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«DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER FAKULTÄT FÜR BIOLOGIE DER LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN vorgelegt von Verena Passerini ...»

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Aneuploidy triggers a conserved global response and

impairs cellular homeostasis

DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER FAKULTÄT FÜR

BIOLOGIE DER LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN

vorgelegt von

Verena Passerini

München, April 2016

Eidesstattliche Erklärung

Hiermit erkläre ich an Eides statt, dass ich die vorliegende Dissertation selbstständig und ohne

unerlaubte Hilfe angefertigt habe. Ich habe weder anderweitig versucht eine Dissertation einzureichen oder eine Doktorprüfung durchzuführen, noch habe ich diese Dissertation oder Teile derselben einer anderen Prüfungskomission vorgelegt.

München, den 25.04.2016 Verena Passerini Erster Gutachter: Prof. Dr. Stefan Jentsch Zweiter Gutachter: Prof. Dr. Peter Becker Tag der Einreichung: 25.04.2016 Tag der mündlichen Prüfung: 10.08.2016 List of Publications Die vorliegende Arbeit wurde zwischen Februar 2012 und April 2016 unter der Anleitung von Zuzana Storchová(Ph.D.) am Max-Planck-Institut für Biochemie in Martinsried durchgeführt.

Wesentliche Teile dieser Arbeit wurden in den folgenden Publikationen veröffentlicht:

Dürrbaum M, Kuznetsova AY, Passerini V, Stingele S, Stoehr G, Storchová Z. Unique features of the transcriptional response to model aneuploidy in human cells. BMC Genomics 2014 Feb 18;15:Pa139.

Donnelly N, Passerini V, Durrbaum M, Stingele S and Storchova Z. HSF1 deficiency and impaired HSP90-dependent protein folding are hallmarks of aneuploid human cells.

The EMBO journal. 2014 Oct 16; 33(20):2374-87 Passerini, V*, Ozeri-Galai E*, de Pagter MS, Donnelly N, Schmalbrock S, Kloosterman WP, Kerem B, Storchová Z The presence of extra chromosomes leads to genomic instability. Nat.

Commun. 7:10754 doi: 10.1038/ncomms10754 (2016).

* these authors contributed equally to this work Declaration of contribution as co-author Dürrbaum M, Kuznetsova AY, Passerini V, Stingele S, Stoehr G, Storchová Z. Unique features of the transcriptional response to model aneuploidy in human cells. BMC Genomics 2014 Feb 18;15:Pa139.

Verena Passerini contributed to this work by culturing the cell lines and preparing the samples for the microarray analysis. She also participated in reading and commenting the manuscript.

Donnelly N, Passerini V, Durrbaum M, Stingele S and Storchova Z. HSF1 deficiency and impaired HSP90-dependent protein folding are hallmarks of aneuploid human cells.

The EMBO journal. 2014 Oct 16; 33(20):2374-87 Verena Passerini contributed to this work by carrying out the experiments showed in figure 1a, 1b, 5a and 5b. She also participated in discussion and interpretation of the results as well as in reading and commenting the manuscript.

Passerini, V*, Ozeri-Galai E*, de Pagter MS, Donnelly N, Schmalbrock S, Kloosterman WP, Kerem B, Storchová Z The presence of extra chromosomes leads to genomic instability. Nat.

Commun. 7:10754 doi: 10.1038/ncomms10754 (2016).

Verena Passerini contributed to this work by designing, planning and performing the experiments showed in main and corresponding supplementary figures 1b, 1c, 1d, 2a, 2b, 2c, 3c, 5 and 6. She created the figures and participated in the interpretation and discussion of obtained results as well as in writing the manuscript.

Martinsried, 22.04.16

–  –  –

Table of contents

1. ABBREVIATIONS

2. SUMMARY

3. ZUSAMMENFASSUNG

4. INTRODUCTION

4.1 ANEUPLOIDY

4.2 CAUSES OF ANEUPLOIDY

4.3 EXPERIMENTAL MODEL SYSTEMS TO STUDY ANEUPLOIDY

4.4 CONSEQUENCES OF ANEUPLOIDY

4.4.1 Global gene expression changes in response to aneuploidy

4.4.2 Gene-dosage imbalance

4.4.3 Protein homeostasis

4.4.4 Proliferation impairment

4.4.5 Metabolic alterations

4.4.6 Genomic instability

4.5 CAUSES OF GENOMIC INSTABILITY

4.5.1 DNA replication as a source of genomic instability

4.5.2 Defective DNA repair

4.5.3 Cell cycle checkpoints

5. AIMS OF THIS STUDY

6. RESULTS

6.1 UNIQUE FEATURES OF THE TRANSCRIPTIONAL RESPONSE TO MODEL ANEUPLOIDY IN HUMAN CELLS.

6.2 HSF1 DEFICIENCY AND IMPAIRED HSP90-DEPENDENT PROTEIN FOLDING ARE HALLMARKS OF

ANEUPLOID HUMAN CELLS.

6.3 THE PRESENCE OF EXTRA CHROMOSOMES LEADS TO GENOMIC INSTABILITY

–  –  –

7. DISCUSSION

7.1 ANEUPLOIDY TRIGGERS A CONSERVED GENE-EXPRESSION PATTERN

7.2 WHOLE CHROMOSOMAL ANEUPLOIDY LEADS TO PROTEOTOXIC STRESS

7.3 WHOLE CHROMOSOMAL ANEUPLOIDY TRIGGERS GENOMIC INSTABILITY IN HUMAN

7.4 ANEUPLOIDY AS A ROAD TO CANCER

7.5 A ROLE FOR GENOMIC INSTABILITY IN TUMORIGENESIS

8. REFERENCES

9. ACKNOWLEDGEMENTS

10. CURRICULUM VITAE

–  –  –

1. Abbreviations 17-AAG 17-N-allylamino-17-demethoxygeldanamycin 53BP1 p53 binding protein 1 AICAR 5-Aminoimidazole-4-carboxamide ribonucleotide APC/C anaphase-promoting complex / cyclosome ARP aneuploidy response pattern ATM ataxia telangiectasia mutated ATR ATM- and RAD3-related BER base excision repair bloom’s syndrome BS CDK cyclin-dependent kinase CIN chromosomal instability CNA copy number aberration CENP-E centromere-associated protein E DDK Dbf4-dependent protein kinase) DDR DNA damage response DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphates down’s syndrome DS DSB double strand break ESR environmental stress response FA fanconi anemia GG-NER global genome nucleotide excision repair GIN genomic instability HR homologous recombination HSF1 Heat shock factor 1 HSP90 heat shock protein 90 HU Hydroxyurea KAR1 karyogamy gene 1 ICL interstrand cross-link iPSC inducible pluripotent stem cells MCM Minichromosome maintenance protein complex MEF Mouse Embryonic Fibroblasts MMBIR microhomology-mediated break-induced replication MMR mismatch repair MVA mosaic variegated aneuploidy NER nucleotide excision repair





11 1. Abbreviations

NGS next generation sequencing NHEJ Non homologous end joining HVP Human Papilloma Virus ORC origin recognition complex PEG Polyethylene glycol PCNA proliferation cell nuclear antigen PIKK Phosphatidylinositol kinase related kinase pre-IC pre-Initiation complex pre-RC pre replication complex RNA Ribonucleic acid ROS Reactive Oxygen Species RPA replication protein A SAC Spindle assembly checkpoint SNP single nucleotide polimorfism SSB single strand breaks ssDNA single stranded DNA TC-NER transcription-coupled nucleotide excision repair TKNEO thymidine kinase with neomycin phosphotransferase reporter gene TLS translesion synthesis XIST X-inactive specific transcript YAC yeast artificial chromosome

–  –  –

2. Summary Aneuploidy, or unbalanced chromosome number, has often detrimental physiological effects in eukaryotic cells. Aneuploidy is associated with congenital trisomy syndromes, e.g. Down syndrome, but it is also linked to several other pathological states such as Alzheimer's disease, schizophrenia and autism. In addition, aneuploidy is often found in cancer cells and high rates of aneuploidy in tumors correlate with poor prognosis and drug resistance. Although it has been proposed that aneuploidy could contribute to tumorigenesis by facilitating genomic instability, whether and how aneuploidy can lead to genomic instability remains elusive.

To study aneuploidy in human cells, we have previously generated cell lines carrying one or two supernumerary chromosomes in an otherwise diploid background by microcell mediated chromosome transfer. Similarly to other aneuploid model systems of earlier studies, our human aneuploid cell lines showed impaired proliferation and a conserved cellular response to the presence of extra chromosomes. Moreover, we found that aneuploidy alters protein homeostasis and impairs induction of heat shock response in human cells. Pathway analysis based on transcriptome and proteome data revealed characteristic gene expression changes called aneuploidy response pattern that is defined, among others, by down-regulation of factors involved in DNA replication and repair. Consistent with these observations we found that aneuploidy increases the frequency of anaphase chromatin bridges, broken chromosomes and ultrafine DNA bridges. Moreover, aneuploid cells accumulate more DNA damage even in unperturbed conditions and display higher sensitivity to replication stress than diploids. Using next generation sequencing we determined that a presence of extra chromosomes elevates frequency of chromosomal rearrangements with a breakpoint junction pattern suggestive of replication defects. Finally, we demonstrated that the observed decreased levels of MCM2-7 contribute to the replication stress and consequent genomic instability detected in aneuploid cells.

Taken together, these results provide a new insight into the possible mechanisms responsible for impaired genomic stability in response to aneuploidy. Our study provides the first evidence that a gain of chromosomes triggers replication defects and accumulation of DNA lesions, thus promoting genomic instability and possibly contributing to tumor development.

–  –  –

3. Zusammenfassung Aneuploidie oder numerische Chromosomenaberration hat meist einen schädlichen Einfluss auf die Physiologie eukaryotischer Zellen. Aneuploidie wird mit angeborenen Trisomien, wie zum Beispiel Down-Syndrom assoziiert, aber auch mit anderen pathologischen Zuständen wie Alzheimer, Schizophrenie und Autismus. Zusätzlich findet man Aneuploidie häufig in Krebszellen.

Komplexe Aneuploidien sind mit schlechter Prognose und Resistenz gegen Krebsmedikamente verbunden. Obwohl hypothetisiert wird, dass Aneuploidie durch genomische Instabilität zur Tumorgenese beiträgt, sind die genauen molekularen Mechanismen wie dies geschieht nicht bekannt.

Um Aneuploidie in menschlichen Zellen zu untersuchen, haben wir mit Hilfe von Mikrozellvermitteltem Chromosomentransfer Zelllinien generiert, welche ein oder zwei zusätzliche Chromosomen, in einem sonst diploiden Hintergrund, haben. Diese humanen aneuploiden Zellen weisen eine beeinträchtige Zellproliferation und eine uniforme Deregulation zellulärer Signalwege auf. Darüber hinaus haben wir gezeigt, dass Aneuploidie die Proteinhomöostase verändert und die Reaktion humaner Zellen auf Hitzeschock beeinträchtigt. Analyse von Transkriptom- und Proteomdaten, offenbarte charakteristische Änderungen in der Genexpression bestimmter Signalwege.

Unter anderem wird dieses wiederkehrende Muster an veränderten Signalwege durch nach unter regulierte Faktoren, die in DNS-Replikation und -Reparatur involviert sind, definiert.

Übereinstimmend mit diesen Beobachtungen haben wir herausgefunden, dass Aneuploidie zur Akkumulation von DNS-Brücken, beschädigten Chromosomen und ultrafeinen DNS-Brücken in der Anaphase führt. Ferner akkumulieren aneuploide Zellen mehr DNS-Schäden, sogar unter normalen Bedingungen, und reagieren sensibler auf Replikationsstress als diploide Zellen. Mit Hilfe von Next Generation Sequencing konnten wir zeigen, dass die Präsenz von zusätzlichen Chromosomen die Frequenz chromosomaler Reorganisation erhöht. Das Muster der Chromosomenbrücke weist auf Replikationsdefekte hin. Darüber hinaus demonstrierten wir, dass die erniedrigten MCM2-7 Proteinlevel zur genomischen Instabilität beitragen, wie sie in aneuploiden Zellen entdeckt wurden.

Unsere Ergebnisse ermöglichen neue Einblicke in die molekulare Mechanismen, die für genomische Instabilität in Aneuploidie verantwortlich sind. Unsere Studie zeigt zum ersten Mal, dass zusätzliche Chromosomen Replikationsdefekte und DNS-Läsionen auslösen, wodurch genomische Instabilität gefördert wird und diese Instabilität möglicherweise zur Entstehung von Tumoren beiträgt.

–  –  –

4. Introduction The hereditary information of each eukaryotic species, coded in the genome, is packed in the nucleus and organized into a defined number of chromosomes. Some organisms, such as fungi and algae, and germ cells have a single set of unpaired chromosomes and they are called haploids (1N). However, the majority of the metazoans cells are diploid, which means that each of their chromosomes is present in two homologues copies, one inherited from the mother and one from the father (2N). A normal human cell contains 46 chromosomes: 22 pairs of autosomes and one pair of sex chromosomes. In every cell division, the genome needs to be accurately duplicated and distributed evenly into the daughter cells. Cells have evolved surveillance mechanisms to ensure that this happens correctly, but sometimes these molecular machineries fail. As a consequence the daughter cells either die or survive with a change in numbers and structure of chromosomes, a scenario also called aneuploidy.

4.1 Aneuploidy

Aneuploidy is a condition where the number of chromosomes in the cell is not a multiple of the haploid set. The term aneuploidy describes very diverse karyotypes that could be classified in two categories: “numerical aneuploidy”, where one or more entire chromosomes are present in abnormal copy number (Figure 1a), and “structural aneuploidy” referring to chromosomal abnormalities that arise from breakage and incorrect rejoining of chromosome segments (Figure 1b) (Storchova, 2012).

Figure 1: Numerical and segmental aneuploidy. Scheme showing examples of (a) whole chromosome gains and losses (numerical aneuploidy) and (b) sub-chromosomal gains, losses, inversions and translocations (structural aneuploidy).

–  –  –

More than a century ago the German zoologist Theodor Boveri discovered the adverse effects of aneuploidy on cell and organism development of sea urchin embryos (Boveri, 2007).



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