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Polycomb repressive complex 2 (PRC2) silences genes responsible for neurodegeneration

Nature Neuroscience volume 19, pages 1321–1330 (2016)Cite this article

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Abstract

Normal brain function depends on the interaction between highly specialized neurons that operate within anatomically and functionally distinct brain regions. Neuronal specification is driven by transcriptional programs that are established during early neuronal development and remain in place in the adult brain. The fidelity of neuronal specification depends on the robustness of the transcriptional program that supports the neuron type-specific gene expression patterns. Here we show that polycomb repressive complex 2 (PRC2), which supports neuron specification during differentiation, contributes to the suppression of a transcriptional program that is detrimental to adult neuron function and survival. We show that PRC2 deficiency in striatal neurons leads to the de-repression of selected, predominantly bivalent PRC2 target genes that are dominated by self-regulating transcription factors normally suppressed in these neurons. The transcriptional changes in PRC2-deficient neurons lead to progressive and fatal neurodegeneration in mice. Our results point to a key role of PRC2 in protecting neurons against degeneration.

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Figure 1: Combined Ezh1 and Ezh2 deficiencies lead to H3K27me3 deficiency in adult neurons.
Figure 2: H3K27me3 is associated with non-neuronal, non-MSN and death-promoting genes in adult MSNs.
Figure 3: Selective effect of PRC2 deficiency on MSN gene expression.
Figure 4: PRC2 deficiency leads to downregulation of MSN-specific genes.
Figure 5: PRC2 deficiency leads to upregulation of death-promoting genes and associated neurodegenerative changes in MSNs.
Figure 6: PRC2 deficiency in adult neurons causes a progressive and fatal neurodegenerative phenotype in mice.
Figure 7: PRC2 deficiency in PCs impairs PC-specific gene expression and function.

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Acknowledgements

We thank T. Jenuwein from the MPI in Freiburg for providing the Ezh1−/− mice, N. Heintz from the Rockefeller University for providing the Pcp2-TRAP mice, D. Reinberg from New York University Medical Center for providing the Ezh1 antibody, K. Uryu for electron microscopy, J. Scarpa and F. Zhang for their assistance with the bioinformatics analyses, M. Akeju, S. Mann and S. Kalik for technical assistance and animal work, and D. Reinberg, P. Greengard, and M. Heiman for discussions. This work was supported by the National Institutes of Health (NIH) Director New Innovator Award DP2 MH100012-01 (A.S.), 1R01NS091574 (A.S.), CURE Challenge Award (A.S.), 5R01GM112811 (A.T.), the Emerald Foundation Inc. (A.T.), NARSAD Young Investigator Award #22802 (M.v.S.), T32AG049688 (A.B.), 5T32MH096678 (J.M.S.), 1RO1MH092306 and 1R01AA022445 (M.-H.H.), J&J/IMHRO Translational Research Star Award (M.-H.H.), NARSAD Independent Investigator Award (M.-H.H.), 1 F31MH108326 and T32MH096678 (S.M.K.), R01GM098316 (A.M.), U54HG008230 (A.M.), and U54CA189201 (A.M.).

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Authors and Affiliations

  1. Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Melanie von Schimmelmann, Philip A Feinberg, Josefa M Sullivan, Stacy M Ku, Ana Badimon, Mary Kaye Duff, Ming-Hu Han & Anne Schaefer

  2. Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Stacy M Ku, Zichen Wang, Alexander Lachmann, Avi Ma'ayan & Ming-Hu Han

  3. BD2K-LINCS Data Coordination and Integration Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Zichen Wang, Alexander Lachmann & Avi Ma'ayan

  4. Genomics Resource Center, The Rockefeller University, New York, New York, USA

    Scott Dewell

  5. Laboratory of Immune Cell Epigenetics and Signaling, The Rockefeller University, New York, New York, USA

    Alexander Tarakhovsky

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  1. Melanie von Schimmelmann
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Contributions

A.S., A.T. and M.v.S. designed the study. M.v.S., P.A.F., J.M.S., A.B. and M.K.D. executed the molecular and behavioral experiments; J.M.S. and S.M.K. performed electrophysiology; M.-H.H. designed and performed electrophysiology; S.D. performed the ChIP-Seq analysis; and Z.W., A.L. and A.M. performed the enrichment and network analysis. A.S., A.T. and M.v.S. wrote the manuscript. All authors discussed results and provided input on the manuscript.

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Correspondence to Anne Schaefer.

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Integrated supplementary information

Supplementary Figure 1 Generation of mice with conditional neuron-specific deficiency in PRC2.

(a) The breeding schemes show the generation of mice with individual Ezh1, Ezh2 or combined Ezh1/Ezh2 (PRC2) deficiency in adult neurons. (Left panel) The Ezh2fl/fl mice were bred to Camk2a-cre mice to generate mice with an Ezh2 deficiency in postnatal forebrain neurons. The Ezh1-/- mice were crossed with Ezh2fl/fl; Camk2a-cre mice to generate mice with combined Ezh1 and Ezh2 deficiencies in postnatal forebrain neurons. (Middle panel) The Ezh1-/-; Ezh2fl/fl mice were bred to either Drd1a-cre, Drd2-cre or Pcp2-cre to generate mice with combined Ezh1 and Ezh2 deficiencies specifically in D1, D2 neurons or Purkinje cells, respectively. (Right panel) 10-week old Ezh1-/-; Ezh2fl/fl mice were stereotaxically injected with AAV-cre; eGFP to obtain adult mice with a dorsal striatal neuron-specific ablation of PRC2. (b) The deficiency of Ezh1 and Ezh2 was confirmed by Western blot analysis of striatal protein extracts derived from Ezh1-/- or Ezh2fl/fl; Camk2a-cre mice. β-Actin is used as loading controls. n = 2 mice per genotype, experiment was performed twice, a representative image is shown. (c) Deficiency in Ezh1 or neuron-specific deficiency in Ezh2 alone has no impact on MSN gene expression (P < 0.05; fold > 2). MSN gene expression analysis was performed using Ezh1-/- and Ezh2fl/fl; Camk2a-cre mice and their respective littermate controls at 12 months of age (n = 4 mice per genotype).

Supplementary Figure 2 MSN-specific ChIP sequencing reveals presence of H3K27me3+H3K4me3+ genes in MSNs.

(a) The scheme shows the FACS based purification of ex vivo isolated MSN nuclei. MSN nuclei are identified as NeuN positive. A representative experiment and gating is shown. (b) Venn diagram shows the number or H3K27me3+ H3K4me3+ gene loci in MSNs (orange) compared to bivalent genes in ES cells22. The profile plots show a similar distribution for H3K4me3, H3K27me3, H3K27acetyl and Pol II over the TSS (+/- 5kb) for MSN-specific bivalent genes (n = 291) and bivalent genes shared between MSNs and ES cells (n = 544 shared with ES cells). Significance of venn diagram was calculated using Chi-square test.

Supplementary Figure 3 Genes upregulated in PRC2-deficient MSNs are direct PRC2 target genes that are enriched in transcriptional regulators with auto- and co-regulatory functions.

(a) Enrichment terms for upregulated PRC2 target genes in MSNs of 3 and 6 months old Ezh1-/- or Ezh2fl/fl; Camk2a-cre mice were determined using the Enrichr gene set enrichment analysis68,69. While both 3 and 6 month upregulated genes are highly enriched in transcriptional regulators (GO MF, blue) controlled by PRC2 (ChEA, red) and H3K27me3 (ENCODE HM, pink), only the 6 month upregulated genes are enriched in cell death-promoting genes (KEGG, yellow) or genes associated with Huntington’s disease (Disease associated genes up, pink). The canvas visualization represent all terms from each library as tiles70. Tiles are arranged by gene set content similarity. Enriched terms are highlighted in color where the brightness indicates a lower p-value. Selective relevant enriched terms plus their respective p-values are listed on the sides on the canvases. (b) Auto-/co-regulatory network formation of upregulated genes in PRC2-deficient MSNs at 3 months of age. The networks connecting the upregulated genes are based on experimental evidence from published ChIP-sequencing datasets obtained from ChEA and ENCODE. Only studies in mice were included. TFs are colored in pink, non-TF genes are colored in blue. Nodes with outgoing links have evidence from ChIP-sequencing data. The size of the nodes is proportional to their connectivity degree.

Supplementary Figure 4 PRC2 deficiency leads to downregulation of MSN-specific genes.

(a) PRC2 deficiency is associated with the downregulation of MSN-specific protein expression. Western blot analysis on striatal protein lysate from 6 month-old Ezh1-/-; Ezh2fl/fl; Camk2a-cre and control mice (n = 2 mice per gentoype; (Adora2a) P = 0.0046, t (2) = 14.65; (Drd2) P = 0.0225, t (2) = 6.559; (Foxp1) P = 0.0256, t (2) = 6.129) is shown. (b) Downregulated genes (n =119) have no/low abundance of H3K27me3 over the TSS, which is not affected by the loss of PRC2. A profile plot of average H3K27me3 coverage over the TSS (+/- 5kb) of downregulated genes in control (black) and Ezh1-/-; Ezh2fl/fl; Camk2a-cre (blue) MSN nuclei analyzed by ChIP sequencing is shown (n = 10 mice each). (c) Genes downregulated in PRC2-deficient MSNs are enriched in genes important for specific MSN functions and behavior, and are associated with MSN-mediated diseases processes in humans. MSN-enrichment terms for downregulated PRC2 target genes were determined using the Enrichr gene set enrichment analysis68,69. The canvas visualization represent all terms from each library as tiles70. Tiles are arranged by gene set content similarity. Enriched terms are highlighted in color where the brightness indicates a lower p-value. Selective relevant enriched terms plus their respective p-values are listed on the sides on the canvases. Data are mean ± s.e.m.. *P ≤ 0.05, **P ≤ 0.01 from two-tailed Student’s t test.

Supplementary Figure 5 PRC2 deficiency in D1 and D2 MSNs leads to cell-intrinsic transcriptional changes.

(a) The gene expression in PRC2-deficient and control D1 neurons (left panel) and D2 neurons (right panel) were measured by TRAP analysis of the striatum derived from control and Ezh1-/-; Ezh2fl/fl; Drd1a-cre; Drd1a-TRAP or Ezh1-/-; Ezh2fl/fl; Drd2-cre; Drd2-TRAP mice, respectively ((Left Panel) D1: n = 2 mutant, 3 control mice; (Top) (Barx1) P < 0.0001, t (3) = 51.75; (Foxd1) P = 0.0009, t (3) = 13.16; (Gfi1) P = 0.0108, t (3) = 5.690; (Hand2) P = 0.001, t (3) = 13.11; (Nkx2-5) P < 0.0001, t (3) = 56.33; (Pitx2) P = 0.0003, t (3) = 20.30; (Pou4f1) P < 0.0001, t (3) = 44.41; (Sfmbt2) P = 0.0007, t (3) = 14.30; (Tal1) P = 0.0002, t (3) = 21.24; (Twist1) P < 0.0001, t (3) = 86.13; (Zic2) P < 0.0001, t (3) = 33.94; (Bottom left) (Ctgf) P = 0.0076, t (3) = 6.435; (Eya1) P = 0.0014, t (3) = 11.60; (Drd1a) P = 0.0077, t (3) = 6.410; (Bottom right) (Arpp21) P = 0.0032, t (3) = 8.689; (Foxp1) P = 0.0044, t (3) = 7.776; (Gpx6) P = 0.0102, t (3) = 5.803; (Ido1) P = 0.0007, t (2) = 38.76; (Rxrg) P = 0.0402, t (3) = 3.475;

(Right panel) D2: n = 3 mice each; (Top) (Barx1) P < 0.0001, t (4) = 74.70; (Foxd1) P = 0.0002, t (4) = 13.09; (Gfi1) P < 0.0001, t (4) = 34.89; (Hand2) P = 0.0069, t (2) = 12.00; (Nkx2-5) P = 0.0057, t (2) = 13.23; (Pitx2) P = 0.0335, t (2) = 5.328; (Pou4f1) P < 0.0001, t (4) = 24.56; (Sfmbt2) P = 0.0088, t (2) = 10.59; (Tal1) P = 0.0008, t (4) = 9.173; (Twist1) P < 0.0001, t (4) = 15.72; (Zic2) P < 0.0001, t (4) = 31.37; (Bottom left) (Drd2) P = 0.0001, t (4) = 14.16; (Adora2a) P = 0.0003, t (4) = 11.34; (Gpr6) P = 0.0039, t (2) = 15.92; (Bottom right) (Arpp21) P = 0.001, t (4) = 8.608; (Foxp1) P < 0.0001, t (4) = 29.32; (Gpx6) P = 0.0003, t (4) = 11.91; (Ido1) P = 0.0004, t (4) = 11.00; (Rxrg) P = 0.0024, t (4) = 6.813). The fold changes of mRNA expression in PRC2-deficient as compared to control D1 and D2 MSNs (log2) are shown (upregulated genes, red; downregulated MSN-specific genes, blue). (b, c) The AAV-Cre mediated PRC2 deficiency in adult MSNs leads to changes in MSN gene expression. (b) Schematic of stereotaxic injection of AAV-Cre; eGFP in the dorsal striatum of 10 week old Ezh1-/-; Ezh2fl/fl and control mice. The validation of H3K27me3 loss (red) in the nucleus (DAPI, blue) of virus infected (GFP, green) PRC2-deficient neurons 3 months post-surgery was determined by immunohistochemistry. Representative image is shown, n = 3 mice per genotype. (c) The fold changes of striatal mRNA expression as compared to control mice is shown 6 months post-surgery (upregulated genes, red; downregulated MSN-specific genes, blue (n = 5 control, 4 mutant mice; (Left) (Dlx4) P = 0.0211, t (7) = 2.960; (Foxd1) P = 0.0024, t (7) = 4.629; (Gata3) P = 0.0211, t (7) = 2.960; (Hand2) P = 0.0439, t (3) = 3.354; (Hoxd8) P < 0.0001, t (7) = 10.68; (Irx5) P = 0.0152, t (7) = 3.194; (Runx3) P = 0.0138, t (3) = 5.196; (Tal1) P = 0.0127, t (3) = 5.362; (Twist1) P = 0.0108, t (7) = 3.441; (Right) (Drd2) P = 0.0007, t (7) = 5.726; (Drd1a) P = 0.0042, t (4) = 5.854; (Ido1) P = 0.0011, t (7) = 5.361; (Rxrg) P < 0.0001, t (7) = 8.443; (Bcl11b) P = 0.0014, t (7) = 5.111; (Foxp1) P = 0.0023, t (7) = 4.655). Data are mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 from two-tailed Student’s t test with Welch’s correction.

Supplementary Figure 6 PRC2 deficiency leads to upregulation of the PRC2-targeted death-promoting genes in MSNs.

The fold induction of death-promoting genes in PRC2-deficient MSN in Ezh1-/-; Ezh2fl/fl; Drd1a-cre; Drd1a-TRAP and Ezh1-/-; Ezh2fl/fl; Drd2-cre; Drd2-TRAP and Ezh1-/-; Ezh2fl/fl; AAV-cre mice as compared to their respective littermate controls is shown ((Left) D1: n = 2 mutant, 3 control mice; (Bid) P = 0.0844, t (3) = 2.544; (Cdkn2b) P < 0.0001, t (3) = 49.57; (Ccnd1) P = 0.0077, t (3) = 6.402; (Gata4) P < 0.0001, t (3) = 114.7; (Hoxa5) P < 0.0001, t (3) = 43.93; (Pmaip1) P < 0.0001, t (3) = 35.07; (Wt1) P < 0.0001, t (3) = 35.07; (Center) D2: n = 3 mice each; (Bid) P = 0.0767, t (4) = 2.371; (Cdkn2b) P = 0.0022, t (2) = 21.48; (Ccnd1) P < 0.0001, t (4) = 30.80; (Gata4) P = 0.0305, t (4) = 3.28; (Hoxa5) P < 0.0001, t (4) = 53.57; (Pmaip1) P = 0.003, t (4) = 6.439; (Wt1) P < 0.0001, t (4) = 35.16; (Right) AAV: n = 4 mutant, 5 control mice; (Bid) P < 0.0001, t (7) = 8.464; (Cdkn2a) P = 0.0004, t (3) = 17.97; (Ccnd1) P < 0.0001, t (7) = 12.00; (Tal1) P = 0.0127, t (3) = 5.362; (Pmaip1) P = 0.0006, t (7) = 5.823). Data are mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 from two-tailed Student’s t test with Welch’s correction.

Supplementary Figure 7 PRC2 deficiency in MSNs causes a progressive and fatal motor disorder in mice.

(a) Impaired hanging wire in Ezh1-/-; Ezh2fl/fl; Camk2a-cre mice at 3 and 5 months of age is shown (3 months: n = 6 mice each; P = 0.6193, t (10) = 0.5126; 5 months: n = 5 mutant, 7 control mice; P = 0.000105, t (6) = 8.974). (b) Premature death of PRC2-deficient mice is preceded by an almost complete cessation of voluntary motor activity in the home cage. Ezh1-/-; Ezh2fl/fl; Camk2a-cre mice were monitored in a 24-hour observation chamber for motor activity prior to their death (n = 3 per genotype). Data are mean ± s.e.m. ***P ≤ 0.001 from two-tailed Student’s t test with Welch’s correction; n.s., nonsignificant.

Supplementary Figure 8 PRC2 deficiency in MSNs causes Huntington’s disease-like changes in gene expression.

(a) Venn diagrams display an overlap of genes upregulated >2 fold in PRC2-deficient MSNs of Ezh1-/-; Ezh2fl/fl; Camk2a-cre mice at 3 (left) or 6 months (right) of age with genes upregulated in two different mouse models of Huntington’s disease. The combined list of genes changes in the R6/2 and YAC Q175 mouse models was generated from published data46,47 after applying a >1.5 fold gene expression change cutoff. (b) Venn diagrams display significant overlap of genes that are upregulated in PRC2-deficient neurons at 3 (left) and 6 months (right) of age and genes induced in the cortex of Huntington’s disease patients14. Significance of venn diagrams was calculated using Chi-square test.

Supplementary Figure 9 Original images of representative western blot images from Figure 1.

Uncropped KODAK films for images in Fig. 1 are shown.

Supplementary Figure 10 Original images of representative western blot images from Figure 5 and Supplementary Figure 4.

Cropped KODAK films for images in Fig. 5 and Supplementary Fig. 4 are shown. Membranes were cut prior to antibody staining to allow for simultaneous detection of proteins running at different sizes on the same membrane.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Table 8 (PDF 2466 kb)

Supplementary Methods Checklist (PDF 597 kb)

Supplementary Table 1: 2057 genes with highest H3K27me3 (FPKM > 1.2) in MSNs ordered by FPKM (H3K27me3).

A FPKM cutoff of >1.2 was used to determine the highest H3K27me3 associated genes in MSNs. The 2057 genes with the highest H3K27me3 abundance are ordered by their H3K27me3 FPKM value. (XLS 304 kb)

Supplementary Table 2: 835 H3K27me3+ H3K4me3+ genes in MSNs.

Bivalent genes were determined by the simultaneous presence of H3K27me3 and H3K4me3 at their transcriptional start site (TSS). 835 genes in MSNs were found. Gene ontology results are shown with selected categories indicated in yellow. (XLSX 121 kb)

Supplementary Table 3: 53 and 190 genes up-regulated upon PRC2 deletion at 3 and 6 months.

Changes in gene expression in PRC2 deficient MSNs were determined at 3 and 6 months of age using P < 0.05, fold change >2. Genes up-regulated at 3 months (n = 53) and 6 months (n = 190) are shown ordered by gene expression fold changes. Gene ontology results for each gene list are shown with selected categories indicated in yellow. (XLSX 159 kb)

Supplementary Table 4: 27 and 87 H3K27me3+ H3K4me3+ genes that are de-repressed in PRC2 deficient MSNs at 3 and 6 months.

Genes changed in PRC2 deficient MSNs with simultaneous presence of H3K27me3 and H3K4me3 at their TSS at 3 months (n = 27) and 6 months (n = 87) of age are shown. (XLSX 14 kb)

Supplementary Table 5: 29 up-regulated PRC2 target genes with potential auto-regulatory functions.

Up-regulated PRC2 target genes with experimental or bioinformatics-predicted auto-regulatory function were determined based on literature search and by using oPossum 3.0 and Genomatix software. Individual analysis and a summary table are shown. (XLSX 622 kb)

Supplementary Table 6: 119 MSN-expressed genes down-regulated upon PRC2 deletion.

Changes in gene expression in PRC2 deficient MSNs were determined at 3 and 6 months of age using P < 0.05, fold change >2. Genes down-regulated at 6 months (n = 190) are shown ordered by fold change. Gene ontology results for are shown with selected categories indicated in yellow. (XLSX 152 kb)

Supplementary Table 7: Overlapping genes between PRC2 KO and HD models.

Genes overlapping between PRC2 deficient MSNs at 3 or 6 months of age with genes that are up-regulated/down-regulated in two different mouse models of Huntington's disease and Huntington's disease patients are shown. (XLSX 10 kb)

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von Schimmelmann, M., Feinberg, P., Sullivan, J. et al. Polycomb repressive complex 2 (PRC2) silences genes responsible for neurodegeneration. Nat Neurosci 19, 1321–1330 (2016). https://doi.org/10.1038/nn.4360

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