Sex effects on chromatin accessibility
We previously studied cell-type specific chromatin structure and genetic regulation of the brain regulome as part of the CommonMind Consortium (CMC)15,16. Here, we present an extensive characterization of sex differences in the human brain epigenome from 469 individuals (172 females, 297 males), consisting of 157 SCZ cases, 77 bipolar disorder (BD) cases, and 235 unaffected controls from the CMC dataset (Fig. 1a and Table S1). We performed the assay for transposase-accessible chromatin followed by sequencing (ATAC-seq) to quantify and characterize sex differences in chromatin accessibility in neuronal (NeuN+) and non-neuronal (NeuN-) nuclei isolated by fluorescence-activated nuclear sorting (FANS) from two brain regions, i.e. the anterior cingulate cortex (ACC) and dorsolateral prefrontal cortex (DLPFC). After a comprehensive quality control analysis of ATAC-seq libraries (Methods), the final dataset consisted of a total of 1,393 samples comprising over 54.8 billion unique reads (39.4 million reads per library) (Table S1). Due to the large chromatin accessibility differences between the two cell types (Fig. 1b), we separated neuronal and non-neuronal samples for downstream analysis. A total of 391,420 and 260,431 neuronal and non-neuronal open chromatin regions (OCRs) were then identified, respectively.
We next quantified sex-specific chromatin accessibility in each cell type. For each cell type, we first fit a linear mixed model using dream17,18 to account for covariates, including diagnosis, cell type, brain region, brain bank, as well as other technical and biological factors (Methods). Consequently, we discovered a total of 8,984 and 5,327 differentially accessible OCRs between males and females (sex-specific OCRs) in neuronal and non-neuronal cells, respectively (FDR < 0.05) (Fig. 1c and Table S2). We identified more female-specific OCRs than male-specific OCRs in both neuronal and non-neuronal cells, the majority of which mapped to the sex chromosomes (Fig. 1c, d). In neurons, 6,072 differentially accessible OCRs (68%) were X-linked, 132 (1%) were Y-linked, and 2,780 (31%) were autosomal; while in non-neurons, 4,305 OCRs (81%) were X-linked, 84 (1%) were Y-linked, and 938 (18%) were autosomal. As expected, the analysis of sex differences also revealed that all Y-linked regulatory elements with increased chromatin accessibility were specific to males, while nearly all X-linked regulatory elements with increased chromatin accessibility were specific to females, potentially due to escape from X-chromosome inactivation (XCI)19 (Table S4). By validating our differential chromatin accessibility analysis against another epigenome study of postmortem brain samples from DLPFC region20, we achieved a high concordance (Spearman correlation coefficients (ρ) of 0.877 and 0.869, p-value < 2.2e-16 for both) in neuronal and non-neuronal samples (Fig. 1e).
Following the observed enrichment of female-specific OCRs on the X chromosome, we next investigated sex-specific changes in chromatin accessibility patterns associated with XCI genes. Out of 277 genes known to escape XCI6,19,21–25, a substantial number – 205 genes in neurons and 206 genes in non-neurons (181 genes shared between the two cell types) – appear to be regulated by sex-specific OCRs (Table S3). Notably, XIST, a non-coding RNA essential for the initiation of XCI26, was found to be up-regulated by female-specific OCRs in neurons and non-neurons (Fig. 1f). FIRRE, an X-linked lncRNA that escapes XCI27, also demonstrated up-regulation in both cell types (Fig. 1f). This complex regulation of XCI highlights the sex-specific interplay between chromatin accessibility and gene expression.
Functions of sex-specific chromatin accessibility
To gain insights into the biological pathways regulated by genes associated with sex-specific OCRs, we performed gene set enrichment analysis (GSEA) in each cell type. We identified enrichment in a wide range of biological pathways, including those involved in response to hormones, reproductive processes, metabolism, immune responses, histone modification, and others (Table S4). Specifically, we discovered that genes associated with sex-specific OCRs were involved in synaptic signaling, neuron projection development, and regulation of axonogenesis, which might contribute to the functional sex differences in brain activity (Fig. 2a). Although significantly associated, sex determination, dosage compensation, and regulation of dosage compensation by inactivation of the X chromosome (Table S4) were not ranked among the most enriched pathways. However, we identified X-linked disease terms that were top ranked and significantly associated with female-specific OCRs (Fig. 2b, Table S4). We also identified heparan sulfate (Hs) glycosaminoglycan (GAG) degradation and metabolism in female-specific OCRs, a previously reported sex difference in the hippocampus known to affect neuronal plasticity and brain development28. A set of pathways involved in histone modifications, regulated by female-specific OCRs, has been reported to result in sex-specific gene expression (Table S4). Trimethylation of Histone H3 at lysine-36 (H3K36me3), driven by female-specific OCRs, is also observed to be less abundant on the male X chromosome29 and is known to induce gene silencing and XCI30. Taken together, these results suggest that sex-specific chromatin accessibility plays a critical role in a range of biological pathways, including some that have not previously been linked to sex.
To further explore the importance of sex-specific regulatory mechanisms and their role in disease, we quantified the overlap of sex-specific OCRs with disease risk variants using the LD-score partitioned heritability (LDsc) approach. We note that LDsc does not support analysis on sex chromosomes; therefore, we conducted partitioned heritability analysis using only non-sex chromosomal OCRs. Enrichment of SCZ risk variants was only observed in male-specific neuronal OCRs (Fig. 2c, Table S5). Furthermore, we observed that ADHD, cognitive performance, and reaction time variants were significantly colocalized with male-specific OCRs in neurons. This finding highlights the critical role of sex-specific regulatory mechanisms of neurons in psychiatric disorders and brain functions, specifically in males.
Because transcription factors (TFs) contribute to sex regulating network structures in various human tissues31, we hypothesized that TFs can affect patterns of differential chromatin accessibility by differential binding of TF motifs at sex-specific OCRs. We employed TF motif enrichment analysis32 in sex-specific OCRs to identify the enrichment of known motifs and to discover novel motifs (Methods). We identified a total of 54 TF motifs enriched for male- and female-specific OCRs (Fig. 2d, Table S6), including 39 TFs enriched for female-specific OCRs and 11 TFs enriched for male-specific OCRs in neurons, as well as 7 TFs enriched for female-specific OCRs and 4 TFs enriched for male-specific OCRs in non-neurons. Of these, 2 neuronal TFs (RFX and RFX2) were shared between males and females, while 5 female-specific TFs (CTCF, CTCFL, MAZ, NF1, and ZFP281) were shared between neurons and non-neurons. We also identified 4 TFs, ELF1, ELK1, KLF5, and YY1, which have been reported to exhibit consistent male-specific enrichment in human tissues6. Among these, ELF1, KLF5, and YY1 were enriched in the brain. The impact of sex on the majority of the remaining TFs has not been fully characterized. Overall, these results suggest that a wide range of TFs, some evidently unrelated to sex hormones, play crucial roles in driving sex-specific regulatory programs in the human brain.
Sex-specific enhancer-promoter regulatory landscape
We next sought to determine the concordance between sex-specific transcriptional activity and genome-wide chromatin accessibility. First, to reconstruct functional enhancer-promoter (E-P) pairs responsible for the regulation of sex-specific genes, we applied the activity-by-contact (ABC)34 model, combining our open chromatin data, histone acetylation data35 and 3D interaction frequency maps36. Using separate models for each sex and cell type, we identified 42,119 − 42,229 E-P links (E-PABC) in neurons and 37,772 − 38,261 E-PABC in non-neurons (Table S7). On average, 64% of E-PABC were shared between sex in neurons, with a moderate correlation of ABC scores (ρ = 0.52; Fig. 3a left panel), whereas 82% of E-PABC were shared in non-neurons, with a high correlation score (ρ = 0.79; Fig. 3a right panel). Additionally, when examining these links on autosomal chromosomes versus the X chromosome, we found a significant drop in the correlation between males and females for X-linked genes (Extended Data Fig. 1). This suggests that X-linked gene activity is controlled by sex-specific enhancer activity, aligning with our initial expectations.
Although a majority of enhancers were predicted to interact with a single gene, 38% were linked to two or more genes (Extended Data Fig. 2a). We found that over 70% of the regulated genes were linked to multiple OCRs (OCRABC) (Extended Data Fig. 2b). Of these, only 22% were linked to the nearest gene (Extended Data Fig. 2c). This finding is consistent with our previous analyses36–38, demonstrating the limitations of proximity-based annotation of regulatory elements. Still, the frequency of E-PABC links sharply decreased with distance to the transcription start site (TSS), with 75% of interactions identified within 100 kb, upstream and downstream, of the TSS (Extended Data Fig. 2d).
To evaluate the sex specificity of the observed E-PABC interactions, we compared sex-specific, but not shared, E-PABC pairs identified in neurons (9,447 female and 9,332 male pairs) and non-neurons (3,459 female and 3,998 male pairs). On average, 96% of OCRABC were autosomal and 4% were X-linked in neurons; 90% of OCRABC were autosomal and 10% were X-linked in non-neurons (Fig. 3b, Table S7). Comparative analysis of sex-specific changes between autosomal and X-linked OCRABC revealed substantially higher effect sizes for X-linked OCRABC in both neurons and non-neurons, respectively (Fig. 3c), further confirming that sex-specific effects are predominantly exhibited on the X chromosome on the enhancer level. When compared to adult human brain chromatin states35, X-linked OCRABC regions showed a relative enrichment in polycomb (H3K27me3) repressed chromatin states as opposed to autosomal OCRABC regions (Fig. 3d, Extended Data Fig. 2e). This observation suggests that X-linked OCRABC play a role in sex-specific silencing of gene expression.
Moreover, our analysis revealed sex-specific distal regulatory landscapes of XCI in the human brain. We identified 313 E-PABC regulating 126 genes known to escape XCI (Table S7). For instance, FIRRE is an X-linked lncRNA that escapes XCI and is involved in chromosome topological organization13,27,39. FIRRE harbors a series of putative intronic enhancers demonstrating female-specific chromatin accessibility through differentially regulated E-PABC (Fig. 3e. Intriguingly, our findings indicate that two enhancers within intron 2 of FIRRE are active in male-specific non-neuronal cells, whereas six enhancers spanning introns 2 to 12 are active only in female-specific cells. Notably, there was no observed activity of FIRRE enhancers in male-specific neurons. NHSL2, a reported XCI gene6, is another illustrative example showing that cell-type specific enhancers can regulate sex-specific activity via XCI (Fig. 3e). Taken together, our results indicate that FIRRE plays a role in maintaining enrichment of a repressive histone mark (H3K27me3) to mediate gene silencing on the X chromosome. Differential accessibility of putative FIRRE’s enhancers between neurons and non-neurons further highlights its contribution to cell-type-specific patterns.
Sex-specific CRDs and TRDs associated with schizophrenia
Cis-regulatory domains (CRDs) are physically interacting regulatory elements that contain multiple locally correlated OCRs and play a crucial role in gene regulation contributing to SCZ risk. Using an in-house analytical framework (Fig. 4a)15,40, we showed that 37% of neuronal OCRs were assembled into 6,706 CRDs and 33% of non-neuronal OCRs were assembled into 4,612 CRDs (Extended Data Fig. 3a). Next, we identified 155 neuronal and 48 non-neuronal CRDs that exhibit significant differential accessibility in their mean between males and females (sex-specific CRDs) (Fig. 4b, Table S8). We observed sex-specific OCRs are more likely to be found inside, rather than outside, sex-specific CRDs in both neurons (odds ratio (OR) = 4.39, p-value < 2.2e-16) and non-neurons (OR = 7.63, p-value < 2.2e-16) (Extended Data Fig. 3a). We previously identified CRDs that exhibit significantly differential accessibility between individuals with SCZ and controls (SCZ CRDs)15. Because OCRs located within neuronal CRDs exhibit significant heritability for SCZ (Extended Data Fig. 3b), we compared the relationship among sex-specific OCRs, SCZ OCRs, sex-specific CRDs and SCZ CRDs in neurons. We found a significant correlation between sex-specific OCRs and SCZ OCRs in neurons, evident in a genome-wide analysis (ρ = 0.59, p-value < 2.2e-16, Extended Data Fig. 4a) and also when focusing on a subset of OCRs inside sex-specific CRDs (ρ = 0.58, p-value < 2.2e-16, Fig. 4c, Extended Data Fig. 4b). The chromatin accessibility of OCRs from male-specific CRDs showed a significant correlation with a SCZ case/control comparison (ρ = 0.52, p-value = 6.2e-5, Fig. 4d, Extended Data Fig. 3c). Conversely, a markedly lower correlation of OCRs from female-specific CRDs (ρ = 0.18, p-value = 0.13) may indicate a different or less direct involvement of these regions with SCZ (Extended Data Fig. 4c, Extended Data Fig. 3c).
We next examined whether the interactions between sex-specific CRDs could identify trans-regulatory domains (TRDs). Using a correlation matrix of expression of sex-specific CRDs across all neuronal samples, we applied hierarchical clustering and Gamma statistics41 to identify a total of 14 neuronal sex-specific TRDs (Fig. 5a). While most TRDs exhibit a mix of female- and male-specific CRDs, there is one particular TRD (TRD1) that stands out with a predominant role in the regulation of male-specific CRDs in neurons. TRD1 also showed the highest proportion of male-specific OCRs compared to other TRDs (Fig. 5b). Conversely, TRD2, TRD4, TRD6, and TRD13 displayed a higher proportion of female-specific OCRs (Fig. 5a and b). For TRD1, TRD2, and TRD13, the majority of sex-specific OCRs showed higher accessibility in SCZ cases compared to controls, whereas for TRD4 and TRD6, the majority of sex-specific OCRs showed lower accessibility (Fig. 5c-d). While we observed a moderate correlation of male-specific- with SCZ-changes (ρ = 0.37; p-value = 0.012) in TRD1, we did not observe a significant correlation of female-specific changes with SCZ-changes in any TRD (Extended Data Fig. 5). Thus, this measurement implies sex-specific chromatin regions are likely to be more accessible in SCZ compared to controls. This suggests a complex relationship between chromatin accessibility and the expression of genes associated with SCZ in females, potentially influenced by other regulatory mechanisms or factors42–44.
Functional pathway analysis in male-specific OCRs that are upregulated in SCZ and TRD1 identified biological processes related to ketone body metabolism and acid thiol ligase activity (Fig. 5e). These findings are further supported by a recent study elucidating sex-dependent modulation of metabolic hormone45, indicating that SCZ may involve sex-specific epigenetic regulation in males, particularly affecting metabolic and enzymatic pathways. Functional pathway analysis in female-specific OCRs that are upregulated in SCZ and TRD13 identified 36 significant associations with biological processes, including regulation of dosage compensation by inactivation of the X chromosome and negative regulation of neural precursor cell proliferation (Fig. 5e; Table S9). This highlights the complexity of female-specific epigenetic regulation in SCZ, particularly emphasizing the role of XCI in neurons46. To evaluate the sex specificity of E-PABC interactions in TRDs, we identified 34 male-specific OCRABC and 95 female-specific OCRABC associated with dysregulation of SCZ in neurons (Table S8). For instance, a male-specific OCRABC (Peak_83969) in TRD1 can regulate four protein-coding genes via E-PABC interactions, including TMEM132B, AACS, BRI3BP, and DHX37 (Fig. 5f). Similarly, a female-specific OCRABC (Peak_391105) in TRD13 can regulate two lncRNA genes, XIST and JPX (Fig. 5f), both of which are known XCI genes21, suggesting that sex-specific enhancers can regulate activity through XCI within SCZ associated TRDs. This highlights the potential of TRDs as key to understanding the regulatory mechanisms driving sex differences in SCZ.