Structural analysis of eukaryotic gene regulation

Abstract

This thesis presents data aimed at deepening our understanding of the mechanisms underlying eukaryotic gene regulation. A comprehensive understanding of these mechanisms should ultimately both allow insight into disease processes that arise from defects in gene regulatory circuits and might enable gene expression to be manipulated for application in health, agriculture and industry. The mechanisms that regulate gene expression include chromatin remodelling, post-translational modification and DNA methylation. These mechanisms are able to work together to ensure that the structure of chromatin is accessible to the machinery used for DNA based processes, such as transcription, and also to shield chromatin containing genes that remain unexpressed. These mechanisms are investigated using blood, or erythropoeisis, as the model system, with GATA1 and the NuRD complex being the elements of interest. The erythroid transcriptional factor GATA1 is subject to acetylation in two lysine rich regions, with acetylation in the second of these (at two specific lysine residues) being associated with increased chromatin occupancy as a result of a direct interaction with the bromodomain protein Brd3. As part of an effort to examine the structural and functional consequences of this interaction, it is necessary to produce site-specifically acetylated GATA1. The NuRD complex is a large multi-subunit complex involved in modulating chromatin structure to regulate gene expression. It has two activities, chromatin remodelling and histone deacetylation. Although many papers have been published on the complex, very little is available on the architecture. In order to gain a greater insight into the workings of the NuRD complex, negative stain transmission electron microscopy is used to determine the structure of a near-complete complex.