The histone H3 lysine 9 methyltransferase G9a/GLP complex activity is required for long-term consolidation of spatial memory in mice

https://doi.org/10.1016/j.nlm.2021.107406Get rights and content

Highlights

  • G9a/GLP histone methyltransferase is required for long-term memory (LTM).

  • Hippocampal G9a/GLP inhibition immediately after learning impairs spatial LTM.

  • G9a/GLP inhibition during initial consolidation window spares short-term memory.

  • G9a/GLP activity regulates H3K9 modifications in both hippocampus and striatum.

Abstract

The G9a/G9a-like protein (GLP) histone lysine dimethyltransferase complex and downstream histone H3 lysine 9 dimethylation (H3K9me2) repressive mark have recently emerged as key transcriptional regulators of gene expression programs necessary for long-term memory (LTM) formation in the dorsal hippocampus. However, the role for hippocampal G9a/GLP complex in mediating the consolidation of spatial LTM remains largely unknown. Using a water maze competition task in which both dorsal hippocampus-dependent spatial and striatum-dependent cue navigation strategies are effective to solve the maze, we found that pharmacological inhibition of G9a/GLP activity immediately after learning disrupts long-term consolidation of previously learned spatial information in male mice, hence producing cue bias on the competition test performed 24 h later. Importantly, the inhibition of hippocampal G9a/GLP did not disrupt short-term memory retention. Immunohistochemical analyses revealed increases in global levels of permissive histone H3K9 acetylation in the dorsal hippocampus and dorsal striatum at 1 h post-training, which persisted up to 24 h in the hippocampus. Conversely, H3K9me2 levels were either unchanged in the dorsal hippocampus or transiently decreased at 15 min post-training in the dorsal striatum. Finally, the inhibition of G9a/GLP activity further increased global levels of H3K9 acetylation while decreasing H3K9me2 in the hippocampus at 1 h post-training. However, both marks returned to vehicle control levels at 24 h. Together, these findings support the possibility that G9a/GLP in the dorsal hippocampus is required for the transcriptional switch from short-term to long-term spatial memory formation.

Introduction

In the last decade, emerging evidence emphasizes dynamic chromatin remodeling through post-translational modifications (PTM) of histone proteins as a key player of gene expression patterns underpinning the formation of long-term memories (LTM) (Peixoto and Abel, 2012, Stilling and Fischer, 2011, Sweatt, 2009). Most of the studies on the dynamic changes in histones PTM during learning and memory processes have focused on acetylation and methylation at multiple lysine residues on the N-terminal tail of specific histones. Studies in rodents have emphasized an essential role of histone acetylation in the dorsal hippocampus (dHPC) in activating the gene expression program necessary for LTM consolidation in several HPC-dependent paradigms, including contextual fear conditioning and spatial learning (Bousiges et al., 2010, Castellano et al., 2012, Castellano et al., 2014, Fischer et al., 2007, Levenson et al., 2004, Peleg et al., 2010, Sweatt, 2009, Vecsey et al., 2007). We previously reported that local infusion of histone deacetylase inhibitor (HDACi) into the dHPC immediately after learning selectively enhances LTM consolidation of spatial water maze training by influencing the strength of functional connectivity between the hippocampus and interconnected structures (Dagnas, Guillou, Prevot, & Mons, 2013). While histone acetylation is a permissive transcriptional mark, changes in lysine methylation of histones are associated with either activation or repression of gene expression, depending on the location of methylated residue and, for a single lysine residue, the degree of methylation status. In recent years, dimethylation of histone H3 lysine 9 (H3K9me2), a repressive mark catalyzed by histone methyltransferases (HMT) G9a (also known as Ehmt2) and G9a-like protein (GLP, also known as Ehmt1), has emerged as a critical epigenetic regulator of HPC-dependent LTM formation and long-lasting forms of synaptic plasticity (for review, see (Benevento et al., 2015, Pang et al., 2019). Several studies have reported, for example, a pivotal role for G9a/GLP-mediated H3K9me2 in mediating transcriptional homeostasis between gene activation and silencing in area CA1 of the dHPC (dCA1) and the entorhinal cortex during contextual fear memory consolidation (Gupta et al., 2010, Gupta-Agarwal et al., 2012, Mahan et al., 2012). Pharmacological inhibition or genetic silencing of G9a/GLP activity in the dCA1 impairs contextual fear LTM and disrupts hippocampal long-term potentiation (LTP) (Gupta et al., 2010, Gupta-Agarwal et al., 2012, Schaefer et al., 2009). In contrast, the same G9a/GLP blockade in the entorhinal cortex had the opposite effect on fear LTM while increasing both repressive H3K9me2 marks at promoter regions of non-memory permissive genes (e.g. COMT) and permissive H3K4me3 and H3K9ac marks at memory-related gene promoters (e.g. Zif268; BDNF exon IV; DNMT3) in the dHPC. Electrophysiology studies in rats also indicated that G9a/GLP complex activity exerts an epigenetic control over BDNF-mediated long-term plasticity and associativity in hippocampal CA1 neurons (Sharma, Dierkes, & Sajikumar, 2017) Collectively, these findings suggest that an accurate regulation of G9a/GLP-mediated H3K9me2 may play a pivotal role in maintaining a fine balance between gene activation and gene silencing during the process of LTM consolidation.

Dual solution tasks are used to study how multiple memory systems interact with one another over time and/or training regimen (Gasser et al., 2020, Martel et al., 2007, Rice et al., 2015). Using a water maze competition task, in which both HPC-based spatial/place and striatum-based cue/response strategies are effective solutions to solve the task, we previously demonstrated that inducing histone hyperacetylation in the dorsal HPC immediately after learning operates as a molecular switch between the HPC- and dorsal striatum-dependent memory systems, biasing mice towards predominant use of a spatial navigational strategy under training conditions that normally results in the use of the cued strategy (Dagnas et al., 2013). The present study used the same water maze task to examine whether immediate post-training infusion of the pharmacological G9a/GLP inhibitor, BIX-01294, administrated into the dHPC of male mice would also influence the consolidation of one particular memory strategies (i.e. spatial versus cue-guided) assessed 24 h later in competition probe tests. Since a proper balance between transcriptionally active H3K9ac and repressive H3K9me2 marks via accurate regulation of the HMT G9a/GLP activity is important for coordinating patterns of gene expression during the process of memory consolidation, we also explored whether differences in performance in the BIX-infused mice would be accompanied by altered patterns of changes of the two histone marks in the dCA1 and the dorsal striatum. Taken together, our results suggest that G9a/GLP complex activity in the dHPC is essential for ensuring the coordinated expression of gene networks that are required for consolidation of long-term spatial memory.

Section snippets

Animals

Male C57BL/6 mice from Charles River Laboratories (L’Arbresle, France) at 4 months old at the beginning of the experiments were used. They were individually housed in a temperature-controlled colony room with a 12:12 h light-dark cycle (lights on at 7:00 am) and were provided with food and water ad libitum. Behavioral tests were performed during the light cycle between 08:00 and 13:00 h. In addition, all mice were handled for 5 days (5 min each time) before any experimental manipulation. All

Pharmacological inhibition of G9a/GLP activity in the dorsal hippocampus impairs consolidation of long-term spatial memory

We first examined the effect of post-training G9a/GLP blockade in the dHPC on consolidation of spatial LTM. Bilateral infusion of the G9a/GLP inhibitor BIX01294 (BIX) or aCSF (Vehicle) was administrated immediately or 1 h after the training session consisting of 8 trials followed 24 h later, by a probe test in which spatial and cue-guided strategies were in competition (Fig. 1A). Given that injection of vehicle, when delayed by 1 h, did not yield any significant effect on any parameters

Discussion

In the present study, we used a dual-solution version of the water maze to examine the role of the H3K9-specific histone methyltransferase (HMT) G9a/G9a-like protein (GLP) activity in LTM consolidation of spatial information and dynamic interaction between HPC-dependent spatial/place and dorsal striatum-dependent cued-response memory systems. First, our behavioural experiments indicate that intra-CA1 infusion of the G9a/GLP HMT inhibitor BIX01294 administrated immediately after a moderate

CRediT authorship contribution statement

Kyrian Nicolay-Kritter: Investigation, Formal analysis, Methodology, Writing - original draft, Visualization. Lassalle Jordan: Investigation, Methodology, Formal analysis. Jean-Louis Guillou: Conceptualization, Formal analysis, Supervision, Writing - review & editing. Nicole Mons: Conceptualization, Funding acquisition, Supervision, Methodology, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The present study was supported by the CNRS, Université de Bordeaux and Observatoire B2V des memoires and PREVAAL Finance to K. Nicolay-Kritter. We would like to thank G. Courtand for guidance with the image analysis, T Lafon for technical assistance and E. Poinama for animals care.

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