神经发育疾病 ATAC

… ATAC-seq enables genome-wide profiling of open chromatin regions, providing insights into active regulatory elements such as enhancers and promoters. It has become a powerful …

Abstract The neocortex is the region that most distinguishes human brain from other mammals and primates [Annu Rev Genet. 2021 Nov;55(1):555–81]. Studying the development of human cortex is important in understanding the evolutionary changes occurring in humans relative to other primates, as well as in elucidating mechanisms underlying neurodevelopmental disorders. Cortical development is a highly regulated process, spatially and temporally coordinated by expression of essential transcriptional factors in response to signaling pathways [Neuron. 2019 Sep;103(6):980–1004]. Enhancers are the most well-understood cis-acting, non-protein-coding regulatory elements that regulate gene expression [Nat Rev Genet. 2014 Apr;15(4):272–86]. Importantly, given the conservation of both DNA sequence and molecular function of the majority of proteins across mammals [Genome Res. 2003 Dec;13(12):2507–18], enhancers [Science. 2015 Mar;347(6226):1155–9], which are far more divergent at the sequence level, likely account for the phenotypes that distinguish the human brain by changing the regulation of gene expression. In this review, we will revisit the conceptual framework of gene regulation during human brain development, as well as the evolution of technologies to study transcriptional regulation, with recent advances in genome biology that open a window allowing us to systematically characterize cis-regulatory elements in developing human brain [Hum Mol Genet. 2022 Oct;31(R1):R84–96]. We provide an update on work to characterize the suite of all enhancers in the developing human brain and the implications for understanding neuropsychiatric disorders. Finally, we discuss emerging therapeutic ideas that utilize our emerging knowledge of enhancer function.

… disorder (ASD) is a prevalent neurodevelopmental disorder (CDC … ATAC-seq analyses underscore the importance of cell-type specific profiling, as between 25 and 40% of 3D chromatin …

The development of the human cerebral cortex is a complex and dynamic process, in which neural stem cell proliferation, neuronal migration, and post-migratory neuronal organization need to occur in a well-organized fashion. Alterations at any of these crucial stages can result in malformations of cortical development (MCDs), a group of genetically heterogeneous neurodevelopmental disorders that present with developmental delay, intellectual disability and epilepsy. Recent progress in genetic technologies, such as next generation sequencing, most often focusing on all protein-coding exons (e.g., whole exome sequencing), allowed the discovery of more than a 100 genes associated with various types of MCDs. Although this has considerably increased the diagnostic yield, most MCD cases remain unexplained. As Whole Exome Sequencing investigates only a minor part of the human genome (1–2%), it is likely that patients, in which no disease-causing mutation has been identified, could harbor mutations in genomic regions beyond the exome. Even though functional annotation of non-coding regions is still lagging behind that of protein-coding genes, tremendous progress has been made in the field of gene regulation. One group of non-coding regulatory regions are enhancers, which can be distantly located upstream or downstream of genes and which can mediate temporal and tissue-specific transcriptional control via long-distance interactions with promoter regions. Although some examples exist in literature that link alterations of enhancers to genetic disorders, a widespread appreciation of the putative roles of these sequences in MCDs is still lacking. Here, we summarize the current state of knowledge on cis-regulatory regions and discuss novel technologies such as massively-parallel reporter assay systems, CRISPR-Cas9-based screens and computational approaches that help to further elucidate the emerging role of the non-coding genome in disease. Moreover, we discuss existing literature on mutations or copy number alterations of regulatory regions involved in brain development. We foresee that the future implementation of the knowledge obtained through ongoing gene regulation studies will benefit patients and will provide an explanation to part of the missing heritability of MCDs and other genetic disorders.

Abstract The human brain is a complex organ comprised of distinct cell types, and the contribution of the 3D genome to lineage specific gene expression remains poorly understood. To decipher cell type specific genome architecture, and characterize fine scale changes in the chromatin interactome across neural development, we compared the 3D genome of the human fetal cortical plate to that of neurons and glia isolated from the adult prefrontal cortex. We found that neurons have weaker genome compartmentalization compared to glia, but stronger TADs, which emerge during fetal development. Furthermore, relative to glia, the neuronal genome shifts more strongly towards repressive compartments. Neurons have differential TAD boundaries that are proximal to active promoters involved in neurodevelopmental processes. CRISPRi on CNTNAP2 in hIPSC-derived neurons reveals that transcriptional inactivation correlates with loss of insulation at the differential boundary. Finally, re-wiring of chromatin loops during neural development is associated with transcriptional and functional changes. Importantly, differential loops in the fetal cortex are associated with autism GWAS loci, suggesting a neuropsychiatric disease mechanism affecting the chromatin interactome. Furthermore, neural development involves gaining enhancer-promoter loops that upregulate genes that control synaptic activity. Altogether, our study provides multi-scale insights on the 3D genome in the human brain.

The human brain develops through a tightly organized cascade of patterning events, induced by transcription factor expression and changes in chromatin accessibility. Although gene expression across the developing brain has been described at single-cell resolution1, similar atlases of chromatin accessibility have been primarily focused on the forebrain2, 3–4. Here we describe chromatin accessibility and paired gene expression across the entire developing human brain during the first trimester (6–13 weeks after conception). We defined 135 clusters and used multiomic measurements to link candidate cis-regulatory elements to gene expression. The number of accessible regions increased both with age and along neuronal differentiation. Using a convolutional neural network, we identified putative functional transcription factor-binding sites in enhancers characterizing neuronal subtypes. We applied this model to cis-regulatory elements linked to ESRRB to elucidate its activation mechanism in the Purkinje cell lineage. Finally, by linking disease-associated single nucleotide polymorphisms to cis-regulatory elements, we validated putative pathogenic mechanisms in several diseases and identified midbrain-derived GABAergic neurons as being the most vulnerable to major depressive disorder-related mutations. Our findings provide a more detailed view of key gene regulatory mechanisms underlying the emergence of brain cell types during the first trimester and a comprehensive reference for future studies related to human neurodevelopment. A study describes chromatin accessibility and paired gene expression across the entire developing human brain during the first trimester in the context of gene regulation and neurodevelopmental disease.

Chromatin accessibility to transcription factors (TFs) strongly influences gene transcription and cell differentiation. However, a mechanistic understanding of the transcriptional control during the neuronal differentiation of human induced pluripotent stem cells (hiPSCs), a promising cellular model for mental disorders, remains elusive. Here, we carried out additional analyses on our recently published open chromatin regions (OCRs) profiling at different stages of hiPSC neuronal differentiation. We found that the dynamic changes of OCR during neuronal differentiation highlighted cell stage-specific gene networks, and the chromatin accessibility at the core promoter region of a gene correlates with the corresponding transcript abundance. Within the cell stage-specific OCRs, we identified the binding of cell stage-specific TFs and observed a lag of a neuronal TF binding behind the mRNA expression of the corresponding TF. Interestingly, binding footprints of NEUROD1 and NEUROG2, both of which induce high efficient conversion of hiPSCs to glutamatergic neurons, were among those most enriched in the relatively mature neurons. Furthermore, TF network analysis showed that both NEUROD1 and NEUROG2 were present in the same core TF network specific to more mature neurons, suggesting a pivotal mechanism of epigenetic control of neuronal differentiation and maturation. Our study provides novel insights into the epigenetic control of glutamatergic neurogenesis in the context of TF networks, which may be instrumental to improving hiPSC modeling of neuropsychiatric disorders.

… , the Assay for Transposase Accessible Chromatin followed by sequencing (ATAC-seq), is being … and function and, in turn, to better understand the genetic basis of psychiatric disease. …

SUMMARY The prevalence of neurodevelopmental disorders (NDDs) in children is increasing, yet their underlying causes remain largely unknown. We identified heterozygous mutations in the Polycomb repressive complex 1 (PRC1) E3 ligases RING1 and RNF2 in individuals with NDDs and revealed distinct mechanisms by which they compromise PRC1 activity. We developed cellular and mouse models carrying the Ring1bR70H variant, which disrupts PRC1/PRC2 recruitment balance and mis-regulates Polycomb target genes. Allele-specific profiling showed that Ring1bR70H preferentially assembles into canonical PRC1 (cPRC1) via the intrinsically disordered region (IDR) of Pcgf2, reducing variant PRC1 (vPRC1) and PRC2.1 binding to chromatin. In Rnf2WT/R70H neuroprecursors, Polycomb complexes aberrantly suppress Wnt signaling, diverting neuroprecursors to non-neuronal lineages and halting neurogenesis. Rnf2R70H/R70H mice are perinatally lethal, while heterozygotes exhibit altered axonal organization, hippocampal and medial prefrontal cortex (mPFC) neuronal imbalances, reduced sociability, and increased anxiety. Our findings reveal an epigenetic mechanism essential for neurodevelopmental integrity and brain function and demonstrate how mutations in Rnf2 disrupt PRC1 occupancy at chromatin, contributing to NDDs.

Prioritizing disease-critical cell types by integrating genome-wide association studies (GWAS) with functional data is a fundamental goal. Single-cell chromatin accessibility (scATAC-seq) and gene expression (scRNA-seq) have characterized cell types at high resolution, and studies integrating GWAS with scRNA-seq have shown promise, but studies integrating GWAS with scATAC-seq have been limited. Here, we identify disease-critical fetal and adult brain cell types by integrating GWAS summary statistics from 28 brain-related diseases/traits (average N  = 298 K) with 3.2 million scATAC-seq and scRNA-seq profiles from 83 cell types. We identified disease-critical fetal (respectively adult) brain cell types for 22 (respectively 23) of 28 traits using scATAC-seq, and for 8 (respectively 17) of 28 traits using scRNA-seq. Significant scATAC-seq enrichments included fetal photoreceptor cells for major depressive disorder, fetal ganglion cells for BMI, fetal astrocytes for ADHD, and adult VGLUT2 excitatory neurons for schizophrenia. Our findings improve our understanding of brain-related diseases/traits and inform future analyses. This study analyzed data from human cells assayed using single-cell technologies, together with data associating genetic variants to disease, to identify fetal and brain cell types whose biologically critically influences the etiology of disease.

Neurodevelopmental disorders (NDDs) arise from disruptions in molecular programs that guide early brain formation, yet the specific epigenetic and transcriptional mechanisms underlying these conditions remain poorly defined. Recent advances in single-cell technologies allow parallel profiling of gene expression, chromatin accessibility, and DNA methylation within individual cells; however, most existing studies remain unimodal and therefore unable to resolve convergent dysregulation across molecular layers. This study presents an integrative single-cell multi-omics framework that combines scRNA-seq, scATAC-seq, and single-cell DNA methylation data from developing human and mouse brain tissue, as well as patient-derived neural progenitor models. By applying canonical correlation analysis, manifold alignment, latent-variable modeling, and network inference, we construct cell-type-specific epigenetic regulatory networks and quantify how chromatin accessibility, methylation state, and transcriptional output collectively deviate in NDD-relevant cell populations. Our analyses reveal that neural progenitors and excitatory neurons exhibit the strongest multimodal alterations, characterized by promoter hypermethylation, loss of enhancer accessibility, and downregulation of neurogenic and synaptic pathways. Integrative network modeling identifies SOX11 and CHD8 as central, multi-layer master regulators whose disrupted activity contributes to aberrant lineage specification. Quantitative evaluation of the Single-Cell Multi-Omics Network demonstrates high enhancer-gene linkage accuracy, consistent cross-species regulatory conservation, and efficient regulatory module reconstruction. Collectively, this integrative approach provides a unified view of epigenetic and transcriptional dysregulation in NDDs, generating mechanistic hypotheses with potential implications for biomarker discovery and targeted therapeutic intervention.

Chromatin-related phenomena regulate gene expression by altering the compaction and accessibility of DNA to relevant transcription factors, thus allowing every cell in the body to attain distinct identities and to function properly within a given cellular context. These processes occur not only in the developing central nervous system, but continue throughout the lifetime of a neuron to constantly adapt to changes in the environment. Such changes can be positive or negative, thereby altering the chromatin landscape to influence cellular and synaptic plasticity within relevant neural circuits, and ultimately behavior. Given the importance of epigenetic mechanisms in guiding physiological adaptations, perturbations in these processes in brain have been linked to several neuropsychiatric and neurological disorders. In this review, we cover some of the recent advances linking chromatin dynamics to complex brain disorders and discuss new methodologies that may overcome current limitations in the field.

DNA sequence variants (single nucleotide polymorphisms or variants, SNPs/SNVs; copy number variants, CNVs) associated to neurodevelopmental disorders (NDD) and traits often map on putative transcriptional regulatory elements, including, in particular, enhancers. However, the genes controlled by these enhancers remain poorly defined. Traditionally, the activity of a given enhancer, and the effect of its possible alteration associated to the sequence variants, has been thought to influence the nearest gene promoter. However, the obtainment of genome-wide long-range interaction maps in neural cells chromatin challenged this view, showing that a given enhancer is very frequently not connected to the nearest promoter, but to a more distant one, skipping genes in between. In this Perspective, we review some recent papers, who generated long-range interaction maps (by HiC, RNApolII ChIA-PET, Capture-HiC, or PLACseq), and overlapped the identified long-range interacting DNA segments with DNA sequence variants associated to NDD (such as schizophrenia, bipolar disorder and autism) and traits (intelligence). This strategy allowed to attribute the function of enhancers, hosting the NDD-related sequence variants, to a connected gene promoter lying far away on the linear chromosome map. Some of these enhancer-connected genes had indeed been already identified as contributive to the diseases, by the identification of mutations within the gene’s protein-coding regions (exons), validating the approach. Significantly, however, the connected genes also include many genes that were not previously found mutated in their exons, pointing to novel candidate contributors to NDD and traits. Thus, long-range interaction maps, in combination with DNA variants detected in association with NDD, can be used as “pointers” to identify novel candidate disease-relevant genes. Functional manipulation of the long-range interaction network involving enhancers and promoters by CRISPR-Cas9-based approaches is beginning to probe for the functional significance of the identified interactions, and the enhancers and the genes involved, improving our understanding of neural development and its pathology.

Genetic variants associated with autism spectrum disorders (ASDs) are enriched in genes encoding synaptic proteins and chromatin regulators. Although the role of synaptic proteins in ASDs is widely studied, the mechanism by which chromatin regulators contribute to ASD risk remains poorly understood. Upon profiling and analyzing the transcriptional and epigenomic features of genes expressed in the cortex, we uncovered a unique set of long genes that contain broad enhancer-like chromatin domains (BELDs) spanning across their entire gene bodies. Analyses of these BELD genes show that they are highly transcribed with frequent RNA polymerase II (Pol II) initiation and low Pol II pausing, and they exhibit frequent chromatin–chromatin interactions within their gene bodies. These BELD features are conserved from rodents to humans, are enriched in genes involved in synaptic function, and appear post-natally concomitant with synapse development. Importantly, we find that BELD genes are highly implicated in neurodevelopmental disorders, particularly ASDs, and that their expression is preferentially down-regulated in individuals with idiopathic autism. Finally, we find that the transcription of BELD genes is particularly sensitive to alternations in ASD-associated chromatin regulators. These findings suggest that the epigenomic regulation of BELD genes is important for post-natal cortical development and lend support to a model by which mutations in chromatin regulators causally contribute to ASDs by preferentially impairing BELD gene transcription.

Autism is highly heritable and diagnosed more frequently in males than females. To identify neurodevelopmental processes that might present sex-biased vulnerability, we generated transcriptomic and epigenomic profiles of cell types present in the prenatally developing human cerebral cortex of 27 males and 21 females. By intersecting sex-biased molecular signatures and genes with de novo mutations in male and female autistic probands, we reveal two points of vulnerability contributing to the sex-biased penetrance in neurodevelopmental disorders (NDDs). First, we show that NDD risk genes are biased towards higher expression in females, identifying the NDD gene MEF2C as a critical transcription factor for female-biased expression. Second, we identify a significant contribution of X chromosome genes to NDD pathobiology. We construct a gene regulatory map of X-linked risk genes to enable functional studies of genetic variants that likely disrupt gene expression in the developing brains of autistic males. Together, these results point towards an outsized contribution of the X-chromosome to both the origin of sex differences in the developing human cortex and NDD vulnerability. We propose a model where female-biased vulnerability is driven by coding variation within genes while male-biased vulnerability is driven by noncoding variation in regulatory elements that affect gene expression.

De novo mutations in POGZ, which encodes the chromatin regulator Pogo Transposable Element with ZNF Domain protein, are strongly associated with autism spectrum disorder (ASD). Here we find that in the developing mouse and human brain POGZ binds predominantly euchromatic loci and these are enriched for human neurodevelopmental disorder genes and transposable elements. We profile chromatin accessibility and gene expression in Pogz−/− mice and find that POGZ promotes chromatin accessibility of candidate regulatory elements (REs) and the expression of clustered synaptic genes. We further demonstrate that POGZ forms a nuclear complex and co-occupies loci with HP1γ and ADNP, another high-confidence ASD risk gene. In Pogz+/− mice, Adnp expression is reduced. We postulate that reduced POGZ dosage disrupts cortical function through alterations in the POGZ-ADNP balance which modifies neuronal gene expression.

… a cellular model for studying neurodevelopmental disorders such as … We mapped open chromatin by ATAC-seq in hiPSCs and … To assure the neuronal identity of the ATAC-seq data, we …

Neurodevelopmental disorders (NDDs), including intellectual disability (ID) and autism spectrum disorders (ASD), are a large group of disorders in which early insults during brain development result in a wide and heterogeneous spectrum of clinical diagnoses. Mutations in genes coding for chromatin remodelers are overrepresented in NDD cohorts, pointing towards epigenetics as a convergent pathogenic pathway between these disorders. In this review we detail the role of NDD-associated chromatin remodelers during the developmental continuum of progenitor expansion, differentiation, cell-type specification, migration and maturation. We discuss how defects in chromatin remodelling during these early developmental time points compound over time and result in impaired brain circuit establishment. In particular, we focus on their role in the three largest cell populations: glutamatergic neurons, GABAergic neurons, and glia cells. An in-depth understanding of the spatiotemporal role of chromatin remodelers during neurodevelopment can contribute to the identification of molecular targets for treatment strategies.

Neurodevelopmental diseases (NDDs), such as autism spectrum disorders, epilepsy, and schizophrenia, are characterized by diverse facets of neurological and psychiatric symptoms, differing in etiology, onset and severity. Such symptoms include mental delay, cognitive and language impairments, or restrictions to adaptive and social behavior. Nevertheless, all have in common that critical milestones of brain development are disrupted, leading to functional deficits of the central nervous system and clinical manifestation in child- or adulthood. To approach how the different development-associated neuropathologies can occur and which risk factors or critical processes are involved in provoking higher susceptibility for such diseases, a detailed understanding of the mechanisms underlying proper brain formation is required. NDDs rely on deficits in neuronal identity, proportion or function, whereby a defective development of the cerebral cortex, the seat of higher cognitive functions, is implicated in numerous disorders. Such deficits can be provoked by genetic and environmental factors during corticogenesis. Thereby, epigenetic mechanisms can act as an interface between external stimuli and the genome, since they are known to be responsive to external stimuli also in cortical neurons. In line with that, DNA methylation, histone modifications/variants, ATP-dependent chromatin remodeling, as well as regulatory non-coding RNAs regulate diverse aspects of neuronal development, and alterations in epigenomic marks have been associated with NDDs of varying phenotypes. Here, we provide an overview of essential steps of mammalian corticogenesis, and discuss the role of epigenetic mechanisms assumed to contribute to pathophysiological aspects of NDDs, when being disrupted.

The nucleosome remodeling and deacetylase (NuRD) complex presents one of the major chromatin remodeling complexes in mammalian cells. Here, we discuss current evidence for NuRD’s role as an important epigenetic regulator of gene expression in neural stem cell (NSC) and neural progenitor cell (NPC) fate decisions in brain development. With the formation of the cerebellar and cerebral cortex, NuRD facilitates experience-dependent cerebellar plasticity and regulates additionally cerebral subtype specification and connectivity in postmitotic neurons. Consistent with these properties, genetic variation in NuRD’s subunits emerges as important risk factor in common polygenic forms of neurodevelopmental disorders (NDDs) and neurodevelopment-related psychiatric disorders such as schizophrenia (SCZ) and bipolar disorder (BD). Overall, these findings highlight the critical role of NuRD in chromatin regulation in brain development and in mental health and disease.

… Chromatin accessibility is elevated at known forebrain enhancers and reduced at … To identify which OCRs are likely to function as neurodevelopmental enhancers, we developed a …

Emerging sequencing studies highlight the critical role of chromatin regulatory mechanisms in human diseases, particularly in neurodevelopmental and neurological disorders. Insights gained from these studies and model organism research reveal the intricate involvement of chromatin regulators in neurodevelopment, raising compelling questions about how mutations in these ubiquitous proteins drive specific dysfunctions in the nervous system. This mini review delves into key chromatin modifiers, including the histone methyl transferases NSD1 and ASH1L, the methyl-CpG-binding repressor MeCP2, and the enzymatic repressor EZH2. While functions of these proteins are relatively well-studied, the roles of many other chromatin modifiers in neurodevelopment remain poorly understood. Existing therapies targeting chromatin modifiers have shown promise, with some achieving significant clinical success. The possibility that neurological dysfunctions may be treatable even later in life underscores the urgency of prioritizing chromatin modifiers as therapeutic targets. In this mini review, we critically evaluate the current understanding of chromatin modifiers, focusing on methylation, and spotlight their pivotal roles in early brain development and neurological disorders. By advancing this field, we aim to inspire progress toward innovative treatments for these challenging conditions.

Organoids recapitulate brain development Gene expression changes and their control by accessible chromatin in the human brain during development is of great interest but limited accessibility. Trevino et al. avoided this problem by developing three-dimensional organoid models of human forebrain development and examining chromatin accessibility and gene expression at the single-cell level. From this analysis, they matched developmental profiles between the organoid and fetal samples, identified transcription factor binding profiles, and predicted how transcription factors are linked to cortical development. The researchers were able to correlate the expression of neurodevelopmental disease risk loci and genes with specific cell types during development. Science, this issue p. eaay1645 Organoids recapitulate the chromatin accessibility profiles of the developing human forebrain. INTRODUCTION The cerebral cortex is responsible for higher-order functions in the nervous system and has undergone substantial expansion in size in primates. The development of the forebrain, including the assembly of the expanded human cerebral cortex, is a lengthy process that involves the diversification and expansion of neural progenitors, the generation and positioning of layer-specific glutamatergic neurons, the cellular migration of γ-aminobutyric acid (GABA)–ergic neurons, and the formation and maturation of glial cells. Disruption of these cellular events by either genetic or environmental factors can lead to neurodevelopmental disease, including autism spectrum disorders and intellectual disability. RATIONALE Human forebrain development is, to a large extent, inaccessible for cellular-level study, direct functional investigation, or manipulation. The lack of availability of primary brain tissue samples—in particular, at later stages—as well as the limitations of conventional in vitro cellular models have precluded a detailed mechanistic understanding of corticogenesis in healthy and disease states. Therefore, tracking epigenetic changes in specific forebrain cell lineages over long time periods, has the potential to unravel the molecular programs that underlie cell specification in the human cerebral cortex and, by temporally mapping disease risk onto these changes, to identify cell types and periods of increased disease susceptibility. RESULTS We used three-dimensional (3D) directed differentiation of human pluripotent stem cells into dorsal and ventral forebrain domains and applied the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) in combination with RNA-sequencing (RNA-seq) to map the epigenetic and gene expression signatures of neuronal and glial cell lineages over 20 months in vitro. We show, through direct comparison with primary brain tissue from our study and several epigenetic datasets, that human stem cell–derived 3D forebrain organoids recapitulated in vivo chromatin accessibility patterns over time. We then integrated these data to discover putative enhancer-gene linkages and lineage-specific transcription factor regulators, including a diverse repertoire of factors that may control cortical specification. We validated protein expression of some of these transcription factors using immunofluorescence, confirming cellular and temporal dynamics in both primary tissue and forebrain organoids. Next, we used this resource to map genes and genetic variants associated with schizophrenia and autism spectrum disorders to distinct accessibility patterns to reveal cell types and periods of susceptibility. Last, we identified a wave of chromatin remodeling during cortical neurogenesis, during which a quarter of regulatory regions are active, and then highlighted transcription factors that may drive these developmental changes. CONCLUSION Using long-term 3D neural differentiation of stem cells as well as primary brain tissue samples, we found that organoids intrinsically undergo chromatin state transitions in vitro that are closely related to human forebrain development in vivo. Leveraging this platform, we identified epigenetic alterations putatively driven by specific transcription factors and discovered a dynamic period of chromatin remodeling during human cortical neurogenesis. Finally, we nominated several key transcription factors that may coordinate over time to drive these changes and mapped cell type–specific disease-associated variation over time and in specific cell types. Developing a human cellular model of forebrain development to study chromatin dynamics. ATAC-seq and RNA-seq studies over long-term differentiation of human pluripotent stem cells into forebrain organoids and in primary brain tissue samples reveal dynamic changes during human corticogenesis, including a wave associated with neurogenesis, and identify disease-susceptible cell types and stages. Forebrain development is characterized by highly synchronized cellular processes, which, if perturbed, can cause disease. To chart the regulatory activity underlying these events, we generated a map of accessible chromatin in human three-dimensional forebrain organoids. To capture corticogenesis, we sampled glial and neuronal lineages from dorsal or ventral forebrain organoids over 20 months in vitro. Active chromatin regions identified in human primary brain tissue were observed in organoids at different developmental stages. We used this resource to map genetic risk for disease and to explore evolutionary conservation. Moreover, we integrated chromatin accessibility with transcriptomics to identify putative enhancer-gene linkages and transcription factors that regulate human corticogenesis. Overall, this platform brings insights into gene-regulatory dynamics at previously inaccessible stages of human forebrain development, including signatures of neuropsychiatric disorders.

Abstract The etiology of neurodevelopmental disorders (NDDs) remains a challenge for researchers. Human brain development is tightly regulated and sensitive to cellular alterations caused by endogenous or exogenous factors. Intriguingly, the surge of clinical sequencing studies has revealed that many of these disorders are monogenic and monoallelic. Notably, chromatin regulation has emerged as highly dysregulated in NDDs, with many syndromes demonstrating phenotypic overlap, such as intellectual disabilities, with one another. Here we discuss epigenetic writers, erasers, readers, remodelers, and even histones mutated in NDD patients, predicted to affect gene regulation. Moreover, this review focuses on disorders associated with mutations in enzymes involved in histone acetylation and methylation, and it highlights syndromes involving chromatin remodeling complexes. Finally, we explore recently discovered histone germline mutations and their pathogenic outcome on neurological function. Epigenetic regulators are mutated at every level of chromatin organization. Throughout this review, we discuss mechanistic investigations, as well as various animal and iPSC models of these disorders and their usefulness in determining pathomechanism and potential therapeutics. Understanding the mechanism of these mutations will illuminate common pathways between disorders. Ultimately, classifying these disorders based on their effects on the epigenome will not only aid in prognosis in patients but will aid in understanding the role of epigenetic machinery throughout neurodevelopment.

In postmitotic neurons, nucleosomal turnover was long considered to be a static process that is inconsequential to transcription. However, our recent studies in human and rodent brain …

Chromatin, a protein–DNA complex, is a dynamic structure that stores genetic information within the nucleus and responds to molecular/cellular changes in its structure, providing conditional access to the genetic machinery. ATP-dependent chromatin modifiers regulate access of transcription factors and RNA polymerases to DNA by either “opening” or “closing” the structure of chromatin, and its aberrant regulation leads to a variety of neurodevelopmental disorders. The chromodomain helicase DNA-binding (CHD) proteins are ATP-dependent chromatin modifiers involved in the organization of chromatin structure, act as gatekeepers of genomic access, and deposit histone variants required for gene regulation. In this review, we first discuss the structural and functional domains of the CHD proteins, and their binding sites, and phosphorylation, acetylation, and methylation sites. The conservation of important amino acids in SWItch/sucrose non-fermenting (SWI/SNF) domains, and their protein and mRNA tissue expression profiles are discussed. Next, we convey the important binding partners of CHD proteins, their protein complexes and activities, and their involvements in epigenetic regulation. We also show the ChIP-seq binding dynamics for CHD1, CHD2, CHD4, and CHD7 proteins at promoter regions of histone genes, as well as several genes that are critical for neurodevelopment. The role of CHD proteins in development is also discussed. Finally, this review provides information about CHD protein mutations reported in autism and neurodevelopmental disorders, and their pathogenicity. Overall, this review provides information on the progress of research into CHD proteins, their structural and functional domains, epigenetics, and their role in stem cell, development, and neurological disorders.

… They found that depletion of Anp32e caused increased genome-wide chromatin accessibility and increased H2A.Z binding at promoters. Importantly, Gene Ontology (GO) analysis …

The human brain is capable of highly complex functions that develops through a tightly organized cascade of patterning events, expressed transcription factors and changes in chromatin accessibility. While extensive datasets exist describing gene expression across the developing brain with single-cell resolution, similar atlases of chromatin accessibility have been primarily focused on the forebrain. Here, we focus on the chromatin landscape and paired gene expression across the developing human brain to provide a comprehensive single cell atlas during the first trimester (6 - 13 post-conceptional weeks). We identified 135 clusters across half a million nuclei and using the multiomic measurements linked candidate cis-regulatory elements (cCREs) to gene expression. We found an increase in the number of accessible regions driven both by age and neuronal differentiation. Using a convolutional neural network we identified putative functional TF-binding sites in enhancers characterizing neuronal subtypes and we applied this model to cCREs upstream of ESRRB to elucidate its activation mechanism. Finally, by linking disease-associated SNPs to cCREs we validated putative pathogenic mechanisms in several diseases and identified midbrain-derived GABAergic neurons as being the most vulnerable to major depressive disorder related mutations. Together, our findings provide a higher degree of detail to some key gene regulatory mechanisms underlying the emergence of cell types during the first trimester. We anticipate this resource to be a valuable reference for future studies related to human neurodevelopment, such as identifying cell type specific enhancers that can be used for highly specific targeting in in vitro models.

The Nucleosome Remodelling and Deacetylase (NuRD) complex represents one of the major chromatin remodelling complexes in mammalian cells, uniquely coupling the ability to “open” the chromatin by inducing nucleosome sliding with histone deacetylase activity. At the core of the NuRD complex are a family of ATPases named CHDs that utilise the energy produced by the hydrolysis of the ATP to induce chromatin structural changes. Recent studies have highlighted the prominent role played by the NuRD in regulating gene expression during brain development and in maintaining neuronal circuitry in the adult cerebellum. Importantly, components of the NuRD complex have been found to carry mutations that profoundly affect neurological and cognitive development in humans. Here, we discuss recent literature concerning the molecular structure of NuRD complexes and how the subunit composition and numerous permutations greatly determine their functions in the nervous system. We will also discuss the role of the CHD family members in an array of neurodevelopmental disorders. Special emphasis will be given to the mechanisms that regulate the NuRD complex composition and assembly in the cortex and how subtle mutations may result in profound defects of brain development and the adult nervous system.

DNA sequencing-based studies of neurodevelopmental disorders (NDDs) have identified a wide range of genetic determinants. However, a comprehensive analysis of these data, in aggregate, has not to date been performed. Here, we find that genes encoding the mammalian SWI/SNF (mSWI/SNF or BAF) family of ATP-dependent chromatin remodeling protein complexes harbor the greatest number of de novo missense and protein-truncating variants among nuclear protein complexes. Non-truncating NDD-associated protein variants predominantly disrupt the cBAF subcomplex and cluster in four key structural regions associated with high disease severity, including mSWI/SNF-nucleosome interfaces, the ATPase-core ARID-armadillo repeat (ARM) module insertion site, the Arp module and DNA-binding domains. Although over 70% of the residues perturbed in NDDs overlap with those mutated in cancer, ~60% of amino acid changes are NDD-specific. These findings provide a foundation to functionally group variants and link complex aberrancies to phenotypic severity, serving as a resource for the chromatin, clinical genetics and neurodevelopment communities. Genes encoding members of mammalian SWI/SNF (BAF) complexes are frequently mutated in individuals with neurodevelopmental disorders (NDDs). Mutant NDD residues include some unique to NDD and those shared with human cancers, impacting key structural hubs.

The fetal brain is adapted to the hypoxic conditions present during normal in utero development. Relatively more hypoxic states, either chronic or acute, are pathologic and can lead to significant long-term neurodevelopmental sequelae. In utero hypoxic injury is associated with neonatal mortality and millions of lives lived with varying degrees of disability. Genetic studies of children with neurodevelopmental disease indicate that epigenetic modifiers regulating DNA methylation and histone remodeling are critical for normal brain development. Epigenetic modifiers are also regulated by environmental stimuli, such as hypoxia. Indeed, epigenetic modifiers that are mutated in children with genetic neurodevelopmental diseases are regulated by hypoxia in a number of preclinical models and may be part of the mechanism for the long-term neurodevelopmental sequelae seem in children with hypoxic brain injury. Thus, a comprehensive understanding the role of DNA methylation and histone modifications in hypoxic injury is critical for developing novel strategies to treat children with hypoxic injury. This review focuses on our current understanding of the intersection between epigenetics, brain development, and hypoxia. Opportunities for the use of epigenetics as biomarkers of neurodevelopmental disease after hypoxic injury and potential clinical epigenetics targets to improve outcomes after injury are also discussed. While there have been many published studies on the epigenetics of hypoxia, more are needed in the developing brain in order to determine which epigenetic pathways may be most important for mitigating the long-term consequences of hypoxic brain injury.

Abstract Neurodevelopment can be precisely regulated by epigenetic mechanisms, including DNA methylations, noncoding RNAs, and histone modifications. Histone methylation was a reversible modification, catalyzed by histone methyltransferases and demethylases. So far, dozens of histone lysine demethylases (KDMs) have been discovered, and they (members from KDM1 to KDM7 family) are important for neurodevelopment by regulating cellular processes, such as chromatin structure and gene transcription. The role of KDM5C and KDM7B in neural development is particularly important, and mutations in both genes are frequently found in human X-linked mental retardation (XLMR). Functional disorders of specific KDMs, such as KDM1A can lead to the development of neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Several KDMs can serve as potential therapeutic targets in the treatment of neurodegenerative diseases. At present, the function of KDMs in neurodegenerative diseases is not fully understood, so more comprehensive and profound studies are needed. Here, the role and mechanism of histone demethylases were summarized in neurodevelopment, and the potential of them was introduced in the treatment of neurodegenerative diseases.

… of the dynamic changes underlying neurodevelopment. Utilizing ATAC-seq, neuronal activity … As many of the disease-associated SNPs in neurodevelopmental disorders are found in …

Neurodevelopmental disorders (NDDs) affect about 1% of the population and can be caused by mutations in genes that affect the epigenetic code. There is limited functional understanding of most of these epigenetic modifiers, and we suggest that associated NDDs are caused, in part, by deficits in epigenetic priming, a prepatterning step that alters the genome in preparation to make cells competent to signaling cues. We provide evidence from high-resolution epigenetic and transcriptomic mapping studies to demonstrate how a failure to adequately prime the genome for neural induction could lead to impairment of terminally differentiated cells. This idea provides a framework for NDD pathogenesis and treatment.

… ATAC-seq was implemented in this study to elucidate chromatin landscape of genes of interest in cells that are intermediate in the reprogramming process, to understand transcription …

The histone variant H3.3 K27M mutation is a defining characteristic of diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). This histone mutation is responsible for major alterations to histone H3 post-translational modification (PTMs) and subsequent aberrant gene expression. However, much less is known about the effect this mutation has on chromatin structure and function, including open versus closed chromatin regions as well as their transcriptomic consequences. Recently, we developed isogenic CRISPR-edited DIPG cell lines that are wild-type for histone H3.3 that can be compared to their matched K27M lines. Here we show via ATAC-seq analysis that H3.3K27M glioma cells have unique accessible chromatin at regions corresponding to neurogenesis, NOTCH, and neuronal development pathways and associated genes that are overexpressed in H3.3K27M compared to our isogenic wild-type cell line. As to mechanisms, accessible enhancers and super-enhancers corresponding to increased gene expression in H3.3K27M cells were also mapped to genes involved in neurogenesis and NOTCH signaling, suggesting that these pathways are key to DIPG tumor maintenance. Motif analysis implicates specific transcription factors as central to the neuro-oncogenic K27M signaling pathway, in particular, ASCL1 and NEUROD1. Altogether our findings indicate that H3.3K27M causes chromatin to take on a more accessible configuration at key regulatory regions for NOTCH and neurogenesis genes resulting in increased oncogenic gene expression, which is at least partially reversible upon editing K27M back to wild-type.

… ATP-dependent chromatin remodeling complex, which disrupts histone-DNA contacts and … of ATP-dependent chromatin remodeling complexes during mammalian development. In …

The generation of individual neurons (neurogenesis) during cortical development occurs in discrete steps that are subtly regulated and orchestrated to ensure normal histogenesis and function of the cortex. Notably, various gene expression programs are known to critically drive many facets of neurogenesis with a high level of specificity during brain development. Typically, precise regulation of gene expression patterns ensures that key events like proliferation and differentiation of neural progenitors, specification of neuronal subtypes, as well as migration and maturation of neurons in the developing cortex occur properly. ATP-dependent chromatin remodeling complexes regulate gene expression through utilization of energy from ATP hydrolysis to reorganize chromatin structure. These chromatin remodeling complexes are characteristically multimeric, with some capable of adopting functionally distinct conformations via subunit reconstitution to perform specific roles in major aspects of cortical neurogenesis. In this review, we highlight the functions of such chromatin remodelers during cortical development. We also bring together various proposed mechanisms by which ATP-dependent chromatin remodelers function individually or in concert, to specifically modulate vital steps in cortical neurogenesis.

… Chromatin modifications occurring at different time points during the life of an organism … regulatory events that affect the development and the function of the brain and other tissues. …

… There is increasing evidence that ATP-dependent chromatin remodeling complexes based … during neural development in both vertebrates and invertebrates. This remodeling complex …

The ATP-dependent BRG1/BRM associated factor (BAF) chromatin remodeling complexes are crucial in regulating gene expression by controlling chromatin dynamics. Over the last decade, it has become increasingly clear that during neural development in mammals, distinct ontogenetic stage-specific BAF complexes derived from combinatorial assembly of their subunits are formed in neural progenitors and post-mitotic neural cells. Proper functioning of the BAF complexes plays critical roles in neural development, including the establishment and maintenance of neural fates and functionality. Indeed, recent human exome sequencing and genome-wide association studies have revealed that mutations in BAF complex subunits are linked to neurodevelopmental disorders such as Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, Kleefstra's syndrome spectrum, Hirschsprung's disease, autism spectrum disorder, and schizophrenia. In this review, we focus on the latest insights into the functions of BAF complexes during neural development and the plausible mechanistic basis of how mutations in known BAF subunits are associated with certain neurodevelopmental disorders.

… roles that chromatin remodelers play in specific tissues and at specific stages of development… roles in metazoan development of 3 major subfamilies of chromatin-remodeling complexes: …

… chromatin is part of this epigenetic memory that restricts or permits differential expression of genes in descendant cells. Establishing a cell-type-specific chromatin … chromatin remodeling …

… chromatin, which consists of DNA and protein condensed into nucleoprotein complexes [5]. The fundamental packaging unit of chromatin … -dependent chromatin remodeling complexes …

… to a range of neurodevelopmental disorders. In this study, we … cell types analyzed by scATAC-seq. E Heatmap showing … browser track represents all scATAC-seq peaks in this study. …

In mammals, early organogenesis begins soon after gastrulation, accompanied by specification of various type of progenitor/precusor cells. In order to reveal dynamic chromatin landscape of precursor cells and decipher the underlying molecular mechanism driving early mouse organogenesis, we performed single-cell ATAC-seq of E8.5-E10.5 mouse embryos. We profiled a total of 101,599 single cells and identified 41 specific cell types at these stages. Besides, by performing integrated analysis of scATAC-seq and public scRNA-seq data, we identified the critical cis-regulatory elements and key transcription factors which drving development of spinal cord and somitogenesis. Furthermore, we intersected accessible peaks with human diseases/traits-related loci and found potential clinical associated single nucleotide variants (SNPs). Overall, our work provides a fundamental source for understanding cell fate determination and revealing the underlying mechanism during postimplantation embryonic development, and expand our knowledge of pathology for human developmental malformations.

Abstract Background Focal cortical dysplasia (FCD) is a heterogeneous group of cortical developmental malformations that constitute a common cause of medically intractable epilepsy. FCD type IIIa (FCD IIIa) refers to temporal neocortex alterations in architectural organisation or cytoarchitectural composition in the immediate vicinity of hippocampal sclerosis. Slight alterations in the temporal neocortex of FCD IIIa patients pose a challenge for the preoperative diagnosis and definition of the resection range. Methods We have performed multimodal integration of single‐nucleus RNA sequencing and single‐nucleus assay for transposase‐accessible chromatin sequencing in the epileptogenic cortex of four patients with FCD IIIa, and three relatively normal temporal neocortex were chosen as controls. Results Our study revealed that the most significant dysregulation occurred in excitatory neurons (ENs) and oligodendrocyte precursor cells (OPCs) in the epileptogenic cortex of FCD IIIa patients. In ENs, we constructed a transcription factor (TF)‐hub gene regulatory network and found DAB1 high ENs subpopulation mediates neuronal immunity characteristically in FCD IIIa. Western blotting and immunofluorescence were used to validate the changes in protein expression levels caused by some of the key genes. The OPCs were activated and exhibited aberrant phenotypes in FCD IIIa, and TFs regulating reconstructed pseudotime trajectory were identified. Finally, our results revealed aberrant intercellular communication between ENs and OPCs in FCD IIIa patients. Conclusions Our study revealed significant and intricate alterations in the transcriptomes and epigenomes in ENs and OPCs of FCD IIIa patients, shedding light on their cell type‐specific regulation and potential pathogenic involvement in this disorder. This work will help evaluate the pathogenesis of cortical dysplasia and epilepsy and explore potential therapeutic targets. Key points Paired snRNA‐seq and snATAC‐seq data were intergrated and analysed to identify crucial subpopulations of ENs and OPCs in the epileptogenic cortex of FCD IIIa patients and explore their possible pathogenic role in the disease. A TF‐hub gene regulatory network was constructed in ENs, and the DAB1high Ex‐1 mediated neuronal immunity was characterstically in FCD IIIa patients. The OPCs were activated and exhibited aberrant phenotypes in FCD IIIa patients, and TFs regulating reconstructed pseudotime traectory were identified. Aberrant intercelluar communications between ENs and OPCs in FCD IIIa patients were identified.

Genomic profiling in postmortem brain from autistic individuals has consistently revealed convergent molecular changes. What drives these changes and how they relate to genetic susceptibility in this complex condition are not well understood. We performed deep single-nucleus RNA sequencing (snRNA-seq) to examine cell composition and transcriptomics, identifying dysregulation of cell type–specific gene regulatory networks (GRNs) in autism spectrum disorder (ASD), which we corroborated using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) and spatial transcriptomics. Transcriptomic changes were primarily cell type specific, involving multiple cell types, most prominently interhemispheric and callosal-projecting neurons, interneurons within superficial laminae, and distinct glial reactive states involving oligodendrocytes, microglia, and astrocytes. Autism-associated GRN drivers and their targets were enriched in rare and common genetic risk variants, connecting autism genetic susceptibility and cellular and circuit alterations in the human brain. INTRODUCTION Historically, psychiatric disorders have been distinguished from neurological disorders by the absence of the associated histological pathology observed in neurological conditions. But, over the past 15 years, epigenetic and transcriptional profiling of postmortem brain samples from multiple psychiatric conditions, including autism spectrum disorder (ASD), have revealed robust underlying molecular differences. In ASD, this reflects up-regulation of immune signaling genes, down-regulation of neuronal markers and synaptic genes, and a blunting of gene expression signatures of cortical regional identity. How a genetically complex condition such as ASD converges on shared transcriptional alterations remains a mystery. This gap is further amplified by the lack of biological insights into the differences in laminar and cell type–specific pathways affected in ASD and the underlying gene regulatory mechanisms. RATIONALE We reasoned that deep molecular profiling at the single-cell level would inform our understanding of changes in cortical cell types and circuits and, when integrated with genetic risk factors, enable identification of candidate drivers of pathways altered in ASD. Further, knowledge of these pathways at a cellular level would inform mechanistically driven therapeutic development. RESULTS We conducted single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) in a large ASD and control (CTL) cohort, as a core component of the PsychENCODE Consortium (https://www.psychencode.org/), to identify cell type–specific changes and the cellular regulatory networks perturbed by genetic risk factors. Our approach (on average >10,000 cells per individual and >1860 genes per cell) enabled identification of all 26 major cortical cell types, validated with published cortical cell atlases. We also identified cell states, primarily activated forms of microglia (MG), oligodendrocytes (ODCs), astrocytes (ASTROs), and blood-brain barrier cells, observed predominantly in ASD. Changes in cell composition in ASD were subtle, involving increases in activated microglia and astrocyte states, which are very rarely observed in controls. In contrast to the minor changes in cell composition, the changes observed in gene expression in ASD were substantial: 2166 down-regulated and 1319 up-regulated genes across 35 cell types, most of which were cell type specific. Through integration of snRNA-seq, snATAC-seq, and spatial transcriptomics, we identified regulatory networks driving cell type–specific transcriptional changes and their location within cortical laminae. These analyses demonstrate concentration of activated microglia, astrocytes, and somatostatin (SST) interneurons in superficial cortical laminae, in conjunction with profound down-regulation of synaptic gene expression and up-regulation of stress-response and proinflammatory pathways in inter- and intrahemispheric projection neurons. A large proportion of these changes could be ascribed to specific transcriptional drivers, and both the drivers and their targets were enriched in genes harboring common and rare genetic risk for ASD. CONCLUSION These analyses refine our knowledge of cellular and circuit alterations in the brain in ASD. By identifying and validating transcriptional drivers enriched in rare and common genetic risk variants, we have discovered a link between autism genetic susceptibility and molecular and cellular circuits and pathways, providing a roadmap for understanding cellular interactions and therapeutic development in ASD. Single-cell genomics reveals cell type–specific and laminar changes in ASD. These changes prominently affect layers 2 and 3 interhemispheric and callosal-projecting excitatory (Ex) neurons, superficial SST interneurons, and reactive glial states in the frontal cortex (FC). By defining gene regulatory networks (GRNs; red, up-regulated; blue, down-regulated) and integrating them with ASD genetic risk variants, we discerned candidate drivers of the transcriptional changes and genetic susceptibility acting in specific cell types. OPCs, oligodendrocyte progenitor cells; INTs, inhibitory neurons; WM, white matter; DEGs, differentially expressed genes. [Created with Biorender.com]

Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN) cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generate single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. We evaluate enhancer activity for 59 elements using an in vivo transgenic assay and validate 44 (75%), demonstrating that single cell accessibility can be a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieve significant reduction in our variant search space and nominate candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 – as well as candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work delivers non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders. Here, the authors present a multi-omic framework using single cell technology to identify non-coding genetic variants in cranial motor neuron disorders, offering insights into their genetic basis and methods for studying other Mendelian diseases.

During mammalian development, differences in chromatin state coincide with cellular differentiation and reflect changes in the gene regulatory landscape1. In the developing brain, cell fate specification and topographic identity are important for defining cell identity2 and confer selective vulnerabilities to neurodevelopmental disorders3. Here, to identify cell-type-specific chromatin accessibility patterns in the developing human brain, we used a single-cell assay for transposase accessibility by sequencing (scATAC-seq) in primary tissue samples from the human forebrain. We applied unbiased analyses to identify genomic loci that undergo extensive cell-type- and brain-region-specific changes in accessibility during neurogenesis, and an integrative analysis to predict cell-type-specific candidate regulatory elements. We found that cerebral organoids recapitulate most putative cell-type-specific enhancer accessibility patterns but lack many cell-type-specific open chromatin regions that are found in vivo. Systematic comparison of chromatin accessibility across brain regions revealed unexpected diversity among neural progenitor cells in the cerebral cortex and implicated retinoic acid signalling in the specification of neuronal lineage identity in the prefrontal cortex. Together, our results reveal the important contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical development. Analysis of chromatin state at a single-cell level in samples of developing human forebrain demonstrate both cell-type-specific and region-specific changes during neurogenesis.

Human cerebellum encompasses numerous neurons, exhibiting a distinct developmental paradigm from cerebrum. Here we conducted scRNA-seq, scATAC-seq and spatial transcriptomic analyses of fetal samples from gestational week (GW) 13 to 18 to explore the emergence of cellular diversity and developmental programs in the developing human cerebellum. We identified transitory granule cell progenitors that are conserved across species. Special patterns in both granule cells and Purkinje cells were dissected multidimensionally. Species-specific gene expression patterns of cerebellar lobes were characterized and we found that PARM1 exhibited inconsistent distribution in human and mouse granule cells. A novel cluster of potential neuroepithelium at the rhombic lip was identified. We also resolved various subtypes of Purkinje cells and unipolar brush cells and revealed gene regulatory networks controlling their diversification. Therefore, our study offers a valuable multi-omics landscape of human fetal cerebellum and advances our understanding of development and spatial organization of human cerebellum.

Neurodevelopmental disorders (NDDs) are associated with a wide range of clinical features, affecting multiple pathways involved in brain development and function. Recent advances in high-throughput sequencing have unveiled numerous genetic variants associated with NDDs, which further contribute to disease complexity and make it challenging to infer disease causation and underlying mechanisms. Herein, we review current strategies for dissecting the complexity of NDDs using model organisms, induced pluripotent stem cells, single-cell sequencing technologies, and massively parallel reporter assays. We further highlight single-cell CRISPR-based screening techniques that allow genomic investigation of cellular transcriptomes with high efficiency, accuracy, and throughput. Overall, we provide an integrated review of experimental approaches that can be applicable for investigating a broad range of complex disorders.

Human cerebellar development is orchestrated by molecular regulatory networks to achieve cytoarchitecture and coordinate motor and cognitive functions. Here, we combined single-cell transcriptomics, spatial transcriptomics and single cell chromatin accessibility states to systematically depict an integrative spatiotemporal landscape of human fetal cerebellar development. We revealed that combinations of transcription factors and cis-regulatory elements (CREs) play roles in governing progenitor differentiation and cell fate determination along trajectories in a hierarchical manner, providing a gene expression regulatory map of cell fate and spatial information for these cells. We also illustrated that granule cells located in different regions of the cerebellar cortex showed distinct molecular signatures regulated by different signals during development. Finally, we mapped single-nucleotide polymorphisms (SNPs) of disorders related to cerebellar dysfunction and discovered that several disorder-associated genes showed spatiotemporal and cell type-specific expression patterns only in humans, indicating the cellular basis and possible mechanisms of the pathogenesis of neuropsychiatric disorders.

… very least, discoveries that contribute to our understanding of novel chromatin features. … in genome-wide association studies (GWAS) which study common variation in complex diseases…

Mutations in gene regulatory elements have been associated with a wide range of complex neuropsychiatric disorders. However, due to their cell-type specificity and difficulties in characterizing their regulatory targets, the ability to identify causal genetic variants has remained limited. To address these constraints, we perform an integrative analysis of chromatin interactions, open chromatin regions and transcriptomes using promoter capture Hi-C, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing, respectively, in four functionally distinct neural cell types: induced pluripotent stem cell (iPSC)-induced excitatory neurons and lower motor neurons, iPSC-derived hippocampal dentate gyrus-like neurons and primary astrocytes. We identify hundreds of thousands of long-range cis-interactions between promoters and distal promoter-interacting regions, enabling us to link regulatory elements to their target genes and reveal putative processes that are dysregulated in disease. Finally, we validate several promoter-interacting regions by using clustered regularly interspaced short palindromic repeats (CRISPR) techniques in human excitatory neurons, demonstrating that CDK5RAP3, STRAP and DRD2 are transcriptionally regulated by physically linked enhancers. An integrative three-dimensional genomic and transcriptional profiling of four human neural cell types links regulatory elements to their target genes and elucidates the function of noncoding variants in neuropsychiatric disorders.

Common genetic variation appears to largely influence risk for neuropsychiatric disorders through effects on gene regulation. It is therefore possible to shed light on the biology of these conditions by testing for enrichment of associated genetic variation within regulatory genomic regions operating in specific tissues or cell types. Here, we have used ATAC-Seq to map open chromatin (an index of active regulatory genomic regions) in bulk tissue, NeuN+ and NeuN− nuclei from the prenatal human frontal cortex, and tested enrichment of SNP heritability for 5 neuropsychiatric disorders (autism spectrum disorder, ADHD, bipolar disorder, major depressive disorder and schizophrenia) within these regions. We observed significant enrichment of SNP heritability for ADHD, major depressive disorder and schizophrenia within open chromatin regions mapped in bulk fetal frontal cortex, and for all 5 tested neuropsychiatric conditions when we restricted these sites to those overlapping histone modifications indicative of enhancers (H3K4me1) or promoters (H3K4me3) in fetal brain. SNP heritability for neuropsychiatric disorders was significantly enriched in open chromatin regions identified in fetal frontal cortex NeuN- as well as NeuN+ nuclei overlapping fetal brain H3K4me1 or H3K4me3 sites. We additionally demonstrate the utility of our mapped open chromatin regions for prioritizing potentially functional SNPs at genome-wide significant risk loci for neuropsychiatric disorders. Our data provide evidence for an early neurodevelopmental component to a range of neuropsychiatric conditions and highlight an important role for regulatory genomic regions active within both NeuN+ and NeuN− cells of the prenatal brain.

genes exhibited dysregulation in excitatory neurons' superficial layers 2/3 influenced by schizophrenia polygenic risk. This study unveils the complex genetic and epigenetic landscape of psychiatric disorders, emphasizing the importance of cell-type-specific analyses in understanding their pathogenesis and contrasting genetic predisposition with clinical diagnosis.

Psychiatric disorders such as schizophrenia, bipolar disorder, and major depression are highly polygenic, with most risk variants mapping to the noncoding genome. Emerging multiomic technologies, including single-cell transcriptomics, epigenomics, proteomics, and spatial profiling, now enable integrative analyses that connect genetic variation to gene regulation, cell-type vulnerability, and circuit dysfunction. This mini-review highlights recent advances in single-cell and spatial omics, functional dissection of regulatory elements, and computational frameworks for causal inference. Together, these approaches move the field from variants to mechanisms and circuits, while also laying the groundwork for biomarker discovery, patient stratification, and precision therapeutic strategies.

Post-traumatic stress disorder (PTSD) is a polygenic disorder occurring after extreme trauma exposure. Recent studies have begun to detail the molecular biology of PTSD. However, given the array of PTSD-perturbed molecular pathways identified so far1, it is implausible that a single cell type is responsible. Here we profile the molecular responses in over two million nuclei from the dorsolateral prefrontal cortex of 111 human brains, collected post-mortem from individuals with and without PTSD and major depressive disorder. We identify neuronal and non-neuronal cell-type clusters, gene expression changes and transcriptional regulators, and map the epigenomic regulome of PTSD in a cell-type-specific manner. Our analysis revealed PTSD-associated gene alterations in inhibitory neurons, endothelial cells and microglia and uncovered genes and pathways associated with glucocorticoid signalling, GABAergic transmission and neuroinflammation. We further validated these findings using cell-type-specific spatial transcriptomics, confirming disruption of key genes such as SST and FKBP5. By integrating genetic, transcriptomic and epigenetic data, we uncovered the regulatory mechanisms of credible variants that disrupt PTSD genes, including ELFN1, MAD1L1 and KCNIP4, in a cell-type-specific context. Together, these findings provide a comprehensive characterization of the cell-specific molecular regulatory mechanisms that underlie the persisting effects of traumatic stress response on the human prefrontal cortex. A comprehensive analysis of the cell-specific molecular regulatory mechanisms underlying post-traumatic stress disorder in the human prefrontal cortex.

BACKGROUND Tourette disorder is characterized by motor hyperactivity and tics that are believed to originate in basal ganglia. Postmortem immunocytochemical analyses previously revealed decreases in cholinergic, parvalbumin, and somatostatin interneurons (IN) within the caudate/putamen of individuals with TS. METHODS We obtained transcriptome and open chromatin datasets by snRNAseq and snATAC-seq, respectively, from caudate/putamen postmortem specimens of 6 adult TS and 6 matched normal control (NC). Differential gene expression and differential chromatin accessibility analyses were performed in identified cell types. RESULTS The data reproduced the known cellular composition of the human striatum, including a majority of medium spiny neurons (MSN) and small populations of GABAergic and cholinergic IN. IN were decreased by ∼50% in TS brains, with no difference in other cell types. Differential gene expression analysis suggested that mitochondrial oxidative metabolism in MSN and synaptic adhesion and function in IN were both decreased in TS subjects, while there was activation of immune response in microglia. Gene expression changes correlated with changes in activity of cis-regulatory elements, suggesting a relationship of transcriptomic and regulatory abnormalities in MSN, OL and AST of TS brains. CONCLUSIONS This initial analysis of the TS basal ganglia transcriptome at the single cell level confirms the loss and synaptic dysfunction of basal ganglia IN, consistent with in vivo basal ganglia hyperactivity. In parallel, oxidative metabolism was decreased in MSN and correlated with activation of microglia cells, attributable at least in part to dysregulated activity of putative enhancers, implicating altered epigenomic regulation in TS.

The nucleus accumbens (NAc) is a key brain region involved in reward processing and is linked to multiple neuropsychiatric conditions such as substance use disorder, depression, and chronic pain. Recent studies have begun to investigate persistent changes in NAc gene expression at a single-cell resolution, however, our understanding of the cellular heterogeneity of the NAc epigenomic landscape remains limited. In this study, we utilize single-nucleus assay for transposase-accessible chromatin using high-throughput sequencing (snATAC-seq) to map cell-type-specific differences in chromatin accessibility in the NAc. Our findings not only reveal the transcription factors and putative gene regulatory elements that may contribute to these cell-type-specific epigenomic differences but also provide a valuable resource for future studies investigating epigenomic changes that occur in neuropsychiatric disorders.

… We performed snATAC-seq using samples from 44 individuals who died during an … psychiatric phenotypes and non-brain-related control traits. The heritable risk for psychiatric disorders …

Abstract Background Despite affecting millions of people worldwide, psychiatric disorders are inadequately treated. Understanding how adversity, one of the major risk factors for these disorders, raises risk for psychopathology is essential for identifying new treatments. The orbitofrontal cortex (OFC), a sub-region of prefrontal cortex (PFC), is involved in higher cognitive processes and is highly vulnerable to adverse experiences. However, the impacts of adversity on various cell-types across the cortical layers of the OFC are largely unknown. Here, we interrogated human postmortem OFC (BA11) samples to unravel adversity-induced cell-type-specific transcriptomic changes in psychiatric disorders. Explorations of single-cell epigenomic, as well as, spatial transcriptomic alterations within the same samples are currently underway. Aims & Objectives The primary objective of this study is to disentangle adversity-induced cell-type- and cortical layer-specific molecular changes in the OFC of psychiatric patients. The overarching goal is to determine if adversity is associated with a transdiagnostic subtype of psychiatric disorders that may benefit from targeted therapy. Method We examined 86 individuals (n=32 controls vs n=54 cases with mixed major psychiatric disorders: schizophrenia, major depression and bipolar disorder) with or without history of profound psychological adversity exposure (n=23 with severe adversity and n=31 without). To capture the cell-type-specific effects, we generated single-nucleus Assay for Transposase-Accessible Chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) datasets using 10x Chromium (10x Genomics, USA). To dissect spatial heterogeneity, we produced spatial transcriptomic dataset using Visium (10x Genomics, USA). We performed compositional and differential gene expression analyses, followed by gene ontology and disease enrichment analyses, on the snRNA-seq data. Results After filtering out low-quality nuclei from the snRNA-seq dataset, about ~800,000 nuclei from 15 different cell types were retained. The proportions of inhibitory neurons and astrocytes were significantly lower in individuals exposed to adversity vs those without adversity, while the proportion of oligodendrocytes was higher (Log2FoldChange of -0.51, -1.02 and 0.33, respectively). We identified a large number of differentially expressed genes (DEGs) in oligodendrocytes and reelin-expressing inhibitory neurons (160 and 265, respectively) only when the contrast was performed among cases based on their history of adversity. Overall, the DEGs showed significant enrichment of psychopathology-associated metabolic processes and phenotypes, including synaptic signaling and inflammatory responses. Discussion & Conclusions These results highlight the striking cell-type-specific impacts of adverse experiences on gene expressions and cellular abundances in the OFC of individuals with psychiatric disorders. The glial cells such as astrocytes and oligodendrocytes, which are known to be involved in stress response, as well as inhibitory neuronal subtypes, known to be affected in stress-related disorders, showed marked impacts of adversity in the context of psychopathology. In general, synaptic signaling, transmembrane transports, energy metabolism and immune response pathways were affected, all of which are implicated in psychopathology. Experiments and analyses are underway to further disentangle the regulatory changes affecting transcription by delineating epigenomic modifications and gene co-expression networks within these cell types and to unmask the changes in transcript and cellular abundances across the cortical layers. The study will thus help decompose adversity-induced transcriptomic and gene regulatory changes into cell-type-specific alterations across cortical layers.

Rats have been widely used as an experimental organism in psychological, pharmacological, and behavioral studies by modeling human diseases such as neurological disorders. It is critical to identify and characterize cell fate determinants and their regulatory mechanisms in single-cell resolutions across rat brain regions. Here, we applied droplet-based single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) to systematically profile the single-cell chromatin accessibility across four dissected brain areas in adult Sprague–Dawley (SD) rats with a total of 59,023 single nuclei and identified 16 distinct cell types. Interestingly, we found that different cortex regions exhibit diversity in both cellular compositions and gene regulatory regions. Several cell-type-specific transcription factors (TFs), including SPI1, KLF4, KLF6, and NEUROD2, have been shown to play important roles during the pathogenesis of various neurological diseases, such as Alzheimer’s disease (AD), astrocytic gliomas, autism spectrum disorder (ASD), and intellectual disabilities. Therefore, our single-nucleus atlas of rat cortex could serve as an invaluable resource for dissecting the regulatory mechanisms underlying diverse cortex cell fates and further revealing the regulatory networks of neuropathogenesis.

The prefrontal cortex subregions—particularly the prelimbic (PLPFC) and infralimbic (ILPFC) cortices in rodents and dorsal anterior cingulate (dACC) and ventromedial prefrontal cortex (vmPFC) in humans—exhibit functionally specialized yet interconnected roles in PTSD pathogenesis. While PLPFC/dACC are implicated in fear memory, ILPFC/vmPFC are associated with fear extinction. However, the inherent difference and cross-species molecular signatures underlying these functional parallels remain unresolved. To bridge the gap, we integrate single-nucleus RNA-seq, ATAC-seq, and spatial transcriptomics across mouse PLPFC/ILPFC and human dACC/vmPFC to construct a cross-species, multi-omic atlas. We then delineate conserved/divergent gene regulatory networks (GRNs), with emphasis on excitatory neuron evolution. By incorporating PTSD GWAS data and gene expression changes from vmPFC of PTSD patients, we identify cell-type-specific PTSD risks, SNP-anchored GRNs linked to PTSD heritability, and stress-induced chromatin-primed genes. This work provides a multiregion atlas and advances translational understanding of PTSD-related gene regulation divergence from mouse transition to human and complement the present multi-omic research of PTSD.

Abstract Background The ganglionic eminences (GE) are fetal-specific structures that give rise to gamma-aminobutyric acid (GABA)- and acetylcholine-releasing neurons of the forebrain. Given the evidence for GABAergic, cholinergic, and neurodevelopmental disturbances in schizophrenia, we tested the potential involvement of GE neuron development in mediating genetic risk for the condition. Study Design We combined data from a recent large-scale genome-wide association study of schizophrenia with single-cell RNA sequencing data from the human GE to test the enrichment of schizophrenia risk variation in genes with high expression specificity for developing GE cell populations. We additionally performed the single nuclei Assay for Transposase-Accessible Chromatin with Sequencing (snATAC-Seq) to map potential regulatory genomic regions operating in individual cell populations of the human GE, using these to test for enrichment of schizophrenia common genetic variant liability and to functionally annotate non-coding variants-associated with the disorder. Study Results Schizophrenia common variant liability was enriched in genes with high expression specificity for developing neuron populations that are predicted to form dopamine D1 and D2 receptor-expressing GABAergic medium spiny neurons of the striatum, cortical somatostatin-positive GABAergic interneurons, calretinin-positive GABAergic neurons, and cholinergic neurons. Consistent with these findings, schizophrenia genetic risk was concentrated in predicted regulatory genomic sequence mapped in developing neuronal populations of the GE. Conclusions Our study implicates prenatal development of specific populations of GABAergic and cholinergic neurons in later susceptibility to schizophrenia, and provides a map of predicted regulatory genomic elements operating in cells of the GE.

… To delineate human-specific molecular profiles, we analyzed snRNA-seq and snATAC-seq data from dissected dACC and vmPFC subregions across 20 postmortem tissues (Methods), …

Major depressive disorder (MDD) associated genetic variants reside primarily in the non-coding, regulatory genome. Here we investigate genome-wide regulatory differences and putative gene-regulatory effects of disease risk-variants by examining chromatin accessibility combined with single-cell gene-expression profiles in over 200,000 cells from the dorsolateral prefrontal cortex (DLPFC) of 84 individuals with MDD and neurotypical controls. MDD-associated accessibility alterations were prominent in deep-layer excitatory neurons characterized by transcription factor (TF) motif accessibility and binding of nuclear receptor (NR)4A2, an activity-dependent TF responsive to pathological stress. The same neurons were significantly enriched for MDD-associated genetic variation disrupting cis-regulatory sites and TF binding associated with genes involved in synaptic communication. Furthermore, a grey matter microglial cluster exhibited differentially closed chromatin in MDD affecting binding sites bound by TFs known to regulate immune homeostasis. In summary, our study points to specific cell types and regulatory mechanisms whereby genetic variation may increase predisposition to MDD.

… Across the 5 cell types we identified 339,857 peaks from snATAC-seq … snATAC-seq peaks overlapped with previously imputed bulk brain ATAC-seq peaks (27). Using the snATAC-seq …

Schizophrenia is a complex neuropsychiatric disorder with a strong genetic component. Genome-wide association studies (GWAS) have identified numerous risk variants, but their functional impact on gene regulation remains largely unknown. A major challenge lies in interpreting the function of non-coding variants, which comprise the majority of GWAS hits, making it difficult to determine their functional consequences, particularly in identifying the target genes and cell types involved. We investigated the disruption and enhancement of transcription factor (TF) binding motifs by schizophrenia-associated GWAS SNPs in 15 cortical cell types of the human brain. We integrated single-nucleus ATAC-seq and RNA-seq data from 71 donors (36 affected by schizophrenia) with GWAS summary statistics to identify TF motifs whose binding affinities are altered by schizophrenia-associated SNPs. We found that risk alleles of schizophrenia-associated SNPs disrupt and enhance TF binding. Furthermore, we demonstrated that disrupted TF motifs can lead to altered expression of target genes, including NAGA in excitatory neurons and SNX19 in protoplasmic astrocytes. These genes have been previously implicated in schizophrenia and our study provides a mechanism for their dysregulation through altered TF binding. Our findings highlight the importance of considering cell type-specific effects and provide a genome-wide map of TF motif disruptions in schizophrenia, offering insights into the regulatory mechanisms underlying disease risk. These findings may inform the development of novel therapeutic strategies targeting specific regulatory mechanisms.

Common genetic risk for neuropsychiatric disorders is enriched in regulatory elements active during cortical neurogenesis. However, it remains poorly understood as to how these variants influence gene regulation. To model the functional impact of common genetic variation on the noncoding genome during human cortical development, we performed the assay for transposase accessible chromatin using sequencing (ATAC-seq) and analyzed chromatin accessibility quantitative trait loci (QTL) in cultured human neural progenitor cells and their differentiated neuronal progeny from 87 donors. We identified significant genetic effects on 988/1,839 neuron/progenitor regulatory elements, with highly cell-type and temporally specific effects. A subset (roughly 30%) of chromatin accessibility-QTL were also associated with changes in gene expression. Motif-disrupting alleles of transcriptional activators generally led to decreases in chromatin accessibility, whereas motif-disrupting alleles of repressors led to increases in chromatin accessibility. By integrating cell-type-specific chromatin accessibility-QTL and brain-relevant genome-wide association data, we were able to fine-map and identify regulatory mechanisms underlying noncoding neuropsychiatric disorder risk loci. Cell-type-specific chromatin accessibility QTL during neurogenesis allow fine mapping of causal variants and more complete underlying of regulatory mechanisms underlying noncoding loci associated with gene expression and neuropsychiatric disorders.

Fibroblast growth factor (FGF) is a neural inducer in many vertebrate embryos, but how it regulates chromatin organization to coordinate the activation of neural genes is unclear. Moreover, for differentiation to progress, FGF signalling must decline. Why these signalling dynamics are required has not been determined. Here, we show that dephosphorylation of the FGF effector kinase ERK1/2 rapidly increases chromatin accessibility at neural genes in mouse embryos, and, using ATAC-seq in human embryonic stem cell derived spinal cord precursors, we demonstrate that this occurs genome-wide across neural genes. Importantly, ERK1/2 inhibition induces precocious neural gene transcription, and this involves dissociation of the polycomb repressive complex from key gene loci. This takes place independently of subsequent loss of the repressive histone mark H3K27me3 and transcriptional onset. Transient ERK1/2 inhibition is sufficient for the dissociation of the repressive complex, and this is not reversed on resumption of ERK1/2 signalling. Moreover, genomic footprinting of sites identified by ATAC-seq together with ChIP-seq for polycomb protein Ring1B revealed that ERK1/2 inhibition promotes the occupancy of neural transcription factors (TFs) at non-polycomb as well as polycomb associated sites. Together, these findings indicate that ERK1/2 signalling decline promotes global changes in chromatin accessibility and TF binding at neural genes by directing polycomb and other regulators and appears to serve as a gating mechanism that provides directionality to the process of differentiation.

To identify chromatin mechanisms of neuronal differentiation, we characterized chromatin accessibility and gene expression in cerebellar granule neurons (CGNs) of the developing mouse. We used DNase-seq to map accessibility of cis-regulatory elements and RNA-seq to profile transcript abundance across postnatal stages of neuronal differentiation in vivo and in culture. We observed thousands of chromatin accessibility changes as CGNs differentiated, and verified, using H3K27ac ChIP-seq, reporter gene assays and CRISPR-mediated activation, that many of these regions function as neuronal enhancers. Motif discovery in differentially accessible chromatin regions suggested a previously unknown role for the Zic family of transcription factors in CGN maturation. We confirmed the association of Zic with these elements by ChIP-seq and found, using knockdown, that Zic1 and Zic2 are required for coordinating mature neuronal gene expression patterns. Together, our data reveal chromatin dynamics at thousands of gene regulatory elements that facilitate the gene expression patterns necessary for neuronal differentiation and function.

CHARGE syndrome, a rare multiple congenital anomaly condition, is caused by haploinsufficiency of the chromatin remodeling protein gene CHD7 (Chromodomain helicase DNA binding protein 7). Brain abnormalities and intellectual disability are commonly observed in individuals with CHARGE, and neuronal differentiation is reduced in CHARGE patient-derived iPSCs and conditional knockout mouse brains. However, the mechanisms of CHD7 function in nervous system development are not well understood. In this study, we asked whether CHD7 promotes gene transcription in neural progenitor cells via changes in chromatin accessibility. We used Chd7 null embryonic stem cells (ESCs) derived from Chd7 mutant mouse blastocysts as a tool to investigate roles of CHD7 in neuronal and glial differentiation. Loss of Chd7 significantly reduced neuronal and glial differentiation. Sholl analysis showed that loss of Chd7 impaired neuronal complexity and neurite length in differentiated neurons. Genome-wide studies demonstrated that loss of Chd7 leads to modified chromatin accessibility (ATAC-seq) and differential nascent expression (Bru-Seq) of neural-specific genes. These results suggest that CHD7 acts preferentially to alter chromatin accessibility of key genes during the transition of NPCs to neurons to promote differentiation. Our results form a basis for understanding the cell stage-specific roles for CHD7-mediated chromatin remodeling during cell lineage acquisition.

Neuronal stimulation induced by the brain‐derived neurotrophic factor (BDNF) triggers gene expression, which is crucial for neuronal survival, differentiation, synaptic plasticity, memory formation, and neurocognitive health. However, its role in chromatin regulation is unclear. Here, using temporal profiling of chromatin accessibility and transcription in mouse primary cortical neurons upon either BDNF stimulation or depolarization (KCl), we identify features that define BDNF‐specific chromatin‐to‐gene expression programs. Enhancer activation is an early event in the regulatory control of BDNF‐treated neurons, where the bZIP motif‐binding Fos protein pioneered chromatin opening and cooperated with co‐regulatory transcription factors (Homeobox, EGRs, and CTCF) to induce transcription. Deleting cis‐regulatory sequences affect BDNF‐mediated Arc expression, a regulator of synaptic plasticity. BDNF‐induced accessible regions are linked to preferential exon usage by neurodevelopmental disorder‐related genes and the heritability of neuronal complex traits, which were validated in human iPSC‐derived neurons. Thus, we provide a comprehensive view of BDNF‐mediated genome regulatory features using comparative genomic approaches to dissect mammalian neuronal stimulation.

Chromatin structure is an essential regulator of gene expression. Its state of compaction contributes to the regulation of genetic programs, in particular during differentiation. Epigenetic processes, which include post-translational modifications of histones, DNA methylation and implication of non-coding RNA, are powerful regulators of gene expression. Neurogenesis and neuronal differentiation are spatio-temporally regulated events that allow the formation of the central nervous system components. Here, we review the chromatin structure and post-translational histone modifications associated with neuronal differentiation. Studying the impact of histone modifications on neuronal differentiation improves our understanding of the pathophysiological mechanisms of chromatinopathies and opens up new therapeutic avenues. In addition, we will discuss techniques for the analysis of histone modifications on a genome-wide scale and the pathologies associated with the dysregulation of the epigenetic machinery.

Neural induction, both in vivo and in vitro, includes cellular and molecular changes that result in phenotypic specialization related to specific transcriptional patterns. These changes are achieved through the implementation of complex gene regulatory networks. Furthermore, these regulatory networks are influenced by epigenetic mechanisms that drive cell heterogeneity and cell-type specificity, in a controlled and complex manner. Epigenetic marks, such as DNA methylation and histone residue modifications, are highly dynamic and stage-specific during neurogenesis. Genome-wide assessment of these modifications has allowed the identification of distinct non-coding regulatory regions involved in neural cell differentiation, maturation, and plasticity. Enhancers are short DNA regulatory regions that bind transcription factors (TFs) and interact with gene promoters to increase transcriptional activity. They are of special interest in neuroscience because they are enriched in neurons and underlie the cell-type-specificity and dynamic gene expression profiles. Classification of the full epigenomic landscape of neural subtypes is important to better understand gene regulation in brain health and during diseases. Advances in novel next-generation high-throughput sequencing technologies, genome editing, Genome-wide association studies (GWAS), stem cell differentiation, and brain organoids are allowing researchers to study brain development and neurodegenerative diseases with an unprecedented resolution. Herein, we describe important epigenetic mechanisms related to neurogenesis in mammals. We focus on the potential roles of neural enhancers in neurogenesis, cell-fate commitment, and neuronal plasticity. We review recent findings on epigenetic regulatory mechanisms involved in neurogenesis and discuss how sequence variations within enhancers may be associated with genetic risk for neurological and psychiatric disorders.

Chromatin architecture influences transcription by modulating the physical access of regulatory factors to DNA, playing fundamental roles in cell identity. Studies on dopaminergic differentiation have identified coding genes, but the relationship with non-coding genes or chromatin accessibility remains elusive. Using RNA-Seq and ATAC-Seq we profiled differentially expressed transcripts and open chromatin regions during early dopaminergic neuron differentiation. Hierarchical clustering of differentially expressed genes, resulted in 6 groups with unique characteristics. Surprisingly, the abundance of long non-coding RNAs (lncRNAs) was high in the most downregulated transcripts, and depicted positive correlations with target mRNAs. We observed that open chromatin regions decrease upon differentiation. Enrichment analyses of accessibility depict an association between open chromatin regions and specific functional pathways and gene-sets. A bioinformatic search for motifs allowed us to identify transcription factors and structural nuclear proteins that potentially regulate dopaminergic differentiation. Interestingly, we also found changes in protein and mRNA abundance of the CCCTC-binding factor, CTCF, which participates in genome organization and gene expression. Furthermore, assays demonstrated co-localization of CTCF with Polycomb-repressed chromatin marked by H3K27me3 in pluripotent cells, progressively decreasing in neural precursor cells and differentiated neurons. Our work provides a unique resource of transcription factors and regulatory elements, potentially involved in the acquisition of human dopaminergic neuron cell identity.

Astrocytes arise from multipotent neural stem cells (NSCs) and represent the most abundant cell type of the central nervous system (CNS), playing key roles in the developing and adult brain. Since the differentiation of NSCs towards a gliogenic fate is a precisely timed and regulated process, its perturbation gives rise to dysfunctional astrocytic phenotypes. Inflammation, which often underlies neurological disorders, including neurodevelopmental disorders and brain tumors, disrupts the accurate developmental process of NSCs. However, the specific consequences of an inflammatory environment on the epigenetic and transcriptional programs underlying NSCs’ differentiation into astrocytes is unexplored. Here, we address this gap by profiling in mice glial precursors from neural tissue derived from early embryonic stages along their astrocytic differentiation trajectory in the presence or absence of tumor necrosis factor (TNF), a master pro-inflammatory cytokine. By using a combination of RNA- and ATAC-sequencing approaches, together with footprint and integrated gene regulatory network analyses, we here identify key differences during the differentiation of NSCs into astrocytes under physiological and inflammatory settings. In agreement with its role to turn cells resistant to inflammatory challenges, we detect Nrf2 as a master transcription factor supporting the astrocytic differentiation under TNF exposure. Further, under these conditions, we unravel additional transcriptional regulatory hubs, including Stat3, Smad3, Cebpb, and Nfkb2, highlighting the interplay among pathways underlying physiological astrocytic developmental processes and those involved in inflammatory responses, resulting in discrete astrocytic phenotypes. Overall, our study reports key transcriptional and epigenetic changes leading to the identification of molecular regulators of astrocytic differentiation. Furthermore, our analyses provide a valuable resource for understanding inflammation-induced astrocytic phenotypes that might contribute to the development and progression of CNS disorders with an inflammatory component.

During the differentiation process of neurons, their gene transcription pattern changes according to both intrinsic and extrinsic stimuli-induced programs. Chromatin regulation at regulatory elements is involved in this precise control of gene expression. However, developmental changes in chromatin accessibility of the cortical neurons in vivo are less understood, partly because there is no convenient method to genetically label neurons of a specific lineage. Here, we establish a method for labeling the differentiating neurons of specific birthdates. Using this method, we traced the four-day differentiation process of in vivo deep-layer excitatory neurons in mouse embryonic cortex and examined the changes in the genome-wide transcription pattern and chromatin accessibility with RNA-seq and DNase-seq, respectively. The genomic regions of genes associated with mature neuron functions, such as deep layer–specific genes and genes responsive to external stimuli, became open even in the embryonic stage. Moreover, genes with bivalent marks in neural precursor/stem cells (NPCs) became open. Together, our data demonstrate the importance of chromatin regulation in vivo differentiating neurons during the embryonic stage to follow activation of neuronal genes in their maturation process.

Adult neural stem cells are largely quiescent, and require transcriptional reprogramming to reenter the cell cycle and undergo neurogenesis. However, the precise mechanisms that underlie the rapid transcriptional overhaul during NSC activation remain undefined. Here, we identify the genome-wide chromatin accessibility differences between primary neural stem and progenitor cells in quiescent and activated states. We show that these distinct cellular states exhibit both shared and unique chromatin profiles, which are both associated with gene regulation. Interestingly, we find that accessible chromatin states specific to quiescent or activated cells are active enhancers bound by pro-neurogenic and quiescence factors, ASCL1 and NFI. In contrast, shared sites are gene promoters harboring constitutively accessible chromatin enriched for particular core promoter elements that are functionally associated with translation and metabolic functions. Together, our findings reveal how accessible chromatin states regulate a transcriptional overhaul and drive the switch between quiescence and proliferation in NSC activation.

Single-cell RNA sequencing can reveal the transcriptional state of cells, yet provides little insight into the upstream regulatory landscape associated with open or accessible chromatin regions. Joint profiling of accessible chromatin and RNA within the same cells would permit direct matching of transcriptional regulation to its outputs. Here, we describe droplet-based single-nucleus chromatin accessibility and mRNA expression sequencing (SNARE-seq), a method that can link a cell’s transcriptome with its accessible chromatin for sequencing at scale. Specifically, accessible sites are captured by Tn5 transposase in permeabilized nuclei to permit, within many droplets in parallel, DNA barcode tagging together with the mRNA molecules from the same cells. To demonstrate the utility of SNARE-seq, we generated joint profiles of 5,081 and 10,309 cells from neonatal and adult mouse cerebral cortices, respectively. We reconstructed the transcriptome and epigenetic landscapes of major and rare cell types, uncovered lineage-specific accessible sites, especially for low-abundance cells, and connected the dynamics of promoter accessibility with transcription level during neurogenesis. SNARE-seq measures expression profiles and chromatin accessibility in the same cell.

Huntington’s Disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion, resulting in a mutant huntingtin protein. While it is now clear that astrocytes are affected by HD and significantly contribute to neuronal dysfunction and pathogenesis, the alterations in the transcriptional and epigenetic profiles in HD astrocytes have yet to be characterized. Here, we examine global transcription and chromatin accessibility dynamics during in vitro astrocyte differentiation in a transgenic non-human primate model of HD. We found global changes in accessibility and transcription across different stages of HD pluripotent stem cell differentiation, with distinct trends first observed in neural progenitor cells (NPCs), once cells have committed to a neural lineage. Transcription of p53 signaling and cell cycle pathway genes was highly impacted during differentiation, with depletion in HD NPCs and upregulation in HD astrocytes. E2F target genes also displayed this inverse expression pattern, and strong associations between E2F target gene expression and accessibility at nearby putative enhancers were observed. The results suggest that chromatin accessibility and transcription are altered throughout in vitro HD astrocyte differentiation and provide evidence that E2F dysregulation contributes to aberrant cell-cycle re-entry and apoptosis throughout the progression from NPCs to astrocytes.