The EN-TEx resource of multi-tissue personal epigenomes & variant-impact models.

Understanding how genetic variants impact molecular phenotypes is a key goal of functional genomics, currently hindered by reliance on a single haploid reference genome. Here, we present the EN-TEx resource of 1,635 open-access datasets from four donors (∼30 tissues × âˆ¼15 assays). The datasets are mapped to matched, diploid genomes with long-read phasing and structural variants, instantiating a catalog of >1 million allele-specific loci. These loci exhibit coordinated activity along haplotypes and are less conserved than corresponding, non-allele-specific ones. Surprisingly, a deep-learning transformer model can predict the allele-specific activity based only on local nucleotide-sequence context, highlighting the importance of transcription-factor-binding motifs particularly sensitive to variants. Furthermore, combining EN-TEx with existing genome annotations reveals strong associations between allele-specific and GWAS loci. It also enables models for transferring known eQTLs to difficult-to-profile tissues (e.g., from skin to heart). Overall, EN-TEx provides rich data and generalizable models for more accurate personal functional genomics.

Large-Scale Topological Changes Restrain Malignant Progression in Colorectal Cancer.

Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.

A Genetic Variant Associated with Five Vascular Diseases Is a Distal Regulator of Endothelin-1 Gene Expression.

Genome-wide association studies (GWASs) implicate the PHACTR1 locus (6p24) in risk for five vascular diseases, including coronary artery disease, migraine headache, cervical artery dissection, fibromuscular dysplasia, and hypertension. Through genetic fine mapping, we prioritized rs9349379, a common SNP in the third intron of the PHACTR1 gene, as the putative causal variant. Epigenomic data from human tissue revealed an enhancer signature at rs9349379 exclusively in aorta, suggesting a regulatory function for this SNP in the vasculature. CRISPR-edited stem cell-derived endothelial cells demonstrate rs9349379 regulates expression of endothelin 1 (EDN1), a gene located 600 kb upstream of PHACTR1. The known physiologic effects of EDN1 on the vasculature may explain the pattern of risk for the five associated diseases. Overall, these data illustrate the integration of genetic, phenotypic, and epigenetic analysis to identify the biologic mechanism by which a common, non-coding variant can distally regulate a gene and contribute to the pathogenesis of multiple vascular diseases.

Genome-wide chromatin state transitions associated with developmental and environmental cues.

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.

Perturbation-specific transcriptional mapping for unbiased target elucidation of antibiotics.

The rising prevalence of antibiotic resistance threatens human health. While more sophisticated strategies for antibiotic discovery are being developed, target elucidation of new chemical entities remains challenging. In the postgenomic era, expression profiling can play an important role in mechanism-of-action (MOA) prediction by reporting on the cellular response to perturbation. However, the broad application of transcriptomics has yet to fulfill its promise of transforming target elucidation due to challenges in identifying the most relevant, direct responses to target inhibition. We developed an unbiased strategy for MOA prediction, called perturbation-specific transcriptional mapping (PerSpecTM), in which large-throughput expression profiling of wild-type or hypomorphic mutants, depleted for essential targets, enables a computational strategy to address this challenge. We applied PerSpecTM to perform reference-based MOA prediction based on the principle that similar perturbations, whether chemical or genetic, will elicit similar transcriptional responses. Using this approach, we elucidated the MOAs of three molecules with activity against Pseudomonas aeruginosa by comparing their expression profiles to those of a reference set of antimicrobial compounds with known MOAs. We also show that transcriptional responses to small-molecule inhibition resemble those resulting from genetic depletion of essential targets by clustered regularly interspaced short palindromic repeats interference (CRISPRi) by PerSpecTM, demonstrating proof of concept that correlations between expression profiles of small-molecule and genetic perturbations can facilitate MOA prediction when no chemical entities exist to serve as a reference. Empowered by PerSpecTM, this work lays the foundation for an unbiased, readily scalable, systematic reference-based strategy for MOA elucidation that could transform antibiotic discovery efforts.

Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells.

Hundreds of chromatin regulators (CRs) control chromatin structure and function by catalyzing and binding histone modifications, yet the rules governing these key processes remain obscure. Here, we present a systematic approach to infer CR function. We developed ChIP-string, a meso-scale assay that combines chromatin immunoprecipitation with a signature readout of 487 representative loci. We applied ChIP-string to screen 145 antibodies, thereby identifying effective reagents, which we used to map the genome-wide binding of 29 CRs in two cell types. We found that specific combinations of CRs colocalize in characteristic patterns at distinct chromatin environments, at genes of coherent functions, and at distal regulatory elements. When comparing between cell types, CRs redistribute to different loci but maintain their modular and combinatorial associations. Our work provides a multiplex method that substantially enhances the ability to monitor CR binding, presents a large resource of CR maps, and reveals common principles for combinatorial CR function.

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

Analyzing histone ChIP-seq data with a bin-based probability of being signal.

Histone ChIP-seq is one of the primary methods for charting the cellular epigenomic landscape, the components of which play a critical regulatory role in gene expression. Analyzing the activity of regulatory elements across datasets and cell types can be challenging due to shifting peak positions and normalization artifacts resulting from, for example, differing read depths, ChIP efficiencies, and target sizes. Moreover, broad regions of enrichment seen in repressive histone marks often evade detection by commonly used peak callers. Here, we present a simple and versatile method for identifying enriched regions in ChIP-seq data that relies on estimating a gamma distribution fit to non-overlapping 5kB genomic bins to establish a global background. We use this distribution to assign a probability of being signal (PBS) between zero and one to each 5 kB bin. This approach, while lower in resolution than typical peak-calling methods, provides a straightforward way to identify enriched regions and compare enrichments among multiple datasets, by transforming the data to values that are universally normalized and can be readily visualized and integrated with downstream analysis methods. We demonstrate applications of PBS for both broad and narrow histone marks, and provide several illustrations of biological insights which can be gleaned by integrating PBS scores with downstream data types.

Defining the core essential genome of Pseudomonas aeruginosa.

Genomics offered the promise of transforming antibiotic discovery by revealing many new essential genes as good targets, but the results fell short of the promise. While numerous factors contributed to the disappointing yield, one factor was that essential genes for a bacterial species were often defined based on a single or limited number of strains grown under a single or limited number of in vitro laboratory conditions. In fact, the essentiality of a gene can depend on both the genetic background and growth condition. We thus developed a strategy for more rigorously defining the core essential genome of a bacterial species by studying many pathogen strains and growth conditions. We assessed how many strains must be examined to converge on a set of core essential genes for a species. We used transposon insertion sequencing (Tn-Seq) to define essential genes in nine strains of Pseudomonas aeruginosa on five different media and developed a statistical model, FiTnEss, to classify genes as essential versus nonessential across all strain-medium combinations. We defined a set of 321 core essential genes, representing 6.6% of the genome. We determined that analysis of four strains was typically sufficient in P. aeruginosa to converge on a set of core essential genes likely to be essential across the species across a wide range of conditions relevant to in vivo infection, and thus to represent attractive targets for novel drug discovery.

Polyploidy can drive rapid adaptation in yeast.

Polyploidy is observed across the tree of life, yet its influence on evolution remains incompletely understood. Polyploidy, usually whole-genome duplication, is proposed to alter the rate of evolutionary adaptation. This could occur through complex effects on the frequency or fitness of beneficial mutations. For example, in diverse cell types and organisms, immediately after a whole-genome duplication, newly formed polyploids missegregate chromosomes and undergo genetic instability. The instability following whole-genome duplications is thought to provide adaptive mutations in microorganisms and can promote tumorigenesis in mammalian cells. Polyploidy may also affect adaptation independently of beneficial mutations through ploidy-specific changes in cell physiology. Here we perform in vitro evolution experiments to test directly whether polyploidy can accelerate evolutionary adaptation. Compared with haploids and diploids, tetraploids undergo significantly faster adaptation. Mathematical modelling suggests that rapid adaptation of tetraploids is driven by higher rates of beneficial mutations with stronger fitness effects, which is supported by whole-genome sequencing and phenotypic analyses of evolved clones. Chromosome aneuploidy, concerted chromosome loss, and point mutations all provide large fitness gains. We identify several mutations whose beneficial effects are manifest specifically in the tetraploid strains. Together, these results provide direct quantitative evidence that in some environments polyploidy can accelerate evolutionary adaptation.

Genetic and epigenetic fine mapping of causal autoimmune disease variants.

Genome-wide association studies have identified loci underlying human diseases, but the causal nucleotide changes and mechanisms remain largely unknown. Here we developed a fine-mapping algorithm to identify candidate causal variants for 21 autoimmune diseases from genotyping data. We integrated these predictions with transcription and cis-regulatory element annotations, derived by mapping RNA and chromatin in primary immune cells, including resting and stimulated CD4(+) T-cell subsets, regulatory T cells, CD8(+) T cells, B cells, and monocytes. We find that ∼90% of causal variants are non-coding, with ∼60% mapping to immune-cell enhancers, many of which gain histone acetylation and transcribe enhancer-associated RNA upon immune stimulation. Causal variants tend to occur near binding sites for master regulators of immune differentiation and stimulus-dependent gene activation, but only 10-20% directly alter recognizable transcription factor binding motifs. Rather, most non-coding risk variants, including those that alter gene expression, affect non-canonical sequence determinants not well-explained by current gene regulatory models.

Integrative analysis of 111 reference human epigenomes.

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Optimization of lag time underlies antibiotic tolerance in evolved bacterial populations.

The great therapeutic achievements of antibiotics have been dramatically undercut by the evolution of bacterial strategies that overcome antibiotic stress. These strategies fall into two classes. 'Resistance' makes it possible for a microorganism to grow in the constant presence of the antibiotic, provided that the concentration of the antibiotic is not too high. 'Tolerance' allows a microorganism to survive antibiotic treatment, even at high antibiotic concentrations, as long as the duration of the treatment is limited. Although both resistance and tolerance are important reasons for the failure of antibiotic treatments, the evolution of resistance is much better understood than that of tolerance. Here we followed the evolution of bacterial populations under intermittent exposure to the high concentrations of antibiotics used in the clinic and characterized the evolved strains in terms of both resistance and tolerance. We found that all strains adapted by specific genetic mutations, which became fixed in the evolved populations. By monitoring the phenotypic changes at the population and single-cell levels, we found that the first adaptive change to antibiotic stress was the development of tolerance through a major adjustment in the single-cell lag-time distribution, without a change in resistance. Strikingly, we found that the lag time of bacteria before regrowth was optimized to match the duration of the antibiotic-exposure interval. Whole genome sequencing of the evolved strains and restoration of the wild-type alleles allowed us to identify target genes involved in this antibiotic-driven phenotype: 'tolerance by lag' (tbl). Better understanding of lag-time evolution as a key determinant of the survival of bacterial populations under high antibiotic concentrations could lead to new approaches to impeding the evolution of antibiotic resistance.

An Experimental Framework for Quantifying Bacterial Tolerance.

Antibiotic tolerance and persistence are often associated with treatment failure and relapse, yet are poorly characterized. In distinction from resistance, which is measured using the minimum inhibitory concentration metric, tolerance and persistence values are not currently evaluated in the clinical setting, and so are overlooked when a course of treatment is prescribed. In this article, we introduce a metric and an automated experimental framework for measuring tolerance and persistence. The tolerance metric is the minimum duration for killing 99% of the population, MDK99, which can be evaluated by a statistical analysis of measurements performed manually or using a robotic system. We demonstrate the technique on strains of Escherichia coli with various tolerance levels. We hope that this, to our knowledge, new approach will be used, along with the existing minimum inhibitory concentration, as a standard for the in vitro characterization of sensitivity to antimicrobials. Quantification of tolerance and persistence may provide vital information in healthcare, and aid research in the field.

Mapping and analysis of chromatin state dynamics in nine human cell types.

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.

ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.

Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals.

In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites.

Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described-one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.

Perturbation-Specific Transcriptional Mapping for unbiased target elucidation of antibiotics.

The rising prevalence of antibiotic resistance threatens human health. While more sophisticated strategies for antibiotic discovery are being developed, target elucidation of new chemical entities remains challenging. In the post-genomic era, expression profiling can play an important role in mechanism-of-action (MOA) prediction by reporting on the cellular response to perturbation. However, the broad application of transcriptomics has yet to fulfill its promise of transforming target elucidation due to challenges in identifying the most relevant, direct responses to target inhibition. We developed an unbiased strategy for MOA prediction, called Perturbation-Specific Transcriptional Mapping (PerSpecTM), in which large-throughput expression profiling of wildtype or hypomorphic mutants, depleted for essential targets, enables a computational strategy to address this challenge. We applied PerSpecTM to perform reference-based MOA prediction based on the principle that similar perturbations, whether chemical or genetic, will elicit similar transcriptional responses. Using this approach, we elucidated the MOAs of three new molecules with activity against Pseudomonas aeruginosa by comparing their expression profiles to those of a reference set of antimicrobial compounds with known MOAs. We also show that transcriptional responses to small molecule inhibition resemble those resulting from genetic depletion of essential targets by CRISPRi by PerSpecTM, demonstrating proof-of-concept that correlations between expression profiles of small molecule and genetic perturbations can facilitate MOA prediction when no chemical entities exist to serve as a reference. Empowered by PerSpecTM, this work lays the foundation for an unbiased, readily scalable, systematic reference-based strategy for MOA elucidation that could transform antibiotic discovery efforts.

"Multiplexed screen identifies a Pseudomonas aeruginosa -specific small molecule targeting the outer membrane protein OprH and its interaction with LPS".

The surge of antimicrobial resistance threatens efficacy of current antibiotics, particularly against Pseudomonas aeruginosa , a highly resistant gram-negative pathogen. The asymmetric outer membrane (OM) of P. aeruginosa combined with its array of efflux pumps provide a barrier to xenobiotic accumulation, thus making antibiotic discovery challenging. We adapted PROSPECT 1 , a target-based, whole-cell screening strategy, to discover small molecule probes that kill P. aeruginosa mutants depleted for essential proteins localized at the OM. We identified BRD1401, a small molecule that has specific activity against a P. aeruginosa mutant depleted for the essential lipoprotein, OprL. Genetic and chemical biological studies identified that BRD1401 acts by targeting the OM ß-barrel protein OprH to disrupt its interaction with LPS and increase membrane fluidity. Studies with BRD1401 also revealed an interaction between OprL and OprH, directly linking the OM with peptidoglycan. Thus, a whole-cell, multiplexed screen can identify species-specific chemical probes to reveal novel pathogen biology.