Aims: Our group is interested in the molecular mechanisms that govern the unraveling of chromatin from histones by chromatin remodeling complexes (CRCs), a process that is key to transcription activities in eukaryotes. To obtain insights into these cellular processes, we use X-ray crystallography in combination with Cryo-electron microscopy (Cryo-EM).
Background: This project aims to contribute to a structural and functional description of mammalian chromatin remodeling ATPases. ATP-dependent chromatin remodeling complexes are evolutionary conserved large (300k-2MDa) multisubunit assemblies, which contain an ATPase protein belonging to the SNF2 subfamily of DEAD/H helicases. There are many members of this family which are grouped into subclasses depending on the domain composition of their ATPase domain. In mammals, the best characterised classes are SWI/SNF, CHD and ISWI. Each has a unique domain (bromo, chromo or sant) which is thought to interact with specific chromatin substrates. One of the current challenges in the study of chromatin regulation is to define the functional and structural differences between the ATPases that are responsible for performing comparable but distinct enzymatic reactions. Prospective students will investigate the structure of chromatin remodelling ATPases on their own and in complex with DNA (either naked DNA or nucleosomes) by a combination of X-ray crystallography and Cryo-electron microscopy. The student will further validate the structural results with biochemical, biophysical and single-molecules studies.
Biological and Medical Relevance: CRCs are key regulators of transcriptional activity. Elucidating the molecular mechanism of ATP-dependent chromatin remodeling has become one of the central challenges in the field of gene regulation3. Accumulating genetic evidence suggests that ATP-dependent chromatin remodeling plays a crucial role in human tumorigenesis. Misregulation of chromatin structure can cause incorrect gene activation or improper gene silencing. Specifically, several subunits of CRCs possess intrinsic tumour-suppressor activity or are required for the activity of other tumour-suppressor genes4.
Training Opportunities: A number of contained projects on the above mentioned CRCs can be suited to the interest of potential candidates. Candidates can be expected to be trained in any of the following aspects: protein cloning, expression and purification; protein analysis - including chromatography and light scattering; protein crystallization, mounting of crystals and collection of diffraction data; determination of structures from diffraction data; methods for preparing samples for Cryo-EM; operation of electron microscopes and the methods for reconstructing electron density from Cryo-EM micrographs.
Location: The Division of Structural Biology at the Wellcome Trust Centre for Human Genetics, has excellent wet lab space, world-class in-house X-ray facilities and a 300 kV field emission gun liquid nitrogen Cryo-EM microscope. In addition, students will be able to benefit from the state-of-the-art Oxford Protein Production Facility bioinformatics tools for target selection and high-throughput cloning, protein expression, purification and crystallization setup.
Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology
Project reference number: 119
| Name | Department | Institution | Country | |
|---|---|---|---|---|
| Dr Erika J Mancini | Structural Biology | Oxford University | UK | erika@strubi.ox.ac.uk |
2004. Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation. Cell, 118 (6), pp. 743-55. Read abstract | Read more
Many viruses package their genome into preformed capsids using packaging motors powered by the hydrolysis of ATP. The hexameric ATPase P4 of dsRNA bacteriophage phi12, located at the vertices of the icosahedral capsid, is such a packaging motor. We have captured crystallographic structures of P4 for all the key points along the catalytic pathway, including apo, substrate analog bound, and product bound. Substrate and product binding have been observed as both binary complexes and ternary complexes with divalent cations. These structures reveal large movements of the putative RNA binding loop, which are coupled with nucleotide binding and hydrolysis, indicating how ATP hydrolysis drives RNA translocation through cooperative conformational changes. Two distinct conformations of bound nucleotide triphosphate suggest how hydrolysis is activated by RNA binding. This provides a model for chemomechanical coupling for a prototype of the large family of hexameric helicases and oligonucleotide translocating enzymes. Hide abstract
2008. ATP-dependent chromatosome remodeling. Biol. Chem., 389 (4), pp. 345-52. Read abstract | Read more
Chromatin serves to package, protect and organize the complex eukaryotic genomes to assure their stable inheritance over many cell generations. At the same time, chromatin must be dynamic to allow continued use of DNA during a cell's lifetime. One important principle that endows chromatin with flexibility involves ATP-dependent 'remodeling' factors, which alter DNA-histone interactions to form, disrupt or move nucleosomes. Remodeling is well documented at the nucleosomal level, but little is known about the action of remodeling factors in a more physiological chromatin environment. Recent findings suggest that some remodeling machines can reorganize even folded chromatin fibers containing the linker histone H1, extending the potential scope of remodeling reactions to the bulk of euchromatin. Hide abstract
2004. Coregulators and chromatin remodeling in transcriptional control. Mol. Carcinog., 41 (4), pp. 221-30. Read abstract | Read more
Despite many years of investigation by numerous investigators, transcriptional regulatory control remains an intensely investigated and continuously evolving field of research. Transcriptional regulation is dependent not only on transcription factor activation and chromatin remodeling, but also on a host of transcription factor coregulators-coactivators and corepressors. In addition to transcription factor activation and chromatin changes, there is an expanding array of additional modifications that titrate transcriptional regulation for the specific conditions of a particular cell type, organ system, and developmental stage, and such events are likely to be greatly influenced by upstream signaling cascades. Here, we will briefly review the highlights and perspectives of chromatin remodeling and transcription controls as affected by cofactor availability, cellular energy state, relative ratios of reducing equivalents, and upstream signaling. We also present the C-terminal binding protein (CtBP) as a novel nuclear receptor (NR) coregulator, which exemplifies the integration of a number of transcriptional regulatory controls. Hide abstract
2003. Chromatin remodeling and human disease. Curr. Opin. Genet. Dev., 13 (3), pp. 246-52. Read abstract | Read more
In the past few years, there has been a nascent convergence of scientific understanding of inherited human diseases with epigenetics. Identified epigenetic processes involved in human disease include covalent DNA modifications, covalent histone modifications, and histone relocation. Each of these processes influences chromatin structure and thereby regulates gene expression and DNA methylation, replication, recombination, and repair. The importance of these processes for nearly all aspects of normal growth and development is illustrated by the array of multi-system disorders and neoplasias caused by their dysregulation. Hide abstract