bioRxiv preprint doi: https://doi.org/10.1101/582908; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Neuronal histone methyltransferase EZH2 regulates neuronal morphogenesis, synaptic plasticity, 2 and cognitive behavior of mice 3 4 Mei Zhang1,5, Yong Zhang2, Qian Xu2, Joshua Crawford3, Cheng Qian1, Guo-Hua Wang4, Eastman Lewis2, 5 Philip Hall, Gül Dolen2, Richard L. Huganir2, Jiang Qian4, Xin-Zhong Dong2, Mikhail V. Pletnikov3, 6 Chang-Mei Liu6, *, Feng-Quan Zhou1,2, * 7 8 1Department of Orthopaedic Surgery, 2The Solomon H. Snyder Department of Neuroscience, 3Department 9 of Psychiatry and Behavioral Sciences, 4Department of Ophthalmology, Johns Hopkins University School 10 of Medicine, Baltimore, Maryland 21205, USA，5School of Life Sciences, University of Science and 11 Technology of China, Hefei 230026, China. 6State Key Laboratory of Reproductive Biology, Institute of 12 Zoology, Chinese Academy of Sciences, Beijing 100190, China 13 14 *Correspondence to Feng-Quan Zhou, Room 291, The John G. Rangos Sr. Bldg., 855 North Wolfe Street, 15 Departments of Orthopaedic Surgery and Neuroscience, Johns Hopkins University School of Medicine, 16 Baltimore, MD 21205, Email: [email protected], or to Chang-Mei Liu, State Key Laboratory of 17 Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100190, China, Email: 18 [email protected] 19 20 21 Running head: Neuronal EZH2 regulates neuronal morphogenesis and cognition 22 23 bioRxiv preprint doi: https://doi.org/10.1101/582908; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 25 Abstract 26 Recent studies showed that in the nervous system histone methyltransferase EZH2-mediated 27 trimethylation of histone H3 lysine 27 (H3K27me3) acts to regulate neural stem cell proliferation and fate 28 specificity through silencing different gene sets. Here we explored the function of EZH2 in early post- 29 mitotic excitatory neurons by generating a neuronal specific Ezh2 conditional knockout mouse line. The 30 results showed that lack of neuronal EZH2 led to delayed neuronal migration, more complex dendritic 31 arborization, and significantly increased dendritic spine density. RNA-sequencing (RNA-seq) 32 experiments comparing control and Ezh2 knockout neurons revealed that neuronal EZH2 regulated genes 33 related to neuronal morphogenesis. In particular, Pak3 was identified as a target gene suppressed by EZH2 34 and H3K27me3, and expression of dominant negative PAK3 reversed Ezh2 knockout-induced higher 35 dendritic spine density. Lastly, lack of neuronal EZH2 resulted in impaired memory behaviors in adult 36 mice. Our results demonstrated that neuronal EZH2 played important roles in controlling multiple steps 37 of neuronal morphogenesis during development, which had long-lasting effects on cognitive function in 38 adult mice. 39 40 41 42 43 44 45 46 bioRxiv preprint doi: https://doi.org/10.1101/582908; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 47 Introduction 48 During the development of the central nervous system (CNS), post-mitotic neurons generated through 49 neurogenesis undergo several steps of neuronal morphogenesis, including neuronal migration, neuronal 50 polarization, axon growth/guidance, dendrite development, and synaptogenesis, to form the functional 51 neural circuits. Disruptions in these processes are believed to cause many neurodevelopmental disorders, 52 such as autism spectrum disorder (ASD), schizophrenia, and intellectual disability. Previous studies have 53 extensively investigated the roles of extracellular guidance cues, their receptors and downstream signaling 54 mediators, as well as cytoskeletal proteins, in regulation of these processes. Epigenetic regulation 55 independent of DNA sequence is emerging to be a key cellular mechanism for coordinated regulation of 56 functionally relevant gene expression in neural development. Interestingly, perturbation of several histone 57 methyltransferases, such as NSD1 (H3K36) and MLL2 (H3K4), causes Sotos Syndrome and Kabuki 58 Syndrome, respectively, both of which show intellectual disability (Douglas et al., 2003; Ng et al., 2010). 59 It appears that these histone-modifying proteins are emerging as causes of neurodevelopmental disorders. 60 The polycomb repressive complex 2 (PRC2) is one of the two polycomb group proteins (PcG) that 61 can methylate the H3 histone lysine 27 (H3K27me3) (Margueron and Reinberg, 2011). The mouse PRC2 62 has 4 subunits: SUZ12, EED, EZH2, and RbAp48, among which EZH2 is the methyltransferase that 63 trimethylates H3K27. In addition to methyltransferase, H3K27 methylation is also negatively regulated 64 by 2 demethylases, JMJD3 and UTX which remove methyl groups specifically from H3K27 (Agger et al., 65 2007; De Santa et al., 2007; Hong et al., 2007; Lan et al., 2007). Previous studies have identified EZH2 66 as one of the causative genes of the Weaver Syndrome, a developmental disorder characterized by 67 macrocephaly, dysmorphic facial features, accelerated skeletal maturation, limb anomalies, development 68 delay and intellectual disability (Gibson et al., 2012; Tatton-Brown et al., 2011). In addition, a genome- 69 wide profiling of histone methylations has shown a significant difference in H3K27 methylation between bioRxiv preprint doi: https://doi.org/10.1101/582908; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 70 olfactory cells (with neuronal traits) obtained from the schizophrenia patients and those from control 71 groups (Kano et al., 2012). Moreover, both EZH2 and JMJD3 have been identified as susceptible genes 72 for ASD (Iossifov et al., 2012; Li et al., 2016). These facts prompted us to study the function of EZH2 and 73 H3K27 methylation in early neuronal development of the brain. 74 The main function of PRC2 and H3K27 methylation is to epigenetically maintain gene silencing 75 (Beisel and Paro, 2011). Previous studies in stem cell maintenance and differentiation suggested that 76 PRC2 function to maintain cell identify at different stages of cell development (Hwang et al., 2014; 77 Margueron and Reinberg, 2011; Pereira et al., 2010; Schoeftner et al., 2006). For instance, in stem cells, 78 H3K27me3 represses the expression of differentiation-related genes to maintain stem cell fate. When stem 79 cells differentiate into specific cell types in response to extracellular signals, H3K27me3 instead represses 80 stem cell genes and genes related to other cell types. Thus, H3K27me3 acts to repress different sets of 81 genes in the same cell lineage at different developmental stages. EZH2 is highly expressed in dividing 82 neural progenitors, and loss of EZH2 in neural progenitor cells disrupts neurogenesis in both embryonic 83 and adult animals (Pereira et al., 2010; Zhang et al., 2014). When neural progenitors differentiate into 84 post-mitotic neurons, the expression of EZH2 is downregulated, but still maintained. Conditionally 85 knocking out Ezh2 in neural progenitors has been shown to regulated directional neuronal migration in 86 the brain stem (Di Meglio et al., 2013). Similarly, knocking down EZH2 in radial glia cells led to impaired 87 cortical neuronal migration (Zhao et al., 2015). Very interestingly, a recent study showed that activating 88 endogenous EZH2 in embryonic stem cells using CRISPRa approach promoted neuronal differentiation, 89 likely by suppressing the expression of endodermal and mesodermal specific gene transcription (Liu et al., 90 2018). Moreover, when combined with other transcription factors, such as POU3F2 and Neurogenin 1, 91 EZH2 expression could induce direct reprogramming of fibroblasts to neurons. In sum, these studies bioRxiv preprint doi: https://doi.org/10.1101/582908; this version posted March 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 92 highlighted the important roles of EZH2 in stem cells to control neuronal differentiation. However, the 93 roles of EZH2 in post-mitotic neurons remain elusive. 94 As mentioned above, EZH2 in neural progenitors and in post-mitotic neurons may regulate 95 different sets of genes and have distinct neuronal phenotypes. More importantly, to date the specific in 96 vivo roles of EZH2 in early post-mitotic neurons during development remain largely unknown. To directly 97 address the in vivo roles of EZH2 specifically in excitatory post-mitotic neurons, we generated neuron- 98 specific Ezh2 conditional knockout mice using Neurod6-Cre mice, in which Cre recombinase is expressed 99 specifically in early post-mitotic excitatory neurons of the developing brain. We found that deletion of 100 neuronal EZH2 delayed but not persistently blocked neuronal migration. More interestingly, EZH2 null 101 neurons displayed more complex dendritic arborization and increased dendritic spine density, indicating 102 that EZH2 acts to suppress dendritic development. RNA-Seq experiments comparing control