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.

Epigenetic Alterations in Keratinocyte Carcinoma.

Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are both derived from epidermal keratinocytes but are phenotypically diverse. To improve the understanding of keratinocyte carcinogenesis, it is critical to understand epigenetic alterations, especially those that govern gene expression. We examined changes to the enhancer-associated histone acetylation mark H3K27ac by mapping matched tumor-normal pairs from 11 patients (five with BCC and six with SCC) undergoing Mohs surgery. Our analysis uncovered cancer-specific enhancers on the basis of differential H3K27ac peaks between matched tumor-normal pairs. We also uncovered biological pathways potentially altered in keratinocyte carcinoma, including enriched epidermal development and Wnt signaling pathways enriched in BCCs and enriched immune response and cell activation pathways in SCCs. We also observed enrichment of transcription factors that implicated SMAD and JDP2 in BCC pathogenesis and FOXP1 in SCC pathogenesis. On the basis of these findings, we prioritized three loci with putative regulation events (FGFR2 enhancer in BCC, intragenic regulation of FOXP1 in SCC, and WNT5A promoter in both subtypes) and validated our findings with published gene expression data. Our findings highlight unique and shared epigenetic alterations in histone modifications and potential regulators for BCCs and SCCs that likely impact the divergent oncogenic pathways, paving the way for targeted drug discoveries.

Prioritizing disease and trait causal variants at the TNFAIP3 locus using functional and genomic features.

Genome-wide association studies have associated thousands of genetic variants with complex traits and diseases, but pinpointing the causal variant(s) among those in tight linkage disequilibrium with each associated variant remains a major challenge. Here, we use seven experimental assays to characterize all common variants at the multiple disease-associated TNFAIP3 locus in five disease-relevant immune cell lines, based on a set of features related to regulatory potential. Trait/disease-associated variants are enriched among SNPs prioritized based on either: (1) residing within CRISPRi-sensitive regulatory regions, or (2) localizing in a chromatin accessible region while displaying allele-specific reporter activity. Of the 15 trait/disease-associated haplotypes at TNFAIP3, 9 have at least one variant meeting one or both of these criteria, 5 of which are further supported by genetic fine-mapping. Our work provides a comprehensive strategy to characterize genetic variation at important disease-associated loci, and aids in the effort to identify trait causal genetic variants.

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.

Detection of Enhancer-Associated Rearrangements Reveals Mechanisms of Oncogene Dysregulation in B-cell Lymphoma.

UNLABELLED: B-cell lymphomas frequently contain genomic rearrangements that lead to oncogene activation by heterologous distal regulatory elements. We used a novel approach called "pinpointing enhancer-associated rearrangements by chromatin immunoprecipitation," or PEAR-ChIP, to simultaneously map enhancer activity and proximal rearrangements in lymphoma cell lines and patient biopsies. This method detects rearrangements involving known cancer genes, including CCND1, BCL2, MYC, PDCD1LG2, NOTCH1, CIITA, and SGK1, as well as novel enhancer duplication events of likely oncogenic significance. We identify lymphoma subtype-specific enhancers in the MYC locus that are silenced in lymphomas with MYC-activating rearrangements and are associated with germline polymorphisms that alter lymphoma risk. We show that BCL6-locus enhancers are acetylated by the BCL6-activating transcription factor MEF2B, and can undergo genomic duplication, or target the MYC promoter for activation in the context of a "pseudo-double-hit" t(3;8)(q27;q24) rearrangement linking the BCL6 and MYC loci. Our work provides novel insights regarding enhancer-driven oncogene activation in lymphoma. SIGNIFICANCE: We demonstrate a novel approach for simultaneous detection of genomic rearrangements and enhancer activity in tumor biopsies. We identify novel mechanisms of enhancer-driven regulation of the oncogenes MYC and BCL6, and show that the BCL6 locus can serve as an enhancer donor in an "enhancer hijacking" translocation.

Identification of nuclear hormone receptor pathways causing insulin resistance by transcriptional and epigenomic analysis.

Insulin resistance is a cardinal feature of Type 2 diabetes (T2D) and a frequent complication of multiple clinical conditions, including obesity, ageing and steroid use, among others. How such a panoply of insults can result in a common phenotype is incompletely understood. Furthermore, very little is known about the transcriptional and epigenetic basis of this disorder, despite evidence that such pathways are likely to play a fundamental role. Here, we compare cell autonomous models of insulin resistance induced by the cytokine tumour necrosis factor-α or by the steroid dexamethasone to construct detailed transcriptional and epigenomic maps associated with cellular insulin resistance. These data predict that the glucocorticoid receptor and vitamin D receptor are common mediators of insulin resistance, which we validate using gain- and loss-of-function studies. These studies define a common transcriptional and epigenomic signature in cellular insulin resistance enabling the identification of pathogenic mechanisms.

Dissecting neural differentiation regulatory networks through epigenetic footprinting.

Models derived from human pluripotent stem cells that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem cell and biomedical community. Notch signalling, particularly through the Notch effector HES5, is a major pathway critical for the onset and maintenance of neural progenitor cells in the embryonic and adult nervous system. Here we report the transcriptional and epigenomic analysis of six consecutive neural progenitor cell stages derived from a HES5::eGFP reporter human embryonic stem cell line. Using this system, we aimed to model cell-fate decisions including specification, expansion and patterning during the ontogeny of cortical neural stem and progenitor cells. In order to dissect regulatory mechanisms that orchestrate the stage-specific differentiation process, we developed a computational framework to infer key regulators of each cell-state transition based on the progressive remodelling of the epigenetic landscape and then validated these through a pooled short hairpin RNA screen. We were also able to refine our previous observations on epigenetic priming at transcription factor binding sites and suggest here that they are mediated by combinations of core and stage-specific factors. Taken together, we demonstrate the utility of our system and outline a general framework, not limited to the context of the neural lineage, to dissect regulatory circuits of differentiation.

Impact of sequencing depth in ChIP-seq experiments.

In a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiment, an important consideration in experimental design is the minimum number of sequenced reads required to obtain statistically significant results. We present an extensive evaluation of the impact of sequencing depth on identification of enriched regions for key histone modifications (H3K4me3, H3K36me3, H3K27me3 and H3K9me2/me3) using deep-sequenced datasets in human and fly. We propose to define sufficient sequencing depth as the number of reads at which detected enrichment regions increase <1% for an additional million reads. Although the required depth depends on the nature of the mark and the state of the cell in each experiment, we observe that sufficient depth is often reached at <20 million reads for fly. For human, there are no clear saturation points for the examined datasets, but our analysis suggests 40-50 million reads as a practical minimum for most marks. We also devise a mathematical model to estimate the sufficient depth and total genomic coverage of a mark. Lastly, we find that the five algorithms tested do not agree well for broad enrichment profiles, especially at lower depths. Our findings suggest that sufficient sequencing depth and an appropriate peak-calling algorithm are essential for ensuring robustness of conclusions derived from ChIP-seq data.

Transcriptional and epigenetic dynamics during specification of human embryonic stem cells.

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.

Genome-wide map of quantified epigenetic changes during in vitro chondrogenic differentiation of primary human mesenchymal stem cells.

BACKGROUND: For safe clinical application of engineered cartilage made from mesenchymal stem cells (MSCs), molecular mechanisms for chondrogenic differentiation must be known in detail. Changes in gene expression and extracellular matrix synthesis have been extensively studied, but the epigenomic modifications underlying these changes have not been described. To this end we performed whole-genome chromatin immunoprecipitation and deep sequencing to quantify six histone modifications, reduced representation bisulphite sequencing to quantify DNA methylation and mRNA microarrays to quantify gene expression before and after 7 days of chondrogenic differentiation of MSCs in an alginate scaffold. To add to the clinical relevance of our observations, the study is based on primary bone marrow-derived MSCs from four donors, allowing us to investigate inter-individual variations. RESULTS: We see two levels of relationship between epigenetic marking and gene expression. First, a large number of genes ontogenetically linked to MSC properties and the musculoskeletal system are epigenetically prepatterned by moderate changes in H3K4me3 and H3K9ac near transcription start sites. Most of these genes remain transcriptionally unaltered. Second, transcriptionally upregulated genes, more closely associated with chondrogenesis, are marked by H3K36me3 in gene bodies, highly increased H3K4me3 and H3K9ac on promoters and 5' end of genes, and increased H3K27ac and H3K4me1 marking in at least one enhancer region per upregulated gene. Within the 7-day time frame, changes in promoter DNA methylation do not correlate significantly with changes in gene expression. Inter-donor variability analysis shows high level of similarity between the donors for this data set. CONCLUSIONS: Histone modifications, rather than DNA methylation, provide the primary epigenetic control of early differentiation of MSCs towards the chondrogenic lineage.

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.

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.