Elife 92393 v1
Elife 92393 v1
Elife 92393 v1
Antipsychotic-induced epigenomic
reorganization in frontal cortex of
individuals with schizophrenia
Bohan Zhu1†, Richard I Ainsworth2†‡, Zengmiao Wang2, Zhengzhi Liu3,
Salvador Sierra4§, Chengyu Deng1, Luis F Callado5, J Javier Meana5, Wei Wang2,6*,
Chang Lu1*, Javier González-Maeso4*
1
Department of Chemical Engineering, Virginia Tech, Blacksburg, United States;
2
Department of Chemistry and Biochemistry, University of California, San Diego,
*For correspondence: La Jolla, United States; 3Department of Biomedical Engineering and Mechanics,
wei-wang@ucsd.edu (WW); Virginia Tech, Blacksburg, United States; 4Department of Physiology and Biophysics,
changlu@vt.edu (CL); Virginia Commonwealth University School of Medicine, Richmond, United States;
javier.maeso@vcuhealth.org 5
Department of Pharmacology, University of the Basque Country UPV/EHU,
(JG-M)
CIBERSAM, Biocruces Health Research Institute, Bizkaia, Spain; 6Department of
†
These authors contributed Cellular and Molecular Medicine, University of California, San Diego, La Jolla, United
equally to this work
States
Present address: ‡Department
of Medicine, Kao Autoimmunity
Institute, Cedars-Sinai
Medical Center, Los Angeles, Abstract Genome-wide association studies have revealed >270 loci associated with schizo-
United States; §Department phrenia risk, yet these genetic factors do not seem to be sufficient to fully explain the molecular
of Neurology, University of
determinants behind this psychiatric condition. Epigenetic marks such as post-translational histone
Michigan, Ann Arbor, Ann Arbor,
modifications remain largely plastic during development and adulthood, allowing a dynamic impact
United States
of environmental factors, including antipsychotic medications, on access to genes and regulatory
Competing interest: See page elements. However, few studies so far have profiled cell-specific genome-wide histone modifi-
22 cations in postmortem brain samples from schizophrenia subjects, or the effect of antipsychotic
Funding: See page 23 treatment on such epigenetic marks. Here, we conducted ChIP-seq analyses focusing on histone
Preprint posted
marks indicative of active enhancers (H3K27ac) and active promoters (H3K4me3), alongside RNA-
29 August 2023 seq, using frontal cortex samples from antipsychotic-free (AF) and antipsychotic-treated (AT) indi-
Sent for Review viduals with schizophrenia, as well as individually matched controls (n=58). Schizophrenia subjects
10 September 2023 exhibited thousands of neuronal and non-neuronal epigenetic differences at regions that included
Reviewed preprint posted several susceptibility genetic loci, such as NRG1, DISC1, and DRD3. By analyzing the AF and AT
07 December 2023 cohorts separately, we identified schizophrenia-associated alterations in specific transcription
Reviewed preprint revised factors, their regulatees, and epigenomic and transcriptomic features that were reversed by anti-
27 March 2024
psychotic treatment; as well as those that represented a consequence of antipsychotic medication
Version of Record published
rather than a hallmark of schizophrenia in postmortem human brain samples. Notably, we also
22 April 2024
found that the effect of age on epigenomic landscapes was more pronounced in frontal cortex of
Reviewing Editor: Charlotte AT-schizophrenics, as compared to AF-schizophrenics and controls. Together, these data provide
Cecil, Erasmus MC, Netherlands important evidence of epigenetic alterations in the frontal cortex of individuals with schizophrenia,
Copyright Zhu, Ainsworth and remark for the first time on the impact of age and antipsychotic treatment on chromatin
et al. This article is distributed organization.
under the terms of the Creative
Commons Attribution License,
which permits unrestricted use
and redistribution provided that eLife assessment
the original author and source The study by Zhu et al. provides important insights into cell-specific genome-wide histone modifi-
are credited. cations in the frontal cortex of individuals with schizophrenia, as well as shedding light on the role of
age and antipsychotic treatment in these associations. The evidence supporting the conclusions is
solid.
Introduction
Schizophrenia has traditionally been viewed as a genetic disorder, with heritability rates estimated
at ~73% (Sawa and Snyder, 2002; Freedman, 2003). However, previous genome-wide association
studies (GWAS) clearly showed a relatively low number of genetic regions associated with schizo-
phrenia risk – these include 108 loci in the first study (Schizophrenia Working Group of the Psychi-
atric Genomics Consortium, 2014) that have been expanded to over 270 regions (Farrell et al.,
2015; Trubetskoy et al., 2022). Most of these genetic variants were located in non-coding regions
and hence, with a few exceptions there is little evidence supporting that coding variants contribute
to schizophrenia risk, which also suggests that genetic factors do not seem to be sufficient to fully
explain the molecular causes underlying this severe psychiatric condition.
Twin studies have provided evidence that environmental factors contribute to schizophrenia
susceptibility. Thus, it had originally been suggested that monozygotic twins, whose DNA sequences
are approximately 100% identical, have a concordance for schizophrenia of nearly 50% (Cardno and
Gottesman, 2000; Hallmayer et al., 2011), yet recent research has revealed a probandwise concor-
dance rate of 33% in monozygotic twins and 7% in dizygotic twins (Hilker et al., 2018). While these
findings underscore the substantial influence of genetic factors on the etiology of schizophrenia,
they also advocate for a significant involvement of environmental events in the intricate develop-
ment of this complex disorder (Li et al., 2021). This concept is further supported by epidemiological
studies suggesting that prenatal environmental insults, such as maternal infection (Brown et al., 2004;
Yudofsky, 2009) and severe adverse life events (Malaspina et al., 2008), increase the risk of schizo-
phrenia in the offspring.
Gene expression is regulated by the ability of the transcriptional machinery to access DNA, which
is tightly packed into chromatin. The status of chromatin organization depends on epigenetic factors,
such as DNA methylation and histone modifications that primarily occur on amino-terminal tails (Gräff
and Tsai, 2013; Onuchic et al., 2018; Bastle and Maze, 2019). Hence, these epigenetic mechanisms
lead to stable changes in gene expression that are mediated via altered chromatin structure without
modification of DNA sequence, and remain largely plastic throughout all periods of brain develop-
ment and aging. It is then tempting to speculate that epigenetic mechanisms mediate, at least in part,
the effects of environmental factors on central nervous system (CNS) gene activity, and are, therefore,
potentially involved in the pathophysiology of schizophrenia and other mental illnesses.
Supporting this concept, previous studies reported alterations in chromatin structure and acces-
sibility in tissue samples from schizophrenic subjects and controls. Most of these previous reports,
however, were focused on DNA methylation differences in peripheral blood (Aberg et al., 2014)
and brain (Jaffe et al., 2016; Mendizabal et al., 2019). Using the assay for transposase-accessible
chromatin sequencing (ATAC-seq) in bulk tissue homogenates of postmortem frontal cortex samples,
only a few differences in chromatin accessibility were observed between schizophrenia subjects and
controls, in contrast to thousands of age-related differential accessible chromatin regions (Bryois et al.,
2018). Histone modifications, including histone H3 acetylation of lysine 27 (H3K27ac) and histone H3
trimethylation of lysine 4 (H3K4me3) are critically involved in epigenomic regulations; H3K27ac marks
active enhancers (Creyghton et al., 2010), whereas H3K4me3 marks active promoters (Bernstein
et al., 2005). Enhancers are highly dynamic cis-regulatory elements with known involvement in neuro-
developmental processes (Won et al., 2019), and the dynamics of promoters are also significantly
connected with the genetic risk of certain psychiatric conditions (Dincer et al., 2015). However, very
few studies have been conducted about potential cell-type-specific genome-wide variations in cova-
lent histone modifications in postmortem human brain samples of individuals with schizophrenia.
As an example, recent work combined fluorescence-activated cell sorting (FACS) of neuronal and
non-neuronal cell nuclei with chromatin immunoprecipitation sequencing (ChIP-seq) assays in two brain
regions (prefrontal cortex and anterior cingulate cortex) from postmortem brain samples of subjects
without any known neurological or psychiatric disease (Girdhar et al., 2018). Besides the identifica-
tion of cell and region-specific histone modification landscapes in this cohort of control subjects, these
findings also compared their datasets with previous GWAS of individuals with psychiatric conditions,
reporting that strong specific enrichments occurred with schizophrenia and weaker associations with
depression in both H3K27ac and H3K4me3 peaks. This correlation was almost exclusively observed
in neuronal chromatin, but not in non-neuronal cell nuclei. More recent investigation conducting
H3K27ac and H3K4me3 ChIP-seq assays in cortical neurons from schizophrenia subjects and controls
has identified rare specific epigenetic variations for a set of non-coding RNA genes (Gusev et al.,
2019) and chromatin domain alterations (Girdhar et al., 2022) that may contribute to the patho-
genesis of schizophrenia. However, these previous epigenomic studies in postmortem human brain
samples did not address the potential effect of previous exposure to antipsychotics on the regulation
of chromatin state. This is particularly relevant considering that repeated administration of antipsy-
chotic medications leads to epigenetic modifications at selected gene regions in mouse (de la Fuente
Revenga et al., 2018; Ma et al., 2018) and postmortem human brain (Kurita et al., 2012; Ibi et al.,
2017) samples. Similarly, whether such type of schizophrenia-associated epigenomic changes are
observable in non-neuronal frontal cortex nuclei remains unexplored; even though there is evidence
that alterations in the glia may contribute to major psychiatric disorders (Liu et al., 2022).
Combining MOWChIP-seq (Cao et al., 2015a; Zhu et al., 2019) and Smart-seq2 (Picelli et al.,
2013) for low-input profiling of H3K27ac and H3K4me3 histone modifications and transcriptomes,
respectively, here we present the first dataset with cell type-specific epigenomic and transcriptomic
landscapes in postmortem frontal cortex samples from two cohorts of schizophrenics either previously
treated or not with antipsychotic medications and control subjects individually matched by sex and
age. Importantly, our analyses allow the identification of transcription factors (TFs), their regulatees,
and genes that may be involved in either the therapeutic effects of antipsychotics or the cause of
undesired antipsychotic-induced epigenomic aberrations.
Results
Quality assessment for ChIP-seq and RNA-seq datasets in frontal
cortex from postmortem human brain samples
Frontal cortex is a brain region involved in processes affected in schizophrenia patients, such as
perception, cognition, and sensorimotor gating (Andreasen et al., 1994). We selected bilateral frontal
cortex (Brodmann area 9) gray matter from 58 brain samples (29 schizophrenia and 29 controls).
Control subjects were individually matched based on sex and age, and to a lower degree on post-
mortem delay (or PMD – time between death and tissue sample collection) (Supplementary files 1
and 2). Nuclei were FACS-sorted using an anti-NeuN antibody as a marker of neuronal nuclei, and
NeuN-positive (NeuN+) and NeuN-negative (NeuN-) nuclei (approximately 60,000 nuclei per NeuN+ or
NeuN- sample) were collected for ChIP-seq (10,000 nuclei per library) and RNA-seq (6000 nuclei per
library) (Figure 1A). After library preparation and sequencing, our MOWChIP-seq technology gener-
ated high-quality ChIP-seq data with average unique reads of ~11 million and ~14 million on histone
modifications H3K27ac and H3K4me3, respectively (Figure 1—figure supplement 1A; and Supple-
mentary file 3). These yields were comparable to those in our previous studies using mouse frontal
cortex and mammalian tissue culture samples (Zhu et al., 2019; de la Fuente Revenga et al., 2021).
We generated saturation curves to validate that our sequencing depth is sufficient, and that a further
increase in the sequencing depth would not lead to significantly more called peaks (Figure 1—figure
supplement 2). MOWChIP-seq datasets have very low background noise (de la Fuente Revenga
et al., 2021) with the fraction of reads in called peaks (FRiP) average at 17.35% and 27.59% for our
H3K27ac and H3K4me3 profiling, respectively (Figure 1—figure supplement 1B; and Supplemen-
tary file 3). The PCR bottleneck coefficient (PBC) was calculated to measure library complexity (0.90
and 0.92 for H3K27ac and H3K4me3, respectively), which indicates that most of our ChIP-seq datasets
have no or mild bottlenecking (Figure 1—figure supplement 1C; and Supplementary file 3).
Using Phantompeakqualtools (Marinov et al., 2014), we calculated the normalized strand cross-
correlation (NSC) and relative strand cross- correlation (RSC) to demonstrate the enrichment of
sequencing reads around the histone modification sites (Figure 1—figure supplement 1D and E; and
Supplementary file 3). The average NSC was 1.15 and 1.23 for H3K27ac and H3K4me3, respectively;
and the average RSC was 3.75 and 2.09 for H3K27ac and H3K4me3, respectively. These NSC and RSC
values were higher than the recommended thresholds of 1.05 and 1.0, respectively. We also compared
our GC metrics with those of previously published ChIP-seq data in postmortem human frontal cortex
Figure 1. Overview of the multi-omics protocol for analyzing frontal cortex of schizophrenia subjects and controls.
(A) Overview of the experimental design starting from postmortem human frontal cortex samples to generate
cell type-specific H3K27ac, H3K4me3, and RNA profiles. (B) Heatmap of the expression of neuronal and glial cell
markers across all NeuN+ and NeuN- frontal cortex samples from 29 control subjects and 29 schizophrenia subjects.
Figure 1 continued on next page
Figure 1 continued
The online version of this article includes the following figure supplement(s) for figure 1:
Figure supplement 1. Distribution of six quality control metrics for ChIP-seq data on H3K4me3 and H3K27ac in
NeuN-positive (NeuN+) and NeuN-negative (NeuN-) nuclei from schizophrenia and control groups, respectively.
Figure supplement 2. Saturation curves on the relationship between the number of sequencing reads and the
number of identified peaks with our ChIP-seq data on H3K4me3 and H3K27ac.
Figure supplement 3. Distribution of four quality control metrics for RNA-seq data in NeuN-positive (NeuN+) and
NeuN-negative (NeuN-) nuclei from schizophrenia and control groups.
Figure supplement 4. Representative results from fluorescence-activated cell sorting (FACS) sorting
demonstrating the separation of neuronal (NeuN+) and non-neuronal (NeuN-) nuclei using fluorescence-labeled
anti-NeuN antibody in postmortem human frontal cortex samples.
Figure supplement 5. Expression of neuronal (NeuN+) and non-neuronal (NeuN-) representative marker genes.
Figure supplement 6. Venn diagrams of the overlap between the identified peaks from our ChIP-seq study
(green) and previous datasets (Girdhar et al., 2022) (cyan).
Figure supplement 7. Visualization of peak-wise or gene-wise means and variances of ChIP-seq and RNA-seq
data, respectively, by voom plots.
samples (Girdhar et al., 2018). This previous study had an average NSC of 1.33 and 2.81, and RSC of
1.18 and 1.06 for H3K27ac and H3K4me3, respectively. The average Pearson’s correlations between
replicates were 0.923 and 0.971 for H3K27ac and H3K4me3, respectively, which compare well with
those of ENCODE data (ENCODE Project Consortium, 2012). Our RNA-seq datasets had an average
of ~6.8 million uniquely mapped reads, and the average mapping rate was 82.5% (Figure 1—figure
supplement 3; and Supplementary file 4), within the recommended range of 70–90% (Conesa et al.,
2016). Our average GC content was 40.73% and exon percentage was 36.52%.
Using all frontal cortex samples from the 29 schizophrenia subjects and 29 controls, we analyzed the
expression of selected neuronal and non-neuronal marker genes. Highly significant (median p-value
= 6×10–7) pair-wise differences in molecular marker expression were observed for all markers ranging
from mature, functional, and synaptic neuron markers to astrocyte, oligodendrocyte, and microglial
markers (Figure 1B; Figure 1—figure supplements 4 and 5; Supplementary file 5) – confirming
neuronal and non-neuronal cell-type identities in the NeuN+ and NeuN- nuclei samples, respectively.
Consequently, it can be concluded that MOWChIP-seq technology offers data quality compa-
rable to that of state-of-the-art standard reference epigenomes while allowing histone modification
profiling using small and highly purified populations of neuronal and non-neuronal nuclei from post-
mortem human frontal cortex samples.
We then investigated the differences in histone modification profiles and gene activity between the
schizophrenia and control cohorts. We regressed demographic (sex, age at death, PMD, antemortem
diagnosis) and technical (align rate, unique rate, FRiP, NSC, RSC, the number of identified peaks,
and PBC) covariates (Sun et al., 2016). We defined differential H3K27ac peaks that have no overlap
regions with promoters as differential enhancers. Our analysis revealed 2,301 differential enhancers,
262 differential promoters, and 802 differentially expressed genes (DEGs) in NeuN+ nuclei between
schizophrenia subjects and controls, while 2,657 differential enhancers, 360 differential promoters, and
1,043 DEGs were discovered in NeuN- nuclei (Figure 2A; Supplementary file 6). We next leveraged
the previously identified promoter-anchored chromatin loops in NeuN+ and NeuN- nuclei to identify
how differential enhancers are linked with genes (Hu et al., 2021). We successfully associated 639 and
714 cell-type-specific enhancers to 328 and 395 genes via enhancer-promoter interactions in NeuN+
and NeuN- nuclei, respectively. The rest of the enhancers were associated with their nearest genes.
We discovered that the schizophrenia group had varied H3K27ac/H3K4me3 levels and RNA-seq reads
compared to controls at various loci involved in processes related to synaptic plasticity and cogni-
tive processes or previously associated with schizophrenia risk (Farrell et al., 2015) – these included
enhancer region of NRG1 (Neuregulin 1) in NeuN+ nuclei, enhancer region of GRM3 (Metabotropic
glutamate 3 receptor) in NeuN- nuclei, promoter region of DRD3 (Dopamine D3 receptor) in NeuN+
nuclei, promoter region of DISC1 in NeuN- nuclei, CDK5 (Cyclin-dependent kinase 5) mRNA in NeuN+
nuclei, and GRIN2A (Glutamate ionotropic receptor NMDA type subunit 2 A) mRNA in NeuN- nuclei
(Figure 2B and C; Supplementary file 6). The accuracy of H3K27ac and H3K4me3 differential peak
calling between schizophrenia subjects and controls identified by MOWChIP-seq was validated by
independent ChIP-qPCR analysis of selected loci (Figure 2—figure supplement 1).
We also constructed QQ plots to examine the validity of our differential analysis (Figure 2—figure
supplement 2). The lambda values of our H3K4me3 ChIP-seq datasets were lower than 1 (i.e. 0.43 and
0.36 for NeuN+ and NeuN- nuclei, respectively), suggesting that there are fewer differential promoters
than in the normal distribution. This matches previous findings on postmortem human brain samples
(Girdhar et al., 2018).
To further explore the association between differential epigenetic modifications and genetic loci
previously associated with schizophrenia risk (Trubetskoy et al., 2022), we examined the overlap
of differential enhancer and promoter peaks with genetic variants using linkage disequilibrium (LD)
score regression (Finucane et al., 2015). Various other brain and non-brain-related traits were also
considered for comparison (Finucane et al., 2018). Schizophrenia was the most significantly enriched
trait for all the differential enhancer and promoter regions in NeuN+ and NeuN- fractions (Supplemen-
tary file 7). Additionally, the level of enrichment was higher in differential enhancers as compared to
promoters, and the highest in differential enhancers of NeuN+ nuclei (Supplementary file 7).
To assess agreement with the literature, we compared the DEGs identified in our study with a
previous single-nucleus RNA sequencing (snRNA-seq) study in the postmortem prefrontal cortex of
schizophrenics and controls (Ruzicka et al., 2020). Importantly, 236 out of our 802 DEGs (p-value =
1.96×10–11) in NeuN+ nuclei, and 63 out of our 1,043 DEGs (p-value = 4.18×10–6) in NeuN- nuclei were
also identified in this previous single-cell dissection work. In NeuN+ nuclei, several genes encoding
metabotropic glutamate receptors (GRM3, GRM5) that are directly associated with schizophrenia risk
(Maj et al., 2016) were found differentially expressed in both studies (Supplementary file 8). We
also identified some novel genes, including LRRTM3, which regulates excitatory synapse develop-
ment (Um et al., 2016), and POU3F2, which is viewed as a key regulator of gene expression in a
schizophrenia-associated gene co-expression module (Chen et al., 2018; Supplementary file 8).
We also overlapped genes identified from differential enhancers/promoters with DEGs from RNA-
seq (Figure 2D; Supplementary file 9). We found that 66 (p-value = 9×10–3) and 148 (p-value =
1.7×10–2) genes were identified in both the DEGs from RNA-seq and differential enhancers/promoters
associated genes in NeuN+ and NeuN- nuclei, respectively. Among these, several schizophrenia-
associated genes were also detected, including NTNG2, which is known to be involved in neurodevel-
opmental disorders (Dias et al., 2019) and GRIN3A, a gene that encodes NMDA receptor subunits in
neuronal nuclei (Yu et al., 2018).
For integrative analysis of these diverse epigenomic and transcriptomic data, we next employed an
unbiased method called EpiSig (Ai et al., 2018), which combines the epigenomic and transcriptomic
dataset into a single analysis to cluster regions with similar epigenomic profiles across all the NeuN+
Figure 2. Comparison of epigenomic and transcriptomic landscapes in the frontal cortex of schizophrenia subjects and controls. (A) Differential
enhancer/promoter peaks and differentially expressed genes (DEGs) obtained by comparing schizophrenia (n=29) and controls (n=29). The
differential peaks or DEGs were identified using FDR <0.05. (B) Exemplar genomic track view of H3K27ac, H3K4me3, and RNA signals for matched
AF-schizophrenia/control and AT-schizophrenia/control pairs in NeuN-positive (NeuN+) and NeuN-negative (NeuN-) cells. 50 Mb region displayed:
Figure 2 continued on next page
Figure 2 continued
chr1:68,000,000–118,000,000 (GRCh38). (C) Volcano plots showing genes associated with differential enhancer and promoter peaks and DEGs.
Candidate genes for schizophrenia or genes involved in significant GO terms are labeled. The horizontal lines indicate FDR of 0.05. (D) Venn diagrams
on the relationship among genes associated with differential enhancer or promoter peaks and DEGs.
The online version of this article includes the following figure supplement(s) for figure 2:
Figure supplement 1. qPCR validation of selected differential ChIP-seq peaks in NeuN+ fraction for H3K27ac (a-c) and H3K4me3 (d-f).
Figure supplement 2. Q-Q plots of the corrected p-value from differential peaks at enhancer/promoter regions and differentially expressed genes.
and NeuN- nuclei samples. 85,462 signal-enriched regions were grouped into 814 epigenomic clus-
ters covering 14.53% of the genome. These clusters were further combined into 6 groups (sections)
using the K-means method (Figure 3A and B; Supplementary files 10 and 11). Section I had high
coverage in the gene annotations for intron (35%) and intergenic regions (29%) indicating inactive
regions. It was also enriched in chromosome X compared to other sections. Section II was anno-
tated as enhancers that are active in NeuN+ nuclei but suppressed in NeuN- nuclei. A hypergeometric
test identified clusters that were significantly enriched in schizophrenia vs control differential histone
marks and differentially expressed genes (Figure 3A and B; Supplementary files 10 and 11). The
top five Section II clusters had schizophrenia-positive association (i.e. activity and expression schizo-
phrenia >controls) in genes enriched in GO terms ‘Trans-synaptic signaling’ (FDR 4.66×10–2). Section III
was highly enriched in enhancers (average of 21% of all regions in each cluster), and had low coverage
in intergenic regions (12%), which is likely associated with active enhancers for both NeuN+ and NeuN-
given the high signal strength for H3K27ac. Top differentially enriched cluster genes, enriched in the
GO term ‘Amyloid fibril formation’ (FDR 5.99×10–2), were found to be negatively associated with
schizophrenia in NeuN+ nuclei, whereas genes enriched in the GO term ‘Neuron projection develop-
ment’ (FDR 3.19×10–2) were positively associated with schizophrenia in NeuN- nuclei (Figure 3A and
B; Supplementary files 10 and 11). Section IV also had high coverage of enhancers (Figure 3A and B;
Supplementary files 10 and 11). However, it had the highest average promoter content with 16% of
all gene annotations being promoter regions, further supported by CpG islands showing the highest
proportion (20%) in this section; indicating active promoters for both NeuN+ and NeuN- nuclei. Both
cell types showed enrichment in respiratory electron transport genes that were negatively associated
with schizophrenia (Figure 3A and B; Supplementary files 10 and 11). As for the average differ-
ential signals across sections, a great variance was observed. For example, the signal of H3K4me3
was higher in schizophrenia subjects compared to controls for NeuN+ nuclei in section V, while it was
lower in schizophrenia subjects for NeuN- samples (Figure 3A and B; Supplementary files 10 and
11). Finally, Section VI was annotated as enhancers that were active in NeuN- nuclei but repressed in
NeuN+ nuclei (Figure 3A and B; Supplementary files 10 and 11).
Transcriptional regulatory processes proceed as a hierarchy of orchestrated events that ultimately
modulate the expression of downstream target genes. Using the recently developed Taiji algorithm
(Zhang et al., 2019), which allows access to information pertaining to transcriptional cascades
deriving from upstream drivers through specific pathway mechanisms to downstream effects, we inte-
grated epigenomic and transcriptomic data to construct 116 individual transcriptional networks in
neuronal and glial nuclei from schizophrenia subjects and controls. We identified active promoters
and enhancers using H3K27ac and then predicted TF binding sites by scanning 1,165 TF motifs linking
putative TF binding sites to their targets using EpiTensor (Zhu et al., 2016), an unsupervised method
to predict enhancer-promoter associations. TFs were subsequently ranked according to regulatory
importance using the Personalized PageRank (PPR) algorithm for each unique network topology (Yu
et al., 2017). Using the differentially expressed TFs (schizophrenia vs controls FDR <0.05), TFs were
ranked by absolute change in schizophrenia vs control PPR score (Figure 4A – top 10 TFs for each
cell type; Supplementary file 12). Of the top 10 TFs of NeuN+ nuclei, all were found to be cell-type
specific. Using the top four TFs, we identified 207 regulatees the were regulated by three or more
TFs and found they were involved in processes such as ‘Neurexins and neuroligins’ (FDR 2.18×10–7)
and ‘Protein-protein interactions at synapses’ (FDR 1.22×10–6) (Figure 4B; Supplementary file 12).
Furthermore, all top 10 TFs of NeuN- nuclei were cell-type specific TFs and the regulatees of the top
four TFs were enriched in signaling pathways including ‘RAF/MAP kinase cascade’ (FDR 2.80×10–2)
and ‘RHO GTPase cycle’ (FDR 3.07×10–2) (Figure 4C; Supplementary file 12).
Figure 3. Genome-wide multidimensional clusters in the frontal cortex of schizophrenia subjects and controls.
(A) Integrative analysis using EpiSig. 814 EpiSig clusters across 348 genome-wide sequencing datasets were
grouped into 6 sections. The heatmap shows the signal in each EpiSig cluster (row: EpiSig cluster; column: marker).
(B) For each EpiSig cluster, from left to right, the heatmaps are: the region percentage in each chromosome;
the genomic annotation; the CpG annotation; the percentage of enhancer; the difference signal between
schizophrenia and controls in NeuN-positive (NeuN+) and NeuN-negative (NeuN-) nuclei.
Figure 4. Transcriptional regulatory processes in the frontal cortex of schizophrenia subjects and controls. (A) Heatmap of z-score Personalized
PageRank (PPR) for top 10 significantly differentially expressed transcription factors (TFs) (FDR <0.05) ranked by absolute change in PPR for NeuN-
positive (NeuN+) (upper panel) and NeuN-negative (NeuN-) (lower panel) schizophrenia vs control nuclei samples. (B) Overrepresented pathway analysis
(FDR <0.05) for 203 downstream regulatees common to the top four schizophrenia vs control NeuN+-specific transcription factors (TFs) (ZNF333, SOX2,
Figure 4 continued on next page
Figure 4 continued
ZEB1, and RBPJ). (C) Overrepresented pathway analysis (FDR <0.05) for 225 downstream regulatees common to the top four schizophrenia vs control
NeuN--specific TFs (FOS, BCL6, IRF1, and KLF15).
Figure 5. Epigenomic alterations affected by antipsychotic treatment. (A) Scatter plot of average pairwise change in Personalized PageRank (PPR)
(PPRschizophrenia – PPRcontrol) for antipsychotic-free (AF) vs antipsychotic-treated (AT) NeuN-positive (NeuN+) cohorts. Orange regions show cohort (AF
– AT)<0.5 (i.e. alterations recovered by antipsychotic treatment), whereas beige regions show cohort (AT – AF)>0.5 (i.e. alterations consequence of
antipsychotic treatment). Transcription factors (TFs) FDR <0.05 highlighted in red. (B) Scatter plot of average pairwise change in PPR (PPRschizophrenia –
Figure 5 continued on next page
Figure 5 continued
PPRcontrol) for AF vs AT NeuN- cohorts. Dark blue regions show cohort (AF – AT)<0.5 (i.e. alterations recovered by antipsychotic treatment), whereas
cyan regions show cohort (AT – AF)>0.5 (i.e. alterations consequence of antipsychotic treatment). TFs FDR <0.05 highlighted in red. (C) Number of
differentially expressed gene (DEG) regulatees by TFs, and number of DEGs in NeuN+ nuclei from AF-schizophrenia/control pairs. (D) Number of DEG
regulatees by TFs, and number of DEGs in NeuN- nuclei from AF-schizophrenia/control pairs. (E) Number of DEG regulatees by TFs, and number of
DEGs in NeuN+ nuclei from AT-schizophrenia/control pairs. (F) Number of DEG regulatees by TFs, and number of DEGs in NeuN- nuclei from AT-
schizophrenia/control pairs. (G) Functional enrichment analysis of union of genes from AF-schizophrenia/control pairs in NeuN+ nuclei. (H) Functional
enrichment analysis of union of genes from AF-schizophrenia/control pairs in NeuN- nuclei. (I) Pairwise expression difference (schizophrenia – control)
of an exemplar AF-schizophrenia/control cohort DEG (TUBB2A) across all 29 schizophrenia-control pairs in NeuN+ nuclei. (J) H3K27ac tracks for
PDK1 (member of the 84 gene set in E) in NeuN+ nuclei. Box highlighting the FOXO1 DNA-binding motif in promoter at position chr2: 172,555,706–
172,555,718 (GRCh38). Two exemplar AT-schizophrenia/control cohort pairs showing differential H3K27ac peak intensity around motif locus and an
example AF-schizophrenia/control cohort patient pair with no difference.
The online version of this article includes the following figure supplement(s) for figure 5:
Figure supplement 1. Uniform manifold approximation and projection (UMAP) visualization of the feature matrix of enhancers, promoters, and RNA
among antipsychotic-free (AF)-schizophrenia, antipsychotic-treated (AT)-schizophrenia, and control subjects.
Figure supplement 2. Differential peak enrichment analysis for antipsychotic-free NeuN-positive (NeuN+) nuclei in EpiSig clusters.
Figure supplement 3. Differential peak enrichment analysis for antipsychotic-treated NeuN-positive (NeuN+) nuclei in EpiSig clusters.
Figure supplement 4. Differential peak enrichment analysis for antipsychotic-treated NeuN-negative (NeuN-) nuclei in EpiSig clusters.
difference between case and control in the AT-schizophrenia group, SOX11 (FDR 1.73×10–2) and MGA
(FDR 9.83×10–3) (Figure 5B; Supplementary file 13). 77 downstream DEG regulatees of these two
TFs were identified in the AF cohort showing significant regulatory case/control change (Figure 5D;
Supplementary file 13). In parallel, 21 cohort-specific DEGs were identified as AF-schizophrenia/
control DEG and having no significant difference in expression in the AT-schizophrenia/control cohort
(Figure 5D; Supplementary file 13). Functional enrichment analysis of the union of 153 genes
included ‘Post NMDA receptor activation events’ (FDR 3.65×10–2), and ‘Long-term potentiation’ (FDR
1.95×10–2) (Figure 5H; Supplementary file 13). EpiSig’s analysis did not show NeuN- alterations in
the AF-schizophrenia cohort.
We also performed the differential analysis with demographic and technical covariates regressed
out on AF-schizophrenia/control and AT-schizophrenia/control cohorts. In NeuN+ nuclei, the results
revealed 2,069 and 574 differential enhancers and promoters, respectively, and 166 DEGs between
AF-schizophrenia and their controls (Figure 6A; Supplementary file 15), while 3,658, 36, and 1,273
differential enhancers, promoters, and DEGs were discovered between AT-schizophrenia and controls
(Figure 6B; Supplementary file 15). In NeuN- nuclei, we identified 891, 19, and 128 differential peaks/
genes between AF-schizophrenia and controls (Figure 6A; Supplementary file 15); 2,651, 775, 776
differential peaks/genes between AT-schizophrenia and controls, in enhancers, promoters, and DEGs,
respectively (Figure 6B; Supplementary file 15). More differential enhances/promoters and genes were
detected between AT-schizophrenia and their matched controls than those between AF-schizophrenia
and their controls with the exception in neuronal promoters (Figure 6A and B; Supplementary file 15).
Similar to our TF analyses (Figure 5), we also identified the genes altered in the AF-schizophrenia/
control group but not in the AT-schizophrenia/control group using differential analyses of enhancers,
promoters, or gene expression. It should be noted that in the differential analyses here, the schizo-
phrenia subjects (whether AF or AT) and their controls were compared at the cohort level, while
matched schizophrenia/control pairs were examined individually in the TF-based analysis. At the
epigenomic level, in NeuN+ nuclei, we identified 687 and 549 genes changed in the AF- but not in
AT-schizophrenics by examining differential enhancers and promoters, respectively (Supplementary
file 16). These genes were linked to epigenomic features restored to their basal level after treat-
ment. In NeuN- nuclei, there were 270 and 17 recovered genes linked with differential enhancers and
promoters, respectively. At the transcriptomic level, 145 DEGs in NeuN+ nuclei and 109 in NeuN-
nuclei were discovered in AF- schizophrenia/control comparison but not in the AT- schizophrenia/
control differential analysis.
Figure 6. Effect of antipsychotic treatment on differential enhancers/promoters and differentially expressed genes (DEGs) in NeuN-positive (NeuN+)
and NeuN-negative (NeuN-) nuclei from the frontal cortex of schizophrenia subjects and controls. (A) Differential enhancer/promoter peaks and DEGs
were obtained by comparing antipsychotic-free (AF)-schizophrenics and individually matched controls. (B) Differential enhancer/promoter peaks and
DEGs obtained by comparing antipsychotic-treated (AT)-schizophrenics and individually matched controls.
Figure 7. Epigenomic effect of age on treated schizophrenia subjects. (A) Violin plots for Pearson’s R correlation coefficients of age vs expression f 742,
622, and 242 genes from control, antipsychotic-free (AF)-schizophrenia, and antipsychotic-treated (AT)-schizophrenia NeuN-positive (NeuN+) nuclei,
respectively. (B) Violin plots for Pearson’s R correlation coefficients of age vs expression for 1031, 389, and 351 genes from control, AF-schizophrenia, and
AT-schizophrenia NeuN-negative (NeuN-) nuclei, respectively. (C) Number of pairwise transcription factor (TF) Personalized PageRank (PPR) and pairwise
Figure 7 continued on next page
Figure 7 continued
gene expression differences correlated with age in NeuN+ nuclei from AF-schizophrenia/control pairs. (D) Number of pairwise TF PPR and pairwise
gene expression differences correlated with age in NeuN+ nuclei from AT-schizophrenia/control pairs. (E) Heatmap for the 14 age positively-correlated
(schizophrenia – control increase with age) and 12 age negatively-correlated (schizophrenia – control decrease with age) genes of the significant
GO term ‘Regulation of kinase activity’ from the AT-schizophrenia/control NeuN+ cohort. (F) Example gene, WNK1 pairwise expression difference
(schizophrenia – control) vs age (Pearson’s R=0.73; p-value = 0.003). (G) Example gene, SFRP2 pairwise expression difference (schizophrenia – control)
vs age (Pearson’s R=–0.70; p-value = 0.005). (H) Example TF, EGR2 pairwise PPR difference (schizophrenia – control) vs age (Pearson’s R=0.69; p-value
= 0.0003) (I) Number of pairwise TF PPR and pairwise gene expression differences correlated with age in NeuN- nuclei from AF-schizophrenia/control
pairs. (J) Number of pairwise TF PPR and pairwise gene expression differences correlated with age in NeuN- nuclei from AT-schizophrenia/control pairs.
(K) Heatmap for the 5 age positively-correlated (schizophrenia – control increase with age) genes of the significant GO term ‘Beta-catenin independent
WNT signaling’ from the AT-schizophrenia/control NeuN- cohort.
marker of psychosis-like behavior (González-Maeso et al., 2007) – that we observed in older subjects
(Figure 7H; Supplementary file 19).
In NeuN- nuclei, 147 and 88 genes were identified with AF-schizophrenia/control and AT-schizo-
phrenia/control expression difference vs age correlations of ≥0.60, respectively (Figure 7I and J;
Supplementary file 19). Enriched pathways in the AT-schizophrenia/control group included: ‘Degra-
dation of DVL’ (p-value 4.04×10–5) and ‘Beta-catenin independent WNT signaling’ (p-value 5.06×10–4)
(Figure 7K; Supplementary file 19). Since dysfunctional WNT signaling is associated with several
CNS disorders including Alzheimer’s (Wan et al., 2014), together, these data also suggest that this
positive correlation between NeuN- gene differences in AT-schizophrenia subjects/control pairs and
age (Figure 7K; Supplementary file 19) may be responsible for some of the negative effects of
antipsychotic treatment on cognitive processes. We also identified 11 and 53 TFs correlated with
age in the AF-schizophrenia/control and AT-schizophrenia/control cohorts, respectively (Figure 7I and
J; Supplementary file 19). However, as in NeuN+ nuclei, the effect of age became more evident in
the AT-schizophrenia/control group with age-related adaptations in NeuN- TF-affected pathways that
included ‘Signaling by NOTCH’ (p-value 3.2×10–4) (Supplementary file 19).
Discussion
Understanding the molecular determinants involved in schizophrenia is critical for devising new treat-
ment strategies and the discovery of the pathogenic mechanisms underlying this psychiatric condi-
tion. In this study, we combined low-input epigenomic and transcriptomic analysis to define how
gene expression and TF regulation vary in schizophrenia subjects relative to controls and in response
to antipsychotic treatment and aging. Our data provide evidence suggesting alterations in cova-
lent histone modifications at different gene regions previously associated with schizophrenia risk,
as well as additional genes involved in pathways related to immunological and neurodevelopmental
processes. Whereas previous studies with postmortem human brain samples compared using indirect
methods differences in chromatin accessibility between schizophrenia subjects and controls (Bryois
et al., 2018), here we provide epigenomic signatures that distinguish between those observed in
AF-schizophrenia subjects as well as alterations that denote previous treatment with antipsychotic
medications.
We conducted a pairwise comparison between schizophrenia and matched controls, which is crucial
to tease out treatment- or age-associated effects. A powerful feature of the Taiji framework is to allow
the analysis of individual samples (Zhang et al., 2019). This integrative analysis of transcriptomic and
epigenomic data at the systems level uncovered key regulators and important pathways based on
their global importance in the genetic networks. We found that transcriptional mechanisms via novel
pathways that had not been previously associated with schizophrenia show alterations in AF-schizo-
phrenia subjects, and that these schizophrenia-linked pathways were statistically unaffected in the
AT-schizophrenia group, consistent with a potential role of these epigenomic signatures in the clinical
efficacy of antipsychotics. These changes appear to impact glutamatergic neurotransmission, IQGAP
scaffold, autophagy, and ubiquitin B expression in neurons; and post NMDA receptor activation and
long-term potentiation in glial cells. Additionally, our data highlight processes related to key pathways
that may represent a consequence of antipsychotic medication, rather than a reversal of the molecular
alterations observed in AF-schizophrenia subjects. These pathways suggest the existence of compen-
satory perturbations that emerge in response to repeated antipsychotic drug administration and
ultimately restrain their therapeutic effects (Kurita et al., 2012; Ibi et al., 2017). Among these, alter-
ations in p53 activity were apparent as a consequence of antipsychotic treatment. Based on our ability
to individually match schizophrenia and control pairs by age, we also revealed the intriguing observa-
tion that the effect of age on TF regulation of gene expression was significantly more pronounced in
AT-schizophrenia subjects as compared to AF-schizophrenia subjects and controls.
Related to the effect of antipsychotic treatment, frontal cortex samples of schizophrenia subjects
were divided into AF and AT based on postmortem toxicological analysis in both blood and when
possible brain samples, which provides information about a longer retrospective drug-free period
due to the high liposolubility of antipsychotic medications (Voicu and Radulescu, 2009). However, we
cannot fully exclude the possibility of previous exposure to antipsychotic medications in the AF-schizo-
phrenia group, and hence that the epigenetic alterations observed exclusively in the AF-schizophrenia
group are a consequence of a potential period of decompensation, which typically occurs following
voluntary treatment discontinuation (Liu-Seifert et al., 2005). It is also worth noting that our find-
ings were established by examining the average characteristics of entire NeuN+ and NeuN- fractions.
Further studies of individual neuronal and glial cell subtypes may yield additional information on the
role of cell-type-subpopulations (Lau et al., 2020; Nagy et al., 2020).
Conclusion
Our ChIP-seq/RNA-seq study in postmortem brain samples from schizophrenia subjects and controls
suggests cell-type specific epigenomic differences in individuals with schizophrenia, as well as cellular
alterations in signaling pathways potentially involved in either the elimination of schizophrenia-related
epigenomic alterations upon antipsychotic drug treatment or the antipsychotic-dependent modula-
tion of alternative epigenetic pathways previously unaffected in the untreated schizophrenia cohort.
Building on our data, future research could test the causal role of specific molecular pathways impli-
cated in schizophrenia pathophysiology, as well as the therapeutic versus compensatory or negative
side epigenomic outcomes induced by chronic treatment with antipsychotic medications.
controls are shown in Supplementary file 1, and the definitive pairs of atypical antipsychotic-treated
schizophrenics and respective matched controls are shown in Supplementary file 2. Presence or
absence of antipsychotic medications was confirmed by toxicological analysis in postmortem brain
samples of a selected group of schizophrenia subjects and controls (Supplementary file 20). Pairs of
schizophrenia and matched controls were processed simultaneously and under the same experimental
conditions. Tissue pH values were within a relatively narrow range (control subjects: 6.7±0.08; schizo-
phrenic subjects: 6.6±0.06). Brain samples were also assayed for RIN (RNA integrity number) values
using the Agilent 2100 Bioanalyzer (Applied Biosystems) – control subjects: 7.87±0.21; schizophrenic
subjects: 7.61±0.32.
After 5 min incubation at room temperature, the beads went through a cleanup procedure with 80%
ethanol. In the end, the DNA library was eluted from the beads into 7 µl of low EDTA TE buffer.
<0.05). The p-values were adjusted by performing a standard Bonferroni correction. The following
covariates were regressed out: demographic covariates (age at death, sex, PMD, and diagnosis) and
technical covariates (align rate, unique rate, FRiP, NSC, RSC, the number of identified peaks, and
PBC) by correlating the top 6 principal components with these covariates. We annotated enhancers
(defined as identified H3K27ac peaks that have no overlap regions with promoters) to genes using
published Hi-C data on neurons and glia (Hu et al., 2021) when possible and the rest of the enhancers
were associated with their nearest genes. We annotated H3K4me3 peaks to genes when they over-
lapped with the promoter regions.
ChIP-qPCR assays
After nuclei extraction, MNase digestion and MOWChIP assays (see above), ChIP DNA was eluted to
10 µl of low EDTA TE buffer. 1 µl of ChIP DNA solution was used for qPCR assays with each primer set.
The following qPCR primer pairs were used:
The following common negative primer set was used in all samples, against which the enrichment
of each positive set was calculated:
GCA G
AA C
CT AGT T
CC TCC T
TC AAC (F); AGT C
AT C
CC T
TC CTA C
AG A
CT GAG A (R)
qPCR primer sets were ordered from IDT, made to lab-ready formulation (100 µM in low EDTA TE
buffer). Ready to use stocks of primer sets were made by combining 10 µl each of both forward and
reverse primers of the same set with 80 µl of low EDTA TE buffer. 10 µl of iQ SYBR Green Supermix,
1.6 µl of primer stock, 1 µl of ChIP DNA, and 7.4 µl of ultrapure water were added to each qPCR well.
Reaction was conducted on a CFX96 real-time PCR machine (Bio-Rad) with C1000 thermal cycler base.
All PCR assays were performed using the following thermal cycling profile: 95 °C for 10 min followed
by 40 cycles of (95 °C for 15 s, 58 °C for 40 s, 72 °C for 30 s). Relative fold enrichment of each positive
primer (P) against the common negative primer (N) was calculated using the following equation:
Enrichment = 2Cq(N) - Cq(P).
Taiji pipeline
Active regulatory elements were first identified via the overlap of high confidence peaks from H3K27ac
with known gene promoter regions (4kbp upstream and 1kbp downstream of the transcription start
sites). The distal H3K27ac peaks were assigned to active promoters using the unsupervised learning
method EpiTensor, and assigned as an enhancer-promoter interaction if one locus overlapped with
the distal peak and the other locus in the pair overlapped with a known promoter. Putative TF binding
motifs were curated from the CIS-BP database (Weirauch et al., 2014). Using FIMO’s algorithm (Grant
et al., 2011). TFs were identified as having binding sites within 150 bp regions centered around
H3K27ac peak summits. 58 unique NeuN+ (29 schizophrenia and 29 control) and 58 unique NeuN- (29
schizophrenia and 29 control) network topologies were thus constructed by forming directed edges
between TF and their regulatees, if the TF had a predicted binding site in the gene’s promoter or
linked enhancer.
EpiSig analysis
To integrate H3K27ac, H3K4me3, and RNA-seq data from two cell types across the postmortem frontal
cortex samples from schizophrenia subjects and controls, EpiSig was employed (Ai et al., 2018). This
algorithm detects the significant signals from sequencing data in 5 kb bins across the whole genome,
and then clusters the regions based on the similar epigenomic profiles across all samples.
Acknowledgements
CL dedicates this paper to his elder brother Lv Wei who passed away in 2023 after a 30 year battle
with schizophrenia. The authors thank the staff members of the Basque Institute of Legal Medicine
for their cooperation in the study. National Institutes of Health R01MH084894 (JG-M), R01MH111940
(JG-M), R01GM143940 (CL), R01GM141096 (CL), R01HG009626 (WW), R01AI50282 (WW), Eusko
Jaurlaritza IT1211-19 (JJM) and IT-1512/22 (LFC), and VCU Presidential Request Fund (JG-M).
Additional information
Competing interests
J Javier Meana: received unrestricted funds from Janssen. Javier González-Maeso: has sponsored
research contracts with Terran Biosciences and Gonogo Solutions. The other authors declare that no
competing interests exist.
Funding
Funder Grant reference number Author
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
Author contributions
Bohan Zhu, Richard I Ainsworth, Data curation, Formal analysis, Investigation, Methodology, Writing
– original draft; Zengmiao Wang, Zhengzhi Liu, Formal analysis, Investigation; Salvador Sierra, Investi-
gation, Methodology; Chengyu Deng, Investigation; Luis F Callado, J Javier Meana, Resources, Inves-
tigation; Wei Wang, Chang Lu, Conceptualization, Resources, Data curation, Software, Formal analysis,
Supervision, Funding acquisition, Methodology, Writing – review and editing; Javier González-Maeso,
Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Meth-
odology, Writing – review and editing
Author ORCIDs
Bohan Zhu http://orcid.org/0009-0003-9823-8630
Richard I Ainsworth http://orcid.org/0000-0002-3350-5692
Luis F Callado https://orcid.org/0000-0001-9941-012X
J Javier Meana https://orcid.org/0000-0002-7913-6714
Wei Wang http://orcid.org/0000-0003-4377-5060
Chang Lu http://orcid.org/0000-0003-0181-5888
Javier González-Maeso https://orcid.org/0000-0003-3105-3204
Ethics
Human brains were obtained during autopsies performed at the Basque Institute of Legal Medi-
cine, Bilbao, Spain. The study was developed in compliance with policies of research and ethical
review boards for post-mortem brain studies (Arias-Diaz et al 2013). The Institutional Review Board
(IRB) of the University of the Basque Country determined that approval was not required for this
research in postmortem samples (M10/2018/283). According to legal requirements, samples were
obtained by opting-out policy, absence of compensation for tissue donation and coded under revers-
ible anonymization.
Additional files
Supplementary files
• Supplementary file 1. Demographic information of antipsychotic-free (AF)-schizophrenia subjects
and controls.
• Supplementary file 2. Demographic information of antipsychotic-treated (AT)-schizophrenia
subjects and controls.
• Supplementary file 3. Quality control measurements on ChIP-seq datasets.
• Supplementary file 4. Quality control measurements on RNA-seq datasets.
• Supplementary file 5. Transcription frequency in transcripts per kilobase million (TPM) on key
marker genes for neuronal and glial nuclei samples from control and schizophrenia subjects.
• Supplementary file 6. Differential histone modification peaks and differentially expressed genes
(DEGs) obtained by comparing schizophrenia and controls.
• Supplementary file 7. Enrichment of various genome-wide association studies (GWAS) traits in
differential enhancers and promoters.
• Supplementary file 8. Overlap of differentially expressed genes (DEGs) identified in our study and
a previous snRNA-seq study.
• Supplementary file 9. Genes identified via differential analyses of enhancers, promoters, and RNA
by comparing schizophrenia and controls.
• Supplementary file 10. EpiSig analysis in NeuN-positive (NeuN+) nuclei from the frontal cortex of
schizophrenia subjects and controls.
• Supplementary file 11. EpiSig analysis in NeuN-negative (NeuN-) nuclei from the frontal cortex of
schizophrenia subjects and controls.
• Supplementary file 12. Taiji analysis in NeuN-positive (NeuN+) and NeuN-negative (NeuN-) nuclei
from the frontal cortex of schizophrenia subjects and controls.
• Supplementary file 13. Taiji analysis in NeuN-positive (NeuN+)and NeuN-negative (NeuN-) nuclei
from the frontal cortex of antipsychotic-free (AF)-schizophrenia subjects, antipsychotic-treated (AT)-
schizophrenia subjects, and controls.
• Supplementary file 14. EpiSig analysis in NeuN-positive (NeuN+) nuclei from the frontal cortex of
antipsychotic-free (AF)-schizophrenia subjects and controls.
• Supplementary file 15. Differential H3K27ac/H3K4me3 peaks and DEGs obtained by comparing
antipsychotic-free (AF)-schizophrenia and antipsychotic-treated (AT)-schizophrenia with their
respective matched controls.
• Supplementary file 16. Genes altered in antipsychotic-free (AF-) but not antipsychotic-treated (AT)-
schizophrenics, and those altered in AT- but not AF-schizophrenics. The lists of genes were obtained
by differential analyses of enhancers, promoters, and RNA in AF-schizophrenia, AT-schizophrenia,
and their respective controls.
• Supplementary file 17. EpiSig analysis in NeuN-positive (NeuN+) nuclei from the frontal cortex of
antipsychotic-treated (AT)-schizophrenia subjects and controls.
• Supplementary file 18. EpiSig analysis in NeuN-negative (NeuN-) nuclei from the frontal cortex of
antipsychotic-treated (AT)-schizophrenia subjects and controls.
• Supplementary file 19. Effect of age on chromatin organization in the frontal cortex of AF-
schizophrenia subjects, AT-schizophrenia subjects, and controls.
• Supplementary file 20. Toxicological analysis in postmortem human brain samples.
• MDAR checklist
Data availability
The raw ChIP-seq and RNA-seq data were deposited in dbGaP under accession number phs002487.
v1.p1. The processed data can be accessed via Gene Expression Omnibus (GEO) under accession
number GSE174407. The code used for the analysis shown in this manuscript was deposited on
GitHub (copy archived at Chang Lu lab, 2023).
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