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.
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.
To study the evolution of recombination rates in apes, we developed methodology to construct a fine-scale genetic map from high-throughput sequence data from 10 Western chimpanzees, Pan troglodytes ...verus. Compared to the human genetic map, broad-scale recombination rates tend to be conserved, but with exceptions, particularly in regions of chromosomal rearrangements and around the site of ancestral fusion in human chromosome 2. At fine scales, chimpanzee recombination is dominated by hotspots, which show no overlap with those of humans even though rates are similarly elevated around CpG islands and decreased within genes. The hotspot-specifying protein PRDM9 shows extensive variation among Western chimpanzees, and there is little evidence that any sequence motifs are enriched in hotspots. The contrasting locations of hotspots provide a natural experiment, which demonstrates the impact of recombination on base composition.
Significance Mammalian chromatin is compartmentalized in topologically associating domains (TADs), genomic regions within which sequences preferentially contact each other. This organization has been ...proposed to be essential to organize the regulatory information contained in mammalian genomes. We show that Six homeobox genes, essential developmental regulators organized in gene clusters across different animal phyla, share a deeply conserved chromatin organization formed by two abutting TADs that predates the Cambrian explosion. This organization is required to generate separate regulatory landscapes for neighboring genes within the cluster, resulting in very different gene expression patterns. Finally, we show that this extremely conserved 3D architecture is associated with a characteristic arrangement of CCCTC-binding factor (CTCF) binding sites in diverging orientations, revealing a genome-wide conserved signature for TAD borders.
Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution.
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.
Display omitted
•Endometrial stromal fibroblasts can be differentiated from human ESCs and iPSCs (PSCs)•PSC-ESFs self-assemble with endometrial epithelial organoids•Cocultured PSC-ESFs and EEOs cyclically respond to hormone treatment and withdrawal•PSC-ESFs respond to epithelial cell signaling while in coculture
Cheung et al. report a 3-dimensional in vitro model of the human decidua composed of hormone-responsive endometrial stromal fibroblasts differentiated through developmental intermediates from pluripotent stem cells (PSC-ESFs) and epithelial cells derived from postpartum decidua. PSC-ESFs associate with and respond to epithelial cells and cyclic hormone treatment and withdrawal.
Genome-wide association studies (GWAS) have consistently implicated noncoding variation within the TCF7L2 locus with type 2 diabetes (T2D) risk. While this locus represents the strongest genetic ...determinant for T2D risk in humans, it remains unclear how these noncoding variants affect disease etiology. To test the hypothesis that the T2D-associated interval harbors cis-regulatory elements controlling TCF7L2 expression, we conducted in vivo transgenic reporter assays to characterize the TCF7L2 regulatory landscape. We found that the 92-kb genomic interval associated with T2D harbors long-range enhancers regulating various aspects of the spatial-temporal expression patterns of TCF7L2, including expression in tissues involved in the control of glucose homeostasis. By selectively deleting this interval, we establish a critical role for these enhancers in robust TCF7L2 expression. To further determine whether variation in Tcf7l2 expression may lead to diabetes, we developed a Tcf7l2 copy-number allelic series in mice. We show that a null Tcf7l2 allele leads, in a dose-dependent manner, to lower glycemic profiles. Tcf7l2 null mice also display enhanced glucose tolerance coupled to significantly lowered insulin levels, suggesting that these mice are protected against T2D. Confirming these observations, transgenic mice harboring multiple Tcf7l2 copies and overexpressing this gene display reciprocal phenotypes, including glucose intolerance. These results directly demonstrate that Tcf7l2 plays a role in regulating glucose tolerance, suggesting that overexpression of this gene is associated with increased risk of T2D. These data highlight the role of enhancer elements as mediators of T2D risk in humans, strengthening the evidence that variation in cis-regulatory elements may be a paradigm for genetic predispositions to common disease.
Display omitted
•Expression of 5 transcription factors mediate reprogramming of term villous trophoblasts to induced trophoblast stem cells.•Induced trophoblast stem cells are capable of long term ...self-renewal and share high transcriptomic similarities to human trophoblast stem cells.•Induced trophoblastic stem cells differentiate into syncytiotrophoblast and extravillus trophoblast under appropriate differentiation conditions in vitro and in vivo.
Human trophoblast stem cells (hTSC) can be isolated from first trimester placenta but not from term placenta. Here we demonstrate that villous cytotrophoblasts (vCTB) from term placenta can be reprogrammed into induced trophoblastic stem-like cells (iTSC) by introducing sets of transcription factors. The iTSCs express TSC markers such as GATA3, TEAD4 and ELF5, and are multipotent, validated by their differentiation into both extravillous trophoblasts (EVT) and syncytiotrophoblasts (STB) in vitro and in vivo. The iTSC can be passaged indefinitely in vitro without slowing of growth. The transcriptome profile of these cells closely resembles the profile of hTSC isolated from first trimester placentae but different from the term placental vCTB from which they originated. The ability to reprogram cells from term placenta into iTSC will allow study of early gestation events which impact placental function later in gestation, including preeclampsia and spontaneous preterm birth.
The mechanisms that underlie the timing of labor in humans are largely unknown. In most pregnancies, labor is initiated at term (≥ 37 weeks gestation), but in a signifiicant number of women ...spontaneous labor occurs preterm and is associated with increased perinatal mortality and morbidity. The objective of this study was to characterize the cells at the maternal-fetal interface (MFI) in term and preterm pregnancies in both the laboring and non-laboring state in Black women, who have among the highest preterm birth rates in the U.S. Using mass cytometry to obtain high-dimensional single-cell resolution, we identified 31 cell populations at the MFI, including 25 immune cell types and six non-immune cell types. Among the immune cells, maternal PD1
CD8 T cell subsets were less abundant in term laboring compared to term non-laboring women. Among the non-immune cells, PD-L1
maternal (stromal) and fetal (extravillous trophoblast) cells were less abundant in preterm laboring compared to term laboring women. Consistent with these observations, the expression of CD274, the gene encoding PD-L1, was significantly depressed and less responsive to fetal signaling molecules in cultured mesenchymal stromal cells from the decidua of preterm compared to term women. Overall, these results suggest that the PD1/PD-L1 pathway at the MFI may perturb the delicate balance between immune tolerance and rejection and contribute to the onset of spontaneous preterm labor.
Mutations in the T-box transcription factor TBX20 are associated with multiple forms of congenital heart defects, including cardiac septal abnormalities, but our understanding of the contributions of ...endocardial TBX20 to heart development remains incomplete. Here, we investigated how TBX20 interacts with endocardial gene networks to drive the mesenchymal and myocardial movements that are essential for outflow tract and atrioventricular septation. Selective ablation of Tbx20 in murine endocardial lineages reduced the expression of extracellular matrix and cell migration genes that are critical for septation. Using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we identified accessible chromatin within endocardial lineages and intersected these data with TBX20 ChIP-seq and chromatin loop maps to determine that TBX20 binds a conserved long-range enhancer to regulate versican (Vcan) expression. We also observed reduced Vcan expression in Tbx20-deficient mice, supporting a direct role for TBX20 in Vcan regulation. Further, we show that the Vcan enhancer drove reporter gene expression in endocardial lineages in a TBX20-binding site-dependent manner. This work illuminates gene networks that interact with TBX20 to orchestrate cardiac septation and provides insight into the chromatin landscape of endocardial lineages during septation.
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.
PDF
