, Issue 3, Pages e8 (February 2018)
DOI: /j.molcel
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2. Molecular Cell 2018 69, 385-397.e8DOI: (10.1016/j.molcel.2017.12.011)
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3. Figure 1
HP1α, HP1β, and HP1γ Form Complexes with the H3K9me3 Dinucleosome
(A) H3K9me3 dinucleosomes mixed with HP1α, HP1αW40A, and HP1αI165E were crosslinked by GraFix (glutaraldehyde), and the peak fractions were analyzed by native PAGE. Lanes 1–4 indicate the experiments with H3K9me3 dinucleosome, H3K9me3 dinucleosome with HP1α, H3K9me3 dinucleosome with HP1αW40A, and H3K9me3 dinucleosome with HP1αI165E, respectively.
(B) Unmodified dinucleosomes were mixed with HP1α. The samples were crosslinked by GraFix (glutaraldehyde), and the peak fractions were analyzed by native PAGE. Lanes 1 and 2 indicate the experiments without and with HP1α, respectively.
(C) H3K9me3 dinucleosomes complexed with HP1β and HP1γ were crosslinked by GraFix (glutaraldehyde), and the peak fractions were analyzed by native PAGE. Lanes 1–3 indicate the experiments with H3K9me3 dinucleosome, H3K9me3 dinucleosome with HP1β, and H3K9me3 dinucleosome with HP1γ, respectively.
(D) Unmodified dinucleosomes mixed with HP1β and HP1γ were crosslinked by GraFix (glutaraldehyde), and the peak fractions were analyzed by native PAGE. Lanes 1–3 indicate the experiments without HP1, with HP1β, and with HP1γ, respectively.
(E) Sedimentation velocity assay. The sedimentation coefficient (S20,W) distributions were calculated by the enhanced van Holde and Weischet method. Unmodified dinucleosome without HP1α, gray squares; unmodified dinucleosome with HP1α, light blue squares; H3K9me3 dinucleosome without HP1α, black circles; H3K9me3 dinucleosome with HP1α, red circles; H3K9me3 dinucleosome with HP1αW40A, green circles; H3K9me3 dinucleosome with HP1αI165E, blue circles.
See also Figure S1.
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4. Figure 2 The Structure of the HP1α-Dinucleosome Complex
(A) Digital micrograph of HP1α-dinucleosome particles in amorphous ice, recorded in-focus with a Volta phase plate. Scale bar, 100 nm.
(B) Selected 2D class averages of 200 classes from 187,784 aligned single-particle images of the HP1α-dinucleosome complex. Box size, 36 nm.
(C) Digital micrograph of dinucleosome particles in amorphous ice, recorded in-focus with a Volta phase plate. Scale bar, 100 nm.
(D) Selected 2D class averages of 40 classes from 35,357 aligned single-particle images of the dinucleosome. Box size, 36 nm.
(E) Iso-surface representation of three orthogonal views of the reconstructed three-dimensional electron potential of the HP1α-dinucleosome complex, contoured at 4.1 sigma above mean density. A model of the nucleosome core particle (PDB: 3LZ0) is docked into each of the two nucleosome densities. The linker DNA and the bridging HP1α dimer are clearly distinguishable. Scale bar, 10 Å.
See also Figures S2 and S3.
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5. Figure 3 HP1α Bridges Two Nucleosomes
(A and B) The HP1α-dinucleosome complexes (15-bp linker DNA) were crosslinked by glutaraldehyde, followed by MNase treatment. The reaction products without proteinase K treatment were analyzed by native PAGE (A). The linker DNA digestion by MNase was confirmed by native PAGE, after treatment of the reaction products with proteinase K (B).
(C) Digital micrograph of the HP1α-dinucleosome complex treated with MNase, recorded in-focus with a Volta phase plate. Scale bar, 100 nm.
(D) Representative single-particle images of the HP1α-dinucleosome complex treated with MNase. Box size, 36 nm.
(E) Representative single-particle images of the HP1α-dinucleosome complex without MNase treatment. Box size, 36 nm.
(F) Digital micrograph of the dinucleosome with MNase treatment, recorded in-focus with a Volta phase plate. Scale bar, 100 nm.
(G and H) The HP1α-dinucleosome complexes (48-bp linker DNA) were crosslinked by glutaraldehyde, followed by XhoI treatment. The reaction products without proteinase K treatment were analyzed by native PAGE (G). The linker DNA digestion by XhoI was confirmed by native PAGE, after treatment of the reaction products with proteinase K (H).
(I and J) The HP1α-dinucleosome complexes (58-bp linker DNA) were crosslinked by glutaraldehyde, followed by XhoI treatment. The reaction products without proteinase K treatment were analyzed by native PAGE (I). The linker DNA digestion by XhoI was confirmed by native PAGE, after treatment of the reaction products with proteinase K (J).
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6. Figure 4
The Structures of the HP1β-Dinucleosome and HP1γ-Dinucleosome Complexes
(A) Digital micrograph of HP1β-dinucleosome particles in amorphous ice, recorded in-focus with a Volta phase plate. Scale bar, 100 nm.
(B) Selected 2D class averages of 100 classes from 86,483 aligned single-particle images of the HP1β-dinucleosome complex. Box size, 36 nm.
(C) Iso-surface representation of three orthogonal views of the reconstructed three-dimensional electron potential of the HP1β-dinucleosome complex, contoured at 3.2 sigma above mean density. Scale bar, 10 Å.
(D) Digital micrograph of HP1γ-dinucleosome particles in amorphous ice, recorded in-focus with a Volta phase plate. Scale bar, 100 nm.
(E) Selected 2D class averages of 80 classes from 71,334 aligned single-particle images of the HP1γ-dinucleosome complex. Box size, 36 nm.
(F) Iso-surface representation of three orthogonal views of the reconstructed three-dimensional electron potential of the HP1γ-dinucleosome complex, contoured at 3.9 sigma above mean density. Scale bar, 10 Å.
(G–I) Iso-surface representations of reconstructed electron potential maps of the HP1-dinucleosome complexes. The HP1α-dinucleosome complex (G), the HP1β-dinucleosome complex (H), and the HP1γ-dinucleosome complex (I) are shown.
(J) Superimposition of three cryo-EM maps of the HP1-dinucleosome complexes. The HP1α-dinucleosome complex, the HP1β-dinucleosome complex, and the HP1γ-dinucleosome complex, colored in gray, orange, and green, are superimposed. The orientations of the left nucleosomes are fixed.
See also Figure S4.
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7. Figure 5 Linker DNA Exposure
(A and B) Sucrose gradient sedimentation assays of the H3K9me3 dinucleosome containing 15-bp linker DNAs at both DNA ends (A) and the H3K9me3 dinucleosome with HP1α (B). The experiments were performed without glutaraldehyde. Aliquots (600 μL) were successively fractionated from the top of the samples. Fractions 1–12 were analyzed by SDS-PAGE.
(C–E) MNase assay. The HP1α-dinucleosome complexes, containing a 15-bp linker DNA (C), a 48-bp linker DNA (D), and a 58-bp linker DNA (E) between nucleosomes, were treated with MNase in the absence of glutaraldehyde.
(F) Hydroxyl radical footprinting without glutaraldehyde. Lanes 1 and 2 represent the H3K9me3 dinucleosome and the H3K9me3 dinucleosome with HP1α, respectively. Densitometric scans of hydroxyl radical cleavage patterns are shown in the right panel. Black and red lines represent scans of lanes 1 and 2, respectively. Dashed lines indicate the linker DNA region.
All experiments were repeated at least twice and the results were reproduced.
See also Figure S5.
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8. Figure 6
HP1α Forms Complexes with H3K9me3 Dinucleosomes Containing 48- and 58-bp Linker DNAs
(A) H3K9me3 dinucleosomes containing a 48-bp linker DNA with or without HP1α were crosslinked by GraFix (glutaraldehyde), and the peak fractions were analyzed by native PAGE. Lanes 1 and 2 indicate the experiments with the H3K9me3 dinucleosome and the H3K9me3 dinucleosome with HP1α, respectively.
(B) Digital micrograph of HP1α-dinucleosome with 48-bp linker DNA particles in amorphous ice, recorded in-focus with a Volta phase plate. Scale bar, 100 nm.
(C) Representative single-particle images of the HP1α-dinucleosome with a 48-bp linker DNA. Box size, 44.5 nm.
(D) H3K9me3 dinucleosomes containing a 58-bp linker DNA with or without HP1α were crosslinked by GraFix (glutaraldehyde), and the peak fractions were analyzed by native PAGE. Lanes 1 and 2 indicate the experiments with the H3K9me3 dinucleosome and the H3K9me3 dinucleosome with HP1α, respectively.
(E) Digital micrograph of HP1α-dinucleosome with 58-bp linker DNA particles in amorphous ice, recorded in-focus with a Volta phase plate. Scale bar, 100 nm.
(F) Representative single-particle images of the HP1α-dinucleosome with a 58-bp linker DNA. Box size, 44.5 nm.
(G) Tomographic slices of a phase plate cryoelectron tomography (cryo-ET) of the HP1α-dinucleosome complex with a 48-bp linker DNA. Particles boxed in white lines and in white dashed lines indicate the dinucleosomes bridged and unbridged by HP1α, respectively. Scale bar, 100 nm.
(H) Boxed particles in (G) correspond to tomographic slices of individual particles of the dinucleosome bridged by HP1α. Scale bar, 74.9 nm.
See also Figure S6 and Movie S1.
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9. Figure 7 ACF Remodels the HP1α-Dinucleosome Complex
(A) Schematic diagram of the HapII sites in the dinucleosome with 15-bp linker DNAs at both DNA ends and between nucleosomes.
(B) Schematic diagram of the nucleosome-remodeling assay. The HP1α-dinucleosome complex was treated with HapII. After an incubation for 5 min, the nucleosome-remodeling reaction was initiated by the addition of the ACF complex. The reaction products were deproteinized and analyzed by native PAGE.
(C) Chromatin-remodeling assay with the HP1α-dinucleosome complex containing a 15-bp linker DNA between two nucleosomes. The HP1α-dinucleosome complex contains additional 15-bp linker DNAs at both ends. Lane 1 indicates 100-bp DNA ladder markers. Lanes 2–5, 6–9, 10–13, and 14–17 represent H3K9me3 dinucleosome, H3K9me3 dinucleosome with HP1α, H3K9me3 dinucleosome with H1, and H3K9me3 dinucleosome with HP1α and H1, respectively. These experiments were repeated three times and the results were reproduced.
(D) Graphic representation of the experiment shown in (C). The average values of three independent experiments (see also Figures S7B and S7C) are plotted with SD values.
(E) Chromatin-remodeling assay with the HP1α-dinucleosome complex containing a 48-bp linker DNA between two nucleosomes. The HP1α-dinucleosome complex contains additional 23-bp linker DNAs at both ends. Lane 1 indicates 100-bp DNA ladder markers. Lanes 2–5 and 6–9 represent H3K9me3 dinucleosome and H3K9me3 dinucleosome with HP1α, respectively. These experiments were repeated twice and the results were reproduced, as shown in Figure S7D.
(F) Chromatin-remodeling assay with the HP1α-dinucleosome complex containing a 58-bp linker DNA between two nucleosomes. The HP1α-dinucleosome complex contains additional 23-bp linker DNAs at both ends. Lane 1 indicates 100-bp DNA ladder markers. Lanes 2–5 and 6–9 represent H3K9me3 dinucleosome and H3K9me3 dinucleosome with HP1α, respectively. These experiments were repeated twice and the results were reproduced, as shown in Figure S7E.
(G) Pull-down assay. FLAG-ACF1 was captured with an anti-FLAG antibody. HP1α co-pelleted with FLAG-ACF1 was detected by western blotting using the anti-HP1α antibody.
See also Figure S7.
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