Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain.

Cytosine DNA methylation is essential in brain development and is implicated in various neurological disorders. Understanding DNA methylation diversity across the entire brain in a spatial context is fundamental for a complete molecular atlas of brain cell types and their gene regulatory landscapes. Here we used single-nucleus methylome sequencing (snmC-seq3) and multi-omic sequencing (snm3C-seq)1 technologies to generate 301,626 methylomes and 176,003 chromatin conformation-methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell taxonomy with 4,673 cell groups and 274 cross-modality-annotated subclasses. We identified 2.6 million differentially methylated regions across the genome that represent potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide spatial transcriptomics data validated the association of spatial epigenetic diversity with transcription and improved the anatomical mapping of our epigenetic datasets. Furthermore, chromatin conformation diversities occurred in important neuronal genes and were highly associated with DNA methylation and transcription changes. Brain-wide cell-type comparisons enabled the construction of regulatory networks that incorporate transcription factors, regulatory elements and their potential downstream gene targets. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a whole-brain SMART-seq2 dataset. Our study establishes a brain-wide, single-cell DNA methylome and 3D multi-omic atlas and provides a valuable resource for comprehending the cellular-spatial and regulatory genome diversity of the mouse brain.

Conserved and divergent gene regulatory programs of the mammalian neocortex.

Divergence of cis-regulatory elements drives species-specific traits1, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains unclear. Here we investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset and mouse using single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome and chromosomal conformation profiles from a total of over 200,000 cells. From these data, we show evidence that divergence of transcription factor expression corresponds to species-specific epigenome landscapes. We find that conserved and divergent gene regulatory features are reflected in the evolution of the three-dimensional genome. Transposable elements contribute to nearly 80% of the human-specific candidate cis-regulatory elements in cortical cells. Through machine learning, we develop sequence-based predictors of candidate cis-regulatory elements in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Finally, we show that epigenetic conservation combined with sequence similarity helps to uncover functional cis-regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Single-cell analysis of chromatin accessibility in the adult mouse brain.

Recent advances in single-cell technologies have led to the discovery of thousands of brain cell types; however, our understanding of the gene regulatory programs in these cell types is far from complete1-4. Here we report a comprehensive atlas of candidate cis-regulatory DNA elements (cCREs) in the adult mouse brain, generated by analysing chromatin accessibility in 2.3 million individual brain cells from 117 anatomical dissections. The atlas includes approximately 1 million cCREs and their chromatin accessibility across 1,482 distinct brain cell populations, adding over 446,000 cCREs to the most recent such annotation in the mouse genome. The mouse brain cCREs are moderately conserved in the human brain. The mouse-specific cCREs-specifically, those identified from a subset of cortical excitatory neurons-are strongly enriched for transposable elements, suggesting a potential role for transposable elements in the emergence of new regulatory programs and neuronal diversity. Finally, we infer the gene regulatory networks in over 260 subclasses of mouse brain cells and develop deep-learning models to predict the activities of gene regulatory elements in different brain cell types from the DNA sequence alone. Our results provide a resource for the analysis of cell-type-specific gene regulation programs in both mouse and human brains.

A comparative atlas of single-cell chromatin accessibility in the human brain.

Recent advances in single-cell transcriptomics have illuminated the diverse neuronal and glial cell types within the human brain. However, the regulatory programs governing cell identity and function remain unclear. Using a single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq), we explored open chromatin landscapes across 1.1 million cells in 42 brain regions from three adults. Integrating this data unveiled 107 distinct cell types and their specific utilization of 544,735 candidate cis-regulatory DNA elements (cCREs) in the human genome. Nearly a third of the cCREs demonstrated conservation and chromatin accessibility in the mouse brain cells. We reveal strong links between specific brain cell types and neuropsychiatric disorders including schizophrenia, bipolar disorder, Alzheimer's disease (AD), and major depression, and have developed deep learning models to predict the regulatory roles of noncoding risk variants in these disorders.

Single-cell DNA methylation and 3D genome architecture in the human brain.

Delineating the gene-regulatory programs underlying complex cell types is fundamental for understanding brain function in health and disease. Here, we comprehensively examined human brain cell epigenomes by probing DNA methylation and chromatin conformation at single-cell resolution in 517 thousand cells (399 thousand neurons and 118 thousand non-neurons) from 46 regions of three adult male brains. We identified 188 cell types and characterized their molecular signatures. Integrative analyses revealed concordant changes in DNA methylation, chromatin accessibility, chromatin organization, and gene expression across cell types, cortical areas, and basal ganglia structures. We further developed single-cell methylation barcodes that reliably predict brain cell types using the methylation status of select genomic sites. This multimodal epigenomic brain cell atlas provides new insights into the complexity of cell-type-specific gene regulation in adult human brains.

Robust enhancer-gene regulation identified by single-cell transcriptomes and epigenomes.

Single-cell sequencing could help to solve the fundamental challenge of linking millions of cell-type-specific enhancers with their target genes. However, this task is confounded by patterns of gene co-expression in much the same way that genetic correlation due to linkage disequilibrium confounds fine-mapping in genome-wide association studies (GWAS). We developed a non-parametric permutation-based procedure to establish stringent statistical criteria to control the risk of false-positive associations in enhancer-gene association studies (EGAS). We applied our procedure to large-scale transcriptome and epigenome data from multiple tissues and species, including the mouse and human brain, to predict enhancer-gene associations genome wide. We tested the functional validity of our predictions by comparing them with chromatin conformation data and causal enhancer perturbation experiments. Our study shows how controlling for gene co-expression enables robust enhancer-gene linkage using single-cell sequencing data.

Single-cell DNA Methylome and 3D Multi-omic Atlas of the Adult Mouse Brain.

Cytosine DNA methylation is essential in brain development and has been implicated in various neurological disorders. A comprehensive understanding of DNA methylation diversity across the entire brain in the context of the brain's 3D spatial organization is essential for building a complete molecular atlas of brain cell types and understanding their gene regulatory landscapes. To this end, we employed optimized single-nucleus methylome (snmC-seq3) and multi-omic (snm3C-seq1) sequencing technologies to generate 301,626 methylomes and 176,003 chromatin conformation/methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell type taxonomy that contains 4,673 cell groups and 261 cross-modality-annotated subclasses. We identified millions of differentially methylated regions (DMRs) across the genome, representing potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide multiplexed error-robust fluorescence in situ hybridization (MERFISH2) data validated the association of this spatial epigenetic diversity with transcription and allowed the mapping of the DNA methylation and topology information into anatomical structures more precisely than our dissections. Furthermore, multi-scale chromatin conformation diversities occur in important neuronal genes, highly associated with DNA methylation and transcription changes. Brain-wide cell type comparison allowed us to build a regulatory model for each gene, linking transcription factors, DMRs, chromatin contacts, and downstream genes to establish regulatory networks. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a companion whole-brain SMART-seq3 dataset. Our study establishes the first brain-wide, single-cell resolution DNA methylome and 3D multi-omic atlas, providing an unparalleled resource for comprehending the mouse brain's cellular-spatial and regulatory genome diversity.

Comparative single cell epigenomic analysis of gene regulatory programs in the rodent and primate neocortex.

Sequence divergence of cis- regulatory elements drives species-specific traits, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains to be elucidated. We investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset, and mouse with single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome, and chromosomal conformation profiles from a total of over 180,000 cells. For each modality, we determined species-specific, divergent, and conserved gene expression and epigenetic features at multiple levels. We find that cell type-specific gene expression evolves more rapidly than broadly expressed genes and that epigenetic status at distal candidate cis -regulatory elements (cCREs) evolves faster than promoters. Strikingly, transposable elements (TEs) contribute to nearly 80% of the human-specific cCREs in cortical cells. Through machine learning, we develop sequence-based predictors of cCREs in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Lastly, we show that epigenetic conservation combined with sequence similarity helps uncover functional cis -regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Cell type-specific enrichment of somatic aneuploidy in the mammalian brain.

Somatic mutations alter the genomes of a subset of an individual's brain cells1-3, impacting gene regulation and contributing to disease processes4,5. Mosaic single nucleotide variants have been characterized with single-cell resolution in the brain2,3, but we have limited information about large-scale structural variation, including whole-chromosome duplication or loss1,6,7. We used a dataset of over 415,000 single-cell DNA methylation and chromatin conformation profiles across the adult mouse brain to identify aneuploid cells comprehensively. Whole-chromosome loss or duplication occurred in <1% of cells, with rates up to 1.8% in non-neuronal cell types, including oligodendrocyte precursors and pericytes. Among all aneuploidies, we observed a strong enrichment of trisomy on chromosome 16, which is syntenic with human chromosome 21 and constitutively trisomic in Down syndrome. Chromosome 16 trisomy occurred in multiple cell types and across brain regions, suggesting that nondisjunction is a recurrent feature of somatic variation in the brain.

Dnmt3a knockout in excitatory neurons impairs postnatal synapse maturation and increases the repressive histone modification H3K27me3.

Two epigenetic pathways of transcriptional repression, DNA methylation and polycomb repressive complex 2 (PRC2), are known to regulate neuronal development and function. However, their respective contributions to brain maturation are unknown. We found that conditional loss of the de novo DNA methyltransferase Dnmt3a in mouse excitatory neurons altered expression of synapse-related genes, stunted synapse maturation, and impaired working memory and social interest. At the genomic level, loss of Dnmt3a abolished postnatal accumulation of CG and non-CG DNA methylation, leaving adult neurons with an unmethylated, fetal-like epigenomic pattern at ~222,000 genomic regions. The PRC2-associated histone modification, H3K27me3, increased at many of these sites. Our data support a dynamic interaction between two fundamental modes of epigenetic repression during postnatal maturation of excitatory neurons, which together confer robustness on neuronal regulation.

Single nucleus multi-omics identifies human cortical cell regulatory genome diversity.

Single-cell technologies measure unique cellular signatures but are typically limited to a single modality. Computational approaches allow the fusion of diverse single-cell data types, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells, we devised single-nucleus methylcytosine, chromatin accessibility, and transcriptome sequencing (snmCAT-seq) and applied it to postmortem human frontal cortex tissue. We developed a cross-validation approach using multi-modal information to validate fine-grained cell types and assessed the effectiveness of computational data fusion methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility, and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling the prediction of cell types that are associated with diseases.

DNA methylation atlas of the mouse brain at single-cell resolution.

Mammalian brain cells show remarkable diversity in gene expression, anatomy and function, yet the regulatory DNA landscape underlying this extensive heterogeneity is poorly understood. Here we carry out a comprehensive assessment of the epigenomes of mouse brain cell types by applying single-nucleus DNA methylation sequencing1,2 to profile 103,982 nuclei (including 95,815 neurons and 8,167 non-neuronal cells) from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas. We identified 161 cell clusters with distinct spatial locations and projection targets. We constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. These features indicate the potential regulatory landscape supporting the assignment of putative cell types and reveal repetitive usage of regulators in excitatory and inhibitory cells for determining subtypes. The DNA methylation landscape of excitatory neurons in the cortex and hippocampus varied continuously along spatial gradients. Using this deep dataset, we constructed an artificial neural network model that precisely predicts single neuron cell-type identity and brain area spatial location. Integration of high-resolution DNA methylomes with single-nucleus chromatin accessibility data3 enabled prediction of high-confidence enhancer-gene interactions for all identified cell types, which were subsequently validated by cell-type-specific chromatin conformation capture experiments4. By combining multi-omic datasets (DNA methylation, chromatin contacts, and open chromatin) from single nuclei and annotating the regulatory genome of hundreds of cell types in the mouse brain, our DNA methylation atlas establishes the epigenetic basis for neuronal diversity and spatial organization throughout the mouse cerebrum.

Epigenomic diversity of cortical projection neurons in the mouse brain.

Neuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain1, neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties.

A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1-3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis.

An atlas of gene regulatory elements in adult mouse cerebrum.

The mammalian cerebrum performs high-level sensory perception, motor control and cognitive functions through highly specialized cortical and subcortical structures1. Recent surveys of mouse and human brains with single-cell transcriptomics2-6 and high-throughput imaging technologies7,8 have uncovered hundreds of neural cell types distributed in different brain regions, but the transcriptional regulatory programs that are responsible for the unique identity and function of each cell type remain unknown. Here we probe the accessible chromatin in more than 800,000 individual nuclei from 45 regions that span the adult mouse isocortex, olfactory bulb, hippocampus and cerebral nuclei, and use the resulting data to map the state of 491,818 candidate cis-regulatory DNA elements in 160 distinct cell types. We find high specificity of spatial distribution for not only excitatory neurons, but also most classes of inhibitory neurons and a subset of glial cell types. We characterize the gene regulatory sequences associated with the regional specificity within these cell types. We further link a considerable fraction of the cis-regulatory elements to putative target genes expressed in diverse cerebral cell types and predict transcriptional regulators that are involved in a broad spectrum of molecular and cellular pathways in different neuronal and glial cell populations. Our results provide a foundation for comprehensive analysis of gene regulatory programs of the mammalian brain and assist in the interpretation of noncoding risk variants associated with various neurological diseases and traits in humans.

Comprehensive analysis of single cell ATAC-seq data with SnapATAC.

Identification of the cis-regulatory elements controlling cell-type specific gene expression patterns is essential for understanding the origin of cellular diversity. Conventional assays to map regulatory elements via open chromatin analysis of primary tissues is hindered by sample heterogeneity. Single cell analysis of accessible chromatin (scATAC-seq) can overcome this limitation. However, the high-level noise of each single cell profile and the large volume of data pose unique computational challenges. Here, we introduce SnapATAC, a software package for analyzing scATAC-seq datasets. SnapATAC dissects cellular heterogeneity in an unbiased manner and map the trajectories of cellular states. Using the Nyström method, SnapATAC can process data from up to a million cells. Furthermore, SnapATAC incorporates existing tools into a comprehensive package for analyzing single cell ATAC-seq dataset. As demonstration of its utility, SnapATAC is applied to 55,592 single-nucleus ATAC-seq profiles from the mouse secondary motor cortex. The analysis reveals ~370,000 candidate regulatory elements in 31 distinct cell populations in this brain region and inferred candidate cell-type specific transcriptional regulators.

Joint profiling of histone modifications and transcriptome in single cells from mouse brain.

Genome-wide profiling of histone modifications can reveal not only the location and activity state of regulatory elements, but also the regulatory mechanisms involved in cell-type-specific gene expression during development and disease pathology. Conventional assays to profile histone modifications in bulk tissues lack single-cell resolution. Here we describe an ultra-high-throughput method, Paired-Tag, for joint profiling of histone modifications and transcriptome in single cells to produce cell-type-resolved maps of chromatin state and transcriptome in complex tissues. We used this method to profile five histone modifications jointly with transcriptome in the adult mouse frontal cortex and hippocampus. Integrative analysis of the resulting maps identified distinct groups of genes subject to divergent epigenetic regulatory mechanisms. Our single-cell multiomics approach enables comprehensive analysis of chromatin state and gene regulation in complex tissues and characterization of gene regulatory programs in the constituent cell types.

Losing Dnmt3a dependent methylation in inhibitory neurons impairs neural function by a mechanism impacting Rett syndrome.

Methylated cytosine is an effector of epigenetic gene regulation. In the brain, Dnmt3a is the sole 'writer' of atypical non-CpG methylation (mCH), and MeCP2 is the only known 'reader' for mCH. We asked if MeCP2 is the sole reader for Dnmt3a dependent methylation by comparing mice lacking either protein in GABAergic inhibitory neurons. Loss of either protein causes overlapping and distinct features from the behavioral to molecular level. Loss of Dnmt3a causes global loss of mCH and a subset of mCG sites resulting in more widespread transcriptional alterations and severe neurological dysfunction than MeCP2 loss. These data suggest that MeCP2 is responsible for reading only part of the Dnmt3a dependent methylation in the brain. Importantly, the impact of MeCP2 on genes differentially expressed in both models shows a strong dependence on mCH, but not Dnmt3a dependent mCG, consistent with mCH playing a central role in the pathogenesis of Rett Syndrome.

An ultra high-throughput method for single-cell joint analysis of open chromatin and transcriptome.

Simultaneous profiling of transcriptome and chromatin accessibility within single cells is a powerful approach to dissect gene regulatory programs in complex tissues. However, current tools are limited by modest throughput. We now describe an ultra high-throughput method, Paired-seq, for parallel analysis of transcriptome and accessible chromatin in millions of single cells. We demonstrate the utility of Paired-seq for analyzing the dynamic and cell-type-specific gene regulatory programs in complex tissues by applying it to mouse adult cerebral cortex and fetal forebrain. The joint profiles of a large number of single cells allowed us to deconvolute the transcriptome and open chromatin landscapes in the major cell types within these brain tissues, infer putative target genes of candidate enhancers, and reconstruct the trajectory of cellular lineages within the developing forebrain.