RESUMEN
RATIONALE: Dextro-transposition of the great arteries (D-TGA) is a severe congenital heart defect which affects approximately 1 in 4,000 live births. While there are several reports of D-TGA patients with rare variants in individual genes, the majority of D-TGA cases remain genetically elusive. Familial recurrence patterns and the observation that most cases with D-TGA are sporadic suggest a polygenic inheritance for the disorder, yet this remains unexplored. OBJECTIVE: We sought to study the role of common single nucleotide polymorphisms (SNPs) in risk for D-TGA. METHODS AND RESULTS: We conducted a genome-wide association study in an international set of 1,237 patients with D-TGA and identified a genome-wide significant susceptibility locus on chromosome 3p14.3, which was subsequently replicated in an independent case-control set (rs56219800, meta-analysis P=8.6x10-10, OR=0.69 per C allele). SNP-based heritability analysis showed that 25% of variance in susceptibility to D-TGA may be explained by common variants. A genome-wide polygenic risk score derived from the discovery set was significantly associated to D-TGA in the replication set (P=4x10-5). The genome-wide significant locus (3p14.3) co-localizes with a putative regulatory element that interacts with the promoter of WNT5A, which encodes the Wnt Family Member 5A protein known for its role in cardiac development in mice. We show that this element drives reporter gene activity in the developing heart of mice and zebrafish and is bound by the developmental transcription factor TBX20. We further demonstrate that TBX20 attenuates Wnt5a expression levels in the developing mouse heart. CONCLUSIONS: This work provides support for a polygenic architecture in D-TGA and identifies a susceptibility locus on chromosome 3p14.3 near WNT5A. Genomic and functional data support a causal role of WNT5A at the locus.
Asunto(s)
Polimorfismo de Nucleótido Simple , Transposición de los Grandes Vasos/genética , Animales , Células Cultivadas , Humanos , Ratones , Herencia Multifactorial , Miocitos Cardíacos/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Transposición de los Grandes Vasos/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Pez CebraRESUMEN
RATIONALE: Mutations in the transcription factor TBX20 (T-box 20) are associated with congenital heart disease. Germline ablation of Tbx20 results in abnormal heart development and embryonic lethality by embryonic day 9.5. Because Tbx20 is expressed in multiple cell lineages required for myocardial development, including pharyngeal endoderm, cardiogenic mesoderm, endocardium, and myocardium, the cell type-specific requirement for TBX20 in early myocardial development remains to be explored. OBJECTIVE: Here, we investigated roles of TBX20 in midgestation cardiomyocytes for heart development. METHODS AND RESULTS: Ablation of Tbx20 from developing cardiomyocytes using a doxycycline inducible cTnTCre transgene led to embryonic lethality. The circumference of developing ventricular and atrial chambers, and in particular that of prospective left atrium, was significantly reduced in Tbx20 conditional knockout mutants. Cell cycle analysis demonstrated reduced proliferation of Tbx20 mutant cardiomyocytes and their arrest at the G1-S phase transition. Genome-wide transcriptome analysis of mutant cardiomyocytes revealed differential expression of multiple genes critical for cell cycle regulation. Moreover, atrial and ventricular gene programs seemed to be aberrantly regulated. Putative direct TBX20 targets were identified using TBX20 ChIP-Seq (chromatin immunoprecipitation with high throughput sequencing) from embryonic heart and included key cell cycle genes and atrial and ventricular specific genes. Notably, TBX20 bound a conserved enhancer for a gene key to atrial development and identity, COUP-TFII/Nr2f2 (chicken ovalbumin upstream promoter transcription factor 2/nuclear receptor subfamily 2, group F, member 2). This enhancer interacted with the NR2F2 promoter in human cardiomyocytes and conferred atrial specific gene expression in a transgenic mouse in a TBX20-dependent manner. CONCLUSIONS: Myocardial TBX20 directly regulates a subset of genes required for fetal cardiomyocyte proliferation, including those required for the G1-S transition. TBX20 also directly downregulates progenitor-specific genes and, in addition to regulating genes that specify chamber versus nonchamber myocardium, directly activates genes required for establishment or maintenance of atrial and ventricular identity. TBX20 plays a previously unappreciated key role in atrial development through direct regulation of an evolutionarily conserved COUPT-FII enhancer.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Atrios Cardíacos/embriología , Miocitos Cardíacos/metabolismo , Proteínas de Dominio T Box/genética , Animales , Línea Celular , Proliferación Celular , Células Cultivadas , Fase G1 , Atrios Cardíacos/citología , Atrios Cardíacos/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Fase S , Proteínas de Dominio T Box/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Homeodominio/genética , Intrones/genética , Oxigenasas de Función Mixta/genética , Obesidad/genética , Oxo-Ácido-Liasas/genética , Proteínas/genética , Factores de Transcripción/genética , Tejido Adiposo/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Animales , Metabolismo Basal/genética , Índice de Masa Corporal , Peso Corporal/genética , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/genética , Dieta , Genes Dominantes/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hipotálamo/metabolismo , Masculino , Ratones , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Delgadez/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
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.
Asunto(s)
Evolución Molecular , Proteínas Represoras/química , Proteínas Represoras/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Sitios de Unión/genética , Factor de Unión a CCCTC , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Secuencia Conservada , ADN/genética , Elementos de Facilitación Genéticos , Genes Homeobox , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Modelos Genéticos , Familia de Multigenes , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , Proteínas Represoras/metabolismo , Strongylocentrotus purpuratus/genética , Pez Cebra/genéticaRESUMEN
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.
Asunto(s)
Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Animales , Cardiomiopatías/genética , Cardiomiopatías/patología , Cardiomiopatías/fisiopatología , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Contracción Miocárdica , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Glucosa/metabolismo , Proteína 2 Similar al Factor de Transcripción 7/genética , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Alelos , Animales , Secuencia de Bases , Glucemia/genética , Cromosomas Artificiales Bacterianos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Elementos de Facilitación Genéticos , Femenino , Expresión Génica , Orden Génico , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Redes y Vías Metabólicas/genética , Ratones , Ratones Noqueados , FenotipoRESUMEN
Plasticity of gene regulatory encryption can permit DNA sequence divergence without loss of function. Functional information is preserved through conservation of the composition of transcription factor binding sites (TFBS) in a regulatory element. We have developed a method that can accurately identify pairs of functional noncoding orthologs at evolutionarily diverged loci by searching for conserved TFBS arrangements. With an estimated 5% false-positive rate (FPR) in approximately 3000 human and zebrafish syntenic loci, we detected approximately 300 pairs of diverged elements that are likely to share common ancestry and have similar regulatory activity. By analyzing a pool of experimentally validated human enhancers, we demonstrated that 7/8 (88%) of their predicted functional orthologs retained in vivo regulatory control. Moreover, in 5/7 (71%) of assayed enhancer pairs, we observed concordant expression patterns. We argue that TFBS composition is often necessary to retain and sufficient to predict regulatory function in the absence of overt sequence conservation, revealing an entire class of functionally conserved, evolutionarily diverged regulatory elements that we term "covert."
Asunto(s)
Secuencia Conservada , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Análisis de Secuencia de ADN/métodos , Animales , Animales Modificados Genéticamente/genética , Biología Computacional/métodos , Evolución Molecular , Sitios Genéticos , Genoma Humano , Humanos , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Alineación de Secuencia , Sintenía , Factores de Transcripción/genética , Pez Cebra/genéticaRESUMEN
The evolutionary transition of the fins of fish into tetrapod limbs involved genetic changes to developmental systems that resulted in novel skeletal patterns and functions. Approaches to understanding this issue have entailed the search for antecedents of limb structure in fossils, genes, and embryos. Comparative genetic analyses have produced ambiguous results: although studies of posterior Hox genes from homology group 13 (Hoxa-13 and Hoxd-13) reveal similarities in gene expression between the distal segments of fins and limbs, this functional homology has not been supported by genomic comparisons of the activity of their cis-regulatory elements, namely the Hoxd Global Control Region. Here, we show that cis-regulatory elements driving Hoxd gene expression in distal limbs are present in fish. Using an interspecies transgenesis approach, we find functional conservation between gnathostome Hoxd enhancers, demonstrating that orthologous sequences from tetrapods, zebrafish and skate can drive reporter gene expression in mouse limbs and zebrafish fins. Our results support the notion that some of the novelties associated with tetrapod limbs arose by modification of deeply conserved cis- and trans-acting mechanisms of Hox regulation in gnathostomes.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Vertebrados/genética , Aletas de Animales/anatomía & histología , Aletas de Animales/embriología , Aletas de Animales/metabolismo , Animales , Animales Modificados Genéticamente , Evolución Biológica , ADN Helicasas/genética , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Extremidades/anatomía & histología , Extremidades/embriología , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/clasificación , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Filogenia , Proteínas de Unión a Poli-ADP-Ribosa , Rajidae/embriología , Rajidae/genética , Factores de Transcripción/clasificación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vertebrados/clasificación , Vertebrados/embriología , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Genome-wide association studies (GWAS) routinely identify risk variants in noncoding DNA, as exemplified by reports of multiple single nucleotide polymorphisms (SNPs) associated with prostate cancer in five independent regions in a gene desert on 8q24. Two of these regions also have been associated with breast and colorectal cancer. These findings implicate functional variation within long-range cis-regulatory elements in disease etiology. We used an in vivo bacterial artificial chromosome (BAC) enhancer-trapping strategy in mice to scan a half-megabase of the 8q24 gene desert encompassing the prostate cancer-associated regions for long-range cis-regulatory elements. These BAC assays identified both prostate and mammary gland enhancer activities within the region. We demonstrate that the 8q24 cancer-associated variant rs6983267 lies within an in vivo prostate enhancer whose expression mimics that of the nearby MYC proto-oncogene. Additionally, we show that the cancer risk allele increases prostate enhancer activity in vivo relative to the non-risk allele. This allele-specific enhancer activity is detectable during early prostate development and throughout prostate maturation, raising the possibility that this SNP could assert its influence on prostate cancer risk before tumorigenesis occurs. Our study represents an efficient strategy to build experimentally on GWAS findings with an in vivo method for rapidly scanning large regions of noncoding DNA for functional cis-regulatory sequences harboring variation implicated in complex diseases.
Asunto(s)
Cromosomas Humanos Par 8/genética , Elementos de Facilitación Genéticos/genética , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Cromosomas Artificiales Bacterianos , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Ratones , Ratones Transgénicos , Polimorfismo de Nucleótido Simple , Proto-Oncogenes MasRESUMEN
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.
Asunto(s)
Obesidad , Retroelementos , Humanos , Femenino , Animales , Ratones , Obesidad/genética , Obesidad/metabolismo , Adiposidad/genética , Índice de Masa Corporal , Relación Cintura-Cadera , Tejido Adiposo/metabolismo , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismoRESUMEN
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.
Asunto(s)
Trabajo de Parto , Trabajo de Parto Prematuro , Nacimiento Prematuro , Embarazo , Humanos , Femenino , Recién Nacido , Antígeno B7-H1/genética , Trabajo de Parto Prematuro/metabolismo , Subgrupos de Linfocitos TRESUMEN
BACKGROUND: Asthma is the most common chronic disease in children, occurring at higher frequencies and with more severe disease in children with African ancestry. METHODS: We tested for association with haplotypes at the most replicated and significant childhood-onset asthma locus at 17q12-q21 and asthma in European American and African American children. Following this, we used whole-genome sequencing data from 1060 African American and 100 European American individuals to identify novel variants on a high-risk African American-specific haplotype. We characterized these variants in silico using gene expression and ATAC-seq data from airway epithelial cells, functional annotations from ENCODE, and promoter capture (pc)Hi-C maps in airway epithelial cells. Candidate causal variants were then assessed for correlation with asthma-associated phenotypes in African American children and adults. RESULTS: Our studies revealed nine novel African-specific common variants, enriched on a high-risk asthma haplotype, which regulated the expression of GSDMA in airway epithelial cells and were associated with features of severe asthma. Using ENCODE annotations, ATAC-seq, and pcHi-C, we narrowed the associations to two candidate causal variants that are associated with features of T2 low severe asthma. CONCLUSIONS: Previously unknown genetic variation at the 17q12-21 childhood-onset asthma locus contributes to asthma severity in individuals with African ancestries. We suggest that many other population-specific variants that have not been discovered in GWAS contribute to the genetic risk for asthma and other common diseases.
Asunto(s)
Asma , Negro o Afroamericano , Negro o Afroamericano/genética , Alelos , Asma/genética , Asma/metabolismo , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Proteínas de Neoplasias/genética , Polimorfismo de Nucleótido Simple , Proteínas Citotóxicas Formadoras de PorosRESUMEN
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.
Asunto(s)
Técnicas de Cocultivo/métodos , Decidua/metabolismo , Endometrio/metabolismo , Fibroblastos/metabolismo , Células Madre Pluripotentes/metabolismo , Células del Estroma/metabolismo , Femenino , HumanosRESUMEN
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.
Asunto(s)
Adipocitos/fisiología , Elementos de Facilitación Genéticos , Pleiotropía Genética , Obesidad/genética , Adipocitos/citología , Arritmias Cardíacas/genética , Arritmias Cardíacas/patología , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Estudio de Asociación del Genoma Completo , Gigantismo/genética , Gigantismo/patología , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/patología , Humanos , Hipotálamo/fisiología , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , MAP Quinasa Quinasa 5/genética , Neuronas/citología , Neuronas/fisiología , Polimorfismo de Nucleótido Simple , Proteínas Quinasas/genética , Sitios de Carácter Cuantitativo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Factores de Transcripción/genética , TranscriptomaRESUMEN
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.
Asunto(s)
Nacimiento Prematuro , Trofoblastos , Diferenciación Celular , Proteínas de Unión al ADN , Femenino , Humanos , Recién Nacido , Proteínas Musculares , Placenta , Embarazo , Células Madre , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Tejido Adiposo/metabolismo , Encéfalo/metabolismo , Proteínas de Homeodominio/genética , Obesidad/genética , Factores de Transcripción/genética , Alelos , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Encéfalo/embriología , Línea Celular , Cromatina/química , Cromatina/metabolismo , Desarrollo Embrionario , Elementos de Facilitación Genéticos , Conducta Alimentaria , Preferencias Alimentarias , Regulación de la Expresión Génica , Haplotipos , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Obesidad/fisiopatología , Polimorfismo de Nucleótido Simple , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Asma/genética , Elementos de Facilitación Genéticos , Interleucina-33/genética , Alelos , Animales , Asma/metabolismo , Cromatina/genética , Cromatina/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Humanos , Interleucina-33/metabolismo , Masculino , Ratones Transgénicos , Factor 1 de Transcripción de Unión a Octámeros/genética , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Pez CebraRESUMEN
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.
Asunto(s)
Nacimiento Prematuro , Transcriptoma , Cromatina/genética , Cromatina/metabolismo , Decidua , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Recién Nacido , Masculino , Embarazo , Nacimiento Prematuro/genética , Nacimiento Prematuro/metabolismo , Células del EstromaRESUMEN
To dissect the genetic architecture controlling blood pressure (BP) regulation in the spontaneously hypertensive rat (SHR) we derived congenic rat strains for four previously mapped BP quantitative trait loci (QTLs) in chromosomes 2, 4, and 16. Target chromosomal regions from the Brown Norway rat (BN) averaging 13-29 cM were introgressed by marker-assisted breeding onto the SHR genome in 12 or 13 generations. Under normal salt intake, QTLs on chromosomes 2a, 2c, and 4 were associated with significant changes in systolic BP (13, 20, and 15 mmHg, respectively), whereas the QTL on chromosome 16 had no measurable effect. On high salt intake (1% NaCl in drinking water for 2 wk), the chromosome 16 QTL had a marked impact on SBP, as did the QTLs on chromosome 2a and 2c (18, 17, and 19 mmHg, respectively), but not the QTL on chromosome 4. Thus these four QTLs affected BP phenotypes differently: 1) in the presence of high salt intake (chromosome 16), 2) only associated with normal salt intake (chromosome 4), and 3) regardless of salt intake (chromosome 2c and 2a). Moreover, salt sensitivity was abrogated in congenics SHR.BN2a and SHR.BN16. Finally, we provide evidence for the influence of genetic background on the expression of the mapped QTLs individually or as a group. Collectively, these data reveal previously unsuspected nuances of the physiological roles of each of the four mapped BP QTLs in the SHR under basal and/or salt loading conditions unforeseen by the analysis of the F2 cross.
Asunto(s)
Mapeo Cromosómico , Cromosomas de los Mamíferos/genética , Hipertensión/genética , Sitios de Carácter Cuantitativo/genética , Animales , Animales Congénicos , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Cruzamientos Genéticos , Femenino , Hipertensión/fisiopatología , Masculino , Modelos Genéticos , Fenotipo , Ratas , Ratas Endogámicas SHR , Cloruro de Sodio Dietético/administración & dosificación , Cloruro de Sodio Dietético/farmacología , Sístole/efectos de los fármacosRESUMEN
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.