DOCSLIB.ORG

Borealin Dimerization Mediates Optimal CPC Checkpoint Function by Enhancing Localization to Centromeres and Kinetochores

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Borealin Dimerization Mediates Optimal CPC Checkpoint Function by Enhancing Localization to Centromeres and Kinetochores ARTICLE Received 2 Dec 2014 | Accepted 26 Feb 2015 | Published 9 Apr 2015 DOI: 10.1038/ncomms7775 Borealin dimerization mediates optimal CPC checkpoint function by enhancing localization to centromeres and kinetochores Michael E. Bekier1,w, Travis Mazur1, Maisha S. Rashid1 & William R. Taylor1 The chromosomal passenger complex (CPC) localizes to centromeres where it activates the mitotic checkpoint in response to inappropriate inter-kinetochore tension. This error correction function is essential for proper chromosome segregation. Here we define several critical features of CPC localization and function. First, the Borealin dimerization domain suppresses dynamic exchange at the centromere to allow optimal CPC function. Second, Borealin dimerization is essential to target a subpopulation of CPC proximal to the kinetochore when the mitotic spindle is disrupted. This subpopulation is also needed for full CPC checkpoint function. The existence of a pool of CPC at the kinetochore suggests that error correction is more complicated than predicted from the Aurora B phosphorylation gradient model. Finally, Haspin kinase plays a key role in maintaining the slowly exchanging centromere Borealin pool, while Aurora B and Mps1 play minimal roles in maintaining CPC localization once cells are in mitosis. 1 Department of Biological Sciences, University of Toledo, 2801 West Bancroft Street, MS 601, Toledo, Ohio 43606, USA. w Present address: Department of Molecular, Cellular and Developmental Biology, University of Michigan, 2127A Natural Science, 830 North University, Ann Arbor, Michigan 48109, USA. Correspondence and requests for materials should be addressed to W.R.T. (email: [email protected]). NATURE COMMUNICATIONS | 6:6775 | DOI: 10.1038/ncomms7775 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7775 ancer cells are commonly aneuploid, a condition defined Sgo1/2 binds pH2AT120, both the N-terminal and central regions by the gain or loss of whole chromosomes or chromo- of Borealin are implicated in centromere targeting of the Csomal segments1,2. Aneuploidy can result from errors in CPC17,18,25,27. We analysed truncated forms of Borealin lacking cell division, such as mis-segregation of individual chromosomes either the dimerization domain (Borealin1–221) or both the 3 during mitosis . Therefore, cell division is monitored by multiple central region and the dimerization domain (Borealin1–110). cell cycle checkpoints to prevent aneuploidy4,5. For example, the Immunofluorescence analysis of FLAG-tagged Borealin spindle assembly checkpoint (SAC) prevents anaphase onset until truncations was performed in cells blocked in mitosis with all chromosomes achieve biorientation on the mitotic spindle6. taxol. Full-length Borealin localized to centromeres with little to 7 The SAC is activated by unattached kinetochores . Anaphase no detectable cytoplasmic staining (Fig. 1a,b). Both Borealin1–221 onset is also delayed in response to insufficient tension generated and Borealin1–110 displayed punctate foci between Hec1-stained between sister kinetochores, for example, when both kinetochores kinetochores; however, truncated forms of Borealin showed attach to the same spindle pole. Insufficient tension triggers elevated cytoplasmic staining compared with full-length the release of bound microtubules, generating unattached Borealin (Fig. 1a,b). Quantification of the centromere/cytoplasm kinetochores that activate the SAC8,9. ratio of Borealin staining indicated a value of 4.5±1.2 The tension-sensing system that regulates kinetochore– (average±s.d.) for Borealin1–280. In contrast, this ratio was microtubule interactions depends on the chromosomal passenger significantly lower for both Borealin1–221 (2.0±0.4, Po0.001 6 complex (CPC) , composed of Borealin, Survivin, INCENP and using a Student’s t-test) and Borealin1–110 (1.7±0.3, Po0.001). Aurora B8. Prior to anaphase, the CPC localizes to inner Punctate staining of truncated forms of Borealin was also centromeres, adjacent to sister kinetochores, to monitor inter- observed when expressed in cells in which the endogenous kinetochore tension. The prevailing model that explains the role protein was knocked down using a untranslated region-targeted of Aurora B in regulating the SAC posits that under low tension, short hairpin RNA (shRNA) (Supplementary Fig. 1). When either Aurora B phosphorylates microtubule-binding kinetochore Borealin1–221 or Borealin1–110 were expressed in cells with proteins resulting in reduced affinity for microtubules leading endogenous Borealin intact, the level of INCENP on to dissociation of kinetochore–microtubule interactions10–14.As chromosomes was significantly reduced compared with cells chromosomes become bioriented and sister kinetochores are transfected with full-length Borealin (Fig. 1b,c). under high tension, Aurora B is spatially separated from the To better understand the elevated cytoplasmic staining of kinetochore and can no longer destabilize kinetochore– Borealin truncations and the reduced INCENP levels, we microtubule interactions. According to this spatial gradient measured centromere-binding dynamics of green fluorescent model, inner centromere localization of the CPC is paramount protein (GFP)-tagged Borealin in nocodazole-arrested mitotic for error correction15. In yeast, however, INCENP can cells, which also exhibit punctate localization of Borealin regulate chromosome segregation independent of centromere truncations (our unpublished data). Full-length Borealin-GFP localization16. recovered to B40% of pre-bleach levels within 60 s with a rate B À 1 CPC localization during metaphase is thought to rely on the constant of 0.020 s (Fig. 1d–f). Borealin1–110 recovered to intersection of two histone modifications at the centromere. B90% of pre-bleach levels with a rate constant of B0.068 s À 1 T3 B Haspin kinase phosphorylates histone H3 on threonine 3 (pH3 ) (Fig. 1d–f). Similarly, Borealin1–221 recovered to 90% of pre- along chromosome arms and at centromeres, which is bound bleach levels with a rate constant of 0.056 s À 1 (Fig. 1d–f). by Survivin17,18. The kinetochore-localized kinase Bub1 A Borealin mutant in which its dimerization domain was fused to T120 phosphorylates histone H2A on threonine 120 (pH2A )at amino acids 1–110 (Borealin110 þ DD) recovered to B90% of pericentromeric chromatin, which recruits Sgo1/2 (refs 18,19). pre-bleach levels with a rate constant of 0.054 s À 1 (Fig. 1d–f). Sgo1/2 can bind CDK1-phosphorylated Borealin, which also These observations suggest that although the N-terminus of contributes to CPC localization to centromeres18,20. Furthermore, Borealin is sufficient for localization to centromeres, the Aurora B regulates pH3T3 by directly activating Haspin via C-terminus suppresses dynamic exchange to maintain stable phosphorylation. Also, both Aurora B and Mps1 regulate Bub1 CPC binding at this location. Furthermore, because Borealin1–221 17,21–23 recruitment to kinetochores . We have analysed CPC and Borealin1–110 behave similarly, the N-terminus likely plays a localization and function using a combination of Borealin dominant role in localization under these conditions. structure–function studies, an FK506-binding protein (FKBP)- Because all Borealin truncations could be detected at mediated dimerization system and kinase inhibitors to deplete centromeres, we tested whether these mutants retained CPC known CPC receptors at the centromere. Our results suggest that function during mitosis. Cells were transfected with Borealin monomeric CPC interacts transiently with the centromere via shRNA along with shRNA-resistant Borealin C-terminal trunca- binding to pH3T3, while dimerization enhances centromere tions and analysed by western blotting to confirm knockdown binding to provide optimal CPC function. Selective reduction of (Fig. 1g,h) and by flow cytometry to determine DNA content pH3T3 reveals a kinetochore-proximal pool of CPC that partly co- (Fig. 1i). Borealin shRNA alone increased the percentage of localizes with pH2AT120, and depends on dimerization for polyploid cells to B13% compared with 1% in control cells, localization. Additional experiments suggest that both the inner indicating impaired CPC function (Fig. 1i). Expression of full- centromere and kinetochore-proximal pool of CPC contribute to length Borealin reduced the incidence of polyploidy to B3.5%, activation of the SAC. while expression of any Borealin truncation partially rescued the incidence of polyploidy to B8% (Fig. 1i). Next, we measured the frequency of mitotic phases in cells expressing Borealin shRNA, Results Borealin truncations and H2BGFP as a screenable marker. Domains of Borealin required for stable centromere binding. Borealin shRNA alone increased the percentage of prometaphase Borealin can be divided into three functional regions, the cells to B95% compared with B20% in control cells, indicating N-terminal alpha helices that interact with Survivin and INCENP impaired mitotic progression and defective CPC function (amino acids 15–76), an unstructured central region that contains (Fig. 1j). Co-expression of full-length Borealin with Borealin CDK1 phosphorylation sites required for interaction with Sgo1/2 shRNA reduced the percentage of prometaphase cells to 60% (amino acids 106–219) and a C-terminal dimerization domain (Fig. 1j). Co-expression of any Borealin C-terminal truncation (amino acids 224–280)24–26. Since Survivin binds pH3T3 and with Borealin
Load more

Recommended publications
  • How Human H1 Histone Recognizes DNA
    molecules Article How Human H1 Histone Recognizes DNA Olesya P. Luzhetskaya, Sergey E. Sedykh and Georgy A. Nevinsky * Institute of Chemical Biology and Fundamental Medicine, SD of Russian Academy of Sciences, 8 Lavrentiev Ave., 630090 Novosibirsk, Russia; [email protected] (O.P.L.); [email protected] (S.E.S.) * Correspondence: [email protected]; Tel.: +7-383-363-51-26; Fax: +7-383-363-51-53 Received: 11 August 2020; Accepted: 1 October 2020; Published: 5 October 2020 Abstract: Linker H1 histone is one of the five main histone proteins (H1, H2A, H2B, H3, and H4), which are components of chromatin in eukaryotic cells. Here we have analyzed the patterns of DNA recognition by free H1 histone using a stepwise increase of the ligand complexity method; the affinity of H1 histone for various single- and double-stranded oligonucleotides (d(pN)n; n = 1–20) was evaluated using their competition with 12-mer [32P]labeled oligonucleotide and protein–oligonucleotide complex delaying on nitrocellulose membrane filters. It was shown that minimal ligands of H1 histone (like other DNA-dependent proteins and enzymes) are different mononucleotides (dNMPs; Kd = (1.30 0.2) 2 ± 10 M). An increase in the length of single-stranded (ss) homo- and hetero-oligonucleotides (d(pA)n, × − d(pT)n, d(pC)n, and d(pN)n with different bases) by one nucleotide link regardless of their bases, leads to a monotonic increase in their affinity by a factor of f = 3.0 0.2. This factor f corresponds ± to the Kd value = 1/f characterizing the affinity of one nucleotide of different ss d(pN)n for H1 at n = 2–6 (which are covered by this protein globule) is approximately 0.33 0.02 M.
    [Show full text]
  • Evolution, Expression and Meiotic Behavior of Genes Involved in Chromosome Segregation of Monotremes
    G C A T T A C G G C A T genes Article Evolution, Expression and Meiotic Behavior of Genes Involved in Chromosome Segregation of Monotremes Filip Pajpach , Linda Shearwin-Whyatt and Frank Grützner * School of Biological Sciences, The University of Adelaide, Adelaide, SA 5005, Australia; fi[email protected] (F.P.); [email protected] (L.S.-W.) * Correspondence: [email protected] Abstract: Chromosome segregation at mitosis and meiosis is a highly dynamic and tightly regulated process that involves a large number of components. Due to the fundamental nature of chromosome segregation, many genes involved in this process are evolutionarily highly conserved, but duplica- tions and functional diversification has occurred in various lineages. In order to better understand the evolution of genes involved in chromosome segregation in mammals, we analyzed some of the key components in the basal mammalian lineage of egg-laying mammals. The chromosome passenger complex is a multiprotein complex central to chromosome segregation during both mitosis and meio- sis. It consists of survivin, borealin, inner centromere protein, and Aurora kinase B or C. We confirm the absence of Aurora kinase C in marsupials and show its absence in both platypus and echidna, which supports the current model of the evolution of Aurora kinases. High expression of AURKBC, an ancestor of AURKB and AURKC present in monotremes, suggests that this gene is performing all necessary meiotic functions in monotremes. Other genes of the chromosome passenger complex complex are present and conserved in monotremes, suggesting that their function has been preserved Citation: Pajpach, F.; in mammals.
    [Show full text]
  • Histone H1 Is Essential for Mitotic Chromosome Architecture
    Published Online: 20 June, 2005 | Supp Info: http://doi.org/10.1083/jcb.200503031 JCB: ARTICLE Downloaded from jcb.rupress.org on May 12, 2019 Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts Thomas J. Maresca, Benjamin S. Freedman, and Rebecca Heald Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 uring cell division, condensation and resolution of spindle. Although functional kinetochores assembled, chromosome arms and the assembly of a func- aligned, and exhibited poleward movement, long and D tional kinetochore at the centromere of each sister tangled chromosome arms could not be segregated in chromatid are essential steps for accurate segregation of anaphase. Histone H1 depletion did not significantly affect the genome by the mitotic spindle, yet the contribution of the recruitment of known structural or functional chromo- individual chromatin proteins to these processes is poorly somal components such as condensins or chromokinesins, understood. We have investigated the role of embryonic suggesting that the loss of H1 affects chromosome archi- linker histone H1 during mitosis in Xenopus laevis egg tecture directly. Thus, our results indicate that linker histone extracts. Immunodepletion of histone H1 caused the assem- H1 plays an important role in the structure and function of bly of aberrant elongated chromosomes that extended off vertebrate chromosomes in mitosis. the metaphase plate and outside the perimeter of the Introduction Correct transmission of the genome by the mitotic spindle complexes condensin and cohesin, which are thought to form during cell division requires dramatic changes in chromosome ring structures that generate chromosome super coiling or architecture.
    [Show full text]
  • Centromere RNA Is a Key Component for the Assembly of Nucleoproteins at the Nucleolus and Centromere
    Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere Lee H. Wong,1,3 Kate H. Brettingham-Moore,1 Lyn Chan,1 Julie M. Quach,1 Melisssa A. Anderson,1 Emma L. Northrop,1 Ross Hannan,2 Richard Saffery,1 Margaret L. Shaw,1 Evan Williams,1 and K.H. Andy Choo1 1Chromosome and Chromatin Research Laboratory, Murdoch Childrens Research Institute & Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Parkville 3052, Victoria, Australia; 2Peter MacCallum Research Institute, St. Andrew’s Place, East Melbourne, Victoria 3002, Australia The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. Here, we demonstrate that centromeric ␣-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I–dependent manner. The nucleolar targeting of CENPC1 and INCENP requires ␣-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following ␣-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, we have identified the nucleolar localization sequences on CENPC1 and INCENP. We present evidence that CENPC1 is an RNA-associating protein that binds ␣-satellite RNA by an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and “RNA replenishment” experiments, we show that ␣-satellite RNA is a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere.
    [Show full text]
  • Snf2h-Mediated Chromatin Organization and Histone H1 Dynamics Govern Cerebellar Morphogenesis and Neural Maturation
    ARTICLE Received 12 Feb 2014 | Accepted 15 May 2014 | Published 20 Jun 2014 DOI: 10.1038/ncomms5181 OPEN Snf2h-mediated chromatin organization and histone H1 dynamics govern cerebellar morphogenesis and neural maturation Matı´as Alvarez-Saavedra1,2, Yves De Repentigny1, Pamela S. Lagali1, Edupuganti V.S. Raghu Ram3, Keqin Yan1, Emile Hashem1,2, Danton Ivanochko1,4, Michael S. Huh1, Doo Yang4,5, Alan J. Mears6, Matthew A.M. Todd1,4, Chelsea P. Corcoran1, Erin A. Bassett4, Nicholas J.A. Tokarew4, Juraj Kokavec7, Romit Majumder8, Ilya Ioshikhes4,5, Valerie A. Wallace4,6, Rashmi Kothary1,2, Eran Meshorer3, Tomas Stopka7, Arthur I. Skoultchi8 & David J. Picketts1,2,4 Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation.
    [Show full text]
  • Transcriptional Regulation by Histone Ubiquitination and Deubiquitination
    Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press PERSPECTIVE Transcriptional regulation by histone ubiquitination and deubiquitination Yi Zhang1 Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina 27599, USA Ubiquitin (Ub) is a 76-amino acid protein that is ubiqui- The fact that histone ubiquitination occurs in the largely tously distributed and highly conserved throughout eu- monoubiquitinated form and is not linked to degrada- karyotic organisms. Whereas the extreme C-terminal tion, in combination with the lack of information regard- four amino acids are in a random coil, its N-terminal 72 ing the responsible enzymes, prevented us from under- amino acids have a tightly folded globular structure (Vi- standing the functional significance of this modification. jay-Kumar et al. 1987; Fig. 1A). Since its discovery ∼28 Recent identification of the E2 and E3 proteins involved years ago (Goldknopf et al. 1975), a variety of cellular in H2B ubiquitination (Robzyk et al. 2000; Hwang et al. processes including protein degradation, stress response, 2003; Wood et al. 2003a) and the discovery of cross-talk cell-cycle regulation, protein trafficking, endocytosis sig- between histone methylation and ubiquitination (Dover naling, and transcriptional regulation have been linked et al. 2002; Sun and Allis 2002) have set the stage for to this molecule (Pickart 2001). Ubiquitylation is pro- functional analysis of histone ubiquitination. In a timely posed to serve as a signaling module, and the informa- paper published in the previous issue of Genes & Devel- tion transmitted by this tag may depend on the nature of opment, Shelley Berger and colleagues (Henry et al.
    [Show full text]
  • Phosphorylation of Threonine 3 on Histone H3 by Haspin Kinase Is Required for Meiosis I in Mouse Oocytes
    ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 5066–5078 doi:10.1242/jcs.158840 RESEARCH ARTICLE Phosphorylation of threonine 3 on histone H3 by haspin kinase is required for meiosis I in mouse oocytes Alexandra L. Nguyen, Amanda S. Gentilello, Ahmed Z. Balboula*, Vibha Shrivastava, Jacob Ohring and Karen Schindler` ABSTRACT a lesser extent. However, it is not known whether haspin is required for meiosis in oocytes. To date, the only known haspin substrates Meiosis I (MI), the division that generates haploids, is prone to are threonine 3 of histone H3 (H3T3), serine 137 of macroH2A and errors that lead to aneuploidy in females. Haspin is a kinase that threonine 57 of CENP-T (Maiolica et al., 2014). Knockdown or phosphorylates histone H3 on threonine 3, thereby recruiting Aurora inhibition of haspin in mitotically dividing tissue culture cell kinase B (AURKB) and the chromosomal passenger complex lines reveal that phosphorylation of H3T3 is essential for the (CPC) to kinetochores to regulate mitosis. Haspin and AURKC, an alignment of chromosomes at the metaphase plate (Dai and AURKB homolog, are enriched in germ cells, yet their significance Higgins, 2005; Dai et al., 2005), regulation of chromosome in regulating MI is not fully understood. Using inhibitors and cohesion (Dai et al., 2009) and establishing a bipolar spindle (Dai overexpression approaches, we show a role for haspin during MI et al., 2009). In mitotic metaphase, phosphorylation of H3T3 is in mouse oocytes. Haspin-perturbed oocytes display abnormalities restricted to kinetochores, and this mark signals recruitment of the in chromosome morphology and alignment, improper kinetochore– chromosomal passenger complex (CPC) (Dai et al., 2005; Wang microtubule attachments at metaphase I and aneuploidy at et al., 2010; Yamagishi et al., 2010).
    [Show full text]
  • A Cytoplasmic Histone H1-Like Protein 2883 Ice-Cold RFB Buffer (160 Mm Kcl, 40 Mm Nacl, 20 Mm Na2egta, According to the Manufacturer’S Instruction
    Research Article 2881 A novel linker histone-like protein is associated with cytoplasmic filaments in Caenorhabditis elegans Monika A. Jedrusik1, Stefan Vogt2, Peter Claus3 and Ekkehard Schulze1,* 1Georg-August University of Göttingen, Third Department of Zoology – Developmental Biology, Humboldtallee 34A, 37073 Göttingen, Germany 2Georg-August University of Göttingen, Institute for X-ray Physics, Geiststraße 11, 37073 Göttingen, Germany 3Hannover Medical School, Neuroanatomy, Carl-Neuberg-Str.1, 30625 Hannover, Germany *Author for correspondence (e-mail: [email protected]) Accepted 1 May 2002 Journal of Cell Science 115, 2881-2891 (2002) © The Company of Biologists Ltd Summary The histone H1 complement of Caenorhabditis elegans structure therein, the tonofilaments. Additionally contains a single unusual protein, H1.X. Although H1.X H1.X::GFP is expressed in the cytoplasm of body and vulva possesses the globular domain and the canonical three- muscle cells, neurons, excretory cells and in the nucleoli of domain structure of linker histones, the amino acid embryonic blastomeres and adult gut cells. RNA composition of H1.X is distinctly different from interference with H1.X results in uncoordinated and egg conventional linker histones in both terminal domains. We laying defective animals, as well as in a longitudinally have characterized H1.X in C. elegans by antibody labeling, enlarged pharynx. These phenotypes indicate a green fluorescent protein fusion protein expression and cytoplasmic role of H1.X in muscle growth and muscle RNA interference. Unlike normal linker histones, H1.X is function. a cytoplasmic as well as a nuclear protein and is not associated with chromosomes. H1.X is most prominently expressed in the marginal cells of the pharynx and is Key words: Caenorhabditis elegans, Histone H1, Chromatin, associated with a peculiar cytoplasmic cytoskeletal Intermediate filaments, Linker histone Introduction essential aspects of higher multicellular life, such as cell Linker histones are highly abundant eukaryotic chromatin differentiation and development.
    [Show full text]
  • A Single Amino Acid Change Converts Aurora-A Into Aurora-B-Like Kinase in Terms of Partner Specificity and Cellular Function
    A single amino acid change converts Aurora-A into Aurora-B-like kinase in terms of partner specificity and cellular function Jingyan Fua, Minglei Biana, Junjun Liub, Qing Jianga, and Chuanmao Zhanga,1 aThe Education Ministry Key Laboratory of Cell Proliferation and Differentiation and the State Key Laboratory of Bio-membrane and Membrane Bio-engineering, College of Life Sciences, Peking University, Beijing 100871, China; and bDepartment of Biological Sciences, California State Polytechnic University, Pomona, CA 91768 Edited by Don W. Cleveland, University of California at San Diego, La Jolla, CA, and approved March 5, 2009 (received for review January 25, 2009) Aurora kinase-A and -B are key regulators of the cell cycle and the original Aurora-A localization at the spindle. Moreover, tumorigenesis. It has remained a mystery why these 2 Aurora Aurora-AG198N prevented the chromosome misalignment and kinases, although highly similar in protein sequence and structure, cell premature exit from mitosis caused by Aurora-B knock- are distinct in subcellular localization and function. Here, we report down, indicating functional substitution for Aurora-B in cell the striking finding that a single amino acid residue is responsible cycle regulation. for these differences. We replaced the Gly-198 of Aurora-A with the equivalent residue Asn-142 of Aurora-B and found that in HeLa Results cells, Aurora-AG198N was recruited to the inner centromere in Aurora-AG198N Colocalizes with Aurora-B at Centromere and Midzone. metaphase and the midzone in anaphase, reminiscent of the We fused the mutants Aurora-AG198N and Aurora-BN142G (Fig. Aurora-B localization. Moreover, Aurora-AG198N compensated for 1A) with GFP and transiently expressed them in HeLa cells the loss of Aurora-B in chromosome misalignment and cell prema- [supporting information (SI) Fig.
    [Show full text]
  • Drosophila Ribosomal Proteins Are Associated with Linker Histone H1 and Suppress Gene Transcription
    Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Drosophila ribosomal proteins are associated with linker histone H1 and suppress gene transcription Jian-Quan Ni,1,3 Lu-Ping Liu,1,3 Daniel Hess,1 Jens Rietdorf,1 and Fang-Lin Sun1,2,4 1Friedrich Miescher Institute for Biomedical Research, Basel CH-4058, Switzerland; 2Institute of Epigenetics and Cancer Research, School of Medicine, Tsinghua University, Beijing 100080, China The dynamics and function of ribosomal proteins in the cell nucleus remain enigmatic. Here we provide evidence that specific components of Drosophila melanogaster ribosomes copurify with linker histone H1. Using various experimental approaches, we demonstrate that this association of nuclear ribosomal proteins with histone H1 is specific, and that colocalization occurs on condensed chromatin in vivo. Chromatin immunoprecipitation analysis confirmed that specific ribosomal proteins are associated with chromatin in a histone H1-dependent manner. Overexpression of either histone H1 or ribosomal protein L22 in Drosophila cells resulted in global suppression of the same set of genes, while depletion of H1 and L22 caused up-regulation of tested genes, suggesting that H1 and ribosomal proteins are essential for transcriptional gene repression. Overall, this study provides evidence for a previously undefined link between ribosomal proteins and chromatin, and suggests a role for this association in transcriptional regulation in higher eukaryotes. [Keywords: Ribosomal protein; L22; histone H1; chromatin; transcription] Supplemental material is available at http://www.genesdev.org. Received September 25, 2005; revised version accepted May 8, 2006. Transcription and translation in eukaryotes are generally discrete nuclear sites was sensitive to inhibitors of both believed to take place within two spatially separated cel- transcription and translation, arguing that the two pro- lular compartments.
    [Show full text]
  • Suppression of Histone H1 Genes in Arabidopsis Results in Heritable Developmental Defects and Stochastic Changes in DNA Methylation
    Copyright © 2005 by the Genetics Society of America DOI: 10.1534/genetics.104.031997 Suppression of Histone H1 Genes in Arabidopsis Results in Heritable Developmental Defects and Stochastic Changes in DNA Methylation Andrzej T. Wierzbicki* and Andrzej Jerzmanowski*,†,1 *Laboratory of Plant Molecular Biology, Warsaw University, 02-106 Warsaw, Poland and †Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland Manuscript received June 3, 2004 Accepted for publication November 8, 2004 ABSTRACT Histone H1 is an abundant component of eukaryotic chromatin that is thought to stabilize higher-order chromatin structures. However, the complete knock-out of H1 genes in several lower eukaryotes has no discernible effect on their appearance or viability. In higher eukaryotes, the presence of many mutually compensating isoforms of this protein has made assessment of the global function of H1 more difficult. We have used double-stranded RNA (dsRNA) silencing to suppress all the H1 genes of Arabidopsis thaliana. Plants with a Ͼ90% reduction in H1 expression exhibited a spectrum of aberrant developmental pheno- types, some of them resembling those observed in DNA hypomethylation mutants. In subsequent genera- tions these defects segregated independently of the anti-H1 dsRNA construct. Downregulation of H1 genes did not cause substantial genome-wide DNA hypo- or hypermethylation. However, it was correlated with minor but statistically significant changes in the methylation patterns of repetitive and single-copy se- quences, occurring in a stochastic manner. These findings reveal an important and previously unrecognized link between linker histones and specific patterns of DNA methylation. N eukaryotes, the complex structural organization of the former.
    [Show full text]
  • Bub1 Is Essential for Assembly of the Functional Inner Centromere
    JCB: ARTICLE Bub1 is essential for assembly of the functional inner centromere Yekaterina Boyarchuk,1,2 Adrian Salic,3 Mary Dasso,1 and Alexei Arnaoutov1 1Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 2Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 199004, Russia 3Department of Cell Biology, Harvard Medical School, Boston, MA 02115 uring mitosis, the inner centromeric region (ICR) Depletion of Bub1 from Xenopus laevis egg extract or recruits protein complexes that regulate sister from HeLa cells resulted in both destabilization and dis- D chromatid cohesion, monitor tension, and modu- placement of chromosomal passenger complex (CPC) from late microtubule attachment. Biochemical pathways that the ICR. Moreover, soluble Bub1 controls the binding of govern formation of the inner centromere remain elusive. Sgo to chromatin, whereas the CPC restricts loading of The kinetochore protein Bub1 was shown to promote Sgo specifi cally onto centromeres. We further provide evi- assembly of the outer kinetochore components, such dence that Bub1 kinase activity is pivotal for recruitment of as BubR1 and CENP-F, on centromeres. Bub1 was also all of these components. Together, our fi ndings demon- implicated in targeting of Shugoshin (Sgo) to the ICR. We strate that Bub1 acts at multiple points to assure the cor- show that Bub1 works as a master organizer of the ICR. rect kinetochore formation. Introduction Attachment of chromosomes to spindle microtubules (MTs) thus, controlling the polymerization/depolymerization state of is performed by kinetochores, which are large proteinaceous tubulin fi laments to achieve correct end-on attachment of MTs structures that assemble at the centromeric regions of each sis- to the kinetochore (Andrews et al., 2004; Lan et al., 2004).
    [Show full text]