Human Poly(ADP-Ribose) Polymerase-1-Expressing Embryonic Stem Cells and Mice:
Generation and Phenotypic Characterization
Dissertation
Zur Erlangung des akademischen Grades
des Doktors der Naturwissenschaften (Dr. rer. nat.) des Fachbereichs Biologie der Universität Konstanz
Vorgelegt von Aswin Mangerich
Konstanz, Mai 2008
Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/6626/
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-66260
Tag der mündlichen Prüfung: 14. Juli 2008
Referenten: Prof. Dr. Alexander Bürkle (Gutachter und Prüfer)
Prof. Dr. Christof Hauck (Gutachter und Prüfer)
Prof. Dr. Thomas von Zglinicki (Prüfer)
Parts of this thesis have been published or are in preparation:
A. Mangerich, H. Scherthan, J. Diefenbach, U. Kloz, F. van der Hoeven, S. Beneke, and A. Bürkle,
“A Caveat in Mouse Genetic Engineering: Ectopic Gene Targeting in ES Cells by Bidirectional Extension of the Homology Arms of a Gene Replacement Vector Carrying Human PARP-1”, submitted
A. Mangerich, O. Popp, J. Diefenbach, U. Kloz, F. van der Hoeven, and A. Bürkle, “Pathological Phenotype and Impaired Survival in a Mouse Model With Ectopic Expression of Human PARP-1”, in preparation
Oral presentations at scientific conferences:
04/2006 Annual Meeting of the “Deutsche Gesellschaft für experimentelle und klinische Pharmakologie und Toxikologie” (DGPT), Mainz, Germany
05/2006 3rd PARP Regio Meeting, Strasbourg, France
09/2006 8th Congress of the “Deutschen Gesellschaft für Gerontologie und Geriatrie“
(DGGD), Freiburg, Germany
05/2007 Spring Meeting of doctoral students of the German National Academic Foundation (“Studienstiftung des deutschen Volkes”), Wittenberg, Germany
03/2008 Annual Meeting of the “Deutsche Gesellschaft für experimentelle und klinische Pharmakologie und Toxikologie” (DGPT), Mainz, Germany
Poster presentations at scientific conferences:
07/2006 8th International Symposium on Neurobiology and Neuroendocrinology of Aging, Bregenz, Austria
09/2006 9th Biennial Meeting (DNA Repair 2006) of the “Deutsche Gesellschaft für DNA Reparaturforschung“ (DGDR), Hamburg, Germany
03/2007 Annual Meeting of the “Deutsche Gesellschaft für experimentelle und klinische Pharmakologie und Toxikologie” (DGPT), Mainz, Germany
09/2007 Gordon Research Conference of Aging and MiMage/Link-AGE Summer School, Les Diablerets, Switzerland
05/2008 PARP 2008, 17th International Symposium on Poly(ADP-ribosyl)ation, Tucson, USA
Awards:
09/2007 Poster award at the MiMage/Link-AGE Summer School, Les Diablerets, Switzerland
Courses within the teaching program of the DFG-funded International Research Training Group 1331, “Cell-based Characterization of Disease Mechanisms in Tissue Destruction and Repair”
02/2005 20th Introductory Course to Clinical Development, Altana Pharma AG, Clinical Development Department, Konstanz, Germany
06/2005 Scientific and Technical Writing, University of Konstanz/Altana Pharma AG Medical Writing Department, Germany
10/2005 Handling of Laboratory Animals (in accordance with FELASA guidelines), University of Konstanz, Animal Care Facility, Konstanz, Germany
11/2005 Generation of Transgenic Mice, German Cancer Research Institute (DKFZ), Transgenic Core Facility, Heidelberg, Germany
11/2006 “Atemwegsforschung, Biopharmaceutical Process Science, Leitstrukturfindung, Nichtklinische Arzneimittelsicherheit“, Boehringer Ingelheim Pharma, Biberach, Germany
02/2007 Presentation Techniques, BioScript International/University of Konstanz, Konstanz, Germany
05/2007 27th Blankenese Conference on Routes to Therapy: From Stem Cell Tailoring to Nano Knitting, Hamburg, Germany
07/2007 DiMI (diagnostic molecular imaging) training: Optical Imaging of Reporter Gene Activity in Transgenic Mice, University of Oslo, Oslo, Norway
08/2007 Aspects of Clinical Cancer Therapy, Norwegian Radium Hospital, Oslo, Norway
11/2007 Alternative Methods to Replace Animal Experiments in Regulatory Toxicology:
Validation Procedures and New Developments in Europe, European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy
Danksagung
Herr Prof. Alexander Bürkle hat mein Interesse am Gebiet der Poly(ADP-Ribose) und Altersforschung geweckt und mir die Bearbeitung dieses spannenden Themas anvertraut. Nicht zuletzt hat seine fachliche, finanzielle und moralische Unterstützung wesentlich zum Gelingen dieser Arbeit beigetragen. Ihm möchte ich meinen ganz besonderen Dank aussprechen.
Herrn Prof. Christof Hauck danke ich herzlich für die freundliche Erstellung des Zweitgutachtens.
Der Studienstiftung des deutschen Volkes, insbesondere Dr. Matthias Frenz, Prof. Jochen Glöckner und Dr. Hans-Ottmar Weyand danke ich für das entgegengebrachte Vertrauen und für die unbürokratische finanzielle Unterstützung, die diese Arbeit ermöglichte.
Dem Internationalen Graduiertenkolleg Konstanz-Zürich, insbesondere Dr. Jutta Schlepper- Schäfer und Prof. Albrecht Wendel danke ich für die persönliche und finanzielle Unterstützung, sowie allen Kollegiaten für die freundschaftliche Begleitung der Promotionszeit.
Bei Katharina Hüttner, Sabine Lehmann, Oliver Popp und Gudrun von Scheven möchte ich mich für die sehr gute, unterhaltsame und freundschaftliche Zusammenarbeit bedanken.
Dem Team der TFA, insbesondere Heidi Henseleit, Klaus Hermann, Dr. Gerald Mende, Birgitt Planitz, und Dr. Dieter Schopper danke ich für die Hilfe bei Tierpflege und Haltung, veterinär- medizinischen Fragen und „Antragswesen“.
Dr. Jörg Diefenbach danke ich für die Vorarbeiten zu diesem Projekt und sein fortlaufendes Interesse am Fortschritt der Arbeit.
Unseren Kooperationspartnern Oliver Bruns, Ulrich Kloz, Dr. Andreas Niemeier, Prof. Harry Scherthan, Frank van der Hoeven und Prof. Rüdiger Wanke danke ich für die produktive und lehrreiche Zusammenarbeit.
Ein herzliches Dankeschön gilt Dr. Sascha Beneke, Dr. Jörg Fahrer, Dr. Dominik Geiger, Andrea Kunzmann und Jens Lutz für viele anregende und bereichernde wissenschaftliche und nicht-wissenschaftliche Diskussionen.
Bei Claudia Hoffmann und Thilo Sindlinger möchte ich mich herzlich für ihre Hilfe bei allen organisatorischen und EDV-Fragen bedanken.
Der gesamten Arbeitsgruppe Molekulare Toxikologie danke ich für die stete Hilfsbereitschaft und die angenehme Arbeitsatmosphäre. Bedanken möchte ich mich hierbei bei Dr. Malgorzata Debiak, Tobias Eltze, Benjamin Hanf, Daniela Hermann, Nina Kaczmarek, Joachim Kienhöfer, Dr. Muriel Marlaise, Dr. Elisa May, Dr. Maria Moreno-Villanueva, Elisabeth Müßig, Rebecca Steinhaus, Christine Strasser, Clara Tandler, Nathalie Veith und Kathrin Weidele.
Ein ganz besonderes Dankeschön gebührt meiner Familie für den uneingeschränkten Rückhalt in allen Lebenslagen.
Zu guter Letzt und doch an erster Stelle möchte ich mich herzlich bei meiner Frau Carole bedanken, für all ihre Unterstützung und viele schöne Jahre.
Summary
Poly(ADP-ribose) polymerase-1 (PARP-1) uses NAD+ as a substrate to modify various nuclear proteins with the biopolymer poly(ADP-ribose), thereby regulating a variety of cellular processes such as DNA repair, gene transcription, and cell death. These diverse functions on the cellular level are also reflected in the contribution of PARP-1 to multiple physiological and pathophysiological conditions on an organismal level.
While several groups have established PARP-1-deficient mice, PARP-1-overexpressing or hypermorphic mice have not been described to date. The latter, however, should also represent a relevant biological model, the rationale being provided by the fact that the poly(ADP- ribosyl)ation capacity of purified human PARP-1 (hPARP-1) is significantly higher than that of its rodent (rat) orthologue. In this thesis, a novel mouse model with ectopic expression of hPARP-1 (comprising two independent congenic lines) was generated, using gene targeting in embryonic stem (ES) cells. The targeting vector was designed to allow replacement of the murine Parp-1 (mParp-1) coding sequence (32 kb) with its human orthologous sequence (46 kb). Unexpectedly though, site-specific homologous recombination was mimicked by bidirectional extension of the vector homology arms, followed by adjacent integration of the targeting vector, thus leaving the murine locus functional. Related to this phenomenon is the so- called „ectopic gene targeting‟ mediated by synthesis-dependent strand annealing (SDSA), which has so far only been described for 'ends-in‟ integration vectors in non-ES cell gene targeting. Therefore, results of this thesis give new insight into the role of SDSA during gene targeting and are of general importance for the design of gene knock-in approaches in mice.
Mutant hPARP-1 ES cells and mice displayed gene-dose-dependent expression levels of hPARP-1, thereby resulting in a moderate overexpression of total PARP-1, while mPARP-1 expression was downregulated to some extent. Consequently, hPARP-1 ES cells exhibited an altered poly(ADP-ribosyl)ation metabolism, but an intact DNA damage response. Phenotypic analyses revealed impaired survival rates in a gene-dose-dependent manner in both sexes of hPARP-1 mice, with females being more affected. Several pathologies were identified in hPARP-1 mice, such as obesity, glomerulopathy, and splenomegaly, all pointing to the development of chronic diseases. Moreover, hPARP-1 mice showed signs of premature aging, such as sporadic kyphosis accompanied by alterations in bone metabolism and impaired regenerative potential of the hair.
In conclusion, this study characterized the occurrence of ectopic gene targeting in murine ES cells transfected with an „ends-out‟ gene replacement vector for the first time. Furthermore, the generated hPARP-1 mice represent a novel model system with unexpected, multifaceted phenotypes, which should be instrumental for the elucidation of the role of PARP-1 in health and disease.
Zusammenfassung
Das Enzym Poly(ADP-Ribose) polymerase-1 (PARP-1) verwendet NAD+ als Substrat, um etliche Zellkernproteine mit dem Biopolymer Poly(ADP-Ribose) zu modifizieren.
Dadurch beeinflusst die PARP-1 eine Vielzahl von zellulären Funktionen, wie z.B. die DNA Reparatur, Gentranskription und Zelltodregulierung. Aufgrund dieser diversen Funktionen auf zellulärer Ebene ist die PARP-1 an mehreren physiologischen sowie pathophysiologischen Prozessen im Organismus beteiligt.
Im Gegensatz zu PARP-1-defizienten Mäusen wurden auf dem Gebiet der Poly(ADP- Ribose) Forschung bislang weder PARP-1 überexprimierende transgene Mäuse noch hypermorphe Mausmutanten beschrieben. Jedoch könnte der Umstand, dass die humane PARP-1 (hPARP-1) im Vergleich zur Nagetier-PARP-1 (Ratte) eine deutlich erhöhte Poly(ADP-ribosyl)ierungskapazität aufweist, dazu ausgenutzt werden, um hypermorphe PARP-1 Knock-in Mäuse zu generieren. In der vorliegenden Arbeit wurde ein neues Mausmodel mit ektopischer Expression der hPARP-1 etabliert. Unter Verwendung der Gene Targeting Technologie in embryonalen Stammzellen (ES-Zellen) wurden dabei zwei unabhängige congene Mauslinien generiert. Das hierbei verwendete Targeting Vektor- Konstrukt sollte einen Austausch der endogenen murinen Parp-1 (mParp-1) kodierenden Sequenz (32 kb) mit der humanen orthologen Sequenz (46 kb) ermöglichen.
Unerwarteterweise wurde durch eine intrazelluläre bidirektionale Verlängerung der homologen Vektor-Sequenzen und die anschließende Integration des Vektors in benachbarte chromosomale Bereiche eine ortsspezische homologe Rekombination des Targeting Vektors vorgetäuscht. Folglich blieb der endogene mPARP-1 Genlokus funktionell intakt. Ein ähnliches Phänomen ist als „ektopisches Gene Targeting“ bekannt, welches durch den zellulären Prozess des Synthesis-dependent Strand Annealing (SDSA) vermittelt wird. Dieses ektopische Gene Targeting wurde bislang ausschließlich bei der Verwendung von „Ends-in“ Integrationskonstrukten, und nicht in ES-Zellen beschrieben.
Die vorliegende Arbeit vermittelt daher einen Einblick in die Rolle des SDSA während des Gene Targeting Prozesses, was für die Konzeption zukünftiger Gen-Knock-in Strategien in der Maus von allgemeiner Bedeutung ist.
Genetisch veränderte hPARP-1 ES-Zellen und Mäuse exprimierten die hPARP-1 in Abhängigkeit von der hPARP-1-Gen-Dosis. Dies führte zu einer moderaten Überexpression der Menge an Gesamt-PARP-1, wobei die Expression der endogenen mPARP-1 erniedrigt war. Folglich zeigten hPARP-1 ES Zellen einen veränderten Poly(ADP-Ribose) Metabolismus bei gleichbleibender Überlebensrate nach gen- toxischen Stimuli. In Abhängigkeit von der hPARP-1 Gen-Dosis wiesen genetisch
veränderte Mäuse eine beeinträchtigte Überlebensrate auf, wobei der Effekt bei Weibchen stärker ausgeprägt war. Mehrere pathologische Veränderungen, insbesondere Adipositas, Glomerulopathie und eine Vergrößerung der Milz, konnten bei hPARP-1 Tieren festgestellt werden, was auf die Ausbildung eines chronisch pathologischen Phänotyps schließen lässt. Diese Hypothese wird gestützt durch Anzeichen auf vorzeitige Alterung bei hPARP-1-exprimierenden Tieren wie dem sporadischen Auftreten einer Kyphose, welche mit Veränderungen des Knochenmetabolismus einherging, sowie einem verminderten regenerativen Fellwachstum.
Die vorliegende Arbeit beschreibt und charakterisiert erstmalig „ektopisches Gene Targeting“ in murinen ES-Zellen, die mit einem „Ends-out“ Genaustauschvektor transfiziert wurden. Weiterhin zeigten die hierbei generierten hPARP-1-expremierende Mäuse unerwartete und vielschichtige phänotypische Veränderungen, welche die Grundlage für ein besseres Verständnis der PARP-1 in physiologischen und pathophysiologischen Prozessen bilden.
Contents
1 INTRODUCTION 1
1.1 POLY(ADP-RIBOSE)POLYMERASE-1 AND POLY(ADP-RIBOSYL)ATION 1
1.1.1 NAD+ and Poly(ADP-Ribose) Metabolism 1
1.1.1.1 NAD+ Metabolism 1
1.1.1.2 Poly(ADP-Ribose) Metabolism 3
1.1.2 Poly(ADP-Ribose) Polymerase-1 5
1.1.2.1 Comparison of Human and Rodent PARP-1 6
1.1.2.2 Regulation of PARP-1 Gene Expression 6
1.1.2.3 Structural and Functional Aspects of PARP-1 7
1.1.2.4 Regulation of PARP-1 Activity 8
1.1.2.5 Cellular Functions of PARP-1 9
1.1.2.5.1 PARP-1 and Chromatin Modification 9
1.1.2.5.2 PARP-1 and Genomic Maintenance 10
1.1.2.5.2.1 PARP-1 and its Interaction Partner PARP-2 in Base Excision Repair 12
1.1.2.5.2.2 PARP-1 in Nucleotide Excision Repair 13
1.1.2.5.2.3 PARP-1 in Double Strand Break Signaling 13
1.1.2.5.2.4 PARP-1, Telomeric Maintenance, and the WRN Protein 14
1.1.2.5.2.5 PARP-1 and Mitotic Regulation 15
1.1.2.5.2.6 PARP-1, P53, and Cell Cycle Control 15
1.1.2.5.3 PARP-1, NF-κB, and Transcription 16
1.1.2.5.4 PARP-1 and its Role in Cell Death 17
1.1.2.5.5 Concluding Remarks on the Cellular Functions of PARP-1 19
1.1.2.6 The Role of PARP-1 in Physiology and Disease 20
1.1.2.6.1 Insights from Mouse Models 20
1.1.2.6.2 The Role of PARP-1 in Aging and Cancer 22
1.1.2.6.3 Pharmacological Inhibition of PARP-1 24
1.2 GENE TARGETING AND SITE-SPECIFIC HOMOLOGOUS RECOMBINATION 24
1.2.1 Homologous Recombination 24
1.2.2 Gene Targeting in Murine Embryonic Stem Cells 26
2 MOTIVATION AND OBJECTIVE 27
3 MATERIALS AND METHODS 28
3.1 MATERIALS 28
3.1.1 Chemicals and Reagents 28
3.1.2 Laboratory Equipment 30
3.1.3 Buffers and Solutions 32
3.1.4 Plasmids 34
3.1.5 Oligonucleotides 34
3.1.6 Molecular Biological Kits 37
3.1.7 Molecular Weight Standards 37
3.1.8 Enzymes 38
3.1.9 Antibodies 39
3.1.10 Cell Culture 39
3.1.10.1 Cells 39
3.1.10.2 Cell Culture Reagents 40
3.1.11 Organisms 41
3.1.12 Cooperation Partners and Service Providers 41
3.1.13 Software 42
3.2 METHODS 42
3.2.1 Cell Culture 42
3.2.1.1 Embryonic Stem Cell Culture 42
3.2.1.1.1 Cell Number Determination 43
3.2.1.1.2 Coating of Cell Culture Plates with Gelatin Solution 43
3.2.1.1.3 Thawing and Plating of Murine Embryonic Fibroblasts and „Feeder‟ Cells 43 3.2.1.1.4 Mitotic Inactivation of Murine Embryonic Fibroblasts and Cryoconservation 43
3.2.1.1.5 Thawing of ES Cells 44
3.2.1.1.6 Cryopreservation of ES Cells 44
3.2.1.1.7 Cell Culture and Passaging of ES Cells 44
3.2.1.1.8 Mycoplasma testing of ES Cell Lines 45
3.2.1.2 Alkaline Phosphatase Detection 45
3.2.1.3 Cell Proliferation Assay and Population Doubling Time 45
3.2.1.4 Excision of the Neomycin Resistance Cassette 45
3.2.1.5 Preparation of Primary Splenocytes 46
3.2.2 Molecular Biological and Biochemical Methods 47
3.2.2.1 Cloning and Microbiology 47
3.2.2.1.1 Cloning of the Human PARP-1 Targeting Vector 47
3.2.2.1.2 Preparation of pturbo-Cre Plasmid DNA for Transfection of ES Cells 47 3.2.2.1.3 Cloning of an mParp-1-specific Fluorescence In Situ Hybridization Probe 47
3.2.2.2 DNA Isolation 48
3.2.2.2.1 DNA Isolation From ES Cells 48
3.2.2.2.2 DNA Isolation From Hair Biopsies 48
3.2.2.2.3 DNA Isolation From Tail Biopsies 48
3.2.2.2.4 DNA Isolation From Mouse Kidney Tissue 49
3.2.2.2.5 Determination of DNA Quantity and Purity 49
3.2.2.3 Southern Blot Analysis 49
3.2.2.3.1 Preparation of ES Cell DNA 49
3.2.2.3.1.1 RNase digestion 49
3.2.2.3.1.2 Phenol-Chloroform Extraction of DNA 49
3.2.2.3.2 DNA Digestion, Gel Electrophoresis, and Blotting Procedure 50
3.2.2.3.3 Generation of Hybridization-probes 51
3.2.2.3.4 Hybridization of Membranes 52
3.2.2.4 Genotyping 52
3.2.2.5 Agarose Gel Electrophoresis 52
3.2.2.6 Determination of Gene Copy Numbers 53
3.2.2.7 Preparation of Metaphase Chromosomes and Karyotyping 55
3.2.2.8 Fluorescence In Situ Hybridization Analyses 55
3.2.2.8.1 Generation of Hybridization Probes 55
3.2.2.8.2 Labeling of Probes, Hybridization, and Data Evaluation. 56
3.2.2.9 RNA Isolation 56
3.2.2.10 Reverse Transcription PCR 56
3.2.2.11 Protein Extraction 57
3.2.2.11.1 From ES Cells 57
3.2.2.11.2 From Mouse Tissue 57
3.2.2.11.3 Determination of Protein Concentration 57
3.2.2.12 SDS-Polyacrylamide Gel Electrophoresis 57
3.2.2.13 Western Blotting 58
3.2.2.14 Generation of Polyclonal Rabbit-anti-murine-PARP-1 Antibody 59
3.2.2.15 Immunofluorescence Analysis 60
3.2.2.15.1 Poly-L-lysine-coating of Coverslips 60
3.2.2.15.2 PARP-1 Immunofluorescence Staining 60
3.2.2.15.3 Immunofluorescence Microscopy 60
3.2.2.16 PARP Activity Measurements 60
3.2.2.16.1 Flow Cytometry Analysis 60
3.2.2.16.1.1 Preparation and Fixation of Cells 60
3.2.2.16.1.2 Poly(ADP-Ribose) Immunofluorescence Staining 61
3.2.2.16.1.3 Flow Cytometry 62
3.2.2.16.2 Immunofluorescence Analysis of Cellular Poly(ADP-Ribose) Formation 62
3.2.2.16.3 NAD+ Cycling Assay 63
3.2.2.17 Clonogenic Survival Assay 64
3.2.3 Generation of Human PARP-1 ES Cells and Mice 64
3.2.3.1 Gene Targeting 64
3.2.3.2 ES Cell Injection and Production of Chimeras. 64
3.2.4 Animal Husbandry 65
3.2.5 Breeding Strategy 67
3.2.6 Phenotypic Analysis of Mice 67
3.2.6.1 Determination of Body Weight 67
3.2.6.2 Blood Sample Collection and Plasma Preparation 67
3.2.6.3 Blood Glucose Determination 67
3.2.6.4 Measurement of Hemoglobin A1c 67
3.2.6.5 Measurement of Plasma Lipids 68
3.2.6.6 Measurement of Plasma Osteocalcin 68
3.2.6.7 Isolation of Embryos 68
3.2.6.8 Necropsy 68
3.2.6.9 Pathology and Histology 69
3.2.6.10 X-ray Examination 69
3.2.6.11 Hair Regrowth Analysis 69
3.2.7 Statistics 69
4 RESULTS 70
4.1 GENERATION OF HPARP-1EMBRYONIC STEM CELLS AND MICE 70
4.1.1 Gene Targeting and Generation of hPARP-1 Mice 70 4.1.1.1 Identification of Targeted ES Cell Clones Using Flanking PCR 71 4.1.1.2 Validation of Targeted ES Cell Clones Using Southern Blot Analyses 73
4.1.1.3 Generation of Chimeras and Germline Transmission 75
4.1.1.4 Breeding of Potentially Homozygous hPARP-1 Mice 76
4.1.2 Genetic Characterization of hPARP-1 ES Cells and Mice 77
4.1.2.1 Localization of hPARP-1 and mParp-1 Using Fluorescence In Situ Hybridization 77 4.1.2.2 Assessment of Gene Copy Numbers Using Quantitative Real-time PCR 80
4.1.2.2.1 Validation of Quantitative Real-time PCR 80
4.1.2.2.2 Analyses of PARP-1 Gene Copy Numbers and Identification of hPARP-1 Homozygotes 82
4.1.2.2.3 Analysis of Murine Parp-1-flanking Genes 84
4.1.3 Excision of the Neomycin Resistance Cassette 85
4.1.3.1 Excision of the NeoR Cassette In Vitro 85
4.1.3.2 Excision of the NeoR Cassette In Vivo 85
4.1.4 Breeding of Two Independent Congenic hPARP-1 Mouse Lines 86
4.1.5 Summary of Mouse Breeding 87
4.2 BIOCHEMICAL CHARACTERIZATION OF HPARP-1ESCELLS AND MICE 87
4.2.1 Expression Analyses 87
4.2.1.1 Analysis of mRNA Expression 87
4.2.1.2 Analyses of Protein Expression 89
4.2.1.2.1 Generation of a Novel Murine-PARP-1-specific Antibody 89
4.2.1.2.2 Expression Analyses in ES Cells 90
4.2.1.2.3 PARP-1 Expression Analyses In Vivo 93
4.2.1.3 Summary of Expression Analyses 94
4.2.2 Analysis of Poly(ADP-ribosyl)ation Metabolism 97
4.2.2.1 Maximum Poly(ADP-ribosyl)ation Capacity in Permeabilized ES Cells 97
4.2.2.2 Poly(ADP-ribosyl)ation Capacity in Living ES Cells 97
4.2.2.3 NAD+ Levels in ES Cells and Primary Splenocytes 100
4.2.2.4 Summary of Poly(ADP-ribosyl)ation Metabolism Analyses 101
4.3 PHENOTYPIC ANALYSES OF HPARP-1ESCELLS AND MICE 101
4.3.1 Analysis of ES Cells 101
4.3.1.1 ES Cell Proliferation and Differentiation Status 101
4.3.1.2 DNA Repair and Genomic Stability 102
4.3.2 Analysis of Mice 103
4.3.2.1 Survival Rates 104
4.3.2.2 Growth Studies 106
4.3.2.3 Organ Weights of 12-week-old Mice 108
4.3.2.3.1 Organ Weight of Spleen 111
4.3.2.4 Analyses of Energy Metabolism 111
4.3.2.4.1 Lipid Metabolism 111
4.3.2.4.2 Glucose Metabolism 114
4.3.2.5 Hair Growth and Regenerative Potential 115
4.3.2.6 Pathological Analyses 116
4.3.2.6.1 Skeletal Abnormalities and Bone Metabolism 116
4.3.2.6.2 Pathological Examination of Moribund Mice 118
4.3.2.7 Summary of Phenotypic Analysis 120
4.4 SUMMARY OF RESULTS 121
5 DISCUSSION 122
5.1 GENERATION OF HPARP-1EMBRYONIC STEM CELLS AND MICE 123
5.1.1 Gene Targeting and Germline Transmission 123
5.1.2 Ectopic Gene Targeting 124
5.2 ESTABLISHMENT OF MODELS TO STUDY THE EFFECTS OF HPARP-1GENE TARGETING 128 5.2.1 ES Cells as an In Vitro Model to Study PARP-1 in General 128
5.2.2 Establishment of hPARP-1 ES Cell Clones 129
5.2.3 Establishment of hPARP-1 Mouse Lines 130
5.3 BIOCHEMICAL CHARACTERIZATION OF HPARP-1ESCELLS AND MICE 131
5.3.1 PARP-1 Expression Analyses 131
5.3.2 Analyses of Poly(ADP-ribosyl)ation Metabolism 132
5.4 PHENOTYPIC ANALYSES OF HPARP-1ESCELLS AND MICE 135
5.4.1 Phenotypic Analysis of ES Cells 136
5.4.2 Phenotypic Analysis of hPARP-1 Mice 137
5.4.2.1 Survival Rates 137
5.4.2.2 Pathological Analyses 138
5.4.2.2.1 Neoplastic Lesions 138
5.4.2.2.2 Energy Metabolism 139
5.4.2.2.3 Splenomegaly 141
5.4.2.2.4 Signs of Premature Aging 142
5.4.2.2.5 Glomerulopathy 143
5.4.2.3 Potential Underlying Molecular Mechanisms of the Observed Phenotype 144
5.5 CONCLUSIONS AND PERSPECTIVES 146
6 REFERENCES 148
7 APPENDIX 163
7.1 ABBREVIATIONS 163
7.2 FIGURES 165
7.3 TABLES 167
1 Introduction
1.1 Poly(ADP-Ribose) Polymerase-1 and Poly(ADP-ribosyl)ation
“We discovered that (…) nicotinamide mononucleotide treatment enhances the activity of a DNA-dependent enzyme which incorporates ATP into a product which is presumably polyadenylic acid; we wish to report some properties of this enzyme which does not seem to have been described as yet.” (Chambon et al. 1963)
This marked the beginning of research in the field of poly(ADP-ribose). Later the same authors showed that the enzyme they discovered actually uses nicotinamide adenine dinucleotide (NAD+) as a substrate to catalyze the formation of the homopolymer poly(ADP- ribose) (D'Amours et al. 1999). Today, this enzyme is known as poly(ADP-ribose) polymerase- 1 (PARP-1). Meanwhile, a total of 17 human and 16 mouse homologues of the PARP-1 gene have been discovered. The function of most of these proteins remains to be assessed and apparently not all of them possess poly(ADP-ribosyl)ation capacity (Otto et al. 2005; Schreiber et al. 2006). The best studied member of the PARP family still is PARP-1 (EC 2.4.2.30) mentioned above. It is a highly abundant nuclear enzyme involved in multiple cellular processes.
1.1.1 NAD
+and Poly(ADP-Ribose) Metabolism
1.1.1.1 NAD+ Metabolism
Poly(ADP-ribosyl)ation is the major NAD+-consuming process in eukaryotic cells that can interfere with other vital NAD+-dependent and cellular functions (Figure 1.1) (Bürkle 2006).
Cellular NAD+ derives from four precursor molecules. The de novo pathway uses tryptophan to synthesize the intermediate nicotinic acid mononucleotide (NaMN), which is then converted to nicotinic acid adenine dinucleotide, and finally to NAD+. In an import pathway, NaMN can also be synthesized from nicotinic acid (Na). In addition, a salvage pathway exists that utilizes nicotinamide (Nam) or nicotinamide riboside to generate nicotinamide mononucleotide (NMN). This intermediate molecule is then converted into NAD+ by nicotinamide mononucleotide adenylyltransferases (NMNAT), which play a central role in NAD+ biosynthesis (Berger et al. 2005; Bürkle 2006; Schreiber et al. 2006).
Figure 1.1. Cellular NAD+ metabolism.
NAD+ biosynthesis uses four different precursors in de novo or salvage pathways. Cellular processes dependent on NAD+ can be NAD+-consuming or non-consuming. The latter ones imply redox reactions, in which NAD+ and its reduced form NADH serve as cofactors for the generation of ATP. Moreover, NAD+ serves as a substrate in four types of ADP-ribose transfer reactions: (i) ADP-ribose cyclization, (ii) deacytylation of proteins resulting in O- acetyl-ADP-ribose, and (iii) mono- or (iv) poly(ADP-ribosyl)ation. Poly(ADP-ribose) can be degraded by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylarginine hydrolases (ARH); O-acetyl-ADP-ribose by Nudix O-acetyl-ADP-ribose hydrolase, resulting in free ADP-ribose and derivatives. Na indicates nicotinic acid;
NaDS, NAD+ synthase; Nam, nicotinamide; NaMN, nicotinic acid mononucleotide; NaMNAT, nicotinic acid mononucleotide adenylyltransferase; Nampt, nicotinamide phosphoribosyl transferase; NaPRTase, nicotinic acid phosphoribosyl transferase; NMNAT, nicotinamide mononucleotide adenylyltransferase; Nrk1, nicotinamide riboside kinase-1. Adapted from (Schreiber et al. 2006).
Cellular processes dependent on NAD+ can be either NAD+-consuming or non-consuming.
The latter ones imply redox reactions catalyzed by NAD+ dehydrogenases, in which NAD+ and its reduced form NADH serve as cofactors for the generation of ATP. Moreover, NAD+ serves as a substrate in four types of ADP-ribose transfer reactions: (i) ADP-ribose cyclization, which is involved in calcium signaling; (ii) deacytylation of proteins results in O-acetyl-ADP-ribose by the family of sirtuins, which are involved in many cellular functions including chromatin remodeling, gene silencing and genomic stability; (iii) mono(ADP-ribosyl)ation, which is associated with intracellular as well as extracellular functions in cell signaling and metabolism;
and (iv) poly(ADP-ribosyl)ation with functions as described in the following sections (Hassa et al. 2006). Poly(ADP-ribose) can be degraded by isoforms of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylarginine hydrolases (ARH), whereas O-acetyl-ADP-ribose is degraded by Nudix O-acetyl-ADP-ribose hydrolase. The resulting free ADP-ribose or its derivatives were shown to cause protein damage by glycation reactions and might be involved in cellular signaling (Cervantes-Laurean et al. 1996; Schreiber et al. 2006).
In conclusion, poly(ADP-ribosyl)ation depends on intracellular NAD+, which is also linked to various other cellular processes ranging from energy metabolism and cellular signaling to chromatin remodeling (Hassa et al. 2006; Schreiber et al. 2006).
1.1.1.2 Poly(ADP-Ribose) Metabolism
Poly(ADP-ribosyl)ation occurs in all metazoan organisms and also in some lower unicellular eukaryotes, but is absent in bacteria and yeast (Bürkle 2005). Up to now, eight enzymes out of the 17-PARP family genes have been reported to possess catalytic activity for the formation of poly(ADP-ribose), i.e. PARP-1, PARP-2, PARP-3, VPARP, tankyrase-1, tankyrase-2, TiPARP- 1, and PARP-10 (Bürkle 2006; Schreiber et al. 2006).
The formation of poly(ADP-ribose) is initiated by an ADP-ribosyl transfer reaction onto glutamic acid or aspartic acid of acceptor proteins (Figure 1.2). The initial, protein-conjugated ADP-ribosyl unit is elongated during repeated cycles of ADP-ribosyl transfer using NAD+ as a substrate leading to the formation of glycosidic 1‟‟2‟ ribose-ribose bonds, resulting in a highly negatively charged poly(ADP-ribose) biopolymer and stoichiometric release of nicotinamide as by-product. The polymer chain lengths range from few units in linear structures to more than 200 subunits in multibranched molecules (Alvarez-Gonzalez and Mendoza-Alvarez 1995;
D'Amours et al. 1999; Bürkle 2005). Branching points result from the formation of glycosidic 1‟‟‟2‟‟ ribose-ribose bonds and occur after every 20 to 50 ADP-ribose subunits (Kawaichi et al. 1981; Miwa et al. 1981; Alvarez-Gonzalez and Jacobson 1987). Minaga and Kun postulated helical conformations of the polymer, similar to those of DNA and RNA. (Minaga and Kun 1983a; Minaga and Kun 1983b).
Figure 1.2: Poly(ADP-ribose) metabolism.
Steps 1-3 and steps 4-7 of the poly(ADP-ribose) cycle represent the anabolic and catabolic reactions, respectively.
The synthesis of poly(ADP-ribose) requires three distinct PARP activities: step 1, initiation or mono(ADP- ribosyl)ation of specific amino acid residues in the corresponding acceptor protein; step 2, elongation of the polymer; and step 3, branching of the polymer. The degradation requires poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosyl protein lyase or hydrolase activities: steps 4 and 5, exoglycosidic and endoglycosidic PARG activities, respectively, that hydrolyze the glycosidic bonds between the ADP-ribose units; step 6 and step 7, mono(ADP-ribosyl) protein lyase or hydrolase activities, respectively. mADPr indicates mono-ADP-ribose.
Adapted from (Hassa et al. 2006).
Most of the poly(ADP-ribose) acceptors are nuclear proteins involved in DNA metabolism and chromatin architecture (D'Amours et al. 1999). The main polymer acceptor molecule is PARP-1 itself, as it catalyses its automodification (Ogata et al. 1981). In vitro automodified PARP-1 may carry as many as 15 polymer molecules with an average chain length of 80 ADP- ribose subunits (Kawaichi et al. 1981). Besides the covalent attachment of polymer to proteins, at least two different evolutionarily conserved poly(ADP-ribose) binding motifs exist in a wide range of proteins which enable non-covalent binding of free or protein-bound poly(ADP-ribose) to proteins (Pleschke et al. 2000; Ahel et al. 2008). Specificity of such non-covalent interactions might be dependent on the degree of polymer branching or polymer chain length (Panzeter et al.
1992; Fahrer et al. 2007).
The cellular occurrence of poly(ADP-ribose) is of transient nature (Figure 1.2). At least two different enzymes were shown to catalyze polymer hydrolysis. The main catabolic activity is attributed to the poly(ADP-ribose) glycohydrolase (PARG) (Hassa et al. 2006). In human cells,
this enzyme is encoded by a single gene, which by alternative splicing gives rise to three isoforms of 111, 102, and 99 kD, respectively. The 111 kD isoform localizes to the nucleus, whereas the isoforms of 102 and 99 kD are restricted to the cytoplasm (Meyer-Ficca et al.
2004). Three phases of polymer degradation by PARG were proposed, with branched polymers being degraded more slowly than linear ones (Braun et al. 1994; Malanga and Althaus 1994).
These phases are (i) endoglycosidic cleavage; (ii) endoglycosidic plus exoglycosidic cleavage with processive degradation; (iii) exoglycosidic cleavage with distributive degradation. The endoglycosidic activity of PARG provides a mechanism by which free poly(ADP-ribose) can be formed, resulting in multiple physiological consequences through non-covalent protein binding and intra- as well as extra-nuclear signaling functions (Hassa et al. 2006). The most proximal protein-bound ADP-ribose residue is removed by PARG itself or by an ADP-ribosyl protein lyase (Oka et al. 1984; Desnoyers et al. 1995). Recently, a second enzyme was identified with potential poly(ADP-ribose) glycohydrolase activity, i.e. ADP-ribose-arginine protein hydrolase 3 (ARH3) (Oka et al. 2006).
1.1.2 Poly(ADP-Ribose) Polymerase-1
Human PARP-1 is an abundant nuclear protein of 113 kD comprising 1014 amino acids. In undamaged cells, the majority of the PARP-1 molecules seem to be in a catalytically inactive state. In cells exposed to genotoxins, PARP-1 is activated upon binding to DNA strand breaks and subsequent dimerization, resulting in an increase of poly(ADP-ribose) levels by 10 to 500- fold (Juarez-Salinas et al. 1979; D'Amours et al. 1999). Under these conditions PARP-1 accounts for 75 to 97% of the cellular poly(ADP-ribose) formation (Shieh et al. 1998).
Figure 1.3: Domain structure of human and murine PARP-1 and the degree of their homology.
Top. Multidomain structure of PARP-1. Percentages indicate the degree of homology between human and murine PARP-1. Bottom. Similarity blot showing degree of homology of full-length human and murine PARP-1 from amino acids 1 to 1014. The WGR domain is named after conserved amino acid motif. AD indicates automodification domain; BRCT, breast cancer 1 protein (BRCA1) C-terminus (BRCT) motif; ZF, zinc finger;
ZRD, zinc ribbon domain.
1.1.2.1 Comparison of Human and Rodent PARP-1
The PARP-1 protein sequence is evolutionarily highly conserved among mammals with an overall homology of about 95% (Figure 1.3). Murine and rat PARP-1 are more closely related to each other than to the human PARP-1 (hPARP-1). All three genes consist of 23 exons spanning 47 kb (human), 31.5 kb (rat), or 32.5 kb (mouse) (Auer et al. 1989; Beneke et al. 2002). The human gene was mapped to chromosome 1q42 and the murine gene to chromosome 1H4 (Ensembl Contig View, http://www.ensembl.org) (Cherney et al. 1987; Baumgartner et al.
1992). Despite similar protein levels, maximum PARP activity is about five times higher in humans compared to rodents (section 1.1.2.6.2, Figure 1.8) (Grube and Bürkle 1992). Although classical enzymatic kinetics parameters, such as Vmax, km, and kcat, do not significantly differ between human and rodent (rat) PARP-1, hPARP-1 displays a twice as high capacity for its automodification than its rodent (rat) orthologue (Figure 1.4). This finding indicates that the intrinsic PARP-1 protein structure itself accounts for the higher human poly(ADP-ribosyl)ation capacity (Beneke et al. 2000). The discrepancy between the Grube and Bürkle study (fivefold increase) and that of Beneke et al. (twofold increase) might be explained by a recent finding that identified a hypomorphic human polymorphism at amino acid position 762 (V762A), decreasing the PARP-1 activity by about 50% (Wang et al. 2007). This is the variant used in the study by Beneke et al.
Figure 1.4: Human PARP-1 displays a higher poly(ADP-ribosyl)ation capacity than its rodent orthologe.
Experiments were performed with recombinant human or rat PARP-1. Y-axis depicts velocity of poly(ADP-ribose) synthesis. Adapted from (Beneke et al. 2000)
1.1.2.2 Regulation of PARP-1 Gene Expression
PARP-1 is constitutively expressed in most rodent organs. Nevertheless, expression levels can differ considerably. High mRNA expression was found in testis, thymus, spleen, and brain;
whereas low expression was observed in liver and kidney (Ogura et al. 1990; Menegazzi et al.
1991). In cell culture, PARP-1 expression levels are dependent on cell density and differentiation status as shown for primary cell cultures (Zaniolo et al. 2005). Although the promoter regions of human, rat, and mouse PARP-1 are different, they share common features
such as multiple GC-rich regions, an initiator element, and the lack of functional TATA and CAAT boxes, which is common in most housekeeping genes. Transcription is oppositely regulated by transcription factors SP1 and NFI (Bergeron et al. 1997; Laniel et al. 1997; Laniel et al. 2004). Interestingly, the transcriptional activator SP1 is negatively regulated by PARP-1 itself (Zaniolo et al. 2007). This finding is consistent with the observation that PARP-1 can elicit its transcriptional repression in an autoregulation-dependent manner by binding to its own promoter sequences (Oei et al. 1994; Soldatenkov et al. 2002).
1.1.2.3 Structural and Functional Aspects of PARP-1
Mammalian PARP-1 comprises three major functional domains: an N-terminal DNA binding domain (DBD) (42 kD), an automodification domain (16 kD), and a C-terminal catalytic domain (55 kD) (Figure 1.3) (D'Amours et al. 1999).
The DBD spans the region from amino acids 1 to 366. It contains two zinc fingers (ZFI and ZFII) at its N-terminal end, which are evolutionarily conserved and belong to the unique group of PARP-like zinc fingers (Petrucco and Percudani 2008). The ZFI and ZFII zinc fingers are encoded by two separate exons, i.e. exons 1 and 3 (Auer et al. 1989). This rather unusual type of zinc finger is also found in DNA ligase III and coordinates the zinc ion via a Cys-X2-Cys- X28,30-His-X2-Cys binding motif. Recently, it was shown that ZFI and ZFII are considerably divergent from an evolutionary point of view, indicating that they are non-redundant and fulfill diverse functions (Petrucco and Percudani 2008). It was shown indeed that activation of PARP- 1 by single-stranded DNA nicks requires ZFI and ZFII, whereas activation by double strand breaks only depends on ZFI (Gradwohl et al. 1990; Ikejima et al. 1990). Apart from its function to detect DNA strand breaks, PARP-1 recognizes other DNA structures such as distortions in the helical structure, hairpins, cruciform structures, and stably unpaired regions in double-stranded DNA. (Sastry and Kun 1990; Lonskaya et al. 2005; Potaman et al. 2005). Two helix-turn-helix motifs located at the C-terminal end of the DBD bind to some of these structures (D'Amours et al. 1999). Recently, a zinc ribbon domain (ZRD) was identified in the C-terminal part of the DBD, which partially overlaps with the helix-turn-helix motifs and seems to enable homodimerization of PARP-1 (Langelier et al. 2008). A caspase cleavage site resides between a bipartite nuclear localization signal, which separates ZFI and ZFII from the ZRD (Schreiber et al. 1992).
The automodification domain comprises the central part of the PARP-1 molecule, spanning amino acids 375 to 525 (Alkhatib et al. 1987; Kurosaki et al. 1987). It is rich in glutamic acid residues, which serve as potential starting points for poly(ADP-ribosyl)ation (Figure 1.2) (Kawaichi et al. 1981; Cherney et al. 1987). Moreover, the automodification domain contains a breast cancer 1 protein (BRCA1) C-terminus (BRCT) motif, which is found in many DNA
repair and cell cycle checkpoint proteins. The BRCT motif enables strong and specific protein- protein interactions (Bork et al. 1997).
The region spanning amino acids 548 to 633 lies within the C-terminal 55-kD NAD+-binding catalytic fragment and comprises a highly conserved WGR domain with unknown function (Otto et al. 2005). This domain is named after its conserved motif of tryptophan (W), glycine (G), and arginine (R). The actual catalytic domain is located at the C-terminal part spanning amino acids 662 to 1014. It is responsible for NAD+ binding, ADP-ribose transfer and polymer branching (Alkhatib et al. 1987; Kurosaki et al. 1987; Simonin et al. 1993). The catalytically active site, which enables NAD+ binding and is known as the „PARP signature‟, consists of a β- α-loop-β-α structural motif (Ruf et al. 1996). The „PARP signature‟ displays 100% conservation among vertebrates (Kraus and Lis 2003). Mutational analyses revealed that lysine 893 and aspartate 993 are involved in the covalent attachment of the initial ADP-ribose unit onto the acceptor amino acid (Simonin et al. 1993). Glutamate 988 seems to function in polymer elongation activity and tyrosine 986 in polymer branching (Marsischky et al. 1995; Rolli et al.
1997). It is worth noting that a single amino acid exchange at position 713 (L713F) resulted in a gain-of-function mutation (“super-PARP-1”) with a more than ninefold increase in PARP-1 activity (Miranda et al. 1995).
1.1.2.4 Regulation of PARP-1 Activity
Catalytic activation of PARP-1 is a dynamic and transient process, regulated by DNA- dependent as well as independent mechanisms.
DNA strand breaks are the most potent activators of PARP-1 catalytic activity. In living cells the synthesis of poly(ADP-ribose) is directly proportional to the amount of single and double strand breaks in the genome (D'Amours et al. 1999). In addition, binding to specific undamaged DNA also triggers PARP-1 catalytic activity as shown by automodification and poly(ADP- ribosyl)ation of histone H1 (section 1.1.2.3) (Lonskaya et al. 2005). The finding that poly(ADP- ribosyl)ation follows second order kinetics with increasing PARP-1 concentration and PARP-1 dimerization is a prerequisite for high enzymatic activity led to the conclusion that the automodification reaction is intermolecular (Mendoza-Alvarez and Alvarez-Gonzalez 1993;
Alvarez-Gonzalez and Mendoza-Alvarez 1995; Pion et al. 2005). Homodimerization via the zinc ribbon domain might be involved in DNA-dependent stimulation of PARP-1 (Langelier et al. 2008).
According to the PARP-1 shuttling model, automodification of PARP-1 leads to increasing negative charges on the molecule until a „point of repulsion‟ is reached, which causes dissociation of PARP-1 from the DNA and subsequent catalytic inactivation. Subsequent hydrolysis of PARP-1-bound poly(ADP-ribose) by PARG reconstitutes the enzymatic activity of PARP-1. Consistent with this model, it was shown that increasing automodification of
PARP-1 lowers its enzymatic activity (Kawaichi et al. 1981). Moreover, it was reported that PARG interacts with PARP-1 via its automodification domain, thereby directly inhibiting PARP-1 activity (Keil et al. 2006).
Meanwhile, several other DNA-independent mechanisms regulating the activity of PARP-1 have been identified. It was suggested that histones and nucleosomes in chromatin are able to act as potent allosteric activators of PARP-1 (Kim et al. 2004; Kun et al. 2004). Moreover, the nuclear isoform of the NAD+ biosynthetic enzyme NMNAT binds to poly(ADP-ribose) and stimulates PARP-1 activity in a process that depends on the phosphorylation state of NMNAT (Berger et al. 2007). In addition, phosphorylation of PARP-1 itself is of general importance, since the „energy sensing‟ enzyme AMP-activated kinase can positively regulate PARP-1 activity by direct phosphorylation (Walker et al. 2006). Furthermore, phosphorylation by the extracellular signal-regulated kinases 1/2 (ERK1/2) is required for maximal PARP-1 activation after DNA damage (Kauppinen et al. 2006). Cohen-Armon et al. reported that PARP-1 phosphorylation by ERK2 is sufficient to stimulate PARP-1 activity even in the absence of DNA damage (Cohen-Armon et al. 2007). ERKs participate in the mitogen-activated protein kinase (MAPK) signaling pathway via the Raf-MEK-ERK phosphorylation cascade. It was hypothesized that this is relevant with regard to the function of PARP-1 in cell proliferation and differentiation (Cohen-Armon 2007).
1.1.2.5 Cellular Functions of PARP-1
Three non-exclusive mechanisms of the cellular functions of PARP-1 can be distinguished:
(i) Functions that rely on the enzymatic activity of PARP-1 and the subsequent covalent or non- covalent interaction of nuclear proteins with poly(ADP-ribose). (ii) Direct interactions of proteins with PARP-1 via protein-protein interaction, e.g. via the BRCT domain.
(iii) Intervention in the cellular NAD+ metabolism by excessive PARP-1 stimulation and potential signaling functions of free poly(ADP-ribose) or its derivatives. The consequences of these actions with regard to modulation of chromatin structure, genomic maintenance, transcription, and cell death are discussed in the following sections.
1.1.2.5.1 PARP-1 and Chromatin Modification
Chromatin is a complex of DNA and proteins with a dynamic structure involved in replication, transcription and other fundamental cellular processes (Felsenfeld and Groudine 2003). PARP-1 seems to act as a structural and regulatory component of chromatin, both in undamaged cells and upon genotoxic stress (Kim et al. 2004). Many poly(ADP-ribose) acceptor proteins were shown to contribute to chromatin and nuclear architecture such as histones, lamins, high- mobility group (HMG) proteins, topoisomerases, and the DEK protein (Figure 1.5) (Gagne et al. 2003; Rouleau et al. 2004; Ditsworth et al. 2007; Gamble and Fisher 2007).
Althaus and colleagues proposed a histone shuttle mechanism, based on the findings that poly(ADP-ribosyl)ation of polynucleosomes causes relaxation of chromatin structure and that activity of PARG degrades poly(ADP-ribose) from modified histones (Poirier et al. 1982; de Murcia et al.; Althaus 1992; Realini and Althaus 1992). According to this model, DNA-bound histones dissociate from DNA upon poly(ADP-ribosyl)ation, causing an open chromatin structure and guiding repair factors to sites of DNA damage. Upon degradation of the ADP- ribose polymer by PARG, DNA reassociates with histones, thereby restoring the condensed chromatin structure. Kim et al. reported that PARP-1 itself is a component of chromatin (Kim et al. 2004). They demonstrated in vitro that histone H1 and PARP-1 bind in a competitive and mutually exclusive manner to nucleosomes. Thereby, PARP-1 promotes the local compaction of chromatin into higher-order structures, which are associated with transcriptional repression.
Those authors suggested that PARP-1 modulates the chromatin architecture and gene transcription through its intrinsic enzymatic activity in a DNA-damage-independent manner; i.e.
PARP-1 automodification through a DNA-damage-independent trigger leads to its release from chromatin, thereby facilitating chromatin decondensation and gene transcription by RNA polymerase II. On the other hand, the same group recently demonstrated a reciprocal binding pattern of PARP-1 and histones H1 at many RNA polymerase II-transcribed promoters. Here, PARP-1 could replace histone H1 in a subset of these promoters, which was associated with actively transcribed genes (Krishnakumar et al. 2008). These findings suggest a functional interplay of PARP-1 with other chromatin-associated factors, implying an active role of PARP-1 in chromatin remodeling and transcriptional regulation. The detailed spatial and temporal characteristics of these mechanisms, however, remain to be determined.
Another mechanism of PARP-1-dependent chromatin regulation arises from the finding that poly(ADP-ribose) and automodified PARP-1 non-covalently interact with the 20S proteasome in the nucleus, which enhances its peptidase activity (Mayer-Kuckuk et al. 1999). It was proposed that this leads to the degradation of oxidatively damaged histones underscoring the function of PARP-1 in maintenance of nuclear stability (Ullrich et al. 1999).
1.1.2.5.2 PARP-1 and Genomic Maintenance
It was estimated that 20000 to 40000 DNA strand breaks occur in a mammalian cell per day, all of which need to be repaired to ensure genomic stability (Vijg 2007). In mammals, at least six, partly overlapping DNA repair pathways exist, i.e. O6-methyl guanine methyltransferase (MGMT), base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and DNA double strand break (DSB) repair including the subpathways homologous recombination (HR) and non-homologous end joining (NHEJ) (Hoeijmakers 2001).
Figure 1.5: PARP-1, its interaction partners, and their role in genomic maintenance.
ATM indicates ataxia telangiectasia mutated; Bub3, Budding uninhibited by benzimidazoles 2; Cenpa/b, centromeric protein a/b; CSB, Cockayne syndrome type B; DEK, DEK oncogene; DNA-Polβ, DNA polymerase β;
DNA-PKCS, DNA-activated protein kinase catalytic subunit; HMGB1, high mobility group box 1; Ku70/80, Ku antigens 70/80 kD subunit; MRE11, meiotic recombination 11; p21, cyclin-dependent kinase inhibitor 1A; p53, tumor protein p53; PCNA, proliferating cell nuclear antigen; TRF2, telomeric repeat binding factor 2; WRN, Werner syndrome protein; XRCC1, X-ray repair complementing defective in Chinese hamster 1; XPA, xeroderma pigmentosum complementation group A.
Except for the MGMT and MMR pathway, PARP-1 is involved in all of these repair pathways and is therefore considered as a general caretaker of genomic stability (Figure 1.5) (Meyer-Ficca et al. 2005a). It was shown that the recruitment of PARP-1 to sites of DNA damage, its activation, and the subsequent production of poly(ADP-ribose) is one of the first responses in mammalian DNA repair (D'Amours et al. 1999). Several studies supported the role of PARP-1 as a cell survival factor upon genotoxic stimuli: Trans-dominant inhibition of PARP- 1 by overexpression of its DNA binding domain potentiates cytotoxicity upon treatment of cells
with alkylating agents and ionizing radiation (Küpper et al. 1995). [N.B. ionizing radiation, such as X-irradiation or γ-irradiation, can introduce various forms of DNA damage, including oxidation of bases, DNA strand breaks and other lesions caused by oxygen radicals (Masutani et al. 2000)]. Moreover, PARP-1-deficient cells exhibit an enhanced sensitivity to alkylating agents (Trucco et al. 1998; Masutani et al. 1999a) and show increased frequencies of sister- chromatid exchanges, spontaneously or upon treatment with alkylating agents (de Murcia et al.
1997; Wang et al. 1997). Consistent with these results, Meyer et al. demonstrated in overexpression studies that PARP-1 acts as a negative regulator of alkylation-induced sister chromatid exchange (Meyer et al. 2000). However, in a different study, overexpression of hPARP-1 sensitized hamster cells to γ-irradiation, indicating an ambiguous role of PARP-1 in DNA damage response (Van Gool et al. 1997).
1.1.2.5.2.1 PARP-1 and its Interaction Partner PARP-2 in Base Excision Repair
Base excision repair is the major repair pathway, acting on damages that occur during cellular metabolism including damages from reactive oxygen species, methylation, deamination, and hydroxylation. The core BER reaction is initiated by a DNA single strand break (SSB) introduced by exogenous or endogenous factors (Hoeijmakers 2001). PARP-1 detects these SSB via its second zinc finger ZFII (Gradwohl et al. 1990; Molinete et al. 1993). Moreover, PARP-1 interacts with the BER loading platform X-ray repair complementing factor 1 (XRCC1) via its BRCT domain (Figure 1.5) (Masson et al. 1998; Dantzer et al. 1999; El-Khamisy et al. 2003). It was shown that PARP-1 is required for the assembly and stability of XRCC1 nuclear foci after DNA damage (El-Khamisy et al. 2003). Here, foci formation was also mediated via the interaction of poly(ADP-ribose) with XRCC1. Furthermore, XRCC1 and PARP-1 interact with DNA polymerase-β and DNA ligase III, forming a multiprotein complex consisting of the major BER factors (Caldecott et al. 1996; Leppard et al. 2003; Confer et al. 2004).
The finding that PARP-1 knock-out cells still synthesized poly(ADP-ribose) led to the identification of an additional nuclear PARP, PARP-2, which was also demonstrated to be activated upon genotoxic stimuli (Shieh et al. 1998; Ame et al. 1999; Bürkle 2006). Up to now, PARP-1 and PARP-2 are the only known DNA damage-dependent PARPs. PARP-1 and PARP- 2 homo- and heterodimerize (Figure 1.5) and work at least partly in a redundant fashion, since only double knock-out mice show embryonic lethality (section 1.1.2.6.1) (Schreiber et al. 2002;
Menissier de Murcia et al. 2003). This notion is supported by the fact that PARP-2 also participates in BER physically and functionally interacting with XRCC1, DNA polymerase-β, and DNA ligase III. Recent experiments using live cell imaging indicated a role of PARP-2 in later steps of BER repair, as proposed by the following model for spatio-temporal accumulation of BER factors: SSBs are detected by the DNA binding domain of PARP-1, leading to its activation, production of poly(ADP-ribose), and chromatin relaxation. Subsequently, additional
PARP-1 molecules are attracted, causing amplification of the signal. At the „point of repulsion‟
PARP-1 then dissociates from the DNA, enabling the recruitment of the BER loading platform XRCC1, PARP-2, and further DNA repair factors. This triggers resealing of the DNA lesion and re-establishment of genomic integrity (Mortusewicz et al. 2007).
1.1.2.5.2.2 PARP-1 in Nucleotide Excision Repair
Nucleotide excision repair is responsible for the removal of bulky DNA adducts, such as pyrimidine dimers, which are caused by UV irradiation (Hoeijmakers 2001).
Although the role of PARP-1 in NER is not very well established, at least two NER factors, the DNA-dependent ATPase Cockayne syndrome group B (CSB) protein and the DNA lesion recognition protein xeroderma pigmentosum group A (XPA), were identified as poly(ADP- ribose) binding enzymes (Figure 1.5) (Thorslund et al. 2005; Fahrer et al. 2007). CSB also physically interacts with PARP-1 and its ATPase activity is inhibited by poly(ADP- ribosyl)ation. Consistently, a third study suggested that PARP-1 is involved in repair of pyrimidine dimers in a CSB-dependent pathway (Flohr et al. 2003).
1.1.2.5.2.3 PARP-1 in Double Strand Break Signaling
DNA double strand breaks (DSBs) arise from ionizing radiation, free radicals, chemicals, or during replication of a SSB through collapsed replication forks. Mammalian cells repair DSBs via two mechanisms: homologous recombination (HR) utilizes the sister chromatid or chromosome for error-free repair of the lesion (for a detailed description of HR see section 1.2.1, Figure 1.9), whereas non-homologous enjoining (NHEJ) simply reattaches free DNA ends without using a template and is therefore error prone (Hoeijmakers 2001). The implementation of one of these pathways depends on the species, cell type, and cell cycle phase (Shrivastav et al. 2008).
In both pathways, PARP-1 already participates in very early phases. PARP-1 and the DSB sensing complexes MRE11/Rad50/NBS1 (involved in HR) and Ku70/80 (involved in NHEJ) were shown to interact with and compete for binding at free DNA ends, with PARP-1 potentially guiding these proteins to the damaged site (Figure 1.5) (Wang et al. 2006a; Haince et al. 2008). PARP-1 also physically and functionally interacts with two phosphatidyl inositol 3-like protein kinases ATM (involved in HR) and DNA-PKcs (involved in NHEJ), which are crucial for DSB signaling (Ruscetti et al. 1998; Aguilar Quesada et al. 2007; Haince et al.
2007). The precise mechanism by which PARP-1 participates in DSB repair has to be elucidated. However, it was suggested that PARP-1 serves as a general DNA damage detecting molecule, which potentially mediates a switch between the NHEJ and the HR pathways (Beneke and Bürkle 2007; Shrivastav et al. 2008). Consistent with this, PARP-1 was shown to function in a NHEJ backup pathway (Audebert et al. 2004; Wang et al. 2006a), whereas several reports demonstrated an anti-recombinogenic activity of PARP-1 (Waldman and Waldman
