Supplementary Figure 7: HDAC3 and MeCP2 regulate the recruitment of FOXO transcription factors to gene promoters.
From: Histone deacetylase 3 associates with MeCP2 to regulate FOXO and social behavior
(a) Upper table: transcription factor-binding motifs enriched at the 194 genes downregulated in Hdac3 cKO mice. Lower table: the FOXO motif (TTGTTT) was enriched within the 1274 genes downregulated in the hippocampus of Mecp2 KO mice27, the 2167 genes downregulated in the hypothalamus of Mecp2 KO mice5, and the 410 genes downregulated in the cerebellum of Mecp2 KO mice6. (b) Frequency probability matrix for de novo motif identified by MEME-ChIP analysis of HDAC3 binding regions; input of 6149 HDAC3 binding sequences of which 1038 sites identified with TRTTTW motif. (c) Foxo1, Foxo3, Foxo4 and Foxo6 gene expression in the CA1 hippocampus of control and Hdac3 cKO mice; n = 2, 2 mice. FPKM, fragments per kilobase of exon per million fragments mapped. (d) Coronal brain slices of control and Hdac3 cKO mice were stained using Hoechst (blue), anti-acetyl-FOXO (green), and anti-NeuN (white). Images of the striatum using a confocal microscope, scale bar, 20 μm. Mean gray value of acetyl-FOXO immunofluorescence quantitated using only NeuN-positive nuclei in 3-month control and Hdac3 cKO mice. Two-tailed t test; t(10) = 0.9065, P = 0.3860; n = 5, 7 mice. (e) Immunoprecipitation of recombinant human FOXO3 with human MeCP2 or human HDAC3 followed by western blot analysis of FOXO3, HDAC3 and MeCP2; n = 3 assays. Full-length blots are presented in Supplementary Fig. 10e. (f) Immunoprecipitation of recombinant human MeCP2 with human HDAC3 followed by western blot analysis of MeCP2 and HDAC3; n = 3 assays. Full-length blots are presented in Supplementary Fig. 10f. (g) Immunostaining of C1 (control), line 4 and line 14 (MECP2R306C), line 6 and line 36 (isogenic control) NPCs using Hoechst (blue), anti-MSI1 (green), and anti-Nestin (red). Imaged using confocal microscope; lines C1, 4 and 14, n = 6, 6, 6 coverslips, 2 experiments; lines 6 and 36, n = 3, 3 coverslips, 1 experiment; scale bar = 40 μm. All values are mean ± s.e.m.
