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Phenotypic and Transcriptomic Consequences of Ribosomal DNA Copy Number Variation in Caenorhabditis Elegans and Saccharomyces Cerevisiae

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Phenotypic and Transcriptomic Consequences of Ribosomal DNA Copy Number Variation in Caenorhabditis Elegans and Saccharomyces Cerevisiae Phenotypic and transcriptomic consequences of ribosomal DNA copy number variation in Caenorhabditis elegans and Saccharomyces cerevisiae Epigenetics ISP, The Babraham Institute, Cambridge Christ’s College, University of Cambridge André Jean Zylstra September 2019 This dissertation is submitted for the degree of Doctor of Philosophy Declaration of Originality This work was carried out at the Babraham Institute under the supervision of Dr Jon Houseley, with co-supervision by Olivia Casanueva, between January 2016 and September 2019. This thesis is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Table of Assistance and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University. This thesis does not exceed 60,000 words, excluding references, tables and appendix. 1 Abstract Phenotypic and transcriptomic consequences of ribosomal DNA copy number variation in Caenorhabditis elegans and Saccharomyces cerevisiae André Zylstra Ribosomal DNA (rDNA) forms the template for mature ribosomal RNA (rRNA) and shows considerable sequence conservation through evolution. In eukaryotic genomes rDNA is present in one or more tandem repeat arrays, with copy number variation (CNV) commonly observed in inter- and intra-species comparisons. rDNA CNV has been primarily studied in the budding yeast Saccharomyces cerevisiae, where an environmentally sensitive copy number amplification mechanism corrects for repeat loss from this inherently unstable locus. Furthermore, there is much evidence implicating rDNA instability and production of extrachromosomal rDNA circles (ERCs) as causative processes in yeast replicative ageing. By contrast, the functional consequences of rDNA CNV have been largely understudied in metazoans and as such rDNA CNV represents a source of poorly understood genomic variation. I undertook this project to investigate possible functional and transcriptomic consequences of rDNA CNV in an animal model, the nematode Caenorhabditis elegans. Additionally, I produced and analysed RNA-seq datasets studying the transcriptomic effects of rDNA CNV, either due to genomic CNV or ERC accumulation, in S. cerevisiae. After developing methods for assaying rDNA copy number in C. elegans by pulsed field gel electrophoresis (PFGE) and quantitative PCR, I determined that there was notable variation between and within laboratory strains. Using a genetic crossing strategy, I derived strains believed to be essentially isogenic with either wild type (WT) or approximately 2.5-fold amplified rDNA. A variety of phenotypic assays found generally small effect sizes for differences unambiguously attributed to rDNA CNV. Some experiments were, however, confounded by unexpected developmental differences and 2 strain pathology, likely the consequence of unidentified background genetic variation. Transcriptomic analyses were consistent with these observations and revealed some genes differentially expressed between samples with different rDNA CN at either young or aged timepoints. Comparison of log-phase yeast samples with divergent rDNA CN similarly revealed few differentially expressed genes, although gene ontology enrichment analysis suggested effects on specific processes such as ribosome biogenesis. I also generated and analysed an ageing RNA-seq dataset which demonstrated that rDNA CNV in the form of ERC accumulation has significant effects on the ageing transcriptome. ERC accumulation influences the expression of hundreds of genes with strong enrichment for several previously identified age-related processes. Finally, using Northern blotting, I identified a common set of changes in rRNA processing intermediates, reminiscent of stress-related effects, which occur with ageing in variety of genetic backgrounds. 3 Table of Assistance 1) Initial training in techniques, laboratory practice, and subsequent mentoring: Dr Jon Houseley and Sharlene Murdoch provided initial lab training. Later JH also provided support during my switch from working with C. elegans to S. cerevisiae. Training in microscopy was provided by Dr Simon Walker. Statistical advice was provided by Dr Anne Segonds-Pichon. Subsequent mentoring and supervision was provided by JH and Dr Olivia Casanueva. My Babraham Institute assessor was Dr Nicholas Ktistakis and official mentors were Dr Cheryl Li, Dr Boo Virk, and Dr Cris Cruz-Bolland. My University supervisor was Dr Philip Zegermann. 2) Data obtained from a technical service provider All sanger sequencing to verify daf-2 C. elegans genotypes was performed by the commercial provider GENEWIZ. All RNA-sequencing was performed by Kristina Tabbada and Nicole Forrester at the Babraham Institute Next Generation Sequencing facility. 3) Data produced jointly Michelle King performed sample processing and Southern blotting for Figure 5.1 A. SM performed outcrossing of C. elegans strain MOC132. 4) Data/materials provided by someone else Several C. elegans strains were provided by the Caenorhabditis Genetics Center or others as noted in Table 2.1. The Mother Enrichment Program wild-type S. cerevisiae strain was kindly provided by the Gottschling lab. Some yeast strains used (Table 2.2) were generated by JH, Dr Carmen Jack, or Monica Della Rosa. JH previously prepared and submitted for sequencing the ‘Houseley dataset’ RNA-seq samples analysed in Section 4.2.3. JH performed the Southern blot analysis for Figure 5.10. Several published sequencing datasets were obtained from the European Nucleotide Archive as noted in the text. 4 Acknowledgements Firstly, I am extremely grateful to my supervisor Jon Houseley for guiding me through the twists and turns of this project. I am extremely privileged to have benefited from four years of Jon’s mentoring. Huge thanks also to Olivia Casanueva for her support, impromptu career advice, and for generally treating me as one of her own lab group. I am very lucky to have been able to become friends with the Houseley and Casanueva lab members – science is far more than just pipetting and they have all made my PhD experience much richer! I should also thank our excellent support staff at Babraham. Kristina Tabbada and Nicole Forrester at the Sequencing facility, as well as Simon Walker and Hanneke Okkenhaug in Imaging, were especially helpful. The assistance of Simon Andrews and Anne Segonds- Pichon in Bioinformatics has also been invaluable. Thanks too to my Babraham assessor Nick Ktistakis and my university supervisor Phil Zegermann. Finally, I must thank my family and friends for keeping me sane over the last four years. My BBSRC ’15 cohort comrades are a bunch of great people I very much hope to keep in contact with! Older friends are all, of course, deeply appreciated and gave me ample excuses to escape Cambridge at weekends. Finally, thanks to the Robinites, without whom this project would certainly have been completed on time without any extra issues. I know they would be extremely disappointed not get a mention. 5 Table of Contents Declaration of Originality ................................................................................................................................ 1 Abstract .................................................................................................................................................................. 2 Table of Assistance ............................................................................................................................................ 4 Acknowledgements ........................................................................................................................................... 5 List of Figures..................................................................................................................................................... 12 List of Tables ...................................................................................................................................................... 15 List of Abbreviations ....................................................................................................................................... 17 1 Introduction .............................................................................................................................................. 18 1.1 The rDNA locus has widespread effects on organismal physiology .......................... 18 1.2 rDNA loci in eukaryotes............................................................................................................... 19 1.3 rDNA transcription and ribosome biogenesis .................................................................... 21 1.3.1 Control of Pol I transcription at the rDNA .................................................................. 24 1.3.2 Post transcriptional processing of pre-rRNA ............................................................. 25 1.3.3 Coordinated regulation of ribosome synthesis and assembly ............................ 28 1.4 rDNA copy number variation .................................................................................................... 30 1.4.1 Mechanisms of directed
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