Downloaded from learnmem.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Research O-GlcNAc and EZH2-mediated epigenetic regulation of gene expression during consolidation of fear memories Anderson A. Butler, Richard G. Sanchez, Timothy J. Jarome, William M. Webb, and Farah D. Lubin Department of Neurobiology, the University of Alabama at Birmingham, Birmingham, Alabama 35294, USA O-GlcNAcylation of serine/threonine residues on target proteins occurs dynamically in postmitotic neurons of the hip- pocampus and may serve to control both the stability and activity of target proteins. Remarkably, the addition and removal of the O-GlcNAc posttranslational modifications are catalyzed by a pair of enzymes, the O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). More than thousands of proteins are modified by O-GlcNAcylation including epigenetic modifying enzymes. A critical target of OGT is the polycomb repressive complex 2 (PRC2) containing the histone lysine methyltrans- ferase EZH2 that mediates trimethylation of lysine 27 on histone H3 (H3K27me3). However, whether OGT and PRC2 ac- tivity in the hippocampus couple to regulate gene transcription mechanisms during memory consolidation remains unknown. Here, we found increases in OGT expression and global O-GlcNAcylation levels in dorsal area CA1 of the hip- pocampus during memory consolidation. Additionally, we observed that OGT exerts control over epigenetic regulation via EZH2-H3K27me3 during memory consolidation. Blocking O-GlcNAc signaling via RNAi within dorsal area CA1 led to the global and site-specific loss of activity-dependent epigenetic plasticity at genes regulated by H3K27me3 and impairment of hippocampus-dependent memory. Together, these findings illustrate a unique epigenetic role of OGT via regulation of histone methylation mediated by EZH2 during memory consolidation of fear conditioned memories. The posttranslational attachment of β-N-glucosamine (GlcNAc) O-GlcNAcylation of histones has been reported in both yeast and to serine/threonine residues of proteins (O-GlcNAcylation) is human cells (Sakabe et al. 2010) and observed to facilitate changes the product of the hexosamine biosynthetic pathway (HBP), which in other epigenetic marks, such as histone monoubiquitination on integrates glucose, amino acid, fatty acid, and nucleotide metabo- H2B (Fujiki et al. 2011). Based on these findings, the significance of lism to generate the donor substrate uridine diphosphate N-acetyl- direct histone O-GlcNAcylation in many mammalian cell types re- glucosamine (UDP-GlcNAc) (Hart and Akimoto 2009; Yang and mains in question (Gagnon et al. 2015). Qian 2017). O-GlcNAc signaling is highly sensitive to various In addition to directly modifying histones and signaling forms of cellular stress, including heat/cold shock (Kazemi et al. proteins, OGT interacts with and regulates numerous chromatin 2010; Yao et al. 2018), hypoxia (Liu et al. 2014), and neuronal exci- modifying enzymes (CMEs) including the TET family of DNA totoxicity (Zhu et al. 2015). Although the O-GlcNAc axis is only hydroxylases (Chen et al. 2013; Dehennaut et al. 2014) and the beginning to be described in neuronal alterations, many upstream H3K27 methyltransferase EZH2 (Chu et al. 2014). Learning- kinases important for memory consolidation are known to inter- induced epigenetic changes mediated by both O-GlcNAc signaling sect with signaling to OGT and O-GlcNAcylation, including (Lagerlöf and Hart 2014; Wang et al. 2016; Yang et al. 2017; Lagerlöf CamKII (Erickson et al. 2013), PKA (Xie et al. 2016), PKC (includ- 2018), as well as OGT-regulated CMEs are critical for long-term ing PKC-zeta [Miura et al. 2018]), CREB (Rexach et al. 2012; Xie memory formation (Jarome and Lubin 2013, 2014); however, the et al. 2016), and many others (Hart et al. 2011). As a result, role of OGT in precipitating learning-induced epigenetic changes O-GlcNAcylation has been observed to function as a signal integra- remains unexplored. In the present study, we observed that OGT tor regulating virtually every aspect of cellular function including is necessary for learning-induced activation of H3K27me3 epige- transcription, translation, signal transduction, and metabolism. netic marks and that OGT activity is critical for memory formation. Consistent with these observations, disruption of OGT signaling and O-GlcNAcylation processes has been implicated in numerous Results neurodegenerative diseases, including epilepsy (Sánchez et al. 2019), Alzheimer’s disease (Xie et al. 2016), and Parkinson’s disease Fear learning triggers increases in O-GlcNAcylation (Wani et al. 2017). and OGT protein levels in the hippocampus In contrast to other forms of posttranslational glycosyla- We therefore sought to investigate the role of this critical signaling tion, which occur largely in the endoplasmic reticulum, pathway in hippocampus-dependent long-term memory. To ascer- O-GlcNAcylation of proteins occurs in the cytoplasm and nucleus tain whether the expression of OGT and protein O-GlcNAcylation (Comer and Hart 2000; Hart and Akimoto 2009). In the nucleus, O-GlcNAcylation occurs on numerous target proteins, consistent with a cellular role as an integrator of signals. In particular, direct # 2019 Butler et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first 12 months after the full-issue publication date (see http://learnmem.cshlp.org/site/misc/terms.xhtml). After 12 months, it is Corresponding author: fl[email protected] available under a Creative Commons License (Attribution-NonCommercial 4.0 Article is online at http://www.learnmem.org/cgi/doi/10.1101/lm.049023.118. International), as described at http://creativecommons.org/licenses/by-nc/4.0/. 26:373–379; Published by Cold Spring Harbor Laboratory Press 373 Learning & Memory ISSN 1549-5485/19; www.learnmem.org Downloaded from learnmem.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press O-GlcNAc and histone methylation in memory consolidation are dynamically regulated during normal memory formation, relative to similarly treated naive controls (Fig. 2F), as observed we compared hippocampal nuclear lysates from Sprague Dawley above in untreated animals (Fig. 1B). Animals treated with OGT rats after training in contextual fear conditioning, a hippo- siRNAs had significantly reduced expression of hippocampal campus-dependent long-term memory paradigm (Fig. 1A). We ob- OGT and O-GlcNAc after training, resembling untrained animals served rapidly increased expression of both OGT and protein (Fig. 2F). Nup62 expression, an OGT substrate, was not sig- O-GlcNAcylation, as soon as 1 h, in trained animals relative to na- nificantly altered during memory consolidation; however, it was ive animals, while animals which received a latent inhibition significantly down-regulated after treatment with OGT siRNAs shock paradigm did not have elevated OGT or O-GlcNAc (Fig. (Fig. 2F). 1B). Similar increases in both OGT and O-GlcNAcylation were ob- served at 24 h posttraining, suggesting that memory-induced O-GlcNAc signaling persists through the memory consolidation Ogt knockdown selectively prevents memory-induced process (Fig. 1C). global and site-specific increases in H3K27me3 levels Several studies have recently reported interactions between Ogt knockdown prevents memory-induced global O-GlcNAc signaling and other histone posttranslational modifica- increases in O-GlcNAcylation tions. Many of these histone marks are known to be important We next sought to examine the necessity of OGT for long-term for hippocampal memory formation (Jarome and Lubin 2014). memory. To determine whether artificially lowering OGT expres- Therefore, we next sought to investigate the potential role of sion increases global levels of O-GlcNacylation, we directly infused O-GlcNAc signaling on memory-related epigenetic crosstalk. To Accell siRNAs (Dharmacon) into the dorsal CA1 and collected tis- determine changes in both memory-relevant changes in bulk fi sue around the target infusion site for downstream analysis (Fig. histone PTMs and site-speci c epigenetic alterations, we assayed 2A). We confirmed infusion into area CA1 with fluorescently la- nuclear histones via both western blots and ChIP-qPCR (Fig. 3A). beled control siRNAs (Fig. 2B). At 5 d postinfusion, we observed sig- We observed global changes in expression of numerous memory- nificant reductions in both OGT protein expression (Fig. 2C) and related histone PTMs after CFC training, including H2BubiK120, OGT mRNA expression in area CA1 but not in the untargeted H3K27me3, H3K4me3, and H3K14ac. H3K27me3 was the only retrosplenial cortex (Fig. 2D). To verify the sufficiency of siRNA in- mark assayed that was sensitive to pretreatment with OGT siRNA fusions to preclude learning-induced increases in OGT and (Fig. 3B). To investigate the necessity of OGT for memory-induced fi O-GlcNAc expression, we assayed hippocampal O-GlcNAc and H3K27me3 in vivo in a gene-speci c manner, we performed OGT in siRNA-treated animals after training in CFC (Fig. 2E). We ChIP-qPCR at numerous gene loci. We observed memory-induced observed that animals treated with a nontargeting siRNA had ele- decreases in promoter H3K27me3 in several memory-relevant fi vated levels of hippocampal O-GlcNAc and OGT after CFC training genes, including Bdnf, and Pkc-zeta. These ndings are consistent with previously observed increases in Bdnf and Pkc-zeta transcrip- tion during memory consolidation, as well as a memory-depen- dent increase at the promoter