PMC

a, hORC1_BAH preferentially binds H4K20me2 peptides. Microarrays spotted with 82 distinct histone peptides as indicated in were probed with glutathione S-transferase (GST) fused to human ORC1 amino acids (aa) 1-185 (hORC1_BAH). Red spots indicate positive binding. b and c, hORC1_BAH specifically binds H4K20me2 peptides. Western blot analysis of histone peptide pull-downs with GST-hORC1_BAH and the indicated biotinylated peptides. d, hORC1_BAH binds with highest affinity to H4K20me2. ITC was used to determine the dissociation constants (K_ds) for the interaction of hORC1_BAH with the indicated peptides: H4K20me1 (green diamonds), H4K20me2 (red squares) and H4K20me3 (blue triangles). Standard deviation (s.d.) was derived from nonlinear fitting. e, hORC1_BAH binds full-length H4K20me2. Top: Western blot of GST-hORC1_BAH and GST control pull-downs from calf thymus histones (CTH) with the indicated antibodies. Bottom: Coomassie blue stain of input (20% of total). f, H4K20me2 binding is a common property of ORC1 BAH domains from diverse metazoan species. Binding assays as in (b) with GST-fused ORC1 BAH domains from the indicated species and using the indicated peptides.

a–c, 1.95 Å crystal structure of murine ORC1 BAH aa 9-170 (mORC1_BAH) complexed with H4(14-25)K20me2 peptide. a, Ribbon representation of mORC1_BAH bound to H4K20me2 peptide. The mORC1_BAH (cyan) and the bound H4K20me2 peptide (yellow) are shown in ribbon and stick representations, respectively. b, Details of intermolecular contacts in the mORC1_BAH-H4K20me2 complex. mORC1_BAH and H4K20me2 peptide residues are colored in pink and yellow, respectively, with hydrogen bonds depicted as red dashed lines, and a water molecular as a red sphere. c, Schematic representation of intermolecular contacts in the mORC1_BAH-H4K20me2 complex. The residues from the H4K20me2 peptide and mORC1_BAH are colored in magenta and black, respectively. Yellow: the hydrophobic contact. d, Positioning of the K20me2 side chain within an aromatic cage of the indicated residues (red) on the surface of mORC1_BAH. The equivalent cage residues in hORC1_BAH are labeled in parenthesis. e, Structural overlay of the mORC1_BAH K20me2-binding aromatic cage in the free (silver) and H4K20me2-bound states (salmon). Arrows indicate binding-induced structural shifts. f, Mutations in the hORC1_BAH H4K20me2-binding channel impair H4K20me2 recognition. ITC analysis of the indicated hORC1_BAH mutants binding to H4K20me2 peptide; s.d. derived from non-linear fitting. g–h, Mutations in the hORC1_BAH dimethyllysine-binding cage abrogate H4K20me2 recognition. g, Binding assays as in () with the indicated hORC1_BAH mutant proteins and biotinylated peptides. h, Top: Western analysis of CTH binding assays as in () with the indicated proteins and antibodies. Bottom: Coomassie blue stain of input GST-fusion proteins and histones (20% of total).

a–c, MGS-associated mutations F89S and E127G impair H4K20me2 binding by hORC1. a, Close-up view of mORC1_BAH bound to H4K20me2, with residues F88 and E126 (equivalent to F89 and E127 in hORC1, respectively) shown in stick representation. b, ITC analysis as in () of F89S and E127G hORC1 _BAH mutants binding to H4K20me2 peptides. *ITC data for wild-type hORC1_BAH from . c, Binding assays as in () with the indicated hORC1_BAH mutant proteins and biotinylated peptides. d–e, The H4K20me2-recognition activity of hORC1 is required to rescue the dwarfism phenotype of orc1 morphants. d, Quantification of dwarf phenotype in zebrafish injected with morphilino oligos (MO) targeting the orc1 translation start site (tss) alone or MO co-injected with the indicated hORC1 mRNAs. Insert on left shows electrophoresis analysis of the indicated hORC1 mRNAs used for reconstitution. Control, uninjected embryos. Dwarfism was defined as a reduction of body length of ≥ 3 s.d. relative to the average size of the control zebrafish. Zebrafish analyzed: control: 26; orc1 MO: 86; MO+hORC1_WT: 67; MO+hORC1_Y64A: 85; MO+hORC1_W88A: 83. P values are calculated with a two-tailed unpaired Student’s t-test. ** P < 0.01. e, Representative images of zebrafish in (b) 1 day post-fertilization (dpf). f, Depletion of H4K20me2/3 in suv4-20h1/suv4-20h2 morphants. Western blot analysis using the indicated antibodies of whole animal extracts 1 dpf from control zebrafish or zebrafish injected with MOs targeting the tss’s of suv4-20h1 and suv4-20h2. Control, uninjected embryos. g–h, Dwarfism in suv4-20h1/suv4-20h2 morphants. g, Quantification of dwarfism in suv4-20h1/suv4-20h2 morphants relative to controls as described in (d). Zebrafish analyzed: control: 38; MO: 60. ** P<0.01. h, representative images of zebrafish in (e) 1 dpf.

a, Western blot analysis with the indicated antibodies of wild-type (WT) and H4K20me2-binding pocket mutants (Y64A and W88A) affinity-purified FLAG-tagged ORC1 complexes from U2OS cells. Control, empty vector control IP. WCE, whole cell extract. b, The ORC1_BAH-H4K20me2 interaction is required for efficient ORC1 chromatin association. Western blot analysis of lysates biochemically separated into chromatin-enriched and soluble fractions from U2OS cells stably expressing the indicated ORC1 protein. Quantitation of FLAG-ORC1 levels is shown. Control, empty vector control lysates. Tubulin and H3 levels are shown as control for the integrity of fractionation. c, Disruption of ORC1 binding to H4K20me2 destabilizes ORC chromatin association. Western blot analysis of biochemically purified chromatin from U2OS cells as in (b) with the indicated antibodies. Total ORC protein levels in WCE are shown. d, H4K20me2 is enriched at DNA replication origins. H4K20me2 signal normalized to total H4 at the MCM4 origin and indicated flanking regions in G1 phase synchronized U2OS cells stably expressing the indicated ORC protein; y-axis: H4K20me2 ChIP/H4 ChIP. e, An intact BAH domain is required for ORC1 occupancy at replication origins. Occupancy of FLAG tagged hORC1, hORC1_Y64A, hORC1_W88A, or control was determined by ChIP analysis (y-axs: % input) as in (d). Error bars in (d, and e,) indicate s.e.m. from three experiments. f, The ORC1-H4K20me2 interaction is required for efficient cell cycle progression. The cell-cycle profile of WI38 cells transiently expressing GFP and the indicated ORC1 protein was determined by flow cytometry. GFP-positive cells were used to ensure that only transfected cells were analyzed. (Left) Percentage of cells in the indicated cell-cycle phase is shown. (Right) The S/G1 ratio for the indicated transfections is shown. Error bars indicate s.d. from two experiments.