Analysis of the physiological role of histone deacetylase 3 (HDAC3) and its regulation by inositol phosphates

Histone deacetylase 3 (HDAC3) acts as the catalytic core of the SMRT/NCoR co-repressor complex which regulates chromatin structure and gene expression. It was recently shown that HDAC3 binds, and is regulated in vitro, by the binding of inositol phosphates (IP). We used transcriptional reporter assays to interrogate whether HDAC3-mediated repression in vivo is dependent of IP. Manipulation of intracellular IP levels through chemical inhibition of enzymes involved in IP metabolism or RNAi-mediated protein knockdown were inconclusive. However, mutation of key IP binding residues in both SMRT and HDAC3 directly impacts the repressive ability of the co-repressor complex, presumably through an impaired ability to bind IP and failure to fully activate the enzyme. Germline deletion of HDAC3 in the mouse results in early embryonic lethality (around e9.5) suggesting it plays an essential role in embryogenesis. To further investigate the role of HDAC3 in embryonic development, I have generated a conditional knockout embryonic stem cell line in which HDAC3 can be specifically inactivated. Loss of the protein occurs within 3 days suggesting a half-life of approximately 24 hours and correlates with concomitant decrease in co-repressor complex components, indicating HDAC3 contributes to co-repressor integrity. Unlike deletion of HDAC1 and -2, loss of HDAC3 does not cause a significant reduction in total deacetylase activity with only minor changes in the acetylation levels of histones. However, the proliferative capacity of knockout cells is inhibited with a delay in cell doubling time. Upon differentiation, we find that embryoid bodies (EBs) lacking HDAC3 are significantly smaller and morphologically different compared to controls. Microarray analysis over a 7-day time course of EB differentiation reveals that endodermal cell markers are over-expressed at both early and late stages of development, suggesting that HDAC3 plays an important role in regulating gene expression during embryonic development.