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, 24 (7), 278-288

Experience-dependent Epigenomic Reorganization in the Hippocampus

Affiliations

Experience-dependent Epigenomic Reorganization in the Hippocampus

Corey G Duke et al. Learn Mem.

Abstract

Using a hippocampus-dependent contextual threat learning and memory task, we report widespread, coordinated DNA methylation changes in CA1 hippocampus of Sprague-Dawley rats specific to threat learning at genes involved in synaptic transmission. Experience-dependent alternations in gene expression and DNA methylation were observed as early as 1 h following memory acquisition and became more pronounced after 24 h. Gene ontology analysis revealed significant enrichment of functional categories related to synaptic transmission in genes that were hypomethylated at 24 h following threat learning. Integration of these data sets with previously characterized epigenetic and transcriptional changes in brain disease states suggested significant overlap between genes regulated by memory formation and genes altered in memory-related neurological and neuropsychiatric diseases. These findings provide a comprehensive resource to aid in the identification of memory-relevant therapeutic targets. Our results shed new light on the gene expression and DNA methylation changes involved in memory formation, confirming that these processes are dynamic and experience-dependent. Finally, this work provides a roadmap for future studies to identify linkage of memory-associated genes to altered disease states.

Figures

Figure 1.
Figure 1.
Next-generation sequencing of mRNA in the rat hippocampus after learning. (A) Experimental design. Rats were either kept naïve, introduced to a novel context, or trained using contextual fear conditioning. (B) Threat recognition memory tests at 24 h and 7 d after training. (C) RNA and DNA were extracted from the CA1 and DG regions of the hippocampus 1 and 24 h after training for transcriptomic (RNA-seq) and DNA methylomic (MBD-seq) analysis. (D) Differentially expressed genes (DEGs) after Context Only and Threat Learning compared with Naïve at 1 and 24 h. (E) RNA-seq read density at the immediate early gene Egr2 increases at 1 h for Context Only and Threat Learning and returns to baseline at 24 h. (F) Genes (1837 total) are differentially expressed at 1 h (black) and 24 h (gray) time points after Threat Learning. Thickness of lines is proportional to the number of genes. In general, 1 h DEGs return to baseline at 24 h, and 24 h DEGs are not significantly altered at the 1 h time point, representing two distinct transcriptional waves post learning. (G) Heat map showing relative expression changes at the 1837 Threat Learning DEGs for all replicates.
Figure 2.
Figure 2.
Sustained gene body methylation changes are associated with Threat Learning, but not Context Only exposure. (A) Venn diagram showing the relative group sizes and overlap of DMGs for Context Only exposure and Threat Learning at 1 and 24 h. (B) Heat map showing relative changes in gene body CpG methylation at the 2097 Threat Learning DMGs for all replicates. (C) The correlation of Threat Learning DMGs that are significant at both 1 and 24 h (R2 = 0.08). (D) Example DMG Dhx16 reveals sustained DNA methylation patterning in Threat Learning but not Context Only exposure; relative changes in RNA-seq and MBD-seq read density for both learning events and time points are shown.
Figure 3.
Figure 3.
Increases in DNA methylation occur at gene exons and correlate with reduced gene expression at 24 h. (A) DMRs are associated with Threat Learning but not Context Only exposure. (B) Threat Learning CpG methylation increases or decreases at DMRs are associated with CpG island density. Depicted are all 5505 24 h Threat Learning DMRs in relation to CpG island density. (C) Significant overlaps between DEGs and DMGs or DMRs after Threat Learning occur at 24 h but not 1 h. (D) 24 h Threat Learning DMR locations relative to gene features compared with random chance. ±95% confidence limits. (E) Schematic of Threat Learning DMR locations relative to gene features for down-regulated DEGs. Each brick represents one DMR (1 or 24 h) that overlaps with Threat Learning DMGs down-regulated at 24 h, and is placed according to the genomic region in which it is located. The length of each region is representative of its average size across all gene elements in the rat genome. (F) Volcano plot of Threat Learning Hyper mC DMR containing DEGs. The vast majority of Threat Learning DEGs that contain exon hypermethylation were decreased in expression when compared with Naïve controls.
Figure 4.
Figure 4.
Coordinated alterations in DNA methylation and gene expression following Threat Learning are genome wide and overlap with genes altered in cognitive disease models. CIRCOS plot depicting Threat Learning gene expression and DNA methylation changes. Labels in the outer band represent chromosomal position (tic marks are in Mb). (A) Gene density is depicted in the outermost plot, with each axis line representing 10 genes per 1 Mb segment. (B) Threat Learning DEGs at 1 h, with orange representing a significant increase in expression and blue a decrease compared with context Naïve. (C) DMGs at 1 h. (D) DEGs at 24 h (E) DMGs at 24 h. (F) Genes that were in both the Threat Learning DMG and DEG sets at 1 h, with green circles representing decreased methylation and increased expression, and red representing the opposite. (G) Genes in both the Threat Learning DMG and DEG sets at 24 h. Generally, DEGs with increased expression were associated with decreased methylation, and decreased expression was associated with an increase in methylation. (H) This track represents the genomic location of genes found to have their mRNA trafficked to dendrites in a previous study (Cajigas et al. 2012). The genomic location of genes up-regulated (I) or down-regulated (J) in an Alzheimer's disease mouse model are represented in the next two tracks (Gjoneska et al. 2015). (K) In the center of the CIRCOS plot, we integrate Threat Learning associated genes with these additional data sets, revealing significant associations. Genes that were both differentially expressed and methylated at 24 h, which were also members of the dendritic trafficking or Alzheimer's disease mouse model gene lists, are shown as arcs stemming from their chromosomal position to a circle colored to represent the additional sets they were members of. Arc colors represent genes that were up-regulated and hypomethylated (green), down-regulated and hypermethylated (red), or noncoordinately expressed (gray). The area of each circle represents the number of members found in that group, and the overlap between circles shows the amount of genes that were members of multiple data groups. Significant overlap between the Threat Learning DEG/DMG gene list and these additional data sets was assessed using Fisher's exact test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 5.
Figure 5.
Gene ontology of Threat Learning differentially expressed genes. (A) Gene ontology term enrichment of Threat Learning DEGs 1 h post-training indicates pathway enrichment for the response to cAMP in up-regulated DEGs. (B) Gene ontology term enrichment of DEGs 24 h post-training indicates enrichment for neuron–neuron synaptic transmission in DEGS that are up-regulated and nucleic acid metabolic processes DEGs that are down-regulated. (C) Gene ontology cluster node-network diagram of genes up-regulated at 24 h post-training.
Figure 6.
Figure 6.
Gene ontology of Threat Learning differentially methylated genes. (A) Gene ontology term enrichment of Threat Learning DMGs 1 h post-training indicates enrichment in gene networks involved in the regulation of neuron differentiation, synapse assembly, and endopeptidase activity. (B) Gene ontology term enrichment of DMGs 24 h post-training indicate enrichment for neuron differentiation and synaptic transmission pathways in hypomethylated DMGs. (C) Gene ontology cluster node-network diagram of genes that are hypomethylated at 24 h post-training.

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