Centromere Protein B Assembles Human Centromeric a-satellite DNA at the 17-bp Sequence, CENPB Box Yoshinao Muro,* Hiroshi Masumoto,t Kinya Yoda,t Naohito Nozaki,f Masaru Ohashi,* and Tuneko Okazakit * Department of Dermatology, Nagoya University School ofMedicine, Showa-ku, Nagoya 466, Japan; and *Department ofMolecular Biology, School of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan Abstract. We purified 15,000-fold from HeLa cell ity-shift assay, Southwestern blotting, and DNase I pro- nuclear extract the centromere antigen that reacts spe- tection analysis. The binding constant of the antigen to cifically with the 17-bp sequence, designated previ- the CENP-B box sequence is 6 x 108 M-' . DNA mo- ously as CENP-B box, in human centromeric u-satel- bility-shift assays indicated that the major complex lite (alphoid) DNA by a two-step procedure including formed between the CENP-B and the DNA contains an oligonucleotide affinity column. The purified protein two DNA molecules, suggesting the importance of the was identified as the centromere protein B (CENRB) CENRB/CENPB box interaction in organization of by its mobility on SDS-PAGE (80 kD), and reactivities higher ordered chromatin structures in the centromere to a monoclonal antibody raised to CENP-B (bacterial and/or kinetochore . Location of DNA binding and fusion protein) and to anticentromere sera from patients dimerization domains in CENP-B was discussed based with autoimmune diseases. Direct binding by CENP-B on the DNA mobility-shift assays performed with a of the CENP-B box sequence in the alphoid DNA has protein fraction containing intact and partial cleavage been proved using the purified CENP-B by DNA mobil- products of CENRB. HE centromere is the essential domain in eukaryotic mammalian centromere is cytologically defined as the pri- chromosomes for proper segregation of chromosomes mary constriction in metaphase chromosome. However, to daughter cells at mitosis and meiosis (7). Sister identification of the DNA segment executing mammalian Tchromatids are held together at the centromere after DNA chromosome segregation has not been straightforward be- replication until the coordinate disjunction takes places at the cause of the high complexity of the DNA in centromere do- metaphase to anaphase transition. The centromere provides main and the difficulty encountered in constructing an activ- attachment sites for spindle microtubules on a chromosome ity assay. by organizing kinetochore (36), a unique proteinaceous ma- The primary constriction of mammalian chromosomes is trix having contact with centromeric chromatin on inner side made up of heterochromatin composed predominantly of and dynamic association with spindle microtubules on its highly repetitive DNA sequence (satellite DNA) (44) . How- outer layer (8, 28) and is seenby electron microscopy as a tri- ever, satellite DNA families located in the primary constric- layered disc-like structure (35, 37). Motor molecules required tion of chromosomes in different mammalian orders (e.g., for positioning of chromosomes during the metaphase and rodents and primates) appear largely unrelated in their se- anaphase stages may also reside within or near the centro- quences and unit length. Thus, all human chromosomes con- mere/kinetochore domain (16, 17, 22, 34, 39) . Thesemultiple tain at the primary constriction the a-satellite (alphoid) centromere functions must in some way be directed by a cis- DNA composed oftandem repeats of 170-bp diverged mono- acting DNA sequence located in the centromere region. In- mers organized into groups of polymeric units that are ar- deed, specific centromere DNA sequences (CEN-DNA) in ranged tandemly in a larger unit repeated further in 300- yeast Saccharomyces cerevisiae and Schizosaccharomyces 5,000-kb area (25, 41, 45, 47) . Human alphoid DNA thus pombe were identified both by physical mapping of centro- varies in amount and sequence from chromosome to chro- mere region DNA and by activity assay to select DNA seg- mosome (44, 45). A kinetochore-related DNA sequence has ment responsible for imparting mitotic and meiotic stability not been identified with human cells. As for the mouse, Mus to plasmids containing autonomously replicating sequences musculus, it has been shown by in situ hybridization that a (9, 12, 18, 21). Although the minimal essential sequence of minor-satellite DNA composed of tandem repeats of 120-bp CEN-DNA for S. pombe is not completely delimited yet, the monomer units localized on the outer surface of the centro- CEN-DNAs of two yeast species are quite different in chain mere corresponding to the kinetochore domain (46). length and lack obvious sequence conservation (11). The With the use of centromere-specific autoantibodies (an- © The Rockefeller University Press, 0021-9525/92/02/585/12 $2 .00 The Journal of Cell Biology, Volume 116, Number 3, February 1992 585-596 585 ticentromere antibodies; ACA') in serum of patients with 0.5 mM EDTA, 0.5 mM PMSF, 0.5,ug/ml Pepstatin A, 4 pM Leupeptin (Sigma Chemical Co ., St. Louis, MO). Buffer A was 20 mM Hepes, pH autoimmune diseases, the localization of specific antigenic 7.6, 0.5 mM EDTA, 10% glycerol, 1 mM DTT, 0.2 mM PMSF. Buffer B proteins has been determined at the centromere-kinetochore was 20 mM Hepes, pH 7.6, 1 mM EDTA, 10% glycerol, 0.05% NP-40, region of chromosomes from a number of mammalian spe- 3 mM DTT, 0.1 mM PMSF. Binding buffer was 20 mM Hepes, pH 7.6, 10% cies (6, 13, 29) . With human cells, three major centromere- glycerol, 1 mM EDTA, 3 mM DTT, 0.05% NP-40, and 150 mM NaCl (final specific proteins (centromere protein [CENP]A, B, and C) concentration) . Binding buffer II was as described by Masumoto et al. (26). TBEbuffer was 12 .5 mM Tris, pH 8.0, 12.5 mM boric acid, 0.5 mM EDTA. have been identified using ACA sera (14, 31). CENP-A (17- kD) is a histone-like protein component of a centromere- specific nucleosome (32, 33). Thegene for CENPB (80-kD) Nuclear Extracts has been cloned and sequenced, and the property of the anti- HeLa cell culture and preparation of HeLa nuclei were performed as de- gen is the most extensively studied among the three centro- scribed by Masumoto et al . (26) . The isolated HeLa nuclei were suspended mere antigens (7, 15). It was reported recently that CENP-B in washing buffer (see above for buffer components) at the concentration of 2 x 108 nuclei/ml, mixed with equal volumes of extraction buffer sup- is a highly conserved protein in mammalian cells (40). The plemented with 0.4 M NaCl and 30% (vol/vol) glycerol, incubated 1 h at property of CENP-C (140-kD) is least known . 0°C with gentle agitation, and then centrifuged for 5 min at 1,500 rpm in Previously, we have shown with HeLa cells that alphoid a rotor (model TS-9; Tomy Seiko, Tokyo, Japan) . The pellet was extracted DNA and centromere antigens colocalize in both mitotic again with extraction buffer supplemented with 0.5 M NaCI and centrifuged by double-label in situ at 25,000 rpm for 1 h in a rotor (type 40; Beckman Instruments Inc., Palo chromosomes and interphase nuclei Alto, CA). The supernatant fraction was recovered and stored at -80°C hybridization and immunofluorescence (27). We have shown (0.2-0 .5 M NaCI nuclear extract) . subsequently that proteins from HeLa cell nuclear extract form a complex containing CENPB with alphoid DNA con- taining the 17-bp motif (PyI'TCGTTMAAPuCGGGA) . Probes The 17-bp motifhas been designated as "CENP-B box", since The following complementary oligonucleotides were synthesized chemi- Southwestern blot analysis on a protein fraction, obtained cally and used for the probes or competitors containing a CENP-B box se- from HeLa cell nuclear extract by immunoprecipitation with quence . an ACA serum, had suggested strongly that CENP-B itself The 56-mer DNA: TTTCTTCATATTATGCT AGACAGAATAATTC-3' binds the 17-bp motif(26) . The CENP-B box is found in only 5'-TCAGAGGC CENP-B box a subset ofalphoid monomers but the CENPB box-contain- CTCCG AAAGAAGTATAATACGA TCTGTCTTATTAAGAGT ing alphoid monomers are detected in all centromeres ofhu- The 23-mer DNA: man chromosomes except Y chromosome (26). The possi- 5'-GGC box TTT-3' bility of presence of a small amount of CENP-B box in Y G CENPB AAACC' chromosome alphoid DNA is still under investigation . In- The 29-mer DNA: terestingly, the CENPB box is also present in mouse minor 5'-TCAGAGGC box TTTC-3' satellite (26, 46). The conserved CENP-B and CENPB box CTCCG CENPB AAAGAGT . interaction in the mammalian centromere might offer a key to solve the common function carried by the centromeric The Py and Pu in the box was C and A, respectively. The defective 23-mer heterochromatin without rigid sequence conservation. DNA had pyrimidine to purine base alterations at the underlined positions in the CENP-B box: 5'-TATCGTTGGAAAA_GGGA-3' . To investigate the alphoid DNA and centromere antigen The end-labeled probe DNA molecules were prepared from the 23-mer, interaction further in molecular level, we purify in this study 29-mer, or 56-mer DNA by repairing the single-stranded ends with DNA the CENP-B box binding protein from HeLa crude nuclear polymerase I Klenow fragment and radioactive nucleotides . Resulting mol- extract and show that CENP-B itself binds CENPB box se- ecules were called the end-labeled 25-bp DNA, 32-bp DNA, or 59-bp quence in alphoid DNA. We then show that a DNA-protein DNA . complex thus formed contains two DNA molecules . This suggests the possible commitment of this DNA-protein in- Preparation ofOligonucleotide-Sepharose teraction to the higher ordered structure formation in centro- The oligonucleotide-sepharose was prepared according to the procedure mere domain.