Genetic regulation of cell type-specific chromatin accessibility shapes brain disease etiology.

Nucleotide variants in cell type-specific gene regulatory elements in the human brain are risk factors for human disease. We measured chromatin accessibility in 1932 aliquots of sorted neurons and non-neurons from 616 human postmortem brains and identified 34,539 open chromatin regions with chromatin accessibility quantitative trait loci (caQTLs). Only 10.4% of caQTLs are shared between neurons and non-neurons, which supports cell type-specific genetic regulation of the brain regulome. Incorporating allele-specific chromatin accessibility improves statistical fine-mapping and refines molecular mechanisms that underlie disease risk. Using massively parallel reporter assays in induced excitatory neurons, we screened 19,893 brain QTLs and identified the functional impact of 476 regulatory variants. Combined, this comprehensive resource captures variation in the human brain regulome and provides insights into disease etiology.

The neuronal chromatin landscape in adult schizophrenia brains is linked to early fetal development.

Non-coding variants increase risk of neuropsychiatric disease. However, our understanding of the cell-type specific role of the non-coding genome in disease is incomplete. We performed population scale (N=1,393) chromatin accessibility profiling of neurons and non-neurons from two neocortical brain regions: the anterior cingulate cortex and dorsolateral prefrontal cortex. Across both regions, we observed notable differences in neuronal chromatin accessibility between schizophrenia cases and controls. A per-sample disease pseudotime was positively associated with genetic liability for schizophrenia. Organizing chromatin into cis- and trans-regulatory domains, identified a prominent neuronal trans-regulatory domain (TRD1) active in immature glutamatergic neurons during fetal development. Polygenic risk score analysis using genetic variants within chromatin accessibility of TRD1 successfully predicted susceptibility to schizophrenia in the Million Veteran Program cohort. Overall, we present the most extensive resource to date of chromatin accessibility in the human cortex, yielding insights into the cell-type specific etiology of schizophrenia.

The neuronal chromatin landscape in adult schizophrenia brains is linked to early fetal development.

Non-coding variants increase risk of neuropsychiatric disease. However, our understanding of the cell-type specific role of the non-coding genome in disease is incomplete. We performed population scale (N=1,393) chromatin accessibility profiling of neurons and non-neurons from two neocortical brain regions: the anterior cingulate cortex and dorsolateral prefrontal cortex. Across both regions, we observed notable differences in neuronal chromatin accessibility between schizophrenia cases and controls. A per-sample disease pseudotime was positively associated with genetic liability for schizophrenia. Organizing chromatin into cis- and trans-regulatory domains, identified a prominent neuronal trans-regulatory domain (TRD1) active in immature glutamatergic neurons during fetal development. Polygenic risk score analysis using genetic variants within chromatin accessibility of TRD1 successfully predicted susceptibility to schizophrenia in the Million Veteran Program cohort. Overall, we present the most extensive resource to date of chromatin accessibility in the human cortex, yielding insights into the cell-type specific etiology of schizophrenia.

Lineage specific 3D genome structure in the adult human brain and neurodevelopmental changes in the chromatin interactome.

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.

Genetic regulation of cell-type specific chromatin accessibility shapes the etiology of brain diseases.

Nucleotide variants in cell type-specific gene regulatory elements in the human brain are major risk factors of human disease. We measured chromatin accessibility in sorted neurons and glia from 1,932 samples of human postmortem brain and identified 34,539 open chromatin regions with chromatin accessibility quantitative trait loci (caQTL). Only 10.4% of caQTL are shared between neurons and glia, supporting the cell type specificity of genetic regulation of the brain regulome. Incorporating allele specific chromatin accessibility improves statistical fine-mapping and refines molecular mechanisms underlying disease risk. Using massively parallel reporter assays in induced excitatory neurons, we screened 19,893 brain QTLs, identifying the functional impact of 476 regulatory variants. Combined, this comprehensive resource captures variation in the human brain regulome and provides novel insights into brain disease etiology.

Population-level variation in enhancer expression identifies disease mechanisms in the human brain.

Identification of risk variants for neuropsychiatric diseases within enhancers underscores the importance of understanding population-level variation in enhancer function in the human brain. Besides regulating tissue-specific and cell-type-specific transcription of target genes, enhancers themselves can be transcribed. By jointly analyzing large-scale cell-type-specific transcriptome and regulome data, we cataloged 30,795 neuronal and 23,265 non-neuronal candidate transcribed enhancers. Examination of the transcriptome in 1,382 brain samples identified robust expression of transcribed enhancers. We explored gene-enhancer coordination and found that enhancer-linked genes are strongly implicated in neuropsychiatric disease. We identified expression quantitative trait loci (eQTLs) for both genes and enhancers and found that enhancer eQTLs mediate a substantial fraction of neuropsychiatric trait heritability. Inclusion of enhancer eQTLs in transcriptome-wide association studies enhanced functional interpretation of disease loci. Overall, our study characterizes the gene-enhancer regulome and genetic mechanisms in the human cortex in both healthy and diseased states.

The three-dimensional landscape of cortical chromatin accessibility in Alzheimer's disease.

To characterize the dysregulation of chromatin accessibility in Alzheimer's disease (AD), we generated 636 ATAC-seq libraries from neuronal and nonneuronal nuclei isolated from the superior temporal gyrus and entorhinal cortex of 153 AD cases and 56 controls. By analyzing a total of ~20 billion read pairs, we expanded the repertoire of known open chromatin regions (OCRs) in the human brain and identified cell-type-specific enhancer-promoter interactions. We show that interindividual variability in OCRs can be leveraged to identify cis-regulatory domains (CRDs) that capture the three-dimensional structure of the genome (3D genome). We identified AD-associated effects on chromatin accessibility, the 3D genome and transcription factor (TF) regulatory networks. For one of the most AD-perturbed TFs, USF2, we validated its regulatory effect on lysosomal genes. Overall, we applied a systematic approach to understanding the role of the 3D genome in AD. We provide all data as an online resource for widespread community-based analysis.

Differential activity of transcribed enhancers in the prefrontal cortex of 537 cases with schizophrenia and controls.

Transcription at enhancers is a widespread phenomenon which produces so-called enhancer RNA (eRNA) and occurs in an activity-dependent manner. However, the role of eRNA and its utility in exploring disease-associated changes in enhancer function, and the downstream coding transcripts that they regulate, is not well established. We used transcriptomic and epigenomic data to interrogate the relationship of eRNA transcription to disease status and how genetic variants alter enhancer transcriptional activity in the human brain. We combined RNA-seq data from 537 postmortem brain samples from the CommonMind Consortium with cap analysis of gene expression and enhancer identification, using the assay for transposase-accessible chromatin followed by sequencing (ATACseq). We find 118 differentially transcribed eRNAs in schizophrenia and identify schizophrenia-associated gene/eRNA co-expression modules. Perturbations of a key module are associated with the polygenic risk scores. Furthermore, we identify genetic variants affecting expression of 927 enhancers, which we refer to as enhancer expression quantitative loci or eeQTLs. Enhancer expression patterns are consistent across studies, including differentially expressed eRNAs and eeQTLs. Combining eeQTLs with a genome-wide association study of schizophrenia identifies a genetic variant that alters enhancer function and expression of its target gene, GOLPH3L. Our novel approach to analyzing enhancer transcription is adaptable to other large-scale, non-poly-A depleted, RNA-seq studies.

DKK1 mediated inhibition of Wnt signaling in postnatal mice leads to loss of TEC progenitors and thymic degeneration.

BACKGROUND: Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of DeltaNP63(+) Foxn1(+) and Aire(+) TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments. CONCLUSIONS/SIGNIFICANCE: Taken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants.