Search

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.

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.

In the version of this article initially published online, the accession code was given as GSE1000333. The correct code is GSE100033. The error has been corrected in the print, HTML and PDF versions of the article.

Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.

ABSTRACT Genome-wide analysis of chromatin accessibility in primary tissues has uncovered millions of candidate regulatory sequences in the human and mouse genomes 1–4 . However, the heterogeneity of biological samples used in previous studies has prevented a precise understanding of the dynamic chromatin landscape in specific cell types. Here, we show that analysis of the transposase-accessible-chromatin in single nuclei isolated from frozen tissue samples can resolve cellular heterogeneity and delineate transcriptional regulatory sequences in the constituent cell types. Our strategy is based on a combinatorial barcoding assisted single cell assay for transposase-accessible chromatin 5 and is optimized for nuclei from flash-frozen primary tissue samples (snATAC-seq). We used this method to examine the mouse forebrain at seven development stages and in adults. From snATAC-seq profiles of more than 15,000 high quality nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell-types in foetal and adult forebrains. We further define cell-type specific cis regulatory sequences and infer potential master transcriptional regulators of each cell population. Our results demonstrate the feasibility of a general approach for identifying cell-type-specific cis regulatory sequences in heterogeneous tissue samples, and provide a rich resource for understanding forebrain development in mammals.

Epidermal growth factor receptor (EGFR) gene amplification and mutations are the most common oncogenic events in glioblastoma (GBM), but the mechanisms by which they promote aggressive tumor growth are not well understood. Here, through integrated epigenome and transcriptome analyses of cell lines, genotyped clinical samples, and TCGA data, we show that EGFR mutations remodel the activated enhancer landscape of GBM, promoting tumorigenesis through a SOX9 and FOXG1-dependent transcriptional regulatory network in vitro and in vivo. The most common EGFR mutation, EGFRvIII, sensitizes GBM cells to the BET-bromodomain inhibitor JQ1 in a SOX9, FOXG1-dependent manner. These results identify the role of transcriptional/epigenetic remodeling in EGFR-dependent pathogenesis and suggest a mechanistic basis for epigenetic therapy.

In mammals, cytosine methylation (5mC) is widely distributed throughout the genome but is notably depleted from active promoters and enhancers. While the role of DNA methylation in promoter silencing has been well documented, the function of this epigenetic mark at enhancers remains unclear. Recent experiments have demonstrated that enhancers are enriched for 5-hydroxymethylcytosine (5hmC), an oxidization product of the Tet family of 5mC dioxygenases and an intermediate of DNA demethylation. These results support the involvement of Tet proteins in the regulation of dynamic DNA methylation at enhancers. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. Our results reveal that DNA demethylation modulates enhancer activity, and its disruption influences the timing of transcriptome reprogramming during cellular differentiation.

The laboratory mouse is the most widely used mammalian model organism in biomedical research. The 2.6 × 10(9) bases of the mouse genome possess a high degree of conservation with the human genome, so a thorough annotation of the mouse genome will be of significant value to understanding the function of the human genome. So far, most of the functional sequences in the mouse genome have yet to be found, and the cis-regulatory sequences in particular are still poorly annotated. Comparative genomics has been a powerful tool for the discovery of these sequences, but on its own it cannot resolve their temporal and spatial functions. Recently, ChIP-Seq has been developed to identify cis-regulatory elements in the genomes of several organisms including humans, Drosophila melanogaster and Caenorhabditis elegans. Here we apply the same experimental approach to a diverse set of 19 tissues and cell types in the mouse to produce a map of nearly 300,000 murine cis-regulatory sequences. The annotated sequences add up to 11% of the mouse genome, and include more than 70% of conserved non-coding sequences. We define tissue-specific enhancers and identify potential transcription factors regulating gene expression in each tissue or cell type. Finally, we show that much of the mouse genome is organized into domains of coordinately regulated enhancers and promoters. Our results provide a resource for the annotation of functional elements in the mammalian genome and for the study of mechanisms regulating tissue-specific gene expression.

We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posting new test results (http://compbio.med.harvard.edu/antibodies/).

Abstract Background To identify potential tumor suppressor genes, genome-wide data from exome and transcriptome sequencing were combined to search for genes with loss of heterozygosity and allele-specific expression. The analysis was conducted on the breast cancer cell line HCC1954, and a lymphoblast cell line from the same individual, HCC1954BL. Results By comparing exome sequences from the two cell lines, we identified loss of heterozygosity events at 403 genes in HCC1954 and at one gene in HCC1954BL. The combination of exome and transcriptome sequence data also revealed 86 and 50 genes with allele specific expression events in HCC1954 and HCC1954BL, which comprise 5.4% and 2.6% of genes surveyed, respectively. Many of these genes identified by loss of heterozygosity and allele-specific expression are known or putative tumor suppressor genes, such as BRCA1, MSH3 and SETX, which participate in DNA repair pathways. Conclusions Our results demonstrate that the combined application of high throughput sequencing to exome and allele-specific transcriptome analysis can reveal genes with known tumor suppressor characteristics, and a shortlist of novel candidates for the study of tumor suppressor activities.

Allelic differences between the two homologous chromosomes can affect the propensity of inheritance in humans; however, the extent of such differences in the human genome has yet to be fully explored. Here we delineate allelic chromatin modifications and transcriptomes among a broad set of human tissues, enabled by a chromosome-spanning haplotype reconstruction strategy (1). The resulting large collection of haplotype-resolved epigenomic maps reveals extensive allelic biases in both chromatin state and transcription, which show considerable variation across tissues and between individuals, and allow us to investigate cis-regulatory relationships between genes and their control sequences. Analyses of histone modification maps also uncover intriguing characteristics of cis-regulatory elements and tissue-restricted activities of repetitive elements. The rich data sets described here will enhance our understanding of the mechanisms by which cis-regulatory elements control gene expression programs., We performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) to generate extensive data sets profiling 6 histone modifications across 16 human tissue types from four individual donors (181 data sets). In [...]

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases., Author(s): Feng Yue [1, 2]; Yong Cheng [3]; Alessandra Breschi [4]; Jeff Vierstra [5]; Weisheng Wu [6]; Tyrone Ryba [7]; Richard Sandstrom [5]; Zhihai Ma [3]; Carrie Davis [8]; Benjamin [...]

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells. [ABSTRACT FROM AUTHOR]