Part 5:Mapping The Epigenomic And Transcriptomic Interplay During Memory Formation And Recall in The Hippocampal Engram Ensemble
Mar 22, 2022
ali.ma@wecistanche.com
Extended Data Fig. 2: Stable DARs are predominantly enriched for enhancers marks
(a) Workflow for the flow cytometry dissection of the different neuronal populations 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, the 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.
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(b) Venn Diagram (left) and table (right), 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 (i.e. all emissions)’ loci and presented as a histogram in the figure. A 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 the z-table. A full analysis is reported in supplement table 3.
(d) Pie chart shows the percentage of different enhancer states, for all stable regions. Overlap of each stable region was performed with previously published H3K4me1 and H3K27ac ChIP-seq data, obtained 1h after FS21. 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 were annotated to promoters (5kb from TSS) and enhancers (>5kb from TSS). Circle size indicates the percentage of enrichment (1–50%). Color indicates –log(P-value).
Extended Data Fig. 3: Coordinated priming of the epigenetic state during memory encoding and
(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 represents 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 show significant common (red rectangle) and unique (arrowed) enhancers that interact 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. The histogram presents 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
(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 (6h) of mouse cultured cortical neurons with KCl33. Analysis was carried out by the 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 = non-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 out by the 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 ofDARs and log2FC DEGs that were annotated to that 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
(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)
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
a 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 ofthe protein levels oftwo 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 presents 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 several 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
(a) Venn diagrams show the 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. The 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 out by the GeneOverlap R package. P-values and Jaccard values (color) from Fisher’s exact test are presented on the heatmap (left). The 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 interactions were with transcriptional repressors (Slug). Upper IGV genome browser tracks (purple - Basal, light green - Activated -early, dark green - Activated -late and orange - Reactivated) present transcriptional changes (ncRNA-seq), middle tracks show chromatin accessibility dynamics (ATAC-seq) on the promoter (red rectangle) and enhancers (gray rectangle). Significant promoter-enhancer interactions 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) were assessed around the center of peaks (−/+ 4000bp) from each cluster.
Extended Data Fig. 8: Proposed model of the chromatin accessibility, promoter-enhancer interaction, and transcriptional dynamics of hippocampal memory engram neurons
regions, which harbor transcriptional activator motifs. This promoter-enhancer reprogramming 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 enhancers that interact with the same promoter, Red - transcriptional repressors, Blue - transcriptional activators.
Supplementary Material
Refer to the Web version on PubMed Central for supplementary material.
Acknowledgments
We thank E. Niederst, J. Penney, S. Barker, R.T. Stott, M. Victor, A. Watson, N. Dedic, E. Lockshin, and the members ofL.H.T. Lab for helpful discussion and suggestions. We thank lab managers Y. Zhou, E. McNamara for mice colony maintenance. We thank P. Autissier (Whitehead Institute) for help with FACS. Funding: These works were supported by NIH grants RF1AG062377, AF1AG054012, RO1NS102730, RF1AG064321, The JPB Foundation, The Alana Down Syndrome Research Foundation, The LuMind Down Syndrome Research 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, UG3NS115064 to M.K. and L-H.T, and R01AG067151, R01MH109978, U01MH119509, R01HG008155, U24HG009446 to M.K. V.D is supported by an AARF-19-618751 grant from Alzheimer’s Association. H.S.M is supported by Burroughs Wellcome Fund and UNCF-Merck postdoctoral fellowship. C. A is supported by the JPB Foundation. R.M.R is supported by NIH T32 grant 5T32HD09806. Hi-C libraries preparation kit was received as a generous gift from DovetailTM (v.1.03, Dovetail Genomics, Chicago, USA). We thank Dovetail team for helpful discussion and suggestions.
