Functional dissection of the genetic interaction network that affects growth of cells with telomere defects: implications for health and disease

The genetic material DNA contains the instructions to make cells and organisms. For these reasons stability of DNA is important to pass on genetic information from one generation to the next. In humans and many simpler organisms such as yeast, DNA is found in chromosomes that contain extremely long, linear, double stranded DNA molecules, in a sense like very long shoelaces. Just as with shoelaces, a chromosome that is broken or damaged in the middle is much less useful than a chromosome that is damaged at the end. Even so, just as with shoelaces, it is normally important that the end of each chromosome is properly "capped". The caps at the ends of shoelaces are called aglets, and those at the caps at the ends of chromosomes are called telomeres. If telomeres fail, then genetic material near the telomeres can be damaged or lost and cells with uncapped telomeres may no longer be permitted to divide. Cellular responses to defective telomeres are largely regulated by the same pathways that respond to DNA damage elsewhere in the genome. In human beings these mechanisms play important roles in ageing and cancer.

This project examines how specific intra-cellular defense mechanisms interact with other mechanisms to respond to telomere defects. Some of these mechanisms can be likened to the mechanisms used to slow down vehicles. When telomere damage occurs inside cells, the cells also slow or even stop cell division. We are particularly interested in measuring and understanding interactions between different mechanisms and seeing what happens if two mechanisms, rather than a single mechanism, are defective. In a car we know that the footbrake, the handbrake and gears provide different mechanisms to reduce speed and all the mechanisms are useful. We also know that there is some redundancy between mechanisms in a car. That is, if the hand brake fails then the foot brake and gears become more important. Taking this analogy further, we are interested in studying how brake mechanisms interact, inside cells.

Yeast is a good experimental system to study these fundamental questions. Yeast has been cultured for thousands of years for making bread, wine and beer and is very easy to grow and study in the lab. Furthermore telomere structure in yeast cells is similar to that in human telomeres, making studies in yeast cells relevant to human health and disease. We will make use of traditional yeast methods and more modern robotic genetic methods to examine large numbers of interactions inside yeast cells. Such experiments are not possible or are too expensive to perform in mammalian cells. We expect that many of our discoveries will also be relevant to human telomere function since many of the proteins and pathways we study in yeast are conserved.

Based on our studies in yeast we will examine genomes from human beings, particularly those who have lived to the age of 85 or more, to see if the same pathways are likely to be affected in human beings. If so, such persons might be particularly resistant or susceptible to telomere related human diseases.

We will use high-throughput robotic genetic procedures to measure fitness of nearly 24,000 genetically different yeast strains at a range of growth temperatures. To ensure that our measurements are accurate we will create 8 independent biological replicates of each of these 24,000 different yeast strains. All the strains will contain cdc13-1 a recessive mutation that causes temperature dependent telomere cap defects, and two null mutations in genes that affect the telomere. We will examine fitness at four temperatures (23, 27, 30 and 33oC), resulting in a spectrum of telomere defects, from minor to severe. We will use appropriate photographic, computational and statistical procedures to measure the fitness of each genetically distinct yeast strain.

From these data we will learn how gene mutations interact (epistasis) in this system and develop new Bayesian statistical methods to identify and quantify epistatic interactions revealed by this novel experimental design. We will also determine how interactions are affected by the severity of the telomere defect (temperature). There is already precedent that very strong genetic interactions occur, allowing in some cases yeast strains to completely bypass the requirement for the normally essential CDC13 gene. Significant genetic interactions identified in yeast will be used to determine if analogous interactions could be relevant in humans using relevant genome-wide association study (GWAS) datasets.

We expect our work will have significant impact in several scientific communities. Perhaps, the most direct beneficiaries of our work are basic scientists who are interested in understanding how telomeres, the ends of chromosomes, affect cell division, ageing and cancer. There are clearly large scientific communities, funded by the MRC and others, interested in these fundamental and medically important areas of biology.

Although we plan to perform experiments using yeast as a model organism, we will also do our best to make the implications of our discoveries clear to those working in areas more directly relevant to human health. For example, in November DL visited Prof Yanick Crow's lab in Manchester. It is Prof Crow's lab that first showed that mutations in CTC1, the human orthologue of Cdc13, are associated with Coats' plus disease. We will do our best to make sure that the implications of our studies in yeast can aid Prof Crow and others interested in human telomere function (see letter of support attached).

We strongly speculate that some of the genetic interactions we identify in yeast will be relevant to human health and therefore the subject of clinical/ epidemiological studies. We will continue to build on our links with the Institute for Ageing and Health in Newcastle, and Prof Crow in Manchester to achieve this aim.

To reach as wide an academic audience as possible we publish in Open Access journals and host data on our web sites.

We will also encourage exploitation of our research by publicising our work at the North East Fungal Forum (NEFF, http://research.ncl.ac.uk/neff). This meeting is held every three months or so and features presentations by students, post docs, group leaders, and sometimes external speakers. NEFF is organised by post docs and students in the various fungal labs in the North East. The RAs appointed on this project will encourage to participate in running NEFF.

NEFF is sponsored, in particular by a major sponsor Formedium. The meetings are advertised on the web in advance and flyers are posted around Newcastle and Durham universities and hospital. By advertising in the local hospital, the Royal Victoria Infirmary, we have engaged with clinical scientists involved in the treatment of human fungal diseases.