Egr1 is a sex-dependent regulator of neuronal chromatin, structural plasticity, and behaviour

Domain: biology
Published: 2025-12-13
Editorial outcome: major_revision

Abstract
Sex differences are found in brain structure and function across species, and across brain disorders in humans. A major source of brain sex differences is differential secretion of steroid hormones from the gonads across the lifespan. Specifically, ovarian hormones oestrogens and progesterone are known to dynamically change structure and function of the adult female brain, having a major impact on psychiatric risk. However, due to limited molecular studies in female rodents, very little is still known about molecular drivers of female-specific brain and behavioural plasticity. Here we show that ventral hippocampal (vHIP) overexpression of Egr1, an oestrous cycle-dependent transcription factor, induces sex-dependent changes in vHIP neuronal chromatin, gene expression, and structural plasticity, along with female-specific effects on vHIP-dependent behaviours. Importantly, Egr1 overexpression and knockdown partially mimic the vHIP chromatin state associated with the high and low-oestrogenic phase of the oestrous cycle, respectively. We demonstrate that Egr1 directs neuronal chromatin opening across the sexes, although with limited genomic overlap. Our study not only reveals a sex-dependent chromatin regulator in the brain, but also provides functional evidence that this sex-dependent gene regulation drives structural and behavioural plasticity, informing sex-based treatments for brain disorders.

Introduction
Sex differences are found in brain structure and function across species, including mice and humans, and have been linked to reproductive and non-reproductive behaviours1. Studies have also reported a significant gender and sex bias in symptoms and prevalence across brain disorders in humans2,3. However, a better understanding of the sex-related factors and mechanisms shaping the healthy and diseased brain is needed to inform novel, sex-based treatments.

Sex-dependent characteristics of the brain are dynamically shaped throughout life, as a result of the actions of gonadal hormones, sex chromosomes, and the environment1,4. Brain sexual differentiation is initiated by prenatal testosterone exposure in males, but gonadal hormones continue to play a major role in shaping the brain across the lifespan. Specifically, during the reproductive period in females, cycling ovarian hormones, oestrogens and progesterone, play a critical role in brain plasticity relevant to various behaviours including mating, feeding5,6, learning7,8, reward processing9,10, and anxiety- and depression-related behaviours11,12. In addition, ovarian hormone shifts are associated with increased risk for neuropsychiatric conditions such as anxiety and depression disorders in women11,13. Thus, revealing molecular mechanisms and drivers underlying female-specific brain plasticity is of crucial importance for understanding sex-biased brain physiology and disease risk.

Historically, molecular studies in neuroscience have focused on the male brain2,12,14,15, and our knowledge of sex-specific molecular mechanisms in the brain is therefore limited. It has been assumed that a specific group of transcription factors, known as immediate early gene (IEG) products, drive chromatin16 and synaptic17 plasticity across sexes, yet the molecular basis of sex differences in synaptic and behavioural plasticity remains obscure. Here, we leveraged previous findings from a physiological mouse model, which indicated that Egr1 is an oestrous cycle-dependent IEG and a candidate chromatin regulator in neurons of the ventral hippocampus (vHIP), driving synaptic and behavioural changes across the oestrous cycle in females18 (Fig. 1a). We focused on the vHIP because of its critical role in emotion regulation and anxiety-related behaviour in mice19.

Specifically, during the high-oestrogenic, proestrus phase of the cycle, we found increased vHIP chromatin accessibility surrounding Egr1 binding sites (Fig. 1a), coupled with increased Egr1 gene expression, increased vHIP dendritic spine density, and reduced anxiety indices, compared to the low-oestrogenic, dioestrus stage18. These findings suggested a model in which rising oestradiol induces neuronal Egr1 expression in the vHIP, after which Egr1 directs chromatin accessibility and transcription to drive oestrous cycle-dependent synaptic and behavioural changes. However, functional evidence validating this model is currently lacking.

Here we perform overexpression and knockdown of Egr1 in vHIP neurons in both sexes and provide functional evidence that Egr1 targets neuronal chromatin sex-dependently, with consequences for gene expression, structural plasticity, and anxiety- and depression-related behaviour. Since Egr1 manipulation mimics changes associated with physiological changes in ovarian hormones, our findings provide the foundation for sex-based treatments for female-biased disorders, strongly influenced by ovarian hormone shifts11, such as anxiety and depression disorders.

Results
Egr1 overexpression in vHIP neurons induces proestrus-like behaviour in female mice
To better understand the physiological, cycle-dependent functions of Egr1, here we first determined Egr1 expression in the vHIP across all phases of the mouse oestrous cycle (Fig. 1a-b). The rodent oestrous cycle is 4-5 days long, comprising four stages (proestrus, oestrus, metestrus, dioestrus), with cyclical oestrogen and progesterone levels (Fig. 1a). While Egr1 shows a complex cyclical expression pattern across the oestrous cycle (Fig. 1b, Supplementary Data 1), its initial rise coincides with the rise in oestrogen in proestrus18, increased expression of its target gene Ppp1r1b (Supplementary Figure 1, Supplementary Data 1) and reduced anxiety-related behaviour, as evidenced by increased time spent in the open arms of the elevated plus maze (EPM, Fig. 1c, Supplementary Data 1).

We further wanted to provide evidence that oestradiol levels directly regulate vHIP Egr1 expression and behaviour across the oestrous cycle (Fig. 1d–f). To this end, we used “cyclical” treatment with oestradiol benzoate (EB; 1 μg s.c. every 4 days) in ovarian hormone-depleted, ovariectomized (OVX) mice for 6 weeks (Fig. 1d) mimicking the physiological rise in oestradiol occurring every 4 days in cycling females20 (Fig. 1a). While removal of ovaries in young adult mice (at 8 weeks) led to reduced vHIP Egr1 mRNA levels (Fig. 1e) and increased anxiety-related behaviour in the open field (Fig. 1f) compared to age-matched, high-estrogenic (proestrus) cycling mice, the cyclical oestrogen replacement restored proestrus-like levels of Egr1 expression (Fig. 1e, Supplementary Data 1) and partially rescued anxiety-related behaviour (Fig. 1f, Supplementary Data 1) in OVX mice. These results confirm the role of cyclical oestradiol in regulating vHIP Egr1 levels and behaviour. Together, these findings imply that oestradiol-induced Egr1 expression and Egr1’s downstream transcriptional activity may drive behavioural changes across the cycle.

To determine whether increasing vHIP Egr1 expression by itself is sufficient to reproduce the proestrus-like behavioural state in the absence of ovarian hormones, we overexpressed Egr1 in vHIP neurons of OVX females (Fig. 1g, Supplementary Fig. 2). In this case, ovariectomy was performed pre-puberty to ensure that females used in these experiments never experienced ovarian hormone cycling (see Methods). These OVX females also show higher anxiety indices compared to high-oestrogenic proestrus females (Supplementary Fig. 3, Supplementary Data 1). Importantly, OVX females lack both fluctuations in ovarian hormones and in vHIP Egr1 levels otherwise present in intact females (Fig. 1a, b), thereby allowing us to isolate the independent effects of this previously identified sex-specific factor. While female gonadectomy precluded a direct comparison between the sexes (see Methods), we included age-matched males for a comparison in all experiments. Strikingly, OVX female mice overexpressing Egr1 in vHIP neurons exhibited reduced anxiety- and depression-related behaviours including increased centre entries in the open field, increased time in the open arms of the EPM, and reduced time spent immobile in the forced swim test, relative to eGFP-overexpressing controls (eGFP, Fig. 1h, Supplementary Data 1). By contrast, none of these behaviours were affected in males following Egr1 overexpression (Fig. 1i, Supplementary Data 1). Further, Egr1 overexpression did not affect overall locomotor activity in either sex (Supplementary Fig. 4, Supplementary Data 1), indicating that its effect is specific to anxiety- and depression-related behavioural indices, inducing proestrus-like behavioural state18 in OVX females with no effect in intact males.

Egr1 drives neuronal gene expression in a sex-dependent manner
With Egr1’s well-defined role as an IEG and a transcription factor21, we reasoned its sex-biased effects on behaviour could arise due to its influence on vHIP neuronal gene expression. vHIP neurons were of special interest because they are known to drive behaviours such as approach-avoidance in the EPM19. Thus, we performed the same AAV-mediated Egr1 overexpression experiment in male and OVX female mice, followed by gene expression analysis (RNA-seq) on purified neuronal (NeuN+) nuclei isolated from the targeted vHIP (Fig. 2a). Egr1 overexpression markedly altered neuronal gene expression in both sexes, with 1209 differentially expressed genes (DEGs) found in females and 1680 DEGs in males (Padj < 0.05, Fig. 2b, Supplementary Data 2). As expected, Egr1 was one such DEG and exhibited an AAV-mediated ~13-fold increase in expression in both females and males (Supplementary Fig. 5a, Supplementary Data 2). Whereas ovariectomy, again, prohibited a direct comparison between the sexes (see Methods), we performed overlaps of Egr1’s transcriptional effects in males with those in OVX females to further isolate female-biased transcriptional mechanisms potentially underlying Egr1’s behavioural effects. This analysis revealed that the majority of DEGs were sex-dependent, with only 37.3% (785/2104) of DEGs being shared between the sexes (Fig. 2c). Gene set enrichment analysis (GSEA) revealed male-biased effects on the expression of genes related to neuropeptide signalling and neurotransmitter transport (Fig. 2d). By contrast, female-biased enrichment includes genes related to excitatory synapses, thyroid hormone signalling, and, importantly, behavioural fear response (Fig. 2d, Supplementary Data 3), consistent with the behavioural effect we observed in females but not males (Fig. 1h, i).

Next, we performed a Gene Ontology (GO) enrichment analysis focused on the 424 DEGs identified exclusively in females (Fig. 2c), and found that these genes were enriched for numerous terms related to synaptic function (Fig. 2e), which implicates Egr1 in sex-dependent regulation of a set of synapse-related genes in females. While effects of Egr1 overexpression likely result from its cumulative impact on gene expression, we identified several anxiety-relevant example genes with expression patterns that are concordant with Egr1’s behavioural effects (Fig. 2f). Atp1a2, for instance, is a gene in the Behavioral Fear Response term enriched specifically in females (Fig. 2d). Atp1a2 deficient-mice exhibit enhanced fear and anxiety behaviours22, and we found that Atp1a2 expression in vHIP neurons is upregulated in females and unchanged in males following Egr1 overexpression (Fig. 2f). Another example is Ppp3r1, encoding calcineurin B, which is a member of multiple synapse-related terms that were enriched in female-specific DEGs (Fig. 2e). Systemic inhibition of calcineurin was found to increase anxiety- and depression-related behaviours in male mice23, and its expression is upregulated in females and unchanged in males after Egr1 overexpression (Fig. 2f). Interestingly, for both of those genes, males seem to show a higher basal level of expression compared to OVX females, while this sex bias is reduced following Egr1 overexpression. Finally, Npsr1, encoding the neuropeptide S receptor 1, was the only gene that exhibited the opposite pattern of expression changes across sex after Egr1 overexpression (Supplementary Fig. 5b). Npsr1 has been implicated in altered anxiety indices in mice24 and humans25, and, notably, its expression is upregulated in females and downregulated in males (Fig. 2f, Supplementary Fig. 5b) following Egr1 overexpression.

Egr1 opens neuronal chromatin in a sex-dependent manner
Given our previous work implicating Egr1 in chromatin regulation within vHIP neurons over the oestrous cycle in females18,26, we wondered whether altered chromatin organization is a feature of Egr1’s sex-dependent gene regulatory functions. To this end, following vHIP-targeted Egr1 overexpression, we performed chromatin accessibility (ATAC-seq) analysis on vHIP neuronal (NeuN+) nuclei purified from male and OVX female mice (Fig. 3a). Across all samples, we identified a total of 222,253 accessible chromatin regions in vHIP neurons. Of these, 16.0% (35,491) in females and 14.0% (31,105) in males were identified as differentially accessible regions (DARs; Padj < 0.05), indicating that Egr1 overexpression led to substantial reorganization of neuronal chromatin (Fig. 3b, Supplementary Data 4). Importantly, the vast majority of DARs in both females (30,565 or 86.1%) and males (26,546 or 85.3%) gained accessibility following Egr1 overexpression. This finding is in line with our hypothesis that Egr1 is an “opener” of chromatin18, and is consistent with previous findings of another IEG, cFos, inducing chromatin accessibility following neuronal activity16. Thus, we focused our subsequent analyses on the regions that gained accessibility following Egr1 overexpression (Egr1 gained-open regions).

Following the same approach we used for RNA-seq data, we performed an overlap of Egr1 gained-open regions in OVX females and males. This analysis revealed that only 56.4% (20,612/36,499) of gained-open regions are shared between females and males, while 27.3% (9953/36,499) were uniquely identified in females and 16.3% (5934/36,499) were uniquely identified in males (Fig. 3b).

We next asked whether these regions gain accessibility indirectly, downstream of Egr1’s transcriptional effects, or alternatively, if they may be targeted by Egr1 specifically, as a function of their DNA sequence. Strikingly, motif analysis revealed, in both sexes, that more than 70% of regions that gain accessibility after Egr1 overexpression contain the Egr1 motif (Fig. 3c, e.g., Supplementary Fig. 6, Supplementary Data 5), providing evidence that Egr1 mediates opening of neuronal chromatin targeted to specific motif-containing regions. While this was previously demonstrated for cFos in the male brain16, our study reveals an IEG product that acts as a sex-dependent molecular driver of chromatin accessibility in neurons.

To explore the functional role of Egr1-induced gained-open regions containing Egr1 binding sites, we overlapped their sequences with previously published ChIP-seq data on histone modifications in hippocampal neurons27. Importantly, we found relative enrichment of H3K4me1 and H3K27ac, marking active enhancers28, and depletion of H3K4me3, marking gene promoters29 (Fig. 3d), indicating that neuronal enhancers are the primary substrate for targeted opening of chromatin by Egr1. Similar to the total Egr1-induced gained-open regions, an overlap between males and females of gained-open regions with Egr1 binding sites revealed that 46.2% (12,911/27,932) are unique to one sex (Fig. 3e). Pathway analysis on genes annotated to these sex-dependent regions show male-biased enrichment for pathways related to BDNF signalling (Fig. 3e), in line with male-biased expression changes related to Neuropeptide Signaling (Fig. 2d), and female-biased enrichment for pathways related to synapse regulation and, importantly, anxiety-related behaviour (Fig. 3e), consistent with the female transcriptional enrichment for Behavioral Fear Response genes (Fig. 2d) and the effects of Egr1 on anxiety-related behaviour in females (Fig. 1h). Together, this indicates that Egr1 initiates chromatin opening near different gene sets with distinct functions in males and females.

Egr1-induced chromatin opening mediates gene expression changes
By overlapping ATAC-seq and RNA-seq data from the Egr1 overexpression experiments, we next explored whether Egr1’s chromatin effects were associated with effects on gene expression. A general overlap revealed that the majority of DEGs in both sexes (Fig. 2b), 62.7% (758/1209) in females and 61.6% (1035/1680) in males, are associated with at least one chromatin region that changes accessibility following Egr1 overexpression (Supplementary Fig. 7). This indicated that, in general, Egr1-induced chromatin changes mediated gene expression changes. However, we wondered which subset of these Egr1-induced DEGs were associated specifically with opening of chromatin targeted to the Egr1 motif. In females, 51.2% (619/1209) of DEGs are associated with gained-open chromatin containing the Egr1 motif (Fig. 3f). Of these DEGs, 40.7% (252/619) are female-specific and enriched for terms and pathways related to synaptic function and cell adhesion (Fig. 3f). An illustrative example demonstrating Egr1’s sex-dependent effects on gene regulation is Nrn1 (Fig. 3g), a neurotrophic factor that has been shown to regulate hippocampal dendritic spine density30. Egr1 overexpression induces chromatin opening within the Egr1 motif-containing region downstream of the Nrn1 locus in females only, which is further associated with a female-specific increase in gene expression (Fig. 3g).

In males, we observed a similar involvement of Egr1’s targeted chromatin opening in gene regulation, where 52.1% of all DEGs (875/1680) are associated with Egr1 motif-containing gained-open chromatin (Fig. 3h). Of these, 58.1% (508/875) are male-specific DEGs and are enriched for terms and pathways related to synapses, cation channels, and neurotransmitter receptors (Fig. 3h). An example is Grin2a, encoding a subunit of the NMDA glutamate receptor which plays an important role in synaptic plasticity31. An Egr1 motif-containing intronic region becomes more accessible after Egr1 overexpression in males but not females, which corresponds to male-specific upregulation of Grin2a expression (Fig. 3i). These results demonstrate that Egr1 overexpression mediates opening of neuronal chromatin surrounding Egr1 binding sites in both sexes, with distinct genomic regions targeted in males and females associated with sex-dependent effects on gene expression.

Egr1 overexpression partially recapitulates the proestrus-associated transcriptional program in vHIP neurons
Having demonstrated that Egr1 overexpression in vHIP neurons reproduces a proestrus-like behavioural phenotype in OVX females (Fig. 1h), we next examined whether Egr1 overexpression similarly recapitulates molecular phenotypes associated with physiological ovarian hormone cycling in vHIP neurons. Leveraging our previously generated RNA-seq data, which measured gene expression in vHIP neurons over the oestrous cycle18, we overlapped the identified cycle-related DEGs with DEGs resulting from Egr1 overexpression in OVX females. This analysis revealed that 18.6% (32/172) of oestrous cycle DEGs similarly undergo expression changes in OVX females overexpressing Egr1 (Fig. 4a, Supplementary Data 2c). As expected, this is a small fraction of total DEGs induced by viral overexpression of Egr1 (32/3023 or 1.1%), yet it is consistent with the 24.1% of oestrous cycle DEGs with chromatin changes that we previously found to have Egr1 binding motifs18. Two example genes which illustrate the role of Egr1-driven gene expression in oestrous cycle-dependent behavioural phenotypes are Arid1a and Gatad2b. Arid1a encodes a subunit of the SWI/SNF chromatin remodelling complex, perturbations of which have been associated with anxiety-related phenotypes32. Importantly, the up-regulation of Arid1a expression observed during the high-estrogenic, proestrus phase in cycling females is mimicked by Egr1 overexpression in OVX females (Fig. 4b). Gatad2b encodes a subunit of the NuRD chromatin remodelling complex, whose levels are associated with anxiety-related behaviour33, and is also concordantly upregulated in proestrus females and OVX females overexpressing Egr1 (Fig. 4b).

Egr1 overexpression partially recapitulates proestrus-associated chromatin changes in vHIP neurons
We next performed overlaps of chromatin accessibility (ATAC-seq data) between the oestrous cycle18 and Egr1 overexpression experiments. Interestingly, compared to gene expression, we see a much greater overlap between the ATAC-seq datasets, where 71.2% (6847/9614) of genes with differential chromatin peaks across the oestrous cycle also show changes in chromatin in OVX females after Egr1 overexpression (Supplementary Fig. 8). Since we specifically implicated Egr1 in mediating the proestrus behavioural and transcriptional state18, we next overlapped Egr1 gained-open regions with proestrus gained-open regions and found 69.7% (4646/6670) of genes with proestrus gained-open chromatin also exhibit gained-open chromatin after Egr1 overexpression (Fig. 4c). Due to the extensive overlapping effects of Egr1 overexpression and proestrus on chromatin accessibility, we wondered whether chromatin effects are more reflective of Egr1’s physiological effects across the cycle than its effects on gene expression, which likely rely on the presence of additional transcription factors that may be absent in the vHIP neurons of OVX females. We therefore explored the overlap between Egr1’s chromatin effects and oestrous cycle DEGs, finding that 55.2% (95/172) of oestrous cycle DEGs have Egr1 gained-open chromatin after Egr1 overexpression in OVX females (Fig. 4d). This larger overlap was expected, in part, due to the large number of genes associated with gained accessibility after Egr1 overexpression (11,315). Importantly, however, we could now further explore the functional significance of genes that simultaneously exhibit Egr1-mediated chromatin regulation and oestrous cycle-mediated transcriptional changes. Of these oestrous cycle DEGs that overlap with Egr1-induced chromatin changes, 68.4% (65/95) also have chromatin changes across the oestrous cycle, indicating the importance of chromatin accessibility in their physiological regulation (Supplementary Fig. 9). Further, consistent with Egr1’s role in both activating34 and repressing35 transcription, we found Egr1 gained-open regions overlap with genes both up- and downregulated in proestrus compared to dioestrus females (Fig. 4d). Overlapping genes upregulated in proestrus (down in dioestrus) are enriched for terms and pathways related to chromatin and transcription regulation, while overlapping genes downregulated in proestrus (up in dioestrus) are enriched for terms related to synaptic function and ion channels (Fig. 4e). These results indicate that Egr1 mediates opening of chromatin near its binding motifs near genes that play a critical role in transcriptional regulation and neuronal function within physiologically cycling female mice.

Egr1 overexpression recapitulates proestrus-associated changes in structural plasticity in vHIP neurons
Notably, we found enrichment of genes relevant to synaptic function among female-specific Egr1-induced DEGs (Fig. 2e) and in the overlap between Egr1’s chromatin effects and oestrous cycle-dependent DEGs (Fig. 4e). Thus, we hypothesized that Egr1’s gene regulatory effects may drive oestrous cycle-dependent changes in the density of dendritic spines, the sites of active synapses. As an example, Gria3 is a putative Egr1 target gene that encodes a subunit of the AMPA glutamate receptor, which is implicated in the regulation of dendritic spines36,37. We previously found that vHIP Gria3 exhibited increased expression and a more open chromatin state during the proestrus (compared to dioestrus) phase of the oestrous cycle18. Notably, Egr1 overexpression mimics proestrus in OVX females, leading to an increase in chromatin accessibility at a region upstream of Gria3 (Fig. 4f), containing an Egr1 binding site, and increased Gria3 gene expression (Fig. 4f). Interestingly, basal Gria3 levels are similar between OVX females and males, and thus Egr1 overexpression leads to similar effects on Gria3 chromatin and gene expression in both sexes (Fig. 4f).

Following up on this regulation of genes involved in synapse regulation, we next assessed whether Egr1 overexpression alters dendritic spine density in vHIP neurons. After delivering Egr1 or control AAVs to the vHIP of OVX females and males, we performed Golgi-Cox staining and imaged dendrites of ventral CA1 pyramidal neurons. We found that Egr1 overexpression increases vHIP dendritic spine density in OVX females (Fig. 4g, Supplementary Data 1), again mimicking the proestrus phase of the cycle18. Egr1 overexpression also increased spine density in males, albeit with a smaller effect size (d=0.4468, compared to d=1.532 in females; Supplementary Data 1).

Egr1 knockdown reduces expression and chromatin accessibility of oestrogen response genes
While overexpression of Egr1 recapitulated a number of oestrous cycle-dependent phenotypes in OVX females, we wondered whether reducing Egr1 expression in intact proestrus females could induce the opposite effect and disrupt the proestrus-driven transcriptional program in vHIP neurons. To this end, we injected a neuron-specific AAV expressing either an Egr1-targeting shRNA or a scramble control into the vHIP of proestrus females and males. We then purified vHIP neuronal (NeuN+) nuclei and performed RNA-seq and ATAC-seq (Fig. 5a). Analysis of the RNA-seq data revealed that Egr1 knockdown led to 91 DEGs (Padj < 0.05; Fig. 5b, Supplementary Data 6), virtually all of which were downregulated in the knockdown group (97.8%; 89/91). This reduced number of DEGs compared to Egr1 overexpression can be explained, in part, by the comparatively smaller effect of knockdown on Egr1 expression (~1.6 fold reduction, Fig. 5b; compared to ~13-fold increase via overexpression, Supplementary Fig. 5a). Importantly, however, the DEGs we did identify were enriched for oestrogen response genes (Fig. 5c, Supplementary Data 6c), including Sh3bp5, which has been previously identified as part of a MAPK-related oestrogen-induced transcriptional program38, and Rara, encoding the retinoic acid receptor alpha which has been shown to cooperate with oestrogen receptors to mediate oestrogen-induced transcriptional regulation39 (Fig. 5d). Further, while proestrus females and males exhibit similar baseline levels of Egr1 expression (Fig. 5b), consistent with our previous findings18, our use of intact females here allowed us to incorporate sex as a factor in our statistical model (see Methods) to directly investigate whether Egr1 knockdown exhibits any sex-biased effects on gene expression. We therefore performed GSEA on ranked gene lists from the analysis of group (i.e., shRNA vs. scramble in both sexes) and group-by-sex interaction effects on gene expression. This revealed that Egr1 knockdown altered expression of genes related to calcium signalling and synapses, with sex-biased effects on the expression of proteins synthesis- and dendrite membrane-related gene sets (Fig. 5e, Supplementary Data 7).

Similar to the gene expression results, analysis of the ATAC-seq data revealed a modest number of DARs (301, Padj < 0.05; Fig. 5f, Supplementary Data 8a). Egr1 knockdown exclusively reduced chromatin accessibility at these DARs (Fig. 5f), and genes annotated to these closed regions were again enriched for oestrogen response genes (Fig. 5g, Supplementary Data 8c). Remarkably, motif analysis of these closed regions revealed that nearly 80% of them contain the Egr1 motif (Fig. 5h, Supplementary Data 9). This result resembles motif enrichment we observed with chromatin opening following Egr1 overexpression (Fig. 3c) and implies that Egr1 expression is required for both the induction and maintenance of accessibility at Egr1 motifs. To more definitively link Egr1 genomic binding to its chromatin regulatory functions, we assessed Egr1 binding activity in regions of interest by leveraging previously published Egr1 ChIP-seq data from the male cortex40. For this analysis, we focused on regions of chromatin that are putatively regulated by Egr1 based on: i) gained accessibility in the overexpression experiment (Supplementary Fig. 10a); ii) lost accessibility in the knockdown experiment (Supplementary Fig. 10b); and iii) gained accessibility in proestrus compared to dioestrus18 (Supplementary Fig. 10c). In all cases, we found that these regions are highly enriched for Egr1 binding in vivo (Supplementary Fig. 10). Further, we again found that Egr1 motif-containing regions were enriched for enhancer marks (Fig. 5i), consistent with our observations from the overexpression experiment (Fig. 3d), along with relatively higher enrichment for the promoter mark H3K4me3 (Fig. 5i).

Since the previous publication on Egr1 binding in the male cortex reported that ~44% of binding sites are at promoters40, we wondered whether the observed enhancer enrichment here was due to a general inaccessibility of cortical Egr1-bound promoters in the ventral hippocampus, or whether they are simply not differential between groups. To answer this question, we overlapped Egr1-bound promoters identified in the cortical ChIP-seq analysis with either all unique ATAC-seq peaks (Supplementary Fig. 11a) or unique Egr1 vs. eGFP DARs (Supplementary Fig. 11b). This analysis demonstrated that while the majority of cortical promoters bound by Egr1 are accessible in vHIP neurons, very few are differentially accessible between the Egr1 and eGFP conditions. This is consistent with our observation that the gained accessibility surrounding Egr1 motifs that we observed following Egr1 overexpression is targeted to neuronal enhancers, rather than promoters.

Taken together, our Egr1 overexpression and knockdown results indicate that Egr1 targets and maintains the accessibility of neuronal enhancers, driving sex-dependent transcriptional programs related to neuronal function and, importantly, the neuronal response to oestrogen.

Egr1 knockdown partially reverses the proestrus chromatin state in females
We next considered the extent to which Egr1 knockdown in proestrus females recapitulates a dioestrus-like chromatin state. In addition to the 301 group DARs we identified (Fig. 5f), we identified regions with female-biased chromatin changes by taking a broader sample of group-by-sex interaction regions (p < 0.05, fold-change > 1.5, Supplementary Data 8b) and filtering them further using divisive hierarchical clustering (Fig. 6a). This analysis revealed 5 total clusters, with the first two clusters containing 864 total regions (287 and 577 regions in Cluster 1 and 2, respectively) that lose accessibility specifically in females following Egr1 knockdown. Importantly, these regions are annotated to genes enriched for synapse related gene ontology (GO) terms (Fig. 6b), further implicating Egr1 in sex-dependent regulation of synaptic genes. Taking the 1165 total regions changed in females (301 sex-invariant, 864 female-specific), we found that nearly half of the genes annotated to these regions (46.4%, 540/1077) gain accessibility in the proestrus phase and after overexpression of Egr1 in OVX females (Fig. 6c). Enrichment analysis on these overlapping genes revealed that Egr1 knockdown partially recapitulates a dioestrus-like chromatin state by reversing the Egr1-mediated gained-accessibility of neuronal chromatin around a subset of genes related to synapse regulation, dendrites, and oestrogen response. Two illustrative examples are Kcnv1 (Supplementary Data 8b), encoding a voltage-gated potassium channel subunit, and Chd4 (Supplementary Data 8a), encoding a chromatin remodelling enzyme (Fig. 6d). Both genes are associated with a chromatin region that contains an Egr1 binding motif and gains accessibility in OVX females following Egr1 overexpression while losing accessibility in proestrus females following Egr1 knockdown (Fig. 6d). Interestingly, Kcnv1 is downregulated and Chd4 is upregulated in proestrus compared to dioestrus females18, which furthers supporting the notion that Egr1 mediates downregulation of ion channels and upregulation of transcriptional regulators during the proestrus phase of the oestrous cycle (Fig. 4e). Together, our data indicate that Egr1 is required to maintain the proestrus chromatin state in vHIP neurons of cycling females, further supporting the role of this IEG in cyclical chromatin control relevant to female-specific neuronal and behavioural plasticity.

Methods
Animals
For this study, we used seven separate cohorts of animals. The first cohort was used for behavioural and gene expression analysis of intact females across the oestrous cycle (Fig. 1b, c, Supplementary Fig. 1) and was comprised of n=48 C57BL/6J female mice that arrived at 5 weeks of age from Jackson Laboratory. The second cohort was used to compare the behaviour of gonadally intact females and ovariectomized (OVX) females that underwent ovariectomy at 4 weeks of age at the Jackson Laboratory (Supplementary Fig. 3) and included n=34 intact and n=12 OVX C57BL/6J female mice. OVX mice were allowed to recover for one week before arriving, together with the intact females, at 5 weeks of age. After habituating for two weeks (5–7 weeks old), intact females from these cohorts underwent oestrous cycle tracking daily in the morning (between 9 AM and 11 AM) for two weeks (7-9 weeks) in order to establish a predictive cycling pattern for each female animal and to ensure that only females with regular cycles were included in the study (see Oestrous cycle monitoring). Vaginal smears were also taken from OVX females for 4 days to verify that ovariectomy was successful. This was followed by behavioural testing at 9–11 weeks of age (see Behavioural testing). Animals were sacrificed at 11 weeks of age, brains were extracted, and bilateral ventral hippocampi were dissected on ice then flash frozen in liquid nitrogen.

The third cohort of mice was used for cyclical oestrogen treatment experiments (Fig. 1d–f) and was comprised of n=32 C57BL/6J female mice that arrived at 6 weeks of age from Jackson Laboratory. Following two weeks of habituation, a subset of mice from this cohort underwent either ovariectomy (n=16) or sham surgery (n=8) at 8 weeks of age in-house at Fordham University (see Ovariectomy surgery). After recovering from surgery for 1 week, OVX mice were cyclically treated with either corn oil vehicle (n=8) or 1 μg oestradiol benzoate (EB) in corn oil (n=8) via subcutaneous injection once every four days for a 6-week treatment period. As above, vaginal smears were taken from OVX females to confirm ovariectomy, while the oestrous cycle of intact control (n=8) and sham females was tracked briefly to ensure proper cyclicity. Mice from this cohort were tested in the open field (see Behavioural testing) at 14–15 weeks of age and sacrificed at 15 weeks of age. Brains were extracted, and bilateral ventral hippocampi were dissected and flash frozen in liquid nitrogen. For the analysis in this cohort, proestrus mice were emerged from sham and control females as no effect of sham surgery of relevance to this study was identified (Supplementary Fig. 12). Intact mice that were not in the proestrus stage, as confirmed by vaginal smear cytology, at the time of behavioural testing or sacrifice were excluded from the relevant analyses.

The following three cohorts (Cohorts 4-6) were used in the Egr1 overexpression experiments (Figs. 1g–i, 2–4, and Supplementary Fig. 2, 4–11) and included OVX C57BL/6J females alongside age-matched males. These cohorts were used for behaviour (n=24/sex), molecular analyses (n=20/sex), and Golgi staining (n=10/sex), respectively. Females underwent ovariectomy at the Jackson Laboratory at 4 weeks of age, just prior to the onset of puberty, to ensure that females used in these experiments never experienced ovarian hormone cycling and do not retain a “cellular memory” of oestrous cycle-related processes. Females recovered for 1 week after the surgery at the Jackson Laboratory, and arrived at Fordham University at 5 weeks of age, together with age-matched males. The final, seventh cohort was used in the Egr1 knockdown experiments (Figs. 5, 6, and Supplementary Fig. 10) and included intact C57BL/6J females and age-matched males (n=6/sex) that arrived at Fordham University at 5 weeks of age. All animals in Cohorts 4–7 were allowed to habituate for two weeks before undergoing stereotaxic surgery (see Stereotaxic surgery) at 7 weeks of age and were allowed to recover for ~3 (overexpression) or 4 (knockdown) weeks to ensure stable transgene expression. During recovery, 4 days of vaginal smears were obtained from OVX females to confirm ovariectomy. The oestrous cycles of intact females used in the knockdown experiments were tracked during the final two weeks of recovery to ensure that all females were sacrificed during the proestrus phase. In the behaviour Egr1 overexpression cohort, animals underwent behavioural testing from 10 to 11 weeks of age before being sacrificed at 11 weeks of age, at which time the whole brain was removed and preserved for cryosectioning (see Immunohistochemistry) so that we could verify viral targeting in every animal that underwent behavioural testing. For the molecular analysis Egr1 overexpression and knockdown cohorts, animals were sacrificed at 10–11 weeks and bilateral hippocampi were dissected on ice and flash frozen in liquid nitrogen. Analysis of RNA-seq data in these cohorts was used to confirm overexpression or knockdown of Egr1. For the Golgi staining Egr1 overexpression cohort, animals were sacrificed at 10 weeks, and the whole brain was removed then underwent Golgi-Cox staining (see Golgi-cox staining for analysis of dendritic spines).

All animals used in this study were housed in same-sex cages (n=3-5/cage) and were kept on a 12:12 h light:dark cycle with ad libitum access to food and water. The temperature of the room is maintained at 21°C, and the humidity ranges from 30 to 70%. All mouse tissue was generated from mice euthanized by cervical dislocation followed by rapid decapitation and dissection of either whole brains or ventral hippocampal tissue. All frozen tissues were stored at −80°C before further processing. All animal procedures were approved by the Institutional Animal Care and Use Committee at Fordham University.

Oestrous cycle monitoring
Oestrous cycle tracking was performed using vaginal smear cytology, a well-established method, as described previously in refs. 18,62,63. Smears were collected by filling a disposable transfer pipette with 100 μl of distilled water, gently placing the tip of the pipette at the vaginal opening, and collecting cells via lavage. The cell-containing water was then applied to a microscope slide and allowed to dry at room temperature for two hours. Once dried, slides were stained with 0.1% crystal violet in distilled water, washed, and then allowed to dry prior to examination with light microscopy. Oestrous cycle stage can be determined by the relative quantities of nucleated epithelial cells, cornified epithelial cells, and leukocytes18. The proestrus phase is characterized by mostly nucleated epithelial cells, while the oestrus phase is characterized by mostly cornified epithelial cells. Metestrus and dioestrus have both forms of epithelial cells as well as leukocytes, and dioestrus exhibits a higher proportion of leukocytes compared to metestrus. After determining the oestrous cycle stage of each female animal daily for two weeks, or three cycles, cycling patterns can be established and stage predictions can be made for the grouping of animals for behavioural tests and molecular analyses. Grouping predictions were confirmed by obtaining vaginal smears of animals following behavioural testing or sacrificing. We previously validated that our predictions correspond to the expected ovarian hormone content in both serum and hippocampal tissue for proestrus (high-oestrogen, low-progesterone) and dioestrus (low-oestrogen, high-progesterone) mice18. Animals with irregular cycles were excluded from behavioural and molecular analyses.

Ovariectomy surgery
In the third cohort of mice (see Animals), ovariectomy was performed at 8 weeks of age in house20. Anaesthesia was achieved with isoflurane using an induction chamber and maintained with a continuous flow of isoflurane (1.5–2%). Aseptic technique was utilized throughout the procedure, which involved a bilateral skin incision followed by excising the exposed ovary at the tip of the uterine horn on each side. Sham surgeries involved identical incisions, as well as manipulation and exposure of the ovaries, but lacked the excision, leaving the ovaries intact. Mice were closely monitored for 3–4 days following surgery to ensure adequate recovery, were administered carprofen for 2 days, and were given Nutra-Gel (Bioserv, S4798) food packs to limit the activity required to obtain food during this recovery period.

Candidate gene expression
To analyse the gene expression of candidate genes, RNA was isolated from the ventral hippocampus with Qiagen’s Allprep DNA/RNA Mini Kit. Reverse transcription was then performed with Invitrogen’s SuperScript III First-Strand Synthesis kit, and qPCR was performed with Applied Biosystems’ FAST SYBR Green and the QuantStudio 3 PCR machine. Relative Egr1 and Ppp1r1b transcript levels were determined using the 2-ΔΔCt method with Cyclophilin A (Ppia) as the endogenous reference gene. The following primer sequences were used in this analysis: Egr1 (forward: 5′- AGCGAACAACCCTATGAGCAC-3′, reverse: 5′-GGATAACTCGTCTCCACCATCG-3′); Ppp1r1b (forward: 5′- GGACGAAGAAGAAGACAGCCA-3′, reverse: 5′-CACTTGGTCCTCAGAGTTTCC-3′); Ppia (forward: 5′-GAGCTGTTTGCAGACAAAGTTC-3′, reverse: 5′-CCCTGGCACATGAATCCTGG-3′).

Behavioural testing
Animals underwent behavioural testing from 9 to 11 weeks of age (Cohorts 1, 2 & 4) or at 14–15 weeks of age (Cohort 3; see Animals). For all behavioural tests, the movement of animals was tracked with a camera and AnyMaze© tracking software. For the open field test, animals were placed in the corner of a 40×40x35 cm open field arena (Stoelting Co.). The animal’s movement was tracked for 10 min, and the total distance travelled by the animal, the time spent in the centre versus the periphery of the maze, and the number of entries into the centre were recorded. For the elevated plus maze test, animals began the test by being placed in the centre of the raised (40 cm) plus-shaped platform (two 35x5 cm arms; Stoelting Co.). The movement of the animal was tracked for 5 min, and the total distance travelled by the animal, as well as the time spent in the open arms versus the closed arms was recorded. For the forced swim test, animals were placed in a 2 L glass beaker with 1 L of lukewarm water. Movement was tracked for 2 minutes and the time spent immobile was recorded.

Adeno-associated viruses
Recombinant adeno-associated viruses (AAVs) were used to overexpress or knock down Egr1 in ventral hippocampal neurons. The AAV9 serotype was selected due to its suitability for gene transduction in brain tissue64. The two overexpression AAVs used in these experiments were an experimental AAV and a control AAV; the experimental AAV contains the Egr1 transgene, an IRES element, and eGFP, with the IRES element ensuring coproduction of Egr1 and eGFP from a single AAV, while the control AAV contains only eGFP. The knockdown AAVs contain either an shRNA targeting Egr1 (experimental AAV) or a scrambled shRNA (control AAV), with each coexpressing eGFP. All AAVs utilize the neuron-specific hSYN promoter65 and contain a WPRE enhancer used to increase the expression of the respective transgenes, with the experimental overexpression AAV containing a smaller iteration of this enhancer (WPRE3) due to space limitations, which retains a high degree of enhancer activity66. All viruses were purchased from Vector Biolabs (overexpression: AAV-258146, eGFP control: 7076; knockdown: shAAV-258146, scramble control: 7045) and were provided in PBS solution with 5% glycerol at a titre of ~1013 genome copies/ml.

Stereotaxic surgery
Animals were anesthetized prior to surgery using isoflurane delivered to an anesthesia induction chamber and kept anesthetized during surgery with a continuous flow of 1.5–2% isoflurane. Either control or experimental AAV solution (overexpression: n=12/group/sex for behaviour, n=6/group/sex for RNA-seq, n=4/group/sex for ATAC-seq, n=5/group/sex for Golgi staining; knockdown: n=3/group/sex for RNA-seq and ATAC-seq) was delivered to the injection site through a glass pipette (15 μm diameter) fixed to a Nanoject III© microinjector pump. The coordinates (relative to bregma) used for ventral hippocampal injections were: A/P: −2.95, M/L: +/− 2.85 (bilateral injections), D/V: −3.8, −3.9. 200 nL was delivered (10 pulses of 20 nL with 15 seconds in between) at D/V −3.9, then again at D/V −3.8 totalling 400 nL of injection/side. Three (overexpression) or four (knockdown) weeks were allowed to pass to permit stable expression levels of the viral transgenes before behavioural testing was conducted or before the animals were sacrificed for molecular analyses or Golgi staining. In order to verify proper viral delivery into the ventral hippocampus of the animals that underwent behavioural testing, immunostaining of brain sections was performed (see Immunohistochemistry). Animals were excluded if there was no viral expression present or if there was evidence of off-target expression or tissue damage (this occurred in n=4 females and n=2 males, or 12.5% of animals tested). For molecular analyses, we were able to confirm efficient Egr1 overexpression or knockdown in ventral hippocampal neurons using Egr1 transcript levels (RNA-seq) and chromatin accessibility data (ATAC-seq).

Immunohistochemistry
Fresh dissected brains were washed with ice-cold 0.1 M PBS and fixed in 4% PFA in 0.1 M PBS at 4°C for 24 h. After fixation, brains were rinsed in cold 0.1 M PBS and underwent sucrose preservation, which involved placing the brains in solutions containing 15% then 30% sucrose dissolved in 0.1 M PBS at 4°C for 24 h and 48 h, respectively. Brains were then frozen in dry ice-cooled hexane and stored at −80°C until sectioning. Cryosectioning was performed by embedding brains in optimal cutting temperature compound (OCT) and cutting serial sections on a rotary cryostat (Leica CM1950, Leica Biosystems GmBH). 20 μm coronal sections containing the ventral hippocampus were collected on SuperFrost Ultra Plus slides (Fisher Scientific) and stored at −80°C prior to immunostaining. Immunostaining involved rehydrating slides in 0.1 M PBS for 30 min at room temperature. After rehydration, blocking buffer (5% BSA and 0.4% Triton X-100 in 0.1 M PBS) was applied for 1 hour at room temperature. Slides were then washed with PBS-T (1% BSA and 0.4% Triton X-100 in 0.1 M PBS), and the Egr1 primary antibody was applied (Rabbit anti-Egr1 mAb Cell Signal #4153, 1:500 in PBS-T) for 24 h at 4°C. Following primary antibody incubation, slides were washed with PBS-T, then the secondary antibody was applied (Donkey anti-Rabbit IgG pAb Invitrogen A-21207, 1:250 in PBS-T) for 2 hours in the dark at room temperature. Following secondary antibody incubation, slides were washed with PBS-T, then DAPI was applied (1:1000 in PBS) for 5 minutes in the dark at room temperature. Following DAPI incubation, slides were washed with 0.1 M PBS and mounted with Mowiol 4-88 and a coverslip. For all staining sessions, sections with no primary antibody (PBS-T applied instead of primary antibody) and sections with no secondary antibody (PBS-T applied instead of secondary antibody) were included to ensure that the observed fluorescent signal corresponded to Egr1 detection rather than autofluorescence of the tissue or non-specific binding of the secondary antibody.

Fluorescence-activated nuclei sorting (FANS)
For overexpression and knockdown experiments, all molecular analyses were performed on neuronal nuclei, which were purified using FANS, as described previously in refs. 67,68. Briefly, for the overexpression ATAC-seq experiment, 4 animals per group (eGFP, Egr1) per sex were included (total n=16). For the overexpression nucRNA-seq experiment, 6 animals per group (eGFP, Egr1) per sex were included (total n=24). The overexpression ATAC-seq experiment used bilateral ventral hippocampi from a single animal for each biological replicate (n=4/group/sex), while for overexpression nucRNA-seq, bilateral ventral hippocampi were pooled from two animals for each biological replicate (n=3 replicates/group/sex). For the knockdown ATAC-seq and nucRNA-seq experiments, 3 animals per group (scrmbl, shRNA) per sex were included (total n=12) with each biological replicate comprised of bilateral ventral hippocampi from a single animal. We performed FANS in batches of 3–4 biological replicates per session and ensured that the groups were evenly dispersed across batches for each experiment. Brain tissue was dissociated in lysis buffer using a tissue douncer, and nuclei were isolated from tissue lysates by ultracentrifugation through a sucrose gradient67. Pelleted nuclei were resuspended in DPBS and incubated for 45 min with the mouse monoclonal antibody against neuronal nuclear marker NeuN conjugated to AlexaFluor 488 (1:1000, MAB377X; Millipore, MA; clone A60). Before sorting, we added DAPI (1:1000) to the incubation mixture and filtered all samples through a 35 μm cell strainer. FANS was performed on a FACSAria instrument (BD Sciences), and data were collected and analysed using BD FACSDiva v8.0.1 software at the Albert Einstein College of Medicine Flow Cytometry Core Facility (Supplementary Fig. 13). In addition to a sample containing NeuN-AlexaFluor 488 and DAPI stain, three controls were used to setup the gates for sorting: DAPI only; IgG1 isotype control-AlexaFluor 488 (1:1000, Mouse monoclonal IgG1-k, FCMAB310A4; Millipore, MA; Clone MOPC-21) and DAPI; and NeuN-AlexaFluor 488 only. We set up the protocol to remove debris, ensure single nuclear sorting (using DAPI), and select the NeuN+ (neuronal) and NeuN- (non-neuronal) nuclei populations (Supplementary Fig. 13). For the ATAC-seq experiments, we collected 50,000 NeuN+ nuclei per biological replicate in BSA-precoated tubes filled with 200 μL of DPBS. For the nucRNA-seq experiments, we collected 50,000–250,000 nuclei directly into Trizol LS reagent (Thermo Fisher Scientific, 10296-010) to protect RNA from degradation. The nuclei used for knockdown nucRNA-seq were the remaining nuclei from the same tissue used for ATAC-seq.

nucRNA-seq
Following sorting of nuclei, chloroform was added to the sample, and the aqueous phase was recovered. nucRNA was then isolated and purified using the RNeasy Micro kit (Qiagen). The quality of nucRNA was assessed using the Fragment Analyzer (Agilent) or Bioanalyzer (Agilent), with each sample having RQN > 7.1 (7.9 average) or RIN > 6.2 (7.5 average) (Supplementary Fig. 14a, Supplementary Data 10). nucRNA quantity was determined using the Qubit RNA High-Sensitivity assay (Thermo Fisher Scientific, Q32852). All purified nucRNA (3–26 ng/sample) was used to construct cDNA sequencing libraries using the KAPA RNA HyperPrep Kit with RiboErase (KK8560, KAPA Biosystems), following the manufacturer’s instructions. rRNA was depleted from nucRNA samples using hybridization of DNA oligonucleotides complementary to rRNA followed by RNase H and DNase treatment. The efficiency of rRNA depletion was confirmed using the Bioanalyzer (Agilent) and with qRT-PCR amplification of the 28S rRNA transcript before and after RiboErase treatment using the following primers: Forward, 5’- CCCATATCCGCAGCAGGTC-3’; Reverse, 5’- CCAGCCCTTAGAGCCAATCC-3’. rRNA-depleted nucRNA was then fragmented at 94°C for 5 minutes in the presence of Mg2+, followed by first and second strand cDNA synthesis and A-tailing. After ligation of KAPA dual-indexed adapters (KK8722), each library was amplified using 13–15 PCR cycles. Following a bead-based clean-up, the length of the libraries (368 bp, on average, Supplementary Data 10) and their quality was assessed using the Bioanalyzer High-Sensitivity DNA Assay (Supplementary Fig. 14b). The libraries were quantified with the Qubit HS DNA kit (Life Technologies, Q32851) and by qPCR (KAPA Biosystems, KK4873) prior to sequencing. 150 bp, paired-end sequencing was performed in one lane of an S1 flow cell on the NovaSeq 6000 instrument at the New York Genome Center, yielding 52–99 million reads per sample (Supplementary Data 10).

RNA-seq analysis
Sequences were adapter-trimmed and aligned to the mouse reference genome (mm39) with the GENCODE M32 gtf (overexpression) or M36 gtf (knockdown) file using StarAligner69. The gene counts produced by Star were collated into a matrix. Differential gene expression analysis was then performed using DESeq270 at significance level Padj < 0.05. For the overexpression experiment, ovariectomy in females precluded a direct comparison across sex, and we therefore analysed the effect of group (Egr1 vs. eGFP) separately in males and ovariectomized females using the Wald statistical test. For the knockdown experiment, use of intact females and males allowed us to include sex as a factor in our analysis; we therefore analysed the effect of group (shRNA vs. Scramble) in males and proestrus females together, with sex included as a factor in the model (~Group + Sex; Wald test), and also performed a group-by-sex interaction analysis (~Group + Sex + Group:Sex; likelihood ratio test). Counts for the knockdown experiment were normalized using the endogenous reference gene Ppia. Volcano plots were created using EnhancedVolcano (https://github.com/kevinblighe/EnhancedVolcano). Enrichment analyses were performed using FUMA71 at significance level Padj < 0.05. Enrichment analysis was also performed using GSEA72 with ranked gene lists and results were plotted using the EnrichmentMap73 and AutoAnnotate apps in Cytoscape74.

ATAC-seq
We performed ATAC-seq according to Buenrostro et al.75 and our previously published protocol on sorted neuronal nuclei18,68, with some modifications76. Following FANS, nuclei were pelleted and resuspended in 50 μL of the transposase reaction mix including 25 μL of the 2xTD reaction buffer, 5 μL of transposase enzyme (Illumina Tagment DNA TDE1 kit, 20034197). We also included additional components: 16.5 μl 0.1 M PBS, 0.5 μl 1% digitonin, and 0.5 μl 10% Tween-20, which were previously shown to enhance transposition efficiency. Transposition occurred at 37°C for 30 minutes and transposed DNA was purified using the MinElute PCR Purification Kit (Qiagen, 28004). Indexing libraries using Nextera i5 and i7 indexed amplification primers (Illumina XT index kit, 15055290) occurred alongside library amplification with NEBNext High-Fidelity PCR Master Mix (New England Biolabs, M0541S). The PCR reaction was carried out with the following conditions: 1 cycle of 72°C for 5 min and 98°C for 30s; followed by 10 cycles of 98°C for 10s, 63°C for 30s, and 72°C for 1 min. Amplified libraries were then purified using the MinElute PCR Purification Kit. Library quality was then assessed using the Bioanalyzer High-Sensitivity DNA Assay (Supplementary Fig. 14c). The ATAC-seq libraries were quantified by Qubit HS DNA kit and by qPCR prior to sequencing. 150 bp, paired-end sequencing was performed in an S1 flow cell on the NovaSeq 6000 instrument at the New York Genome Center, yielding 40–150 million paired-end reads per sample (Supplementary Data 11).

ATAC-seq analysis
Sequences were adapter-trimmed and aligned to the mouse reference genome (mm39) using BWA-MEM software77. Duplicates and mitochondrial reads were removed, then fragment length distribution plots were generated to confirm that insert sizes corresponded primarily to nucleosome-free, mono- and di-nucleosomal DNA (Supplementary Fig. 14d). Next, peak-calling was performed using MACS278 as previously reported75 and called peaks were filtered using the mm39 blacklist79. We ensured all samples had ≥ 20% of reads in peaks, indicative of high-quality ATAC-seq samples (Supplementary Data 11). Differential accessibility of open chromatin regions was assessed using DiffBind as described previously80. Briefly, reads were counted in consensus peaks (shared by at least two replicates), normalized, then quantitatively compared using DESeq270 with a significance threshold of Padj < 0.05 for the effect of group. The DESeq2 analysis of ATAC-seq data followed the same strategy as the RNA-seq analysis, with overexpression data analysed by evaluating the effect of group separately in males and ovariectomized females. Then, for the knockdown data, we analysed the effect of group together in males and proestrus females (~Group + Sex), along with a group-by-sex interaction (~Group + Sex + Group:Sex) analysis. To identify sex-specific patterns of chromatin accessibility in the knockdown experiment, we applied unbiased clustering to a broad group of DARs as described previously81. Specifically, we clustered group-by-sex interaction DARs (Padj < 0.05 and fold-change > 1.5) using DEGreport (https://lpantano.github.io/DEGreport/) which utilizes the DIANA divisive clustering algorithm82. Annotation of differentially accessible regions (DARs) was performed using ChIPSeeker83 on the TxDb.Mmusculus.UCSC.mm39.refGene annotation, and we identified overlapping peaks using the findOverlapsOfPeaks function of ChIPpeakAnno84. Motif analysis was performed using HOMER85 (hypergeometric test), and enrichment analysis on lists of annotated genes was performed with FUMA71 (hypergeometric test) and EnrichR86 (hypergeometric test) at a significance threshold of Padj < 0.05. Plots of aligned ATAC-seq data were created using SparK87. Heatmaps and profile plots of H3K4me1, H3K27ac, and H3K4me3 enrichment within gained-open (overexpression) or -closed (knockdown) Egr1 binding sites were performed using deepTools88 using the publicly available ChIP-seq data GSE7496427 generated using neuronal (NeuN+) CA1 hippocampal neurons from male mice. The same deepTools analysis was performed for Egr1 ChIP-seq signal enrichment using the publicly available ChIP-seq data GSE10876840 generated using prefrontal cortical tissue from male mice.

Integration of genomics data
Our ATAC-seq and nucRNA-seq data previously generated from ventral hippocampal neurons (GSE114036) of proestrus female, dioestrus female, and male mice18 were integrated with the data used in this study. For the purposes of overlapping the RNA-seq data, we applied the significance threshold used in the previous study18 (Pnominal < 0.05). Plots of aligned ATAC-seq data were created using SparK87.

Golgi-cox staining for analysis of dendritic spines
Whole brains of mice injected with either control or Egr1 AAV (see Adeno-associated viruses, n=5/sex/group) were rinsed with distilled water and underwent the Golgi-Cox staining procedure (Golgi-Cox OptimStain Kit, Hitobiotec Inc. #HTKNS1125) following the manufacturer’s instructions. Briefly, whole brains were submerged in premixed Golgi-Cox impregnation solution and stored in the dark at room temperature for 24 h, after which the solution was replaced and the brains remained submerged for an additional two weeks. Samples were then transferred to a tissue-protectant solution and kept in the dark at 4°C for 12 h, after which the solution was changed and the brains remained submerged for an additional 72 h. Samples were then frozen in dry ice-cooled hexane and stored at −80°C until sectioning. 100 μm sections of OCT-embedded brains were cut using a cryostat (Leica CM1950) and collected on gelatin-coated slides. After drying overnight, slides were stained according to the kit protocol and mounted using DPX (Sigma-Aldrich). Brightfield Z-stacks of dendrites from ventral hippocampal CA1 pyramidal neurons were taken at 60x magnification and analysed using ImageJ (https://imagej.nih.gov/ij/index.html).

Statistics
Statistics were performed using SPSS and R. For the analysis of candidate gene expression and behaviour i) across the oestrous cycle (Fig. 1b, c; Supplementary Fig. 1), ii) in OVX females undergoing cyclical oestradiol or vehicle treatment alongside untreated proestrus females (Fig. 1e, f), and iii) in prepubertally ovariectomized females alongside intact proestrus and dioestrus females (Supplementary Fig. 3), one-way ANOVA was used with Holm’s post hoc test. For behavioural and dendritic spine density analysis of animals from the overexpression experiments, Welch two-sample t-tests were performed separately in males and ovariectomized females, since ovariectomy precluded the analysis across sexes. The effect size for the dendritic spines experiment was reported for each sex as Cohen’s d, calculated as the difference between the group means of Egr1 and eGFP groups divided by the pooled standard deviation. For each of these statistical tests, p < 0.05 was considered significant. Detailed results of all ANOVA and t-tests are available in Supplementary Data 1.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Overall, the authors have done an outstanding job of responding to my previous critiques, as well as those of my fellow Reviewers. The new data & analyses provided nicely support the authors' assertions, and the text revisions have greatly improved the clarity of the manuscript, along with providing more precise language (particularly with respect to the structural morphology experiments) so as to not over interpret their findings. I continue to feel that this manuscript is robust and will be of broad interest to the field. As such, I now support publication of this work at Nature Communications.", "reviewer_2": "The manuscript by Rocks et al describes the effects of overexpressing the transcription factor Erg1 in ventral hippocampus of male and female mice in studies testing the possibility that this IEG regulate chromatin structure and gene expression changes underlying synaptic plasticity and behavioral changes across the female estrous cycle. The original submission received three detailed and extensive reviews that brought up issues of experimental design, the strength of support for some of the conclusions reached and wording. The authors have gone to great lengths to modify the manuscript in response to the review comments including the addition of additional experiments and analyses of publicly available data (response to Reviewer #1), revision of statistical analysis and wording (response to all three reviewers), and changes in interpretation (all three reviewers). The revisions increased support for the conclusions reached and improved language so as to not overinterpret the results. Moreover, specific elements of experimental design were explained and some of the experimental findings were more clearly acknowledged. Overall, it is a strong manuscript, providing novel evidence for consequences of Erg1 overexpression in females which indicate mechanisms through which normal fluctuations in Erg1 expression across the estrous cycle in females may control gene expression.\n\nRegarding response to Reviewer #1 point 3, I agree with the authors in that analysis of overexpression in non-OVX females could give rise to complex and difficult-to-interpret results. In those animals there would be a hugely different hormonal environment that could provide counter balancing influences.\n\nThe authors made a number of wording changes, reflecting review comments (e.g., the changes from ‘synaptic plasticity’ to ‘structural plasticity’ and from sex-specific to sex-dependent is important). Also important is the recognition, and now fuller description, that many of the effects of Erg1 overexpression are similar between males and females – this is now note and more fully considered in the manuscript.\n\nI do think the authors are still selling their results a bit hard at the end of the abstract and discussion but the changes in wording are an improvement.\n\nLine 95: I think “across sex” should be changed to “between the sexes” (also line 116)", "reviewer_3": "In their revised manuscript, the authors have added substantial new analyses and an entirely new Egr1 knockdown experiment as suggested by Reviewer 1. I appreciate that they provided the complete lists of their DEGs, GO terms and DARs for their genomic experiments formatted as Supplementary Tables. However, I still have concerns with the overall study design, interpretation and conclusions. As I stated previously, the Egr1 overexpression experiment does not effectively model Egr1 levels in proestrus. Partial rescue of spine and behavior phenotypes with Egr1 overexpression suggest that additional proestrus-dependent mechanisms beyond Egr1 expression contribute to increased spines and decreased anxiety-related behaviors during proestrus. Egr1 overexpression only captures a fraction of the authors’ published proestrus-specific changes in gene expression and chromatin accessibility. Extended Data 9 shows very minimal overlap between DARs that open with Egr1 overexpression and DARs that change across the estrous cycle. Taken together, these results suggest that the Egr1 overexpression experiments are not modeling the physiological increase in Egr1 expression with proestrus. Overexpression will persistently boost Egr1 expression in all neuronal cell types as is apparent in Extended Data Figure 2. The mild effects of Egr1 knockdown experiments in proestrus additionally demonstrate that the overexpression experiments are a bit of a sledgehammer approach.\n\nI dispute the claim that ovariectomy precludes the inclusion of sex as a variable. Female gonadectomy does not preclude the ability to perform statistical comparisons with males. The authors repeatedly use the term “sex-dependent” and their primary conclusion is that Egr1 acts differently in males and females. Accordingly, the Egr1 overexpression and control comparisons should be made in the males and the ovariectomized females in the same statistical analysis, using a 2 way ANOVA. This should be done throughout the manuscript whenever there are adjacent pairwise comparisons (spines, behavior, genes/DARs). Figure 4 shows that Egr1 overexpression in OVX females boosts spine numbers to similar numbers as in control and OE males. Reviewer 2 makes the same argument in their point 7.\n\nAdditional points:\nIn the Egr1 knockdown experiment, the authors claim that genes and DARs that decrease or close with Egr1 knockdown are enriched for estrogen response genes, but the Figures show minimal enrichment. Figure 5c shows perhaps 5 genes associated with the term “estrogen response early” and Figure 5g indicates between 7.5 and 10 genes located near regions that lose accessibility with Egr1. What are these genes and sites? Are the closing sites near the down-regulated genes? The downregulated genes Sh3bp5 and Rara do not have DARs annotated to them in Supp Table 8a.\n\nKcnv1 is shown in Figure 6, but there is no DAR associated with this gene in Supp Table 8a.\n\nExtended Data 10 should include controls. The authors should plot signal from the published Egr1 ChIP-seq in all groups. For panel a), this includes the ATAC peaks for GFP control-injected males and females. For panel b), this includes ATAC peaks from the scrambled RNA controls and all phases of the estrous cycle.\n\nIn the publication for the Egr1 ChIP-seq data used here, it was reported that ~44% of binding sites are at promoters, while here the authors report that “neuronal enhancers are the primary substrate for targeted opening of chromatin by Egr1” in their overexpression experiments. Are the promoter sites not open in the ventral hippocampus, or are they simply not differential between groups?\n\nThe example ATAC-seq library in Supplementary Figure 2 looks under-transposed. Although a nucleosomal ladder is apparent, very little of the library consists of accessible chromatin, defined by Buenrostro et al (2013) as mono- and di-nucleosomes and subnucleosomal fragments. The authors should generate plots of their fragment distribution to ensure that the reads they aligned are predominantly fragments of accessible chromatin, as in Nature Methods papers PMID: 24097267 and PMID: 28846090.\n\nOverstatements throughout the manuscript are distracting. It is not that Egr1 is “sex-dependent” but that its expression and consequent effects are increased in proestrus. It is likely that many IEGs show sex differences in expression due to sex differences in sex hormones and neural activity. Fos was first reported to show sex differences in the rat brain 20 years ago. PMID: 15842237\n\nAdditional overstatements include the following:\nLine 52. “So far, molecular studies in neuroscience have focused on the male brain. Please omit this sentence. Many if not most modern molecular neuroscience studies include females, in line with SABV requirements from major funding institutions, and there has been extensive work over the past decade on mechanisms of estrogen signaling in the female rodent brain. The cited reference 14 is from 2011.\n\nLines 182-184: “Pathway analysis on genes annotated to these sex-dependent regions show male-biased enrichment for pathways related to BDNF signalling, in line with male-biased expression changes related to Neuropeptide Signaling”.\nBDNF is not a neuropeptide\n\nLines 323-325: “Remarkably, motif analysis of these closed regions revealed that nearly 80% of them contain the Egr1 motif (Figure 5h, Suppl.Table 9)”. This is not remarkable, this is expected. Knocking down a TF should result in the closure of sites that contain that TF’s motif.\n\nLines 378-382: “While earlier studies emphasized epigenetic regulation of IEGs as part of their activity-dependent regulation, evidence for the role of IEGs as direct epigenetic regulators of their target genes is a newer finding that provides a novel mechanistic insight into IEGs’ regulatory role in neuronal adaptation and plasticity.” This is simply false. The study of IEG TFs and their target genes goes back decades. AP-1 (Fos/Jun) heterodimers were extensively investigated going back to the 90s and more recently the Greenberg lab and others have comprehensively characterized the targets of Fos, Npas4, and other IEGs in the brain, even with single-cell resolution.\n\nLines 385-387: “In addition, this is reminiscent of the role of cFos, another IEG, shown in males to direct chromatin opening in a neuronal activity-dependent manner, although whether this occurs in the female brain is not known.” Are the authors suggesting that none of the published work on Fos targets was performed in cells from females?\n\nLines 406-409: “In fact, this cyclical expression in females may also be indicative of Egr1’s role in driving the biological clocks in the body more generally, since Egr1 is also induced in the suprachiasmatic nucleus by light stimulation, and has been implicated in circadian rhythm-driven brain plasticity.” This connection between oestrus and circadian cycles is speculative.\n\nLines 425-428: “Considering its sensitivity to sex hormone levels and sex specificity of its actions, Egr1 therefore represents a master molecular adaptor at the interface of the external and internal environment, with a critical role in sex-dependent neurobiology and psychiatric risk.” The data presented here do not show that changes in Egr1 expression are directly due to sex hormones."}, "author_response": "Response to Reviewer’s Comments\nWe would like to express our continued gratitude to the reviewers for their careful review of this\nmanuscript and general enthusiasm for the study. Here, we address all remaining critiques\nthrough both changes to the manuscript, including 5 new figure panels (2 main, 3 supplemental)\nand ~900 words of additional text, as well as in our point-by-point responses below. We feel that\nthe additional data and text included in this revision further strengthen our conclusions and have\nimproved the overall quality of the study.\nReviewer #1 (Remarks to the Author):\nOverall, the authors have done an outstanding job of responding to my previous critiques, as well\nas those of my fellow Reviewers. The new data & analyses provided nicely support the authors'\nassertions, and the text revisions have greatly improved the clarity of the manuscript, along with\nproviding more precise language (particularly with respect to the structural morphology\nexperiments) so as to not over interpret their findings. I continue to feel that this manuscript is\nrobust and will be of broad interest to the field. As such, I now support publication of this work at\nNature Communications.\nWe thank the reviewer again for their previous suggestions and for their overall support for the\nrevised manuscript.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Rocks et al describes the effects of overexpressing the transcription factor\nErg1 in ventral hippocampus of male and female mice in studies testing the possibility that this\nIEG regulate chromatin structure and gene expression changes underlying synaptic plasticity and\nbehavioral changes across the female estrous cycle. The original submission received three\ndetailed and extensive reviews that brought up issues of experimental design, the strength of\nsupport for some of the conclusions reached and wording. The authors have gone to great lengths\nto modify the manuscript in response to the review comments including the addition of additional\nexperiments and analyses of publicly available data (response to Reviewer #1), revision of\nstatistical analysis and wording (response to all three reviewers), and changes in interpretation\n(all three reviewers). The revisions increased support for the conclusions reached and improved\nlanguage so as to not overinterpret the results. Moreover, specific elements of experimental\ndesign were explained and some of the experimental findings were more clearly acknowledged.\nOverall, it is a strong manuscript, providing novel evidence for consequences of Erg1\noverexpression in females which indicate mechanisms through which normal fluctuations in Erg1\nexpression across the estrous cycle in females may control gene expression.\nWe would like to thank the reviewer for their enthusiastic support for the revised manuscript and\nfor their previous recommendations which were critical toward improving the original draft.\nRegarding response to Reviewer #1 point 3, I agree with the authors in that analysis of\noverexpression in non-OVX females could give rise to complex and difficult-to-interpret results. In\nthose animals there would be a hugely different hormonal environment that could provide counter\nbalancing influences.\nWe thank the reviewer for their support on this point. While it is certainly a complex question, it is\nencouraging that other experts in the field share our view on the appropriate way to analyze and\ninterpret data from this experimental paradigm.\nfrom ‘synaptic plasticity’ to ‘structural plasticity’ and from sex-specific to sex-dependent is\nimportant). Also important is the recognition, and now fuller description, that many of the effects\nof Erg1 overexpression are similar between males and females – this is now note and more fully\nconsidered in the manuscript.\nI do think the authors are still selling their results a bit hard at the end of the abstract and\ndiscussion but the changes in wording are an improvement.\nLine 95: I think “across sex” should be changed to “between the sexes” (also line 116)\nWe again want to thank the reviewer for their appreciation for the revised manuscript, and we fully\nagree that the changes in wording and the framing of the results has improved the study\nsignificantly. To continue improving the manuscript, we have altered the end of the discussion\nand the lines mentioned above to address the reviewer’s remaining concerns:\n(Lines 464-467):\nSince ovarian hormone shifts are an established but underexplored psychiatric risk factor\nin humans3,11,12,56–58, our study represents an important contribution toward the discovery\nof sex-based drug targets and treatments for neuropsychiatric disorders.\n(Lines 108-109):\nWhile female gonadectomy precluded a direct comparison between the sexes (see\nMethods), we included age-matched males for a comparison in all experiments.\n(Lines 128-131):\nWhereas ovariectomy, again, prohibited a direct comparison between the sexes (see\nMethods), we performed overlaps of Egr1’s transcriptional effects in males with those in\nOVX females to further isolate female-biased transcriptional mechanisms potentially\nunderlying Egr1’s behavioural effects.\nReviewer #3 (Remarks to the Author):\nIn their revised manuscript, the authors have added substantial new analyses and an entirely new\nEgr1 knockdown experiment as suggested by Reviewer 1. I appreciate that they provided the\ncomplete lists of their DEGs, GO terms and DARs for their genomic experiments formatted as\nSupplementary Tables.\nWe thank the reviewer for their recognition of the efforts dedicated toward revising the original\nmanuscript and the appreciation for the availability of the data that we generated for the reader.\nWe also thank the reviewer for their continued efforts in carefully reviewing our work and\narticulating their critiques of the study. We want to emphasize that we understand the reviewer’s\nconcerns and have done our best to address the remaining critiques through additional\nexperiments, analyses, and changes to the text, as well as with point-by-point responses below.\nWe feel that these additional changes have further improved the quality of the manuscript.\nHowever, I still have concerns with the overall study design, interpretation and conclusions. As I\nstated previously, the Egr1 overexpression experiment does not effectively model Egr1 levels in\nproestrus. Partial rescue of spine and behavior phenotypes with Egr1 overexpression suggest\nthat additional proestrus-dependent mechanisms beyond Egr1 expression contribute to increased\nspines and decreased anxiety-related behaviors during proestrus. Egr1 overexpression only\n2\ncaptures a fraction of the authors’ published proestrus-specific changes in gene expression and\nchromatin accessibility.\nWe agree with the reviewer that the proestrus behavioral and vHIP molecular state is complex,\nand that while our data indicate that Egr1 is an important driver of this state, we agree that other\nmechanisms are almost certainly involved as well, and may function either independently or\nsynergistically with Egr1. This is why we mention, throughout the text, that Egr1 manipulations\nelicit partial, rather than total, recapitulations of estrous cycle-dependent states (please see\nbelow). As a note, manipulation experiments typically dissect only one component of the system\nunder study so we do not find this concern so worrisome. We always emphasize that the estrous\ncycle is a complex phenomenon with multiple hormones (not only estradiol!) and underlying\nmechanisms (not only Egr1) certainly being involved.\n(Lines 27-29):\nImportantly, Egr1 overexpression and knockdown partially mimic the vHIP chromatin state\nassociated with the high and low-oestrogenic phase of the oestrous cycle, respectively.\n(Lines 230-231):\nEgr1 overexpression partially recapitulates the proestrus-associated transcriptional\nprogram in vHIP neurons\n(Lines 252-253):\nEgr1 overexpression partially recapitulates proestrus-associated chromatin changes in\nvHIP neurons\n(Line 365):\nEgr1 knockdown partially reverses the proestrus chromatin state in females\n(Lines 377-380):\nEnrichment analysis on these overlapping genes revealed that Egr1 knockdown partially\nrecapitulates a dioestrus-like chromatin state by reversing the Egr1-mediated gained-\naccessibility of neuronal chromatin around a subset of genes related to synapse\nregulation, dendrites, and oestrogen response.\n(Lines 899-900):\nEgr1 overexpression partially recapitulates proestrus-associated changes in gene\nexpression, chromatin, and synaptic plasticity.\n(Lines 430-434):\nIt is also worth noting that, beyond Egr1’s cyclical expression, we previously found a\nsimilar cyclic gene expression pattern for other epigenetic regulators, including multiple\nATP-dependent chromatin remodelling factors, in vHIP neurons across the oestrous\ncycle18. Thus, cycling transcriptional regulators appear to be a distinctive feature of the\noestrous cycle-driven gene regulation18 that is likely to establish “the oestrous rhythm” in\nthe cell.\n(Lines 427-429):\nHowever, the receptor signalling pathway leading to Egr1 induction remains to be\nestablished and, considering the hormonal complexity of the oestrous cycle, multiple\nhormones and receptors may be regulators of cyclical Egr1 levels.\nexpression levels that mimic the estrous cycle will be valuable for modeling Egr1 and other\nimportant factors with the appropriate temporal dynamics. We already developed cyclical\nestrogen regimen and these are now new data added to the manuscript (Fig. 1d-f). As for Egr1,\nwe now make this point explicitly in the discussion:\n(Lines 437-442):\nWhile the long-lasting viral manipulations of Egr1 levels performed here gave important\ninsights into Egr1’s sex-dependent effects on gene regulation and behaviour, the\ndevelopment of new molecular tools that allow for finer temporal control of gene\nexpression will be important for future studies to model the cyclical expression of Egr1 and\nother epigenetic regulators involved in driving the proestrus state.\nExtended Data 9 shows very minimal overlap between DARs that open with Egr1 overexpression\nand DARs that change across the estrous cycle. Taken together, these results suggest that the\nEgr1 overexpression experiments are not modeling the physiological increase in Egr1 expression\nwith proestrus.\nExtended Data 9 (now Supplementary Figure 9) does not show the overlap between DARs across\nthe two datasets mentioned by the reviewer. To clarify, this figure takes the overlap between Egr1\nDARs and estrous cycle DEGs shown in Figure 4d and shows that the majority of these DEGs\nalso undergo chromatin changes across the estrous cycle, indicating that chromatin regulation is\nan important driver of their expression levels in the context of the estrous cycle. This is now\nclarified in the text:\n(Lines 272-274):\nOf these oestrous cycle DEGs that overlap with Egr1 chromatin changes, 68.4% (65/95)\nalso have chromatin changes across the oestrous cycle, indicating the importance of\nchromatin accessibility in their physiological regulation (Suppl. Fig. 9).\nOverexpression will persistently boost Egr1 expression in all neuronal cell types as is apparent in\nExtended Data Figure 2. The mild effects of Egr1 knockdown experiments in proestrus\nadditionally demonstrate that the overexpression experiments are a bit of a sledgehammer\napproach.\nAs with other overexpression methodologies used throughout the neuroscience field, our\noverexpression of Egr1 expectedly resulted in supraphysiological levels of Egr1. This allowed us\nto definitively characterize the behavioral and molecular consequences of raised Egr1 levels\nwithout the additional variability one would expect from attempting to raise levels to those\nbordering physiological. Such approaches are commonly used not only in the context of\noverexpression but also, relevant to this field of study, hormone treatments. For example, in the\nstudy by Su et al (Nat. Neurosci, PMID: 28166220), the researchers employed AAVs to\noverexpress cFos for one week and used this “continuous overexpression” experiment to show\nthat cFos overexpression partially mimics neuronal activity-induced chromatin opening at regions\nwith cFos-binding sites. This is a very similar approach to one we took in our overexpression\nstudy. In addition, we and others (e.g. Gegenhuber et al. Nature 2022 PMID: 35508660 and Rocks\net al Nat Commun, PMID: 35705546) treated mice with 5 µg estradiol benzoate, a dose which\nhas been shown by HPLC/MS to result in supraphysiological levels of serum estradiol (PMID:\n35705546). Such manipulations are particularly warranted in the context of ovariectomized female\nmice, where too low of a dose may be insufficient to overcome lasting adaptations associated\nwith ovariectomy. Our recent strategy of cyclical oestradiol dosing (PMID: 39946826; also used\n4\nhere as described below) overcomes some of these limitations, though an identical strategy for\nmanipulating expression levels of a specific gene do not yet exist, as mentioned above and now\nin the text:\n(Lines 437-442):\nWhile the long-lasting viral manipulations of Egr1 levels performed here gave important\ninsights into Egr1’s sex-dependent effects on gene regulation and behaviour, the\ndevelopment of new molecular tools that allow for finer temporal control of gene\nexpression will be important for future studies to model the cyclical expression of Egr1 and\nother epigenetic regulators involved in driving the proestrus state.\nAs the reviewer notes, the knockdown experiments were a subtler manipulation with expectedly\nmore subtle results and therefore serve to complement the overexpression experiments by\ndemonstrating the importance of Egr1’s effects in the context of physiologically cycling females.\nWe are very glad that we included this additional experiment that provides critical complementary\nsupport to our hypothesis.\nI dispute the claim that ovariectomy precludes the inclusion of sex as a variable. Female\ngonadectomy does not preclude the ability to perform statistical comparisons with males. The\nauthors repeatedly use the term “sex-dependent” and their primary conclusion is that Egr1 acts\ndifferently in males and females. Accordingly, the Egr1 overexpression and control comparisons\nshould be made in the males and the ovariectomized females in the same statistical analysis,\nusing a 2 way ANOVA. This should be done throughout the manuscript whenever there are\nadjacent pairwise comparisons (spines, behavior, genes/DARs). Figure 4 shows that Egr1\noverexpression in OVX females boosts spine numbers to similar numbers as in control and OE\nmales. Reviewer 2 makes the same argument in their point 7.\nWe agree with the general sentiment of the reviewer, as demonstrated by our analysis of the\nknockdown data which included sex as a factor in the clustering analyses, as well as our prior\nwork which analyzes sex as a factor whenever comparisons are made between intact females\nand males (e.g. PMID: 37777968). However, we hope that the reviewer will agree and consider\nfor their future work, too, that “sex” is a complex biological variable that consists of multiple sex-\nrelated variables, among which gonadal hormone status is likely the most important for shaping\nsex-biased neurobiology. In the experiment in which we remove gonads from females, we also\n“strip” sex as a variable from its major component and cannot just use it simply as a “whole” factor\nin the statistical analysis. In addition, OVX females went through the surgical procedure while\nmales did not, which further complicates “sex” as a variable in this case. After consulting with our\nstatistician colleague, who is an author on the study, we have determined that the analysis\ndescribed by the reviewer here is not appropriate for the overexpression experiments. We would\nalso like to note that Reviewers 1 and 2 are satisfied with our revisions and have not raised any\nissues with the analysis in the second round of reviews. In fact, the choice of using OVX animals\nin the case of overexpression experiment was warranted, as agreed by Reviewer 2 in their\ncomment above. With the multifaceted approach that includes the integration of physiological,\npharmacological, and gene manipulation data within the ovarian hormone-depleted and naturally-\ncycling background we provide robust evidence that the effects of Egr1 are indeed sex-\ndependent.\nAdditional points:\nIn the Egr1 knockdown experiment, the authors claim that genes and DARs that decrease or close with Egr1 knockdown are enriched for estrogen response genes, but the Figures show\n5\nminimal enrichment. Figure 5c shows perhaps 5 genes associated with the term “estrogen response early” and Figure 5g indicates between 7.5 and 10 genes located near regions that lose accessibility with Egr1. What are these genes and sites? Are the closing sites near the down-regulated genes? The downregulated genes Sh3bp5 and Rara do not have DARs annotated to them in Supp Table 8a.\nWe would like to clarify that our claim that “genes and DARs that decrease or close with Egr1\nknockdown are enriched for estrogen response genes” is supported by statistical tests, which\nwere significant at p < 0.05. In the case of enrichment analysis, these tests assess whether the\nadj\noverlap between a list of genes (e.g. DEGs or genes annotated to DARs) and an annotated gene\nset (e.g. “Estrogen response early”) exceeds what would be expected by chance. The\ncomponents of these analyses which contribute to statistical significance are the size of each list,\nthe size of the overlap, and the size of the background. Statistically significant gene enrichment\ncan therefore be observed for even overlaps of relatively small magnitude. We have been very\ntransparent with the way we present our data so there is nothing misleading or inaccurate in our\ndata interpretation, as we can indeed see by reviewer’s ability to predict the number of genes\npresent in enriched terms. However, we agree that the particular genes and sites that are enriched\nwould be of interest to the reader, and we therefore now included them in the Supplementary\nTable 6c and Supplementary Table 8c. We also note that not all DEGs have an associated DAR,\nand not all genes annotated to DARs are differentially expressed, as was observed for the\noverexpression experiment.\nKcnv1 is shown in Figure 6, but there is no DAR associated with this gene in Supp Table 8a.\nWe thank the reviewer for pointing this out. The DAR associated with Kcnv1 is listed in Suppl.\nTable 8b, which shows regions identified as having sex-biased chromatin changes based on our\nclustering analysis. We now clarify in the text where the region associated with Kcnv1 can be\nfound:\n(Lines 380-385):\nTwo illustrative examples are Kcnv1 (Suppl. Table 8b), encoding a voltage-gated\npotassium channel subunit, and Chd4 (Suppl. Table 8a), encoding a chromatin\nremodelling enzyme (Fig. 6d). Both genes are associated with a chromatin region that\ncontains an Egr1 binding motif and gains accessibility in OVX females following Egr1\noverexpression while losing accessibility in proestrus females following Egr1 knockdown\n(Fig. 6d).\nExtended Data 10 should include controls. The authors should plot signal from the published Egr1\nChIP-seq in all groups. For panel a), this includes the ATAC peaks for GFP control-injected males\nand females. For panel b), this includes ATAC peaks from the scrambled RNA controls and all\nphases of the estrous cycle.\nThis request indicates a potential misinterpretation of the figure which we now clarify both here\nand in the text. Specifically, Extended Data 10 (Now Supplementary Figure 10) currently shows\nthe Egr1 ChIP-seq signal in the following regions: a) DARs more accessible in the Egr1-\noverexpression group that contain Egr1 binding sites, b) DARs less accessible in the Egr1\nknockdown condition that contain Egr1 binding sites, and c) DARs more accessible in proestrus\nthan diestrus containing Egr1 binding sites. The rationale for this analysis is that in each case,\nthe relevant DARs are enriched for Egr1 binding motifs as determined by motif analysis. We\ntherefore sought to determine whether these motif-containing regions represent regions of actual\nEgr1 binding determined by ChIP-seq. Such motif enrichment was not observed for the\n6\ncorresponding control DARs (of which there are none for the knockdown experiment, for example)\nthat the reviewer is requesting. We have now clarified this in the text:\n(Line 339-346):\nTo more definitively link Egr1 genomic binding to its chromatin regulatory functions, we\nassessed Egr1 binding activity in regions of interest by leveraging previously published\nEgr1 ChIP-seq data from the male cortex40. For this analysis, we focused on regions of\nchromatin that are putatively regulated by Egr1 based on: i) gained accessibility in the\noverexpression experiment (Suppl. Fig. 10a); ii) lost accessibility in the knockdown\nexperiment (Suppl. Fig. 10b); and iii) gained accessibility in proestrus compared to\ndioestrus18 (Suppl. Fig. 10c). In all cases, we found that these regions are highly enriched\nfor Egr1 binding in vivo (Suppl. Fig. 10).\nIn the publication for the Egr1 ChIP-seq data used here, it was reported that ~44% of binding sites\nare at promoters, while here the authors report that “neuronal enhancers are the primary substrate\nfor targeted opening of chromatin by Egr1” in their overexpression experiments. Are the promoter\nsites not open in the ventral hippocampus, or are they simply not differential between groups?\nWe thank the reviewer for pointing this out. To answer this question, we overlapped Egr1-bound\npromoters identified in the cortical ChIP-seq analysis with either all unique ATAC-seq peaks (a,\nleft) or unique Egr1 vs. eGFP DARs (b, right). Below is the Figure that is now included as\nSuppl.Fig. 11:\nWe also included the following text:\n(Line 350-360):\nSince the previous publication on Egr1 binding in the male cortex reported that ~44% of\nbinding sites are at promoters40, we wondered whether the observed enhancer enrichment\nhere was due to a general inaccessibility of cortical Eg1-bound promoters in the ventral\nhippocampus, or whether they are simply not differential between groups. To answer this\nquestion, we overlapped Egr1-bound promoters identified in the cortical ChIP-seq analysis\nwith either all unique ATAC-seq peaks (Suppl Fig. 11a) or unique Egr1 vs. eGFP DARs\n(Suppl. Fig. 11b). This analysis demonstrated that while the majority of cortical promoters\nbound by Egr1 are accessible in vHIP neurons, very few are differentially accessible\nbetween the Egr1 and eGFP conditions. This is consistent with our observation that the\ngained accessibility surrounding Egr1 motifs that we observed following Egr1\noverexpression is targeted to neuronal enhancers, rather than promoters.\nnucleosomal ladder is apparent, very little of the library consists of accessible chromatin, defined\nby Buenrostro et al (2013) as mono- and di-nucleosomes and subnucleosomal fragments. The\nauthors should generate plots of their fragment distribution to ensure that the reads they aligned\nare predominantly fragments of accessible chromatin, as in Nature Methods papers PMID:\n24097267 and PMID: 28846090.\nAs explained in the Nature Protocols ATAC-seq paper (PMID: 35478247), the presence of higher\nmolecular weight fragments in the electrophoretic trace is not necessarily indicative of under-\ntransposition, and the trace we present in Supplementary Figure 2 (now Supplementary Figure\n14) actually resembles the example shown if Fig 3d of that publication which is used to exemplify\nthe clear nucleosomal periodicity representative of a high-quality ATAC-seq library. We have\ngenerated the fragment distribution plot for the same library, which is now included as\nSupplementary Figure 14d. This plot demonstrates this point clearly, as we see that insert sizes\npredominantly correspond to nucleosome-free, mono-, and di-nucleosomal DNA.\nOverstatements throughout the manuscript are distracting. It is not that Egr1 is “sex-dependent”\nbut that its expression and consequent effects are increased in proestrus. It is likely that many\nIEGs show sex differences in expression due to sex differences in sex hormones and neural\nactivity. Fos was first reported to show sex differences in the rat brain 20 years ago. PMID:\n15842237\nWe agree with the reviewer that the difference in Egr1 levels (and in particular fluctuating levels!)\nis one source of sex difference. It is worth mentioning, however, that we previously found\nnumerous examples of proestrus-specific accessibility of Egr1 binding sites despite similar Egr1\nexpression levels in proestrus females and males (PMID: 31253786), which was the first time\nsomeone reported IEG-driven sex differences in chromatin accessibility in the literature. In\naddition, we demonstrate that, for example, an identical manipulation of Egr1 expression in males\nand ovariectomized females has differing effects on behavior, gene expression, and chromatin\norganization. We think it is appropriate to refer to these as “sex-dependent” effects of Egr1. We\nmention that Egr1’s effects for certain outcomes are not female-exclusive in the discussion:\n(Line 451-453):\nThese findings explain why we see extensive chromatin and gene expression changes in\nboth sexes with partial overlaps in molecular and structural plasticity, indicating sex-\ndependent but not female-exclusive effects by Egr1.\nLine 52. “So far, molecular studies in neuroscience have focused on the male brain. Please omit\nthis sentence. Many if not most modern molecular neuroscience studies include females, in line\nwith SABV requirements from major funding institutions, and there has been extensive work over\nthe past decade on mechanisms of estrogen signaling in the female rodent brain. The cited\nreference 14 is from 2011.\nWhile we agree that some progress has been made since the cited reference was published, this\nis still a pervasive problem in neuroscience and we would argue that “many” modern neuroscience\nstudies including females still falls short of the goals of the SABV initiative. It is also worth\nmentioning that while SABV requires researchers to mention inclusion of females in their grants,\nthere is no mechanism requiring researchers to actually follow through on this, and studies\nincluding males only are still published everywhere including in major journals. Take a recent\nneuroepigenetics paper in Science (PMID: 39052786) as an example. Lastly, the consequences\nof females being historically excluded from neuroscience studies has lasting effects that can’t be\ndiscounted simply because more recent studies include females, considering the corpus of\nknowledge is still primarily derived from studies focusing on the male brain. To clarify that this\nstatement concerns primarily the latter point, we have altered it as follows and included three\nmore recent articles that are addressing the issue of female exclusion from the neuroscience\nliterature.\n(Line 52-53):\nHistorically, molecular studies in neuroscience have focused on the male brain2,12,14,15, and\nour knowledge of sex-specific molecular mechanisms in the brain is therefore limited.\nLines 182-184: “Pathway analysis on genes annotated to these sex-dependent regions show\nmale-biased enrichment for pathways related to BDNF signalling, in line with male-biased\nexpression changes related to Neuropeptide Signaling”. BDNF is not a neuropeptide\nThis is a semantic point and while peptides are usually smaller in size than BDNF, experts in the\nfield typically still refer to BDNF as a neuropeptide considering it is a gene product that transmits\nsignals across synapses. Consider, for example, the first line of the BDNF signaling review article\npublished in Cell by Wang, Kavalali, and Monteggia (PMID: 34963057):\n“Brain-derived neurotrophic factor (BDNF) is a neuropeptide that plays numerous important roles\nin synaptic development and plasticity.”\nLines 323-325: “Remarkably, motif analysis of these closed regions revealed that nearly 80% of\nthem contain the Egr1 motif (Figure 5h, Suppl.Table 9)”. This is not remarkable, this is expected.\nKnocking down a TF should result in the closure of sites that contain that TF’s motif.\nWe disagree with the statement that “Knocking down a TF should result in the closure of sites that\ncontain that TF’s motif”. First, we still know very little about pioneering factors, particularly in\nneuroscience. After reviewing the literature, we have concluded that this has only been\ndemonstrated for select pioneering factors in drosophila (e.g. Zelda: PMIDs 26335634) and cell\nculture (e.g. Sox2: PMID 37691488). The knockdown of an immediate early gene transcription\nfactor in neurons of the mammalian brain leading to chromatin closure surrounding its motif was\nonly shown in the manuscript of Su et al which we already cite (PMID: 28166220). They showed\nthat knockdown of cFos attenuated neuronal activity induced-chromatin opening at cFos-binding\n9\nsites. We hope the reviewer can recognize the novelty and the difficulty of our approach in which\nwe performed the knockdown of an IEG in the naturally-cycling hormonal background in which\nthe expression of the studied IEG varies. This is a difficult experiment where we needed to track\nthe estrous cycle phase following surgery and sacrifice the animals in the right phase of the cycle\n(proestrus). Another compensatory mechanisms could have easily interfered with our results and\nit is remarkable to be able to show closure in chromatin after a 50% knock-down of a TF under\nphysiological conditions in neurons of the live brain. In fact, Su et al induced neuronal activation\nvia electroconvulsive stimulation and while this is an outstanding experiment and result, our\napproach was the only viable option within the specific demands of our study, which has not\npreviously been explored in this context.\nLines 378-382: “While earlier studies emphasized epigenetic regulation of IEGs as part of their\nactivity-dependent regulation, evidence for the role of IEGs as direct epigenetic regulators of their\ntarget genes is a newer finding that provides a novel mechanistic insight into IEGs’ regulatory role\nin neuronal adaptation and plasticity.” This is simply false. The study of IEG TFs and their target\ngenes goes back decades. AP-1 (Fos/Jun) heterodimers were extensively investigated going\nback to the 90s and more recently the Greenberg lab and others have comprehensively\ncharacterized the targets of Fos, Npas4, and other IEGs in the brain, even with single-cell\nresolution.\nThe examples given by the reviewer refer to the target genes of TFs encoded by IEGs being\nidentified. We agree that there has been quite a lot of work on this in the field already. However,\nas quoted by the reviewer, we are referring specifically to “the role of IEGs as direct epigenetic\nregulators of their target genes”. To our knowledge, the only other studies to show something\nsimilar are Su et al. Nat Neurosci 2017 (PMID: 28166220) which shows that Fos overexpression\nin males increases accessibility around Fos binding sites, and Sharma et al. Neuron 2019 (PMID:\n30846309), now cited in our manuscript, which shows that cKO of Npas4 prevents activity-\ndependent H3K27ac at Npas4 binding sites. If there are additional examples that we are unaware\nof, we would be happy to include them as references in our manuscript.\nLines 385-387: “In addition, this is reminiscent of the role of cFos, another IEG, shown in males\nto direct chromatin opening in a neuronal activity-dependent manner, although whether this\noccurs in the female brain is not known.” Are the authors suggesting that none of the published\nwork on Fos targets was performed in cells from females?\nWe thank the reviewer for pointing this out, we mistakenly left the appropriate citation from this\nsentence, which is the above-mentioned study by Su et al. (PMID: 28166220), which was\nperformed on dentate gyrus tissue from male mice. We have now included this reference in the\ntext:\n(Line 411-413):\nIn addition, this is reminiscent of the role of cFos, another IEG, shown in males to direct\nchromatin opening in a neuronal activity-dependent manner15, although whether this\noccurs in the female brain is not known.\nAs stated above, other than this study by Su et al., none of the published work on Fos targets that\nwe are aware of describes the ability of Fos to “direct chromatin opening”, and are therefore not\nrelevant to the sentence.\nLines 406-409: “In fact, this cyclical expression in females may also be indicative of Egr1’s role in\n10\ndriving the biological clocks in the body more generally, since Egr1 is also induced in the\nsuprachiasmatic nucleus by light stimulation, and has been implicated in circadian rhythm-driven\nbrain plasticity.” This connection between oestrus and circadian cycles is speculative.\nWe agree that this is a speculative sentence. However, the word “may” in the sentence makes it\nclear that this is an intriguing possibility, rather than a factual claim. Egr1 has been implicated,\nthough, in circadian rhythm-driven brain plasticity, so our discussion is well-placed.\nLines 425-428: “Considering its sensitivity to sex hormone levels and sex specificity of its actions,\nEgr1 therefore represents a master molecular adaptor at the interface of the external and internal\nenvironment, with a critical role in sex-dependent neurobiology and psychiatric risk.” The data\npresented here do not show that changes in Egr1 expression are directly due to sex hormones.\nWe thank the reviewer for this important point. While we have shown that Egr1 expression levels\nfluctuate over the estrous cycle, we did not directly implicate any specific sex hormones in cycle-\ndependent regulation. To address this, we performed an additional experiment in which proestrus\nfemales were compared to 2 groups of ovariectomized females: OVX-EB received 1ug estradiol\nbenzoate (EB) every 4 days for 6 weeks to model cyclical estradiol replacement, and OVX-Veh\nreceived vehicle injections along the same schedule. We found that this cyclical treatment\nrestores proestrus-like levels of Egr1 in the ventral hippocampus, directly linking estradiol levels\nto the regulation of vHIP Egr1 expression. Further, cyclical EB treatment partially rescued OVX-\ninduced deficits in the time spent in the center of the open field, which is consistent with the partial\nbehavioral rescue observed with Egr1 overexpression in OVX females. The newly generated data\ncan be found in Figure 1d-f and the results are described in the text:\n(Line 88-98):\nWe further wanted to provide evidence that oestradiol levels directly regulate vHIP Egr1\nexpression and behaviour across the oestrous cycle (Fig. 1d-f). To this end, we used\n“cyclical” treatment with oestradiol benzoate (EB; 1μg s.c. every 4 days) in ovarian\nhormone-depleted, ovariectomized (OVX) mice for 6 weeks (Fig. 1d) mimicking the\nphysiological rise in oestradiol occurring every 4 days in cycling females20 (Fig. 1a). While\nremoval of ovaries in young adult mice (at 8 weeks) led to reduced vHIP Egr1 mRNA\nlevels (Fig. 1e) and increased anxiety-related behaviour in the open field (Fig. 1f)\ncompared to age-matched, high estrogenic (proestrus) cycling mice, the cyclical\noestrogen replacement restored proestrus-like levels of Egr1 expression (Fig. 1e, Suppl.\nTable 1) and partially rescued anxiety-related behaviour (Fig. 1f, Suppl. Table 1) in OVX\nmice. These results confirm the role of cyclical oestradiol in regulating vHIP Egr1 levels\nand behaviour.\ndata which has further strengthened the conclusions of the manuscript and our overall\nunderstanding of how hormones drive changes in chromatin and gene expression. We hope that\nthe reviewer will appreciate all the effort put into this manuscript, its importance to the field, and\nthe integration of physiological, pharmacological, and gene manipulation data within the ovarian\nhormone-depleted and naturally-cycling background which is setting an example for our field, And\nwe again thank all the reviewers for helping us produce such comprehensive and strong\nmanuscript.\nWe thank Reviewer 3 for their support and acceptance of our previous revision, which aimed to\naddress all of their remaining concerns. Below, we address the one outstanding concern from the\nreviewer.\nReviewer #3 (Remarks to the Author):\nWhile I maintain that reports of sex differences should be supported by inclusion of data from\nmales and females in the same statistical analysis, I have no further comments for the authors.\nThank you for making this point. We maintain our position that the covariate “sex” in a statistical\ntest is misleading when one sex is gonadectomized, and that the knockdown experiment which\nwas performed in intact females and does include a group-by-sex analysis addresses this\nconcern. However, upon reviewing the manuscript we noted that this limitation of the\noverexpression experiment and corresponding strength of the knockdown experiment was not\nclearly addressed in our discussion. We have therefore extended the discussion to address this\npoint more clearly.\nLines 417-431:\n“While ovariectomy allowed us to isolate the effect of ovarian hormones in females, it also\n“stripped” sex as a variable of its major component and precluded using sex as a factor in\nour statistical analysis of the overexpression experiments. However, the knockdown\nexperiment provided a complementary approach in cycling females, allowing for analysis\nacross sex in intact male and female mice. With the multifaceted approach that includes\nthe integration of physiological, pharmacological, and gene manipulation data within the\novarian hormone-depleted (overexpression) and naturally-cycling (knockdown)\nbackground we provide robust evidence that the effects of Egr1 are indeed sex-\ndependent.\nIn fact, our results show that the cyclicity of ovarian hormone levels may provide a critical,\nsex-specific signal for Egr1’s effect on chromatin regulation in females. Egr1’s response\nto ovarian hormones seems to be optimized by oestrogen’s regulation of Egr1 expression,\nas reflected in the cyclical Egr1 expression pattern across the oestrous cycle in the vHIP,\nbut also in other brain regions such as the prefrontal cortex47 or peripheral tissues such\nas uterus48, as well as in the restoration of proestrus-like vHIP Egr1 levels observed when\nwe treated OVX females with cyclical oestradiol regimen.”"}, {"round": 2, "reviewer_comments": {"reviewer_3": "While I maintain that reports of sex differences should be supported by inclusion of data from males and females in the same statistical analysis, I have no further comments for the authors."}, "author_response": ""}]

Decision letter