Relative roles of UBF and RRN3 in the transcription of the ribosomal RNA genes and ribosome biogenesis determined using in vivo mouse models

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Relative roles of UBF and RRN3 in the transcription of

the ribosomal RNA genes and ribosome biogenesis

determined using in vivo mouse models

Thèse

Chelsea Herdman

Doctorat en biologie cellulaire et moléculaire

Philosophiae doctor (Ph.D.)

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Relative roles of UBF and RRN3 in the transcription of

the ribosomal RNA genes and ribosome biogenesis

determined using in vivo mouse models

Thèse

Chelsea Herdman

Sous la direction de :

Tom Moss, directeur de recherche

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Résumé

La biogenèse des ribosomes, aussi appelée la synthèse ribosomale, est un processus cellulaire important se déroulant dans le nucléole et implique la transcription par les trois ARN polymérases nucléaires. L’étape initiale et limitante de ce processus est la transcription des ARNs ribosomaux catalytiques, 28S, 18S and 5.8S, sous la forme d’un long précurseur d’ARN ribosomal (pre-ARNr/47S) par l’ARN polymérase I (RPI). RPI possède un ensemble de facteurs de transcription généraux responsables de son activation. Ces facteurs sont la protéine architecturale UBF, le facteur SL1 qui contient TBP, le facteur d’initiation RRN3 et le facteur de terminaison TTF1. La synthèse de l’ARN ribosomale est finement régulée et correspond à 30-50% de l’ensemble de la transcription de la cellule. De plus, ce processus est lié à la croissance cellulaire, la transformation, la prolifération et à l’activité des facteurs suppresseurs de tumeurs et des oncogènes. UBF et RRN3 sont notamment activés par plusieurs voies de signalisation de croissance cellulaire. Dans les cellules de mammifère, il existe ~200 copies d’ADNr par génome haploïde. Les fragments répétés d’ADN ribosomal sont arrangés en répétition en tandem sur les bras courts des chromosomes acrocentriques. De façon intéressante, dans les cellules somatiques, seulement la moitié des copies d’ADNr sont actives, alors que les autres sont maintenues dans une forme inactive par les modifications épigénétiques et la formation d’hétérochromatine. La raison pour laquelle le génome contient autant de copies et la régulation de leur activité ne sont pas bien comprises.

Cette thèse présente l’analyse de l’importance in vivo d’UBF et de RRN3 pour la régulation de la transcription de l’ARNr et pour le maintien de la structure chromatinienne de l’ADNr. Nous avons précédemment analysé la perte de fonction de UBF dans les fibroblastes embryonnaires de souris en utilisant le système de perte de fonction conditionnelle dépendante du tamoxifène. Puisque l’un de nos objectifs était de comparer la fonction de RRN3 dans un modèle similaire, nous avons réanalysé la perte de fonction de RRN3 chez la souris et généré des lignées cellulaires comme préalablement réalisées avec la perte de fonction d’UBF. Nous avons déterminé que RRN3 est essentiel à la préimplantation et le développement est arrêté à E3.5, ce qui contredit les résultats obtenus par un autre groupe qui avait obtenu un arrêt du développement beaucoup plus tardif, à E9.5. Une lignée de fibroblastes embryonnaires de souris inductible au tamoxifène a été créée pour RRN3 de façon similaire à ce qui avait été fait pour UBF. La perte de fonction d’UBF ou de RRN3 inhibe la transcription par RPI. Par contre, nous démontrons que UBF est responsable du recrutement à l’ADNr des autres facteurs associés à RPI et du maintient de l’état ouvert de la chromatine. En comparaison, RRN3 est requis simplement pour le recrutement de RPI. Dans cette étude, nous avons également identifié une région frontalière en amont de l’ADNr formée de H2A.Z, TTF1, CTCF et des modifications d’histones activatrices. Nous avons également découvert que la perte d’UBF entraine une mort cellulaire synchronisée par apoptose,

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qu’il pourrait être possible de cibler UBF dans le traitement contre le cancer puisque la perte de UBF dans les lignées cellulaires primaires cause un arrêt de prolifération sans entrainer l’apoptose. Finalement, nous avons observé que le niveau d’activité de l’ADNr dans les cellules pluripotentes est différent que dans les cellules différenciées. Des lignées de cellules souches embryonnaires (ESCs) ont été générées à partir des souris conditionnelles pour UBF et RRN3 et nos résultats préliminaires suggèrent que la totalité des gènes de l’ADNr est active dans les cellules pluripotentes. Ce modèle est idéal pour étudier la régulation de l’ADNr ainsi que le rôle de UBF et RRN3 dans cette régulation après l’induction de la différentiation. En résumé, ces résultats permettront de clarifier le rôle in vivo de UBF et RRN3 dans la transcription de l’ARN ribosomal et dans le maintien de l’intégrité de l’ADNr.

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Abstract

Ribosome biogenesis, or the synthesis of ribosomes, is an important cell process occurring in the nucleolus that utilizes transcription by all three nuclear RNA polymerases. The initial and rate-limiting step is the transcription of the catalytic ribosomal RNAs 28S, 18S and 5.8S in the form of a precursor ribosomal RNA (pre-rRNA/47S) by RNA polymerase I (RPI, also known as Pol1 and POLR1). RPI has a dedicated set of basal factors responsible for its activation. These are the architectural factor UBF, the TBP containing factor SL1, the initiation factor RRN3, and the termination factor TTF1. Ribosomal RNA synthesis is tightly regulated and accounts for 30-50% of total gene transcription. As such, this process is linked to cell growth, transformation, proliferation and the actions of tumour suppressors and oncogenes. Notably, UBF and RRN3 are activated by many of the same growth signaling pathways.

The human and mouse haploid genome contain ~200 copies of the ribosomal RNA genes, the ribosomal DNA (rDNA). These ribosomal DNA copies are arranged in tandem repeats on the short arms of acrocentric chromosomes. Interestingly, only a fraction of the rDNA copies are active, and a significant number are epigenetically silenced and heterochromatic. The reason for having so many copies and their regulation in vivo by silencing is not yet understood, though it has been connected with genome stability.

This thesis presents the analysis of the in vivo requirements for UBF and RRN3 in rRNA transcription and rDNA chromatin structure. We had previously analyzed the loss of UBF in mouse embryonic fibroblasts using tamoxifen-dependent conditional knockout. As we wanted to compare the loss of RRN3 in a similar model, we re-analyzed the RRN3 knockout mice and created cell lines as was performed for the UBF knockout. Importantly, we find that RRN3 is essential for preimplantation and its loss arrests development at E3.5, contrary to previous work that showed a late E9.5 developmental arrest. Using mouse embryonic fibroblast (MEF) cell lines conditional for UBF or RRN3, we found that the loss of either factor prevented RPI transcription. However, we found that UBF was essential for the recruitment of the other RPI transcription factors and the formation of the preinitiation complex, as well as to maintain an open rDNA chromatin structure, while RRN3 was required only for RPI recruitment. These studies allowed us to identify an upstream boundary element on the rDNA formed of H2A.Z, TTF1, CTCF and activating histone marks, which is independent of RPI activity. We also found that UBF loss, but not RRN3 loss, led to a synchronous and massive p53-independent apoptosis, specifically in oncogenically transformed cells. This strongly suggests that drug targeting UBF could be a viable cancer treatment. Finally, we have observed that the rDNA activity status in pluripotent cells differs from that of differentiated cells. Embryonic stem cells (ESCs) were also generated from the mice conditional for UBF and RRN3. Preliminary results indicate that, unlike somatic cells, all the rRNA genes in these and other pluripotent cell lines are potentially active. This makes ESCs and their

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differentiation an ideal model in which to study the establishment of rDNA silencing and the role of UBF and/or RRN3 in this process. Together these data define the in vivo roles of UBF and RRN3 in ribosomal RNA transcription and suggest mechanisms by which they maintain rDNA integrity and may drive cell differentiation.

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List of Tables

Table 1.1 Identified phosphorylation sites of UBF ... 38 Table 1.2 Identified phosphorylation sites of RRN3 ... 39 Table 1.3 Anticancer drugs in use that inhibit rRNA synthesis ... 48

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List of Figures

Figure 1.1 Structure of the nucleolus as seen through an electron microscope ... 2

Figure 1.2 The position of NORs on human and mouse chromosomes ... 4

Figure 1.3 Mammalian acrocentric chromosome carrying a NOR ... 5

Figure 1.4 UBF is found on active NORs throughout the cell cycle ... 6

Figure 1.5 Ribosome biogenesis involves all three nuclear polymerases ... 8

Figure 1.6 Nuceolar stress ... 10

Figure 1.7 Model for eNoSC function in the nucleolus ... 11

Figure 1.8 Ribosomal RNA is co-transcriptionally assembled ... 13

Figure 1.9 Ribosomal DNA organization in mammals... 14

Figure 1.10 The epigenetic readers, writers and erasers ... 16

Figure 1.11 Psoralen crosslinking of active and inactive rDNA ... 18

Figure 1.12 Model of the mechanism of rDNA silencing by NoRC ... 20

Figure 1.13 Transcription of a lncRNA from the intergenic spacer ... 21

Figure 1.14 TTF1 as a central regulator of rDNA activity status ... 22

Figure 1.15 DNA damage response pathway ... 23

Figure 1.16 Pre-initiation complex assembly in yeast and mammals ... 25

Figure 1.17 The enhancesome model of UBF binding ... 27

Figure 1.18 rDNA transcription is cell cycle dependant... 36

Figure 1.19 rRNA transcription is linked to cell growth... 37

Figure 1.20 Main apoptotic signaling pathways ... 41

Figure 1.21 Oncogenes and tumor suppressor regulation of RPI basal factors ... 43

Figure 1.22 c-Myc regulates multiple levels of ribosome biogenesis ... 44

Figure 1.23 ARF is a negative regulator of ribosome biogenesis ... 46

Figure 1.24 NPM and ARF regulate TTF1 localization and activity ... 47

Figure 1.25 Preimplantation development in the mouse ... 51

Figure 1.26 Gene expression regulation during early development ... 52

Figure 1.27 UBF is essential for NPB formation in early embryos ... 56

Figure 2.1 RNA polymerase I basal factor occupancy across the rDNA ... 64

Figure 2.2 RRN3 is essential for RNA polymerase I recruitment to the rDNA ... 67

Figure 2.3 UBF is necessary for RPI machinery recruitment ... 69

Figure 2.4 Loss of UBF induces nucleosomal formation and shut down of rRNA genes ... 71

Figure 2.5 A chromatin boundary element is found upstream of the rDNA unit ... 73

Figure 2.6 Activating histone marks at the spacer promoter are increased after loss of UBF ... 74

Figure 2.7 A stalled RNA polymerase I is found just downstream of the spacer promoter ... 75

Figure 2.8 UBF occupancy correlates with GC-rich sequences in the rRNA gene body ... 80

Figure 2.9 Conditional knockout models of RRN3 and UBF ... 81

Figure 2.10 Deletion of the Rrn3/Tif1a gene arrests mouse development during early cleavage divisions ... 83

Figure 2.11 qPCR analysis after RRN3 or UBF knockout ... 85

Figure 2.12 TTF1 diminishes its presence through the gene body after knockout ... 86

Figure 2.13 MNase digestion of UBF and RRN3 knockout MEFs ... 87

Figure 3.1 Cisplatin treatment of Ubfwt/wt_/Er-cre+/+_{/SvT iMEFS induces displacement of UBF from the nucleolus ... 94}

Figure 3.2 Cisplatin coordinately displaces UBF from the rRNA genes and arrests their transcription... 95

Figure 3.3 UBF loss induces synchronous apoptotic cell death selectively in oncogenically transformed iMEFs ... 97

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Figure 3.5 UBF loss arrests cell proliferation and leads to a cell cycle arrest ... 100

Figure 3.6 Apoptosis of oncogenically transformed cells after Ubf gene inactivation is p53 independent ... 102

Figure 3.7 p53 independent apoptosis is a general response to UBF loss in an oncogenic stress context ... 103

Figure 3.8 Cell cycle distribution of p53-null cells during UBF depletion ... 105

Figure 3.9 Cisplatin treatment of MEFs induces displacement of UBF from the nucleolus ... 114

Figure 3.10 Analysis of UBF loss in primary MEFs ... 115

Figure 3.11 Primary MEFs survive UBF loss while SV40Tt transformed iMEFs suffer cell death... 117

Figure 3.12 TIF1A loss does not induce TUNEL positive apoptosis in SV40Tt transformed MEFs ... 118

Figure 3.13 UBF loss leads to a cell cycle arrest and to a loss of mitotic cells ... 119

Figure 3.14 p53-independent apoptosis is a general response to UBF loss in an oncogenic stress context ... 120

Figure 4.1 rRNA genes in embryonic stem cells are fully unmethylated in the gene and control regions ... 128

Figure 4.2 Psoralen crosslinking analysis of the rRNA genes... 129

Figure 4.3 ChIP-seq analysis of the RPI machinery in ESCs ... 130

Figure 4.4 CTCF and cohesin are found upstream of the spacer promoter in ESCs ... 131

Figure 4.5 Preliminary 4-HT treatment of ES cells to induce CRE-mediated excision of Ubf and Rrn3 ... 132

Figure 4.6 Knockout of UBF leads to the formation of a closed conformation of the rDNA ... 132

Figure 4.7 Retinoic acid induced differentiation of embryonic stem cells ... 133

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Abbreviations

A adenine

A adenosine

a.a amino acid

ActD actinomycin D

AgNOR argyrophilic nucleolar organiser region ALU Arthrobacter luteus (element)

AML acute myeloid leukemia AP alkaline phosphatase ARF alternative reading frame ARN acide ribonucléique ARNr ARN ribosomal

ATM ataxia-telangiectasia mutated ATP adenosine triphosphate

bp base pair

BrUTP bromouridine-triphosphate

C cytosine

c-Myc avian myelocytomatosis virus oncogene cellular homolog CDK-2/4 cyclin-dependent kinase 2/4

CF core factor

ChIP chromatin immunoprecipitation CK2 casein kinase II

CpG cytosine-phosphate-guanine Cre causes recombination CSB Cockayne syndrome group B CTCF CCCTC-binding factor Ctrl control CV crystal violet DAPI 4′,6-diamidino-2-phenylindole DDR DNA-damage response DDX5 DEAD-box helicase 5 DFC dense fibrillar component

DMEM Dulbecco’s modified eagle’s medium DNA deoxyribonucleic acid

DNMT DNA (cytosine-5)-methyltransferase 1 DPC days post coitum

DSB double strand breaks dsDNA double-stranded DNA E-Syt extended-synaptotagmin EDTA ethylenediaminetetraacetic acid EF3 elongation factor 3

EGTA ethylene glycol-bis(β-aminoethyl ether EM electron microscope

eNoSC energy-dependent nucleolar silencing complex

EPI epiblast

EpiSCs epiblast stem cells ER-Cre estrogen receptor-Cre

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ESC embryonic stem cell EtBr ethidium bromide

ETS external transcribed sequences FACS fluorescence-activated cell sorting FBI fibrillarin

FBS fetal bovine serum FC fibrillar center

FCP1 TFIIF-associating component of CTD phosphatase FGF fibroblast growth factor

FGFR2 FGF receptor

FISH fluorescence in-situ hybridization Fob1p fork blocking less 1 protein

G guanine

GC granular component GC granular component

gDNA genomic DNA

GSK3 glycogen synthetase kinase 3 GTP guanosine-5'-triphosphate

H histone

h hour

H2A.Z histone 2 A family member Z HDAC histone deacetylases

HEAT Huntingtin, elongation factor 3, protein phosphatase 2A, TOR1 HMG high-mobility group

HR homologous recombination ICM inner cell mass

IF immunofluorescence IGS intergenic spacer

IGV integrative genomics viewer iMEF immortalized MEF

IP immunoprecipitation

ITS internal transcribed sequences KLF Krüppel-like factor 2

KO knockout

LBD ligand binding domain LIF leukemia inhibitory factor

LINE long interspersed nuclear element lncRNA long non-coding RNA

MAPK mitogen-activated protein kinase

MDa mega dalton

MDM2 murine E3 ligase murine double minute 2

me methylation

MEF mouse embryonic fibroblast

MEK MAPK/ERK kinase

miRNA microRNA

mRNA messenger RNA

mTOR mammalian target of rapamycin MYMMP1A protein MYB binding protein 1a MZT maternal-to-zygotic transition NaCl sodium chloride

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NAD nicotinamide adenine dinucleotide NBP nucleolar precursor bodies NCL nucleolin

ncRNA non-coding RNA

NHEJ non-homologous end-joining NML nucleomethylin

NOR nucleolar organizer region NoRC nucleolar remodling complex NPM nucleophosmin

nt.sec-1 _{nucleotides per second}

OCT4 octamer-binding transcription factor 4 PARP poly-(ADP-ribose) polymerase PAX6 paired box 6

PBS phosphate buffered saline PE primitive endoderm PFA paraformaldehyde pHT post 4-hydroxytamoxifen PI propidium iodide

PI3K phosphatidyl inositol-3 kinase PIC pre-initiation complex

PK proteinase K

PMSF phenylmethylsulfonyl fluoride

PP1 𝛾 protein phosphatase 1 isoform gamma PP2A subunit A of protein phosphatase 2A ppRb hyperohosphorylated Rb

PTEN phosphatase and tensin homolog qPCR real-time polymerase chain reaction r-proteins ribosomal proteins

r-proteins ribosomal proteins r.p.m. revolutions per minute

Ras retrovirus-associated DNA sequences from murine sarcoma viruses RB retinoblastoma protein

rDNA ribosomal DNA, a group term for the rRNA genes RFB replication fork barrier

RNA ribonucleic acid

RNP ribonucleoprotein particle

RPI RNA polymerase I, also known as POL1 and POLR1 RPL ribosomal protein large subunit

RRN3 regulation of RNA polymerase I rRNA ribosomal RNA

s/sec second

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SFES serum free embryonic stem cell media

SINE short interspersed nuclear element Sir2p silent information regulator 2 protein SIRT1 NAD-dependent deacetylase sirtuin-1 SL1 selectivity factor

SMAD1 mothers against decapentaplegic homolog 1 Smc1/3 structural maintenance of chromosomes protein 1/3

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snoRNPs small nucleolar ribonucleoprotein SNP single-nucleotide polymorphisms SOX (sex determining region Y)-box SpPr spacer promoter

SRP signal recognition particle SSC saline-sodium citrate buffer SSU small subunit

STAT signal transducer and activator of transcription 3 STS staurosporin

SvT simian vacuolating virus 40 TAg T1 terminator site 1

TAFs TBP-associated factors TBP TATA binding protein TCS Treacher-Collins syndrome

TE trophoectoderm

TIF-IA transcription initiation factor IA TIF-IB transcription initiation factor IB TIP5 TTF1-interacting protein 5 Topo I topoisomerase I

TOR1 target of rapamycin 1 TSS transcription start site

TTF1 transcription termination factor 1

TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling UAF upstream activating factor

UBF upstream binding factor UCE upstream control element UV ultra violet

VE visceral endoderm

WGBS whole-genome bisulfite sequencing

wt wild-type

µCi microcurie

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Acknowledgements

In the final steps of preparing this thesis, I have been able to step back and take the time to think about the five years I spent in the Moss Lab and realize just how grateful I am for this time in my life. So much of my experience in the lab and in Québec, in general, has shaped my professional and personal life.

Firstly, I would like to thank my thesis director Tom Moss for taking a chance on me and allowing me the opportunity to study in his lab. His enthusiasm for science is contagious and motivating. His patience, especially when I would become bogged down in details, and his encouragement to always keep pushing to find the answer were what helped me to persevere through the more challenging aspects of my Ph.D. Tom is a great mentor and a scientist to admire, especially in a field that is becoming obsessed with getting the end result quickly but not necessarily answering the “big questions”.

I would also like to thank my colleagues in the Moss Lab that I have worked with closely, Victor Stefanovsky, Nourdine Hamdane, Joël Boutin, Prakash Mishra and Jean-Clément Mars. I appreciated the discussion (scientific or not), the laughs, the crosswords and the drinks.

Je remercie Michel Tremblay en particulier, qui m’a enseigné la grosse majorité des protocoles en laboratoire. Il m’a aussi aidé avec plusieurs aspects de la vie de tous les jours qui sont normalement demandés aux parents. Du premier voyage au Costco, jusqu’à être témoin pour notre entrevue avec le greffier avant le mariage, Mike a toujours été là comme deuxième papa pendant mon séjour à Québec et je lui en serai toujours reconnaissante.

Of course, I must thank my husband, Gabriel, for his love, his support, his patience, his everything. His calm demeanor and his ability to encourage me at the same time as challenging me to do better has influenced how I approach research and many other aspects of life. Thank you as well for believing that I can be a student, a mother, a wife, and a future researcher.

I am very grateful for my family, both the old and the new. Thank you to my parents that have always supported me in my endeavors and are always just a phone call away. I owe my love of learning to them as they encouraged my love of reading and allowed me the space to pursue independent learning from a very young age. I also thank them for putting me in French Immersion even though no one in our family spoke the language. Merci aussi à ma nouvelle famille qui m’a accueilli à bras ouverts. Je vous aime beaucoup.

I very much appreciated my time at the research center and would like to thank the St. Patrick community at large. Whether it was to share protocols, borrow antibodies or discuss results, I always found a welcoming and collaborative environment. I’ve found the researchers very available and I appreciate the discussions about

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career development and the reference letters, in particular from Dr. Jacques Côté, Dr. Lucie Jeannotte, Dr. Nicolas Bisson and Dr. Josée Lavoie. I also thank my good friends in the center and out, including Jana Krietsch, Niraj Joshi, and Jean-Clément Mars, for making these years pass too quickly.

I also thank the Canadian Institutes of Health Research for funding my Ph.D. studies and for the opportunity to participate in multiple international conferences.

Finally, I would like to thank my thesis committee, Dr. Marlene Oeffinger, Dr. Lucie Jeannotte, and Dr. Darren Richard for agreeing to read and evaluate this thesis.

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Preface

The work presented in this thesis is the product of nearly four years of work as a doctoral student in the laboratory of Tom Moss. The appendices contain work related to my first project in the lab, which was not sustainable as a Ph.D. project and therefore is not presented within the chapters of this dissertation. In total, my time in the Moss lab has led to the publication of one co-first-author paper, two second-author papers, one third-author paper and one first-author review manuscript as well as one more co-first author paper that has been submitted.

The Moss lab is interested in the regulation of cell growth and in particular, ribosome biogenesis. Ribosome biogenesis is activated by mitogenic pathways such as ERK/MAPK and therefore the lab also is interested in the regulation of these growth-activating pathways.

I arrived in the lab summer 2011 and began studying the role of mammalian Extended-Synaptotagmin (E-Syt), a factor that the lab had previously characterized in Xenopus. They found that E-Syt is a negative regulator of ERK activation through its capacity to block internalization of the FGF receptor at the plasma membrane (Jean et al. 2010). My role in this project was to investigate E-Syt in human cell lines and in mouse, and whether the phenotypes observed in Xenopus were conserved. Unfortunately, most of these phenotypes are not conserved in cell culture, and the knockout of one of the E-Syt family members (E-Syt2) gave no phenotype.

Though I continued to collaborate on the E-Syt project throughout the years, I soon shifted my focus to study the regulation of ribosomal RNA transcription. By 2012 I had started characterizing the knockout of RRN3 and I presented this project at my doctoral exam in 2013 when I transitioned from the M.Sc. to the Ph.D. program. I continued my work on RRN3 and UBF, another RNA Polymerase I transcription factor, the results of which are presented in Chapters 2, 3 and 4 of this thesis.

My principle work in comparing the loss of RRN3 to that of UBF and studying the role of these factors in transcription and chromatin regulation of the ribosomal DNA is included in this thesis as Chapter 2. This work entitled “An enhancer adjacent chromatin boundary is maintained on the ribosomal RNA gene repeats even in the absence of the basal factors and active transcription” (Herdman C*_{, Mars JC}*_{, Stefanovsky VY, Tremblay}

MG, Sabourin-Felix M, and Moss T) has been submitted as a co-first author paper.

Chapter 3 of this dissertation is a second author manuscript published in Oncotarget in 2015: “Depletion of the cisplatin targeted HMGB-box factor UBF selectively induces p53-independent apoptotic death in transformed cells” (Hamdane N, Herdman C, Mars JC, Stefanovsky V, Tremblay MG, and Moss T). I played an integral part

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in the preparation of this article, completing experiments leading to multiple figures and answering major revisions. This article in its published form is also presented as Annex 1.

The most recent aspect of my doctoral research, the development of a model by which to study rDNA transcription in pluripotent cells and preliminary results using these cells, is presented in Chapter 4.

Though my involvement in the E-Syt project was short-lived, it was productive in the end. Firstly I participated in the revision process of the manuscript “The endocytic adapter E-Syt2 recruits the p21 GTPase activated kinase PAK1 to mediate actin dynamics and FGF signalling” (Jean S, Tremblay MG, Herdman C, Guillou F and Moss T), which was published in Biology Open in 2012. In this publication, I was responsible for investigating the role of Xenopus E-Syt in cell migration which resulted in Figures 4b, 4c, and 5b. This publication is included in Annex 2.

Secondly, the negative results demonstrating the lack of phenotype in the mouse after the loss of E-Syt2 and E-Syt3 were published as a co-first-author paper in Cell Cycle in 2014. “Loss of Extended Synaptotagmins ESyt2 and ESyt3 does not affect mouse development or viability, but in vitro cell migration and survival under stress are affected” (Herdman C*, Tremblay MG*, Mishra PK, and Moss T) is included in Annex 3. All figures except 6c and 6d were produced by Michel Tremblay and myself. I participated in the writing and editing of the article.

The third publication that arose from this collaboration was a second author paper in the Journal of Biological Chemistry in 2015. Michel Tremblay did the majority of the work with contributions from the co-authors. I performed the immunofluorescence analysis that accompanied all of the mutants used in the immunoprecipitation experiments but these controls were not included in the paper. I also optimized an immunofluorescence protocol that analyzed endocytosis which translated into Figures 4e-g in the paper. This manuscript entitled “Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity” (Tremblay MG, Herdman C, Guillou F, Mishra PK, Baril J, Bellenfant S and Moss T) is included in Annex 4.

Finally, I had the opportunity to write an invited review of the literature for Pharmacological Research in 2016. This paper, “Extended-Synaptotagmins (E-Syts); the extended story” (Herdman C and Moss T), is presented as Annex 5.

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Chapter 1 Introduction

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1 Introduction

Cell growth, or increase in cell mass, requires a significant number of ribosomes to produce the cell’s translational requirement. The biogenesis of ribosomes is therefore tightly linked to the cell’s capacity to grow and therefore proliferate (Lempiäinen & Shore 2009). In fact, 1-2 million ribosomes are produced every cell generation, utilizing all three RNA polymerases and a significant proportion of the cells translational activity. In growing cells, the ribosomal RNAs (rRNAs) account for 35% to 60% of all gene transcription (Moss & Stefanovsky 2002). As the manufacture of ribosomes is so energetically demanding, it is not surprising therefore that it is a major limiting task for cancer cell proliferation. In fact, changes in rRNA levels and the deregulation of many proteins involved in ribosome biogenesis have been linked with cancer and the nucleolus has long been used as a prognostic marker for tumor cells (Amsterdam et al. 2004, Derenzini et al. 2009, Drygin et al. 2010, Ruggero 2012, Ruggero & Pandolfi 2003).

The mammalian ribosome is a 4-MDa complex whose subunits are assembled in the nucleolus, the largest subnuclear organelle, from four rRNAs and ~82 ribosomal proteins (r-proteins). Several hundred other proteins and small RNAs are also needed to assemble the ribosome. Three rRNAs are transcribed from hundreds of nucleolar rDNA repeats by RNA polymerase I (RPI, also known as Pol I, POL1, POLR1), which has a set of dedicated transcription factors, namely UBF, RRN3/TIF-IA, SL1/TIF-IB and TTF1 (Moss & Stefanovsky 2002, Moss et al. 2007, Pederson 2011). These factors play a central role in the regulation of rRNA transcription, and two in particular, UBF and RRN3, have been linked to cell growth and proliferation. The roles these two proteins play in vivo is the primary focus of this thesis and hence will be introduced in the following chapter.

1.1 The nucleolus: site of ribosome biogenesis

The nucleolus is the largest body in the nucleus where the machinery necessary for ribosome biogenesis is concentrated. As the nucleolus is visible in brightfield microscopy, it was identified in the early 19th_-century

(Pederson 2011). However, it was a century later before McClintock and Heitz independently associated the nucleolus with a particular chromosomal locus, thus named the nucleolar organizer region (NOR) (Heitz 1931, McClintock 1934) and still decades before the NOR was associated specifically with the rRNA genes or ribosomal DNA (rDNA) (Pederson 2011). We now know that the nucleolus is the site of ribosomal RNA (rRNA) transcription, maturation and assembly of the ribosomal subunits.

1.1.1 The structure of the nucleolus

The nucleolus is formed of three visible compartments that were first described by their density of fibrils or granules observed using the electron microscope (EM). These are the fibrillar centers (FCs) of lower electron density surrounded by the dense fibrillar components (DFCs), which are then further surrounded by the

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granular components (GCs) (Figure 1.1) (Farley et al. 2015, Pederson 2011). These compartments are thought to partition the different steps of ribosome biogenesis. For instance, the first step of rDNA transcription likely occurs at the interface between the FC and the DFC, whereas early processing of the rRNA precursor (pre-rRNA) happens in the DFC. The location of the actively transcribed rDNA is, however, still debated and some believe that transcription by RPI also occurs in the DFC while inactive factors are stored in the FC (Raška et al. 2006). Finally, ribosome subunit maturation takes place in the GC, and these subunits are then exported to the cytoplasm, where they undergo the final processing steps to render them translationally active. One may observe the different compartments of the nucleolus by immunolabelling specific ribosome biogenesis factors. For example, upstream binding factor (UBF), an RPI transcription factor, can indicate the FC and DFC regions whereas a late processing factor such as Nop52 can mark the GC (Savino et al. 2001). There can be multiple FCs per nucleolus, even up to two hundred as is the case with human fibroblast cell lines or mouse oocytes depending on their activity (Jordan & McGovern 1981, Mirre & Stahl 1981).

Though there are three visible components of the nucleolus, in fact, its architecture is not stable, but rather depends on a precise regulation of ongoing ribosome biogenesis and the interplay between the multitude of proteins that dynamically exchange in and out of the nucleolus. The nucleolus appears as a distinct body due to the proteins that associate with the rDNA that form a core structure that other nucleolar and non-nucleolar proteins can interact with stably or transiently, which in some cases can even be just for a few tenths of seconds. Furthermore, even the typical nucleolar proteins can migrate out of the nucleolus depending on the cell cycle stage or environmental cues (Pederson 2011). As the proteins that are present in the nucleolus are

Figure 1.1 Structure of the nucleolus as seen through an electron microscope

Electron micrograph of a thin-sectioned nucleolus from a mouse cell. The fibrillar centers, dense fibrillary components and granular components are indicated with f, d and g respectively. The arrows indicate perinucleolar heterochromatin and the asterisk denotes a DFC clump within the FC. Reprinted with permission from Raska I., Trends in Cell Biology, 2003.

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so dynamic, the structure of the nucleolus appears to be a consequence of rRNA transcription and processing (Misteli 2001, Misteli & Phair 2000, Raška et al. 2006). For instance, inhibiting RPI transcription with Actinomycin D (ActD), a DNA intercalator with a particular specificity for rDNA due to its G/C content, causes a loss of nucleolar structure (Floutsakou et al. 2013, Sirri et al. 2008). Another study used 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB), a nucleoside analog that can indirectly and reversibly inhibit rRNA synthesis. They concluded that it is the production of rRNA transcripts that maintains the localization of rRNA processing factors in the nucleolus and not the rDNA (Louvet et al. 2005).

In addition to the multitude of proteins and the rDNA found in the nucleolus, there are, of course, many copies of the rRNA transcripts. It was previously shown that a small quantity of non-ribosomal RNA was also present in the FC (Thiry 1988). Recently, RPII transcripts from intronic Alu insertion elements were observed at high concentration in the human nucleolus and that they contribute to its structure through interaction with nucleolar protein nucleolin (NCL) (Caudron-Herger et al. 2015). Therefore, it is not only RNA produced from the rDNA that maintains the nucleolar structure but also noncoding RPII transcripts.

On the whole, the nucleolus is a stable entity but with a highly dynamic composition that responds to signaling pathways, drug treatments, cell cycle status, etc. So, to describe its structure as simply the three visible structural components, FC, DFC, and GC, is oversimplifying this complex multifunctional organelle.

1.1.2 Nucleolar organizer regions

During mitosis, rRNA transcription arrests and the nucleolus disassembles in mammals and plants but not in yeast. At the end of mitosis, the nucleolus is reformed, remaining stable throughout interphase until the next cell cycle (Boisvert et al. 2007, Németh & Längst 2011, Raška et al. 2006). Nucleolar organizer regions (NORs) are the morphological sites where nucleoli form at the end of mitosis (McClintock 1934). These sites are the chromosomal loci where the rDNA arrays are found and are located on the short arms of acrocentric chromosomes (13, 14, 15, 21 and 22 in human cells), between the heterochromatic centromeric and telomeric DNA. In contrast, mice have all acrocentric chromosomes, but the NORs are still found on particular chromosomes (12, 15, 16, 18, 19) and are close to centromeric and telomeric DNA (Figure 1.2). Furthermore, the number of NORs varies between species, and the rDNA composition of NORs can vary between cells of an individual or between individuals of the same species (Stults et al. 2009, 2008). Notably, NORs are thought to contain only the rDNA and no other DNA sequences (Sakai et al. 1995).

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Electron microscopy studies revealed that NORs appear as gaps on metaphase chromosomes, and are now referred to as secondary constrictions since centromeres are known as primary constrictions (Figure 1.3) (Heitz 1931, McClintock 1934). Heliot and colleagues discovered that these gaps were due to the low compaction of rDNA repeats in the active NOR. In fact, the satellite DNA repeats adjacent to the NORs referred to as perinucleolar heterochromatin, are ten times more condensed than active rDNA (Héliot et al. 1997). Even though NORs are decondensed, they have a tight link with heterochromatin. For example, heterochromatin from non-NOR chromosomes associates with nucleoli throughout the cell cycle. Also, the silent X-chromosome needs to temporarily associate with nucleoli during S phase to maintain its heterochromatic nature (Manuelidis & Borden 1988, Zhang et al. 2007). One reason for the proximity to heterochromatin could be to separate NORs from genes transcribed by RPII and RPIII (McStay 2016). Additionally, heterochromatin could prevent homologous recombination between rDNA repeats, therefore protect against genomic instability (McStay & Grummt 2008). The role of the rDNA and NORs in genomic instability and DNA damage will be discussed further at a later point in this chapter.

Diagram of the telocentric mouse chromosomes and the acrocentric human chromosomes indicating the position of the NORs. Modified and reprinted with permission from McStay B. and Grummt I., Annu Rev Cell Dev Biol, 2008.

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Another identifying feature of active NORs during metaphase is that they can be stained with silver nitrate, the AgNOR staining technique, due to the acidic or argyrophilic domains found in the RPI transcription machinery (Goodpasture & Bloom 1975, McClintock 1934, Ploton et al. 1994)). For example, RPI transcription factors UBF, SL1, and TTF1 have been identified as AgNOR proteins on mitotic NORs (Figure 1.4) (Jordan et al. 1996, Leung et al. 2004, Roussel et al. 1993, Sirri et al. 1999). These factors stay associated with the rDNA during mitosis, thereby allowing a rapid restart of transcription at the start of G1. In fact, UBF can induce an open chromatin conformation and therefore, may in part be responsible for inducing secondary constrictions on mitotic chromosomes (Chen et al. 2004). Furthermore, random insertions of large arrays of the 60/81 bp rDNA enhancer repeats from Xenopus known to bind UBF into human chromosomes induce visible secondary constrictions referred to as pseudo-NORs (Mais et al. 2005), and their formation depends on UBF (Grob et al. 2014). Interestingly, these pseudo-NORs, like natural NORs, associate with UBF and many other factors including RPI and even ribosome processing factors throughout the cell cycle and are stained with silver nitrate (Prieto & McStay 2007).

However, not all NORs are actively transcribed and chromosomes carrying such silent NORs do not display an associated secondary constriction, nor AgNOR staining. These are generally believed to be heterochromatic, and their inactivity appears to be inherited through many cell generations (Kurihara et al. 1994, Roussel et al. 1993).

The silver staining of metaphase spreads technique has been used to identify the number of active NORs in human cells. It is estimated that there is an average of eight active NORs and that this can vary from seven to ten (Héliot et al. 2000). This is curious since around 50% of the rDNA is heterochromatically silenced, which would mean that there should be five active NORs per cell (half of the total number of short arms on acrocentric chromosomes). This leads to the hypothesis that NORs must be in fact variable in their rDNA content and have both active and silent repeats (McStay 2016). Indeed, variation in rDNA content of NORs Diagram of an acrocentric DAPI-stained human chromosome with the secondary constriction or NOR in red on the short arms. Reprinted with permission from McStay B. and Grummt I., Annu Rev Cell Dev Biol, 2008.

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these products using pulsed field gel electrophoresis and then hybridizing using rDNA probes (Sakai et al. 1995). This technique has been done by numerous groups using EcoRV and has shown that most human NORs are made up of 70 copies of the rDNA but more recently the Pierce group found differences in NOR size all the way from just one repeat (40kb) to over 130 repeats (6Mb) in different human cell lines (Stults et al. 2009, 2008).

1.1.3 Functions of the nucleolus

Over ten years ago, mass spectrometry studies had purified over 700 proteins from nucleoli of human cells and approximately 90% of human nucleolar proteins have yeast homologues, indicating the high level of conservation of the nucleolar proteome (Andersen et al. 2005, 2002; Scherl et al. 2002). Since 2014, more than 4500 proteins are named in the nucleolar protein database (http://lamondlab.com/NOPdb3.0/) (Quin et al. 2014). It is estimated that only around 30% of these proteins play a role in ribosome biogenesis, which is the most studied function of the nucleolus. However, it is less well known that there are in fact many other cellular processes that involve the nucleolus. These include cell cycle regulation, cellular stress response, control of aging, and the maturation of non-ribosomal RNAs and RNP complexes. One general function of the nucleolus is protein sequestration or release that can affect all the above listed processes (Emmott & Hiscox 2009).

Immuno-FISH labeling of the rDNA (green) and UBF (red) in HeLa cells during metaphase and interphase. The nuclei are stained with DAPI (blue). Reprinted with permission from McStay B. and Grummt I., Annu Rev Cell Dev Biol, 2008.

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1.1.3.1 Ribosome biogenesis

Ribosome biogenesis is the main function of the nucleolus and the most well-studied. This process consumes much of the cell’s energy and is tightly linked to cell growth. In eukaryotic cells, assembly of a mature ribosome involves bringing together ~80 ribosomal proteins (r-proteins) and the four rRNAs (28S, 18S, 5.8S and 5S), which are the ribozymes in the ribosome core that carry out catalyzation of decoding and amino acid polymerization during translation (Lafontaine 2015, Tschochner & Hurt 2003). In fact, the cell utilizes all three RNA polymerases (Pol I, RPII and Pol III) to accomplish the task of generating the cell’s protein factory, the ribosome. RPI transcribes the precursor rRNA (pre-rRNA) which will become the mature 28S, 18S, and 5.8S, RPII produces the messenger RNAs (mRNAs) that encode the r-proteins and RPIII transcribes the 5S rRNA (Figure 1.5). The initial and rate-limiting step of ribosome biogenesis is the transcription of the rDNA to produce the pre-rRNA, named 35S in yeast and 47S in human (Mougey et al. 1993, Tschochner & Hurt 2003). In addition to the components that form the ribosomal subunits, ribosome biogenesis involves numerous other factors such as assembly factors and small nucleolar ribonucleoprotein complexes (snoRNPs) that contain the snoRNAs that target these complexes to the rRNA. These include methyltransferases and endo- or exonucleases that process the pre-rRNA, chaperones, and helicases to facilitate folding of the RNP subunits and ATP/GTPases to help assemble the complexes. In fact, the pre-rRNA undergoes hundreds of post-transcriptional modifications, namely methylation, and pseudouridylation. The early processing steps occur co-transcriptionally at the FC/DFC interface. These processing factors, the rRNAs, and the r-proteins come together to form the 90S ribosomal subunit in the GC of the nucleolus, which is then divided into the pre-40S and pre-60S subunits. These pre-ribosome subunits undergo the last modifications in the nucleoplasm and are then exported to the cytoplasm as the mature 40S and 60S subunits ready to be assembled into the ribosome (Tschochner & Hurt 2003).

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1.1.3.2 Cell cycle regulation

The nucleolus is a dynamic structure that disassembles during mitosis, which corresponds to a decrease in rDNA transcription, and reassembles at the start of the next cell cycle (Hein et al. 2012, Sirri et al. 2002). In fact, this reassembly and rDNA transcription are prerequisites for G1-S progression. The major role for the nucleolus in cell cycle regulation seems to be in the sequestration and release of factors that directly affect the cell cycle (Boisvert et al. 2007). For example, the tumor suppressor retinoblastoma protein’s (RB) location is temporally regulated throughout the cell cycle. Nucleolin/C23 (NCL) associates with active RB (pRB) during G1 and could have a role in RB’s regulation of the G1-S checkpoint (Angus et al. 2003, Bartek et al. 1996, Grinstein et al. 2006). Hyperphosphorylated RB (ppRB) is released from the nucleolus until late S or G2, at which point nucleophosmin/B23 (NPM) interacts with RB mediating its renewed retention in the nucleolus (Takemura et al. 2002). Though the mechanisms or functional purpose of the retention and release of certain factors is unclear, it does show that the nucleolus has a role in the cellular distribution of factors that are important for cell cycle regulation (Boisvert et al. 2007).

Figure 1.5 Ribosome biogenesis involves all three nuclear polymerases

Ribosomal biogenesis starts with transcription of the 47S rRNA precursor by RPI in the nucleolus (in brown). The pre-rRNA undergoes processing in the granular component of the nucleolus (light brown) by snoRNPs and processing factors produced from mRNAs transcribed by RPII. The mature rRNAs along with the 5S rRNA transcribed by RPIII are assembled into the ribosomal subunits and then exported from the nucleus.

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1.1.3.3 Stress response

The protein components of the nucleolus are significantly altered under various stress conditions, and the release of nucleolar factors is an important characteristic of the tumor-suppressor protein p53-related stress response (Boulon et al. 2010, Moore et al. 2011, Rubbi & Milner 2003). In normal cells, p53 is continually transcribed but short-lived and found at low levels due to its ubiquitination by the murine E3 ligase murine double minute 2 (MDM2, HDM2 in human cells) marking it for nuclear export and proteasomal degradation (Olson 2004). Upon cellular stress, such as DNA damage, inhibition of ribosome biogenesis or oncogene activation, p53 is stabilized, leading to activation or repression of genes important for cell cycle regulation. Notably, the increased level of p53 activates p21, which in turn inhibits cyclin D1-CDK4 and cyclin E1-CDK2 leading to a G1 arrest (Prives 1998). Subsequently, if the stress or damage is not rectified, this process leads to apoptosis.

Two main types of cellular stress, in particular, induce a response involving ribosome biogenesis factors; oncogenic stress and nucleolar stress. Stress induced by oncogene activation leads to upregulation of the nucleolar factor murine p19ARF (p14ARF in human cells; alternative reading frame), which can sequester its binding partner MDM2 in the nucleolus. This sequestration favors the accumulation of p53 in the nucleus and subsequent cell cycle arrest (Weber et al. 2000, Wsierska-Gadek & Horky 2003). However, ARF levels are variable during the cell cycle and it is not found in all cell types, nor is it conserved in all species, therefore, other factors likely can replace ARF as MDM2 binding partners (David-Pfeuty & Nouvian-Dooghe 2002, Olson 2004, Zhang & Xiong 2001). Other nucleolar proteins that may have a role in stress-induced p53 stabilization include NPM and NCL, which both have been shown to interact directly with p53 (Colombo et al. 2002, Daniely & Borowiec 2000).

In the case of nucleolar stress, which is caused by defects in ribosome biogenesis, it appears that p53 is activated by r-proteins (Figure 1.6). Multiple r-proteins (RPS3, RPS5, RPS7, RPL5, RPL11, RPL23, RPL26), as well as the 5.8S and 5S rRNAs, have been shown to interact with MDM2 or p53 (Deisenroth & Zhang 2010, Fontoura et al. 1992, Riley & Maher 2007, Zhang & Lu 2009). Upon inhibition of ribosome biogenesis, a pool of uncomplexed ribosomal proteins could be available for interaction with other non-ribosomal partners. For example, in growing cells, normally L11 would be assembled into ribosomes and no longer present in the nucleolus. However, if ribosome biogenesis was inhibited, L11 would be in excess and free to bind MDM2 and inhibit the export of p53 (Zhang et al. 2003). Therefore, depending on the cellular stress, oncogenic or nucleolar, either ARF or r-proteins can stabilize p53 and induce a cell cycle arrest.

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1.1.3.4 Control of aging

Aging is the time-dependent decline of cellular functioning and is characterized by certain hallmarks including cellular senescence, stem cell exhaustion, genomic instability, telomere attrition, epigenetic alterations, and deregulated nutrient sensing (López-Otín et al. 2013). The nucleolus and ribosomal DNA are involved in many of these hallmarks and appear to be an important aspect of cellular aging, which has been demonstrated mostly through studies in yeast.

The main body of work involving the nucleolus and aging involves the sirtuins, a family of NAD+-dependent histone deacetylases that were linked to prolonging life span in yeast due to their role as silencers of the rDNA (Hein et al. 2012, Kennedy et al. 1995, 1997). Ribosomal gene arrays are very unstable due to their repetitive nature, which can lead to the loss of copies due to homologous recombination (HR). They are the most

(A) In a normal cell, p53 levels are maintained by the interaction with MDM2 which ubiquitinates p53 targeting it for export from the nucleolus and proteosomal degradation. (B) Upon nucleolar stress, uncomplexed ribosomal proteins are released from the nucleolus and bind to MDM2, allowing the accumulation of p53 in the nucleus. Reprinted with permission from Quin J. et al., BBA – Molecular Basis of Disease, 2014.

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unstable regions of the yeast genome (Kobayashi 2008) but there is a gene amplification system in place to regain lost copies. Fob1p blocks replication forks by binding to a replication fork barrier (RFB) downstream of the 35S coding sequence, leading to stalled forks and double strand breaks (DSBs) (Brewer et al. 1992, Burkhalter & Sogo 2004, Kobayashi et al. 1992, 2004; Weitao et al. 2003). Amplification occurs when the breaks are repaired by unequal sister chromatid exchange, due to transcripts originating from the promoter E-pro in the IGS. This transcription causes the displacement of the cohesin complex holding the sister chromatids in place and therefore promotes slippage and unequal crossovers (Kobayashi & Ganley 2005). A sirtuin family member and histone deacetylase, Sir2p, represses transcription from E-pro once amplification has restored any rDNA copy loss (Fritze et al. 1997, Saka et al. 2013). The deletion of Sir2p leads to hyper-recombination of the rDNA and subsequently a shorter lifespan (Kaeberlein et al. 1999). It is this balance of gene amplification and deleterious recombination regulated by Fob1p and Sir2p respectively that maintains the appropriate number of rRNA genes and their stability. These concepts constitute the ‘rDNA theory of aging’ where rDNA locus instability translates to genome-wide instability (Kobayashi 2008). This theory also describes the timing of senescence as being determined by the rDNA. As the rDNA forms the most unstable loci in the genome, the cell can likely discern this increasing instability throughout cell divisions or replicative stress and therefore, sense when the limit of divisions is being reached. Cells will enter senescence rather than continue to accumulate damage and risk becoming transformed (Kobayashi 2011a, 2014; MacInnes 2016).

Figure 1.7 Model for eNoSC function in the nucleolus

A model depicting the proposed function of the energy-dependent nucleolar silencing complex (eNoSC). SIRT1, NML, and SUV39H1 form a complex on the rDNA and modify H3K9 methylation, which could influence silencing and heterochromatin formation. This complex is dependent on the energy status of the cell, as depicted by the high or low glucose situations. Reprinted with permission from Murayama A et al., Cell, 2008.

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Though ribosomal DNA stability has been linked to aging and senescence in yeast, studies in mammals have just recently begun to show the link between ribosomal biogenesis and aging. Sirtuin 1 (SIRT1) is part of the eNoSC, or energy-dependent nucleolar silencing complex, that potentially epigenetically silences the rDNA based on the energy status of the cell (Figure 1.7) (Murayama et al. 2008). Interestingly, overexpression of SIRT1 in mouse brains prolongs life expectancy (Satoh et al. 2013). The nucleolus has been linked to senescence in mammalian cells as changes in nucleolar morphology are observed in senescent cells, which only have one large nucleolus as opposed to several smaller ones (Mehta et al. 2007). This typical change in nucleolar morphology in response to nucleolar stress often leads to cell cycle arrest. Senescent cells arrest at the G1-S checkpoint which is regulated in part by p53. As described in the previous section, some nucleolar factors, such as p19ARF mediate p53 stability and are therefore also involved in regulation of the senescent phenotype (Hein et al. 2012). The 5S ribonucleoprotein particle (RNP) which consists of RPL11, RPL5, and the 5S rRNA was recently shown to induce senescence upon oncogenic or replicative stress through p53 activation (Nishimura et al. 2015).

1.1.3.5 Other RNA maturation

The nucleolus has also been implicated in the processing and maturation of RNA species other than the rRNA precursor. This includes the assembly of RNP complexes such as telomerase, spliceosomal small nuclear RNPs (snRNPs) and RNA modifications on tRNAs, RNAse P RNA, signal recognition particle (SRP) RNA and more recently some microRNAs (miRNAs) (Boisvert et al. 2007). The SRP complex, composed of six proteins and an RNA, are found in the nucleolus prior to their export to the cytoplasm which indicates a potential function for the nucleolus in the maturation of this complex (Jacobson & Pederson 1998). Other examples are RNA species transcribed by RPIII in the nucleus, which are then imported into the nucleolus, potentially for modification or maturation, similarly to the 5S rRNA. These include tRNA, RNase P RNA and the U6 spliceosomal snRNA (Ganot et al. 1999, Jacobson et al. 1997).

1.2 Ribosomal DNA

In a cell, the r-proteins and the rRNA transcripts are equimolar and their amounts are tightly regulated to fill the cell’s requirement for ribosomes, but the number of rDNA copies that are transcribed to produce the rRNA is very different. The number of rDNA repeats differs between organisms, from less than 100 to more than 10,000 repeats (McStay & Grummt 2008). In Saccharomyces cerevisiae (S. cerevisiae) there are ~200 copies of a 9.1 kb repeat located on chromosome XII (Petes 1979) whereas Arabidopsis thaliana (A. thaliana) has 700-800 copies of a ~10 kb repeat found close to telomeres on chromosomes 2 and 4 (Copenhaver & Pikaard 1996). Drosophila melanogaster (D. melanogaster) has two NORs, one in the centromeric heterochromatin of the X chromosome and the other on the short arm of the heterochromatic Y chromosome. Each NOR contains

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about 200 copies of an 11-17kb repeat, but copy number can vary greatly (Williams & Robbins 1992). Humans and mice each have around 200 rDNA repeats on the short arms of five acrocentric chromosomes (Britton-Davidian et al. 2012, Henderson et al. 1972), though in each genome, segments of rDNA are found inserted on other chromosomes and probably represent pseudogenes.

1.2.1 Organization of the rDNA

Each mammalian rDNA repeat is quite large, spanning ~43 kb in humans and ~45 kb in mice and these are arranged in a head-to-tail manner (Gonzalez & Sylvester 1995, Grozdanov et al. 2003). The rDNA is the region in the cell that is the most actively transcribed and the pre-rRNA is assembled co-transcriptionally, meaning that the processing machinery and the transcription machinery are working closely together. This mechanism can be observed by electron microscopy using the Miller chromatin spreading technique (Miller spread) which produces "Christmas tree" structures (Moss et al. 2007). The rDNA forms the trunk of the tree and the multiple rRNA transcripts are the branches. The round, dark spots at the end of the nascent pre-rRNA transcripts are referred to as terminal knobs, which are thought to be made up of the SSU processome (Figure 1.8) (Mougey et al. 1993).

The mouse and human rDNA repeat is composed of a long intergenic spacer (IGS) of ~30 kb and the coding sequences for the pre-rRNA (13-14 kb). The pre-rRNA or 47S is made up of the external transcribed sequences (ETS), internal transcribed sequences (ITS) and the rRNAs 18S, 5.8S, and 28S (Figure 1.9). In most species, the fourth rRNA, 5S, and the tRNAs are transcribed by RPIII at the nucleolar periphery, except Electron micrograph depicting a ‘Miller’ spread or Christmas tree from a mouse Ltk-_{cell. The rDNA is being}

transcribed by many closely packed polymerases. The inset shows the magnified rDNA, many rRNAs and the 3’ terminal knobs at the extremities. Reprinted with permission from Moss T., Cell Mol Life Sci, 2007.

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in yeast where the 5S sequence is linked to the rDNA and therefore transcribed in the nucleolus (Moss et al. 2007).

The IGS houses the regulatory elements such as promoters, spacer promoters, enhancer repeats, and terminators. The promoter is made up of two elements, the core promoter at the transcription start site (TSS) and the upstream control element (UCE), which lies ~100 nucleotides upstream (Haltiner et al. 1986, Learned et al. 1986). There are multiple terminator elements found at the 3’ end of the rDNA repeat (T1-T10) as well as

one terminator (T0) 170 bp upstream of the promoter UCE in mouse (Figure 1.9) (Grummt et al. 1985, 1986a;

Henderson & Sollner-Webb 1986, McStay & Reeder 1986, Moss 1983). Early studies in Xenopus demonstrated that the same sequence just upstream of the rRNA coding sequence was found repeated in the IGS (Moss 1983, Moss & Birnstiel 1979). This sequence was found to be the RPI promoter and its upstream repeats were therefore referred to as ‘spacer promoters’. These spacer promoters (SpPr) stimulate efficient pre-rRNA production and act in conjunction with adjacent arrays of short repeated enhancer sequences (Caudy & Pikaard 2002, De Winter & Moss 1986, 1987; Dunaway & Dröge 1989, Moss 1983, Osheim et al. 1996). Several functions of the T0 promoter-proximal terminator and IGS transcripts were originally suggested,

mostly based on work done in Xenopus. These include a process called ‘readthrough enhancement’, by which polymerases recruited by the spacer promoter and transcribing the IGS are arrested at T0 and then handed

over to the pre-rRNA promoter (Grimaldi et al. 1990, Längst et al. 1998, Moss 1983). If the T0 or the SpPr are

mutated, termination of the IGS transcripts is impaired, and the amount of pre-rRNA transcripts produced from A representation of the mammalian rDNA repeat (not to scale) is pictured with the transcribed region highlighted in yellow and the intergenic spacer in grey. The regulatory elements are indicated; terminator elements (T), spacer promoter (SP), promoter-proximal terminator (T0), promoter (P) formed of the

upstream control element (UCE) and core promoter (Core), and the transcription start site (+1). Reprinted with permission from Russell J., Zomerdijk JCBM., Trends in Biochemical Sciences, 2005.

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the adjacent gene is reduced. Therefore, transcription from the IGS is clearly important for regulating the neighboring gene’s production (Grummt et al. 1985, 1986a; Henderson & Sollner-Webb 1986, McStay & Reeder 1986, Moss 1983). More recently, this region has been implicated in regulating the active chromatin state and gene silencing (Mayer et al. 2006, Santoro et al. 2009). The role of the T0 terminator and the

transcripts originating from the SpPr in gene silencing will be discussed in more detail in another section of this chapter. The remainder of the IGS possesses many intermediate repetitive and transposable elements, and may potentially contain further regulatory elements, but to date, these appear to be of minor importance (McStay & Grummt 2008).

Due to the large number of rRNA genes and the repetitive nature of these sequences, there is not currently complete sequencing data available for the entire region. The coding region of the rDNA was sequenced and is available in GenBank for humans (Acc. No. U13369) and for mice (BK000964), however, there are many possible small variations in this sequence (Grozdanov et al. 2003, Kuo et al. 1996). The coding sequences for 18S, 5.8S and 28S are very highly conserved between species, but promoter and other rDNA sequences (ITS, ETS, IGS) are not. This is one reason why RPI machineries are incompatible between most animal species. For example, mouse and human systems are functionally incompatible (Moss et al. 1985, 2007).

1.2.2 Epigenetic regulation of the rDNA

The epigenetic regulation of genes is manifested by the addition of epigenetic marks either on the DNA itself such as methylation or on the histones that make up the chromatin such as acetylation, methylation, ubiquitination, sumoylation, and phosphorylation. These modifications are performed by various enzymes such as histone acetyltransferases and methyltransferases called writers, are removed by erasers (e.g. histone deacetylases) and bound by readers (e.g. bromodomains, chromodomains) (Figure 1.10). Over the years, specific modifications have become associated either with active transcription or gene silencing however it is the complex pattern of these marks, the histone code, that can give an indication of gene activity (Wang et al. 2007). The rDNA’s transcriptional status is also demonstrated by epigenetic marks. Silenced genes are methylated and the affiliated histones have repressive marks, whereas the active genes lack DNA methylation and are bound by RPI and RPI-specific transcription factors (Németh & Längst 2011).

1.2.2.1 rDNA activity status and chromatin

Growing cells actually have three populations of rDNA; (1) genes that are actively being transcribed and therefore are euchromatic and coupled with pre-rRNA molecules and the RPI machinery, (2) inactive genes that are in a potentially active state and still euchromatic and (3) silent genes that are methylated and are heterochromatic. The intergenic spacer of active, inactive or silent rRNA genes is nucleosomal but the presence of histones in the coding region of the active rDNA repeats is still debated. Some studies in yeast