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1.
PLoS Genet ; 16(1): e1008538, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31917787

RESUMO

Genome-wide association studies have identified multiple novel genomic loci associated with vascular diseases. Many of these loci are common non-coding variants that affect the expression of disease-relevant genes within coronary vascular cells. To identify such genes on a genome-wide level, we performed deep transcriptomic analysis of genotyped primary human coronary artery smooth muscle cells (HCASMCs) and coronary endothelial cells (HCAECs) from the same subjects, including splicing Quantitative Trait Loci (sQTL), allele-specific expression (ASE), and colocalization analyses. We identified sQTLs for TARS2, YAP1, CFDP1, and STAT6 in HCASMCs and HCAECs, and 233 ASE genes, a subset of which are also GTEx eGenes in arterial tissues. Colocalization of GWAS association signals for coronary artery disease (CAD), migraine, stroke and abdominal aortic aneurysm with GTEx eGenes in aorta, coronary artery and tibial artery discovered novel candidate risk genes for these diseases. At the CAD and stroke locus tagged by rs2107595 we demonstrate colocalization with expression of the proximal gene TWIST1. We show that disrupting the rs2107595 locus alters TWIST1 expression and that the risk allele has increased binding of the NOTCH signaling protein RBPJ. Finally, we provide data that TWIST1 expression influences vascular SMC phenotypes, including proliferation and calcification, as a potential mechanism supporting a role for TWIST1 in CAD.


Assuntos
Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/genética , Proteína 1 Relacionada a Twist/genética , Doenças Vasculares/genética , Células Cultivadas , Vasos Coronários/citologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Transcriptoma , Proteína 1 Relacionada a Twist/metabolismo
2.
Am J Hum Genet ; 103(3): 377-388, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30146127

RESUMO

Coronary artery disease (CAD) is the leading cause of death globally. Genome-wide association studies (GWASs) have identified more than 95 independent loci that influence CAD risk, most of which reside in non-coding regions of the genome. To interpret these loci, we generated transcriptome and whole-genome datasets using human coronary artery smooth muscle cells (HCASMCs) from 52 unrelated donors, as well as epigenomic datasets using ATAC-seq on a subset of 8 donors. Through systematic comparison with publicly available datasets from GTEx and ENCODE projects, we identified transcriptomic, epigenetic, and genetic regulatory mechanisms specific to HCASMCs. We assessed the relevance of HCASMCs to CAD risk using transcriptomic and epigenomic level analyses. By jointly modeling eQTL and GWAS datasets, we identified five genes (SIPA1, TCF21, SMAD3, FES, and PDGFRA) that may modulate CAD risk through HCASMCs, all of which have relevant functional roles in vascular remodeling. Comparison with GTEx data suggests that SIPA1 and PDGFRA influence CAD risk predominantly through HCASMCs, while other annotated genes may have multiple cell and tissue targets. Together, these results provide tissue-specific and mechanistic insights into the regulation of a critical vascular cell type associated with CAD in human populations.


Assuntos
Doença da Artéria Coronariana/genética , Vasos Coronários/fisiologia , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Miócitos de Músculo Liso/fisiologia , Locos de Características Quantitativas/genética , Linhagem Celular , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Humanos , Polimorfismo de Nucleotídeo Único/genética , Risco
3.
PLoS Biol ; 8(9)2010 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-20856902

RESUMO

Expression levels of human genes vary extensively among individuals. This variation facilitates analyses of expression levels as quantitative phenotypes in genetic studies where the entire genome can be scanned for regulators without prior knowledge of the regulatory mechanisms, thus enabling the identification of unknown regulatory relationships. Here, we carried out such genetic analyses with a large sample size and identified cis- and trans-acting polymorphic regulators for about 1,000 human genes. We validated the cis-acting regulators by demonstrating differential allelic expression with sequencing of transcriptomes (RNA-Seq) and the trans-regulators by gene knockdown, metabolic assays, and chromosome conformation capture analysis. The majority of the regulators act in trans to the target (regulated) genes. Most of these trans-regulators were not known to play a role in gene expression regulation. The identification of these regulators enabled the characterization of polymorphic regulation of human gene expression at a resolution that was unattainable in the past.


Assuntos
Regulação da Expressão Gênica/fisiologia , Polimorfismo Genético , Alelos , Ligação Genética , Humanos
4.
Clin Transl Sci ; 14(4): 1369-1379, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34156146

RESUMO

Hepatocytes store triglycerides (TGs) in the form of lipid droplets (LDs), which are increased in hepatosteatosis. The regulation of hepatic LDs is poorly understood and new therapies to reduce hepatosteatosis are needed. We performed a siRNA kinase and phosphatase screen in HuH-7 cells using high-content automated imaging of LDs. Changes in accumulated lipids were quantified with developed pipeline that measures intensity, area, and number of LDs. Selected "hits," which reduced lipid accumulation, were further validated with other lipid and expression assays. Among several siRNAs that resulted in significantly reduced LDs, one was targeted to the nuclear adapter protein, transformation/transcription domain-associated protein (TRRAP). Knockdown of TRRAP reduced triglyceride accumulation in HuH-7 hepatocytes, in part by reducing C/EBPα-mediated de novo synthesis of TGs. These findings implicate TRRAP as a novel regulator of hepatic TG metabolism and nominate it as a potential drug target for hepatosteatosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Proteínas Nucleares/metabolismo , Triglicerídeos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Técnicas de Silenciamento de Genes , Ensaios de Triagem em Larga Escala , Humanos , Gotículas Lipídicas/metabolismo , Proteínas Nucleares/análise , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Triglicerídeos/análise
5.
Hepatol Commun ; 4(9): 1316-1331, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32923835

RESUMO

Establishment of a physiologically relevant human hepatocyte-like cell system for in vitro translational research has been hampered by the limited availability of cell models that accurately reflect human biology and the pathophysiology of human disease. Here we report a robust, reproducible, and scalable protocol for the generation of hepatic organoids from human induced pluripotent stem cells (hiPSCs) using short exposure to nonengineered matrices. These hepatic organoids follow defined stages of hepatic development and express higher levels of early (hepatocyte nuclear factor 4A [HNF4A], prospero-related homeobox 1 [PROX1]) and mature hepatic and metabolic markers (albumin, asialoglycoprotein receptor 1 [ASGR1], CCAAT/enhancer binding protein α [C/EBPα]) than two-dimensional (2D) hepatocyte-like cells (HLCs) at day 20 of differentiation. We used this model to explore the biology of the pleiotropic TRIB1 (Tribbles-1) gene associated with a number of metabolic traits, including nonalcoholic fatty liver disease and plasma lipids. We used genome editing to delete the TRIB1 gene in hiPSCs and compared TRIB1-deleted iPSC-HLCs to isogenic iPSC-HLCs under both 2D culture and three-dimensional (3D) organoid conditions. Under conventional 2D culture conditions, TRIB1-deficient HLCs showed maturation defects, with decreased expression of late-stage hepatic and lipogenesis markers. In contrast, when cultured as 3D hepatic organoids, the differentiation defects were rescued, and a clear lipid-related phenotype was noted in the TRIB1-deficient induced pluripotent stem cell HLCs. Conclusion: This work supports the potential of genome-edited hiPSC-derived hepatic 3D organoids in exploring human hepatocyte biology, including the functional interrogation of genes identified through human genetic investigation.

6.
Genetics ; 173(1): 215-23, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16510787

RESUMO

Sexual isolating mechanisms that act before fertilization are often considered the most important genetic barriers leading to speciation in animals. While progress has been made toward understanding the genetic basis of the postzygotic isolating mechanisms of hybrid sterility and inviability, little is known about the genetic basis of prezygotic sexual isolation. Here, we map quantitative trait loci (QTL) contributing to prezygotic reproductive isolation between the sibling species Drosophila santomea and D. yakuba. We mapped at least three QTL affecting discrimination of D. santomea females against D. yakuba males: one X-linked and one autosomal QTL affected the likelihood of copulation, and a second X chromosome QTL affected copulation latency. Three autosomal QTL also affected mating success of D. yakuba males with D. santomea. No epistasis was detected between QTL affecting sexual isolation. The QTL do not overlap between males and females and are not disproportionately concentrated on the X chromosome. There was some overlap in map locations of QTL affecting sexual isolation between D. santomea and D. yakuba with QTL affecting sexual isolation between D. simulans and D. mauritiana and with QTL affecting differences in pigmentation between D. santomea and D. yakuba. Future high-resolution mapping and, ultimately, positional cloning, will reveal whether these traits do indeed have a common genetic basis.


Assuntos
Drosophila/genética , Drosophila/fisiologia , Locos de Características Quantitativas/genética , Comportamento Sexual Animal/fisiologia , Zigoto/metabolismo , Animais , Mapeamento Cromossômico , Cromossomos/genética , Drosophila/citologia , Marcadores Genéticos , Reprodução/genética , Reprodução/fisiologia
7.
Genetics ; 173(1): 225-33, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16510788

RESUMO

A major unresolved challenge of evolutionary biology is to determine the nature of the allelic variants of "speciation genes": those alleles whose interaction produces inviable or infertile interspecific hybrids but does not reduce fitness in pure species. Here we map quantitative trait loci (QTL) affecting fertility of male hybrids between D. yakuba and its recently discovered sibling species, D. santomea. We mapped three to four X chromosome QTL and two autosomal QTL with large effects on the reduced fertility of D. yakuba and D. santomea backcross males. We observed epistasis between the X-linked QTL and also between the X and autosomal QTL. The X chromosome had a disproportionately large effect on hybrid sterility in both reciprocal backcross hybrids. However, the genetics of hybrid sterility differ between D. yakuba and D. santomea backcross males, both in terms of the magnitude of main effects and in the epistatic interactions. The QTL affecting hybrid fertility did not colocalize with QTL affecting sexual isolation in this species pair, but did colocalize with QTL affecting the marked difference in pigmentation between D. yakuba and D. santomea. These results provide the basis for future high-resolution mapping and ultimately, molecular cloning, of the interacting genes that contribute to hybrid sterility.


Assuntos
Drosophila/genética , Drosophila/fisiologia , Infertilidade Masculina/genética , Infertilidade Masculina/fisiopatologia , Zigoto/metabolismo , Animais , Mapeamento Cromossômico , Cromossomos/genética , Cruzamentos Genéticos , Drosophila/classificação , Epistasia Genética , Marcadores Genéticos , Hibridização Genética , Masculino , Pigmentação/genética , Locos de Características Quantitativas/genética , Reprodução/genética , Reprodução/fisiologia , Especificidade da Espécie
8.
Evolution ; 60(2): 279-91, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16610320

RESUMO

The desaturase-2 (desat2) locus of Drosophila melanogaster has two alleles whose frequencies vary geographically: one (the "Z" allele) is found primarily in east Africa and the Caribbean, and the other (the "M" allele) occurs in other parts of the world. It has been suggested that these alleles not only cause sexual isolation between races, but that their distribution reflects differential adaptation to climate: Z alleles are supposedly adapted to tropical conditions and M alleles to temperate ones. This has thus been viewed as a case of reproductive isolation evolving as a pleiotropic byproduct of adaptation. Here we reinvestigate this presumed climatic adaptation, using transgenic lines differing in the nature of their desat2 alleles. We were unable to replicate earlier results showing that carriers of M alleles are uniformly more cold resistant and less starvation resistant than carriers of Z alleles. It is thus doubtful whether the distribution of these alleles reflects natural selection involving climate. Mating studies of transgenic lines show some evidence for sexual isolation due to desat2. However, work on other, wild-type lines, as well as observations on the nature of sexual isolation, suggest that this conclusion--and thus the relationship between this locus and mating discrimination between races of D. melanogaster--may also be doubtful.


Assuntos
Adaptação Fisiológica/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Ácidos Graxos Dessaturases/genética , Comportamento Sexual Animal , Animais , Tamanho Corporal , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Ácidos Graxos Dessaturases/metabolismo , Feminino , Privação de Alimentos , Regulação Enzimológica da Expressão Gênica , Genótipo , Masculino , Temperamento
9.
Evolution ; 59(12): 2588-601, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16526506

RESUMO

Many studies of speciation rely critically on estimates of sexual isolation obtained in the laboratory. Here we examine the sensitivity of sexual isolation to alterations in experimental design and mating environment in two sister species of Drosophila, D. santomea and D. yakuba. We use a newly devised measure of mating frequencies that is able to disentangle sexual isolation from species differences in mating propensity. Variation in fly density, presence or absence of a quasi-natural environment, degree of starvation, and relative frequency of species had little or no effect on sexual isolation, but one factor did have a significant effect: the possibility of choice. Designs that allowed flies to choose between conspecific and heterospecific mates showed significantly more sexual isolation than other designs that did not allow choice. These experiments suggest that sexual isolation between these species (whose ranges overlap on the island of São Tomé) is due largely to discrimination against D. yakuba males by D. santomea females. This suggestion was confirmed by direct observations of mating behavior. Drosophila santomea males also court D. yakuba females less ardently than conspecific females, whereas neither males nor females of D. yakuba show strong mate discrimination. Thus, sexual isolation appears to be a result of evolutionary changes in the derived island endemic D. santomea. Surprisingly, as reported in a companion paper (Llopart et al. 2005), the genotypes of hybrids found in nature do not accord with expectations from these laboratory studies: all F1 hybrids in nature come from matings between D. santomea females and D. yakuba males, matings that occur only rarely in the laboratory.


Assuntos
Drosophila/fisiologia , Projetos de Pesquisa , Comportamento Sexual Animal , Animais , Planejamento Ambiental , Feminino , Especiação Genética , Hibridização Genética , Masculino
10.
Evolution ; 56(11): 2262-77, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12487356

RESUMO

Drosophila yakuba is a species widespread in Africa, whereas D. santomea, its newly discovered sister species, is endemic to the volcanic island of São Tomé in the Gulf of Guinea. Drosophila santomea probably formed after colonization of the island by its common ancestor with D. yakuba. The two species differ strikingly in pigmentation: D. santomea, unlike the other eight species in the D. melanogaster subgroup, almost completely lacks dark abdominal pigmentation. D. yakuba shows the sexually dimorphic pigmentation typical of the group: both sexes have melanic patterns on the abdomen, but males are much darker than females. A genetic analysis of this species difference using morphological markers shows that the X chromosome accounts for nearly 90% of the species difference in the area of abdomen that is pigmented and that at least three genes (one on each major chromosome) are involved in each sex. The order of chromosome effects on pigmentation area are the same in males and females, suggesting that loss of pigmentation in D. santomea may have involved the same genes in both sexes. Further genetic analysis of the interspecific difference between males in pigmentation area and intensity using molecular markers shows that at least five genes are responsible, with no single locus having an overwhelming effect on the trait. The species difference is thus oligogenic or polygenic. Different chromosomal regions from each of the two species influenced pigmentation in the same direction, suggesting that the species difference (at least in males) is due to natural or sexual selection and not genetic drift. Measurements of sexual isolation between the species in both light and dark conditions show no difference, suggesting that the pigmentation difference is not an important cue for interspecific mate discrimination. Using DNA sequence differences in nine noncoding regions, we estimate that D. santomea and D. yakuba diverged about 400,000 years ago, a time similar to the divergences between two other well-studied pair of species in the subgroup, both of which also involved island colonization.


Assuntos
Drosophila/genética , Pigmentação/genética , Animais , Biomarcadores , Cromossomos/genética , Cruzamentos Genéticos , Drosophila/anatomia & histologia , Drosophila/fisiologia , Feminino , Genes de Insetos , Genótipo , Masculino , Fenótipo , Comportamento Sexual Animal/fisiologia , Especificidade da Espécie
11.
Evolution ; 56(12): 2424-34, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12583583

RESUMO

Drosophila yakuba is widespread in Africa, whereas D. santomea, its newly discovered sister species, is endemic to the volcanic island of São Tomé in the Gulf of Guinea. Drosophila santomea probably formed after colonization of the island by a D. yakuba-like ancestor. The species presently have overlapping ranges on the mountain Pico do São Tomé, with some hybridization occurring in this region. Sexual isolation between the species is uniformly high regardless of the source of the populations, and, as in many pairs of Drosophila species, is asymmetrical, so that hybridizations occur much more readily in one direction than the other. Despite the fact that these species meet many of the conditions required for the evolution of reinforcement (the elevation of sexual isolation by natural selection to avoid maladaptive interspecific hybridization), there is no evidence that sexual isolation between the species is highest in the zone of overlap. Sexual isolation is due to evolutionary changes in both female preference for heterospecific males and in the vigor with which males court heterospecific females. Heterospecific matings are also slower to take place than are homospecific matings, constituting another possible form of reproductive isolation. Genetic studies show that, when tested with females of either species, male hybrids having a D. santomea X chromosome mate much less frequently with females of either species than do males having a D. yakuba X chromosome, suggesting that the interaction between the D. santomea X chromosome and the D. yakuba genome causes behavioral sterility. Hybrid F1 females mate readily with males of either species, so that sexual isolation in this sex is completely recessive, a phenomenon seen in other Drosophila species. There has also been significant evolutionary change in the duration of copulation between these species; this difference involves genetic changes in both sexes, with at least two genes responsible in males and at least one in females.


Assuntos
Drosophila/fisiologia , Comportamento Sexual Animal , Animais , Evolução Biológica , Drosophila/genética , Feminino , Geografia , Hibridização Genética , Masculino , Reprodução
12.
Nature ; 419(6905): 360; discussion 360, 2002 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-12353025

RESUMO

Many species of the fruitfly Drosophila are either sexually dimorphic for abdominal pigmentation (the posterior segments in males are black and those of females have thin dark stripes) or sexually monomorphic for this pigmentation (both sexes show striping). Kopp et al. report a correlation in two Drosophila clades between the expression of the bric-à-brac (bab) gene, which represses male-specific pigmentation in D. melanogaster females, and the presence of sexually dimorphic pigmentation. They suggest that sexual selection acted to produce sexual dichromatism in Drosophila by altering the regulation of bab, on the grounds that D. melanogaster males show a strong mate preference for females with lightly pigmented abdomens, and that this discrimination helps to maintain sexual dichromatism by preventing males from wasting time by courting other (darkly pigmented) males. Here we show that the mate discrimination observed by Kopp et al. may in fact have resulted from the nature of the strains and comparisons they used in their study and so could be irrelevant to mate choice in nature.


Assuntos
Drosophila melanogaster/fisiologia , Pigmentação , Caracteres Sexuais , Comportamento Sexual Animal/fisiologia , Fatores de Transcrição , Abdome/anatomia & histologia , Animais , Evolução Biológica , Cor , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Feminino , Genes de Insetos/genética , Masculino , Mutação
13.
Genet Res ; 84(1): 11-26, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15663255

RESUMO

We performed genetic analysis of hybrid sterility and of one morphological difference (sex-comb tooth number) on D. yakuba and D. santomea, the former species widespread in Africa and the latter endemic to the oceanic island of São Tomé, on which there is a hybrid zone. The sterility of hybrid males is due to at least three genes on the X chromosome and at least one on the Y, with the cytoplasm and large sections of the autosomes having no effect. F1 hybrid females carrying two X chromosomes from either species are perfectly fertile despite their genetic similarity to completely sterile F1 hybrid males. This implies that the appearance of Haldane's rule in this cross is at least partially due to the faster accumulation of genes causing male than female sterility. The larger effects of the X and Y chromosomes than of the autosomes, however, also suggest that the genes causing male sterility are recessive in hybrids. Some female sterility is also seen in interspecific crosses, but this does not occur between all strains. This is seen in pure-species females inseminated by heterospecific males (probably reflecting incompatibility between the sperm of one species and the female reproductive tract of the other) as well as in inseminated F1 and backcross females, probably reflecting genetically based incompatibilities in hybrids that affect the reproductive system. The latter 'innate' sterility appears to involve deleterious interactions between D. santomea chromosomes and D. yakuba cytoplasm. The difference in male sex-comb tooth number appears to involve fairly large effects of the X chromosome. We discuss the striking evolutionary parallels in the genetic basis of sterility, in the nature of sexual isolation, and in morphological differences between the D. santomea/D. yakuba divergence and two other speciation events in the D. melanogaster subgroup involving island colonization.


Assuntos
Drosophila/genética , Hibridização Genética , Animais , Extremidades/anatomia & histologia , Feminino , Infertilidade Feminina/genética , Infertilidade Masculina/genética , Masculino , Filogenia , Razão de Masculinidade , Espermatogênese/genética
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