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. Author manuscript; available in PMC: 2012 May 13.
Published in final edited form as: Cell. 2011 May 13;145(4):596–606. doi: 10.1016/j.cell.2011.04.013

Figure 6. HDAC4 activates FOXO, also see Figure S6.

Figure 6

(A) Immunoblot of FOXO recovered from immunoprecipitates of HDAC4 or control IgG prepared from Drosophila S2 cells. Input amounts of transfected HDAC4 and FOXO shown.

(B) Q-PCR analysis of mRNA amounts for fasting inducible genes (PEPCK, bmm, CPTI) in HDAC4 hypomorphic mutant flies (top) or in flies expressing phosphorylation-defective, constitutively active HDAC4.3A in fat-body (bottom). Genotypes are: FB>(FB-GAL4/+) and FB>HDAC4.3A (FB-GAL4/+;UAS-HDAC4.3A/+) for over-expression assays. For HDAC4 hypomorphic mutant flies, genotypes are HDAC4+ (HDAC4KG09091/+) and HDAC4− (HDAC4KG09091/HDAC4e04575). Effects of HDAC4− (top) examined in 24 hour fasted flies. Effects of HDAC4.3A examined under ad libitum feeding conditions.

(C) Immunoblot showing time course of FOXO phosphorylation and acetylation during refeeding in wild-type (w), SIK3 mutant (SIK348). Ac-FOXO protein detected using anti ac-FOXO1 (K242/245) antibody after immunoprecipitation with anti- acetyl-lysine antibody. Extract from FOXO mutant (FOXO21/FOXO25) flies (FOXO−) included to show specificity of ac-FOXO1 and non-discriminating FOXO antisera. (D) Top: schematic showing domain organization of FOXO, including DNA-binding domain (DBD), nuclear localization sequence (NLS), nuclear export sequence (NES), and trans-activation domain (TA). Middle: alignment of conserved lysine residues in the DBDs of Drosophila FOXO and human FOXO1. Conserved lysine residues that undergo acetylation (Lys 179,182 in Drosophila; Lys 242, 245 in human) shown. Bottom, Immunoblot showing Ac-FOXO, and unphosphorylated (FOXO) or phospho-FOXO (pFOXO) protein amounts in 24 hour fasted or 1 hour refed wild-type (HDAC4+) and HDAC4-mutant (HDAC4−) flies. Relative amounts of acetylated FOXO, determined by densitometric analysis, indicated above each lane.

Genotypes are: HDAC4+ (HDAC4KG09091/+) and HDAC4− (HDAC4KG09091/HDAC4e04575).

(E) In vitro deacetylation assay showing effect of purified HDAC4 on deacetylation of a GST-FOXO (aa.95–195) polypeptide acetylated in vitro with the histone acetyl transferase P300. Immunoblot shows amounts of acetylated FOXO (Ac-FOXO) using anti-acetyl lysine antibody. Input protein amounts for GST-FOXO (aa. 95–195) and HDAC4 proteins determined by Ponceau S staining.

(F) Effect of oral supplementation with class I/II HDAC inhibitor trichostatin A (TSA) in the food (10μM) on fat body lipid levels in SIK3 mutant flies. (n=10 per genotype). Genotypes are: w− (w) and SIK3− (SIK348). Data are averages ± SEM (**, P<0.01; *, P<0.05).