Structure and function of chromatin remodelling ATPase's and their dysfunction in in human disease.

The genomes of eukaryotes are associated with histone proteins to form a DNA protein complex called chromatin. The regulation of chromatin structure provides one means by which eukaryotic organisms regulated access to DNA. One means by which this is achieved involves the action of an extended family molecular motor proteins, known as chromatin remodelling ATPases. Recent advances to electron microscopy provide an opportunity to gain major new insights into how these motor proteins interact with chromatin. In this proposal we will apply advanced electron microscopy and image processing together with other complementary techniques to study how the shape of these motor proteins changes as they act to reconfigure chromatin. This will provide new insight into the process by which access to genes is regulated. It has recently become apparent that the genes that encode these motor proteins are often altered in cancer cells. We have noticed that these alterations repeatedly occur at a similar position on several different subtypes of motor proteins. Related alterations are also detected in some congenital diseases. As a result, we will systematically characterise how these alterations affect the way the motors work and determine which stage in the motors activity is blocked. We will also generate cell lines that will enable us to study the difference in between the loss of genes that encode motor proteins and the repeated alterations that are observed in cancers. We will find out whether cells are able to function normally when an altered copy of the motor gene is removed. If this is the case, blocking the altered motor and letting the cells carry on using the normal copy of the motor gene they typically retain may provide a way of treating patients. The proposal will generate a platform of knowledge from which new ways to target the defective motors can be devised.

Recent high resolution structures of chromatin remodelling enzymes bound to nucleosomes provide a snapshot of these enzymes in action. In order to gain insight into domain motions during the course of ATP-hydrolysis, we will determine structures in different nucleotide bound states and apply advanced image analysis to identify likely domain motions. Single molecule FRET and pulsed electron paramagnetic resonance will be used to measure these changes directly. Structural and biochemical studies provide a context in which to understand how these enzymes function and malfunction in disease states. Therefore, we will also characterise the defects to enzyme action that arise as a result of mutations that are found repeatedly across many ATPases in tumour tissues. The effects of several shared mutations will be assessed across a panel of purified ATPases. To determine whether the downstream consequences of different ATPases mutations are related, we will characterise how and where chromatin is altered when different ATPase mutations are introduced into mouse ES cells.

The high rate at which components of the human SWI/SNF complex are mutated in cancer makes it attractive to develop therapeutics that target deficiencies this loss of function. Most mutations to the catalytic ATPase subunit are missense mutations. This proposal sets out to characterise these mutants in some detail, focussing on the most frequently occurring mutations. As several of these occur within the ATP-binding pocket, we believe that it will be possible to identify compounds that target these mutants.

Dundee Drug Discovery Unit (DDU) have agreed to work with us to identify compounds that target ATPase mutants. This work will be funded separately by an MRC confidence in concept award to the Dundee drug discovery unit. Although this is a distinct project, there will be significant synergy with this proposal. It is clear that the work proposed here will have impact in that it will provide the background structural and mechanistic framework upon which compounds targeting mutant enzymes can be developed.

As the sites that are frequently mutated in the SMARCA4 subunit of the human SWI/SNF complex are conserved in many remodelling ATPases and we have noticed that related sites are mutated in different enzymes, we believe that the insights gained from one ATPase are likely to have relevance across multiple members of the protein family. Compounds identified in collaboration with the DDU may be effective against malignancies driven by mutations to different members of the protein family. In addition, the strategy of targeting mutant ATPase's may have relevance to congenital syndromes in associated with heterozygous mutations in the same domains.

Chromatin remodelling ATPases act to reconfigure chromatin during the course of most genetic processes. These include transcription, DNA replication, and DNA repair. As a result providing insight into the function of these enzymes is important in that it improves understanding of a process which is fundamental to many biological processes. As a result of improving knowledge of how genes are normal regulated it will become easier to understand how to identify and correct defects to gene regulation associated with diseases.