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A unique binding mode enables MCM2 to chaperone histones H3–H4 at replication forks

Nature Structural & Molecular Biology volume 22, pages 618–626 (2015)Cite this article

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Abstract

During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3–H4. Our first structure shows an H3–H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3–H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2–7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3–H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication.

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Figure 1: Structure of the MCM2 HBD–H3–H4 tetramer complex.
Figure 2: Structure of the MCM2 HBD–H3–H4 dimer–ASF1 complex.
Figure 3: MCM2 binding stabilizes non-nucleosomal H3.
Figure 4: MCM2 chaperones H3–H4 as part of the MCM2–7 helicase in chromatin.
Figure 5: MCM2 histone-chaperone function is required for cell proliferation.
Figure 6: MCM2 can handle new and old histones, including all H3 variants.
Figure 7: Structure-based model for how MCM2 handles parental histones H3 and H4 genome wide during DNA replication.

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Acknowledgements

We thank the beamline staff at the synchrotrons at the Argonne National Laboratory (NE-CAT) and the Brookhaven National Laboratory (XL-29) for technical assistance. We thank H. Wu for access to SEC-MALS equipment for molecular-weight measurements. We thank Y. Feng and C. Alabert for help with immunofluorescence and PLA analysis. We thank C. Alabert for comments on the manuscript and Z. Jasencakova for help with experiments and for input in the manuscript and the model. We also thank P. Meraldi (University of Geneva) and L. Jansen (Gulbenkian Institute) for reagents and K. Labib (University of Dundee) for sharing information before publication. D.J.P. was supported in part by grants from the Leukemia and Lymphoma Society (LLS 7006-13) and the Starr foundation (I5-A554). A.G. is supported as a European Molecular Biology Organization Young Investigator, and her research is supported by the Danish National Research Foundation to the Center for Epigenetics (DNRF82), the European Commission ITN FP7 'aDDRess', a European Research Council Starting Grant (ERC2011StG, no. 281,765), the Danish Cancer Society, the Danish Medical Research Foundation and the Lundbeck Foundation.

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  1. Hongda Huang and Caroline B Strømme: These authors contributed equally to this work.

Authors and Affiliations

  1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Hongda Huang, Shoudeng Chen & Dinshaw J Patel

  2. Biotech Research and Innovation Centre (BRIC) Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark

    Caroline B Strømme, Giulia Saredi, Martina Hödl, Anne Strandsby, Cristina González-Aguilera & Anja Groth

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Contributions

H.H. conceived and led the generation of cassettes for crystallization of the complexes, and H.H. solved the structures under the supervision of D.J.P. C.B.S. and A.G. conceived and led the functional studies. G.S. performed immunoprecipitation of HA–H3.1 from chromatin. M.H. analyzed histone incorporation by SNAP-tag assay. A.S. carried out lentiviral transduction and cell characterization. C.G.-A. helped with cloning and data analysis. S.C. performed SEC-MALS molecular-weight measurements. H.H., C.B.S., A.G. and D.J.P. wrote the manuscript, and all authors commented on the manuscript.

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Correspondence to Anja Groth or Dinshaw J Patel.

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Integrated supplementary information

Supplementary Figure 1 Biochemical characterization of different MCM2 HBD–H3–H4 complexes and structural comparisons.

(a) His-pulldown of MCM2 HBD with recombinant histones. (b) The MCM2 HBD(61–130)–H3.3(Δ56) –H4 tetramer (complex 1) and MCM2 HBD(43–160)–H3.3(Δ56) –H4 tetramer (complex 2) were analyzed by gel-filtration assay. (c) SDS-PAGE analysis of the peak fractions from panel b. (d) The MCM2 HBD(43–160)–H3.2–H4 tetramer (complex 6), MCM2 HBD(43–160)–H3.2–H4 dimer–ASF1a(1-172) (complex 5) and H3.2–H4 tetramer were analyzed as in panel b. (e) SDS-PAGE analysis of the peak fractions from panel d. (f) SEC-MALS assay. The apparent Mw determined by SEC-MALS of MCM2 HBD–H3–H4 tetramer complex is 93.5 kDa (errors 3%, with the expected Mw about 80.0 kDa). The apparent Mw of MCM2 HBD–H3–H4 dimer–ASF1 complex is 64.3 kDa (errors 4%, with the expected Mw about 60.0 kDa). (g) A stereo view following superposition of the two halves of the MCM2 HBD–H3–H4 tetramer complex. One half was in color and the other half was in silver. (h) The reported structure (Richet, N. et al., Nucleic Acids Res 43, 1905-1917, 2015) observed a 1:1:1 MCM2 HBD–H3.2–H4 dimer complex in the asymmetric unit (AU) and a ‘2:2:2’ complex could be generated through crystallographic symmetric operation, while our structure directly observed a 2:2:2 MCM2 HBD–H3.3–H4 tetramer complex in the AU. Panel h showing the superposition of the generated ‘2:2:2’ structure (in silver) with our observed 2:2:2 structure (in color) with an rmsd of 1.3 Å. The major differences in conformations are pointed out with arrows. (i) A stereo view following superposition of the H3–H4 tetramers from the structures of MCM2 HBD–H3-H4 tetramer complex (in color) and nucleosome (in silver). The H2A docking domain from the nucleosome was also shown.

Supplementary Figure 2 Selective details of intermolecular contacts of our crystal structures and biochemical characterizations of different MCM2 HBD–H3–H4 –ASF1 complexes.

(a–c) Each panel highlighting the key interactions in the S1 (panel a), S2 (panel b) and S3 (panel c) regions of MCM2 HBD in the MCM2 HBD–H3–H4 tetramer complex. (d) GST-pulldown of ASF1 WT and V94R mutant with recombinant H3–H4 tetramers and prepurified MCM2 HBD–H3–H4 tetramer complex. (e) The prepurified MCM2 HBD(43–160)–H3–H4 tetramer (P1), ASF1b(1–158) (P3) and MCM2 HBD(43–163)–H3(EE) –H4 dimer (a constitutive H3–H4 dimer with dual mutation L126E I130E on H3) (P4) were analyzed by gel filtration and served as controls; the prepurified MCM2 HBD(43–160)–H3–H4 tetramer (20 nmole) was incubated with ASF1b(1–158) (12 nmole) and the reaction mixture was analyzed. Two peaks P1’ (not reacted complex) and P2 (reaction product) were expected and observed. (f) The peak fractions from panel e were analyzed on SDS-PAGE. (g) The MCM2 HBD(61–130)-linker-ASF1b(1–158)–H3.3(Δ56) –H4 dimer (complex 3) and MCM2 HBD(43-160)–H3.2(Δ55) –H4 dimer–ASF1a(1–172) (complex 4) were analyzed as in panel b of Supplementary Fig. 1. (h) SDS-PAGE analysis of the peak fractions from panel g. (i–k) Each panel highlighting the key interactions in the S1 (panel i), S2 (panel j) and S3 (panel k) regions of MCM2 HBD in the MCM2 HBD–H3–H4 dimer–ASF1 complex. For clarity, the ASF1 segment was omitted in panel k.

Supplementary Figure 3 Details of intermolecular contacts of ASF1–H3–H4 dimer complex and structural comparisons of different chaperone–H3–H4 complexes.

(a, b) Views of details of the intermolecular interactions between ASF1 and H3–H4 dimer from PDB 2IO5 (Natsume, R. et al., Nature 446, 338-341, 2007). Panel a highlighting the interactions of ASF1 with the helices α2 and α3 of H3, and panel b highlighting the interactions of ASF1 with the βc strand and C-terminus of H4. (c) Structural comparison of the ASF1–H3–H4 dimer complex (colored in silver) and our MCM2 HBD–H3–H4 dimer–ASF1 complex (in color). (d, e) Following the comparison shown in panel c, the RMSDs of residues in H3 (panel d) and H4 (panel e) were plotted respectively. The conformational differences on H3–H4 dimer caused upon binding of MCM2 HBD are spread out and include the L1 loop, the α2 helix and the L2 loop of H3 (panel d), as well as the α1 helix, the tip of α2 helix, the L2 loop and especially the α3 helix of H4 (panel e). (f) Structural comparison of one half of the MCM2 HBD–H3–H4 tetramer complex (colored in silver) with the MCM2 HBD–H3–H4 dimer–ASF1 complex (in color). (g, h) Following the comparison shown in panel f, the RMSDs of residues in H3 (panel g) and H4 (panel h) were plotted respectively.

Supplementary Figure 4 Structural comparison emphasizing the local differences and biochemical characterizations of the linker complex used for crystallization.

(a) Structural comparison as in panel c of Supplementary Fig. 3, emphasizing the local differences on H3 and H4. All the differences were seen along the MCM2 HBD binding interface on H3–H4. (b, c) Structural comparison as in panel f of Supplementary Fig. 3, emphasizing a 12o rotational difference in the α3 helix of H3 between conformations (panel b); and emphasizing the difference in the C-terminal tail of H4 between conformations, which undergoes a disorder to order transition (formation of a βc strand) with ASF1 (panel c). (d) Gel-filtration analysis of the complex with linker (complex 3) and without linker (complex 3’). The complex 3’ was similar to complex 3 except lacking the 12-mer covalent linker. (e) The peak fractions from panel d were analyzed on SDS-PAGE. (f) The tetrasome assembly assay. Lane 1-4, controls. Lane 5, excess H3–H4 tetramer mixed with DNA in physiological salt condition (150 mM NaCl) forming high aggregation. Lane 6, the prepurified complex of MCM2 HBD(43–160)–H3–H4 dimer–ASF1a(1–172) had tetrasome assembly activity. Lane 7, the prepurified complex of MCM2 HBD(61–130)-linker-ASF1b(1–158)–H3–H4 dimer had tetrasome assembly activity on linear Widom 601 DNA, though it shifted the balance among tetrasome and unassigned band (‘*’).

Supplementary Figure 5 Input controls for the immunoprecipitation reactions.

(a – f), Western blot of inputs for the immunoprecipitation reactions shown in Fig. 3a (panel a), Fig. 4a (panel b), Fig. 4c (panel c), Fig. 4e (panel d), Fig. 6a (panel e) and Fig. 6f (panel f).

Supplementary Figure 6 MCM2 binding is not required for incorporation of H3.1–H4 into chromatin but is important for stability of H3.1–H4.

(a) Representative images of newly synthesized TMR* labelled SNAP-HA-H3.1 wt and R63A K64A as described in Fig. 3b. Scale bar, 50 μm. (b) Analysis of total chromatin-bound and soluble SNAP-HA-H3.1 in stable cell lines used in Fig. 3b&c. Total SNAP-HA-H3.1 wt or R63A K64A were measured by TMR* labelling and direct fixation. The dot-plot showed median TMR* intensities from 3 independent experiments each including 5 technical replicates of total > 5,000 TMR* positive cells. Error bars, s.d. n=3. Unpaired t-test: *, P < 0.05. (c) SNAP-HA-H3.1 mRNA levels in stable cell lines used to measure the histone stability in Fig. 3c. Expression of e-H3 mRNA levels was measured by qPCR using two different primer sets, normalized to GAPDH and shown relative to wt. Error bars, s.d n=3 (for primer-set 2 wt, n=2). (d , e) Immunofluorescence of U-2-OS cells conditionally expressing Flag-HA-MCM2 WT or Y81A Y90A mutant. Cells were induced by tetracyclin (tet) for 24 hours and fixed directly (d) or pre-extracted (e) by CSK-T. HA staining confirmed induction of FLAG-HA-MCM2 in non-extracted cells (d) and showed typical cell cycle dependent MCM2 staining patterns in pre-extracted cells (e). Representative images of typical G1, early S and mid S phase were shown. Note: because MCM2-7 is unloaded as S phase progresses, late S and G2 cells are negative for MCM2. Flag-HA-MCM2 patterns were scored in 3 independent experiments (n=3), error-bars represent standard deviation, > 200 HA positive cells were scored per experiment. Scale bars, 20 μm (d) and 5 μm (e).

Supplementary Figure 7 ASF1a colocalizes with MCM2 and CDC45.

(a –c) PLA of endogenous chromatin-bound ASF1a with MCM2 (panels a, c) and CDC45 (panel b) as described in Fig. 6 b–d. Images showed a large field of pre-extracted cells to complement the single cells shown in Fig. 6. Scale bars were 16 μm. (d) Interactions of MCM2 HBD and H3.2–H4 in the MCM2 HBD–H3.2–H4dimer–ASF1 complex, highlighting the region S2 as in Fig. 1e. (e) Alignment of the MCM2 HBD binding region in histone H3 variants, showing strict conservation in red and conservation of properties in green. (f) Superposition of the H3–H4 tetramer from the MCM2 HBD–H3–H4 tetramer structure (in color) and the CENPA–H4 tetramer from the CENPA nucleosome structure (in silver).

Supplementary Figure 8 Biochemical characterizations of the MCM2–CENPA–H4 tetramer complex.

(a) Gel-filtration analyses of the CENPA–H4 tetramer and MCM2 HBD(43–160)–CENPA–H4 tetramer complex (complex 7) in high salt buffer (1 M NaCl). (b) The peak fractions from panel a were analyzed by SDS-PAGE. (c) The same analyses as in panel a, but with a low salt buffer (0.3 M NaCl). (d, e) IP of Flag-HA-MCM2 WT and its H3–H4-binding mutant from cells stably expressing EGFP-CENPA (Amaro, A.C. et al., Nat Cell Biol 12, 319-329, 2010).

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Huang, H., Strømme, C., Saredi, G. et al. A unique binding mode enables MCM2 to chaperone histones H3–H4 at replication forks. Nat Struct Mol Biol 22, 618–626 (2015). https://doi.org/10.1038/nsmb.3055

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