We are studying multi-component histone chaperone complexes, which contain the Rb-associated proteins RbAp46 and RbAp48. RbAp46 is an essential subunit of the histone acetyl-transferase Hat1 complex. RbAp48 on the other hand is a component of the heterotrimeric p48/p60/p150 chromatin assembly factor-1 (CAF-1) that is responsible for deposition of histones H3/H4 in nucleosome assembly. Both proteins are found in the nucleosome remodelling and deacetylase complex NuRD, which plays a key role in controlling the differentiation of embryonic stem (ES) cells. Several different projects are available to study various aspects of the structure and assembly of the NuRD complex, and its interactions with nucleosomes. Projects could involve tagging different components of the NuRD complex, purifying the complex and carrying out a combination of EM and chemical cross-linking/mass spectrometry (MS) to study the structure of different in vivo NuRD complexes. Secondly, we are systematically co-expressing and purifying different sub- and intact NuRD complexes for studies of their structures and interactions with nucleosomes using either X-ray crystallography or EM. Thirdly, we are using single molecule fluorescence methods to study how CHD4, and NuRD complexes, remodel nucleosomes in vitro.
We are also carrying out single molecule studies of chromatin assembly using super-resolution fluorescence microscopy. In recent years a number of techniques have been developed for studying single molecules using fluorescence microscopy at a higher resolution than that limited by the diffraction of light (so called super-resolution methods). Several research groups have been able to apply these methods to studies in live bacteria, or on the surface of mammalian cells, but we would like to extend this to studies of nuclear processes in higher eukaryotic organisms. In particular, we aim to develop systems to study chromatin disassembly/assembly at a single replication fork using single molecule methods and super-resolution fluorescence microscopy. The project would involve developing and characterising a system to study the deposition of CENP-A (a histone H3 variant) at centromeres in S. pombe. On the one hand we would further develop and investigate the utility of novel FRET cassettes for single molecule two-colour co-localisation studies of CENP-A with different histone chaperones involved in the assembly of CENP-A nucleosomes. However, we would also investigate the use of these fluorophores to see if we could carry out pulse-chase experiments at super-resolution and study the turnover of CENP-A and other proteins during the cell cycle.
In order to gain a more system-wide view of chromatin structure within the cell we are exploiting similar computational approaches (to those used to determine structural models of protein complexes) to studies of the global structure of chromatin. A chromatin conformation capture (3C) experiment, called Hi-C, is being used to provide spatial restraints on the structure. In these experiments restriction fragment digestion of DNA in intact nuclei is followed by the ligation of free DNA ends that are in close 3D spatial proximity. These ligated DNA junctions are then identified by high-throughput sequencing to provide distance restraints for structure calculations of chromatin architecture in the nucleus. Our initial studies have focussed on calculating the 3D structure of the X chromosome in stably differentiated cells, but current studies are focussed on determining chromosome structure in haploid embryonic stem (ES) cells.
Applicants should have a good honours degree (at least a 2:1 or equivalent) in either Biochemistry, Computer Science or one of the Physical sciences.
In general: Applications are welcome from EU and International students. Funding is available through a BBSRC Doctoral Training Grant (please check the Department of Biochemistry website) or other funding schemes such as the Gates Foundation, as well as various college funds (please check individual college websites).