The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot ...precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance.
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► Genome-wide Tet-assisted bisulfite sequencing for single-base detection of 5hmC ► Single-base resolution maps of 5hmC versus 5mC in human and mouse ESCs ► 5hmC is enriched at distal-regulatory elements and promoters with low CpG content ► 5hmC deposition is asymmetric and strand biased toward G-rich sequences
A method utilizing Tet oxidization in combination with bisulfite sequencing now enables distinction between 5mC and 5hmC on a genome-wide scale and at base resolution. Analysis of the distribution of these two marks in mouse and human genomes suggests a sequence bias and strand asymmetry at 5hmC sites.
The study of enhancers has been hampered by the scarcity of methods to systematically quantify their endogenous activity. We develop Mosaic-seq to systematically perturb enhancers and measure ...their endogenous activities at single-cell resolution. Mosaic-seq uses a CRISPR barcoding system to jointly measure a cell’s transcriptome and its sgRNA modulators, thus quantifying the effects of dCas9-KRAB-mediated enhancer repression in single cells. Applying Mosaic-seq to 71 constituent enhancers from 15 super-enhancers, our analysis of 51,448 sgRNA-induced transcriptomes finds that only a small number of constituents are major effectors of target gene expression. Binding of p300 and RNAPII are key features of these constituents. We determine two key parameters of enhancer activity in single cells: their penetrance in a population and their contribution to expression in these cells. Through combinatorial interrogation, we find that simultaneous repression of multiple weak constituents can alter super-enhancer activity in a manner greatly exceeding repression of individual constituents.
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•Mosaic-seq enables high-throughput endogenous interrogation of enhancers•71 constituent enhancers from 15 super-enhancers are analyzed•Mosaic-seq measures enhancer usage in single cells•Mosaic-seq enables systematic evaluation of combinatorial enhancer activity
Xie et al. develop a technology called Mosaic-seq. This approach enables the interrogation of enhancers at high throughput, at single-cell resolution, and in combination.
Limited access to embryos has hampered the study of human embryogenesis and disorders that occur during early pregnancy. Human pluripotent stem cells provide an alternative means to study human ...development in a dish
. Recent advances in partial embryo models derived from human pluripotent stem cells have enabled human development to be examined at early post-implantation stages
. However, models of the pre-implantation human blastocyst are lacking. Starting from naive human pluripotent stem cells, here we developed an effective three-dimensional culture strategy with successive lineage differentiation and self-organization to generate blastocyst-like structures in vitro. These structures-which we term 'human blastoids'-resemble human blastocysts in terms of their morphology, size, cell number, and composition and allocation of different cell lineages. Single-cell RNA-sequencing analyses also reveal the transcriptomic similarity of blastoids to blastocysts. Human blastoids are amenable to embryonic and extra-embryonic stem cell derivation and can further develop into peri-implantation embryo-like structures in vitro. Using chemical perturbations, we show that specific isozymes of protein kinase C have a critical function in the formation of the blastoid cavity. Human blastoids provide a readily accessible, scalable, versatile and perturbable alternative to blastocysts for studying early human development, understanding early pregnancy loss and gaining insights into early developmental defects.
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.
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•Base resolution maps of 5hmC and 5mC in WT, Tet1−/−, and Tet2−/− mESCs•Reduced 5hmC and increased 5mC at enhancers in Tet2−/− mESCs•Hypermethylated enhancers exhibit reduced activity•Hypermethylation and delayed gene induction during Tet2−/− mESC differentiation
Active transcriptional enhancers coordinate gene regulation and exhibit characteristic epigenetic signatures, including depletion of DNA methylation. Hon et al. show that Tet2 mediates enhancer demethylation to modulate enhancer activity, which influences the timing of transcriptome reprogramming during differentiation of mouse embryonic stem cells.
Integrating results from diverse experiments is an essential process in our effort to understand the logic of complex systems, such as development, homeostasis and responses to the environment. With ...the advent of high-throughput methods--including genome-wide association (GWA) studies, chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA sequencing (RNA-seq)--acquisition of genome-scale data has never been easier. Epigenomics, transcriptomics, proteomics and genomics each provide an insightful, and yet one-dimensional, view of genome function; integrative analysis promises a unified, global view. However, the large amount of information and diverse technology platforms pose multiple challenges for data access and processing. This Review discusses emerging issues and strategies related to data integration in the era of next-generation genomics.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here, we describe ...a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single-copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes establishes evidence for composition-based hierarchical organization. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally regulated super-enhancers reveals spatial features that causally control gene transcription. Thus, comprehensive and unbiased analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.
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•dCas9 capture enables analysis of locus-specific chromatin-regulating proteome•dCas9 capture identifies high-resolution locus-specific long-range DNA interactions•Capture of disease-associated CREs uncovers mechanisms for β-globin disorders•Capture of super-enhancers reveals composition-based hierarchical structure
A biotinylated dCas9-based method for simultaneously studying long-range DNA interactions and chromatin-associated proteins at any genomic location.
Mammalian development requires cytosine methylation, a heritable epigenetic mark of cellular memory believed to maintain a cell's unique gene expression pattern. However, it remains unclear how ...dynamic DNA methylation relates to cell type-specific gene expression and animal development. Here, by mapping base-resolution methylomes in 17 adult mouse tissues at shallow coverage, we identify 302,864 tissue-specific differentially methylated regions (tsDMRs) and estimate that >6.7% of the mouse genome is variably methylated. Supporting a prominent role for DNA methylation in gene regulation, most tsDMRs occur at distal cis-regulatory elements. Unexpectedly, some tsDMRs mark enhancers that are dormant in adult tissues but active in embryonic development. These 'vestigial' enhancers are hypomethylated and lack active histone modifications in adult tissues but nevertheless exhibit activity during embryonic development. Our results provide new insights into the role of DNA methylation at tissue-specific enhancers and suggest that epigenetic memory of embryonic development may be retained in adult tissues.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract
Motivation
Single cell RNA-Seq (scRNA-Seq) has broadened our understanding of cellular heterogeneity and provided valuable insights into cellular functions. Recent experimental strategies ...extend scRNA-Seq readouts to include additional features, including cell surface proteins and genomic perturbations. These ‘feature barcoding’ strategies rely on converting molecular and cellular features to unique sequence barcodes, which are then detected with the transcriptome.
Results
Here, we introduce FBA, a flexible and streamlined package to perform quality control, quantification, demultiplexing, multiplet detection, clustering and visualization of feature barcoding assays.
Availabilityand implementation
FBA is available on PyPi at https://pypi.org/project/fba and on GitHub at https://github.com/jlduan/fba.
Supplementary information
Supplementary data are available at Bioinformatics online.
Simultaneously detecting CRISPR-based perturbations and induced transcriptional changes in the same cell is a powerful approach to unraveling genome function. Several lentiviral approaches have been ...developed, some of which rely on the detection of distally located genetic barcodes as an indirect proxy of sgRNA identity. Since barcodes are often several kilobases from their corresponding sgRNAs, viral recombination-mediated swapping of barcodes and sgRNAs is feasible. Using a self-circularization-based sgRNA-barcode library preparation protocol, we estimate the recombination rate to be ~50% and we trace this phenomenon to the pooled viral packaging step. Recombination is random, and decreases the signal-to-noise ratio of the assay. Our results suggest that alternative approaches can increase the throughput and sensitivity of single-cell perturbation assays.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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•Oncogenic EGFRvIII mutation remodels the enhancer landscape in GBM•EGFRvIII induces SOX9 and FOXG1 transcription in GBM•SOX9 and FOXG1 collaborate to activate an oncogenic gene regulatory program•EGFRvIII-dependent transcription sensitizes GBM cells to JQ1
Epidermal growth factor receptor (EGFR) gene amplification and mutations are the most common oncogenic events in glioblastoma (GBM). Through an integrative genomics analysis, Liu et al., identify a role for transcriptional/epigenetic remodeling in EGFR-dependent pathogenesis and suggest a mechanistic basis for epigenetic therapy.
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