Temporally specific gene expression and chromatin remodeling programs regulate a conserved Pdyn enhancer

Reviewer #1 (Public Review):

Summary:
In this manuscript, the authors use ATAC-seq to find regions of the genome of rat embryonic striatal neurons in culture that show changes in regulatory element accessibility following stimulation by KCl-mediated membrane depolarization. The authors compare 1hr and 4hr transcriptomes to see both rapid and late response genes. When they look at ATAC-seq data they see no changes in accessibility at 1hr but strong changes at 4hr. The differentially accessible sites were enriched for the AP-1 site, suggesting regulation by Fos-Jun family members, and consistent with the requirement for IEG expression, anisomycin blocked the increase in accessibility. To test the functional importance of this regulation the authors focus on a putative enhancer 45kb upstream of the activity-induced gene encoding the neuromodulator dynorphin (Pdyn). To test the function of this region, the authors recruited CRISPRi to the site, which blocked KCL-dependent Pdyn induction, or CRISPRa, which selectively increased Pdyn expression in the absence of KCl. Finally, the authors reanalyze other human and rat datasets to show cell-type specific function of this enhancer correlated to Pdyn expression.

Strengths:
The idea that stimuli that induce expression of Fos in neurons can change the accessibility of regulatory elements bound from Fos has been shown before, but almost all the data are from hippocampal neurons so it is nice to see the different cell type used here. The most interesting part of the study is the identification of the Pdyn enhancer because of the importance of this gene product in the function of striatal neurons. Overall the conclusions appear to be well supported by the data.

Weaknesses:
The timing and the location of the accessibility changes are meaningfully different from other similar studies, which should be discussed. The authors provide good data for the function of a single enhancer near Pdyn, but could contextualize this with respect to other regulatory elements nearby.

Reviewer #2 (Public Review):

In this manuscript, the authors characterize activity-dependent transcriptional and epigenetic changes at two different time points (1h and 4hrs) after neuronal activation using rat striatal primary cultures. They show that while at 1h post-stimulation mostly a selective set of IEGs are up-regulated, at 4hrs a wider set of genes, identified as late-response genes (LRGs), are upregulated, with distinct functional signatures. By using ATAC-seq, the authors show how chromatin accessibility is mostly spared at 1h post-stimulation, while a prominent set of differentially accessible regions (DARs) could be identified at 4hrs post-stimulation, enriched in motifs for TFs upregulated at their initial time-point. These chromatin changes appear to be dependent on the earlier translation of proteins, as they are avoided when neuronal cultures are pre-treated with the protein synthesis inhibitor Anisomycin. Afterwards, the authors characterized a set of regulatory regions of a particular LRG, Pdyn, associated with neuropsychiatric disorders, by using CRISRPR to activate or inactivate an enhancer that increases its accessibility at 4hrs post-stimulation, showing that the expression of Pdyn is highly dependent on this regulatory region both, at basal level and for its proper activity-dependent stimulation and that it is enriched in motifs for IEGs upregulated at 1h post-stimulation. Using publicly available data from human GABAergic and glutamatergic neurons similarly stimulated with KCl, the authors show that this enhancer is conserved in humans and that it is mostly modified in GABAergic neurons in response to neuronal stimulation, but not in glutamatergic neurons. Finally, the authors suggest that the regulatory role of the Pdyn enhancer they focus on it might be cell-type specific, as single-nuclei ATAC-seq data generated in rat Nucleus Accumbens (NAc) shows that its coaccessibility score together with Pdyn promoter is more prominent in Drd1- and Grm8-MSNs.

Among the major strengths of the article, there is the generation of neuronal RNA-seq and ATAC-seq data in a model system, rat striatal neuronal cells, that hasn't been so broadly characterized as other more common ones such as mouse hippocampal neuronal cells and the functional characterization of an enhancer of the Pdyn gene that might be of interest for translational applications in which alterations of this gene might be occurring in neurological disorders.

On the other hand, the manuscript presents several weaknesses to consider. First of all, at a conceptual level, most of the findings related to the induction of particular transcriptional programs upon neuronal activation the changes in chromatin state, and the need for protein translation for proper induction of LRGs have been broadly characterized previously in the literature (Tyssowski et al., Neuron, 2018; Ibarra et al., Mol. Syst. Biol., 2022; and also reviewed by Yap and Greenberg, Neuron, 2018). In addition, it is not so obvious why to focus on Pdyn gene regulatory regions among the thousands of genes upregulated and with modified chromatin landscape after neuronal activation. The authors highlight three particular traits of this gene as the reason to choose it, but those traits are probably shared by most of the genes that are part of the LRGs set.

At the methodological level, some attention should be put into the timings chosen for generating the data. The authors claim that these time points (1h and 4hrs) identify the first (i.e IEGs) and second (i.e LRGs) waves of transcription. However, at 4hrs the highest over-expressed genes are still IEGs, as shown in the volcano plots of Figure 1B and 1C, showing a high overlap with up-regulated genes found at 1h (Figure 1D). This might suggest that the 4hrs time point is somewhere in between the first and second wave of transcription, probably missing some of the still-to-be-induced LRGs of the latest one.

Finally, while only prosed as a suggestion, the assumption that from the data generated in this article, we can envision a mechanism by which AP-1 family of transcription factors interacts with the SWI/SNF chromatin remodeling complex is going too far, as no evidence is provided implicated SWI/SNF in the data presented in the manuscript.

Reviewer #3 (Public Review):

This work contributes to the literature characterizing early and late waves of transcription and associated chromatin remodeling following neuronal depolarization, here in cultured embryonic striatum. While they find IEG transcription 1h after depolarization, they find chromatin remodeling is slower (opening at the 4h time point). This may be due to chromatin at IEG regulatory regions already being open (in embryonic striatum), although previous work has found remodeling occurring at the 1h time point (in adult dentate gyrus). The authors next show that the chromatin remodeling that occurs at the late (4h) stage is largely in putative regulatory regions of the genome (rather than gene bodies), and is dependent on translation, which validates and extends the prior literature. The authors then transition from genome-wide basic neuroscience to focus on a specific gene of interest, prodynorphin (Pdyn), and a putative enhancer they identify from their chromatin analysis. They target CRISPR-activating and -inhibiting complexes to the putative enhancer and demonstrate that accessibility of this locus is necessary and sufficient for Pdyn transcription. They then show that at least one PDYN enhancer is conserved from rodents to humans, and is only activity-regulated in human GABAergic but not glutamatergic neurons. Finally, the authors generate snATAC-seq and show Pdyn gene and enhancer activity are also cell-type-specific in the rat striatum. The Pdyn work in particular is thorough and novel.

Strengths:
This work integrates multiple cutting-edge methods (multiple forms of genome-wide sequencing, combining new and published data across species, applying new forms of bioinformatic analysis, and targeted epigenome editing) to repeatedly and convincingly demonstrate these waves of chromatin remodeling and transcription. The figures and visual representations of data in particular set a new standard for the field. Although several findings within this paper are not novel, this paper ties previous findings all together in one place and goes on to show potential relevance for neuropsychiatric disorders beyond basic cellular neuroscience. The conclusions are mostly supported by the data.

Results and conclusions that would benefit from clarification/extension.
1. Throughout the paper, the authors emphasize a "temporal decoupling" of transcriptional and chromatin response to depolarization, based on a lack of significant chromatin changes at 1h, despite IEG transcription. However, previous publications show significant chromatin remodeling at 1h (e.g. Su et al., NN 2017 in adult dentate gyrus) or 2h (Kim et al., Nature 2010; Malik et al., NN 2014 in cultured embryonic cortical neurons). The discussion briefly mentions this contrast, but it remains difficult to conclude decisively whether there is temporal decoupling when such decoupling is not found consistently. If one is to make broad conclusions about basic neural chromatin response to depolarization, it would be ideal to know under which conditions there is temporal decoupling, or if this is a region-specific phenomenon.

2. The UMAP analysis is a novel way to probe transcription factor enrichment, but it's unclear what this is actually showing. The authors sought to ask whether "DARs could be separated based on transcription factor motifs in these regions." However, the motifs present in any genomic stretch are fixed based on genomic sequence, so it seems like this analysis might be asking whether certain motifs are more likely to be physically clustered together in the genome, in activity-regulated regions (rather than certain transcription factors acting in concert, as is implied in discussion). While still potentially interesting, this analysis does not seem to give much additional insight into activity-dependent chromatin remodeling beyond the motif enrichment analysis already performed. Nevertheless, to draw stronger conclusions, it would be necessary to compare clustering to a random set of genomic regions of the same length/size to interpret the clustering here. It would also be useful to know whether the ISL1 motif is also enriched in ubiquitously accessible genomic regions in the striatum (and not just DARs).

3. The authors identify late-response gene enhancers by 3 criteria. However, only Pdyn was highlighted thereafter. How many putative DARs met these three criteria in striatum? Only Pdyn?