Causal Factors in Genome Control

Abstract

The aim of this thesis is to study how genes are switched on and off in a coordinated way across an entire genome. In order to do this yeast is used as a model organism. The mechanisms that control gene expression in yeast are very similar to those of human cells. Chapter 1 provides a general introduction to these topics. Even though yeast cells are easier to work with than human cells, studying gene expression is still technically difficult because all of the factors involved cannot be directly observed. In order to get enough DNA and protein for experiments, millions of living rapidly growing cells are typically destroyed to extract their components. The measurements that are taken are then representative of a hypothetical average cell. In chapter 2 it is shown that gene expression changes associated with many previous experiments reported in the literature are related to a change in the growth rate of the cells. A method is presented for mathematically correcting this indirect effect. To understand how something works it is often necessary to first break it. In order to discover what a protein does in a cell, a common approach is to delete from a genome the gene coding for that protein. The effect that loss of a specific protein has on cell function can then be observed. In chapter 3 an alternative approach is employed to solve the problem of secondary effects associated with gene deletion strains. Here cells are used where a kinase protein (Cdk8) can be rapidly removed from the cell nucleus upon addition of an inducing chemical. Genome-wide measurements are then taken minutes after loss of protein function in the nucleus. The knock on effects to gene expression are also followed through time. Genes are not only simply switched on or off in cells. Controlling the expression levels of proteins can also be important for efficient cell functioning and disease prevention. In chapter 4 the activity of a DNA binding protein (Hsf1) is controlled by exploiting a modified version of the conditional protein depletion strategy used in chapter 3. There are two main pathways used in the yeast genome for activating genes. One of the pathways (TFIID) typically controls expression of proteins that serve housekeeping functions in a cell. The other pathway (SAGA) controls expression of genes with functions important in rapid responses to changing environmental conditions and developmental processes. By precisely controlling Hsf1 levels in the nucleus and directly measuring the effects on gene expression it was found that Hsf1 can have very different effects in each of the two gene activation pathways. Chapter 5 discusses the wider implications of the findings in the previous chapters and points out some further caveats relating to indirect effects.