Comparison of open chromatin regions between dentate granule cells and other tissues and neural cell types.
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1 Supplementary Figure 1 Comparison of open chromatin regions between dentate granule cells and other tissues and neural cell types. (a) Pearson correlation heatmap among open chromatin profiles of different cell types and tissues (See Supplementary Table 2 for sources of data used). (b) UCSC genome browser visualization of open chromatin profiling coverage at the Prox1 locus for the adult mouse dentate gyrus (red) and other subtypes of neuronal cells and tissues (gray). Bottom panel shows the UCSC genome browser visualization of RNA-seq coverage for dentate gyrus (blue).
2 Supplementary Figure 2 Characterization of open chromatin regions identified by ATAC-seq. (a) Distribution of open chromatin regions in different genomic regions before (E0; n = 89, 946) and 1 h (E1; n = 114,959) after neuronal activation (b) ATAC-seq reads at E0 across all annotated genes are stratified by their mrna levels (left panel) or plotted on gene bodies with their expression levels ranked in descending order for the heatmap view (right panel).
3 Supplementary Figure 3 Characterization of the chromatin state of regions with neuronal activity-induced chromatin-accessibility changes at E1 compared to E0. (a) UCSC genome browser visualization of open chromatin profiling coverage at E0 and E1 and H3K4me1, H3K27Ac and H3K4me3 ChIP-seq coverage at the Arc and cfos loci for the adult mouse dentate gyrus. Red bar indicates the gained-open sites at E1. Blue bar indicates the previously functionally identified enhancer regions 1-3. (b) Aggregate plots of histone modifications and CTCF ChIP-seq signals centered at gained-open (left panel) and gained-closed (right panel) sites at E1. The histone ChIP-seq data used for plots are from a previously published dataset from the adult mouse hippocampus 4 and a CTCF ChIP-seq dataset from the adult mouse cortex 5.
4 The significance of overlap between peaks was calculated using a hypergeometric model. (c) Shown are the emission matrix, state annotation and enrichment of biological features from ChromHMM6. Refer to caption of Fig. 2c.
5 Supplementary Figure 4 Characterization of H3K4me1, H3K4me3, H3K27Ac and H3K27me3 changes before and after stimulation at regions with neuronal activity-induced chromatin-accessibility changes at E1 compared to E0. Shown are aggregate plots of H3K4me1, H3K4me3, H3K27Ac and H3K27me3 signals before and after KCl stimulation centered at gained-open (up) and gained-closed (bottom) sites at E1. The histone ChIP-seq data used for plots are from a previously published dataset 7 (n = 2-3).
6 Supplementary Figure 5 Motif prediction of neuronal activity-induced gained-open and gained-closed sites at E1 compared to E0 by two different algorithms, HOMER and MEME-ChIP. (a) De novo motif identified by MEME-ChIP analysis of neuronal activity-induced gained-open regions at E1 resembles AP-1 complex, including cfos, JunB, Jun and JunD. Refer to Fig. 3a. (b) De novo motif identified by MEME-ChIP analysis of neuronal activationinduced gained-closed regions at E1 does not recover motifs similar to known transcription factor binding motifs (P = 4.5e -51). (c) De novo motif identified by HOMER from neuronal activity-induced gained-open and gain-closed sites at E1. ChIP-seq data for cfos, FosB, JunB and CREB used for plots were from a published dataset 7.
7 Supplementary Figure 6 Characterization of c-fos expression in the adult mouse dentate gyrus. (a, b) Expression of endogenous cfos in response to transient synchronous neuronal stimulation. Shown are summaries of time-course analyses of mrna (a) and protein (b) expression of cfos in the adult dentate gyrus after electroconvulsive stimulation. Values represent mean ± s.e.m. (n = 3 for a, n = 2 for b; *P < 0.05; **P < 0.01; ANOVA). Full-length western blots are presented in Supplementary Fig. 10. (c) Schematic diagrams of AAV constructs used to express shrna or exogenous protein in the adult mouse dentate gyrus (top panel) and the experimental paradigm (bottom panel). (d, e) Q-PCR analysis of expression of cfos in the adult dentate gyrus expressing shrna-ctrl and shrna-cfos (d) or cfos and/or EYFP (e). Values represent mean ± s.e.m. (n = 3 mice; *P < 0.05; permutation test).
8 Supplementary Figure 7 Effect of cfos knockdown in neuronal activity-induced chromatin opening and gene expression. (a) Confirmation of cfos knockdown efficacy upon AAV-mediated expression of shrna-cfos in samples for RNA-seq. Values represent mean + s.e.m. (n = 3; *P < 0.05; permutation test). (b) Principal components analysis of ATAC-seq data under different experimental conditions. (c, d) Venn diagrams of differential peaks (c) and genes (d) in the adult dentate gyrus expressing shrna-ctrl and shrna-cfos in response to neuronal activation. (e) Venn diagrams of gained-open regions induced by cfos overexpression and neuronal activation at E1 without exogenous cfos expression.
9 Supplementary Figure 8 Characterization of neuronal activation-induced chromatin-accessibility changes at E1, E4 and E24. (a) Comparison of open chromatin profiles of the dentate gyrus of adult mice at different time points after synchronous neuronal activation. Differential regions are shown in a volcano plot. Significantly gained-open sites are shown in red and gained-closed sites are shown in blue (T-test; P < 1e -5; Fold changes > 2). (b, c) Distribution of differential peaks after neuronal activation in different genomic regions. Distal binding sites are defined as > 1 kb from an NCBI annotated RefSeq TSS (b).
10 Supplementary Figure 9 ChIP-qPCR analysis at the sustained gained-open regions at E4 for Kcnj6, Nr3c1, Kcnv2 and Nrxn3 in the adult dentate gyrus at E0, E1 and E4. Data represent normalized percentage input (n = 3 mice in each group; #P > 0.05; *P < 0.05; permutation test).
11 Supplementary Figure 10 Full-length Western blot image for sample Western blot shown in Supplementary Figure 6b.
12 Supplementary Tables Supplementary Table 1. Summary of open chromatin regions at E0, E1, E4 and E24 (See Excel file). Supplementary Table 2. Sources of chromatin accessibility datasets and ChIP-Seq datasets used in the current study. Dataset Accession number Reference Cerebellum DNase-seq GEO: GSE51336 Ref 8 Astrocyte FAIRE-seq GEO: GSE73262 Ref 9 excitatory neurons ATAC-seq GEO: GSE63137 Ref 10 PV neurons ATAC-seq GEO: GSE63137 Ref 10 VIP neuron ATAC-seq GEO: GSE63137 Ref 10 Retina DNase-seq GEO: GSE51336 Ref 8 Liver DNase-seq GEO: GSE51336 Ref 8 Kidney DNase-seq GEO: GSE51336 Ref 8 CTCF ChIP-seq GEO: GSE49847 Ref 5 cfos ChIP-seq (± KCl) GEO: GSE60192 Ref 7 FosB ChIP-seq (± KCl) GEO: GSE60192 Ref 7 JunB ChIP-seq (± KCl) GEO: GSE60192 Ref 7 CREB ChIP-seq (± KCl) GEO: GSE60192 Ref 7 H3K4me1 ChIP-seq (± KCl) GEO: GSE60192 Ref 7 H3K4me3 ChIP-seq (± KCl) GEO: GSE60192 Ref 7 H3K27Ac ChIP-seq (± KCl) GEO: GSE60192 Ref 7 H3K27me3 ChIP-seq (± KCl) GEO: GSE60192 Ref 7 H3K4me1 ChIP-seq GEO: GSE65159 Ref 4 H3K4me3 ChIP-seq GEO: GSE65159 Ref 4 H3K27Ac ChIP-seq GEO: GSE65159 Ref 4 H3K27me3 ChIP-seq GEO: GSE65159 Ref 4 H3K9me3 ChIP-seq GEO: GSE65159 Ref 4 H3K36me3 ChIP-seq GEO: GSE65159 Ref 4 H4K20me1 ChIP-seq GEO: GSE65159 Ref 4 Supplementary Table 3. Summary of dentate granule neuron-specific open chromatin regions and their associated genes compared to other neuronal subtype cells (See Excel file). Supplementary Table 4. Summary of differential chromatin open regions after synchronous neuronal activation from ATAC-seq analysis. (a) Gained-open peaks at E1 compared to E0. (b) Gained-closed peaks at E1 compared to E0. (c) Sustained gained-open peaks at E1 and E4. (d) Sustained gained-closed peaks at E1 and E4. (e) Sustained gained-open peaks at E1, E4 and E24. (f) Sustained gained-closed peaks at E1, E4 and E24 (See Excel file). Supplementary Table 5. Summary of RNA-seq analyses. (a-c) Differentially expressed genes in the adult mouse dentate gyrus at E1 (a), E4 (b) and E24 (c) compared to E0. (d, e) Differentially expressed genes in the adult mouse dentate gyrus expressing shrna-ctrl (d) or shrna-cfos (e) at E1 compared to E0. (f) Differentially expressed genes in the adult mouse dentate gyrus expressing cfos compared to those expressing EYFP (See Excel file). Supplementary Table 6. List of gained-open peaks with cfos binding sites at E1 compared to E0 (See Excel file).
13 Supplementary Table 7. Summary of differential peaks under cfos knockdown and overexpression conditions from ATAC-seq analysis. (a, b) Gained-open (a) and gained-closed (b) peaks in neurons expressing shrna-ctrl at E1 compared to E0. (c, d) Gained-open (c) and gainedclosed (d) peaks in neurons expressing shrna-cfos at E1 compared to E0. (e, g) Gained-open (e) and gained-closed (g) peaks in neurons expressing cfos compared to those expressing EYFP at E0 (See Excel file).
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