Fig.1

Expression and subcellular localization of HDAC4^WT and mutants. A Schematic of the domain structure of Drosophila HDAC4. Green, MEF2 binding site; blue, NLS; red, NES; purple, ankyrin repeat binding domain; yellow circles, phosphorylated serine residues that bind 14–3-3 proteins; black, catalytic domain; pink, location of the Y1142 residue in the catalytic site; yellow Myc tag. Shaded area depicts the α helix region important in tetramerization. B Amino acid substitutions present in each of the HDAC4 mutants are shown in red. All residues in red were mutated to alanines. C Cartoon of the mushroom body showing the Kenyon cells (kc), peduncle (p), calyx (c) and α, α', β, β' and γ lobes of the mushroom body. The location of the lobes in the anterior region of the brain (red square) and the Kenyon cells at the posterior (blue square) is also shown. D–G All genotypes were generated by crossing OK107-GAL4; tubP-GAL80ts females to males carrying the indicated UAS-HDAC4 transgene. Flies were raised at 18 °C, at which temperature GAL80ts represses GAL4, until after eclosion when the temperature was raised to 30 °C. At this temperature, GAL80ts is inactivated, allowing GAL4 to induce transgene expression. Brains were dissected after 48 h at 30 °C. D Immunohistochemistry on whole mount brains with anti-Myc (green) counterstained with DAPI (magenta). Representative images from n = 16–19 brains per genotype are shown. The top panel shows uninduced HDAC4^WT and the panels below show expression of each of the transgenes following induction. The left panel shows a Z-stack maximum projection spanning the mushroom body lobes. Scale bar = 100 μm. The middle panel is a Z-stack through the Kenyon cell layer of the same brain. Scale bar = 100 μm. The right panel is a single 1 μm optical section through the cell bodies of the Kenyon cells Scale bar = 10 μm. Arrows indicate small puncta in HDAC4^ΔNLS nuclei that are substantially smaller than those in HDAC4^WT nuclei. E Western blot showing expression of each of the transgenes at 18 °C (uninduced) and 30 °C (induced) as detected by anti-Myc with α-tubulin as a loading control. Line through the center indicates joining of two blots that were processed simultaneously. F Quantification of Western blot. HDAC4^WT and mutant protein levels were normalized to tubulin. Error bars indicate SEM of blots from three independent fly crosses. There were no significant differences in protein levels at 18 °C (ANOVA F_(5,12) = 1.974, p = 0.156) or 30 °C (ANOVA, F_(5,12) = 0.787, p = 0.579). G Quantification of nuclear aggregates. The number of aggregates per section were counted from three sections per brain, n = 16–19 brains/genotype. The number of aggregates was significantly increased in the Kenyon cell nuclei of HDAC4^3SA and HDAC4^ΔANK brains and was significantly reduced in HDAC4^ΔMEF2 and HDAC4^ΔNLS brains in comparison to HDAC4^WT (ANOVA, F_(5,97) = 38.46, p = 1.11 × 10.⁻¹⁶, Tukey’s post hoc test, *p < 0.05, ***p < 0.001)