The epigenome and three-dimensional (3D) genomic architecture are emerging as key factors in the dynamic regulation of different transcriptional programs required for neuronal functions. In this study, we used an activity-dependent tagging system in mice to determine the epigenetic state, 3D genome architecture and transcriptional landscape of engram cells over the lifespan of memory formation and recall. Our findings reveal that memory encoding leads to an epigenetic priming event, marked by increased accessibility of enhancers without the corresponding transcriptional changes. Memory consolidation subsequently results in spatial reorganization of large chromatin segments and promoter–enhancer interactions. Finally, with reactivation, engram neurons use a subset of de novo long-range interactions, where primed enhancers are brought in contact with their respective promoters to upregulate genes involved in local protein translation in synaptic compartments. Collectively, our work elucidates the comprehensive transcriptional and epigenomic landscape across the lifespan of memory formation and recall in the hippocampal engram ensemble.
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Supplementary Tables 1–11 provide direct access to the main results derived from the transcriptome and epigenome assays presented in this study. In addition, raw and processed data sets generated during the study are available in the Gene Expression Omnibus repository using the accession number GSE152956. Any other data that support the findings of this study are available from the corresponding author upon reasonable request.
Codes used for the analysis of this study are available in the supplementary software appendix. A live version of the custom R scripts generated during this study is available in the Github repository at https://github.com/vishnudileep2000/IntronVsExon_RNAseq
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We thank E. Niederst, J. Penney, S. Barker, R.T. Stott, M. Victor, A. Watson, N. Dedic, E. Lockshin and the members of the L.-H.T. Lab for helpful discussion and suggestions. We thank lab manager Y. Zhou and E. McNamara for mice colony maintenance. We thank P. Autissier (Whitehead Institute) for help with FACS. This work was supported by National Institutes of Health (NIH) grants RF1AG062377, AF1AG054012, RO1NS102730 and RF1AG064321, the JPB Foundation, the Alana Foundation, the LuMind Down Syndrome Foundation, the Cure Alzheimer’s Fund CIRCUITS consortium and the Robert A. and Renee E. Belfer Family Foundation to L.-H.T. This work was also supported, in part, by NIH grants R01AG058002, U01NS110453, R01AG062335 and UG3NS115064 to M.K. and L.-H.T. and R01AG067151, R01MH109978, U01MH119509, R01HG008155 and U24HG009446 to M.K. V.D. is supported by the AARF-19-618751 grant from the Alzheimer’s Association. H.S.M. is supported by the Burroughs Wellcome Fund and a UNCF–Merck postdoctoral fellowship. C.A. is supported by the JPB Foundation. R.M.R. is supported by NIH T32 grant 5T32HD09806. The Hi-C libraries preparation kit was received as a generous gift from DovetailTM (v.1.03, Dovetail Genomics). We thank the Dovetail team for helpful discussion and suggestions.
The authors declare no competing interests.
Peer review Information Nature Neuroscience thanks Kaoru Inokuchi, Hongjun Song, and Jason Stein for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Schematic of the Targeted Recombination in Active Populations (TRAP), which requires two transgenes, one that expresses CreERT2 from an activity-dependent Arc promoter (ArcCreERT2) and one that allows expression of the eYFP reporter, in a Cre- dependent manner. Administration of TAM to TRAP mice results in a permanent eYFP label in the initially activated Arc neurons. Without TAM, CreERT2 is retained in the cytoplasm of active neurons in which it is expressed, so no recombination can occur. b, Representative images and co-localization analysis between endogenous Arc protein (Cyan) and the Arc:eYFP reporter (Magenta), 1.5 h after the FS. Upper panel shows images from the whole hippocampus and lower panel images shows neurons from the DG. Magenta arrows—neurons with only Arc:eYFP reporter signal, Cyan arrows—neurons with only endogenous Arc signal, White arrows—neurons with signal from both populations. Analysis was performed using IMARIS module (co-localization tools) and revealed an average of 84% overlap between the two populations. Scale bar, 100 μm. n = 3 mice /5 slices per animal, boxplot indicates the mean, interquartile range and the minimum and maximum. c, Contextual fear conditioning (CFC) freezing test. Percentage of the time freezing levels were measured during habituation (Day 0, Pre-FS) and during the re-exposure (Day 5) to the fear-inducing cue. n = 30 mice, boxplot indicates the mean, interquartile range and the minimum and maximum, two-sided unpaired Student’s t-test, t = 15.63, df=16, ****P < 0.0001. d, Representative images of Activated -late and Reactivated neurons in the hippocampus in two experimental groups; CFC with (TAM) or without (NO-TAM) tamoxifen administration. Scale bar, 100 μm. e, Contextual fear conditioning (CFC) freezing test. Percentage of the time freezing levels were measured during the re-exposure to the fear inducing cue (A-A) and during the exposure to a novel neutral environment (context B). n = 15 mice, boxplot indicates the mean, interquartile range and the minimum and maximum, two-sided unpaired Student’s t-test, t = 8.506, df=14, ****P < 0.0001. f, Representative images of Activated -late and Reactivated neurons from the DG in the A-A’ or A-B group.
a, Workflow for the flow cytometry dissection of different neuronal population from the hippocampus, during memory formation and retrieval. Representative FACS plots showing expression of all population (left panel). Further selection was made on single nuclei and NeuN + /DAPI + population (middle panel). Last, selection was made on the gated sub-population; GFP + (adjusted to ~2.5% from all cells), ARC + /GFP + (~0.15% from all cells) and nuclei were sorted to 1.5 ml Eppendorf tubes coated with 200ul of 1% PBS. b, Venn Diagram (left) and table (right), which illustrate the overlap between the DARs identified in the different pairwise comparisons during memory formation and recall. c, Resampling-based statistical analysis was performed to determine if the enrichments of chromatin states over ChromHMM emissions (observed) are statistically significant. Expected enrichment was calculated by performing 10,000 randomized sets of overlaps (permutations) between ‘all accessible sites’ and ‘all histone modifications sites (that is all emissions)’ and presented as histogram in the figure. Sample size of each randomized set was determined by the size of DARs from each state. The mean and standard deviation of the sample was calculated (Supplementary Table 3). The number of observed overlaps between DARs and each emission was calculated and presented as lines. z-score was calculated as fallows; Z = (observed values (X)—mean of the sample (μ))/(standard deviation of the sample (σ)). z-score Basal vs. Early; S.E 10.5, W.E 6.9. Stable; S.E 16.9, W.E 12.7, all p < 0.0001, z-score Early vs. Late; S.P 91.7, W.P 38.7. Late vs. Reactivated; S.P 26.8, W.P 28.9, all p < 0.0001. p-values (Two-Sided) were calculated from z-table. Full analysis is reported in Supplementary Table 3. d, Pie chart shows percentage of different enhancer states, for all stable regions. Overlap of each individual stable region was performed with previously published H3K4me1 and H3K27ac ChIP-seq data, obtained 1 h after FS 2921. Enhancers states were classified as ‘primed’—overlap with regions marked with only H3K4me1, ‘active’—with H3K4me1/H3K27ac or ‘latent’—no overlap. e, Motifs identified from nucleosome free regions (NFR) on the ATAC-seq tracks from each state (Basal, Activated-early, Activated-late and Reactivated). Peaks were divided into positions that annotated to promoters (5 kb from TSS) and enhancers (>5 kb from TSS). Circle size indicate percentage of enrichment (1–50%). Color indicates –log(P-value).
Extended Data Fig. 3 Coordinated priming of the epigenetic state during memory encoding and consolidation facilitates long-range interactions during reactivation.
a, The properties of CHiCAGO-detected interactions in each phase (Basal, Activated -early, Activated -late and Reactivated). Default settings and a score threshold of 5 were used in significant interaction calling, performed jointly on all replicates. b, Pie chart represent the percentage of all CHiCAGO-detected interactions that are demarcated by either H3K27ac/H3K4me1 (67.5% enhancers marks), H3K4me3/H3K9ac (46.2% promoters mark) or H3K27me3 (1.1% repressive marks). c, WashU epigenome browser image, encompassing ~ 500 Kb region around the Eif4e2 genes. Arcs shows significant common (red rectangle) and unique (arrowed) enhancers that interacts with promoters (blue rectangle). d, Examples of interactions called by CHiCAGO. Plots showing all the read counts from bait-other-end (enhancer), within 500–700 kb (upstream and downstream) of the Grink3 and Wwc2 promoters. Significant interactions detected by CHiCAGO (score ≥5) are shown in red, and sub-threshold interactions (3 ≤ score < 5) are shown in blue. Grey lines show expected counts and dashed lines the upper bound of the 95 % confidence intervals. e, Overlap enrichment analysis between interacting enhancers and DARs, using a permutation procedure on 10,000 randomized sets of accessible sites. Histogram present random sampling distribution of accessible sites for each condition (Basal vs. Activated-early, Activated-early vs. Activated-late, Activated-late vs. Reactivated, Stable). The number of overlapped loci is presented in colored lines from Basal, Activated-early, Activated-late and Reactivated neurons. DARs of BAS vs. Activated-early (z-score; 7.1, 7.7, 8.5, 10.9). DARs of Activated-early vs. Activated-late (z-score; −0.1, −0.8, −2.4, −3.2). DARs of Activated-late vs. Reactivated (0.4, 1.8, 0.4, −0.2). DARs of stable (z-score; 1.7, 2.0, 6.5, 7.3).
Extended Data Fig. 4 Chromatin changes that occur during the early phase enable transcriptional changes observed at a later time point, primarily in reactivated engram cells.
a, Overlap analysis between gene names from the pair-wise differential analysis and previously published data of: i) activated DG granule cells 1 h after novel exposure32 ii) 24 h after FS20 and iii) after prolonged stimulation (6 h) of mouse cultured cortical neurons with KCl33. Analysis was carried by GeneOverlap R package. b, Exonic (red) and intronic (blue) reads were quantified separately across all conditions and compared to transcriptional activity as measured by DEseq2. Reads were normalized (RPKM) and the log2FC changes are presented for each state. Violin plot indicates the mean, interquartile range and the minimum and maximum, one-way ANOVA (parametric, unpaired), Basal vs. Early; F (5, 248) = 389.9. Early vs. Late; F (5, 2374) = 2183. Late vs. Reactivated. F (5, 1357) = 945.5, All Ps < 0.0001. Bonferroni’s multiple comparisons. n.s = not significant, ***P < 0.0001. c, Exon/Intron ratios were measured in each cluster across all conditions (Log2FC scale). Violin plot indicates the mean, interquartile range and the minimum and maximum, one-way ANOVA (parametric, unpaired), F (5, 1143) = 260.2, P < 0.0001. Bonferroni’s multiple comparisons test. n.s = non-significant, ***P < 0.0001. d, Overlap analysis between DARs and DEGs during different memory phases. DARs on Intergenic and introns regions were mapped to their respective genes with the pc-HiC interaction maps. Overlap analysis was carried by GeneOverlap R package. P-value (numbers) and odds ratio (color) from Fisher’s exact test are presented in the heatmap. n.s, not significant. e, Pearson correlation between log2FC values of DARs and log2FC of DEGs that were annotated to those region (intergenic regions were mapped via the pc-HiC data set). Chromatin accessibility changes were compared with parsed exonic reads (red line), intronic reads (blue lines) and total transcriptional changes (both intronic and exonic reads) as measured by Desq2 (gray line). All r and p-values are reported in Supplementary Table 9.
Extended Data Fig. 5 Transcriptional changes in the activated-late neurons correlated higher with intronic reads and reactivated neurons presented higher correlation with exonic reads.
a, Exonic (red) and intronic (blue) reads were quantified separately across all conditions for each of the clusters identified in Fig. 5b. Reads were normalized (RPKM) and the log2FC changes are presented for each cluster. b, Exon/intron ratios were measured in each cluster across all conditions. Violin plot indicates the mean, interquartile range and the minimum and maximum, n = 3 biologically independent samples one-way ANOVA (parametric, unpaired), Dw–Late cluster; F (3, 968) = 139.4, P < 0.0001. Up -Late cluster; F (3, 734) = 15.95, P < 0.0001. Stable-cluster; F (3, 652) = 93.97, P < 0.0001. Reactivation cluster; F (3, 1600) = 485.2, P < 0.0001. Bonferroni’s multiple comparisons test to Deseq2 reads. ***P < 0.0001.
Extended Data Fig. 6 Distinct temporally transcriptional programs are being synchronized to maintain neuronal excitability, structural changes and protein translation in synapses in the engram ensemble.
a, Schematics representation of the experimental design. Three weeks prior to the CFC test, ArcCreERT2 mice were bilaterally injected to the DG with AAV9-EF1a-DIO-hChR2-EYFP. In a similar manner to the TRAP system, eYFP reporter is only expressed in a Cre- dependent manner in the presence of tamoxifen. In the right panel, representative IHC images of the DG. Green—AAV-eYFP, Red—endogenous Arc. The scale bar represents 50 μm. b, Spines morphology assessment during different memory phases. Right panel shows a single eYFP+ dendritic shaft with different types of spines (Stubby, Thin, Mushroom, Enlarged mushroom). The scale bar represents 5 μm. boxplot indicates the mean, interquartile range and the minimum and maximum, Activated-early: n = 4 mice /5 section per animal, Activated-late: n = 4 mice /4 section per animal, Reactivated: n = 4 mice /2 section per animal. one-way ANOVA (parametric, unpaired), Stubby; F (2, 36) = 2.313, P = 0.1135. Thin; F (2, 36) = 35.12, P < 0.0001. Mushroom; F (2, 36) = 38.42, P < 0.0001. Bonferroni’s multiple comparisons test, ***P < 0.0001. c, Representative IHC images and quantification of the protein levels of two members of the EIF family; (left) Eif2a and (right) Eif3e. The scale bar represents 10 μm. Data for dendritic shaft is presented as a ratio between number and the length (μM). n = 4 mice /5 section per animal, boxplot indicates the mean, interquartile range and the minimum and maximum, one-way ANOVA (parametric, unpaired) with Bonferroni’s multiple comparisons test, n.s = not significant, Eif2a Shaft; F (2, 20) = 4.484, P = 0.0246. Soma; F (2, 21) = 19.58, P < 0.0001. (Activated-early vs. Activated-late *P = 0.0142, Activated-early vs. Reactivated *P < 0.0001, Activated-late vs. Reactivated *P = 0.0303). Eif3e Shaft; F (2, 14) = 1.983, P = 0.1745. Soma; F (2, 23) = 8.309, P = 0.0019, (Activated-early vs. Reactivated *P = 0.0057, Activated-late vs. Reactivated *P = 0.0055). d, Pie chart present the percentage of enlarged mushroom spines (Dh ≥ 3Dn) and mushroom spines from Activated-late and Reactivated neurons. e, Representative images (left panel) and quantification (right panel) of Gria1 mRNA levels, during different phases of memory. Data is presented as a ratio between number of puncta and the dendritic shaft length. The scale bar represents 10 μm. N = 4 mice /5 section per animal, boxplot indicates the mean, interquartile range and the minimum and maximum, Shaft; one-way ANOVA (parametric, unpaired) F (2, 15) = 10.41, P = 0.0015. Bonferroni’s multiple comparisons test, **P = 0.0011. lower panel - Soma; one-way ANOVA F (2, 12) = 0.13, P = 0.88.
Extended Data Fig. 7 Interactions with distinct combinatorial enhancers leads to a directional change in gene expression.
a, Venn diagrams shows percentage of overlap between chromatin accessibility (DARs) across all memory phases (BAS vs. Activated-early, light green grid circle; Activated -early vs. Activated-late, dark green grid circle; Activated-late vs. Reactivated, orange grid circle) and total transcriptional changes from all identified clusters (Dw–late, Up –late, Stable, Reactivation, blue grid circle). Intergenic and introns DARs were mapped to their respective genes with the pc-HiC interaction maps. Percentage of overlap was calculated from all identified DEGs in the clusters (n = 1095). b, Overlap analysis between DARs (pairwise) and DEGs from each cluster. Intergenic and introns DARs were mapped to their respective genes with the pc-HiC interaction maps. Overlap analysis was carried by GeneOverlap R package. P-values and Jaccard values (color) from Fisher’s exact test are presented on the heat map (left). Percentage of overlap was calculated from all identified DEGs in the clusters (right). c, Representative image of chromatin and transcriptional changes of the Gabrb3 locus from the Dw-late cluster. While early state interactions were between promoter and enhancers with transcriptional activators (Ap1), late state interaction were with transcriptional repressors (Slug). Upper IGV genome browser tracks (purple - Basal, light green - Activated-early, dark green - Activated-late and orange - Reactivated) presenting transcriptional changes (nRNA-seq), middle tracks shows chromatin accessibility dynamics (ATAC-seq) on promoter (red rectangle) and enhancers (gray rectangle). Significant promoter-enhancer interaction are represented as arcs (WashU browser tracks). Lower track present motifs that were identified via HOMER tools (Slug, Ap1 and Rest). d, Aggregation plots for individual motifs. The enrichment values (motifs per bp/ per peak) of six selected motifs (two repressors, two activators and two bivalent) was assessed around the center of peaks (-/+ 4000 bp) from each cluster.
Extended Data Fig. 8 Proposed model of the chromatin accessibility, promoter-enhancer interaction and transcriptional dynamics of hippocampal memory engram neurons.
Over the course of memory formation and recall. Basal state - gene promoters interact with enhancers that carry transcriptional repressor cargo and express low levels of mRNA. Early phase - leads to a priming event, in which enhancers that harbor transcriptional activator cargo become more accessible, but most of them remain isolated and lack interactions with respective gene promoters. Late phase - gene promoters shift their interaction to the primed regions, which harbor transcriptional activator motifs. This promoter-enhancer reprograming results in increased gene expression that presumably allows the stabilization of the memory. Recall - reactivated engram neurons utilized a subset of primed promoter-enhancer interactions, which is associated with transcriptional changes involved in mRNA transport to synaptic compartments and protein translation. Transcription factor - TF, E(1–3)—different enhancer that interact with the same promoter, Red - transcriptional repressors, Blue - transcriptional activators.
Number of isolated nuclei from each group via FACS. The numbers recorded in this table were obtained for ATAC-seq analysis.
DARs between all populations (ATAC-seq)
Resampling-based statistical analysis to determine the enrichments of chromatin states over ChromHMM emissions (observed). Expected enrichment was calculated by performing 10,000 randomized sets of overlaps (permutations) between ‘all accessible sites’ and ‘all histone modifications sites’ (that is, all emissions).
HOMER motif enrichment analysis.
Dynamic changes in compartments organization.
Promoter–enhancer interaction score as defined by CHiCAGO for each population.
Pearson’s correlation between log2FC values of DARs and log2FC DEGs.
Matrix table for k-means cluster shows the (a) promoter–enhancer interaction scores, (b) chromatin accessibility (normalized RPKM values) and (c) transcriptional changes (normalized counts).
Primary antibodies list.
Sequences of primer pairs for ChIP-qPCR assays.
ATAC-seq library statistics.
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Marco, A., Meharena, H.S., Dileep, V. et al. Mapping the epigenomic and transcriptomic interplay during memory formation and recall in the hippocampal engram ensemble. Nat Neurosci (2020). https://doi.org/10.1038/s41593-020-00717-0