Biophysical Characterization of the Centromere-specific Nucleosome from Budding Yeast

The centromeric DNA of all eukaryotes is assembled upon a specialized nucleosome containing a histone H3 variant known as CenH3. Despite the importance and conserved nature of this protein, the characteristics of the centromeric nucleosome are still poorly understood. In particular, the stoichiometry and DNA-binding properties of the CenH3 nucleosome have been the subject of some debate. We have characterized the budding yeast centromeric nucleosome by biochemical and biophysical methods and show that it forms a stable octamer containing two copies of the Cse4 protein and wraps DNA in a left-handed supercoil, similar to the canonical H3 nucleosome. The DNA-binding properties of the recombinant nucleosome are identical to those observed in vivo demonstrating that the octameric structure is physiologically relevant. The centromeric DNA of all eukaryotes is assembled upon a specialized nucleosome containing a histone H3 variant known as CenH3. Despite the importance and conserved nature of this protein, the characteristics of the centromeric nucleosome are still poorly understood. In particular, the stoichiometry and DNA-binding properties of the CenH3 nucleosome have been the subject of some debate. We have characterized the budding yeast centromeric nucleosome by biochemical and biophysical methods and show that it forms a stable octamer containing two copies of the Cse4 protein and wraps DNA in a left-handed supercoil, similar to the canonical H3 nucleosome. The DNA-binding properties of the recombinant nucleosome are identical to those observed in vivo demonstrating that the octameric structure is physiologically relevant. IntroductionThe centromere is the chromosomal locus responsible for attachment to the mitotic spindle via the kinetochores, and it is essential for correct chromosome segregation in all eukaryotes (1Allshire R.C. Karpen G.H. Nat. Rev. Genet. 2008; 9: 923-937Crossref PubMed Scopus (422) Google Scholar). It has been recognized for some time that centromeric chromatin has somewhat different properties to bulk chromatin; in particular, centromeric nucleosomes are distinguished by containing a histone H3 variant, known as CENP-A in humans (2Earnshaw W.C. Rothfield N. Chromosoma. 1985; 91: 313-321Crossref PubMed Scopus (642) Google Scholar), Cse4 in budding yeast (3Meluh P.B. Yang P. Glowczewski L. Koshland D. Smith M.M. Cell. 1998; 94: 607-613Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar), Cnp1 in fission yeast (4Takahashi K. Chen E.S. Yanagida M. Science. 2000; 288: 2215-2219Crossref PubMed Scopus (313) Google Scholar), and CID in fruit flies (5Henikoff S. Ahmad K. Platero J.S. van Steensel B. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 716-721Crossref PubMed Scopus (252) Google Scholar). For simplicity, we shall refer to these generically as CenH3 nucleosomes. This histone H3 variant includes a conserved histone fold domain (HFD) 2The abbreviations used are: HFD, histone fold domain; MNase, micrococcal nuclease; AUC, analytical ultracentrifugation. preceded by an N-terminal extension that is highly divergent in both size and amino acid composition (Fig. 1A). The HFD also contains a CENP-A targeting domain that appears to be responsible for the special mechanical properties of the centromere-specific nucleosome (6Black B.E. Foltz D.R. Chakravarthy S. Luger K. Woods Jr., V.L. Cleveland D.W. Nature. 2004; 430: 578-582Crossref PubMed Scopus (317) Google Scholar), and it is sufficient to impart centromere targeting and functionality in hybrid H3-CENP-A targeting domain nucleosomes (7Black B.E. Jansen L.E. Maddox P.S. Foltz D.R. Desai A.B. Shah J.V. Cleveland D.W. Mol. Cell. 2007; 25: 309-322Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Although there are now numerous crystal structures of histone H3-containing nucleosomes, there are currently no crystal structures of an intact CenH3 nucleosome, and it is unclear exactly how these specific modifications contribute to the assembly and overall structure of the chromatin in which they are embedded.Recently, it has been proposed that CenH3 nucleosomes might be tetrameric instead of octameric (8Dalal Y. Wang H. Lindsay S. Henikoff S. PLoS Biol. 2007; 5: e218Crossref PubMed Scopus (193) Google Scholar, 9Furuyama T. Dalal Y. Henikoff S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 6172-6177Crossref PubMed Scopus (121) Google Scholar) and wrap DNA in the reverse direction to canonical nucleosomes, introducing positive supercoiling (10Furuyama T. Henikoff S. Cell. 2009; 138: 104-113Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), suggestions that have proved somewhat controversial (11Dechassa M.L. D'Arcy S. Luger K. Cell. 2009; 138: 22-24Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 12Lavelle C. Recouvreux P. Wong H. Bancaud A. Viovy J.L. Prunell A. Victor J.M. Cell. 2009; 139: 1216-1218Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). It is also unclear whether a centromeric nucleosome octamer might contain two copies of CenH3 (homotypic) or one each of CenH3 and H3 proteins (heterotypic), as shown in Fig. 1B. In addition, the non-histone protein Scm3 has also been shown to bind Cse4 and H4 (13Mizuguchi G. Xiao H. Wisniewski J. Smith M.M. Wu C. Cell. 2007; 129: 1153-1164Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) and may be involved in recruitment of the centromeric nucleosome. Despite original suggestions (13Mizuguchi G. Xiao H. Wisniewski J. Smith M.M. Wu C. Cell. 2007; 129: 1153-1164Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), it does not appear that it remains part of the core nucleosome once at the active centromere (14Camahort R. Shivaraju M. Mattingly M. Li B. Nakanishi S. Zhu D. Shilatifard A. Workman J.L. Gerton J.L. Mol. Cell. 2009; 35: 794-805Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), although it may remain associated with it. In vivo studies of centromeric nucleosome composition, for example by immunoprecipitation analyses, may be complicated by the chromatin preparation procedure or co-purification of other non-CenH3 components bound to chromatin. Here, we present the results of a rigorous biophysical analysis of the budding yeast centromeric nucleosome, using purified recombinant proteins to try and clarify some of these issues.DISCUSSIONIn this study, we have demonstrated that the budding yeast centromeric nucleosome forms a stable octamer, containing two copies of the Cse4 protein and that this structure is maintained upon DNA binding. At no stage in our work have we seen any evidence for a species corresponding to a tetrameric hemisome, and the Cse4-H3 mixing experiments strongly argue that a symmetrical CenH3-CenH3 (or H3-H3) interaction is strongly favored over a CenH3-H3 heterodimer.While this manuscript was in preparation, the structure of the core nucleosomal CenH3/H4 tetramer was described, using a truncated version of the human CENP-A protein (20Sekulic N. Bassett E.A. Rogers D.J. Black B.E. Nature. 2010; 467: 347-351Crossref PubMed Scopus (162) Google Scholar). The conclusions reached by analysis of the crystal structure are essentially the same as those presented in this study, which together show that both a subnucleosomal structure and an intact DNA-bound nucleosome contain a (CenH3-H4)2 tetramer that acts as a core for the full octameric complex.This study, together with previously published results (14Camahort R. Shivaraju M. Mattingly M. Li B. Nakanishi S. Zhu D. Shilatifard A. Workman J.L. Gerton J.L. Mol. Cell. 2009; 35: 794-805Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 20Sekulic N. Bassett E.A. Rogers D.J. Black B.E. Nature. 2010; 467: 347-351Crossref PubMed Scopus (162) Google Scholar), unequivocally demonstrates that the centromeric nucleosome is octameric in vitro. We believe that this is also true in vivo for several reasons. The symmetric octamer is the favored and most stable form of the complex, and this must be true regardless of the pathway by which it was formed. The presence in vivo of an intrinsically unstable tetrameric hemisome could only occur if it were stabilized by another non-histone factor. This would have to bind tightly in stoichiometric quantities, be an essential gene product, as well as being highly conserved throughout evolution. No such factor has yet been found.A second reason to think that the reconstituted nucleosomes are equivalent to those found at the centromere is provided by analysis of the DNA-binding properties. Whereas the Cse4 octamer is similar in size to canonical H3 nucleosomes, the site size of DNA protected from MNase digestion is somewhat reduced and less well defined. This altered protection relative to canonical nucleosomes has previously been seen in multiple in vivo and in vitro studies (8Dalal Y. Wang H. Lindsay S. Henikoff S. PLoS Biol. 2007; 5: e218Crossref PubMed Scopus (193) Google Scholar, 21Polizzi C. Clarke L. J. Cell Biol. 1991; 112: 191-201Crossref PubMed Scopus (98) Google Scholar, 22Yoda K. Ando S. Morishita S. Houmura K. Hashimoto K. Takeyasu K. Okazaki T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7266-7271Crossref PubMed Scopus (192) Google Scholar) and has been interpreted as evidence for a tetrameric nucleosome (8Dalal Y. Wang H. Lindsay S. Henikoff S. PLoS Biol. 2007; 5: e218Crossref PubMed Scopus (193) Google Scholar). Our results clearly show that this smaller site size is compatible with octameric nucleosomes, and it is not necessary to invoke alternative assembly models. Crucially, this demonstrates that the DNA-binding properties of the recombinant nucleosomes reflect the situation in vivo and argues against the possibility that experiments on reconstituted nucleosomes are artifactual.There are probably multiple reasons for the reduced DNA protection exhibited by CenH3 nucleosomes. A more compact structure is probably partly responsible, but we feel the main cause is weaker protein-DNA interactions. Crystal structures of intact, DNA-bound nucleosomes have shown that the interaction closest to the entrance-exit points of the DNA is formed with the N-terminal section of histone H3 (16Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6791) Google Scholar, 23White C.L. Suto R.K. Luger K. EMBO J. 2001; 20: 5207-5218Crossref PubMed Scopus (312) Google Scholar), which is highly diverged in all known CenH3 orthologs. Weaker interactions here would allow increased access to nucleases and explain the species-specific differences seen in MNase protection assays, as well as explain the more diffuse appearance of the protected bands. It is unclear what the physiological relevance of this might be, but it may well involve the as-yet unknown role of the extended CenH3 N-terminal tails.Our results and those of others (20Sekulic N. Bassett E.A. Rogers D.J. Black B.E. Nature. 2010; 467: 347-351Crossref PubMed Scopus (162) Google Scholar) show that the octameric CenH3 nucleosome is only capable of wrapping DNA in a conventional left-handed manner, with induction of negative supercoils. It is possible that that previous results from the in vivo minichromosome assay indicating Cse4-induced positive supercoiling in yeast (10Furuyama T. Henikoff S. Cell. 2009; 138: 104-113Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) could be explained by transcriptional artifacts or nonspecific binding of Ndc10 to the centromeric DNA complicating the analysis. This work, together with previously published observations on a range of CenH3 orthologs, strongly suggests that the octamer is the physiologically relevant form. Further work will be required to understand how the CenH3-specific histone contributes to the overall structure of the centromeric nucleosome and what effect this has on the mechanical properties of the centromere. IntroductionThe centromere is the chromosomal locus responsible for attachment to the mitotic spindle via the kinetochores, and it is essential for correct chromosome segregation in all eukaryotes (1Allshire R.C. Karpen G.H. Nat. Rev. Genet. 2008; 9: 923-937Crossref PubMed Scopus (422) Google Scholar). It has been recognized for some time that centromeric chromatin has somewhat different properties to bulk chromatin; in particular, centromeric nucleosomes are distinguished by containing a histone H3 variant, known as CENP-A in humans (2Earnshaw W.C. Rothfield N. Chromosoma. 1985; 91: 313-321Crossref PubMed Scopus (642) Google Scholar), Cse4 in budding yeast (3Meluh P.B. Yang P. Glowczewski L. Koshland D. Smith M.M. Cell. 1998; 94: 607-613Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar), Cnp1 in fission yeast (4Takahashi K. Chen E.S. Yanagida M. Science. 2000; 288: 2215-2219Crossref PubMed Scopus (313) Google Scholar), and CID in fruit flies (5Henikoff S. Ahmad K. Platero J.S. van Steensel B. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 716-721Crossref PubMed Scopus (252) Google Scholar). For simplicity, we shall refer to these generically as CenH3 nucleosomes. This histone H3 variant includes a conserved histone fold domain (HFD) 2The abbreviations used are: HFD, histone fold domain; MNase, micrococcal nuclease; AUC, analytical ultracentrifugation. preceded by an N-terminal extension that is highly divergent in both size and amino acid composition (Fig. 1A). The HFD also contains a CENP-A targeting domain that appears to be responsible for the special mechanical properties of the centromere-specific nucleosome (6Black B.E. Foltz D.R. Chakravarthy S. Luger K. Woods Jr., V.L. Cleveland D.W. Nature. 2004; 430: 578-582Crossref PubMed Scopus (317) Google Scholar), and it is sufficient to impart centromere targeting and functionality in hybrid H3-CENP-A targeting domain nucleosomes (7Black B.E. Jansen L.E. Maddox P.S. Foltz D.R. Desai A.B. Shah J.V. Cleveland D.W. Mol. Cell. 2007; 25: 309-322Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Although there are now numerous crystal structures of histone H3-containing nucleosomes, there are currently no crystal structures of an intact CenH3 nucleosome, and it is unclear exactly how these specific modifications contribute to the assembly and overall structure of the chromatin in which they are embedded.Recently, it has been proposed that CenH3 nucleosomes might be tetrameric instead of octameric (8Dalal Y. Wang H. Lindsay S. Henikoff S. PLoS Biol. 2007; 5: e218Crossref PubMed Scopus (193) Google Scholar, 9Furuyama T. Dalal Y. Henikoff S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 6172-6177Crossref PubMed Scopus (121) Google Scholar) and wrap DNA in the reverse direction to canonical nucleosomes, introducing positive supercoiling (10Furuyama T. Henikoff S. Cell. 2009; 138: 104-113Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), suggestions that have proved somewhat controversial (11Dechassa M.L. D'Arcy S. Luger K. Cell. 2009; 138: 22-24Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 12Lavelle C. Recouvreux P. Wong H. Bancaud A. Viovy J.L. Prunell A. Victor J.M. Cell. 2009; 139: 1216-1218Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). It is also unclear whether a centromeric nucleosome octamer might contain two copies of CenH3 (homotypic) or one each of CenH3 and H3 proteins (heterotypic), as shown in Fig. 1B. In addition, the non-histone protein Scm3 has also been shown to bind Cse4 and H4 (13Mizuguchi G. Xiao H. Wisniewski J. Smith M.M. Wu C. Cell. 2007; 129: 1153-1164Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) and may be involved in recruitment of the centromeric nucleosome. Despite original suggestions (13Mizuguchi G. Xiao H. Wisniewski J. Smith M.M. Wu C. Cell. 2007; 129: 1153-1164Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), it does not appear that it remains part of the core nucleosome once at the active centromere (14Camahort R. Shivaraju M. Mattingly M. Li B. Nakanishi S. Zhu D. Shilatifard A. Workman J.L. Gerton J.L. Mol. Cell. 2009; 35: 794-805Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), although it may remain associated with it. In vivo studies of centromeric nucleosome composition, for example by immunoprecipitation analyses, may be complicated by the chromatin preparation procedure or co-purification of other non-CenH3 components bound to chromatin. Here, we present the results of a rigorous biophysical analysis of the budding yeast centromeric nucleosome, using purified recombinant proteins to try and clarify some of these issues.