RCS1, a Substrate of APC/C, Controls the Metaphase to Anaphase Transition

Total Page:16

File Type:pdf, Size:1020Kb

RCS1, a Substrate of APC/C, Controls the Metaphase to Anaphase Transition RCS1, a substrate of APC/C, controls the metaphase to anaphase transition Wei-meng Zhao*, Judith A. Coppinger†, Akiko Seki*, Xiao-li Cheng*, John R. Yates III†, and Guowei Fang*‡ *Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020; and †Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037 Edited by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved June 26, 2008 (received for review September 28, 2007) The anaphase-promoting complex/cyclosome (APC/C) controls the ing of its function in the cell cycle. We report here the identification onset of anaphase by targeting securin for destruction. We report of a substrate of APC/C through a powerful functional genomic here the identification and characterization of a substrate of APC/C, analysis (5) and the characterization of the physiological function of RCS1, as a mitotic regulator that controls the metaphase-to-anaphase this substrate at the metaphase-to-anaphase transition. transition. We showed that the levels of RCS1 fluctuate in the cell cycle, peaking in mitosis and dropping drastically as cells exit into G1. Results Indeed, RCS1 is efficiently ubiquitinated by APC/C in vitro and de- Functional Genomic Analyses Identified Substrates of APC/C. To graded during mitotic exit in a Cdh1-dependent manner in vivo. develop a systematic approach for the identification of APC/C APC/C recognizes a unique D-box at the N terminus of RCS1, as substrates (5), we analyzed 16 known substrates of the APC/C that mutations of this D-box abolished ubiquitination in vitro and stabi- have been characterized at the onset of this study. These substrates lized the mutant protein in vivo. RCS1 controls the timing of the included cyclin A, cyclin B, Plk1, Aurora A, Aurora B, Nek2, Cdc20, anaphase onset, because the loss of RCS1 resulted in a faster pro- UbcH10, securin, GTSE1, anillin, TPX2, Cdc6, geminin, ribonu- gression from the metaphase to anaphase and accelerated degrada- cleotide reductase M2, and SnoN (1–4, 7, 8). The first 15 substrates tion of securin and cyclin B. Biochemically, mitotic RCS1 associates are cell-cycle regulators (1), whereas the last one, SnoN, acts in cell with the NuRD chromatin-remodeling complex, and this RCS1 com- signaling (7). Genes involved in cell cycle regulation have been plex is likely involved in regulating gene expression or chromatin shown to covary transcriptionally during tumorigenesis, as these structure, which in turn may control anaphase onset. Our study regulators are under selective pressures to act coordinately as one uncovers a complex regulatory network for the metaphase-to- module for cell proliferation (9, 10). Indeed, the expression of the anaphase transition. first 15 cell-cycle substrates of the APC/C covaries transcriptionally across hundreds of tumor tissues/cell lines (10), whereas the ex- HDAC1/2 ͉ mitosis ͉ ubiquitin-dependent proteolysis ͉ Cdh1 pression of SnoN does not (9, 10). Furthermore, of the 15 cell cycle substrates, the first 12 act in mitosis and cytokinesis (1–4, 7), and all biquitin-mediated proteolysis regulates key transitions in bi- 12 genes are transcriptionally induced in G2 or in mitosis (11). We Uology. For example, the anaphase-promoting complex/ concluded that a common theme for cell cycle substrates is their cyclosome (APC/C) is an essential ubiquitin ligase that controls the transcriptional covariation with each other across hundreds of ordered degradation of over 20 cell-cycle regulators in mitosis and tumor tissues. Substrates that only function in mitosis and cytoki- nesis are likely to be induced in G2 or in mitosis. Thus, we searched G1 (1). Degradation of securin by the APC/C at the onset of anaphase allows activation of separase and cleavage of the cohesin published microarray databases for genes that fulfill these two complex, leading to segregation of sister chromatids. Similarly, criteria (10, 11) and then tested the corresponding proteins for their APC/C-dependent destruction of cyclin B at anaphase inactivates ability to be ubiquitinated by the active APC/C-Cdh1 in a recon- the cyclin B/Cdk1 kinase and results in mitotic exit. At telophase stituted assay (4, 12). From these experiments, we identified eight and cytokinesis, APC/C controls the degradation of a group of new substrates of the APC/C: ECT2, KIF2C/MCAK, KIFC1/ cell-cycle proteins, such as Aurora A/B, Plk1, TPX2, CKAP2 and HSET, MELK, MKLP2, Par-1a/MARK3, and two proteins, anillin (2–5), to ensure the ordered mitotic exit and cytokinesis. FLJ20354 and FLJ10156, all of which are efficiently ubiquitinated by the APC/C in a Cdh1-dependent manner [Fig. 1 and supporting Beyond mitosis, APC/C is also involved in the G1/S transition and DNA replication. information (SI) Fig. S1]. We named FLJ10156 as a regulator of The ordered destruction of APC/C substrates is at least in part chromosome segregation (RCS1) and reported here the charac- controlled by the association of APC/C with its regulators, Cdc20 terization of FLJ10156 as a substrate of APC/C that controls the and Cdh1 (1). Cdc20 binds to APC/C in mitosis and activates its metaphase-to-anaphase transition. ligase activity at the anaphase onset to trigger the degradation of securin, cyclin B, and Xkid, whereas Cdh1 is required for the RCS1 Is a Substrate of the APC/C in Vitro. Two activators, Cdc20 and Cdh1, control the activity of APC/C in anaphase and from activation of APC/C during mitotic exit and in G1. Cdc20 and Cdh1 recognize two motifs in the substrates: the destruction box (D-box; mitotic exit to G1, respectively (1, 7). In vitro ubiquitination assay RxxL) and the KEN box. indicated that RCS1 was ubiquitinated by both APC/C-Cdc20 The temporal control of APC/C activation, and hence the and APC/C-Cdh1 (Fig. 2A), similar to securin and cyclin B (data anaphase onset, is under complex regulation (1). Upon mitotic entry, APC/C is phosphorylated by mitotic kinases, such as Plk1, Author contributions: W.-m.Z., A.S., X.-l.C., J.R.Y., and G.F. designed research; W.-m.Z., which is necessary but not sufficient for its activation. On the other J.A.C., A.S., and X.-l.C. performed research; W.-m.Z., J.A.C., A.S., X.-l.C., and J.R.Y. contrib- hand, the spindle checkpoint, which delays the anaphase onset until uted new reagents/analytic tools; W.-m.Z., J.A.C., A.S., X.-l.C., J.R.Y., and G.F. analyzed data; all of the chromosomes are aligned at the metaphase plate, prevents and W.-m.Z., A.S., and G.F. wrote the paper. the activation of APC/C through association of inhibitory check- The authors declare no conflict of interest. point proteins Mad2 and BubR1 with APC/C-Cdc20 (6). This This article is a PNAS Direct Submission. checkpoint mechanism ensures the fidelity of chromosome segre- ‡To whom correspondence should be addressed. E-mail: [email protected]. gation and determines the timing of anaphase onset. This article contains supporting information online at www.pnas.org/cgi/content/full/ Given the central role of the APC/C in mitotic regulation, it is 0709227105/DCSupplemental. CELL BIOLOGY essential to identify its substrates to gain a mechanistic understand- © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709227105 PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 ͉ 13415–13420 Downloaded by guest on October 1, 2021 Fig. 1. Substrates are specifically ubiquitinated by ECT2 RCS1 FLJ20345 APC/C-Cdh1 in vitro. iAPC/C was immunopurified from - Cdh1 + Cdh1 - Cdh1 + Cdh1 - Cdh1 + Cdh1 Xenopus interphase extracts and activated by in vitro translated Cdh1. APC/C-dependent ubiquitination of 35S-labeled substrates was analyzed in the presence of E1, E2, ubiquitin, ubiquitin-aldehyde, and ATP at indi- Ubiquitin cated times. The arrow points to the input substrates, Conjugates and the bracket indicates the position of ubiquitin conjugates. The extent of ubiquitination can be judged by the disappearance of the input substrates and by the formation of ubiquitin conjugates migrat- 0 15 30 60 0 15 30 60 0 15 30 60 0 15 30 60 0 15 30 60 0 15 30 60 (min) ing slower than the input substrates. not shown), whereas substrates degraded at the M-to-G1 tran- RCS1 protein stability during mitotic exit. The level of RCS1 was sition, such as Plk1, were efficiently ubiquitinated by APC/C- reduced before that of Cdh1 (Fig. 3A), which occurs in G1.To Cdh1, but weakly by APC/C-Cdc20 (Fig. 2A). determine the exact cell-cycle stage for RCS1 destruction, HeLa S3 RCS1 contains two putative D-boxes at amino acids 14–17 and cells were synchronized at prometaphase by a thymidine- 53–56, but no KEN box (Fig. 2B). The second D-box is conserved nocodazole block and then released into fresh media (Fig. 3B and among human, mouse, and Xenopus RCS1, whereas the first one is Fig. S2B). As expected, RCS1 levels were down-regulated during not. Mutation of the first D-box (DB1) (RxxL to AxxA) did not or mitotic exit, and the bulk of RCS1 was degraded after the destruc- only weakly affected the ubiquitination efficiency, whereas muta- tion of securin at anaphase but before the degradation of Plk1 in G1. tion of the second D-box (DB2) greatly reduced its ubiquitination To confirm that the APC/C-Cdh1 is required for the destruction by the APC/C-Cdh1 (Fig. 2C). Mutations of both D-boxes (DB1/2) of RCS1, we inhibited the activity of the APC/C-Cdh1 by RNAi.
Recommended publications
  • Mad1 Kinetochore Recruitment by Mps1-Mediated Phosphorylation of Bub1 Signals the Spindle Checkpoint
    Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint Nitobe London1,2 and Sue Biggins1,3 1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA; 2Molecular and Cellular Biology Program, University of Washington/Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA The spindle checkpoint is a conserved signaling pathway that ensures genomic integrity by preventing cell division when chromosomes are not correctly attached to the spindle. Checkpoint activation depends on the hierarchical recruitment of checkpoint proteins to generate a catalytic platform at the kinetochore. Although Mad1 kinetochore localization is the key regulatory downstream event in this cascade, its receptor and mechanism of recruitment have not been conclusively identified. Here, we demonstrate that Mad1 kinetochore association in budding yeast is mediated by phosphorylation of a region within the Bub1 checkpoint protein by the conserved protein kinase Mps1. Tethering this region of Bub1 to kinetochores bypasses the checkpoint requirement for Mps1-mediated kinetochore recruitment of upstream checkpoint proteins. The Mad1 interaction with Bub1 and kinetochores can be reconstituted in the presence of Mps1 and Mad2. Together, this work reveals a critical mechanism that determines kinetochore activation of the spindle checkpoint. [Keywords: kinetochore; spindle checkpoint; Mps1 kinase; Mad1; Bub1 kinase] Supplemental material is available for this article. Received October 25, 2013; revised version accepted December 13, 2013. Successful eukaryotic cell division requires accurate the conserved checkpoint proteins Mps1, Bub1, Bub3, chromosome segregation, which relies on correct attach- Mad1, and Mad2 as well as the Mad3 homolog BubRI in ments of spindle microtubules to chromosomes.
    [Show full text]
  • The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression
    International Journal of Molecular Sciences Review The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression Tingting Zou and Zhenghong Lin * School of Life Sciences, Chongqing University, Chongqing 401331, China; [email protected] * Correspondence: [email protected] Abstract: The cell cycle is a collection of events by which cellular components such as genetic materials and cytoplasmic components are accurately divided into two daughter cells. The cell cycle transition is primarily driven by the activation of cyclin-dependent kinases (CDKs), which activities are regulated by the ubiquitin-mediated proteolysis of key regulators such as cyclins, CDK inhibitors (CKIs), other kinases and phosphatases. Thus, the ubiquitin-proteasome system (UPS) plays a pivotal role in the regulation of the cell cycle progression via recognition, interaction, and ubiquitination or deubiquitination of key proteins. The illegitimate degradation of tumor suppressor or abnormally high accumulation of oncoproteins often results in deregulation of cell proliferation, genomic instability, and cancer occurrence. In this review, we demonstrate the diversity and complexity of the regulation of UPS machinery of the cell cycle. A profound understanding of the ubiquitination machinery will provide new insights into the regulation of the cell cycle transition, cancer treatment, and the development of anti-cancer drugs. Keywords: cell cycle regulation; CDKs; cyclins; CKIs; UPS; E3 ubiquitin ligases; Deubiquitinases (DUBs) Citation: Zou, T.; Lin, Z. The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression. 1. Introduction Int. J. Mol. Sci. 2021, 22, 5754. https://doi.org/10.3390/ijms22115754 The cell cycle is a ubiquitous, complex, and highly regulated process that is involved in the sequential events during which a cell duplicates its genetic materials, grows, and di- Academic Editors: Kwang-Hyun Bae vides into two daughter cells.
    [Show full text]
  • The APC/C in Female Mammalian Meiosis I
    REPRODUCTIONREVIEW The APC/C in female mammalian meiosis I Hayden Homer1,2 1Mammalian Oocyte and Embryo Research Laboratory, Cell and Developmental Biology, UCL, London WC1E 6BT, UK and 2Reproductive Medicine Unit, Institute for Women’s Health, UCLH Elizabeth Garrett Anderson Wing, London NW1 2BU, UK Correspondence should be addressed to H Homer; Email: [email protected] Abstract The anaphase-promoting complex or cyclosome (APC/C) orchestrates a meticulously controlled sequence of proteolytic events critical for proper cell cycle progression, the details of which have been most extensively elucidated during mitosis. It has become apparent, however, that the APC/C, particularly when acting in concert with its Cdh1 co-activator (APC/CCdh1), executes a staggeringly diverse repertoire of functions that extend its remit well outside the bounds of mitosis. Findings over the past decade have not only earmarked mammalian oocyte maturation as one such case in point but have also begun to reveal a complex pattern of APC/C regulation that underpins many of the oocyte’s unique developmental attributes. This review will encompass the latest findings pertinent to the APC/C, especially APC/CCdh1, in mammalian oocytes and how its activity and substrates shape the stop–start tempo of female mammalian first meiotic division and the challenging requirement for assembling spindles in the absence of centrosomes. Reproduction (2013) 146 R61–R71 Introduction associated somatic follicular compartment at the time of ovulation. Significantly, although primordial germ cells Meiosis is the unique cell division that halves the in the ovary commit to meiosis during fetal life, it is chromosome compliment by coupling two successive not until postnatal adulthood that mature oocytes (or nuclear divisions with a single round of DNA replication.
    [Show full text]
  • 1 Spindle Assembly Checkpoint Is Sufficient for Complete Cdc20
    Spindle assembly checkpoint is sufficient for complete Cdc20 sequestering in mitotic control Bashar Ibrahim Bio System Analysis Group, Friedrich-Schiller-University Jena, and Jena Centre for Bioinformatics (JCB), 07743 Jena, Germany Email: [email protected] Abstract The spindle checkpoint assembly (SAC) ensures genome fidelity by temporarily delaying anaphase onset, until all chromosomes are properly attached to the mitotic spindle. The SAC delays mitotic progression by preventing activation of the ubiquitin ligase anaphase-promoting complex (APC/C) or cyclosome; whose activation by Cdc20 is required for sister-chromatid separation marking the transition into anaphase. The mitotic checkpoint complex (MCC), which contains Cdc20 as a subunit, binds stably to the APC/C. Compelling evidence by Izawa and Pines (Nature 2014; 10.1038/nature13911) indicates that the MCC can inhibit a second Cdc20 that has already bound and activated the APC/C. Whether or not MCC per se is sufficient to fully sequester Cdc20 and inhibit APC/C remains unclear. Here, a dynamic model for SAC regulation in which the MCC binds a second Cdc20 was constructed. This model is compared to the MCC, and the MCC-and-BubR1 (dual inhibition of APC) core model variants and subsequently validated with experimental data from the literature. By using ordinary nonlinear differential equations and spatial simulations, it is shown that the SAC works sufficiently to fully sequester Cdc20 and completely inhibit APC/C activity. This study highlights the principle that a systems biology approach is vital for molecular biology and could also be used for creating hypotheses to design future experiments. Keywords: Mathematical biology, Spindle assembly checkpoint; anaphase promoting complex, MCC, Cdc20, systems biology 1 Introduction Faithful DNA segregation, prior to cell division at mitosis, is vital for maintaining genomic integrity.
    [Show full text]
  • Kinetochores, Microtubules, and Spindle Assembly Checkpoint
    Review Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling 1 1,2,3 Carlos Sacristan and Geert J.P.L. Kops 1 Molecular Cancer Research, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands 2 Center for Molecular Medicine, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands 3 Cancer Genomics Netherlands, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands Error-free chromosome segregation relies on stable and cell division. The messenger is the SAC (also known as connections between kinetochores and spindle microtu- the mitotic checkpoint) (Figure 1). bules. The spindle assembly checkpoint (SAC) monitors The transition to anaphase is triggered by the E3 ubiqui- such connections and relays their absence to the cell tin ligase APC/C, which tags inhibitors of mitotic exit cycle machinery to delay cell division. The molecular (CYCLIN B) and of sister chromatid disjunction (SECURIN) network at kinetochores that is responsible for microtu- for proteasomal degradation [2]. The SAC has a one-track bule binding is integrated with the core components mind, inhibiting APC/C as long as incorrectly attached of the SAC signaling system. Molecular-mechanistic chromosomes persist. It goes about this in the most straight- understanding of how the SAC is coupled to the kineto- forward way possible: it assembles a direct and diffusible chore–microtubule interface has advanced significantly inhibitor of APC/C at kinetochores that are not connected in recent years. The latest insights not only provide a to spindle microtubules. This inhibitor is named the striking view of the dynamics and regulation of SAC mitotic checkpoint complex (MCC) (Figure 1).
    [Show full text]
  • Bub1 Positions Mad1 Close to KNL1 MELT Repeats to Promote Checkpoint Signalling
    ARTICLE Received 14 Dec 2016 | Accepted 3 May 2017 | Published 12 June 2017 DOI: 10.1038/ncomms15822 OPEN Bub1 positions Mad1 close to KNL1 MELT repeats to promote checkpoint signalling Gang Zhang1, Thomas Kruse1, Blanca Lo´pez-Me´ndez1, Kathrine Beck Sylvestersen1, Dimitriya H. Garvanska1, Simone Schopper1, Michael Lund Nielsen1 & Jakob Nilsson1 Proper segregation of chromosomes depends on a functional spindle assembly checkpoint (SAC) and requires kinetochore localization of the Bub1 and Mad1/Mad2 checkpoint proteins. Several aspects of Mad1/Mad2 kinetochore recruitment in human cells are unclear and in particular the underlying direct interactions. Here we show that conserved domain 1 (CD1) in human Bub1 binds directly to Mad1 and a phosphorylation site exists in CD1 that stimulates Mad1 binding and SAC signalling. Importantly, fusion of minimal kinetochore-targeting Bub1 fragments to Mad1 bypasses the need for CD1, revealing that the main function of Bub1 is to position Mad1 close to KNL1 MELTrepeats. Furthermore, we identify residues in Mad1 that are critical for Mad1 functionality, but not Bub1 binding, arguing for a direct role of Mad1 in the checkpoint. This work dissects functionally relevant molecular interactions required for spindle assembly checkpoint signalling at kinetochores in human cells. 1 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Correspondence and requests for materials should be addressed to G.Z.
    [Show full text]
  • The Closed Form of Mad2 Is Bound to Mad1 and Cdc20 at Unattached Kinetochores
    bioRxiv preprint doi: https://doi.org/10.1101/305763; this version posted April 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. The closed form of Mad2 is bound to Mad1 and Cdc20 at unattached kinetochores. Gang Zhang1,2,3 and Jakob Nilsson1 1 The Novo Nordisk Foundation Center for Protein Research, Faculty of health and medical sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark 2 Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266061, China 3 Qingdao Cancer Institute, Qingdao, Shandong 266061, China For correspondence: [email protected] or [email protected] Keywords: Mad2, Cdc20, Kinetochore, Spindle Assembly Checkpoint, Mad1 1 bioRxiv preprint doi: https://doi.org/10.1101/305763; this version posted April 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. ABSTRACT The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying anaphase onset in response to unattached kinetochores. Anaphase is delayed by the generation of the mitotic checkpoint complex (MCC) composed of the checkpoint proteins Mad2 and BubR1/Bub3 bound to the protein Cdc20. Current models assume that MCC production is catalyzed at unattached kinetochores and that the Mad1/Mad2 complex is instrumental in the conversion of Mad2 from an open form (O-Mad2) to a closed form (C-Mad2) that can bind to Cdc20.
    [Show full text]
  • BUB3 That Dissociates from BUB1 Activates Caspase-Independent Mitotic Death (CIMD)
    Cell Death and Differentiation (2010) 17, 1011–1024 & 2010 Macmillan Publishers Limited All rights reserved 1350-9047/10 $32.00 www.nature.com/cdd BUB3 that dissociates from BUB1 activates caspase-independent mitotic death (CIMD) Y Niikura1, H Ogi1, K Kikuchi1 and K Kitagawa*,1 The cell death mechanism that prevents aneuploidy caused by a failure of the spindle checkpoint has recently emerged as an important regulatory paradigm. We previously identified a new type of mitotic cell death, termed caspase-independent mitotic death (CIMD), which is induced during early mitosis by partial BUB1 (a spindle checkpoint protein) depletion and defects in kinetochore–microtubule attachment. In this study, we have shown that survived cells that escape CIMD have abnormal nuclei, and we have determined the molecular mechanism by which BUB1 depletion activates CIMD. The BUB3 protein (a BUB1 interactor and a spindle checkpoint protein) interacts with p73 (a homolog of p53), specifically in cells wherein CIMD occurs. The BUB3 protein that is freed from BUB1 associates with p73 on which Y99 is phosphorylated by c-Abl tyrosine kinase, resulting in the activation of CIMD. These results strongly support the hypothesis that CIMD is the cell death mechanism protecting cells from aneuploidy by inducing the death of cells prone to substantial chromosome missegregation. Cell Death and Differentiation (2010) 17, 1011–1024; doi:10.1038/cdd.2009.207; published online 8 January 2010 Aneuploidy – the presence of an abnormal number of of spindle checkpoint activity.20,21
    [Show full text]
  • The Genome of Schmidtea Mediterranea and the Evolution Of
    OPEN ArtICLE doi:10.1038/nature25473 The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms Markus Alexander Grohme1*, Siegfried Schloissnig2*, Andrei Rozanski1, Martin Pippel2, George Robert Young3, Sylke Winkler1, Holger Brandl1, Ian Henry1, Andreas Dahl4, Sean Powell2, Michael Hiller1,5, Eugene Myers1 & Jochen Christian Rink1 The planarian Schmidtea mediterranea is an important model for stem cell research and regeneration, but adequate genome resources for this species have been lacking. Here we report a highly contiguous genome assembly of S. mediterranea, using long-read sequencing and a de novo assembler (MARVEL) enhanced for low-complexity reads. The S. mediterranea genome is highly polymorphic and repetitive, and harbours a novel class of giant retroelements. Furthermore, the genome assembly lacks a number of highly conserved genes, including critical components of the mitotic spindle assembly checkpoint, but planarians maintain checkpoint function. Our genome assembly provides a key model system resource that will be useful for studying regeneration and the evolutionary plasticity of core cell biological mechanisms. Rapid regeneration from tiny pieces of tissue makes planarians a prime De novo long read assembly of the planarian genome model system for regeneration. Abundant adult pluripotent stem cells, In preparation for genome sequencing, we inbred the sexual strain termed neoblasts, power regeneration and the continuous turnover of S. mediterranea (Fig. 1a) for more than 17 successive sib- mating of all cell types1–3, and transplantation of a single neoblast can rescue generations in the hope of decreasing heterozygosity. We also developed a lethally irradiated animal4. Planarians therefore also constitute a a new DNA isolation protocol that meets the purity and high molecular prime model system for stem cell pluripotency and its evolutionary weight requirements of PacBio long-read sequencing12 (Extended Data underpinnings5.
    [Show full text]
  • Transcriptional Recapitulation and Subversion Of
    Open Access Research2007KaiseretVolume al. 8, Issue 7, Article R131 Transcriptional recapitulation and subversion of embryonic colon comment development by mouse colon tumor models and human colon cancer Sergio Kaiser¤*, Young-Kyu Park¤†, Jeffrey L Franklin†, Richard B Halberg‡, Ming Yu§, Walter J Jessen*, Johannes Freudenberg*, Xiaodi Chen‡, Kevin Haigis¶, Anil G Jegga*, Sue Kong*, Bhuvaneswari Sakthivel*, Huan Xu*, Timothy Reichling¥, Mohammad Azhar#, Gregory P Boivin**, reviews Reade B Roberts§, Anika C Bissahoyo§, Fausto Gonzales††, Greg C Bloom††, Steven Eschrich††, Scott L Carter‡‡, Jeremy E Aronow*, John Kleimeyer*, Michael Kleimeyer*, Vivek Ramaswamy*, Stephen H Settle†, Braden Boone†, Shawn Levy†, Jonathan M Graff§§, Thomas Doetschman#, Joanna Groden¥, William F Dove‡, David W Threadgill§, Timothy J Yeatman††, reports Robert J Coffey Jr† and Bruce J Aronow* Addresses: *Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA. †Departments of Medicine, and Cell and Developmental Biology, Vanderbilt University and Department of Veterans Affairs Medical Center, Nashville, TN 37232, USA. ‡McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706, USA. §Department of Genetics and Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA. ¶Molecular Pathology Unit and Center for Cancer Research, Massachusetts deposited research General Hospital, Charlestown, MA 02129, USA. ¥Division of Human Cancer Genetics, The Ohio State University College of Medicine, Columbus, Ohio 43210-2207, USA. #Institute for Collaborative BioResearch, University of Arizona, Tucson, AZ 85721-0036, USA. **University of Cincinnati, Department of Pathology and Laboratory Medicine, Cincinnati, OH 45267, USA. ††H Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA. ‡‡Children's Hospital Informatics Program at the Harvard-MIT Division of Health Sciences and Technology (CHIP@HST), Harvard Medical School, Boston, Massachusetts 02115, USA.
    [Show full text]
  • BUB3 Antibody
    Efficient Professional Protein and Antibody Platforms BUB3 Antibody Basic information: Catalog No.: UPA63666 Source: Rabbit Size: 50ul/100ul Clonality: Monoclonal Concentration: 1mg/ml Isotype: Rabbit IgG Purification: Protein A affinity purified Useful Information: WB:1:500-1:2000 ICC:1:50-1:200 Applications: IHC:1:50-1:200 IP:1:10-1:50 FC:1:50-1:100 Reactivity: Human, Mouse, Rat Specificity: This antibody recognizes BUB3 protein. Immunogen: Recombinant protein corresponding to the N-terminus of human Bub3. BUB3 (budding uninhibited by benzimidazoles 3 homolog), also known as BUB3L or hBUB3, is a conserved component of the mitotic spindle assembly complex (MCC). It contains five WD repeat domains and forms cell cycle constitutive complexes with BUB1 and BUBR1. BUB3 is essential for the ki- netochore localization of BUB1 and BUBR1. As a component of the MCC, BUB3 is involved in the essential spindle checkpoint pathway that operates Description: during early embryogenesis. The spindle checkpoint pathway functions to postpone the initiation of anaphase until chromosomes are properly at- tached to the spindle. This acts to ensure accurate chromosome segrega- tion. In addition, BUB3 plays a role in regulating the establishment of cor- rect kinetochore-microtubule attachments. BUB3 is also thought to bind Tctex1L (or DYNLT3), a dynein light chain. Uniprot: O43684(Human) Q9WVA3(Mouse) BiowMW: 37 kDa Buffer: 1*TBS (pH7.4), 1%BSA, 40%Glycerol. Preservative: 0.05% Sodium Azide. Storage: Store at 4°C short term and -20°C long term. Avoid freeze-thaw cycles. Note: For research use only, not for use in diagnostic procedure. Data: Gene Universal Technology Co.
    [Show full text]
  • HDAC3: Taking the SMRT-N-Correct Road to Repression
    Oncogene (2007) 26, 5439–5449 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW HDAC3: taking the SMRT-N-CoRrect road to repression P Karagianni and J Wong1 Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA Known histone deacetylases (HDACs) are divided into repression when targeted to promoters, as well as different classes, and HDAC3 belongs to Class I. Through physical association with the DNA-binding factor forming multiprotein complexes with the corepressors YY1 (Yang et al., 1997; Dangond et al., 1998; Emiliani SMRT and N-CoR, HDAC3 regulates the transcription et al., 1998). Together, these findings suggested that this of a plethora of genes. A growing list of nonhistone ubiquitously expressed protein could be involved in the substrates extends the role of HDAC3 beyond transcrip- regulation of mammalian gene expression. On the other tional repression. Here, we review data on the composi- hand, HDAC3 contains an intriguingly variable C tion, regulation and mechanism of action of the SMRT/ terminus, with no apparent similarity with other N-CoR-HDAC3 complexes and provide several examples HDACs. This observation led to the hypothesis that of nontranscriptional functions, to illustrate the wide HDAC3 may have some unique properties and may variety of physiological processes affected by this deacety- not be completely redundant with the other HDACs lase. Furthermore, we discuss the implication of HDAC3 (Yang et al., 1997). This is also suggested by the in cancer, focusing on leukemia. We conclude with some differential localization of HDAC3, which, unlike the thoughts about the potential therapeutic efficacies of predominantly nuclear HDACs 1 and 2, can be found in HDAC3 activity modulation.
    [Show full text]