Understanding the essential requirement for HDAC1 and HDAC2 in tissue development and homeostasis: implications for disease and therapy.

'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 need an improved understanding of how individual HDAC enzymes work in normal cells. In this study we intend study two closely related HDAC enzymes, HDAC1 and HDAC2. 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.

Histone deacetylases (HDAC) are important drug targets in the treatment of cancer and mental illness. However, the existing HDAC inhibitors used in the clinic (SAHA, valproic acid) inhibit all 11 Zinc-dependent HDAC enzymes (HDAC1-11) and their prescription is associated with a number of debilitating side-effects, including: fatigue, diarrhea, low platelet counts, and hyperammonemia, which can lead to brain damage. Knock-out (KO) mice and RNAi studies implicate HDAC1 and 2 (HDAC1/2) as the most clinically relevant targets, particularly in terms of growth inhibition and apoptosis. We propose that targeting HDAC1/2, rather than HDAC1-11, might retain the therapeutic benefits of current HDAC inhibitors while avoiding the side-effects associated with inhibiting the entire sub-family of enzymes. To explore this, we intend to identify the key pathways regulated by HDAC1/2 and determine the therapeutic value of specific inhibitors. Objective 1: Since HDAC inhibitors are used clinically to treat cutaneous T-cell lymphoma, we will assess the role of HDAC1/2 during T-cell development, using existing HDAC1/2 knock-out mice and a novel catalytically inactive HDAC1 mutant line. Objective 2: HDAC1/2 activity in vivo is regulated by incorporation into three specific co-repressor complexes: Sin3A, NuRD and CoREST. To determine molecular details of complex assembly we will solve the structures of MTA1-HDAC1/2 and Sin3A-HDAC1/2 using X-ray crystallography. Using this data, the functional necessity of complex incorporation will then be assessed by testing the ability HDAC1/2 mutants to rescue the viability of HDAC1/2 double KO cells. Objective 3: Define the physiological substrates and genomic targets of HDAC1/2 activity, using quantitative mass-spectrometry and ChIP-seq assays, in double HDAC1/2 KO cells. This multi-disciplinary program of work will clarify the essential roles of HDAC1/2, their mechanism of action and establish the therapeutic value of specific HDAC inhibitors.

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 with data from the novel HDAC1-Y303H mice and interaction studies, 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.