DOCSLIB.ORG

The Ski Oncoprotein Interacts with Skip, the Human Homolog of Drosophila Bx42

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Ski Oncoprotein Interacts with Skip, the Human Homolog of Drosophila Bx42 Oncogene (1998) 16, 1579 ± 1586 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 The Ski oncoprotein interacts with Skip, the human homolog of Drosophila Bx42 Richard Dahl, Bushra Wani and Michael J Hayman Department of Microbiology, State University of New York, Stony Brook, New York 11794, USA The v-Ski avian retroviral oncogene is postulated to act Little is known about how Ski functions. Both the as a transcription factor. Since protein ± protein interac- cellular and the viral genes share transforming and tions have been shown to play an important role in the muscle inducing ability. Ski localizes to the nucleus and transcription process, we attempted to identify Ski is observed to associate with chromatin (Barkas et al., protein partners with the yeast two-hybrid system. Using 1986; Sutrave et al., 1990a). Because of its nuclear v-Ski sequence as bait, the human gene skip (Ski localization and its ability to induce expression of Interacting Protein) was identi®ed as encoding a protein muscle speci®c genes in quail cells (Colmenares and which interacts with both the cellular and viral forms of Stavnezer, 1989), Ski has been assumed to be a Ski in the two-hybrid system. Skip is highly homologous transcription factor. Consistent with this assumption, to the Drosophila melanogaster protein Bx42 which is c-Ski has been shown to bind DNA in vitro with the found associated with chromatin in transcriptionally help of an unknown protein factor (Nagase et al., active pus of salivary glands. The Ski-Skip interaction 1990), and has been recently shown to enhance MyoD is potentially important in Ski's transforming activity dependent transactivation of a myosin light chain since Skip was demonstrated to interact with a highly enhancer linked reporter gene in muscle cells (Engert conserved region of Ski required for transforming et al., 1995). activity. Like Ski, Skip is expressed in multiple tissue Protein-protein interactions are an established types and is localized to the cell nucleus. mechanism of transcriptional regulation. As discussed above, Ski has been proposed to be involved in Keywords: Ski oncogene transcriptional regulation and presumably achieves this regulation by binding to DNA following interac- tion with an unknown protein(s). Thus in order to understand the mechanism of action of the Ski protein Introduction it will be necessary to identify these putative binding partners. In this report the yeast two-hybrid system The v-Ski oncogene was originally identi®ed as the was used to identify the human gene skip encoding a transforming protein of the SKV avian retroviruses Ski interacting protein which is highly homologous to which were isolated by their ability to transform avian the Drosophila melanogaster nuclear protein Bx42. ®broblasts in vitro (Stavnezer et al., 1981). V-Ski is truncated at the amino and carboxyl termini compared to the cellular proto-oncogene c-Ski. Ski has been Results cloned from several species including human, chicken, and Xenopus (Nomura et al., 1989; Sleeman and Identi®cation of a Ski binding partner Laskey, 1993; Sutrave et al., 1990a); and is the founding member of a family of proteins which To identify putative Ski binding proteins the yeast two- includes itself and the Ski related protein Sno (Boyer hybrid system was utilized. We chose to use Ski et al., 1993). sequence derived from v-Ski as bait since all identi®ed For an oncogene Ski has the unusual property of biological activity is shared between c-Ski and the being able to aect cellular proliferation, and also smaller v-Ski (Colmenares et al., 1991a; Sutrave et al., initiate dierentiation. Quail embryo ®broblasts infected 1990a). V-Ski sequence was cloned into the two-hybrid with a retrovirus expressing Ski dierentiate into GAL4 DNA binding vector pGBT9 and the resultant myoblasts capable of forming myotubes in culture plasmid was used to screen a previously constructed (Colmenares and Stavnezer, 1989), and transgenic mice two-hybrid library made from cDNA of EBV overexpressing a truncated c-Ski protein display transformed human lymphoid cells (Durfee et al., enlarged skeletal muscle mass compared to their normal 1993). litter mates (Sutrave et al., 1990b). Despite evidence for The yeast strain Y153 was used for the screening a role in muscle development, c-Ski is only expressed at since it allows for double selection using two GAL4 very low levels in normal skeletal muscle tissue, similar regulatable reporter genes: lacZ and HIS3. Colonies to its expression levels detected in other adult tissues positive for an interaction will result in a phenotype (Colmenares and Stavnezer, 1990; Nomura et al., 1989). that allows Y153 to grow on His7 media and express lacZ. Approximately 26106 yeast colonies were screened, and four colonies were isolated that were consistently His+ and lacZ+. The library plasmids from Correspondence: MJ Hayman these colonies were isolated and assayed to determine if Received 4 December 1996; revised 29 October 1997; accepted 29 the activation of the reporter genes was dependent on October 1997 the presence of the Ski sequence in the DNA binding Identification of a Ski interacting protein RDahlet al 1580 vector. The isolated plasmids were either transformed Identi®cation of the Skip binding domain of Ski into Y153 alone or co-transformed with one of the following: GBT9, VA3 (murine p53 aa's 70-390), or We were next interested in determining if Skip pGTB9-vSki. Three of the four isolated plasmids were associated with any of Ski's de®ned structural dependent on Ski for their activation of lacZ (data not domains. Ski has several putative domains including a shown). These three clones were shown also to interact proline rich region, two cysteine rich regions, two with the full length c-Ski in the two-hybrid system. putative amphipathic helices, a basic domain and a Subsequent analysis of the three isolated plasmids coiled-coil domain (Sleeman and Laskey, 1993; Stavne- revealed that they all contained the same 2.2 kb insert. zer et al., 1989). Two of these, the coiled-coil domain This insert was fully sequenced and found to code for a and the putative amphipathic helices domain have novel gene, skip (Ski Interacting Protein). Because the known functional activity. The coiled-coil domain has two-hybrid clone lacked an initiating methionine, a been shown to mediate homo-multimerization of Ski, RACE (Rapid Ampli®cation of cDNA ends) procedure and hetero-multimerization of Ski and the Ski related was used to isolate additional 5' sequence containing protein Sno (Heyman and Stavnezer, 1994; Nagase et an ATG surrounded by a minimal Kozak consensus al., 1993). By mutational analysis, the amphipathic sequence (Kozak, 1989). The composite nucleotide helices have been shown to be required for Ski sequence and deduced amino acid sequence of the transformation of ®broblasts and induction of quail skip clone from the two-hybrid and RACE search is ®broblasts into myoblasts (Colmenares et al., 1991b). shown in Figure 1. To identify potential Ski domains involved with A blast (Altschul et al., 1990) search of the mediating Skip interaction, Ski truncation mutants GenBank database demonstrated that Skip is highly were fused to the GAL4 DNA binding domain and homologous to the Drosophila melanogaster Bx42 assayed in the two-hybrid system using the originally protein. Skip and Bx42 share almost 60% identity isolated Skip-GAL4 activation domain plasmid. Ex- at the amino acid level. Recently other Skip homologous sequences have been deposited in GenBank from Caenorhabditis elegans, Schizosacchar- omyces pombe, and Dictyostelium discoideum; (Folk et a al., 1996) and Skip homologous expressed sequence tags (ESTs) have been identi®ed from mouse, Arabidopsis thaliana, and rice root. An amino acid alignment of Skip, Bx42 and the S. pombe Skip homolog is shown in Figure 2a. Additionally an essential gene from the yeast Saccharomyces cerevisiae, Fun20 (Diehl and Pringle, 1991; Harris et al., 1992), shares two regions of homology with Skip. An alignment of these two homologous regions with Skip is shown in Figure 2b. b Figure 2 (a) Alignment of the predicted amino acid sequences of Skip homologs from human, Drosophila, and S. pombe.(b) Alignment of Skip with two homologous regions from the S. Figure 1 Nucleotide sequence of human skip and predicted cerivisiae protein FUN20. Sequences were aligned using the amino acid (single letter code) sequence. Numbers on the left refer ClustalW algorithm. Capital letters in consensus sequence to nucleotide position, and numbers on the right refer to amino represent amino acids shared by all three sequences and lower acid position. (GenBank accession number U51432) case letters represent similar amino acids in all three sequences Identification of a Ski interacting protein RDahlet al 1581 pression of Ski fusion proteins was veri®ed by performed. C-Ski and v-Ski were expressed as immunoblot (data not shown). glutathione S-transferase (GST) fusion proteins in E. As shown in Figure 3, the last 566 carboxyl terminal coli. GST alone and GST-chicken SHP2 (SH2 domain amino acids of c-Ski were not required for Skip tyrosine phosphatase) were used as non-speci®c interaction (see mutant CT2). These amino acids controls in the binding reactions. Approximately 5 mg contain the coiled-coil domain, the Ski basic region, of each fusion protein bound to equivalent amounts of and one of Ski's cysteine rich regions. However, when glutathione agarose was incubated with an equivalent Ski was truncated to contain only the proline rich amount of cell lysate of COS1 cells transfected with a region Ski no longer could bind to Skip (CT3). When hemagglutinin (HA) epitope tagged version of Skip Ski was truncated at the amino end so that the proline (HA-Skip). After extensive washing proteins were rich region was deleted Ski still associated with Skip eluted and run on an SDS ± PAGE gel and Western (NT1), but when Ski was deleted further to remove the blotted.
Load more

Recommended publications
  • Mutations in SKI in Shprintzen–Goldberg Syndrome Lead to Attenuated TGF-B Responses Through SKI Stabilization
    RESEARCH ARTICLE Mutations in SKI in Shprintzen–Goldberg syndrome lead to attenuated TGF-b responses through SKI stabilization Ilaria Gori1, Roger George2, Andrew G Purkiss2, Stephanie Strohbuecker3, Rebecca A Randall1, Roksana Ogrodowicz2, Virginie Carmignac4, Laurence Faivre4, Dhira Joshi5, Svend Kjær2, Caroline S Hill1* 1Developmental Signalling Laboratory, The Francis Crick Institute, London, United Kingdom; 2Structural Biology Facility, The Francis Crick Institute, London, United Kingdom; 3Bioinformatics and Biostatistics Facility, The Francis Crick Institute, London, United Kingdom; 4INSERM - Universite´ de Bourgogne UMR1231 GAD, FHU-TRANSLAD, Dijon, France; 5Peptide Chemistry Facility, The Francis Crick Institute, London, United Kingdom Abstract Shprintzen–Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys–Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-b signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional co-repressor SKI, which is a negative regulator of TGF-b signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing, and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-b responses, both in knockin cells expressing an SGS mutation and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-b- *For correspondence: induced transcriptional responses, and not enhancement, which has important implications for [email protected] other Marfan-related syndromes. Competing interests: The authors declare that no competing interests exist.
    [Show full text]
  • The New Melanoma: a Novel Model for Disease Progression
    DISS. ETH NO. 17606 The new melanoma: A novel model for disease progression A dissertation submitted to SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZÜRICH for the degree of DOCTOR OF SCIENCES presented by Natalie Schlegel Master of Science, Otago University (New Zealand) born on January 20th 1976 citizen of Zürich (ZH) accepted on the recommendation of Professor Sabine Werner, examinor Professor Reinhard Dummer, co-examinor Professor Josef Jiricny, co-examinor 2008 22 Table of Contents Abstract ...................................................................................................................................... 6 Résumé....................................................................................................................................... 8 Abbreviations ........................................................................................................................... 10 1. Introduction ...................................................................................................................... 13 1.1 Definition ................................................................................................................. 14 1.2 Clinical features........................................................................................................ 14 1.3 Pathological features and staging............................................................................. 16 1.3.2 Clark’s level of invasion and Breslow’s thickness........................................... 16 1.3.3 TNM staging ...................................................................................................
    [Show full text]
  • The Role of Drosophila Bx42/SKIP in Cell Cycle
    The Role of Drosophila Bx42/SKIP in Cell Cycle D i s s e r t a t i o n zur Erlangung des akademischen Grades d o c t o r r e r u m n a t u r a l i u m (Dr. rer. nat.) im Fach Biologie eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin von Diplom-Biologin Shaza Dehne Präsidentin der Humboldt-Universität zu Berlin Prof. Dr.-Ing. Dr. Sabine Kunst Dekanin/Dekan der Lebenswissenschaftlichen Fakultät Prof. Dr. Richard Lucius Gutachter/innen: 1. Prof. Dr. Harald Saumweber 2. Prof. Dr. Christian Schmitz-Linneweber 3. Prof. Dr. Achim Leutz Tag der mündlichen Prüfung: 30.11.2016 Contents Abstract Abstrakt 1 Introduction .................................................................. 10 1.1 Bx42: Identification, protein structure and function .................................................. 11 1.2 Bx42/SNW/SKIP is an essential protein family, conserved in evolution and involved in several biological processes ............................................................................................ 13 1.2.1 Involvement of Bx42/SNW/SKIP in signaling pathways ................................ 16 1.2.1.1 Involvement in nuclear receptor pathways ................................................ 16 1.2.1.2 Involvement in the Notch signaling pathway ............................................ 17 1.2.1.3 Involvement in the TGF-ß/Dpp signal pathway ........................................ 19 1.2.2 Involvement of Bx42/SNW/SKIP in RNA splicing ......................................... 20 1.2.3 Bx42/SNW/SKIP protein family and cell cycle regulation .............................. 22 1.2.3.1 Short overview on cell cycle regulation. ................................................... 22 1.2.3.2 Evidence for Bx42/SNW/SKIP contribution to cell cycle regulation ....... 24 1.3 Drosophila eye imaginal disc: a model to study proliferation and cell cycle regulation during development.
    [Show full text]
  • (Cxxc5) a Thesis Submitted to the Graduate
    STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE CXXC-TYPE ZINC FINGER PROTEIN 5 (CXXC5) A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF MIDDLE EAST TECHNICAL UNIVERSITY BY GAMZE AYAZ ŞEN IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOLOGY NOVEMBER 2018 Approval of the thesis: STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE CXXC-TYPE ZINC FINGER PROTEIN 5 (CXXC5) submitted by GAMZE AYAZ ŞEN in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology Department, Middle East Technical University by, Prof. Dr. Halil Kalıpçılar Dean, Graduate School of Natural and Applied Sciences Prof. Dr. Orhan Adalı Head of Department, Biological Sciences Prof. Dr. Mesut Muyan Supervisor, Biological Sciences Dept., METU Examining Committee Members: Prof. Dr. A. Elif Erson Bensan Biological Sciences Dept., METU Prof. Dr. Mesut Muyan Biological Sciences Dept., METU Asst. Prof. Dr. Murat Alper Cevher Molecular Biology and Genetics Dept., Bilkent University Assoc. Prof. Dr. Nurcan Tunçbağ Informatics Institute, METU Asst. Prof. Dr. Onur Çizmecioğlu Molecular Biology and Genetics Dept., Bilkent University Date: 16.11.2018 I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work. Name, Last name: Gamze Ayaz Şen Signature: iv To All Women Who Pursue Their Dreams In Science v ABSTRACT STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE CXXC-TYPE ZINC FINGER PROTEIN 5 (CXXC5) Ayaz Şen, Gamze Ph.D., Biology Department Supervisor: Prof.
    [Show full text]
  • The SMAD-Binding Domain of SKI: a Hotspot for De Novo Mutations Causing Shprintzen&Ndash
    European Journal of Human Genetics (2015) 23, 224–228 & 2015 Macmillan Publishers Limited All rights reserved 1018-4813/15 www.nature.com/ejhg ARTICLE The SMAD-binding domain of SKI: a hotspot for de novo mutations causing Shprintzen–Goldberg syndrome Dorien Schepers1,20, Alexander J Doyle2,3,20,GretchenOswald2, Elizabeth Sparks2,LorethaMyers2,PatrickJWillems4, Sahar Mansour5, Michael A Simpson6,HelenaFrysira7, Anneke Maat-Kievit8,RickVanMinkelen8, Jeanette M Hoogeboom8, Geert R Mortier1, Hannah Titheradge9,LouiseBrueton9,LoisStarr10, Zornitza Stark11, Charlotte Ockeloen12, Charles Marques Lourenco13,EdBlair14, Emma Hobson15,JaneHurst16, Isabelle Maystadt17, Anne Destre´e17, Katta M Girisha18, Michelle Miller19,HarryCDietz2,3,BartLoeys1,20 and Lut Van Laer*,1,20 Shprintzen–Goldberg syndrome (SGS) is a rare, systemic connective tissue disorder characterized by craniofacial, skeletal, and cardiovascular manifestations that show a significant overlap with the features observed in the Marfan (MFS) and Loeys–Dietz syndrome (LDS). A distinguishing observation in SGS patients is the presence of intellectual disability, although not all patients in this series present this finding. Recently, SGS was shown to be due to mutations in the SKI gene, encoding the oncoprotein SKI, a repressor of TGFb activity. Here, we report eight recurrent and three novel SKI mutations in eleven SGS patients. All were heterozygous missense mutations located in the R-SMAD binding domain, except for one novel in-frame deletion affecting the DHD domain. Adding our new findings to the existing data clearly reveals a mutational hotspot, with 73% (24 out of 33) of the hitherto described unrelated patients having mutations in a stretch of five SKI residues (from p.(Ser31) to p.(Pro35)).
    [Show full text]
  • Uva-DARE (Digital Academic Repository)
    UvA-DARE (Digital Academic Repository) Inside out Behavioral phenotyping in genetic syndromes Mulder, P.A. Publication date 2020 Document Version Other version License Other Link to publication Citation for published version (APA): Mulder, P. A. (2020). Inside out: Behavioral phenotyping in genetic syndromes. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:25 Sep 2021 6 Further delineation of Malan syndrome Authors: Priolo M, Schanze D, Tatton-Brown K, Mulder PA, Tenorio J, Kooblall K, Acero IH, Alkuraya FS, Arias P, Bernardini L, Bijlsma EK, Cole T, Coubes C, Dapia I, Davies S, Di Donato N, Elcioglu NH, Fahrner JA, Foster A, González NG, Huber I, Iascone M, Kaiser AS, Kamath A, Liebelt J, Lynch SA, Maas SM, Mammì C, Mathijssen IB, McKee S, Menke LA, Mirzaa GM, Montgomery T, Neubauer D, Neumann TE, Pintomalli L, Pisanti MA, Plomp AS, Price S, Salter C, Santos-Simarro F, Sarda P, Segovia M, Shaw-Smith C, Smithson S, Suri M, Valdez RM, Van Haeringen A, Van Hagen JM, Zollino M, Lapunzina P, Thakker RV, Zenker M, Hennekam RC.
    [Show full text]
  • Roles of the NFI/CTF Gene Family in Transcription and Development
    Gene 249 (2000) 31–45 www.elsevier.com/locate/gene Review Roles of the NFI/CTF gene family in transcription and development Richard M. Gronostajski * Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation and Department of Biochemistry, 9500 Euclid Ave., Case Western Reserve University, Cleveland, OH 44195, USA Received 20 December 1999; received in revised form 15 February 2000; accepted 13 March 2000 Received by A.J. van Wijnen Abstract The Nuclear Factor I (NFI) family of site-specific DNA-binding proteins (also known as CTF or CAAT box transcription factor) functions both in viral DNA replication and in the regulation of gene expression. The classes of genes whose expression is modulated by NFI include those that are ubiquitously expressed, as well as those that are hormonally, nutritionally, and developmentally regulated. The NFI family is composed of four members in vertebrates (NFI-A, NFI-B, NFI-C and NFI-X ), and the four NFI genes are expressed in unique, but overlapping, patterns during mouse embryogenesis and in the adult. Transcripts of each NFI gene are diVerentially spliced, yielding as many as nine distinct proteins from a single gene. Products of the four NFI genes diVer in their abilities to either activate or repress transcription, likely through fundamentally diVerent mechanisms. Here, we will review the properties of the NFI genes and proteins and their known functions in gene expression and development. © 2000 Elsevier Science B.V. All rights reserved. Keywords: CAAT box transcription factor; Cancer; CTD; Gene expression; Nuclear Factor I 1. Introduction 2. Discovery of viral and cellular NFI-binding sites The Nuclear Factor I (NFI) family of site-specific After the initial observation that NFI protein isolated DNA-binding proteins plays wide reaching roles in from nuclear extracts of human HeLa cells greatly animal physiology, biochemistry and pathology.
    [Show full text]
  • Exosome-Mediated Recognition and Degradation of Mrnas Lacking A
    R EPORTS wild-type PGK1 mRNA was synthesized with a poly(A) tail of approximately 70 residues and Exosome-Mediated Recognition was subsequently deadenylated slowly (Fig. 2A) (12). In contrast, nonstop-PGK1 transcripts dis- and Degradation of mRNAs appeared rapidly without any detectable dead- enylation intermediates (Fig. 2B). In addition, in Lacking a Termination Codon a ski7⌬ strain, the nonstop mRNA persisted as a fully polyadenylated species for 8 to 10 min Ambro van Hoof,1,2* Pamela A. Frischmeyer,1,3 Harry C. Dietz,1,3 before disappearing (Fig. 2C). These data indi- Roy Parker1,2* cate that exosome function is required for rapid degradation of both the poly(A) tail and the One role of messenger RNA (mRNA) degradation is to maintain the fidelity of body of the mRNA. Based on these observa- gene expression by degrading aberrant transcripts. Recent results show that tions, we suggest that nonstop mRNAs are rap- mRNAs without translation termination codons are unstable in eukaryotic cells. idly degraded in a 3Ј-to-5Ј direction by the We used yeast mutants to demonstrate that these “nonstop” mRNAs are exosome, beginning at the 3Ј end of the poly(A) degraded by the exosome in a 3Ј-to-5Ј direction. The degradation of nonstop tail (13). transcripts requires the exosome-associated protein Ski7p. Ski7p is closely Two observations suggest a mechanism by related to the translation elongation factor EF1A and the translation termi- which nonstop mRNAs are specifically recog- nation factor eRF3. This suggests that the recognition of nonstop mRNAs nized and targeted for destruction by the exo- involves the binding of Ski7p to an empty aminoacyl-(RNA-binding) site (A site) some.
    [Show full text]
  • Mecp2 Nuclear Dynamics in Live Neurons Results from Low and High
    RESEARCH ARTICLE MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions Francesco M Piccolo1*, Zhe Liu2, Peng Dong2, Ching-Lung Hsu2, Elitsa I Stoyanova1, Anjana Rao3, Robert Tjian4, Nathaniel Heintz1* 1Laboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States; 2Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States; 3La Jolla Institute for Allergy and Immunology, La Jolla, United States; 4Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, CIRM Center of Excellence, University of California, Howard Hughes Medical Institute, Berkeley, United States Abstract Methyl-CpG-binding-Protein 2 (MeCP2) is an abundant nuclear protein highly enriched in neurons. Here we report live-cell single-molecule imaging studies of the kinetic features of mouse MeCP2 at high spatial-temporal resolution. MeCP2 displays dynamic features that are distinct from both highly mobile transcription factors and immobile histones. Stable binding of MeCP2 in living neurons requires its methyl-binding domain and is sensitive to DNA modification levels. Diffusion of unbound MeCP2 is strongly constrained by weak, transient interactions mediated primarily by its AT-hook domains, and varies with the level of chromatin compaction and cell type. These findings extend previous studies of the role of the MeCP2 MBD in high affinity DNA binding to living neurons, and identify a new role for its AT-hooks domains as critical determinants of its kinetic behavior. They suggest that limited nuclear diffusion of MeCP2 in live neurons contributes to its local impact on chromatin structure and gene expression.
    [Show full text]
  • The Role of Mecp2 in Learning and Memory
    Downloaded from learnmem.cshlp.org on August 21, 2019 - Published by Cold Spring Harbor Laboratory Press Review The role of MeCP2 in learning and memory Holly A. Robinson and Lucas Pozzo-Miller Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama 35294, USA Gene transcription is a crucial step in the sequence of molecular, synaptic, cellular, and systems mechanisms underlying learning and memory. Here, we review the experimental evidence demonstrating that alterations in the levels and function- ality of the methylated DNA-binding transcriptional regulator MeCP2 are implicated in the learning and memory deficits present in mouse models of Rett syndrome and MECP2 duplication syndrome. The significant impact that MeCP2 has on gene transcription through a variety of mechanisms, combined with well-defined models of learning and memory, make MeCP2 an excellent candidate to exemplify the role of gene transcription in learning and memory. Together, these studies have strengthened the concept that precise control of activity-dependent gene transcription is a fundamental mech- anism that ensures long-term adaptive behaviors necessary for the survival of individuals interacting with their congeners in an ever-changing environment. The roles of gene transcription and mRNA translation in learning DNA-associated histones, which leads to transient or enduring and memory throughout the animal kingdom have been exten- regulation of gene transcription without changes in the gene cod- sively and very well defined over the past five decades. Studies in- ing sequence itself. The most common occurrence of epigenetic hibiting gene transcription demonstrate its necessity for learning modification is during the differentiation of specific cell types by and memory in both invertebrates and vertebrates (Brink et al.
    [Show full text]
  • Elucidation of the Pathogenesis
    ELUCIDATION OF THE PATHOGENESIS OF TGF-BETA VASCULOPATHIES IDENTIFIES NOVEL THERAPEUTIC STRATEGIES by Jefferson J. Doyle A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland October 2015 © 2015 Jefferson J. Doyle All Rights Reserved ABSTRACT Marfan syndrome (MFS) is one of several related disorders that is driven by increased TGFβ signaling. While TGFβ can stimulate both canonical (Smad2/3) and noncanonical (MAPK; ERK, JNK, p38) cascades, this work has identified a novel and critical role for ERK1/2 activation in aortic aneurysm pathogenesis in Marfan mice, while inhibition of ERK1/2 activation represents a novel therapeutic strategy for the disorder. Angiotensin II influences aortic growth in Marfan mice, but the contribution of its type 1 receptor (AT1R) and type 2 receptor (AT2R) was unknown. This has direct clinical relevance, as the relative therapeutic merits of selective AT1R blockade using losartan versus inhibition of signaling through both receptors using the ACE inhibitor enalapril remained unknown. We find that losartan shows superior efficacy to enalapril in Marfan mice, this is mediated by their differential effects on ERK1/2 activation, and losartan’s full protective effect requires intact AT2R signaling. Calcium channel blockers (CCBs) are used in Marfan patients, although evidence for their efficacy is limited. Surprisingly, we find that CCBs accelerate aortic growth, dissection and premature lethality in Marfan mice, while Marfan patients taking CCBs show increased odds for aortic dissection and need for aortic surgery. CCBs enhance ERK1/2 activation via PKCβ, while PKCβ inhibition using enzastaurin or the clinically available agent hydralazine rescues aortic growth and ERK1/2 activation in Marfan mice, and hence represents a novel therapeutic strategy for the disorder.
    [Show full text]
  • Figures S1&S2 3-08
    Table S5 Transgenerational Female Hippocampus Regulated Genes Apoptosis Sample F3-Cont F3-Vinc Vin/Con Gene Symbol Raw Raw Ratio Genbank Gene Title Bag3 163 103 0.63 AI231792 Bcl2-associated athanogene 3 Cbl27 16 160 9.99 AF275151 androgen receptor-related apoptosis-associated protein CBL27 Dapk1_predicted 55 149 2.69 BI281823 death associated protein kinase 1 (predicted) Faim2 278 425 1.53 AF044201 Fas apoptotic inhibitory molecule 2 Pdcd6ip 457 706 1.54 BF389407 programmed cell death 6 interacting protein RGD1306214_predicted19 100 5.32 BE107277 similar to TGF-beta induced apotosis protein 2 (predicted) Tegt 358 559 1.56 NM_019381 testis enhanced gene transcript (Bax inhibitor-1) Cell Cycle Sample F3-Cont F3-Vinc Vin/Con Gene Symbol Raw Raw Ratio Genbank Gene Title Ccnd1 66 176 2.67 BI295861 cyclin D1 Ccnd2 41 546 13.21 L09752 cyclin D2 Ccnd3 87 139 1.60 NM_012766 cyclin D3 Cdc2l5 65 153 2.37 AI170800 cell division cycle 2-like 5 (cholinesterase-related cell division F3-Contler) Cdc2l6_predicted 275 480 1.75 BG376309 cell division cycle 2-like 6 (CDK8-like) (predicted) Cdkn1c 66 105 1.59 AI013919 cyclin-dependent kinase inhibitor 1C (P57) Ddb1 53 245 4.58 AJ277077 damage-specific DNA binding protein 1 Dab2 117 207 1.77 NM_024159 disabled homolog 2 (Drosophila) Gas6 156 541 3.47 NM_057100 growth arrest specific 6 Numa1 100 168 1.67 AI599394 nuclear mitotic apparatus protein 1 RGD1560358_predicted75 381 5.11 AW534965 similar to cell division cycle and apoptosis regulator 1 (predicted) Unc5b 54 108 1.98 BI303989 Unc-5 homolog B (C.
    [Show full text]