DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three ...quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The human genome is folded into regulatory units termed 'topologically-associated domains' (TADs). Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal ...looping and the topological constraint of TAD boundaries. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, partially-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing. Finally, we apply these strategies to simulate an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence.
We introduce an approach to identify disease-relevant tissues and cell types by analyzing gene expression data together with genome-wide association study (GWAS) summary statistics. Our approach uses ...stratified linkage disequilibrium (LD) score regression to test whether disease heritability is enriched in regions surrounding genes with the highest specific expression in a given tissue. We applied our approach to gene expression data from several sources together with GWAS summary statistics for 48 diseases and traits (average N = 169,331) and found significant tissue-specific enrichments (false discovery rate (FDR) < 5%) for 34 traits. In our analysis of multiple tissues, we detected a broad range of enrichments that recapitulated known biology. In our brain-specific analysis, significant enrichments included an enrichment of inhibitory over excitatory neurons for bipolar disorder, and excitatory over inhibitory neurons for schizophrenia and body mass index. Our results demonstrate that our polygenic approach is a powerful way to leverage gene expression data for interpreting GWAS signals.
Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background, yet the landscapes of in vivo tissues remain largely uncharted. Here, we ...mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages, lineages, and cellular environments. They also reveal global features of the epigenome, related to nuclear architecture, that also vary across cellular phenotypes. Specifically, developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture, and discuss their implications for lineage fidelity, cellular senescence, and reprogramming.
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► A resource of chromatin state maps for phenotypically diverse human tissues ► Annotation of regulatory elements across developmental stages and environments ► Developmental specification is accompanied by progressive chromatin restriction ► Chromatin architecture changes in cultured cells have implications for reprogramming
A large collection of chromatin state maps, representing human cells and tissues in vivo, reveals tissue-specific enhancer-like elements as well as repressive chromatin domains that arise during development or in response to nonphysiologic cellular environments and may present a hindrance to cellular reprogramming.
A large fraction of the mammalian genome is organized into inactive chromosomal domains along the nuclear lamina. The mechanism by which these lamina associated domains (LADs) are established remains ...to be elucidated. Using genomic repositioning assays, we show that LADs, spanning the developmentally regulated IgH and Cyp3a loci contain discrete DNA regions that associate chromatin with the nuclear lamina and repress gene activity in fibroblasts. Lamina interaction is established during mitosis and likely involves the localized recruitment of Lamin B during late anaphase. Fine-scale mapping of LADs reveals numerous lamina-associating sequences (LASs), which are enriched for a GAGA motif. This repeated motif directs lamina association and is bound by the transcriptional repressor cKrox, in a complex with HDAC3 and Lap2β. Knockdown of cKrox or HDAC3 results in dissociation of LASs/LADs from the nuclear lamina. These results reveal a mechanism that couples nuclear compartmentalization of chromatin domains with the control of gene activity.
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► Lamina-associating DNA sequences (LASs) direct chromatin to the nuclear lamina ► In mammalian cells, LASs are bound by the transcriptional repressor cKrox ► cKrox recruits DNA to the lamina via HDAC3 and lamina-associated protein Lap2β ► Lamina interaction is established during mitosis, likely through recruitment of Lamin B
Binding of a transcriptional repressor complex to DNA sequences enriched in GAGA motifs directs the association of specific chromosome domains to the nuclear lamina during mitosis. These lamin-associated sequences thus couple nuclear compartmentalization of chromatin domains with gene silencing.
Recurrent somatic ASXL1 mutations occur in patients with myelodysplastic syndrome, myeloproliferative neoplasms, and acute myeloid leukemia, and are associated with adverse outcome. Despite the ...genetic and clinical data implicating ASXL1 mutations in myeloid malignancies, the mechanisms of transformation by ASXL1 mutations are not understood. Here, we identify that ASXL1 mutations result in loss of polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) tri-methylation. Through integration of microarray data with genome-wide histone modification ChIP-Seq data, we identify targets of ASXL1 repression, including the posterior HOXA cluster that is known to contribute to myeloid transformation. We demonstrate that ASXL1 associates with the PRC2, and that loss of ASXL1 in vivo collaborates with NRASG12D to promote myeloid leukemogenesis.
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► ASXL1 mutations are loss-of-function mutations ► ASXL1 loss results in a genome-wide reduction in H3K27me3 occupancy ► ASXL1 interacts with the PRC2 complex and is important for PRC2 recruitment ► ASXL1 collaborates with co-occurring oncogenes in vivo to promote leukemogenesis
Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we ...performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition, we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches, leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions.
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•Epigenetic and transcriptional dynamics in hESCs and hESC-derived populations•Lineage-specific remodeling at regions bound by OCT4, SOX2, and NANOG in hESCs•Germ-layer-specific switch to H3K4me1 or H3K27me3 at sites of high DNA methylation•Epigenetic dynamics frequently precede transcriptional activation
The epigenetic and transcriptional landscapes of three cell types representing each embryonic lineage derived from human embryonic stem cells are profiled, revealing distinct histone modification and DNA methylation dynamics that accompany lineage specification.
Tumors can evolve and adapt to therapeutic pressure by acquiring genetic and epigenetic alterations that may be transient or stable. A precise understanding of how such events contribute to ...intratumoral heterogeneity, dynamic subpopulations, and overall tumor fitness will require experimental approaches to prospectively label, track, and characterize resistant or otherwise adaptive populations at the single-cell level. In glioblastoma, poor efficacy of receptor tyrosine kinase (RTK) therapies has been alternatively ascribed to genetic heterogeneity or to epigenetic transitions that circumvent signaling blockade.
We combine cell lineage barcoding and single-cell transcriptomics to trace the emergence of drug resistance in stem-like glioblastoma cells treated with RTK inhibitors. Whereas a broad variety of barcoded lineages adopt a Notch-dependent persister phenotype that sustains them through early drug exposure, rare subclones acquire genetic changes that enable their rapid outgrowth over time. Single-cell analyses reveal that these genetic subclones gain copy number amplifications of the insulin receptor substrate-1 and substrate-2 (IRS1 or IRS2) loci, which activate insulin and AKT signaling programs. Persister-like cells and genomic amplifications of IRS2 and other loci are evident in primary glioblastomas and may underlie the inefficacy of targeted therapies in this disease.
A method for combined lineage tracing and scRNA-seq reveals the interplay between complementary genetic and epigenetic mechanisms of resistance in a heterogeneous glioblastoma tumor model.
Human cancers are complex ecosystems composed of cells with distinct phenotypes, genotypes, and epigenetic states, but current models do not adequately reflect tumor composition in patients. We used ...single-cell RNA sequencing (RNA-seq) to profile 430 cells from five primary glioblastomas, which we found to be inherently variable in their expression of diverse transcriptional programs related to oncogenic signaling, proliferation, complement/immune response, and hypoxia. We also observed a continuum of stemness-related expression states that enabled us to identify putative regulators of stemness in vivo. Finally, we show that established glioblastoma subtype classifiers are variably expressed across individual cells within a tumor and demonstrate the potential prognostic implications of such intratumoral heterogeneity. Thus, we reveal previously unappreciated heterogeneity in diverse regulatory programs central to glioblastoma biology, prognosis, and therapy.
Chromatin profiling has emerged as a powerful means of genome annotation and detection of regulatory activity. The approach is especially well suited to the characterization of non-coding portions of ...the genome, which critically contribute to cellular phenotypes yet remain largely uncharted. Here we map nine chromatin marks across nine cell types to systematically characterize regulatory elements, their cell-type specificities and their functional interactions. Focusing on cell-type-specific patterns of promoters and enhancers, we define multicell activity profiles for chromatin state, gene expression, regulatory motif enrichment and regulator expression. We use correlations between these profiles to link enhancers to putative target genes, and predict the cell-type-specific activators and repressors that modulate them. The resulting annotations and regulatory predictions have implications for the interpretation of genome-wide association studies. Top-scoring disease single nucleotide polymorphisms are frequently positioned within enhancer elements specifically active in relevant cell types, and in some cases affect a motif instance for a predicted regulator, thus suggesting a mechanism for the association. Our study presents a general framework for deciphering cis-regulatory connections and their roles in disease.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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