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• W2480057469 abstract "•CENP-A nucleosomal ends are highly flexible in solution•Dynamic CENP-A nucleosomal ends prevent H1 recruitment•Flexibility of DNA ends allows active kinetochore complex assembly•Open CENP-A nucleosomal structure is essential for its mitotic function CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway. CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway. Chromatin is composed of repetitive structures of the basic unit, a nucleosome, which consists of histone octamer composed of core histones (two of each H2A, H2B, H3, and H4) around which 167 base pairs (bp) of DNA are wrapped to form two superhelices (Luger et al., 1997Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 A resolution.Nature. 1997; 389: 251-260Crossref PubMed Scopus (6912) Google Scholar, Van Holde et al., 1980Van Holde K.E. Allen J.R. Tatchell K. Weischet W.O. Lohr D. DNA-histone interactions in nucleosomes.Biophys. J. 1980; 32: 271-282Abstract Full Text PDF PubMed Scopus (38) Google Scholar). Individual nucleosomes interspersed with linker DNA form the 10 nm chromatin filament (Thoma et al., 1979Thoma F. Koller T. Klug A. Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin.J. Cell Biol. 1979; 83: 403-427Crossref PubMed Scopus (1179) Google Scholar, Van Holde et al., 1980Van Holde K.E. Allen J.R. Tatchell K. Weischet W.O. Lohr D. DNA-histone interactions in nucleosomes.Biophys. J. 1980; 32: 271-282Abstract Full Text PDF PubMed Scopus (38) Google Scholar). A fifth histone, termed “linker histone”, interacts with this linker DNA and assists in the assembly, condensation, and stability of the 30 nm chromatin fiber (Makarov et al., 1983Makarov V.L. Dimitrov S.I. Petrov P.T. Salt-induced conformational transitions in chromatin. A flow linear dichroism study.Eur. J. Biochem. 1983; 133: 491-497Crossref PubMed Scopus (43) Google Scholar, Thoma et al., 1979Thoma F. Koller T. Klug A. Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin.J. Cell Biol. 1979; 83: 403-427Crossref PubMed Scopus (1179) Google Scholar). In addition to the conventional core histones, each cell expresses histone variants. Histone variants are non-allelic isoforms of conventional histones and all histones, except H4, have variants (Van Holde et al., 1980Van Holde K.E. Allen J.R. Tatchell K. Weischet W.O. Lohr D. DNA-histone interactions in nucleosomes.Biophys. J. 1980; 32: 271-282Abstract Full Text PDF PubMed Scopus (38) Google Scholar). Incorporation of these variants confers novel structural and functional properties to chromatin (reviewed in Boulard et al., 2007Boulard M. Bouvet P. Kundu T.K. Dimitrov S. Histone variant nucleosomes: structure, function and implication in disease.Subcell. Biochem. 2007; 41: 71-89PubMed Google Scholar). The histone CENP-A is a textbook example of a histone variant that upon incorporation changes the properties of a nucleosome (Goutte-Gattat et al., 2013Goutte-Gattat D. Shuaib M. Ouararhni K. Gautier T. Skoufias D.A. Hamiche A. Dimitrov S. Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function.Proc. Natl. Acad. Sci. USA. 2013; 110: 8579-8584Crossref PubMed Scopus (46) Google Scholar, Kingston et al., 2011Kingston I.J. Yung J.S. Singleton M.R. Biophysical characterization of the centromere-specific nucleosome from budding yeast.J. Biol. Chem. 2011; 286: 4021-4026Crossref PubMed Scopus (66) Google Scholar, Mizuguchi et al., 2007Mizuguchi G. Xiao H. Wisniewski J. Smith M.M. Wu C. Nonhistone Scm3 and histones CenH3-H4 assemble the core of centromere-specific nucleosomes.Cell. 2007; 129: 1153-1164Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, Tachiwana et al., 2011Tachiwana H. Kagawa W. Shiga T. Osakabe A. Miya Y. Saito K. Hayashi-Takanaka Y. Oda T. Sato M. Park S.Y. et al.Crystal structure of the human centromeric nucleosome containing CENP-A.Nature. 2011; 476: 232-235Crossref PubMed Scopus (285) Google Scholar). CENP-A belongs to the H3 family of histones (Earnshaw and Migeon, 1985Earnshaw W.C. Migeon B.R. Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome.Chromosoma. 1985; 92: 290-296Crossref PubMed Scopus (191) Google Scholar, Palmer et al., 1987Palmer D.K. O’Day K. Wener M.H. Andrews B.S. Margolis R.L. A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones.J. Cell Biol. 1987; 104: 805-815Crossref PubMed Scopus (318) Google Scholar) and is exclusively localized to centromeres and defines the specific centromere structure and function (Buscaino et al., 2010Buscaino A. Allshire R. Pidoux A. Building centromeres: home sweet home or a nomadic existence?.Curr. Opin. Genet. Dev. 2010; 20: 118-126Crossref PubMed Google Scholar). CENP-A epigenetically marks the centromeres, where it is required for the assembly of active kinetochores. The constitutive centromere associated network (CCAN), a complex consisting of 16 proteins (termed generally as CENPs), recognizes and directly interacts with centromeric chromatin (Perpelescu and Fukagawa, 2011Perpelescu M. Fukagawa T. The ABCs of CENPs.Chromosoma. 2011; 120: 425-446Crossref PubMed Scopus (149) Google Scholar). Importantly, two of the CCAN members, CENP-C and CENP-T, assemble a platform to direct kinetochore formation (Perpelescu and Fukagawa, 2011Perpelescu M. Fukagawa T. The ABCs of CENPs.Chromosoma. 2011; 120: 425-446Crossref PubMed Scopus (149) Google Scholar). CENP-A depletion results in numerous mitotic and cytokinetic defects and subsequent aneuploidy (Goutte-Gattat et al., 2013Goutte-Gattat D. Shuaib M. Ouararhni K. Gautier T. Skoufias D.A. Hamiche A. Dimitrov S. Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function.Proc. Natl. Acad. Sci. USA. 2013; 110: 8579-8584Crossref PubMed Scopus (46) Google Scholar, Régnier et al., 2005Régnier V. Vagnarelli P. Fukagawa T. Zerjal T. Burns E. Trouche D. Earnshaw W. Brown W. CENP-A is required for accurate chromosome segregation and sustained kinetochore association of BubR1.Mol. Cell. Biol. 2005; 25: 3967-3981Crossref PubMed Scopus (144) Google Scholar). CENP-A loss leads to altered composition and organization of the kinetochore, including the delocalization of the inner kinetochore proteins CENP-C, CENP-I, and CENP-H as well as the outer kinetochore components HEC1, Mad2, and CENP-E (Goutte-Gattat et al., 2013Goutte-Gattat D. Shuaib M. Ouararhni K. Gautier T. Skoufias D.A. Hamiche A. Dimitrov S. Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function.Proc. Natl. Acad. Sci. USA. 2013; 110: 8579-8584Crossref PubMed Scopus (46) Google Scholar, Régnier et al., 2005Régnier V. Vagnarelli P. Fukagawa T. Zerjal T. Burns E. Trouche D. Earnshaw W. Brown W. CENP-A is required for accurate chromosome segregation and sustained kinetochore association of BubR1.Mol. Cell. Biol. 2005; 25: 3967-3981Crossref PubMed Scopus (144) Google Scholar). The crystal structure of the CENP-A nucleosome particle was recently solved (Tachiwana et al., 2011Tachiwana H. Kagawa W. Shiga T. Osakabe A. Miya Y. Saito K. Hayashi-Takanaka Y. Oda T. Sato M. Park S.Y. et al.Crystal structure of the human centromeric nucleosome containing CENP-A.Nature. 2011; 476: 232-235Crossref PubMed Scopus (285) Google Scholar). In contrast to the conventional nucleosome structure, only 121 bp of DNA are resolved in the crystal structure of the CENP-A nucleosome, suggesting that 13 bp of DNA at each nucleosomal end display marked flexibility. In agreement with this suggestion, CENP-A nucleosomal ends exhibited higher accessibility to nucleases (Kingston et al., 2011Kingston I.J. Yung J.S. Singleton M.R. Biophysical characterization of the centromere-specific nucleosome from budding yeast.J. Biol. Chem. 2011; 286: 4021-4026Crossref PubMed Scopus (66) Google Scholar). Experiments in solution point to some crystal packing artifacts, which might affect the central part of the CENP-A nucleosomes, but not the dynamics of their ends (Falk et al., 2015Falk S.J. Guo L.Y. Sekulic N. Smoak E.M. Mani T. Logsdon G.A. Gupta K. Jansen L.E. Van Duyne G.D. Vinogradov S.A. et al.Chromosomes. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere.Science. 2015; 348: 699-703Crossref PubMed Scopus (134) Google Scholar). However, whether the CENP-A driven nucleosomal end DNA flexibility has any physiological consequences is totally unknown. To determine the dynamics of nucleosomal DNA ends in solution, both conventional and CENP-A nucleosomes were analyzed by using electron cryomicroscopy (ECM) combined with 3D reconstruction. ECM data clearly show that the CENP-A nucleosomal ends, as suggested by the crystal structure, exhibit a high degree of flexibility. The αN helix of H3 and the preceding loop, which is in contact with DNA, plays a role in stabilizing the conventional nucleosomal DNA ends. This specific rigid orientation of the exit and entry angle of the nucleosomal DNA ends, in addition to the linker histone H1 binding modes and condensation of nucleosomal arrays, is regulating the interaction of H1 with conventional nucleosomes (Song et al., 2014Song F. Chen P. Sun D. Wang M. Dong L. Liang D. Xu R.M. Zhu P. Li G. Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units.Science. 2014; 344: 376-380Crossref PubMed Scopus (395) Google Scholar, Syed et al., 2010Syed S.H. Goutte-Gattat D. Becker N. Meyer S. Shukla M.S. Hayes J.J. Everaers R. Angelov D. Bednar J. Dimitrov S. Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome.Proc. Natl. Acad. Sci. USA. 2010; 107: 9620-9625Crossref PubMed Scopus (157) Google Scholar, Zhou et al., 2013Zhou B.R. Feng H. Kato H. Dai L. Yang Y. Zhou Y. Bai Y. Structural insights into the histone H1-nucleosome complex.Proc. Natl. Acad. Sci. USA. 2013; 110: 19390-19395Crossref PubMed Scopus (147) Google Scholar, Zhou et al., 2015Zhou B.R. Jiang J. Feng H. Ghirlando R. Xiao T.S. Bai Y. Structural mechanisms of nucleosome recognition by linker histones.Mol Cell. 2015; 59: 628-638Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Thus, highly dynamic CENP-A nucleosomal ends would likely preclude the binding of H1, which might in turn be important for centromere function and kinetochore assembly (Figures 1A and 1B ). Since the fixed entry/exit angle and rigidity of DNA ends of conventional nucleosome are governed by the interaction of the αN helix of H3, we created a hybrid CENP-A nucleosome, wherein the αN helix of CENP-A was replaced with that of H3, which is one helical turn longer. ECM analysis reveals that this hybrid H3-CENP-A particle, in contrast to the wild-type (WT) CENP-A nucleosome, exhibits a compact structure very similar to that of the conventional nucleosome. We then studied the properties of these hybrid nucleosomes in vitro and in vivo and compared them with those of the WT CENP-A nucleosomes. Our biochemical and cell biological data demonstrate that the flexible DNA ends of CENP-A nucleosome are essential for the structural integrity of the centromere, which is required for the fidelity of the mitotic process. We used ECM to analyze the dynamics of CENP-A nucleosome in solution. We purified recombinant human core histone octamers containing conventional H3 or CENP-A (Figure S1) and reconstituted centrally positioned nucleosomes on 197 bp 601 DNA in order to have 25 bp free DNA ends. The reconstituted particles were studied in frozen hydrated conditions (Figure S2). Frames showing well-separated nucleosomes were selected and 29,711 and 24,286 molecular images were extracted for conventional and CENP-A nucleosomes, respectively. These images were used for building ab initio a 3D model of the particles (Figure 1C). The final ECM map is in full agreement with the crystal structure of the nucleosome core particle (NCP) (Luger et al., 1997Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 A resolution.Nature. 1997; 389: 251-260Crossref PubMed Scopus (6912) Google Scholar). To analyze the conformational variability of the particle population, a 3D classification scheme based on maximum likelihood optimization was used to separate different structural variants (Scheres, 2012Scheres S.H. RELION: implementation of a Bayesian approach to cryo-EM structure determination.J. Struct. Biol. 2012; 180: 519-530Crossref PubMed Scopus (3034) Google Scholar; Table S1). While the nucleosome core showed little variation, we observed distinct configurations of the DNA ends for both conventional and CENP-A particles (Figure 1C). For each 3D class, the angles between each linker arm and the dyad axis were determined in the front and the site view of the nucleosome (Figure S3). The analyses indicated that, in particular, the CENP-A particles have open conformations with much higher entry/exit angles (higher DNA end orientation fluctuations) compared to the conventional ones. We conclude that in solution, the CENP-A nucleosomal ends exhibited, as suggested by the X-ray diffraction studies, a very high degree of flexibility. The αN helix of CENP-A is one helical turn shorter than that of conventional H3 nucleosome and the preceding region, in contrast to that of H3, is completely disordered (Tachiwana et al., 2011Tachiwana H. Kagawa W. Shiga T. Osakabe A. Miya Y. Saito K. Hayashi-Takanaka Y. Oda T. Sato M. Park S.Y. et al.Crystal structure of the human centromeric nucleosome containing CENP-A.Nature. 2011; 476: 232-235Crossref PubMed Scopus (285) Google Scholar; see also Figure 1B). However, both the αN helix length and the loop segment preceding the αN helix (which directly interacts with the DNA ends in H3 nucleosome; Luger et al., 1997Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 A resolution.Nature. 1997; 389: 251-260Crossref PubMed Scopus (6912) Google Scholar) are required for maintaining the DNA orientation at the entrance and exit of H3 nucleosomes. Thus, the specific organization of these regions in CENP-A might be responsible for the inherent flexibility of the DNA ends of the CENP-A nucleosome (Tachiwana et al., 2011Tachiwana H. Kagawa W. Shiga T. Osakabe A. Miya Y. Saito K. Hayashi-Takanaka Y. Oda T. Sato M. Park S.Y. et al.Crystal structure of the human centromeric nucleosome containing CENP-A.Nature. 2011; 476: 232-235Crossref PubMed Scopus (285) Google Scholar; Figure 1B). Therefore, we hypothesized that swapping the CENP-A αN helix and the segment preceding it with those of conventional H3 would rigidify the ends of nucleosomal CENP-A DNA. To test this, we generated a hybrid H3-CENP-A mutant (αNH3-CENP-A) containing the αN helix and the preceding loop region of H3. Next, we expressed this construct in bacteria and, after purification, we used it for reconstitution of αNH3-CENP-A nucleosomes on 197 bp 601 DNA (Figure S1). The structure and dynamics of the mutant αNH3-CENP-A nucleosomes were studied by ECM as described above for both conventional and CENP-A nucleosome. There were 155,000 molecular images that were extracted and used for building ab initio a 3D model of the hybrid particles (Figures 1C, bottom, S2 and S3). As seen, the mutant αNH3-CENP-A particle exhibits a structure very similar to those of conventional H3 nucleosome conformations, with smaller entry/exit angles of the DNA ends. Therefore, the defectiveαN helix of CENP-A is the main determinant for the highly flexible CENP-A nucleosomal ends. The binding of histone H1 to the nucleosome is regulated by the entry/exit angle of the nucleosomal DNA ends and is favored by rigid DNA (Bednar et al., 1998Bednar J. Horowitz R.A. Grigoryev S.A. Carruthers L.M. Hansen J.C. Koster A.J. Woodcock C.L. Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin.Proc. Natl. Acad. Sci. USA. 1998; 95: 14173-14178Crossref PubMed Scopus (447) Google Scholar, Syed et al., 2010Syed S.H. Goutte-Gattat D. Becker N. Meyer S. Shukla M.S. Hayes J.J. Everaers R. Angelov D. Bednar J. Dimitrov S. Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome.Proc. Natl. Acad. Sci. USA. 2010; 107: 9620-9625Crossref PubMed Scopus (157) Google Scholar). The flexibility of the ends within the CENP-A nucleosome might therefore interfere with the binding of histone H1. To analyze the ability of histone H1 to interact in vitro with the CENP-A nucleosome, we have used a combination of two techniques, namely electro-mobility shift assay (EMSA) and hydroxyl radical (⋅OH) footprinting. EMSA allows the visualization of H1 binding to the nucleosome, but does not differentiate between specific and non-specific association, while ⋅OH footprinting detects the specific H1 binding at 1 bp resolution (Menoni et al., 2012Menoni H. Shukla M.S. Gerson V. Dimitrov S. Angelov D. Base excision repair of 8-oxoG in dinucleosomes.Nucleic Acids Res. 2012; 40: 692-700Crossref PubMed Scopus (55) Google Scholar, Syed et al., 2010Syed S.H. Goutte-Gattat D. Becker N. Meyer S. Shukla M.S. Hayes J.J. Everaers R. Angelov D. Bednar J. Dimitrov S. Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome.Proc. Natl. Acad. Sci. USA. 2010; 107: 9620-9625Crossref PubMed Scopus (157) Google Scholar). A physiologically relevant linker histone chaperone (NAP-1) was used to deposit histone H1 on centrally positioned conventional or CENP-A nucleosomes (Syed et al., 2010Syed S.H. Goutte-Gattat D. Becker N. Meyer S. Shukla M.S. Hayes J.J. Everaers R. Angelov D. Bednar J. Dimitrov S. Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome.Proc. Natl. Acad. Sci. USA. 2010; 107: 9620-9625Crossref PubMed Scopus (157) Google Scholar). The particle solutions were incubated with increasing amounts of NAP-1/H1 complex and run on native PAGE (Figure 2A). As seen, at the NAP-1/H1 concentration, when a complete shift for the H3-nucleosome was found, only a very weak shifted band reflecting the H1-CENP-A-nucleosomal complex was detected. These data showed that the presence of CENP-A interferes with the binding of histone H1 to the nucleosome. The ⋅OH footprinting patterns of conventional and CENP-A di-nucleosome were very similar; i.e., no enhanced cleavage at the DNA ends of the CENP-A nucleosome was observed (Figure 2B). This reflects the lack of sensitivity of the method to detect the dynamics and the transient dissociation of the ends from the core histone octamer. Some perturbations (increase of the ⋅OH cleavage “noise”) in the ⋅OH cleavage pattern can be observed only when the DNA nucleosomal ends are permanently and completely dissociated from the histone octamer as in the case of the histone variant H2A.Bbd and the chimeric H2A.ddBbd nucleosomes (see Figures 4 and S1; Shukla et al., 2011Shukla M.S. Syed S.H. Goutte-Gattat D. Richard J.L. Montel F. Hamiche A. Travers A. Faivre-Moskalenko C. Bednar J. Hayes J.J. et al.The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling.Nucleic Acids Res. 2011; 39: 2559-2570Crossref PubMed Scopus (42) Google Scholar). In agreement with our earlier data (Syed et al., 2010Syed S.H. Goutte-Gattat D. Becker N. Meyer S. Shukla M.S. Hayes J.J. Everaers R. Angelov D. Bednar J. Dimitrov S. Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome.Proc. Natl. Acad. Sci. USA. 2010; 107: 9620-9625Crossref PubMed Scopus (157) Google Scholar), the binding of histone H1 to conventional H3 di-nucleosomes results in: (1) clear protection of the nucleosomal dyad due to the strong interaction of the globular domain of H1 with the dyad, and (2) generation of 10 bp repeat of the linker DNA (Figure 2B, footprinting gel and the scans), which reflects the H1-induced formation of the stem structure (H1 interacts with both linkers and brings them in close vicinity and, thus, induces the assembly of the stem (Hamiche et al., 1996Hamiche A. Schultz P. Ramakrishnan V. Oudet P. Prunell A. Linker histone-dependent DNA structure in linear mononucleosomes.J. Mol. Biol. 1996; 257: 30-42Crossref PubMed Scopus (157) Google Scholar, Menoni et al., 2012Menoni H. Shukla M.S. Gerson V. Dimitrov S. Angelov D. Base excision repair of 8-oxoG in dinucleosomes.Nucleic Acids Res. 2012; 40: 692-700Crossref PubMed Scopus (55) Google Scholar, Syed et al., 2010Syed S.H. Goutte-Gattat D. Becker N. Meyer S. Shukla M.S. Hayes J.J. Everaers R. Angelov D. Bednar J. Dimitrov S. Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome.Proc. Natl. Acad. Sci. USA. 2010; 107: 9620-9625Crossref PubMed Scopus (157) Google Scholar). However, only very faint protection and 10 bp repeats were observed in CENP-A-di-nucleosomes incubated with the NAP1/H1 complex (Figure 2B, footprinting gel and the scans). These data revealed weak interaction of histone H1 with the CENP-A particles. Taken together, our in vitro experiments demonstrate that the CENP-A nucleosomal templates are poor substrates for H1 binding. A low degree of flexibility of the nucleosomal DNA ends is required for the efficient and specific binding of histone H1. The swapped αNH3-CENP-A mutant particle has rigid DNA ends (Figure 1C) as conventional H3 nucleosomes, and thus, would allow H1 binding (see schematics, Figures 1A and 1B). To test this, we analyzed the interaction of H1 with αNH3-CENP-A nucleosomes with both EMSA and ⋅OH footprinting as described for WT CENP-A (Figure 2). H1.5 subtype was used in these experiments. Of note, H1.5 exhibits very similar to H1.2 binding efficiency to the nucleosome (Figure S4). The deposition of H1 was performed by using the NAP-1/H1 complex, as detailed in Figure 2. The EMSA experiment clearly shows that histone H1 binds to both H3 and αNH3-CENP-A particles with the same efficiency (Figure 3A, compare upper and lower). ⋅OH footprinting revealed identical localization of histone H1 on both control H3 and αNH3-CENP-A nucleosomes as evidenced by a clear protection at the nucleosomal dyad and the generation of the typical ⋅OH cleavage 10 bp-repeat of the linker DNA (Figure 3B). Therefore, the hybrid αNH3-CENP-A nucleosomes, in contrast to WT CENP-A nucleosomes, are able, as predicted, to bind H1 with higher specificity and affinity. To determine whether H1 binding to CENP-A nucleosomes was also negatively affected in vivo as our in vitro experiments demonstrated, we used a proteomic approach coupled to mass spectrometry using cell culture models. We generated stable HeLa cell lines expressing double HA and FLAG tagged CENP-A (e-CENP-A) (Goutte-Gattat et al., 2013Goutte-Gattat D. Shuaib M. Ouararhni K. Gautier T. Skoufias D.A. Hamiche A. Dimitrov S. Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function.Proc. Natl. Acad. Sci. USA. 2013; 110: 8579-8584Crossref PubMed Scopus (46) Google Scholar, Shuaib et al., 2010Shuaib M. Ouararhni K. Dimitrov S. Hamiche A. HJURP binds CENP-A via a highly conserved N-terminal domain and mediates its deposition at centromeres.Proc. Natl. Acad. Sci. USA. 2010; 107: 1349-1354Crossref PubMed Scopus (147) Google Scholar). Stable HeLa cell lines expressing double-tagged conventional histone H3.1 (e-H3.1) or the histone variant H3.3 (e-H3.3) were used as positive controls. We isolated the nucleosomal e-CENP-A as well as both e-H3.1 and e-H3.3 nucleosomal complexes by double immunoaffinity purification (Goutte-Gattat et al., 2013Goutte-Gattat D. Shuaib M. Ouararhni K. Gautier T. Skoufias D.A. Hamiche A. Dimitrov S. Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function.Proc. Natl. Acad. Sci. USA. 2013; 110: 8579-8584Crossref PubMed Scopus (46) Google Scholar). The composition of the complexes was analyzed by mass spectrometry, SDS PAGE, and western blotting (Figure 4). The e-CENP-A complex, in agreement with the available data (Foltz et al., 2006Foltz D.R. Jansen L.E. Black B.E. Bailey A.O. Yates 3rd, J.R. Cleveland D.W. The human CENP-A centromeric nucleosome-associated complex.Nat. Cell Biol. 2006; 8: 458-469Crossref PubMed Scopus (521) Google Scholar, Goutte-Gattat et al., 2013Goutte-Gattat D. Shuaib M. Ouararhni K. Gautier T. Skoufias D.A. Hamiche A. Dimitrov S. Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function.Proc. Natl. Acad. Sci. USA. 2013; 110: 8579-8584Crossref PubMed Scopus (46) Google Scholar), contained several proteins from the CCAN as well as other proteins (Figure 4; Table S2). Importantly, no histone H1 was found associated within the complex as shown by electrophoretic analysis, western blotting, and mass spectrometry (Figures 4B–4D), although e-H3.1 and e-H3.3 nucleosomal complexes contained both isoforms H1.1 and H1.2 of histone H1 (Figure 4A). Therefore, histone H1 does not associate in vivo with CENP-A chromatin. These in vivo data fully agree with the poor non-specific in vitro binding of H1 to reconstituted CENP-A nucleosomes (Figure 1). Does artificially rigidifying CENP-A nucleosomal ends allow H1 binding in vivo? To analyze this, we generated stable HeLa cell lines expressing double HA and FLAG epitope tagged αNH3-CENP-A. The αNH3-CENP-A nucleosomal complex was purified as above and compared to the WT CENP-A nucleosomal complex. Its members were characterized by SDS PAGE, western blot, and mass spectrometry (Figures 4B–4D). Unlike the CENP-A nucleosomal complex, all methods identified histone H1 present in the αNH3-CENP-A nucleosomal complex. The characteristic histone H1 doublet was present in the electrophoretic pattern of the αNH3-CENP-A complex, but not in that of WT CENP-A one (Figure 4B). The anti-H1 antibody revealed a clear doublet corresponding to H1 (Figure 4C) and 15 H1 peptides (in total) were found by mass spectrometry in the αNH3-CENP-A nucleosomal complex (Figure 4D). Taken as a whole, our data reveal that the swapping the αN helix, and the preceding region of CENP-A with those of H3, primarily drives the generation of a particle with rigid orientation of the entry/exit DNA ends, which then allows efficient and specific association with histone H1 both in vitro and in vivo. A hybrid αNH3-CENP-A with H1 bound to it would in turn lead to the formation of condensed chromatin fibers similar to those established with canonical H3. Could this compact structure of the αNH3-CENP-A chromatin then affect the function of centromeres? To test this hypothesis, endogenous CENP-A was knocked down by using specific small interfering (si)RNAs in HeLa cell lines, and we analyzed the effect of expressing a siRNA resistant GFP-αNH3-CENP-A hybrid transcript as compared to a siRNA resistant GFP-CENP-A transcript (see schematics in Figure 5A). This was followed by quantitative analysis of mitotic progression in all cell lines. Treatment with siRNA results in very strong ablation of endogenous CENP-A: more than 85%–90% of endogenous CENP-A was depleted in each of the cell lines used and, as expected, the expression of the siRNA-resistant GFP-fusions was not affected (Figure 5B). Both GFP-fusions were found localized to the centromeres (Figure 5C). In agreement with the reported data (Goutte-Gattat et al., 2013Goutte-Gattat D. Shuaib M. Ouararhni K. Gautier T. Skoufias D.A. Hamiche A. Dimitrov S. Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function.Proc. 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• W2480057469 title "The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity" @default.
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• W2480057469 doi "https://doi.org/10.1016/j.molcel.2016.06.023" @default.
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