Mechanosensitive super-enhancers regulate genes linked to atherosclerosis in endothelial cells.

Vascular homeostasis and pathophysiology are tightly regulated by mechanical forces generated by hemodynamics. Vascular disorders such as atherosclerotic diseases largely occur at curvatures and bifurcations where disturbed blood flow activates endothelial cells while unidirectional flow at the straight part of vessels promotes endothelial health. Integrated analysis of the endothelial transcriptome, the 3D epigenome, and human genetics systematically identified the SNP-enriched cistrome in vascular endothelium subjected to well-defined atherosclerosis-prone disturbed flow or atherosclerosis-protective unidirectional flow. Our results characterized the endothelial typical- and super-enhancers and underscored the critical regulatory role of flow-sensitive endothelial super-enhancers. CRISPR interference and activation validated the function of a previously unrecognized unidirectional flow-induced super-enhancer that upregulates antioxidant genes NQO1, CYB5B, and WWP2, and a disturbed flow-induced super-enhancer in endothelium which drives prothrombotic genes EDN1 and HIVEP in vascular endothelium. Our results employing multiomics identify the cis-regulatory architecture of the flow-sensitive endothelial epigenome related to atherosclerosis and highlight the regulatory role of super-enhancers in mechanotransduction mechanisms.

Genetics of sexually dimorphic adipose distribution in humans.

Obesity-associated morbidity is exacerbated by abdominal obesity, which can be measured as the waist-to-hip ratio adjusted for the body mass index (WHRadjBMI). Here we identify genes associated with obesity and WHRadjBMI and characterize allele-sensitive enhancers that are predicted to regulate WHRadjBMI genes in women. We found that several waist-to-hip ratio-associated variants map within primate-specific Alu retrotransposons harboring a DNA motif associated with adipocyte differentiation. This suggests that a genetic component of adipose distribution in humans may involve co-option of retrotransposons as adipose enhancers. We evaluated the role of the strongest female WHRadjBMI-associated gene, SNX10, in adipose biology. We determined that it is required for human adipocyte differentiation and function and participates in diet-induced adipose expansion in female mice, but not males. Our data identify genes and regulatory mechanisms that underlie female-specific adipose distribution and mediate metabolic dysfunction in women.

A pleiotropic hypoxia-sensitive EPAS1 enhancer is disrupted by adaptive alleles in Tibetans.

In Tibetans, noncoding alleles in EPAS1-whose protein product hypoxia-inducible factor 2α (HIF-2α) drives the response to hypoxia-carry strong signatures of positive selection; however, their functional mechanism has not been systematically examined. Here, we report that high-altitude alleles disrupt the activity of four EPAS1 enhancers in one or more cell types. We further characterize one enhancer (ENH5) whose activity is both allele specific and hypoxia dependent. Deletion of ENH5 results in down-regulation of EPAS1 and HIF-2α targets in acute hypoxia and in a blunting of the transcriptional response to sustained hypoxia. Deletion of ENH5 in mice results in dysregulation of gene expression across multiple tissues. We propose that pleiotropic adaptive effects of the Tibetan alleles in EPAS1 underlie the strong selective signal at this gene.

Asthma-associated genetic variants induce IL33 differential expression through an enhancer-blocking regulatory region.

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as an enhancer-blocking element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that the asthma-associated single nucleotide polymorphism (SNP) rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a mechanism through which a regulatory SNP contributes to genetic risk of asthma.

A functional genomics pipeline identifies pleiotropy and cross-tissue effects within obesity-associated GWAS loci.

Genome-wide association studies (GWAS) have identified many disease-associated variants, yet mechanisms underlying these associations remain unclear. To understand obesity-associated variants, we generate gene regulatory annotations in adipocytes and hypothalamic neurons across cellular differentiation stages. We then test variants in 97 obesity-associated loci using a massively parallel reporter assay and identify putatively causal variants that display cell type specific or cross-tissue enhancer-modulating properties. Integrating these variants with gene regulatory information suggests genes that underlie obesity GWAS associations. We also investigate a complex genomic interval on 16p11.2 where two independent loci exhibit megabase-range, cross-locus chromatin interactions. We demonstrate that variants within these two loci regulate a shared gene set. Together, our data support a model where GWAS loci contain variants that alter enhancer activity across tissues, potentially with temporally restricted effects, to impact the expression of multiple genes. This complex model has broad implications for ongoing efforts to understand GWAS.

Extensive pleiotropism and allelic heterogeneity mediate metabolic effects of IRX3 and IRX5.

Whereas coding variants often have pleiotropic effects across multiple tissues, noncoding variants are thought to mediate their phenotypic effects by specific tissue and temporal regulation of gene expression. Here, we investigated the genetic and functional architecture of a genomic region within the FTO gene that is strongly associated with obesity risk. We show that multiple variants on a common haplotype modify the regulatory properties of several enhancers targeting IRX3 and IRX5 from megabase distances. We demonstrate that these enhancers affect gene expression in multiple tissues, including adipose and brain, and impart regulatory effects during a restricted temporal window. Our data indicate that the genetic architecture of disease-associated loci may involve extensive pleiotropy, allelic heterogeneity, shared allelic effects across tissues, and temporally restricted effects.

Pluripotent stem cell-derived endometrial stromal fibroblasts in a cyclic, hormone-responsive, coculture model of human decidua.

Various human diseases and pregnancy-related disorders reflect endometrial dysfunction. However, rodent models do not share fundamental biological processes with the human endometrium, such as spontaneous decidualization, and no existing human cell cultures recapitulate the cyclic interactions between endometrial stromal and epithelial compartments necessary for decidualization and implantation. Here we report a protocol differentiating human pluripotent stem cells into endometrial stromal fibroblasts (PSC-ESFs) that are highly pure and able to decidualize. Coculture of PSC-ESFs with placenta-derived endometrial epithelial cells generated organoids used to examine stromal-epithelial interactions. Cocultures exhibited specific endometrial markers in the appropriate compartments, organization with cell polarity, and hormone responsiveness of both cell types. Furthermore, cocultures recapitulate a central feature of the human decidua by cyclically responding to hormone withdrawal followed by hormone retreatment. This advance enables mechanistic studies of the cyclic responses that characterize the human endometrium.

Transcriptome and regulatory maps of decidua-derived stromal cells inform gene discovery in preterm birth.

While a genetic component of preterm birth (PTB) has long been recognized and recently mapped by genome-wide association studies (GWASs), the molecular determinants underlying PTB remain elusive. This stems in part from an incomplete availability of functional genomic annotations in human cell types relevant to pregnancy and PTB. We generated transcriptome (RNA-seq), epigenome (ChIP-seq of H3K27ac, H3K4me1, and H3K4me3 histone modifications), open chromatin (ATAC-seq), and chromatin interaction (promoter capture Hi-C) annotations of cultured primary decidua-derived mesenchymal stromal/stem cells and in vitro differentiated decidual stromal cells and developed a computational framework to integrate these functional annotations with results from a GWAS of gestational duration in 56,384 women. Using these resources, we uncovered additional loci associated with gestational duration and target genes of associated loci. Our strategy illustrates how functional annotations in pregnancy-relevant cell types aid in the experimental follow-up of GWAS for PTB and, likely, other pregnancy-related conditions.

Altered transcriptional and chromatin responses to rhinovirus in bronchial epithelial cells from adults with asthma.

There is a life-long relationship between rhinovirus (RV) infection and the development and clinical manifestations of asthma. In this study we demonstrate that cultured primary bronchial epithelial cells from adults with asthma (n = 9) show different transcriptional and chromatin responses to RV infection compared to those without asthma (n = 9). Both the number and magnitude of transcriptional and chromatin responses to RV were muted in cells from asthma cases compared to controls. Pathway analysis of the transcriptionally responsive genes revealed enrichments of apoptotic pathways in controls but inflammatory pathways in asthma cases. Using promoter capture Hi-C we tethered regions of RV-responsive chromatin to RV-responsive genes and showed enrichment of these regions and genes at asthma GWAS loci. Taken together, our studies indicate a delayed or prolonged inflammatory state in cells from asthma cases and highlight genes that may contribute to genetic risk for asthma.

A promoter interaction map for cardiovascular disease genetics.

Over 500 genetic loci have been associated with risk of cardiovascular diseases (CVDs); however, most loci are located in gene-distal non-coding regions and their target genes are not known. Here, we generated high-resolution promoter capture Hi-C (PCHi-C) maps in human induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes (CMs) to provide a resource for identifying and prioritizing the functional targets of CVD associations. We validate these maps by demonstrating that promoters preferentially contact distal sequences enriched for tissue-specific transcription factor motifs and are enriched for chromatin marks that correlate with dynamic changes in gene expression. Using the CM PCHi-C map, we linked 1999 CVD-associated SNPs to 347 target genes. Remarkably, more than 90% of SNP-target gene interactions did not involve the nearest gene, while 40% of SNPs interacted with at least two genes, demonstrating the importance of considering long-range chromatin interactions when interpreting functional targets of disease loci.

Obesity-associated variants within FTO form long-range functional connections with IRX3.

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.

Dual transcriptional activator and repressor roles of TBX20 regulate adult cardiac structure and function.

The ongoing requirement in adult heart for transcription factors with key roles in cardiac development is not well understood. We recently demonstrated that TBX20, a transcriptional regulator required for cardiac development, has key roles in the maintenance of functional and structural phenotypes in adult mouse heart. Conditional ablation of Tbx20 in adult cardiomyocytes leads to a rapid onset and progression of heart failure, with prominent conduction and contractility phenotypes that lead to death. Here we describe a more comprehensive molecular characterization of the functions of TBX20 in adult mouse heart. Coupling genome-wide chromatin immunoprecipitation and transcriptome analyses (RNA-Seq), we identified a subset of genes that change expression in Tbx20 adult cardiomyocyte-specific knockout hearts which are direct downstream targets of TBX20. This analysis revealed a dual role for TBX20 as both a transcriptional activator and a repressor, and that each of these functions regulates genes with very specialized and distinct molecular roles. We also show how TBX20 binds to its targets genome-wide in a context-dependent manner, using various cohorts of co-factors to either promote or repress distinct genetic programs within adult heart. Our integrative approach has uncovered several novel aspects of TBX20 and T-box protein function within adult heart. Sequencing data accession number (http://www.ncbi.nlm.nih.gov/geo): GSE30943.

Genome-wide maps of transcription regulatory elements.

Expression of eukaryotic genes with complex spatial-temporal regulation during development requires finer regulation than that of genes with simpler expression patterns. Given the high degree of conservation of the developmental gene set across distantly related phylogenetic taxa, it is argued that evolutionary variation has occurred by tweaking regulation of expression of developmental genes, rather than by changes in genes themselves. Complex regulation is often achieved through the coordinated action of transcription regulatory elements spread across the genome up to tens of kilobases from the promoters of their target genes. Disruption of regulatory elements has been implicated in several diseases and studies showing associations between disease traits and nonprotein coding variation hint for a role of regulatory elements as cause of diseases. Therefore, the identification and mapping of regulatory elements in genome scale is crucial to understand how gene expression is regulated, how organisms evolve, and to identify sequence variation causing diseases. Previously developed experimental techniques have been adapted to identify regulatory elements in genome scale and high-throughput, allowing a global view of their biological roles. We review methods as chromatin immunoprecipitation, DNase I hypersensitivity, and computational approaches and how they have been employed to generate maps of histone modifications, open chromatin, nucleosome positioning, and transcription factor binding regions in whole mammalian genomes. Given the importance of non-promoter elements in gene regulation and the recent explosion in the number of studies devoted to them, we focus on these elements and discuss the insights on gene regulation being obtained by these studies.

Genome-wide discovery of human heart enhancers.

The various organogenic programs deployed during embryonic development rely on the precise expression of a multitude of genes in time and space. Identifying the cis-regulatory elements responsible for this tightly orchestrated regulation of gene expression is an essential step in understanding the genetic pathways involved in development. We describe a strategy to systematically identify tissue-specific cis-regulatory elements that share combinations of sequence motifs. Using heart development as an experimental framework, we employed a combination of Gibbs sampling and linear regression to build a classifier that identifies heart enhancers based on the presence and/or absence of various sequence features, including known and putative transcription factor (TF) binding specificities. In distinguishing heart enhancers from a large pool of random noncoding sequences, the performance of our classifier is vastly superior to four commonly used methods, with an accuracy reaching 92% in cross-validation. Furthermore, most of the binding specificities learned by our method resemble the specificities of TFs widely recognized as key players in heart development and differentiation, such as SRF, MEF2, ETS1, SMAD, and GATA. Using our classifier as a predictor, a genome-wide scan identified over 40,000 novel human heart enhancers. Although the classifier used no gene expression information, these novel enhancers are strongly associated with genes expressed in the heart. Finally, in vivo tests of our predictions in mouse and zebrafish achieved a validation rate of 62%, significantly higher than what is expected by chance. These results support the existence of underlying cis-regulatory codes dictating tissue-specific transcription in mammalian genomes and validate our enhancer classifier strategy as a method to uncover these regulatory codes.

CTdatabase: a knowledge-base of high-throughput and curated data on cancer-testis antigens.

The potency of the immune response has still to be harnessed effectively to combat human cancers. However, the discovery of T-cell targets in melanomas and other tumors has raised the possibility that cancer vaccines can be used to induce a therapeutically effective immune response against cancer. The targets, cancer-testis (CT) antigens, are immunogenic proteins preferentially expressed in normal gametogenic tissues and different histological types of tumors. Therapeutic cancer vaccines directed against CT antigens are currently in late-stage clinical trials testing whether they can delay or prevent recurrence of lung cancer and melanoma following surgical removal of primary tumors. CT antigens constitute a large, but ill-defined, family of proteins that exhibit a remarkably restricted expression. Currently, there is a considerable amount of information about these proteins, but the data are scattered through the literature and in several bioinformatic databases. The database presented here, CTdatabase (http://www.cta.lncc.br), unifies this knowledge to facilitate both the mining of the existing deluge of data, and the identification of proteins alleged to be CT antigens, but that do not have their characteristic restricted expression pattern. CTdatabase is more than a repository of CT antigen data, since all the available information was carefully curated and annotated with most data being specifically processed for CT antigens and stored locally. Starting from a compilation of known CT antigens, CTdatabase provides basic information including gene names and aliases, RefSeq accession numbers, genomic location, known splicing variants, gene duplications and additional family members. Gene expression at the mRNA level in normal and tumor tissues has been collated from publicly available data obtained by several different technologies. Manually curated data related to mRNA and protein expression, and antigen-specific immune responses in cancer patients are also available, together with links to PubMed for relevant CT antigen articles.