Characterization of temporally and functionally distinct activity-dependent gene expression programs in cultured striatal neurons. a, Experimental design. DIV11 cultures were depolarizated for 1 or 4 h with 10mM KCl. Following treatment, RNA-seq libraries were constructed. b-c, Volcano plots displaying gene expression changes after 1 and 4 h of neuronal depolarization. d-e, Venn diagrams comparing 1 and 4 h upregulated and downregulated DEGs. f, Top 10 cellular component GO terms for 1 and 4 h upregulated DEGs.

Activity-dependent chromatin remodeling in cultured primary rat striatal neurons. a, DIV11 primary rat striatal neurons were treated with 10mM KCl for 1 or 4 h. Following treatment, ATAC-seq libraries were prepared. b-c, Volcano plots displaying differentially accessible regions (DARs) after 1 and 4 h of neuronal depolarization. d, Genomic location of vehicle and 4 h DAR ATAC peaks. e, Odds ratio for genomic annotations of 4 h DARs or vehicle peaks.

Motifs for activity-dependent transcription factors are significantly enriched in 4 h DARs. a-b, Plots showing enrichment of specific transcription factor motifs in vehicle peaks and 4 h DARs. Motifs with significant adjusted p-values and a percent enrichment greater than 50% are shown in red and labeled with the corresponding transcription factor. c, Representative results from HOMER known motif enrichment analysis conducted using 4 h DAR peak set. d, Motifs for activity-dependent TFs (AP1, FOS, FOSL2, JUNB), MEF2C, and ISL1 are enriched at the center of DARs. e, UMAP generated using transcription factor motif enrichment within the 4 h DARs. f, UMAPs colored by the presence of absence of specific transcription factor motifs. KLF10 and OCT6 specifically mark clusters 2 and 1, respectively.

Activity-dependent chromatin remodeling requires protein translation. a, Experimental design. DIV11 primary rat striatal neurons were treated with DMSO or anisomycin for 30 minutes followed by 4 h of depolarization with 10mM KCl. b, Western blot for FOS and β-tubulin from striatal neurons treated with vehicle, 10mM KCl, or 10mM KCl + 40 μM Anisomycin. c, Boxplots demonstrating the effects of anisomycin on activity-dependent chromatin remodeling. One way ANOVA with Tukey’s multiple comparisons test, ****p<0.0001. d, Heatmaps and mean accessibility plots from 4 h DARs. For heatmaps, each row represents a single DAR. CPM = counts per million.

Transcriptional regulation of Pdyn mRNA. a, ATAC-seq tracks at the Pdyn gene locus of embryonic striatal neurons treated with DMSO + Vehicle, DMSO + KCl, or anisomycin + KCl. A DAR 45 kb upstream of the Pdyn TSS in a non-coding region becomes accessible with depolarization only with intact protein translation. b, RT-qPCR for Pdyn mRNA from DIV 11 rat striatal neurons treated with vehicle, KCl, anisomycin, or anisomycin + KCl. Induction of Pdyn mRNA by KCl is blocked by anisomycin pretreatment (One-way ANOVA with Tukey’s multiple comparisons test **p<0.01, ****p<0.0001). c, Targeted activation of dopamine-regulated immediate early genes with CRISPR activation (data from reference 9). dCas9-VPR was transduced with multiplexed sgRNAs targeting 16 immediate early genes. d, Pdyn mRNA is upregulated following CRISPR-based activation of 16 IEGs. Mann-Whitney Test **p<0.01. e, Pseudotime analysis to predict regulators of Pdyn expression in Drd1-MSNs was performed with avaialable snRNA-seq data from the rat nucleus accumbens9. f-g, Feature plots for Pseudotime, Fos, Fosb, and Pdyn in Drd1-MSNs. h, Gene regulatory network reconstruction from single cell trajectories identifies predicted regulators of Pdyn in Drd1-MSNs.

CRISPR-based functional validation of a novel Pdyn enhancer. a, Viral strategy for functional validation of putative enhancers using CRISPR interference with dCas9-KRAB-MeCP2 and CRISPR activation with dCas9-VPR. b, CRISPR sgRNAs (4x) were designed to target the activity-regulated DAR 45.1 kb upstream of Pdyn in the rat genome. c, CRISPRi at the Pdyn DAR blocks activity-dependent induction of Pdyn mRNA. Cultured embryonic striatal neurons were transduced with dCas9-KRAB-MeCP2 and sgRNAs targeting lacZ (non-targeting control) or the Pdyn DAR. Neurons were then treated with vehicle or 10mM KCl for 4 h prior to RT-qPCR. One-way ANOVA with Tukey’s multiple comparisons test **p<0.01,***p<0.001. d, CRISPRa at the Pdyn DAR selectively upregulates Pdyn mRNA without altering expression of the nearest upstream and downstream genes. Striatal neurons were transduced with dCas9-VPR and sgRNAs targeting lacZ or the Pdyn DAR. Folowing RNA extraction at DIV 11, RT-qPCR was used to measure expression of Pdyn, Sirpa, and Stk35. Mann-Whitney test ****p<0.0001.

Identification and validation of a conserved PDYN enhancer in the human genome. a, Experimental design for published RNA-seq and ATAC-seq datasets from human GABAergic and glutamatergic neurons treated with 55mM KCl. b-c, Volcano plots for human GABAergic neurons treated with 55mM KCl for 0.75 or 4 h. PDYN is a significant DEG at 4 h, but not 0.75 h. d, PDYN is significanly upregulated 4 h after a KCl stimulus, but only in GABAergic neurons. Two-way ANOVA with Tukey’s multiple comparison’s test. *p<0.05. e, Linear synteny view of the rat and human Pdyn/PDYN locus reveals shared conservation of 4 distinct activity-dependent DARs identified in rat striatal neurons. f, ATAC-seq tracks of GABAergic neurons treated with 55mM KCl for 0, 0.75, or 1.5 h at the human PDYN locus. Regions conserved between rats and humans undergo dynamic remodeling in human GABAergic cells at 1.5 h after stimulation. A region homologous to the rat Pdyn enhancer characterized in Figure 6 is 63.7 kb upstream of the human PDYN gene. g, Location of CRISPR sgRNAs in the human genome for CRISPR-based activation of the PDYN DAR in human cells. h, CRISPRa at the human PDYN DAR is sufficient to upregulate PDYN mRNA in HEK293T cells. Mann-Whitney test. *p<0.05.

Characterization of the rat Pdyn DAR in the adult nucleus accumbens at single cell resolution. a, Experimental design. b, UMAP of 10,085 nuclei from adult rat nucleus accumens (NAc). c, Genome tracks displaying snATAC-seq data at the Pdyn locus. The experimentally validated Pdyn DAR (highlighted in blue) exhibits high co-accessibility with the proximal promoter for Pdyn as well as other nearby accessible regions.

IEGs and LRGs are temoporally and functionally distinct. a-b, Plots demonstrating activation of IEGs at both 1 and 4 hours after depolarization, while LRGs are only activated after 4 hours of stimulation. Two-way ANOVA with Tukey’s multiple comparisons test.****p<0.0001. c, Venn diagram comparing molecular function GO terms enriched in 1 and 4 hour DEG lists.

Transcription factor binding and histone modifications in 4 h DARs, random regions, and vehicle peaks. Enrichment plots for a, H3K27ac, b, H3K4me1, c, H3K4me3, d, H3K4me1/H3K4me3. e, ΔFosB. Data from Yeh, et al., 2023 Biological Psychiatry.

Enhancers for Fos are accessible at baseline and do not undergo activity-dependent chromatin remodeling. ATAC-seq tracks at the Fos locus for neurons treated with DMSO + Veh, DMSO + KCl, or Anisomycin + KCl for 4 h.

Predicted regulators of Pdyn and Scg2 in Drd1-MSNs. Reconstruction of gene regulatory networks in Drd1-MSNs predicts AP-1 family members such as Fos and Fosb regulate both Scg2 and Pdyn.

Conserved activity-regulated PDYN DAR is selective for GABAergic neurons. ATAC-seq tracks from human induced GABAergic and Glutamaterigc neurons treated with 55mM KCl for 0, 0.75, or 1.5 h. Data from Sanchez-Priego, et al., 2022 Cell Reports.