- Email: [email protected]
A Different Twist on Centromeric Chromatin
Structure
Previews presented here (Boughton et al., 2019), will provide increasingly deep insight into the important question of how effector molecules translate the intriguing language of ubiquitylation into efficient and precise signal transduction. DECLARATION OF INTERESTS M.R. is co-founder and consultant to Nurix, a biotech company in the ubiquitin space. REFERENCES Blythe, E.E., Olson, K.C., Chau, V., and Deshaies, R.J. (2017). Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP,NPLOC4,UFD1L is enhanced by a mutation that causes multisystem proteinopathy. Proc. Natl. Acad. Sci. USA 114, E4380–E4388.
Boughton, A.J., Krueger, S., and Fushman, D. (2020). Branching via K11 and K48 bestows ubiquitin chains with a unique interdomain interface and enhanced affinity for proteasomal subunit Rpn1. Structure 28, this issue, 29–43. Dikic, I. (2017). Proteasomal and autophagic degradation systems. Annu. Rev. Biochem. 86, 193–224. Husnjak, K., Elsasser, S., Zhang, N., Chen, X., Randles, L., Shi, Y., Hofmann, K., Walters, K.J., Finley, D., and Dikic, I. (2008). Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature 453, 481–488. Kulathu, Y., and Komander, D. (2012). Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat. Rev. Mol. Cell Biol. 13, 508–523. Meyer, H.J., and Rape, M. (2014). Enhanced protein degradation by branched ubiquitin chains. Cell 157, 910–921.
Nakasone, M.A., Livnat-Levanon, N., Glickman, M.H., Cohen, R.E., and Fushman, D. (2013). Mixed-linkage ubiquitin chains send mixed messages. Structure 21, 727–740. Shi, Y., Chen, X., Elsasser, S., Stocks, B.B., Tian, G., Lee, B.H., Shi, Y., Zhang, N., de Poot, S.A., Tuebing, F., et al. (2016). Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome. Science 351, https:// doi.org/10.1126/science.aad9421. Yau, R., and Rape, M. (2016). The increasing complexity of the ubiquitin code. Nat. Cell Biol. 18, 579–586. Yau, R.G., Doerner, K., Castellanos, E.R., Haakonsen, D.L., Werner, A., Wang, N., Yang, X.W., Martinez-Martin, N., Matsumoto, M.L., Dixit, V.M., and Rape, M. (2017). Assembly and function of heterotypic ubiquitin chains in cell-cycle and protein quality control. Cell 171, 918– 933.e20.
A Different Twist on Centromeric Chromatin Fabrizio Martino1 and Alessandro Costa1,* 1Macromolecular Machines Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK *Correspondence: [email protected] https://doi.org/10.1016/j.str.2019.12.005
Nucleosomes at the centromere contain CENP-A, a histone H3 variant that establishes a specific, yet poorly defined centromeric chromatin architecture. In this issue of Structure, Takizawa et al. (2019) describe an untwisted configuration for an H3-CENP-A-H3 tri-nucleosome that mimics centromeric chromatin. Untwisting may increase centromeric-protein accessibility to CENP-A in compacted chromatin. Eukaryotic chromosomes are packaged in arrays of nucleosomes forming chromatin, which is responsible for the protection and regulation of the genome. In its canonical form, the nucleosome core particle contains a histone H3-H4 tetramer, which is symmetrically capped by two histone H2A-H2B dimers. The resulting histone octamer is wrapped by 147 base pairs of DNA arranged as a lefthanded superhelix. Chromatin accessibility is regulated by epigenetic markers, which include covalent modifications of nucleosome components and the incorporation of specific histone variants (Zhou et al., 2019). One of these markers serves a pivotal role during cell division, ensuring that each daughter cell inherits the correct chromosome copy number, in a highly regulated process that prevents the rise of genome instability. This epigenetic mark is CENP-A, a
histone H3 variant that functions as a determinant for centromere specification. The centromere is a chromosomal region that serves as the assembly point for kinetochores, protein complexes that connect chromosomes to spindle microtubules during chromosome segregation. Unlike S. cerevisiae that contain one CENP-A nucleosome in the middle of the centromere, connected to one microtubule, vertebrate centromeres contact multiple microtubules and feature multiple CENP-A nucleosomes positioned by alpha satellite DNA sequence repeats, interspersed between H3 nucleosomes (Bloom and Costanzo, 2017). CENP-A partakes in defining the architecture of centromeric chromatin; however, the molecular basis is poorly understood. Previous crystallographic analysis of an isolated human CENP-A nucleosome revealed two notable features
distinct from the canonical H3 nucleosome. First, two amino acid residues unique to the L1 loop in CENP-A are solvent exposed. As amino acid substitutions in L1 reduce CENP-A retention at centromeres in human cells, this CENP-A loop has been postulated to form a docking site for trans-acting factors. Second, the nucleosomal DNA arms are markedly flexible in the CENP-A nucleosome, likely due to the limited length of the aN helix, which appears too short to orient and stabilize the DNA ends, as seen in the canonical H3 nucleosome (Tachiwana et al., 2011). These two unique CENP-A features first described by biochemical and X-ray diffraction studies appear as recurring themes in more recent cryo-EM analyses that captured the dynamics of CENP-A recognition by kinetochore components. To make one example, three cryo-EM studies of human CENP-A
Structure 28, January 7, 2020 ª 2019 Elsevier Ltd. 3
Structure
Previews NCP
Nucleosome
NCP stacking disrupted NCP
NCP
NCP
NCP UNTWISTING
NCP
NCP
NCP
NCP
NCP NCP Stacked H3 nucleosomes
CENP-A becomes accessible
Interspersed CENP-A nucleosome
Figure 1. CENP-A Might Disrupt Nucleosome Alignment at Centromeres A cartoon model for stacked H3-containing nucleosomes, resulting in compacted chromatin. CENP-A nucleosomes in the center of a tri-nucleosome are untwisted and might alter nucleosome alignment at centromeres, hence becoming accessible for the interaction to kinetochore proteins.
nucleosome bound to kinetochore protein CENP-N reveal that kinetochore binding involves the L1 loop of CENP-A, while this association concomitantly limits DNA arm mobility (Chittori et al., 2018; Pentakota et al., 2017; Tian et al., 2018). In a more recent, tour-de-force study, the cryo-EM structure of the Saccharomyces cerevisiae inner kinetochore module was solved, bound to a CENP-A nucleosome. While the mode of CENP-N interaction with a CENP-A nucleosome is different in the yeast structure, the DNA gyre in the CENP-A nucleosome is symmetrically unwrapped at each DNA terminus (Yan et al., 2019), as originally observed in the CENP-A nucleosome crystal structure (Tachiwana et al., 2011). Importantly, unwrapping of one of the two arms provides a new binding site for several components in the inner kinetochore module. Thus, the flexible DNA wrapping observed in CENP-A can serve a direct role in recruiting centromere interactors. Despite these important advances, it remains to be established how the CENP-A nucleosome is embedded in the higher-order chromatin architecture at the centromere, and how it can be accessed by the kinetochore proteins. In this issue of Structure, Takizawa et al. (2019) investigate the quaternary structure of CENP-A containing nucleosome arrays by comparing and contrasting the cryo-EM reconstructions of an H3 trinucleosome (H3-H3-H3) and a tri-nucleo4 Structure 28, January 7, 2020
some that mimics the composition of centromeric chromatin. The model centromeric tri-nucleosome contains a CENP-A nucleosome flanked by two histone H3 nucleosomes (H3-CENP-A-H3). Using nucleosome reconstitution, followed by ligation of cohesive ends, tri-nucleosomes were generated containing a 22-bp linker DNA (matching the predicted linker length in human centromeric chromatin) or a slightly longer 30-bp linker DNA (matching the length of alpha-satellite centromeric repeats in plants). In the presence of close-to-physiological concentrations of magnesium, the peripheral H3 nucleosomes were stacked against each other, in a configuration typical of compacted chromatin. As judged by 2D particle analysis, the H3-H3-H3 tri-nucleosomes featured the central nucleosome with linker DNA connected to the innerfacing strand of the peripheral nucleosomes for the 30-bp linker DNA constructs, and a mixture of inner- and outer-facing conformers for the 22-bp strands. Conversely, the H3-CENP-A-H3 tri-nucleosome featured the outer-facing conformer, in both the 22-bp and 30-bp linker length, and only when CENP-A was positioned centrally. Comparison between the 3D structures of H3-H3-H3 and H3-CENP-A-H3 tri-nucleosomes reveals a uniquely untwisted configuration for the central CENP-A nucleosome (irrespective of the linker DNA length). By overlaying H3-H3-H3 tri-nucleosomes, a model for compacted chromatin can be
generated, which closely matches the 30-nm fiber (Song et al., 2014), while the untwisted CENP-A nucleosome strongly departs from the stacked and compacted chromatin configuration. Thus, central CENP-A nucleosomes display a propensity to disrupt nucleosome stacking in chromatin (Figure 1). Nucleosome untwisting would therefore expose CENP-A nucleosome surfaces that are generally protected in compacted chromatin, hence facilitating the recruitment of centromeric proteins (Takizawa et al., 2019). Single-particle cryo-EM analysis continues to provide tremendous novel insights into nucleosome biology (Zhou et al., 2019), for example informing us about chromatin remodeling and the structural changes triggered by the introduction of epigenetic marks. At the same time, the increasing complexity of the in vitro reconstituted systems requires that we adapt our structural biology tools to capture the broader context of complex and dynamic chromatin transactions. A global analysis of nucleosome array architecture will be needed to understand the mechanisms that modulate conformational transitions of chromatin, which underlie chromosome reconfigurations. The first reconstituted centromere chromatin tools developed in the present study by Takizawa et al. (2019) move exactly in this direction. While artificial strong positioning sequences were used for nucleosome reconstitution, a frontier task will be poly-nucleosome reconstitution with native centromeric alpha satellite DNA sequences (Takizawa et al., 2019). From the image analysis viewpoint, traditional single particle approaches might not suffice to describe the chromosome transactions in the context of nucleosome arrays. In silico reconstitution approaches that reposition averaged structures back into the original micrograph can be useful to describe long-range nucleic acid transactions at single-particle cryo-EM resolution (Miller et al., 2019). In summary, structural characterization of CENP-A containing poly-nucleosomes (Takizawa et al., 2019) and entire kinetochore modules (Yan et al., 2019) provides key novel insights into centromere function. An exciting future challenge will be visualizing kinetochore association to dynamic centromere chromatin.
Structure
Previews ACKNOWLEDGMENTS The authors would like to thank Thomas Miller and Marcus Wilson for critical reading of the manuscript. Work in the Costa lab is supported by the Wellcome Trust, MRC and CRUK at the Francis Crick Institute (FC001065), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 820102). We apologize to colleagues whose work could not be cited due to space constraints. REFERENCES Bloom, K., and Costanzo, V. (2017). Centromere structure and function. Prog. Mol. Subcell. Biol. 56, 515–539. Chittori, S., Hong, J., Saunders, H., Feng, H., Ghirlando, R., Kelly, A.E., Bai, Y., and Subramaniam, S. (2018). Structural mechanisms of centromeric nucleosome recognition by the
kinetochore 339–343.
protein
CENP-N.
Science
359,
Miller, T.C.R., Locke, J., Greiwe, J.F., Diffley, J.F.X., and Costa, A. (2019). Mechanism of headto-head MCM double-hexamer formation revealed by cryo-EM. Nature 575, 704–710.
Takizawa, Y., Ho, C.H., Tachiwana, H., Matsunami, H., Kobayashi, W., Suzuki, M., Arimura, Y., Hori, T., Fukagawa, T., Ohi, M.D., et al. (2020). Cryo-EM structures of centromeric tri-nucleosomes containing a central CENP-A nucleosome. Structure 28, this issue, 44–53.
Pentakota, S., Zhou, K., Smith, C., Maffini, S., Petrovic, A., Morgan, G.P., Weir, J.R., Vetter, I.R., Musacchio, A., and Luger, K. (2017). Decoding the centromeric nucleosome through CENP-N. eLife 6, https://doi.org/10.7554/eLife.33442.
Tian, T., Li, X., Liu, Y., Wang, C., Liu, X., Bi, G., Zhang, X., Yao, X., Zhou, Z.H., and Zang, J. (2018). Molecular basis for CENP-N recognition of CENP-A nucleosome on the human kinetochore. Cell Res. 28, 374–378.
Song, F., Chen, P., Sun, D., Wang, M., Dong, L., Liang, D., Xu, R.M., Zhu, P., and Li, G. (2014). Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344, 376–380.
Yan, K., Yang, J., Zhang, Z., McLaughlin, S.H., Chang, L., Fasci, D., Ehrenhofer-Murray, A.E., Heck, A.J.R., and Barford, D. (2019). Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome. Nature 574, 278–282.
Tachiwana, H., Kagawa, W., Shiga, T., Osakabe, A., Miya, Y., Saito, K., Hayashi-Takanaka, Y., Oda, T., Sato, M., Park, S.Y., et al. (2011). Crystal structure of the human centromeric nucleosome containing CENP-A. Nature 476, 232–235.
Zhou, K., Gaullier, G., and Luger, K. (2019). Nucleosome structure and dynamics are coming of age. Nat. Struct. Mol. Biol. 26, 3–13.
Structure 28, January 7, 2020 5
Previews presented here (Boughton et al., 2019), will provide increasingly deep insight into the important question of how effector molecules translate the intriguing language of ubiquitylation into efficient and precise signal transduction. DECLARATION OF INTERESTS M.R. is co-founder and consultant to Nurix, a biotech company in the ubiquitin space. REFERENCES Blythe, E.E., Olson, K.C., Chau, V., and Deshaies, R.J. (2017). Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP,NPLOC4,UFD1L is enhanced by a mutation that causes multisystem proteinopathy. Proc. Natl. Acad. Sci. USA 114, E4380–E4388.
Boughton, A.J., Krueger, S., and Fushman, D. (2020). Branching via K11 and K48 bestows ubiquitin chains with a unique interdomain interface and enhanced affinity for proteasomal subunit Rpn1. Structure 28, this issue, 29–43. Dikic, I. (2017). Proteasomal and autophagic degradation systems. Annu. Rev. Biochem. 86, 193–224. Husnjak, K., Elsasser, S., Zhang, N., Chen, X., Randles, L., Shi, Y., Hofmann, K., Walters, K.J., Finley, D., and Dikic, I. (2008). Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature 453, 481–488. Kulathu, Y., and Komander, D. (2012). Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat. Rev. Mol. Cell Biol. 13, 508–523. Meyer, H.J., and Rape, M. (2014). Enhanced protein degradation by branched ubiquitin chains. Cell 157, 910–921.
Nakasone, M.A., Livnat-Levanon, N., Glickman, M.H., Cohen, R.E., and Fushman, D. (2013). Mixed-linkage ubiquitin chains send mixed messages. Structure 21, 727–740. Shi, Y., Chen, X., Elsasser, S., Stocks, B.B., Tian, G., Lee, B.H., Shi, Y., Zhang, N., de Poot, S.A., Tuebing, F., et al. (2016). Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome. Science 351, https:// doi.org/10.1126/science.aad9421. Yau, R., and Rape, M. (2016). The increasing complexity of the ubiquitin code. Nat. Cell Biol. 18, 579–586. Yau, R.G., Doerner, K., Castellanos, E.R., Haakonsen, D.L., Werner, A., Wang, N., Yang, X.W., Martinez-Martin, N., Matsumoto, M.L., Dixit, V.M., and Rape, M. (2017). Assembly and function of heterotypic ubiquitin chains in cell-cycle and protein quality control. Cell 171, 918– 933.e20.
A Different Twist on Centromeric Chromatin Fabrizio Martino1 and Alessandro Costa1,* 1Macromolecular Machines Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK *Correspondence: [email protected] https://doi.org/10.1016/j.str.2019.12.005
Nucleosomes at the centromere contain CENP-A, a histone H3 variant that establishes a specific, yet poorly defined centromeric chromatin architecture. In this issue of Structure, Takizawa et al. (2019) describe an untwisted configuration for an H3-CENP-A-H3 tri-nucleosome that mimics centromeric chromatin. Untwisting may increase centromeric-protein accessibility to CENP-A in compacted chromatin. Eukaryotic chromosomes are packaged in arrays of nucleosomes forming chromatin, which is responsible for the protection and regulation of the genome. In its canonical form, the nucleosome core particle contains a histone H3-H4 tetramer, which is symmetrically capped by two histone H2A-H2B dimers. The resulting histone octamer is wrapped by 147 base pairs of DNA arranged as a lefthanded superhelix. Chromatin accessibility is regulated by epigenetic markers, which include covalent modifications of nucleosome components and the incorporation of specific histone variants (Zhou et al., 2019). One of these markers serves a pivotal role during cell division, ensuring that each daughter cell inherits the correct chromosome copy number, in a highly regulated process that prevents the rise of genome instability. This epigenetic mark is CENP-A, a
histone H3 variant that functions as a determinant for centromere specification. The centromere is a chromosomal region that serves as the assembly point for kinetochores, protein complexes that connect chromosomes to spindle microtubules during chromosome segregation. Unlike S. cerevisiae that contain one CENP-A nucleosome in the middle of the centromere, connected to one microtubule, vertebrate centromeres contact multiple microtubules and feature multiple CENP-A nucleosomes positioned by alpha satellite DNA sequence repeats, interspersed between H3 nucleosomes (Bloom and Costanzo, 2017). CENP-A partakes in defining the architecture of centromeric chromatin; however, the molecular basis is poorly understood. Previous crystallographic analysis of an isolated human CENP-A nucleosome revealed two notable features
distinct from the canonical H3 nucleosome. First, two amino acid residues unique to the L1 loop in CENP-A are solvent exposed. As amino acid substitutions in L1 reduce CENP-A retention at centromeres in human cells, this CENP-A loop has been postulated to form a docking site for trans-acting factors. Second, the nucleosomal DNA arms are markedly flexible in the CENP-A nucleosome, likely due to the limited length of the aN helix, which appears too short to orient and stabilize the DNA ends, as seen in the canonical H3 nucleosome (Tachiwana et al., 2011). These two unique CENP-A features first described by biochemical and X-ray diffraction studies appear as recurring themes in more recent cryo-EM analyses that captured the dynamics of CENP-A recognition by kinetochore components. To make one example, three cryo-EM studies of human CENP-A
Structure 28, January 7, 2020 ª 2019 Elsevier Ltd. 3
Structure
Previews NCP
Nucleosome
NCP stacking disrupted NCP
NCP
NCP
NCP UNTWISTING
NCP
NCP
NCP
NCP
NCP NCP Stacked H3 nucleosomes
CENP-A becomes accessible
Interspersed CENP-A nucleosome
Figure 1. CENP-A Might Disrupt Nucleosome Alignment at Centromeres A cartoon model for stacked H3-containing nucleosomes, resulting in compacted chromatin. CENP-A nucleosomes in the center of a tri-nucleosome are untwisted and might alter nucleosome alignment at centromeres, hence becoming accessible for the interaction to kinetochore proteins.
nucleosome bound to kinetochore protein CENP-N reveal that kinetochore binding involves the L1 loop of CENP-A, while this association concomitantly limits DNA arm mobility (Chittori et al., 2018; Pentakota et al., 2017; Tian et al., 2018). In a more recent, tour-de-force study, the cryo-EM structure of the Saccharomyces cerevisiae inner kinetochore module was solved, bound to a CENP-A nucleosome. While the mode of CENP-N interaction with a CENP-A nucleosome is different in the yeast structure, the DNA gyre in the CENP-A nucleosome is symmetrically unwrapped at each DNA terminus (Yan et al., 2019), as originally observed in the CENP-A nucleosome crystal structure (Tachiwana et al., 2011). Importantly, unwrapping of one of the two arms provides a new binding site for several components in the inner kinetochore module. Thus, the flexible DNA wrapping observed in CENP-A can serve a direct role in recruiting centromere interactors. Despite these important advances, it remains to be established how the CENP-A nucleosome is embedded in the higher-order chromatin architecture at the centromere, and how it can be accessed by the kinetochore proteins. In this issue of Structure, Takizawa et al. (2019) investigate the quaternary structure of CENP-A containing nucleosome arrays by comparing and contrasting the cryo-EM reconstructions of an H3 trinucleosome (H3-H3-H3) and a tri-nucleo4 Structure 28, January 7, 2020
some that mimics the composition of centromeric chromatin. The model centromeric tri-nucleosome contains a CENP-A nucleosome flanked by two histone H3 nucleosomes (H3-CENP-A-H3). Using nucleosome reconstitution, followed by ligation of cohesive ends, tri-nucleosomes were generated containing a 22-bp linker DNA (matching the predicted linker length in human centromeric chromatin) or a slightly longer 30-bp linker DNA (matching the length of alpha-satellite centromeric repeats in plants). In the presence of close-to-physiological concentrations of magnesium, the peripheral H3 nucleosomes were stacked against each other, in a configuration typical of compacted chromatin. As judged by 2D particle analysis, the H3-H3-H3 tri-nucleosomes featured the central nucleosome with linker DNA connected to the innerfacing strand of the peripheral nucleosomes for the 30-bp linker DNA constructs, and a mixture of inner- and outer-facing conformers for the 22-bp strands. Conversely, the H3-CENP-A-H3 tri-nucleosome featured the outer-facing conformer, in both the 22-bp and 30-bp linker length, and only when CENP-A was positioned centrally. Comparison between the 3D structures of H3-H3-H3 and H3-CENP-A-H3 tri-nucleosomes reveals a uniquely untwisted configuration for the central CENP-A nucleosome (irrespective of the linker DNA length). By overlaying H3-H3-H3 tri-nucleosomes, a model for compacted chromatin can be
generated, which closely matches the 30-nm fiber (Song et al., 2014), while the untwisted CENP-A nucleosome strongly departs from the stacked and compacted chromatin configuration. Thus, central CENP-A nucleosomes display a propensity to disrupt nucleosome stacking in chromatin (Figure 1). Nucleosome untwisting would therefore expose CENP-A nucleosome surfaces that are generally protected in compacted chromatin, hence facilitating the recruitment of centromeric proteins (Takizawa et al., 2019). Single-particle cryo-EM analysis continues to provide tremendous novel insights into nucleosome biology (Zhou et al., 2019), for example informing us about chromatin remodeling and the structural changes triggered by the introduction of epigenetic marks. At the same time, the increasing complexity of the in vitro reconstituted systems requires that we adapt our structural biology tools to capture the broader context of complex and dynamic chromatin transactions. A global analysis of nucleosome array architecture will be needed to understand the mechanisms that modulate conformational transitions of chromatin, which underlie chromosome reconfigurations. The first reconstituted centromere chromatin tools developed in the present study by Takizawa et al. (2019) move exactly in this direction. While artificial strong positioning sequences were used for nucleosome reconstitution, a frontier task will be poly-nucleosome reconstitution with native centromeric alpha satellite DNA sequences (Takizawa et al., 2019). From the image analysis viewpoint, traditional single particle approaches might not suffice to describe the chromosome transactions in the context of nucleosome arrays. In silico reconstitution approaches that reposition averaged structures back into the original micrograph can be useful to describe long-range nucleic acid transactions at single-particle cryo-EM resolution (Miller et al., 2019). In summary, structural characterization of CENP-A containing poly-nucleosomes (Takizawa et al., 2019) and entire kinetochore modules (Yan et al., 2019) provides key novel insights into centromere function. An exciting future challenge will be visualizing kinetochore association to dynamic centromere chromatin.
Structure
Previews ACKNOWLEDGMENTS The authors would like to thank Thomas Miller and Marcus Wilson for critical reading of the manuscript. Work in the Costa lab is supported by the Wellcome Trust, MRC and CRUK at the Francis Crick Institute (FC001065), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 820102). We apologize to colleagues whose work could not be cited due to space constraints. REFERENCES Bloom, K., and Costanzo, V. (2017). Centromere structure and function. Prog. Mol. Subcell. Biol. 56, 515–539. Chittori, S., Hong, J., Saunders, H., Feng, H., Ghirlando, R., Kelly, A.E., Bai, Y., and Subramaniam, S. (2018). Structural mechanisms of centromeric nucleosome recognition by the
kinetochore 339–343.
protein
CENP-N.
Science
359,
Miller, T.C.R., Locke, J., Greiwe, J.F., Diffley, J.F.X., and Costa, A. (2019). Mechanism of headto-head MCM double-hexamer formation revealed by cryo-EM. Nature 575, 704–710.
Takizawa, Y., Ho, C.H., Tachiwana, H., Matsunami, H., Kobayashi, W., Suzuki, M., Arimura, Y., Hori, T., Fukagawa, T., Ohi, M.D., et al. (2020). Cryo-EM structures of centromeric tri-nucleosomes containing a central CENP-A nucleosome. Structure 28, this issue, 44–53.
Pentakota, S., Zhou, K., Smith, C., Maffini, S., Petrovic, A., Morgan, G.P., Weir, J.R., Vetter, I.R., Musacchio, A., and Luger, K. (2017). Decoding the centromeric nucleosome through CENP-N. eLife 6, https://doi.org/10.7554/eLife.33442.
Tian, T., Li, X., Liu, Y., Wang, C., Liu, X., Bi, G., Zhang, X., Yao, X., Zhou, Z.H., and Zang, J. (2018). Molecular basis for CENP-N recognition of CENP-A nucleosome on the human kinetochore. Cell Res. 28, 374–378.
Song, F., Chen, P., Sun, D., Wang, M., Dong, L., Liang, D., Xu, R.M., Zhu, P., and Li, G. (2014). Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344, 376–380.
Yan, K., Yang, J., Zhang, Z., McLaughlin, S.H., Chang, L., Fasci, D., Ehrenhofer-Murray, A.E., Heck, A.J.R., and Barford, D. (2019). Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome. Nature 574, 278–282.
Tachiwana, H., Kagawa, W., Shiga, T., Osakabe, A., Miya, Y., Saito, K., Hayashi-Takanaka, Y., Oda, T., Sato, M., Park, S.Y., et al. (2011). Crystal structure of the human centromeric nucleosome containing CENP-A. Nature 476, 232–235.
Zhou, K., Gaullier, G., and Luger, K. (2019). Nucleosome structure and dynamics are coming of age. Nat. Struct. Mol. Biol. 26, 3–13.
Structure 28, January 7, 2020 5