Structural and functional characterization of a mammalian chromatin remodeling ATPase

The DNA in every cell of our body is compressed 20,000 times in length into a compact, highly ordered structure called chromatin, enabling it to fit into the tiny cell nucleus. This compression is achieved by protein complexes, called histones, which spool the DNA around themselves into higher order structures. However, this tight packaging is a barrier to factors that need to gain access to the DNA during the fundamental processes of replication and repair. To solve this problem cells use very specialized multi-protein assemblies called chromatin remodeling complexes. These complexes not only need to locate the right piece of DNA that is to be made accessible, but once they find it, they also need to slide the DNA string along the spools and unravel it. This work requires energy, which is the reason why these complexes are also called molecular motors and burn energy in the process.

Because of their important role, when components of these complexes are absent or mutated, cells lose the ability to properly control their fates and growth. Accumulating evidence suggests that malfunctioning ATP-dependent chromatin remodeling complexes cause highly imparing genetic diseases (Alpha-thalassemia X-linked mental retardation syndrome, X-linked Rett syndrome, Cocakyne syndrome, Schimke immuno-osseous dysplasia, Rubinstein-Taybi syndrome, Coffin-Lowry syndrome, etc.) and various types of cancer.

My group at the Wellcome Trust Centre for Human Genetics (Oxford), in collaboration with Dr. Roman Tuma at the University of Helsinki, is interested in the fundamental question of how chromatin remodeling complexes work and how their activity regulates and controls genes. I am hoping to use two techniques called X-ray crystallography and electron microscopy, which allow you to look with amazing details at very small objects, to have a closer look at the shape of these chromatin remodeling complexes. The outcome of this project is to provide snapshots at atomic level that show how chromatin remodeling complexes perform their very important task within the cell.

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. Despite progress in recent years, the molecular mechanism of ATP-dependent chromatin remodeling remains elusive and progress is hampered by the almost complete lack of structural information on chromatin remodeling ATPases.

This project aims to contribute to a structural and functional description of mammalian chromatin remodeling ATPases. Recently, we have successfully expressed various constructs of CHD4 a highly conserved and key member of the CHD remodeling ATPase family. Starting from these promising results we will investigate the structure of CHD4 on its own and in complex with DNA (either naked DNA or nucleosomes) by a combination of X-ray crystallography and Cryo-electron microscopy. In collaboration with Roman Tuma?s group we will further validate and add value to our structural results with biochemical, biophysical and single-molecules studies.

The research objectives of the proposed project are:

1. to detail the mechanism by which chromatin remodeling ATPase CHD4 converts the conformational changes induced by ATP hydrolysis into disruption of histone-DNA interactions, by solving the atomic structure of the full length protein, or its ATPase domain, on its own and in key nucleotide bound states.
2. to characterize at a molecular level how the conserved DNA targeting domains, tandem chromodomains and PHD fingers, direct CHD4 to specific chromatin structures and how these domains interact with and regulate the ATPase domain
3. to visualize and characterize the interactions of CHD4 with substrate DNA, either naked or nucleosomal, by a combination of structural, biophysical and single molecules studies.