Human Centromere Protein B Induces Translational Positioning of Nucleosomes on α-Satellite Sequences

The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other α-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the α-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box. The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other α-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the α-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box. The centromere is a chromosomal locus that plays an essential role in chromosome segregation at mitosis and meiosis. These specific loci are located in the primary constriction of each chromosome and are organized into a specialized chromatin structure composed of α-satellite DNA repeats and their associated proteins (reviewed in Refs. 1Pluta A.F. Mackay A.M. Ainsztein A.M. Goldberg I.G. Earnshaw W.C. 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CENP-A and CENP-C do not show any sequence specificity in DNA binding; however, in contrast, CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears in every other α-satellite repeat (171 base pairs) in human centromeres (12Masumoto H. Masukata H. Muro Y. Nozaki N. Okazaki T. J. Cell Biol. 1989; 109: 1963-1973Crossref PubMed Scopus (538) Google Scholar, 13Masumoto H. Properties of CENP-B and Its Target Sequence in α Satellite DNA. 1993; (Springer-Verlag, Berlin, Germany)Google Scholar, 14Ikeno M. Masumoto H. Okazaki T. Hum. Mol. Genet. 1994; 3: 1245-1257Crossref PubMed Scopus (142) Google Scholar). centromere protein A B and C respectively nitrilotriacetic acid micrococcal nuclease. centromere protein A B and C respectively nitrilotriacetic acid micrococcal nuclease. CENP-A shares sequence similarity with histone H3 and binds to α-satellite DNA in human centromeres in vivo (7Palmer D.K. O'Day K. Trong H.L. Charbonneau H. Margolis R.L. Proc. Natl. Acad. Sci. 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Yang H. Nozaki N. Okazaki T. Yoda K. Mol. Cell. Biol. 2002; 22: 2229-2241Crossref PubMed Scopus (141) Google Scholar, 30Suzuki N. Nakano M. Nozaki N. Egashira S. Okazaki T. Masumoto H. J. Biol. Chem. 2004; 279: 5934-5946Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). CENP-B is a dimeric protein composed of 80-kDa subunits (6Earnshaw W.C. Sullivan K.F. Machlin P.S. Cooke C.A. Kaiser D.A. Pollard T.D. Rothfield N.F. Cleveland D.W. J. Cell Biol. 1987; 104: 817-829Crossref PubMed Scopus (320) Google Scholar) and contains DNA-binding and dimerization domains at its N terminus and C terminus, respectively (31Yoda K. Kitagawa K. Masumoto H. Muro Y. Okazaki T. J. Cell Biol. 1992; 119: 1413-1427Crossref PubMed Scopus (108) Google Scholar, 32Kitagawa K. Masumoto H. Ikeda M. Okazaki T. Mol. Cell. Biol. 1995; 15: 1602-1612Crossref PubMed Google Scholar). CENP-B specifically binds the CENP-B box within α-satellite DNA (12Masumoto H. Masukata H. Muro Y. Nozaki N. Okazaki T. J. Cell Biol. 1989; 109: 1963-1973Crossref PubMed Scopus (538) Google Scholar, 33Muro Y. Masumoto H. Yoda K. Nozaki N. Ohashi M. Okazaki T. J. Cell Biol. 1992; 116: 585-596Crossref PubMed Scopus (187) Google Scholar, 34Kipling D. Mitchell A.R. Masumoto H. Wilson H.E. Nicol L. Cooke H.J. Mol. Cell. Biol. 1995; 15: 4009-4020Crossref PubMed Scopus (96) Google Scholar, 35Yoda K. Okazaki T. Chromosome Res. 1997; 5: 207-211Crossref PubMed Scopus (9) Google Scholar). We previously determined the crystal structures of the CENP-B N-terminal DNA-binding domain (amino acid residues 1-129) complexed with the CENP-B box DNA and the C-terminal dimerization domain (amino acid residues 540-599). The C-terminal dimerization domain consists of two α-helices, which are folded into an antiparallel configuration, and forms a dimer with a symmetrical, antiparallel, four-helix bundle structure (36Tawaramoto M.S. Park S.Y. Tanaka Y. Nureki O. Kurumizaka H. Yokoyama S. J. Biol. Chem. 2003; 278: 51454-51461Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). On the other hand, the N-terminal DNA-binding domain forms two helix-turn-helix motifs, which are bound to adjacent major grooves of the CENP-B box DNA (37Tanaka Y. Nureki O. Kurumizaka H. Fukai S. Kawaguchi S. Ikuta M. Iwahara J. Okazaki T. Yokoyama S. EMBO J. 2001; 20: 6612-6618Crossref PubMed Scopus (70) Google Scholar). The existence of the CENP-B box sequence within the α-satellite sequence is required for the formation of a functional centromere in vivo (38Ikeno M. Grimes B. Okazaki T. Nakano M. Saitoh K. Hoshino H. McGill N. Cooke H. Masumoto H. Nat. Biotech. 1998; 16: 431-439Crossref PubMed Scopus (349) Google Scholar, 39Ohzeki J. Nakano M. Okada T. Masumoto H. J. Cell Biol. 2002; 159: 765-775Crossref PubMed Scopus (218) Google Scholar). However, CENP-B null mice appeared to be normal (40Hudson D.F. Fowler K.J. Earle E. Saffery R. Kalitsis P. Trowell H. Hill J. 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Therefore, CENP-B may a that the formation of centromere-specific chromatin the α-satellite DNA repeat at the nucleosome In the present study, we the centromere-specific nucleosome histones H2A, H2B, H4, and CENP-A and the role of CENP-B binding in nucleosome formation α-satellite DNA in recombinant human histones H2A, H2B, H4, and CENP-A previously Y. Tawaramoto M.S. Kawaguchi S. Ohta T. Yoda K. Kurumizaka H. Yokoyama S. Methods. 2004; 33: 3-11Crossref PubMed Scopus (133) Google Scholar). The DNA histones H2A, H2B, and CENP-A into the H. J.J. 2002; Scopus Google Scholar), and that histone into the The of the histone and CENP-A for in cells Y. Tawaramoto M.S. Kawaguchi S. Ohta T. Yoda K. Kurumizaka H. Yokoyama S. Methods. 2004; 33: 3-11Crossref PubMed Scopus (133) Google Scholar). These recombinant proteins N-terminal The histones and CENP-A in the The in and The recombinant histones and CENP-A acid The the and the and the the of recombinant histones and CENP-A a with The recombinant human in E. cells and in the of previously (37Tanaka Y. Nureki O. Kurumizaka H. Fukai S. Kawaguchi S. Ikuta M. Iwahara J. Okazaki T. Yokoyama S. EMBO J. 2001; 20: 6612-6618Crossref PubMed Scopus (70) Google Scholar). The DNA human α-satellite DNA Y. Tawaramoto M.S. Kawaguchi S. Ohta T. Yoda K. Kurumizaka H. Yokoyama S. Methods. 2004; 33: 3-11Crossref PubMed Scopus (133) Google Scholar) DNA the and into the The pair of the the and its sequence is The CENP-B box sequence is The pair with the This pair contains at of the pair These α-satellite DNA the an The α-satellite DNA the and into the The previously Y. Tawaramoto M.S. Kawaguchi S. Ohta T. Yoda K. Kurumizaka H. Yokoyama S. Methods. 2004; 33: 3-11Crossref PubMed Scopus (133) Google Scholar). The dimer and the the with the α-satellite DNA at in the of and The and for at and for at the to and with a this the and The and for at the nucleosomes in the of with the dimer and the the with the α-satellite DNA at in the of and The and for at and for at the to and with a this the and and The and for at The nucleosomes with the recombinant histones and CENP-A in and The at for and with a I α-satellite DNA in the of at the The nucleosomes the α-satellite DNA and for at the the to with the of I. DNA and the with of I of DNA. with of of DNA. I at for and the of to the the the nucleosomes and the in and and the DNA the J.J. D. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). DNA and and of and for at the the to with the of DNA with and for at The nucleosomes with and and the the of to the DNA with and The DNA in of and a used for the in the of the DNA In the translational positioning a of the DNA to and the pair DNA the The pair at and with and The DNA The in of a histones H2B, H4, and the α-satellite and the the nucleosomes histones with of and the with of and The proteins with the The CENP-A into the with the nucleosome core particle is composed of a histone octamer two and an and a pair DNA which is wrapped the histone octamer C.A. D.F. Luger K. J. Mol. Biol. 2002; PubMed Scopus Google Scholar, C.A. Nature. 2003; PubMed Scopus Google Scholar). This pair is and can be the α-satellite DNA is wrapped the histone octamer CENP-A forms we the CENP-A nucleosome with recombinant histones H2A, H2B, H4, and CENP-A in the of a pair human α-satellite DNA shown in when the CENP-A with the pair human α-satellite DNA with a pair DNA and a pair DNA also when H3 nucleosomes with and not when the DNA used a for this and Therefore, indicate that the CENP-A nucleosome wraps 147 base pairs of the α-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. CENP-B to we the nucleosome in the of the DNA-binding domain of human CENP-B K. Masumoto H. Ikeda M. Okazaki T. Mol. Cell. Biol. 1995; 15: 1602-1612Crossref PubMed Google Scholar, Y. Nureki O. Kurumizaka H. Fukai S. Kawaguchi S. Ikuta M. Iwahara J. Okazaki T. Yokoyama S. EMBO J. 2001; 20: 6612-6618Crossref PubMed Scopus (70) Google Scholar, Y. Kurumizaka H. Yokoyama S. J. PubMed Scopus Google Scholar). CENP-B specifically binds the CENP-B box 17-base pair DNA with this DNA-binding The pair α-satellite DNA used in this contains a CENP-B and is located in the base pair to the nucleosome core particle 147 base pairs of DNA the CENP-B box sequence be located within the nucleosome core particle, the nucleosome an of this α-satellite DNA shown in bound to the H3 and CENP-A The the nucleosomes without a A of binding to the CENP-B box sequence in the H3 and CENP-A nucleosomes I and However, the CENP-B of the nucleosomal DNA with that of the DNA and that the histones and not the I and to that the not two with without we the and the nucleosomes I the of the CENP-B not Therefore, specifically binds to the CENP-B box sequence within the nucleosomal the of the nucleosomal CENP-B box not with that of the CENP-B box DNA. The CENP-B binding to the nucleosomal DNA may the CENP-B box to the binding may not be with that with the CENP-B box DNA. CENP-B the of the the nucleosomal DNA is the histone octamer a pair of is observed J.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar). In H3 and CENP-A nucleosomes without the pair observed I and that the α-satellite DNA in H3 and CENP-A the the pair of the not in the H3 and CENP-A the CENP-B box and These indicate that the rotational setting of the α-satellite nucleosomal DNA is not the CENP-B of we CENP-B binding the translational of the histone octamer the α-satellite DNA. The H3 and CENP-A nucleosomes with without the pair α-satellite DNA and the nucleosomal with shown in a pair DNA observed when bound to the nucleosomal DNA. the translational positioning of the pair DNA the pair a such and shown in B and in the of two major translational A and in the H3 and CENP-A nucleosomes the pair α-satellite A is located the of the DNA with respect to the CENP-B and B is base pairs A. The to the DNA the probably due to of the nucleosome The of the in the H3 and CENP-A nucleosomes in the of for H3 nucleosomes and for CENP-A suggesting that the histone octamer may be A and B. However, in the of the of the B nucleosomes the to the A nucleosomes observed for H3 and for CENP-A CENP-B binding to the nucleosomal CENP-B box sequence induces the formation of the A suggesting that the translational positioning of nucleosomes is CENP-B binding α-satellite DNA. CENP-B binding to the nucleosomal CENP-B box may the of the histone octamer the DNA like histones K. K. EMBO J. 15: PubMed Scopus Google Scholar). of the the of the CENP-B box sequence in the α-satellite DNA CENP-B binding to nucleosomal we α-satellite These α-satellite and In the α-satellite the 17-base pair CENP-B box the sequence which is for CENP-B In the and the CENP-B box and base the of the α-satellite DNA On the other hand, in the and α-satellite the CENP-B box and base the of the α-satellite DNA In the α-satellite the CENP-B box to a to the of the nucleosome. These α-satellite DNA base pairs) and shown in bound to of α-satellite for the α-satellite DNA. The showed a probably due to the of the α-satellite sequence and the DNA the CENP-B binding (37Tanaka Y. Nureki O. Kurumizaka H. Fukai S. Kawaguchi S. Ikuta M. Iwahara J. Okazaki T. Yokoyama S. EMBO J. 2001; 20: 6612-6618Crossref PubMed Scopus (70) Google Scholar, D.M. 1991; PubMed Scopus Google Scholar). CENP-B to CENP-A CENP-B binding to the centromere-specific the CENP-A α-satellite do the CENP-A nucleosomes with the α-satellite base in the of and the nucleosomes Surprisingly, bound to of the α-satellite nucleosomes the CENP-B box the of the bound nucleosomal α-satellite DNA and and that binding to α-satellite nucleosomal the of the CENP-B box In to the binding to the α-satellite we a In this the α-satellite nucleosomes histones and CENP-A in the of bound to the nucleosomal CENP-B box DNA with histones and CENP-A the and an with a the histone with the histones in the of the α-satellite DNA not in the of the α-satellite DNA These indicate that bound to the CENP-B box DNA not to histones and DNA. proteins in experiments without histones B and that the bound to the CENP-B box DNA not Consistent with the the binding with the and nucleosomes B and and C and However, the binding not be with the and nucleosomes and their with in the and We this and that the Therefore, CENP-B may to the nucleosomes the and its for be The of the CENP-B Sequence for the we CENP-B induces the translational positioning of CENP-A nucleosomes the α-satellite like the of the α-satellite DNA. shown in translational of the CENP-A nucleosomes the α-satellite observed without and two major translational observed with the α-satellite DNA Surprisingly, translational positioning of the CENP-A nucleosome in the and nucleosomes and Therefore, the of the histone octamer may be in the and In contrast, like the the of translational positioning binding not observed in the and nucleosomes nucleosomes have the to The nucleosome may the CENP-B box in a not in the nucleosome core particle, the nucleosome located at the of the DNA However, of the CENP-B box of the nucleosome be located within the nucleosome core particle, the nucleosome at A. the CENP-B box be located within the nucleosome core particle in the and nucleosomes the of nucleosomal DNA is base C.A. D.F. Luger K. J. Mol. Biol. 2002; PubMed Scopus Google Scholar, K. R.K. D.F. Nature. 1997; PubMed Scopus Google Scholar), the CENP-B box in the and nucleosomes may have rotational to that of the α-satellite DNA with respect to the histone octamer surface. Therefore, the of the nucleosome CENP-B may only when the CENP-B box is located with the proper rotational with respect to the of the histone octamer within the nucleosome. CENP-B may be a determinant for the translational positioning of centromere-specific nucleosomes its functional binding to the nucleosomal CENP-B box In the present study, we showed that CENP-B the to to the CENP-B box sequence located within the nucleosome core This that CENP-B binds to nucleosomal DNA without the the histone octamer and DNA. CENP-B binding not the nucleosome. We two of a centromere-specific CENP-A nucleosome histones H2A, H2B, H4, and CENP-A and a canonical H3 nucleosome histones H2A, H2B, and with mouse cells that the of CENP-A significant of CENP-B (28Howman E.V. Fowler K.J. Newson A.J. Redward S. MacDonald A.C. Kalitsis P. Choo K.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1148-1153Crossref PubMed Scopus (326) Google Scholar), suggesting that CENP-B binding to the centromere may the of However, in vitro not show any for CENP-B binding to the CENP-A nucleosome the H3 nucleosome. an may be in the functional CENP-A and CENP-B. to the crystal structure of complexed with CENP-B box essential within the 17-base pair CENP-B box sequence (37Tanaka Y. Nureki O. Kurumizaka H. Fukai S. Kawaguchi S. Ikuta M. Iwahara J. Okazaki T. Yokoyama S. EMBO J. 2001; 20: 6612-6618Crossref PubMed Scopus (70) Google Scholar). of and DNA are only of the DNA and DNA of Therefore, we a the nucleosome core particle and the structures In this we that the nucleosome is at A, CENP-B binding nucleosome formation at A. shown in to the nucleosomal DNA without in this This structure not the DNA CENP-B significant in observed the nucleosome and the that the nucleosome core particle the CENP-B-induced DNA and nucleosome CENP-B of the are required to the functional of CENP-B within the nucleosome core The CENP-B box sequence a for nucleosome α-satellite DNA K. Ando S. A. A. Okazaki T. Genes 1998; 3: PubMed Scopus Google Scholar). Consistent with this in the present study, we showed that CENP-B binding to the nucleosomal CENP-B box translational positioning of the nucleosome core experiments with the α-satellite showed that of the CENP-B box within the nucleosomal α-satellite DNA CENP-B binding to the nucleosomal CENP-B box sequence not translational positioning of the nucleosome core These indicate that the proper rotational setting of the CENP-B box within the nucleosome is essential for the CENP-B-induced translational which may in the centromere-specific chromatin The I showed that the α-satellite DNA sequence in the H3 and CENP-A probably due to its A. A. M. J. P. J. PubMed Scopus Google Scholar, J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, T. H. H. K. T. Y. Hashimoto K. Res. 1998; PubMed Scopus Google Scholar). This rotational setting of the α-satellite sequence is to the CENP-B box rotational which the CENP-B-induced translational positioning of the nucleosome. Therefore, the rotational setting and translational positioning of the centromere-specific nucleosomes are in the of CENP-B and may be for the formation of a functional centromere-specific chromatin been that of DNA cells in the of a DNA in the of CENP-B A.R. P. Nicol L. H. D. J. Cell Sci. 109: PubMed Google Scholar). the CENP-B box sequence contains two within its essential for CENP-B binding (37Tanaka Y. Nureki O. Kurumizaka H. Fukai S. Kawaguchi S. Ikuta M. Iwahara J. Okazaki T. Yokoyama S. EMBO J. 2001; 20: 6612-6618Crossref PubMed Scopus (70) Google Scholar). We previously that CENP-B binds to an CENP-B and the DNA-binding of CENP-B is to the of binding at Y. Kurumizaka H. Yokoyama S. J. PubMed Scopus Google Scholar). In the present study, we showed that CENP-B binding to the nucleosomal CENP-B box induces the translational positioning of nucleosomes the α-satellite Therefore, at the CENP-B box may be an of centromere-specific chromatin through the CENP-B-induced nucleosome positioning the α-satellite DNA We K. and E. for

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