Research rDNAome - GANLEY Laboratory

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Research

Our lab is primarily interested in the function and evolution of the ribosomal DNA repeats (rDNA) in eukaryotes. In particular, we are interested in the so-called extra-coding functions of the rDNA. These are functions that the rDNA exhibits in addition to the primary function of encoding ribosomal RNA. These extra-coding functions affect many aspects of cell biology and some are responsible for the unusual evolutionary behaviour of the rDNA, known as concerted evolution. We investigate the rDNA using both molecular and computational approaches. We also maintain interests in fungal genome evolution, polyploidy, human genetics, and mushrooms.

Introduction

Current research projects

Organisation and structure of the rDNA

The rDNA is present as head-to-tail tandem repeats in most eukaryotes (Figure 1). A single rDNA repeat unit can range in size from ~8-45kb, depending on the organism. It encodes the major species of ribosomal RNA that make up the ribosome and the rDNA repeats are found within the nucleolus, the site of primary ribosome biogenesis. They are an essential and ancient component of genomes.

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Figure 1: Organisation of the ribosomal DNA repeats in eukaryotes.

A single repeat unit consists of the 18S, 5.8S and 28S ribosomal RNA genes separated by internal transcribed spacers (ITS) 1 and 2. Between adjacent coding regions is a large spacer region, the intergenic spacer (IGS). In some species, the IGS harbours another ribosomal RNA gene, the 5S. Although detailed IGS characterization has been performed for few organisms, an origin of replication (ori) and replication fork barrier site (RFB) have been found in the IGS of organisms that have been examined.

The region separating adjacent coding regions is called the intergenic spacer (IGS). Although initially thought to be “junk” DNA, we now know that the IGS is packed full of functional, noncoding elements (NOCs). We are particularly interested in the roles these NOCs play. Although copy number of the rDNA varies widely between species, each species maintains its own characteristic number of rDNA copies. These may be present at one-or-more chromosomal locations. The repetitive nature of the rDNA makes it prone to unequal recombination between repeats, resulting in copy number variation, and there is an extensive regulation system in place to control rDNA recombination. Therefore, while copy number in a species is held roughly constant, the precise copy number varies between individuals due to recombination-based copy number fluctuation. This recombination has a powerful influence on the evolution of the rDNA repeats. It results in the rDNA evolving via the evolutionary pattern of concerted evolution that is characterized by a high level of sequence identity between the repeats. In the yeast, Saccharomyces cerevisiae, we have shown that non-coding transcription from a bidirectional promoter in the rDNA, called E-pro, is a key regulator of rDNA recombination, with the level of this noncoding transcription being modulated by the silencing gene, SIR2. This recombination is also dependent on the fork blocking protein, Fob1p, which binds to the replication fork barrier (RFB) site in the rDNA. Binding of Fob1p to the RFB blocks the replication fork in one direction, and induces double strand break formation that is repaired by homologous recombination with another repeat. This recombination appears to depend on replication, and each rDNA unit contains a replication origin. Therefore replication initiates within the rDNA, although origins only fire in a subset of rDNA repeat units in each cell cycle. We have shown that these functional elements are all conserved in Saccharomyces yeasts, and that there are additional conserved elements for which we do not yet know the function of (Figure 2).

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Figure 2: Conserved/functional elements in the IGS of Saccharomyces yeasts.

This diagram plots the level of sequence conservation across the IGS region in yeast. Conserved regions are indicated by coloured boxes, and known functional elements are named above. The rARS (autonomously replicating sequence) is the yeast rDNA origin of replication. It and the RFB site both consist of three conserved sequence elements. E-pro is the bidirectional noncoding promoter responsible for regulating unequal recombination in conjunction with Fob1p, which binds to the RFB site. Figure adapted from Ganley et al., 2005 PNAS 102: 11787-92. Refer to this paper for more details on other IGS functional elements shown.

Finally, the rDNA is an extremely interesting epigenetic system, as although there are a large number of rDNA copies, at least in most conditions only a subset (half or less) are actually being transcribed. For those that are being transcribed (the rRNA genes are transcribed by RNA polymerase I), the transcription level is extremely high. Indeed, it is the most highly-transcribed gene in the genome, and this high level of transcription influences the behaviour of the rDNA. However, the remaining repeats are completely transcriptionally silent. We have shown that rDNA units within a genome are all essentially identical at the DNA sequence level, therefore this mosaic pattern of active/inactive units must be determined epigenetically. There is great interest in understanding how this occurs, as rRNA transcription level correlates with growth rate, and many cancer cells increase the number of active rDNA units they have.

rDNA as a target for anti-cancer therapies

rDNAome - GANLEY LaboratoryNucleoli and the rDNA undergo dramatic changes in many malignant cells, something that was first observed over 100 years ago. However, the role that these changes play in cancer have remained largely unexplored. Interestingly, inhibiting rRNA transcription is a potent killer of cancer cells, yet leaves normal surprisingly untouched. This effect does not seem to be a simple consequence of preventing proliferation, and instead seems to result from underlying chromatin structure changes that occur when RNA polymerase I is inhibited. We have a Marsden funded project to explore the suite of changes that occur in the rDNA during tumorigenesis, and to investigate what underlies the addictive nature of these changes in the rDNA. We are doing this using a combination of experimental and bioinformatic approaches, using both mouse and human model systems. This is collaboration with Prof. Ross Hannan at the Australian National University in Canberra, and Dr. Sebastian Schmeier at Massey University (Auckland).

Developing tools for the analysis of rDNA in mammalian cells

rDNAome - GANLEY LaboratoryThe rDNA is a very dynamic region of the genome, and several aspects of rDNA dynamics have been relatively well studied in yeast. In contrast, very little is known about many aspects of mammalian rDNA, because of technical limitations for studying them. We have a project funded by the University of Auckland that aims to address this by developing new tools to analyse aspects of rDNA dynamics in mammalian cells. These include the ability to accurately measure the total number of rDNA copies, and the ability to determine the rate of recombination in the rDNA repeats.

The genomic consequences of polyploidy

rDNAome - GANLEY LaboratoryPolyploidy is a widespread and powerful evolutionary, particularly allopolyploidy which involves the merger of two different species to form a “hybrid” genome with both progenitor genomes present. Polyploidy is found in many eukaryote lineages, and is proposed to fuel adaptive evolution by providing instant genetic redundancy, yet combining two genomes also provides a major challenge for the new polyploid species (known as “genome shock”). Despite the widespread nature of polyploidy, we understand little of the genome-wide responses that occur when genomes merge. The advent of next-generation sequencing technologies is finally providing the tools to look at these responses, and we have a Marsden funded project to document these genome-wide responses in a model fungal polyploid system, the Epichloë endophytes. Our goal is to not just document these responses, but to determine whether they are stochastic or deterministic in nature. This is collaboration with Prof. Murray Cox at Massey University (Palmerston North) who leads the project, and Dr. Carolyn Young at the Noble Foundation in the USA.

Virtual hub for national sharing of environmental DNA data

rDNAome - GANLEY LaboratoryThe ability to sequence DNA cheaply and easily has led to a revolution in our ability to determine the organisms present in environments, particularly microorganisms that are very difficult to detect by other means. DNA extraction from environmental samples, followed by PCR amplification and next-generation sequencing of marker genes, is increasingly being used as a measure of biodiversity. The Biological Heritage National Science Challenge has recognized that the value of these data can be greatly increased by sharing it across the country. To this end, we are developing a “virtual hub” for the sharing of environmental DNA data. The objective is not just to make a tool that is useful to researchers, but that anyone, regardless of expertise, can use to understand biodiversity in this country using environmental DNA data.

New beer yeast for the craft beer industry

The yeast Saccharomyces cerevisiae is a core ingredient of non-lager beers that adds to the flavour and character profile. Interestingly, most beer yeasts have lost the ability to complete the sexual cycle, meaning traditional breeding techniques are not available for developing new strains of beer yeast. We are working with Dr. Sylvie Hermann-Le Denmat at the Paris SUD to isolate wild yeast in an effort to develop new strains with novel characters for use in the burgeoning craft beer industry.

Role of the rDNA in chromosome missegregation

rDNAome - GANLEY LaboratoryThe rDNA is a distinctive feature of genomes that has its own unique properties. Several of these properties, such as high rates of transcription and recombination, may interfere with the orderly separation of the chromosomes during cell division. Consistent with this, the rDNA maintains a novel form of cohesion (the forces that hold sister chromatids together until their separation during mitosis/meiosis). Suggestively, chromosome 21 in human harbours the rDNA, and the frequent missegregation of this chromosome can result, of course, in Down syndrome. We have been investigating what role the rDNA plays in chromosome missegregation, using S. cerevisiae as a model system to explore this. We have found that the rDNA indeed does influence chromosome segregation, and are working to define the molecular mechanisms that underlie this effect. This work has been supported by the Marsden Fund, and is collaboration with Prof. Takehiko Kobayashi at the National Institute of Genetics in Japan.

Characterisation of functional elements in the human rDNA

rDNAome - GANLEY LaboratoryThe basic structure of the rDNA in human is conserved with yeast, but the organization is more complex. The rDNA is multi-chromosomal: it is located on the short arms of the five acrocentric human chromosomes (13, 14, 15, 21, and 22). Furthermore, the repeating unit is considerably longer: ~45kb in length compared to ~9kb in yeast. Most of this increased length is in the intergenic spacer. Despite this, little is known about functional elements in the human IGS. We are using computational approaches to identify functional elements in the human IGS region, and this work is supported by the Auckland Medical Research Foundation.

Role of the rDNA in ageing

rDNAome - GANLEY LaboratoryS. cerevisiae has a finite lifespan, and shows a characteristic set of aging phenotypes. In many ways this yeast aging is similar to that seen in higher eukaryotes, and therefore it is of interest to understand the genetic and epigenetic factors that underlie aging in yeast. We have recently shown that the level of unequal recombination in the rDNA correlates negatively with lifespan of S. cerevisiae, and, with the help of funding from Massey University, we are working towards understanding how rDNA recombination influences lifespan, and whether factors that are known to influence lifespan, such as diet, do so through effects on the rDNA. This is collaboration with Prof. Takehiko Kobayashi at the National Institute of Genetics in Japan (http://www.nig.ac.jp/labs/CytoGen/e_index.html) and Prof. David Raubenheimer, now at the University of Sydney.

Concerted evolution of the ribosomal DNA

Concerted evolution describes an evolutionary pattern displayed by some repeats whereby the sequences of the repeats are more similar to each other within a genome than they are to orthologous repeats in related species. This pattern arises from a process that acts to maintain repeats within a genome with the same sequence over time, known as homogenization. It is now widely accepted that the driving force behind homogenization is unequal recombination between repeats. We (and others) have previously shown that concerted evolution is extremely efficient in the rDNA. While the theory of concerted evolution/homogenisation was established over 30 years ago, many details of the dynamics that govern how rDNA recombination leads to concerted evolution remain unknown. We are using a unique dual approach, combining experimental evolution-based wet-lab approaches in S. cerevisiae with computer simulation approaches to try and answer major outstanding questions concerning how unequal recombination leads to concerted evolution. This dual approach allows us to simultaneously advance our theoretical and experimental knowledge in a synergistic fashion, and is being done in collaboration with Dr. Eric Libby, now at the Santa Fe Institute, USA.

The genetics of caffeine consumption in New Zealand

rDNAome - GANLEY LaboratoryCaffeine is an incredibly widely used stimulant. It is present in an increasing number of products, some of which caffeine presence is obvious, some of which it is less so, and the consumption levels in children are increasing. Much caffeine consumption is tied to its beneficial effects, but serious caffeine-induced medical events are on the rise. Furthermore, there is great inter-individual variability in the response to caffeine. We are working on genetic aspects of caffeine consumption and physiological response in the New Zealand population as part of the larger Caffeine Team at Massey University. This work is in collaboration with the Caffeine Team at Massey University (Auckland), which is being led by Dr. Ajmol Ali and Dr. Kay Rutherford.

Organisation of the rDNA in the nucleolus

The repetitive nature of the rDNA makes it both a boon and a handicap for experimental research. Notably, the extremely high sequence similarity of the rDNA repeats means that we have little idea of how the rDNA is arranged in three dimensions within the nucleolus, or of the spatial organization of active and inactive repeats. The inability to distinguish individual repeats from one-another in the array also means we have little idea about the interrelationships of the various activities that occur in the rDNA, such as RNA polymerase I transcription, recombination, replication, and RNA polymerase II transcription. We are collaborating with Dr. Justin O’Sullivan at the Liggins Institute, University of Auckland, with the support of a Life Technologies $10k Genome Prize, rDNAome - GANLEY Laboratoryto explore the spatial organization of the rDNA and how this alters when the rDNA is moved between chromosomes. We are also looking at the functional consequences of these changes. Furthermore, we are developing systems that allow us to distinguish individual rDNA repeats so we can build a picture of the rDNA as a system of dynamically-regulated repeats within a defined spatial framework.

Function of the Tar1 gene

Hemiascomycete yeasts curiously encode a gene, known as Tar1, that is located antisense to the large ribosomal RNA subunit gene in the rDNA. This gene is therefore multi-copy, yet is expressed at very low levels. The protein it encodes, Tar1p, is localized to mitochondria, but the role of Tar1p remains unknown. Myself, Dr. Ant Poole at the University of Auckland, and Dr. Sylvie Hermann-Le Denmat at the Paris SUD are working on establishing the function of Tar1 and the significance of its localization in the rDNA repeats.

Previous research projects

rDNAome - GANLEY LaboratoryI was involved in a collaboration with Prof. Brian McStay at the National University of Ireland (Galway) that identified the flanking regions of the rDNA in human. These regions of the genome were uncharacterized and missing from the human genome sequence. We characterized over 350kb of sequence on the centromere distal side of the rDNA array, and over 200kb of sequence on the centromere proximal side. These sequences were highly similar amongst all five acrocentric chromosomes, implying ongoing recombination between them. The two regions have contrasting patterns, with the proximal flanking region consisting almost exclusively of segmental duplicated DNA, while the distal flanking region is similar in profile to other, “unique” regions of the genome. However, it is characterized by a large (over 200kb) inverted repeat, and a large array of tandem 48bp satellite repeats.

rDNAome - GANLEY LaboratoryI was involved in a project led by Prof. Rosie Bradshaw at Massey University (Palmerston North). This project looked at the genome of a pathogen of pine trees called Dothistroma septosporum. We used genomic sequence data to determine the effects that repeats within the genome of this fungus have had on the evolution and clustering of secondary metabolite biosynthetic genes. This work was supported the Bio-Protection Centre of Research Excellence

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