Understanding the recruitment of Class I HDACs into diverse repression complexes: implications for physiological activity and therapeutic devlopment

'Histone deacetylase' (HDAC) enzymes are present in all cells of the body. Their function is to switch genes 'off', and make sure they stay 'off'. My lab studies how HDACs do this and which, amongst the 25,000 genes in each cell, are selected for inactivation. HDAC enzymes also represent an exciting medical opportunity because they are 'druggable'. Already, drugs which inhibit HDAC activity are being used in the clinic as anti-cancer agents, and are being further developed for their beneficial effects on dementia and anti-inflammatory properties. There is therefore a compelling applied, as well as academic, motivation for studying their physiological roles in order to assess their potential as pharmacological targets. We intend to study how three different HDAC enzymes (HDACs 1, 2 and 3) work in normal cells. One of the best methods for understanding how an enzyme works is to generate mutant cells in which the specific enzyme has been inactivated, or 'knocked-out'. These 'knock-out' cells can then be examined for changes in their characteristics, lack of growth for instance, which can then be attributed to the function of that particular enzyme. Previously, we have generated 'knock-out' cells for HDAC1 and HDAC2 alone, but their function is overlapping, and so the effects on cell growth were small. To get around this, we have generated cells in which HDAC1 and 2 can be removed at the same time, so called 'double knock-out' cells. Early experiments indicate that loss of both enzymes causes cells to die, indicating that their activity is essential. Using DNA technology it is possible to add back normal or mutated forms of HDAC1 to prevent the double knock-out cells from dying and then ask, which parts of the enzyme are important for its function? In related experiments, we also intend to visualize the actual molecular structure of HDAC1 bound to a molecule called MTA1, using a technique called X-ray crystallography. The interaction of HDAC1 with other molecules in the cell is fundamental to their function. By understanding the molecular basis of these interactions we can better understand how HDAC enzymes work in normal and cancer cells, and potentially use that knowledge to design new drugs to prevent them from working. The ability to stop HDAC1 and 2 from working, as seen in our double knock-out cells, causes cells to stop growing and die, making them excellent drug targets in the search for improved anti-cancer agents.

Class-1 histone deacetylases (HDACs 1, 2, 3 and 8) are essential enzymes present in the nucleus of all mammalian cells where they help regulate chromatin structure as the catalytic component of co-repressor complexes such as Sin3A, NuRD, CoREST and SMRT. The function of HDACs in vivo is dependent upon recruitment into specific multi-protein co-repressor complexes, which regulate both substrate specificity and enzymatic activity. Despite this, little attention has been paid to the molecular assembly of individual complex components, and in particular there are no structural data to explain the specificity of assembly. We aim to combine in vivo (Cowley lab) and structural (Schwabe lab) approaches to understand the molecular determinants of HDAC1/2 recruitment to the Sin3A, NuRD and CoREST complexes, identify areas of similarity and diversity between these different complexes and understand how specificity is maintained so as to exclude the highly related enzyme, HDAC3. To address these questions we have three specific objectives: Objective 1: Using the known structures of HDAC2 and HDAC3 we will design mutations to map the Sin3A/MTA1/CoREST1 interaction surface(s) of HDAC1 and 2. Objective 2: Determine structures of the HDAC1/2 interaction domains of Sin3A, MTA1 and CoREST1, with and without HDAC1/2, using NMR and X-ray crystallographic approaches. Objectives 1 and 2 will run simultaneously and are highly interdependent. Both approaches will identity critical residues in HDAC1/2 required for complex incorporation. Objective 3: We will make mutations in these critical residues and test their importance for the physiological activity of HDAC1/2 by testing their ability to rescue the viability of HDAC1/2 double knock-out ES cells (part i), and be recruited to target genes (part ii). This work will directly establish the molecular determinants of HDAC1/2 complex assembly and to what degree this determines their physiological activity

1. Commercial / Industrial -
There is a growing awareness within the pharmaceutical industry of histone modifying enzymes as potential drug targets. HDAC1 and 2 function has been implicated in almost all cellular processes including, cell cycle progression, DNA repair, differentiation and cancer. Furthermore, mouse knock-out studies have demonstrated that HDAC1/2 have essential roles in the development of the heart, neurons, skin and B-cells. Given these essential biological roles, HDAC1 and 2 enzymes are strong candidates for pharmacological manipulation. The novel structural data of HDAC:co-repressor complexes, coupled to the physiological validation of key determinants, will be of great value in the design of conventional HDAC inhibitors (focussed on the active site); and the long-term goal of using small molecules to inhibit the protein-protein interactions of these complexes to perturb HDAC1/2 function. The University of Leicester has a vigorous and experienced "Enterprise & Business Development" team and an embedded unit ("The Biobator"), dedicated to exploitation of activities arising from work in biomedical research. Outputs from the project will be used by BIOBATOR to establish partnerships with industrial collaborators to exploit these findings.

2. Societal -
Inhibitors of HDAC1 and 2 are currently used in the clinic to treat depression and cancer. It is only a matter of time before their application becomes more extensive, enhancing the well-being of society as a whole. In the laboratory, inhibition of HDAC1 and 2 reactivates alpha-globin (the foetal globin isoform) in human erythroid progenitors, making them potential therapeutic targets for the treatment of sickle cell disease. Inhibition of HDAC activity has ameliorative effects in mice models of dementia and muscular dystrophy. The essence of our project is basic science, and the therapeutic payoff long-term. However, an understanding of HDAC enzymes in their cellular context, incorporated into diverse co-repressor complexes, will be necessary to understand the action of existing HDACi used clinically, and in the design of small molecules which inhibit HDAC function.

3. Animal Welfare - 3R's.
Reduction, refinement and replacement (3R's) of animals in experimental research is a commitment made by each of the research councils and major research institutes within the United Kingdom. This project will use cells with an inducible knock-out of HDAC1 and 2 (Objective 3), which were generated using 'embryo free' methodology. This contrasts with the traditional method of generating knock-out cell lines from mouse embryos. Recently, the large scale production of knock-out mice embryonic stem (ES) cell lines was begun by the KOMP (Knock-out Mouse Project) and EUCOMM (European Community Mutant Mouse project) consortia, in their attempt to make a mutant cell line for every protein-coding gene in the mouse genome. The KO cells generated by these schemes will be made freely available to the academic community. This project will promote the same ES cell knock-out technology, their suitability for study of gene expression and raise awareness of this alternative to animal based systems.