Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A
Aaron A. Van Hooser1,
Ilia I. Ouspenski1,*,
Heather C. Gregson2,
Daniel A. Starr3,
Tim J. Yen4,
Michael L. Goldberg3,
Kyoko Yokomori2,
William C. Earnshaw5,
Kevin F. Sullivan6 and
B. R. Brinkley1,
1 Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
2 Department of Biological Chemistry, College of Medicine, University of California, Irvine, CA 92697, USA
3 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
4 The Fox Chase Cancer Institute, Philadelphia, PA 19111, USA
5 Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, EH9 3JR, UK
6 Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
* Present address: Laboratory of Biosystems and Cancer, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Fig. 1. CENP-A is targeted to the kinetochore region of centromeres and, at higher levels of expression, along chromosome arms. (A) HA-tagged CENP-A was immunolocalized with anti-HA antibody (red) in a stable HeLa cell line that expresses the gene in trans from a CMV-based tetracycline-regulated promoter (Shelby et al., 1997). Cells were arrested in mitosis with Colcemid and harvested by mitotic shake-off. Centromeres were detected using SH-CREST auto-antiserum (green), which recognizes antigens in both the pairing and kinetochore domains of centromeres. DNA was counterstained with DAPI (blue). Bar, 1 µm. (B) HA-epitope-tagged human CENP-A cDNA was re-transfected transiently into the stable HeLa cell line. Under these conditions, HA-tagged CENP-A was found to target the lengths of chromosomes (red). Following  24 hours of expression, CENP-A-(HA) was predominately found on the euchromatic arms of chromosomes, with the pericentric heterochromatin of certain chromosomes containing reduced levels of staining (arrows). Phosphorylated histone H3 was detected using a specific antibody (green) and was found to be interspersed with the mistargeted CENP-A along the arms and within the pericentric heterochromatin of all chromosomes. Bar, 10 µm.
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Fig. 4. CENP-C is mistargeted with CENP-A during interphase. To determine if CENP-C is recruited to non-centromeric regions of chromatin to which CENP-A has been mistargeted, CENP-A was overexpressed and co-localized with other CENPs recognized by human autoimmune sera. A stable CHO cell line was used that expresses an HA-tagged human CENP-A cDNA from a CMV-based tetracycline-regulated promoter. (A) SH-CREST antiserum, which primarily recognizes CENPs-A, -B, and -C, stains exclusively centromeres in uninduced control interphase CHO cells. A low level of anti-HA antibody background staining is observed. DNA was counterstained with DAPI. (B) SH-CREST was found to localize throughout the interphase chromatin of CHO cells that were induced to express the HA-tagged CENP-A transgene. CENP-A-(HA) was detected using anti-HA antibody. (C) To determine if the non-centromeric staining of CREST observed following CENP-A overexpression was in part due to the mistargeting of factors other than CENP-A recognized by the serum, CENP-A was overexpressed in the CHO cell line, and then eluted from fixed cell preparations by salt and DNase treatment prior to immunostaining. Exogenous centromeric and non-centromeric CENP-A was significantly reduced in extracted preparations when compared with unextracted (B), as detected by immunofluorescence using anti-HA antibody. Speckles of CENP-A remained at non-centromeric sites in the extracted preparations, perhaps corresponding to matrix-associated regions of the genome. Importantly, centromeric and non-centromeric SH-CREST staining remained following the elution of CENP-A, indicating the presence of CENP-B and/or -C at the ectopic sites. Bars, 10 µm.
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Fig. 5. Mistargeted CENP-C does not recruit CENP-A to ectopic loci. Similar to CENP-A, CENP-C can be mistargeted to non-centromeric regions of chromatin by overexpression. To determine if mistargeted CENP-C is capable of recruiting CENP-A to non-centromeric chromatin, a tetracycline-regulated CENP-C expression vector was constructed and transfected into a stable HeLa cell line in which HA-tagged CENP-A is expressed at near-physiological levels and faithfully targeted to centromeres (Fig. 1A). CENP-C and CENP-A were coexpressed for 48 hours in the cells. Cells were grown and harvested on coverslips. Overexpressed CENP-C was observed to mistarget throughout interphase chromatin (cell on left), visualized using an anti-CENP-C antibody that stains exclusively centromeres in untransfected cells (cell on right). CENP-A was immunolocalized with anti-HA antibody and was found to be faithfully targeted to centromeres despite the overexpression and mistargeting of CENP-C. DNA was counterstained with DAPI. Bar, 10 µm.
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Fig. 6. Ectopic expression of CENP-A does not alter the level of CENP-B, CENP-C or hSMC1 that is soluble in the cell or associated with chromatin. The levels of various centromere-kinetochore proteins were compared between a stable CHO cell line induced to express HA-tagged CENP-A over 50 passages and the same cell line uninduced over the same number of passages. Protein samples from whole cells (50,000 cell-equivalents per lane) and detergent-extracted nuclear enrichments (100,000 cell-equivalents per lane) were compared by western analysis. Expression of CENP-A-(HA) was determined using anti-HA antibody and SH-CREST antiserum. SH-CREST also recognizes a non-centromeric 20-25 kDa antigen of unknown identity that displays certain biochemical properties similar to CENP-A (Earnshaw and Rothfield, 1985; Valdivia and Brinkley, 1985; Ouspenski and Brinkley, 1993; He and Brinkley, 1996). CENP-C was detected using a specific antibody and with SH-CREST antiserum. hSMC1 was detected using a specific antiserum. CENP-B was detected using SH-CREST. The subcellular localization of CENP-B was unaltered by CENP-A overexpression and serves as a control for the relative amount of protein loaded in each lane.
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Fig. 7. The CENP-A N-terminal tail is required for the recruitment of CENP-C to ectopic sites. A DNA construct encoding the C-terminal core domain of CENP-A with an HA-epitope tag (referred to as CENP-A-core) was transiently transfected into HeLa cells and detected by immunofluorescence using anti-HA antibody. Cells were grown and processed on coverslips. CENP-C was co-stained with a specific antibody. (A) Anti-CENP-C staining is centromere-specific in untransfected control cells (cell on right), in which CENP-A-core expression was undetectable. Similarly, CENP-C localization remained centromere-specific in transfected cells (cell on left), which demonstrated the localization of CENP-A-core throughout the nucleus and most predominately at centromeres. DNA was counterstained with DAPI. (B) Cells expressing the CENP-A-core peptide appeared to progress through mitosis normally. As during interphase, CENP-C was found exclusively at centromeres. The core domain of CENP-A appears to be targeted to chromatin less efficiently than the full-length protein and some accumulations were observed in the cytoplasm (Sullivan et al., 1994). Bars, 10 µm.
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Fig. 8. hSMC1 is mislocalized along chromosome arms with CENP-A specifically during mitosis. (A) hSMC1 was immunolocalized in HeLa cells that were transiently transfected with HA-tagged CENP-A 48 hours prior to arrest in mitosis. Cells overexpressing CENP-A-(HA), detected with anti-HA antibody, demonstrated anti-hSMC1 mislocalized along the arms of chromosomes (cell on right). Control untransfected cells within the same preparation (cell on left) demonstrated the characteristic centromere-kinetochore-exclusive staining of anti-hSMC1 during mitosis. DNA was counterstained with DAPI. (B) CHO cells were grown and processed on coverslips to examine ectopic and centromeric hSMC1 at all stages of mitosis. hSMC1 was observed along the lengths of chromosomes as early as prometaphase. SH-CREST was used to detect the mistargeted CENP-A. (C) Centromeric and non-centromeric anti-hSMC1 staining was redistributed to perichromosomal foci during late anaphase. Centromeric and non-centromeric SH-CREST staining, however, was observed at all stages. Bars, 10 µm.
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Fig. 11. Mislocalized CENP-A does not support the mitotic movement of non-centromeric chromosome fragments. (A) CHO cells expressing HA-tagged CENP-A were arrested at the G1/S-phase boundary with hydroxyurea, then induced to prematurely enter mitosis and fragment their genome by caffeine treatment. Centromere-kinetochore subunits, which stain intensely with SH-CREST antiserum, were observed to congress at the metaphase plate (arrow). Whereas non-centromeric genome fragments containing mistargeted CENP-A, detected using anti-HA antibody, stained less intensely with SH-CREST antiserum, were immotile and lay in clusters around the cell periphery. DNA was counterstained with DAPI. (B) CENP-A was transiently transfected and overexpressed in COLO 320DM cells, which contain numerous acentric double-minute (DM) chromosomes (arrows). Targeting of CENP-A to the DMs was not sufficient to assemble kinetochores, as demonstrated by an apparent lack of anti-ZW10 antibody reaction. (C) hSMC1, detected with a specific antibody, was observed to be recruited to the DMs (arrows) following the mistargeting of CENP-A, detected with anti-HA antibody, to these acentric chromosomes. Bars, 10 µm.
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Fig. 12. Mistargeting of CENP-A to the inactive centromere of dicentric chromosomes is not sufficient for kinetochore formation. CENP-A was overexpressed for 48 hours in a cell line (COLO 320DM) carrying multiple dicentric chromosomes and was immunolocalized using SH-CREST autoimmune serum. Cells were arrested in mitosis with Colcemid and harvested by mitotic shake-off. In untransfected control cells, CENP-A is only observed at active and not inactive centromeres (not shown). In cells overexpressing the protein, CENP-A was observed along the lengths of chromosome arms and at inactive centromeres. Despite the mistargeting of CENP-A, anti-ZW10 antibody localized only at the active centromere (arrow) and not at the inactive centromere (arrowhead) of dicentric chromosomes. DNA was counterstained with DAPI. Bar, 10 µm.
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