Analysis of Class-I Histone Deacetylase Function in Embryonic Development, Tissue Formation and Homeostasis.

‘Histone deacetylase‘ (HDAC) enzymes are present in all cells of the body. Their function is to switch genes ‘off‘, and making sure they stay ‘off‘. In many respects shutting a gene down is every bit as important as switching a gene on. I intend to study how three different HDAC enzymes (HDACs 1, 2 and 8) do this. One of the best methods for understanding how an enzyme works is to generate mutant cells in which the specific enzyme has been inactivated. 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. If we use mouse stem cells to this then we can create HDAC knock-out mice.

HDAC enzymes represent an exciting medical opportunity because they are ‘druggable‘. Already, inhibitors of HDAC activity are being tested in the clinic as anti-cancer agents, and in the laboratory for their beneficial affects on an Alzheimer‘s disease and their 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 for use in treating patients.

Our aim is to understand the function and utilization of class-1 ‘histone deacetylase‘ (HDAC) enzymes during embryonic development, tissue formation and homeostasis. Hundreds of different transcriptional repressors employ class-1 HDACs (HDACs 1, 2, 3 and 8) to remove the acetyl moiety from Lysine residues within the N-termini of histones. Loss of the acetyl group restores the lysine‘s positive charge, thus increasing the natural affinity of the histone tail for the phosphate backbone of DNA, creating a repressive chromatin conformation. HDACs are therefore, common mechanistic components used for the regulation of global gene transcription in virtually all cells, from yeast to man. HDAC enzymes also represent an exciting medical opportunity because they are ‘druggable‘. Already, generic small molecule inhibitors of HDAC activity are being tested in the clinic as anti-cancer agents, and in the laboratory for their ameliorative effects on an Alzheimer‘s disease mouse model and their anti-inflammatory properties. There is therefore a compelling applied, as well as academic, motivation for studying the physiological roles of individual HDAC enzymes.

As a starting point for a comprehensive analysis of class-1 HDAC function we are generating conditional knock-out (cKO) mice for HDAC1, HDAC2 and HDAC8. The deletion of each particular HDAC enzyme in a physiological setting allows us to identify the cell type, or developmental step, that is dependent upon HDAC activity. We can then use these primary cells (isolated from the mouse) and biochemical approaches to decipher the mechanism for the observed phenotype. If homozygous deletion of HDAC2 and HDAC8 causes early embryonic lethality, as previous work has shown that HDAC1 does, we will generate and analyze a T cell specific deletion for each HDAC enzyme. In addition, we will also utilize embryonic stem cells derived from the HDAC cKO mice to examine how loss of HDAC function affects cell cycle and endogenous gene expression. A Chip-on-chip approach will then be employed to define which of these genes are directly regulated by HDAC function. Finally, we will address a gap in our understanding of HDAC8 function by purifying HDAC8 protein complexes from cells.

By inactivating HDAC1, HDAC2 and HDAC8 in the mouse, analyzing their gene networks and protein-protein interaction partners we hope to uncover the fundamental mechanisms of class-1 HDAC function. And in doing so, help realize their potential as pharmacological targets for use in treating human disease.