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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References
Hobert, O. Regulation of terminal differentiation programs in the nervous system. Annu. Rev. Cell Dev. Biol. 27, 681–696 (2011).
Molyneaux, B.J., Arlotta, P., Menezes, J.R. & Macklis, J.D. Neuronal subtype specification in the cerebral cortex. Nat. Rev. Neurosci. 8, 427–437 (2007).
Deneris, E.S. & Hobert, O. Maintenance of postmitotic neuronal cell identity. Nat. Neurosci. 17, 899–907 (2014).
Corley, M. & Kroll, K.L. The roles and regulation of polycomb complexes in neural development. Cell Tissue Res. 359, 65–85 (2015).
Margueron, R. et al. Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms. Mol. Cell 32, 503–518 (2008).
Margueron, R. & Reinberg, D. The Polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011).
Laible, G. et al. Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres. EMBO J. 16, 3219–3232 (1997).
Swigut, T. & Wysocka, J. H3K27 demethylases, at long last. Cell 131, 29–32 (2007).
He, X.B. et al. Vitamin C facilitates dopamine neuron differentiation in fetal midbrain through TET1- and JMJD3-dependent epigenetic control manner. Stem Cells 33, 1320–1332 (2015).
Palomer, E., Carretero, J., Benvegnù, S., Dotti, C.G. & Martin, M.G. Neuronal activity controls Bdnf expression via Polycomb de-repression and CREB/CBP/JMJD3 activation in mature neurons. Nat. Commun. 7, 11081 (2016).
Li, J. et al. EZH2-mediated H3K27 trimethylation mediates neurodegeneration in ataxia-telangiectasia. Nat. Neurosci. 16, 1745–1753 (2013).
Södersten, E. et al. Dopamine signaling leads to loss of Polycomb repression and aberrant gene activation in experimental parkinsonism. PLoS Genet. 10, e1004574 (2014).
Seong, I.S. et al. Huntingtin facilitates polycomb repressive complex 2. Hum. Mol. Genet. 19, 573–583 (2010).
Dong, X. et al. The role of H3K4me3 in transcriptional regulation is altered in Huntington's disease. PLoS One 10, e0144398 (2015).
Biagioli, M. et al. Htt CAG repeat expansion confers pleiotropic gains of mutant huntingtin function in chromatin regulation. Hum. Mol. Genet. 24, 2442–2457 (2015).
Hoss, A.G. et al. MicroRNAs located in the Hox gene clusters are implicated in Huntington's disease pathogenesis. PLoS Genet. 10, e1004188 (2014).
Labadorf, A. et al. RNA sequence analysis of human Huntington disease brain reveals an extensive increase in inflammatory and developmental gene expressioN. PLoS One 10, e0143563 (2015).
Su, I.H. et al. Polycomb group protein ezh2 controls actin polymerization and cell signaling. Cell 121, 425–436 (2005).
Santos-Rosa, H. et al. Active genes are tri-methylated at K4 of histone H3. Nature 419, 407–411 (2002).
Voigt, P., Tee, W.W. & Reinberg, D. A double take on bivalent promoters. Genes Dev. 27, 1318–1338 (2013).
Bernstein, B.E. et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006).
Ku, M. et al. Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. PLoS Genet. 4, e1000242 (2008).
Heiman, M. et al. A translational profiling approach for the molecular characterization of CNS cell types. Cell 135, 738–748 (2008).
Lein, E.S. et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176 (2007).
Hersch, S.M. et al. Electron microscopic analysis of D1 and D2 dopamine receptor proteins in the dorsal striatum and their synaptic relationships with motor corticostriatal afferents. J. Neurosci. 15, 5222–5237 (1995).
Fridman, J.S. & Lowe, S.W. Control of apoptosis by p53. Oncogene 22, 9030–9040 (2003).
Kranenburg, O., van der Eb, A.J. & Zantema, A. Cyclin D1 is an essential mediator of apoptotic neuronal cell death. EMBO J. 15, 46–54 (1996).
Marathe, S., Liu, S., Brai, E., Kaczarowski, M. & Alberi, L. Notch signaling in response to excitotoxicity induces neurodegeneration via erroneous cell cycle reentry. Cell Death Differ. 22, 1775–1784 (2015).
Lovell, M.A., Xie, C., Xiong, S. & Markesbery, W.R. Wilms' tumor suppressor (WT1) is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer's disease. Brain Res. 983, 84–96 (2003).
Lu, X.H. et al. Targeting ATM ameliorates mutant Huntingtin toxicity in cell and animal models of Huntington's disease. Sci. Transl. Med. 6, 268ra178 (2014).
Illuzzi, J., Yerkes, S., Parekh-Olmedo, H. & Kmiec, E.B. DNA breakage and induction of DNA damage response proteins precede the appearance of visible mutant huntingtin aggregates. J. Neurosci. Res. 87, 733–747 (2009).
Anne, S.L., Saudou, F. & Humbert, S. Phosphorylation of huntingtin by cyclin-dependent kinase 5 is induced by DNA damage and regulates wild-type and mutant huntingtin toxicity in neurons. J. Neurosci. 27, 7318–7328 (2007).
Turmaine, M. et al. Nonapoptotic neurodegeneration in a transgenic mouse model of Huntington's disease. Proc. Natl. Acad. Sci. USA 97, 8093–8097 (2000).
Orlando, V. Polycomb, epigenomes, and control of cell identity. Cell 112, 599–606 (2003).
Doyle, J.P. et al. Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell 135, 749–762 (2008).
Savas, J.N., Toyama, B.H., Xu, T., Yates, J.R. III & Hetzer, M.W. Extremely long-lived nuclear pore proteins in the rat brain. Science 335, 942 (2012).
Duerre, J.A. & Lee, C.T. In vivo methylation and turnover of rat brain histones. J. Neurochem. 23, 541–547 (1974).
Culhane, J.C. & Cole, P.A. LSD1 and the chemistry of histone demethylation. Curr. Opin. Chem. Biol. 11, 561–568 (2007).
Dobenecker, M.W. et al. Coupling of T cell receptor specificity to natural killer T cell development by bivalent histone H3 methylation. J. Exp. Med. 212, 297–306 (2015).
Mohn, F. et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30, 755–766 (2008).
Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).
Wachter, E. et al. Synthetic CpG islands reveal DNA sequence determinants of chromatin structure. eLife 3, e03397 (2014).
Mendenhall, E.M. et al. GC-rich sequence elements recruit PRC2 in mammalian ES cells. PLoS Genet. 6, e1001244 (2010).
Kaneko, S., Son, J., Bonasio, R., Shen, S.S. & Reinberg, D. Nascent RNA interaction keeps PRC2 activity poised and in check. Genes Dev. 28, 1983–1988 (2014).
Jadhav, U. et al. Acquired tissue-specific promoter bivalency is a basis for PRC2 necessity in adult cells. Cell 165, 1389–1400 (2016).
Langfelder, P. et al. Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice. Nat. Neurosci. 19, 623–633 (2016).
Vashishtha, M. et al. Targeting H3K4 trimethylation in Huntington disease. Proc. Natl. Acad. Sci. USA 110, E3027–E3036 (2013).
Klapstein, G.J. et al. Electrophysiological and morphological changes in striatal spiny neurons in R6/2 Huntington's disease transgenic mice. J. Neurophysiol. 86, 2667–2677 (2001).
Menalled, L.B. & Chesselet, M.F. Mouse models of Huntington's disease. Trends Pharmacol. Sci. 23, 32–39 (2002).
Reynolds, J.P. et al. Transcriptional response of Polycomb group genes to status epilepticus in mice is modified by prior exposure to epileptic preconditioning. Front. Neurol. 6, 46 (2015).
Casanova, E. et al. A CamKIIalpha iCre BAC allows brain-specific gene inactivation. Genesis 31, 37–42 (2001).
Schaefer, A. et al. Cerebellar neurodegeneration in the absence of microRNAs. J. Exp. Med. 204, 1553–1558 (2007).
Zhang, X.M. et al. Highly restricted expression of Cre recombinase in cerebellar Purkinje cells. Genesis 40, 45–51 (2004).
Ezhkova, E. et al. EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair. Genes Dev. 25, 485–498 (2011).
Maze, I. et al. G9a influences neuronal subtype specification in striatum. Nat. Neurosci. 17, 533–539 (2014).
Maze, I. et al. Essential role of the histone methyltransferase G9a in cocaine-induced plasticity. Science 327, 213–216 (2010).
Kriaucionis, S. & Heintz, N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929–930 (2009).
Gao, Z. et al. An AUTS2-Polycomb complex activates gene expression in the CNS. Nature 516, 349–354 (2014).
Mullen, R.J., Buck, C.R. & Smith, A.M. NeuN, a neuronal specific nuclear protein in vertebrates. Development 116, 201–211 (1992).
Goodarzi, H., Elemento, O. & Tavazoie, S. Revealing global regulatory perturbations across human cancers. Mol. Cell 36, 900–911 (2009).
Tan, C.L. et al. MicroRNA-128 governs neuronal excitability and motor behavior in mice. Science 342, 1254–1258 (2013).
Schaefer, A. et al. Control of cognition and adaptive behavior by the GLP/G9a epigenetic suppressor complex. Neuron 64, 678–691 (2009).
Cartharius, K. et al. MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 21, 2933–2942 (2005).
Kwon, A.T., Arenillas, D.J., Worsley Hunt, R. & Wasserman, W.W. oPOSSUM-3: advanced analysis of regulatory motif over-representation across genes or ChIP-Seq datasets. G3 (Bethesda) 2, 987–1002 (2012).
Sandelin, A., Alkema, W., Engström, P., Wasserman, W.W. & Lenhard, B. JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 32, D91–D94 (2004).
Dermitzakis, E.T. & Clark, A.G. Evolution of transcription factor binding sites in mammalian gene regulatory regions: conservation and turnover. Mol. Biol. Evol. 19, 1114–1121 (2002).
Lenhard, B. et al. Identification of conserved regulatory elements by comparative genome analysis. J. Biol. 2, 13 (2003).
Chen, E.Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14, 128 (2013).
Kuleshov, M.V. et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 44 W1: W90–W97 (2016).
Tan, C.M., Chen, E.Y., Dannenfelser, R., Clark, N.R. & Ma'ayan, A. Network2Canvas: network visualization on a canvas with enrichment analysis. Bioinformatics 29, 1872–1878 (2013).
Lachmann, A. et al. ChEA: transcription factor regulation inferred from integrating genome-wide ChIP-X experiments. Bioinformatics 26, 2438–2444 (2010).
ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).
Sullivan, J.M. et al. Autism-like syndrome is induced by pharmacological suppression of BET proteins in young mice. J. Exp. Med. 212, 1771–1781 (2015).
Crawley, J.N. What's Wrong with My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice 2nd edn. (Wiley-Interscience, 2007).
Lobo, M.K. et al. Cell type-specific loss of BDNF signaling mimics optogenetic control of cocaine reward. Science 330, 385–390 (2010).
Wallace, D.L. et al. CREB regulation of nucleus accumbens excitability mediates social isolation-induced behavioral deficits. Nat. Neurosci. 12, 200–209 (2009).
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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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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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 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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DOI: https://doi.org/10.1038/nn.4360
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