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Histone deacetylase 3 associates with MeCP2 to regulate FOXO and social behavior

Abstract

Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). The RTT missense MECP2R306C mutation prevents MeCP2 from interacting with the NCoR/histone deacetylase 3 (HDAC3) complex; however, the neuronal function of HDAC3 is incompletely understood. We found that neuronal deletion of Hdac3 in mice elicited abnormal locomotor coordination, sociability and cognition. Transcriptional and chromatin profiling revealed that HDAC3 positively regulated a subset of genes and was recruited to active gene promoters via MeCP2. HDAC3-associated promoters were enriched for the FOXO transcription factors, and FOXO acetylation was elevated in Hdac3 knockout (KO) and Mecp2 KO neurons. Human RTT-patient-derived MECP2R306C neural progenitor cells had deficits in HDAC3 and FOXO recruitment and gene expression. Gene editing of MECP2R306C cells to generate isogenic controls rescued HDAC3-FOXO-mediated impairments in gene expression. Our data suggest that HDAC3 interaction with MeCP2 positively regulates a subset of neuronal genes through FOXO deacetylation, and disruption of HDAC3 contributes to cognitive and social impairment.

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Figure 1: Hdac3 cKO mice exhibit social and cognitive impairments similar to models of RTT.
Figure 2: HDAC3 regulates the transcription of a subset of genes in common with MeCP2.
Figure 3: MeCP2 modulates HDAC3 localization to the promoters of transcribed genes.
Figure 4: HDAC3 regulates FOXO3 deacetylation and recruitment to gene promoters in neurons.
Figure 5: FOXO3 and HDAC3 recruitment to gene regulatory regions is abrogated in NPCs harboring a patient-derived MECP2 mutation.

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Acknowledgements

We thank E.N. Olson (UT Southwestern Medical Center) for kindly providing the HDAC3f/f mice. We thank M. Sur and A. Banerjee (both at Massachusetts Institute of Technology for providing Mecp2 KO mice. We thank G.-L. Ming (Johns Hopkins University) for kindly providing the C1 iPSC cell line. We thank R. Madabhushi, A. Watson and J. Penney for comments on the manuscript. We thank E. Demmons for help with mouse colony maintenance. This work was supported by US National Institutes of Health grants (MH102690 and NS079625) and Rettsyndrome.org to P.J., and NIH grant NS78839 and the JPB Foundation to L.-H.T.

Author information

Authors and Affiliations

Authors

Contributions

A.N. and L.-H.T. designed the study, and L.-H.T. directed and coordinated the study. A.N. initiated, planned and performed experimental work. J.C. helped with ChIP experiments. F.G. and J.M. contributed to bioinformatic analysis. P.M. helped with some behavioral experiments. A.V.Z. helped with validation of the RNA-seq. E.G. prepared sequencing libraries for the ChIP-seq experiment. T.K. derived the NPC lines. T.K. and Y.-T.L. helped to maintain the iPSCs. F.Z. and P.J. provided Mecp2 KO tissue. A.N. and L.-H.T. wrote the manuscript with critical input from all of the authors.

Corresponding author

Correspondence to Alexi Nott.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Hdac3 forebrain-specific conditional knockout from excitatory neurons.

(a) Coronal brain slices of 3-month control and Hdac3 cKO male mice were stained using Hoechst (blue) and anti-HDAC3 (green), and images captured of the hippocampus using tile scan of a confocal microscope. Scale bar, 200 μm. (b) Mean gray value quantification of HDAC3 immunofluorescence was compared between control and Hdac3 cKO mice in the CA1 and dentate gyrus (DG) of the hippocampus; two-tailed t test; CA1, t(8) = 12.88, **** P < 0.0001; DG, t(8) = 5.958, *** P = 0.0003; n = 4, 6 mice. (c) Coronal brain slices of 3-month control and Hdac3 cKO male mice were stained using Hoechst (blue), anti-HDAC3 (red), anti-GFAP (green), and anti-NeuN (white), and images captured of hippocampal CA1 using a confocal microscope. Scale bar, 20 μm; n = 3, 3 mice. (d) Coronal brain slices of 3-month control and Hdac3 cKO male mice were stained using Hoechst (blue), anti-HDAC3 (red), anti-Parvalbumin (green), and anti-NeuN (white), and images captured of hippocampal CA1 using a confocal microscope. Scale bar, 20 μm; n = 3, 3 mice. (e) Western blot analysis of HDAC3, HDAC2 and actin protein levels using hippocampal whole cell lysates from control and Hdac3 cKO mice. Full-length WB’s are presented in Supplementary Fig. 10b. (f) Mean gray value quantification of HDAC3 protein levels normalized to actin of western blot in c. Two-tailed t test; t(6) = 9.663, **** P < 0.0001; n = 4, 4 mice. (g) Western blot analysis of HDAC3 and actin protein levels using cortex whole cell lysates from control and Hdac3 cKO mice. Full-length WB’s are presented in Supplementary Fig. 10c. (h) Mean gray value quantification of HDAC3 protein levels normalized to actin of western blot in e. Two-tailed t test; t(6) = 3.460, * P = 0.0135; n = 4, 4 mice. (i), Western blot analysis of HDAC3 and actin protein levels using striatum whole cell lysates from control and Hdac3 cKO mice. Full-length WB’s are presented in Supplementary Fig. 10d. (j) Mean gray value quantification of HDAC3 protein levels normalized to actin of western blot in g. Two-tailed t test; t(6) = 0.9516, P = 0.3780; n = 4, 4 mice. All values are mean ± s.e.m.

Supplementary Figure 2 Hdac3 cKO mice exhibit locomotor abnormalities.

(a) (f), Control and Hdac3 cKO mice were subjected to open-field test for 60 minutes and the data was analyzed in 5 minute bins using two-way ANOVA with Bonferroni post hoc. Two-way ANOVA for, (a) vertical activity; Genotype: F(1,44) = 150.1, **** P < 0.0001, Time: F(11, 484) = 18.45, **** P < 0.0001 (Bonferroni post hoc **** P < 0.0001), (b) total distance traveled; Genotype: F(1,44) = 57.18, **** P < 0.0001, Time: F(11, 484) = 47.40, **** P < 0.0001 (Bonferroni post hoc **** P < 0.0001), (c) horizontal activity; Genotype: F(1,44) = 136.26, **** P < 0.0001, Time: F(11, 484) = 38.26, **** P < 0.0001 (Bonferroni post hoc **** P < 0.0001), (d) margin distance traveled Genotype: F(1,44) = 95.66, **** P < 0.0001, Time: F(11, 484) = 63.44, **** P < 0.0001 (Bonferroni post hoc **** P < 0.0001), (e) center distance traveled; Genotype: F(1,44) = 72.76, **** P < 0.0001, Time: F(11, 484) = 5.077, **** P < 0.0001 (Bonferroni post hoc **** P < 0.0001, * P < 0.05), and (f) time spent in the margin; Genotype: F(1,44) = 24.82, **** P < 0.0001, Time: F(11, 484) = 2.131, * P = 0.0171 (Bonferroni post hoc **** P < 0.0001, * P < 0.05); for a-f, n = 22, 24 mice. All values are mean ± s.e.m.

Supplementary Figure 3 Hdac3 cKO mice exhibit deficits in sociability and hippocampal-dependent learning.

(a) Time spent in chambers during 10 minute habituation phase of the sociability task (Fig. 1d) for control and Hdac3 cKO mice. Control; one-way ANOVA, F(2, 24) = 0.1687, P = 0.8457; Bonferroni post hoc, Left versus Center, P > 0.99; Left versus Right, P > 0.99; Center versus Right, P > 0.99; n = 9 mice. Hdac3 cKO; one-way ANOVA, F(2, 24) = 2.793, P = 0.0812; Bonferroni post hoc, Left versus Center, P = 0.4200; Left versus Right, P > 0.99; Center versus Right, P = 0.0864; n = 9, 9 mice. (b) Total time spent with both objects (s) during the object location memory task (Fig. 1e); two-tailed t test; t(26) = 3.883; *** P = 0.0006; n = 16, 12 mice. (c) The average swim speed (m s−1) of the mice was recorded during the training days (day 1 – 7) and on the probe trial day (day 8) of the MWM task. Two-way ANOVA; Genotype, F(1,133) = 9.530, ** P =0.0025; Day, F(6,133) = 3.050, ** P = 0.0079; Bonferroni post hoc; day 1, P > 0.99; day 2, P > 0.99; day 3, P > 0.99; day 4, P > 0.99; day 5, P = 0.0677; day 6, P > 0.99; day 7, P = 0.2703; n = 10, 11 mice. (d) Mean velocity (cm s−1) measured during the habituation and the e-stimulation phases on the training day of fear-conditioned learning; Control: two-tailed t test; t(20) = 15.13; **** P < 0.0001; Hdac3 cKO: t(20) = 17.92; **** P < 0.0001; n = 11, 11 mice. (e) Activity suppression of control and Hdac3 cKO mice during testing of contextual memory; two-tailed t test; t(20) = 3.973; *** P = 0.0007; n = 11, 11 mice. (f) Activity suppression of control and Hdac3 cKO mice during testing of cued (tone) memory; two-tailed t test; t(20) = 5.069; **** P < 0.0001; n = 11, 11 mice. c, report mean value ± s.e.m. a, b, d - f, report median, 25th and 75th percentile, min and max value.

Supplementary Figure 4 Transcriptional regulation in the Hdac3 cKO.

(a) qRT-PCR analysis of RNA extracted from the CA1 region of control and Hdac3 cKO mice. Primers designed within exon 10 (non-floxed) and exon 11 (floxed). Two-tailed t test; t(6) = 7.390, *** P = 0.0003; n = 4, 4 mice. Values are mean ± s.e.m. (b) RT-PCR analysis of RNA extracted from the CA1 region of control and Hdac3 cKO mice. Primers designed within exons 10 and 15, flanking floxed exons 11 – 14, n = 2, 2 mice. (c) log2fold change analysis of Hdac3 exon read counts between control and Hdac3 cKO mice following RNA-sequencing of the CA1 region of the hippocampus; n = 2, 2 mice. (d) UCSC genome browser visualization of RNA-seq coverage at the Hdac3 locus for control (blue) and Hdac3 cKO (red). The y-axis is number of reads mapped.

Supplementary Figure 5 HDAC3 is recruited to promoters of transcribed genes.

(a) Enrichment of HDAC3 binding peaks at annotated regions of the genome analyzed using Homer (enrichment over genome). TTS, transcriptional termination site; UTR, untranslated region. (b) Enrichment of HDAC3 binding peaks at genomic regions analyzed using chromatin state profiling of the hippocampus. TSS, transcriptional start site. (c) Aggregate plots of average HDAC3 ChIP-seq intensity signal in 3-month wild-type hippocampus. Aggregate plots were generated at the TSS of genes upregulated (green) and downregulated (blue) in the CA1 of Hdac3 cKO mice. HDAC3 ChIP-seq intensity signal at the TSS of genes with FPKM expression values below 0.1 (black; negative control). TSS, transcriptional start site. (d) UCSC genome browser view of Tle1 and Klf10 loci showing RNA-seq coverage from control and Hdac3 cKO hippocampus, and HDAC3 ChIP-seq coverage and peak calling from wild-type hippocampus. (e) ChIP of HDAC3 and IgG followed by qPCR analysis at the promoters of Arrdc2, Dusp4, Klf10, Tle1, Bdnf, and Nr4a1 in hippocampus of wild-type mice (% input); two-tailed t test; Arrdc2, t(4) = 6.944, ** P = 0.0023; Dusp4, t(4) = 5.074, ** P = 0.0071; Klf10, t(4) = 7.636, ** P = 0.0016; Tle1, t(4) = 2.917, * P = 0.0434; Bdnf, t(4) = 3.350, * P = 0.0286; Nr4a1, t(3) = 6.237, ** P = 0.0083; n = 3, 3 mice. (f) ChIP of HDAC3 and IgG followed by qPCR analysis at the promoters of Arrdc2, Dusp4, Klf10, Tle1, Bdnf, and Nr4a1 in hippocampal CA1 region of control and Hdac3 cKO mice (% input); One-way ANOVA; Arrdc2, F(2,6) = 9.653, * P = 0.0133; Dusp4, F(2,6) = 20.38, ** P = 0.0021; Klf10, F(2,6) = 77.61, **** P < 0.0001; Tle1, F(2,6) = 19.07, ** P = 0.0025; Bdnf, F(2,6) = 15.94, ** P = 0.0040; Nr4a1, F(2,6) = 54.27, *** P = 0.0001. Bonferroni post hoc; control versus Hdac3 KO: Arrdc2, * P = 0.0472; Dusp4, * P = 0.0246; Klf10, *** P = 0.0006; Tle1, * P = 0.0368; Bdnf, * P = 0.0201; Nr4a1, *** P = 0.0006; control versus IgG: Arrdc2, * P = 0.0105; Dusp4, ** P = 0.0014; Klf10, **** P < 0.0001; Tle1, ** P = 0.0017; Bdnf, ** P = 0.0029; Nr4a1, *** P = 0.0001; n = 3, 3, 3 mice. e, f, report mean value ± s.e.m.

Supplementary Figure 6 HDAC3 and MeCP2 regulate the recruitment of FOXO transcription factors to gene promoters.

(a) UCSC genome browser view at the Arrdc2 locus of HDAC3 ChIP-seq coverage in the hippocampus of P45 wild-type and Mecp2 KO mice. (b) Aggregate plots of average HDAC3 ChIP-seq signal in P45 wild-type hippocampus (blue) and Mecp2 KO hippocampus (purple). Aggregate plots were generated at the TSS of genes upregulated in the hippocampus of Mecp2 KO mice27. HDAC3 ChIP-seq signal at the TSS of genes with the lowest expression in P45 hippocampus27 (black; negative control; 10% of total genes). TSS, transcriptional start site. (c) Aggregate plots of average HDAC3 ChIP-seq signal in P45 wild-type hippocampus (blue) and Mecp2 KO hippocampus (purple). Aggregate plots were generated at the TSS of genes downregulated in the hippocampus of Mecp2 KO mice. HDAC3 ChIP-seq signal at the TSS of genes with the lowest expression in P45 hippocampus27 (black; negative control; 10% of total genes). TSS, transcriptional start site. (d) ChIP of MeCP2 and IgG followed by qPCR at the promoters of Arrdc2, Dusp4, Klf10, Tle1, Bdnf and Nr4a1 in hippocampus of wildt-ype and Mecp2 KO mice (% input). One-way ANOVA; Arrdc2, F(2,9) = 12.62, ** P = 0.0024; Dusp4, F(2,9) = 28.96, **** P < 0.0001; Klf10, F(2,9) = 14.84, ** P = 0.0014; Tle1, F(2,9) = 14.39, ** P = 0.0016; Bdnf, F(2,9) = 8.119, ** P = 0.0097; Nr4a1, F(2,9) = 5.996, * P = 0.0221. Bonferroni post hoc; wild-type versus Mecp2 KO: Arrdc2, ** P = 0.0028; Dusp4, *** P = 0.0003; Klf10, ** P = 0.0053; Tle1, ** P = 0.0037; Bdnf, * P = 0.0307; Nr4a1, * P = 0.0231; wild-type versus IgG: Arrdc2, ** P = 0.0051; Dusp4, *** P = 0.0001; Klf10, ** P = 0.0012; Tle1, ** P = 0.0017; Bdnf, ** P = 0.0080; Nr4a1, * P = 0.0410; n = 4, 4, 4 mice. (e) ChIP of H4K12ac and IgG followed by qPCR analysis at the promoters of Arrdc2, Dusp4, Klf10, Tle1, Bdnf, and Nr4a1 in hippocampal CA1 region of control and Hdac3 cKO mice (% input); one-way ANOVA; Arrdc2, F(2,6) = 18.79, ** P = 0.0026; Dusp4, F(2,6) = 7.802, * P = 0.0214; Klf10, F(2,6) = 10.09, ** P = 0.0120; Tle1, F(2,6) = 6.464, * P = 0.0318; Bdnf, F(2,6) = 8.743, * P = 0.0167; Nr4a1, F(2,6) = 5.209, * P = 0.0488. Bonferroni post hoc; Control versus Hdac3 cKO: Arrdc2, P = 0.5511; Dusp4, P > 0.99; Klf10, P = 0.4278; Tle1, P > 0.99; Bdnf, P > 0.99; Nr4a1, P > 0.99; Control versus IgG: Arrdc2, ** P = 0.0023; Dusp4, * P = 0.0168; Klf10, * P = 0.0478; Tle1, * P = 0.0310; Bdnf, * P = 0.0245; Nr4a1, * P = 0.0448; n = 3, 3, 3 mice. (f) ChIP of H3K9ac and IgG followed by qPCR analysis at the promoters of Arrdc2, Dusp4, Klf10, Tle1, Bdnf, and Nr4a1 in hippocampal CA1 region of control and Hdac3 cKO mice (% input); one-way ANOVA; Arrdc2, F(2,5) = 7.810, * P = 0.0290; Dusp4, F(2,5) = 18.34, ** P = 0.0050; Klf10, F(2,5) = 8.792, * P = 0.0231; Tle1, F(2,5) = 9.315, * P = 0.0206; Bdnf, F(2,5) = 43.53, * P = 0.0007; Nr4a1, F(2,5) = 30.06, ** P = 0.0016. Bonferroni post hoc; Control versus Hdac3 cKO: Arrdc2, P > 0.99; Dusp4, P > 0.99; Klf10, P > 0.99; Tle1, P > 0.99; Bdnf, P = 0.8642; Nr4a1, P = 0.9790; Control versus IgG: Arrdc2, * P = 0.0317; Dusp4, ** P = 0.0058; Klf10, * P = 0.0248; Tle1, * P = 0.0318; Bdnf, *** P = 0.0007; Nr4a1, ** P = 0.0016; n = 3, 3, 2 mice. All values are mean ± s.e.m.

Supplementary Figure 7 HDAC3 and MeCP2 regulate the recruitment of FOXO transcription factors to gene promoters.

(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.

Supplementary Figure 8 Genotyping of iPSCs and NPCs derived from a MECP2R306C RTT patient and corresponding isogenic control lines generated by CRISPR/Cas9 gene editing.

(a) - (d) Chromatogram view of MECP2R306C locus following sequencing of genomic DNA (gDNA) and complementary DNA (cDNA) from iPSC and NPC lines as indicated. Sequences were analyzed for MECP2R306C iPSCs (a, b), line 4 and 14 MECP2R306C NPCs (c), line 6 and 36 iPSCs, and line 6 and 36 NPCs (d). Purple inset indicates the nucleotide position of the R306C mutation. Green inset indicates the nucleotide position of the corrected R306C mutation following CRISPR/Cas9 gene editing. Red insets indicate intentional introduction of heterozygous silent mutations following CRISPR/Cas9 gene editing.

Supplementary Figure 9 Chromatogram view of the five genomic regions with the highest probability for off-targeted CRISPR/Cas9-mediated gene editing.

Chromatograms show sequencing of gDNA from isogenic control lines 6 and 36. Off-target CRISPR/Cas9 genomic editing was not detected in either line. Black inset indicates the nucleotide region predicted for potential off-target gene editing.

Supplementary Figure 10 Full-length Western Blots

(a) Full-length western blots of Snap25 and Actin for cropped images in Fig. 2e. (b) Full-length western blots of HDAC3, HDAC2 and Actin for cropped images in Supplementary Fig. 1e. (c) Full-length western blots of HDAC3 and Actin for cropped images in Supplementary Fig. 1g. (d) Full-length western blots of HDAC3 and Actin for cropped images in Supplementary Fig. 1i. (e) Full-length western blots of HDAC3, FOXO3 and MeCP2 for cropped images in Supplementary Fig. 7e. (f) Full-length western blots of HDAC3 and MeCP2 for cropped images in Supplementary Fig. 7f.

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Nott, A., Cheng, J., Gao, F. et al. Histone deacetylase 3 associates with MeCP2 to regulate FOXO and social behavior. Nat Neurosci 19, 1497–1505 (2016). https://doi.org/10.1038/nn.4347

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