SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION doi: /nature10258 Supplementary Figure 1 Reconstitution of the CENP-A nucleosome with recombinant human histones H2A, H2B, H4, and CENP-A. a, Purified recombinant human histones H2A, H2B, H4, and CENP-A were analyzed by 16% SDS-PAGE with Coomassie Brilliant Blue staining. Lane 1 indicates the molecular mass markers. Lanes 2-5 are the purified H2A, H2B, CENP-A, and H4, respectively. b, The CENP-A nucleosome reconstitution. A 147 base pair palindromic α-satellite derivative was incubated with purified H2A, H2B, CENP-A, and H4, and the CENP-A nucleosome was reconstituted by a salt dialysis method. The reconstituted nucleosome was analyzed by non-denaturing 6% PAGE. c, The histone composition of the purified CENP-A nucleosome was analyzed by 16% SDS-PAGE with Coomassie Brilliant Blue staining. 1

2 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 2 The human α-satellite DNA derivative used in the crystallization of the CENP-A nucleosome. a, The human α-satellite DNA (sat4) is represented at the top. The red ellipse indicates the previously mapped translational position of the CENP-A nucleosome on this α-satellite DNA (1). The 71-mer DNA fragment containing one-half of the sat4 α-satellite sequence, including the CENP-B box at the edge, was prepared (second row). This 71-mer DNA fragment contained an extra 5-base overhang, 5 GTAAC 3, at the distal edge of the CENP-B box. The 71-mer DNA fragment was self-ligated (third row) with this cohesive 5-base overhang, to yield a palindromic 147 base pair α-satellite DNA derivative (bottom row). In this palindromic 147 base pair α-satellite DNA derivative, the CENP-B box sequence was located at both DNA ends, and an adenine-adenine mismatch was introduced at the center of the 147 bp DNA fragment (bottom row). The disordered DNA regions in the crystal structure of the CENP-A nucleosome are indicated. b, Alignment of the palindromic 147 base pair α-satellite DNA derivative (CA-NCP147), used in the structural analysis of the CENP-A nucleosome, with the palindromic 147 base pair DNA (H3-NCP147). The H3-NCP147 DNA is identical to that used in the previous structural analysis of the H3 nucleosome (2). Black and orange letters indicate residues facing the major and minor grooves, respectively. 2

3 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 3 Negative supercoils are introduced by CENP-A nucleosome formation (salt dialysis). a, Schematic representation of the topological assay for nucleosome assembly. The CENP-A octamer (H2A, H2B, CENP-A, and H4) or the H3 octamer (H2A, H2B, H3, and H4) was incubated with relaxed pgsat4 DNA (10 ng/µl) (1) in the presence of calf topoisomerase I (Invitrogen). Nucleosomes were then assembled by a salt dialysis method. After the nucleosome assembly, the proteins were removed by an incubation with 50 µl of a proteinase K solution (20 mm Tris-HCl (ph 8.0), 20 mm EDTA, 0.5% SDS, and 0.5 mg/ml proteinase K) at 37 C for 30 min, followed by phenol-chloroform extraction. b, The CENP-A nucleosome assembly on the plasmid DNA was analyzed by 1% agarose gel electrophoresis in 1 TAE buffer (1.3 V/cm for 15.5 hr), and the gel was stained with SYBR Gold (Invitrogen). The concentrations of the CENP-A octamer (H2A, H2B, CENP-A, and H4) were 0 µm (lane 1), 0.02 µm (lane 2), 0.04 µm (lane 3), 0.09 µm (lane 4), and 0.18 µm (lane 5). c, Schematic diagram of the two-dimensional gel electrophoresis. Migration patterns for negatively and positively supercoiled DNA molecules are represented. d, Two-dimensional gel electrophoretic analysis of the H3 nucleosome formation by the salt dialysis method. e, Two-dimensional gel electrophoretic analysis of the CENP-A nucleosome formation by the salt dialysis method. The sample shown in panel b (lane 5) was subjected to two-dimensional gel electrophoresis. 3

4 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 4 Negative supercoils are introduced by CENP-A nucleosome formation (histone chaperones). H2A/H2B (15 ng/µl) and CENP-A/H4 (15 ng/µl) were preincubated with hnap1 or snasp at 37 C for 15 min. The nucleosome assembly reaction was initiated by the addition of relaxed pgsat4 DNA (10 ng/µl), which was previously incubated with 2 U wheat germ topoisomerase I (Promega) per 100 ng DNA at 37 C for 150 min, under physiological salt conditions (140 mm NaCl). The reaction (10 µl) was incubated in 20 mm Tris-HCl buffer (ph 8.0), containing 140 mm NaCl, 2 mm MgCl 2, and 5 mm DTT, at 37 C for 60 min. After the nucleosome assembly, the samples were incubated at 42 C for 60 min, to eliminate nonspecific DNA binding by the histones, and the proteins were then removed by an incubation with 60 µl of a proteinase K solution (20 mm Tris-HCl (ph 8.0), 20 mm EDTA, 0.25% SDS, and 0.5 mg/ml proteinase K) at 37 C for 15 min, followed by phenol-chloroform extraction. a, The samples were analyzed by 1% agarose gel electrophoresis in 1 TBE buffer (1.3 V/cm for 15 hr), and the gel was stained with SYBR Gold (Invitrogen). Lanes 1 and 2 indicate negative control experiments without histones and chaperones, in the absence and presence of wheat germ topoisomerase I, respectively. Lanes 3 and 4 indicate control experiments without histones in the presence of hnap1 and snasp, respectively. The concentrations of hnap1 or snasp were 0 µm (lane 5), 0.25 µm (lanes 6 and 9), 0.5 µm (lanes 7 and 10), and 1 µm (lanes 8 and 11). b, Two-dimensional gel electrophoretic analysis of CENP-A nucleosome formation by hnap1. The sample shown in panel a (lane 8) was subjected to two-dimensional gel electrophoresis. c, Two-dimensional gel electrophoretic analysis of CENP-A nucleosome formation by snasp. The sample shown in panel a (lane 11) was subjected to two-dimensional gel electrophoresis. 4

5 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 5 Small-angle X-ray scattering (SAXS) measurements of CENP-A and H3 nucleosomes. a, Normalized small-angle X-ray scattering (SAXS) curves of the nucleosomes. Red and black lines show the SAXS curves as a function of q (q = 4πsinθ/λ) for the CENP-A nucleosome and the H3 nucleosome, respectively. b, Guinier plots of the CENP-A nucleosome (red) and the H3 nucleosome (black). c, The radii of gyration R g of the CENP-A nucleosome and the H3 nucleosome, estimated by fitting the SAXS curves using the Guinier approximation. d, Distance distribution functions P(r) of the CENP-A nucleosome and the H3 nucleosome. e, The maximum dimensions D max of the CENP-A nucleosome and the H3 nucleosome, estimated from the P(r) function as the distance r, where P(r) =

6 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 6 Comparison of the compact CENP-A nucleosome and the CENP-A nucleosome models. a, The CENP-A nucleosome model. The disordered DNA regions of the crystal structure were modeled using the structure of the CENP-B box DNA (magenta) in the CENP-B-DNA complex (PDB ID: 1HLV) (4). Two CENP-A molecules are shown in yellow. b, The compact CENP-A nucleosome model. The model was constructed by starting with the CENP-A/H4 tetramer structure reported by Black and colleagues (PDB ID: 3NQJ) (3), and placing ~70 bp of DNA around the tetramer. For H2A/H2B dimers and the remaining DNA, the corresponding coordinates from a were used, and placed so that the octamer becomes wider when viewed from the 6

7 SUPPLEMENTARY INFORMATION RESEARCH edge of the nucleosome, as proposed by Black and colleagues. Two CENP-A molecules are shown in yellow. c, Normalized SAXS curves of the CENP-A nucleosome and the compact CENP-A nucleosome models. SAXS curves of these two models were generated by program CRYSOL (5). Blue and black lines show the SAXS curves as a function of q (q = 4πsinθ/λ) for the CENP-A nucleosome model and the compact CENP-A nucleosome model, respectively. Red line shows the actual SAXS curve of the CENP-A nucleosome shown in Supplementary Fig. 5a. d, Distance distribution functions P(r) of the CENP-A nucleosome and the compact CENP-A nucleosome models. 7

8 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 7 Crystal packing differences between the CENP-A and H3 nucleosomes. Four nucleosomes in the CENP-A nucleosome crystal (panel a, left) and the H3 nucleosome crystal (panel b, left; PDB ID: 3AFA) are shown. The DNA is shown in a surface representation, and is colored orange and magenta. Histones are shown in cartoon representations, and are colored dark green. Circles indicate the DNA ends in the crystal structures. Schematic diagrams of the crystal packing are shown on the right sides of both panels. The DNA in the H3 nucleosome engages in end-to-end stacking interactions, and forms an essential lattice contact. In the CENP-A nucleosome, the disordered DNA ends (panel a, right, colored grey) may or may not engage in end-to-end stacking interactions. 8

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10 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 8 Reconstitution of the CENP-A nucleosome with recombinant human histones H2A, H2B, H4, and CENP-A. a, The CENP-A nucleosome was reconstituted with a 121 base pair palindromic α-satellite derivative, which lacks 13 base pair regions from both edges of the 147 base pair palindromic α-satellite derivative used in the crystallography of the CENP-A nucleosome. The rest of the 121 base pair DNA sequence is identical to the 147 base pair palindromic α-satellite derivative. The CENP-A nucleosome reconstitution was performed by the salt dialysis method, and the reconstituted nucleosomes were analyzed by non-denaturing 6% PAGE. b, The histone compositions of the purified CENP-A nucleosomes with the 121 base pair DNA (lane 3) and the 147 base pair DNA (lane 2) were analyzed by 18% SDS-PAGE with Coomassie Brilliant Blue staining. c, Competitive nucleosome assembly assay. H2A, H2B, CENP-A, and H4 were incubated in the presence of both the 147 base pair DNA (24 µg) and 121 base pair DNA (20 µg), and the CENP-A nucleosomes were reconstituted by the salt dialysis method. The amounts of the CENP-A octamer were 14 µg (lane 5), 28 µg (lane 6), 42 µg (lane 7), and 56 µg (lane 8). The reconstituted nucleosomes were analyzed by non-denaturing 6% PAGE. d, Graphic representation of the experiments shown in panel c. Averages of three independent experiments are represented with the standard deviation values. e, Schematic representation of the nucleosome disruption assay with the CENP-A nucleosome. f, The thermal stability of the CENP-A nucleosome with the 121 base pair DNA was compared to that of the CENP-A nucleosome with the 147 base pair DNA. The averages of three independent experiments are represented with the standard deviation values. 10

11 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 9 The CENP-A nucleosome induces supercoils into plasmid DNA less efficiently than the H3 nucleosome. a, The CENP-A octamer (H2A, H2B, CENP-A, and H4) or the H3 octamer (H2A, H2B, H3, and H4) was incubated with relaxed pgsat4 DNA (10 ng/µl) (1) in the presence of wheat germ topoisomerase I (Promega). Nucleosomes were then assembled by the salt dialysis method. After the nucleosome assembly, the proteins were removed by an incubation with 50 µl of proteinase K solution (20 mm Tris-HCl (ph 8.0), 20 mm EDTA, 0.5% SDS, and 0.5 mg/ml proteinase K) at 37 C for 30 min, followed by phenol-chloroform extraction. The nucleosome assembly on the plasmid DNA was analyzed by 1% agarose gel electrophoresis in 1 TAE buffer (1.3 V/cm for 15 hr), and the gel was stained with SYBR Gold (Invitrogen). The concentrations of the H3 octamer and the CENP-A octamer were 18 nm (lanes 3 and 7), 27 nm (lanes 4 and 8), 36 nm (lanes 5 and 9), and 44 nm (lanes 6 and 10). b, Densitometric analysis of the topoisomer distributions produced by the H3 nucleosome assembly and the CENP-A nucleosome assembly. Blue and red lines indicate the densitometric profiles of the H3 nucleosome (panel a, lane 6) and the CENP-A nucleosome (panel a, lane 10), respectively. 11

12 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 10 Exonuclease assay. a, Schematic representation of the exonuclease assay. The reconstituted CENP-A nucleosome (400 ng) or the H3 nucleosome (400 ng) was treated with 3 units of Escherichia coli exonuclease III in 10 µl of 50 mm Tris-HCl (ph 8.0), 5 mm MgCl 2, and 1 mm DTT. After an incubation for the indicated time at 37 C, the reaction was stopped by the proteinase K solution, as described in the Supplementary Figure 8 legend. The DNA was extracted with phenol/chloroform, precipitated with ethanol, dissolved in Hi-Di Formamide (Applied Biosystems), and then fractionated on a 10% denaturing polyacrylamide gel containing 7 M urea in 0.5 TBE buffer (21 V/cm for 1.5 hr). b, Schematic representations of the DNAs used in the exonuclease assay. The DNA sequences of CA-NCP147 and H3-NCP147 are represented in Supplementary Fig. 2b. CA/H3-NCP147 is a chimeric DNA, in which the CENP-B box sequences of CA-NCP147 were replaced by the H3-NCP147 sequence (5 AATATCCACCTGCAGAT 3 ). c-e, Gel images of the exonuclease assays. Lane 1 indicates molecular mass markers. Lanes 2-5 and 6-9 indicate experiments with the H3 nucleosome and the CENP-A nucleosome, respectively. c, Experiments with nucleosomes containing CA-NCP147. d, Experiments with nucleosomes containing H3-NCP147-2, in which the HinfI sequence (5 GGAATCC 3 ) at the dyad of H3-NCP147 was replaced by the BstP1 sequence (5 GGTAACC 3 ) of CA-NCP147. e, Experiments with nucleosomes containing CA/H3-NCP

13 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 11 Stereo view of the loop 1 region of CENP-A. A composite omit map (contoured at 1.0 σ) surrounds a stick representation of residues of CENP-A. Supplementary Figure 12 Comparison of the CENP-A B factors between the CENP-A nucleosome and the free CENP-A/H4 tetramer (PDB ID: 3NQJ) (3). The relative B factors of the CENP-A main chain atoms are plotted for each residue. Red and blue lines indicate the CENP-A B factors of the CENP-A nucleosome and the free CENP-A/H4 tetramer, respectively. 13

14 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 13 The CENP-A mutants form less stable associations with the centromere. htert-rpe1 cells were transfected with RFP-tagged wild type CENP-A and a GFP-tagged mutant (CENP-A(A80A81), CENP-A(del82-83), or CENP-A(A82A83)). In the CENP-A(A80A81) mutant, the CENP-A Arg80 and Gly81 residues are replaced by Ala80 and Ala81 residues. In the CENP-A(del82-83) mutant, the Val82 and Asp83 residues are deleted. In the CENP-A(A82A83) mutant, the CENP-A Val82 and Asp83 residues are replaced by Ala82 and Ala83 residues. Experiments were performed as shown in Fig. 4. The numbers of transfected htert-rpe1 cells exhibiting RFP-CENP-A, GFP-CENP-A mutants, or both at centromeres were counted (n >20), and the average percentages from five independent transfections are plotted with the standard deviations. a, CENP-A(A80A81). b, CENP-A(del82-83). c, CENP-A(A82A83). 14

15 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 1 Data collection and refinement statistics (Molecular replacement) CENP-A nucleosome Data collection Space group P21 Cell dimensions a, b, c (Å) 65.84, 83.29, α, β, γ ( ) 90, 100.7, 90 Resolution (Å) 3.6 ( ) * R sym or R merge 10.2 (42.7) I/σI 8.3 (3.2) Completeness (%) 95 (87) Redundancy 3.5 (3.2) Refinement Resolution (Å) No. reflections 21,021 R work/ R free 27.1/32.8 No. atoms Protein 5,592 Ligand/ion 4,956 Water - B-factors Protein 72 Ligand/ion 145 Water - R.m.s. deviations Bond lengths (Å) Bond angles (º) 1.27 PDB ID 3AN2 The structure of the CENP-A nucleosome was determined from a single crystal. *Highest resolution shell is shown in parentheses. 15

16 RESEARCH SUPPLEMENTARY INFORMATION Supplementary References 1. Tanaka, Y. et al. Human centromere protein B induces translational positioning of nucleosomes on alpha-satellite sequences. J. Biol. Chem. 280, (2005). 2. Davey, C.A., Sargent, D.F., Luger, K., Maeder, A.W., Richmond, T.J. Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 Å resolution. J. Mol. Biol. 319, (2002). 3. Sekulic, N., Bassett, E. A., Rogers, D. J. & Black, B. E. The structure of (CENP-A-H4) 2 reveals physical features that mark centromeres. Nature 467, (2010). 4. Tanaka, Y. et al. Crystal structure of the CENP-B protein-dna complex: The DNA-binding domains of CENP-B induce kinks in the CENP-B box DNA. EMBO J. 20, (2001). 5. Svergun, D.I., Barberato, C. & Koch, M.H.J. CRYSOL - a Program to Evaluate X-ray Solution Scattering of Biological Macromolecules from Atomic Coordinates J. Appl. Cryst., 28, (1995). 16