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1.
PLoS Genet ; 19(3): e1010680, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36928188

RESUMO

Genome-wide association studies have identified >250 genetic variants associated with coronary artery disease (CAD), but the causal variants, genes and molecular mechanisms remain unknown at most loci. We performed pooled CRISPR screens to test the impact of sequences at or near CAD-associated genetic variants on vascular endothelial cell functions. Using CRISPR knockout, inhibition and activation, we targeted 1998 variants at 83 CAD loci to assess their effect on three adhesion proteins (E-selectin, ICAM1, VCAM1) and three key endothelial functions (nitric oxide and reactive oxygen species production, calcium signalling). At a false discovery rate ≤10%, we identified significant CRISPR perturbations near 42 variants located within 26 CAD loci. We used base editing to validate a putative causal variant in the promoter of the FES gene. Although a few of the loci include genes previously characterized in endothelial cells (e.g. AIDA, ARHGEF26, ADAMTS7), most are implicated in endothelial dysfunction for the first time. Detailed characterization of one of these new loci implicated the RNA helicase DHX38 in vascular endothelial cell senescence. While promising, our results also highlighted several limitations in using CRISPR perturbations to functionally dissect GWAS loci, including an unknown false negative rate and potential off-target effects.


Assuntos
Doença da Artéria Coronariana , Humanos , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Estudo de Associação Genômica Ampla , Locos de Características Quantitativas , Células Endoteliais/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Polimorfismo de Nucleotídeo Único/genética , Predisposição Genética para Doença , Fatores de Processamento de RNA/genética , RNA Helicases DEAD-box/genética
2.
BMC Med Genet ; 19(1): 97, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29884117

RESUMO

BACKGROUND: Genome-wide association studies (GWAS) have identified a variant (rs9349379) at the phosphatase and actin regulator 1 (PHACTR1) locus that is associated with coronary artery disease (CAD). The same variant is also an expression quantitative trait locus (eQTL) for PHACTR1 in human coronary arteries (hCA). Here, we sought to characterize PHACTR1 splicing pattern in atherosclerosis-relevant human cells. We also explored how rs9349379 modulates the expression of the different PHACTR1 splicing isoforms. METHODS: We combined rapid amplification of cDNA ends (RACE) with next-generation long-read DNA sequencing to discover all PHACTR1 transcripts in many human tissues and cell types. We measured PHACTR1 transcripts by qPCR to identify transcript-specific eQTLs. RESULTS: We confirmed a brain-specific long transcript, a short transcript expressed in monocytes and four intermediate transcripts that are different due to alternative splicing of two in-frame exons. In contrast to a previous report, we confirmed that the PHACTR1 protein is present in vascular smooth muscle cells. In 158 hCA from our collection and the GTEx dataset, rs9349379 was only associated with the expression levels of the intermediate PHACTR1 transcripts. CONCLUSIONS: Our comprehensive transcriptomic profiling of PHACTR1 indicates that this gene encodes six main transcripts. Five of them are expressed in hCA, where atherosclerotic plaques develop. In this tissue, genotypes at rs9349379 are associated with the expression of the intermediate transcripts, but not the immune-specific short transcript. This result suggests that rs9349379 may in part influence CAD by modulating the expression of intermediate PHACTR1 transcripts in endothelial or vascular smooth muscle cells found in hCA.


Assuntos
Processamento Alternativo , Aterosclerose/genética , Aterosclerose/patologia , Regulação da Expressão Gênica , Proteínas dos Microfilamentos/genética , Músculo Liso Vascular/metabolismo , Locos de Características Quantitativas , Células Cultivadas , Humanos , Músculo Liso Vascular/citologia , Isoformas de Proteínas
3.
Breast Cancer ; 27(4): 594-606, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31993937

RESUMO

BACKGROUND: Breast cancer is the most common cancer in women. Despite high survival rates in Western countries, treatments are less effective in metastatic cases and triple-negative breast cancer (TNBC) patient survival is the shortest across breast cancer subtypes. High expression levels of stearoyl-CoA desaturase-1 (SCD1) have been reported in breast cancer. The SCD1 enzyme catalyzes the formation of oleic acid (OA), a lipid stimulating the migration of metastatic breast cancer cells. Phospholipase activity is also implicated in breast cancer metastasis, notably phospholipase D (PLD). METHODS: Kaplan-Meier survival plots generated from gene expression databases were used to analyze the involvement of SCD1 and PLD in several cancer subtypes. SCD1 enzymatic activity was modulated with a pharmaceutical inhibitor or by OA treatment (to mimic SCD1 over-activity) in three breast cancer cell lines: TNBC-derived MDA-MB-231 cells as well as non-TNBC MCF-7 and T47D cells. Cell morphology and migration properties were characterized by various complementary methods. RESULTS: Our survival analyses suggest that SCD1 and PLD2 expression in the primary tumor are both associated to metastasis-related morbid outcomes in breast cancer patients. We show that modulation of SCD1 activity is associated with the modification of TNBC cell migration properties, including changes in speed, direction and cell morphology. Cell migration properties are regulated by SCD1 activity through a PLD-mTOR/p70S6K signaling pathway. These effects are not observed in non-TNBC cell lines. CONCLUSION: Our results establish a key role for the lipid desaturase SCD1 and delineate an OA-PLD-mTOR/p70S6K signaling pathway in TNBC-derived MDA-MB-231 cell migration.


Assuntos
Movimento Celular , Estearoil-CoA Dessaturase/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Metástase Neoplásica , Ácido Oleico/metabolismo , Fosfolipase D/antagonistas & inibidores , Fosfolipase D/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/cirurgia
4.
Genome Biol ; 20(1): 133, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31287004

RESUMO

BACKGROUND: Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD) and blood pressure (BP) or hypertension. Many of these loci are not linked to traditional risk factors, nor do they include obvious candidate genes, complicating their functional characterization. We hypothesize that many GWAS loci associated with vascular diseases modulate endothelial functions. Endothelial cells play critical roles in regulating vascular homeostasis, such as roles in forming a selective barrier, inflammation, hemostasis, and vascular tone, and endothelial dysfunction is a hallmark of atherosclerosis and hypertension. To test this hypothesis, we generate an integrated map of gene expression, open chromatin region, and 3D interactions in resting and TNFα-treated human endothelial cells. RESULTS: We show that genetic variants associated with CAD and BP are enriched in open chromatin regions identified in endothelial cells. We identify physical loops by Hi-C and link open chromatin peaks that include CAD or BP SNPs with the promoters of genes expressed in endothelial cells. This analysis highlights 991 combinations of open chromatin regions and gene promoters that map to 38 CAD and 92 BP GWAS loci. We validate one CAD locus, by engineering a deletion of the TNFα-sensitive regulatory element using CRISPR/Cas9 and measure the effect on the expression of the novel CAD candidate gene AIDA. CONCLUSIONS: Our data support an important role played by genetic variants acting in the vascular endothelium to modulate inter-individual risk in CAD and hypertension.


Assuntos
Doença da Artéria Coronariana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Sistemas CRISPR-Cas , Células Endoteliais/metabolismo , Epigenômica , Estudo de Associação Genômica Ampla , Humanos , Elementos Reguladores de Transcrição , Transcriptoma
5.
PLoS One ; 12(6): e0178700, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28570605

RESUMO

The introduction of frameshift indels by genome editing has emerged as a powerful technique to study the functions of uncharacterized genes in cell lines and model organisms. Such mutations should lead to mRNA degradation owing to nonsense-mediated mRNA decay or the production of severely truncated proteins. Here, we show that frameshift indels engineered by genome editing can also lead to skipping of "multiple of three nucleotides" exons. Such splicing events result in in-frame mRNA that may encode fully or partially functional proteins. We also characterize a segregating nonsense variant (rs2273865) located in a "multiple of three nucleotides" exon of LGALS8 that increases exon skipping in human erythroblast samples. Our results highlight the potentially frequent contribution of exonic splicing regulatory elements and are important for the interpretation of negative results in genome editing experiments. Moreover, they may contribute to a better annotation of loss-of-function mutations in the human genome.


Assuntos
Éxons , Mutação da Fase de Leitura , Edição de Genes , Mutação INDEL , Linhagem Celular Transformada , Humanos
6.
Lipids ; 52(2): 129-150, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27838812

RESUMO

Berardinelli-Seip congenital lipodystrophy (BSCL) is an autosomal recessive disorder. The more severe form, designated BSCL2, arises due to mutations in the BSCL2 gene. Patients with BSCL2, as well as Bscl2 -/- mice, have a near total absence of body fat, an organomegaly, and develop metabolic disorders including insulin resistance and hepatic steatosis. The function of the Seipin (BSCL2) protein remains poorly understood. Several lines of evidence have indicated that Seipin may have distinct functions in adipose versus non-adipose cells. Here we present evidence that BSCL2/Bscl2 plays a role in lipid droplet (LD) biogenesis and homeostasis in primary and cultured hepatocytes. Our results show that decreasing BSCL2/Bscl2 expression in hepatocytes increases the number and size of LD, as well as the expression of genes implicated in their formation and stability. We also show that knocking down SCD1 expression reverses the phenotype associated with Seipin deficiency. Interestingly, BSCL2 knockdown induces SCD1 expression and activity, potentially leading to increased basal phosphorylation of proteins involved in the insulin signaling cascade, as well as further increasing fatty acid uptake and de novo lipogenesis. In conclusion, our results suggest that a hepatic BSCL2/Bscl2 deficiency induces the increase and expansion of LD, potentially via increased SCD1 activity.


Assuntos
Subunidades gama da Proteína de Ligação ao GTP/deficiência , Hepatócitos/citologia , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Estearoil-CoA Dessaturase/genética , Animais , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Células Hep G2 , Hepatócitos/metabolismo , Homeostase , Humanos , Insulina/metabolismo , Tamanho das Organelas , Fosforilação , Ratos , Estearoil-CoA Dessaturase/metabolismo
7.
PLoS One ; 10(6): e0130230, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26083030

RESUMO

Transgenic mice (Tg) overexpressing human apolipoprotein D (H-apoD) in the brain are resistant to neurodegeneration. Despite the use of a neuron-specific promoter to generate the Tg mice, they expressed significant levels of H-apoD in both plasma and liver and they slowly develop hepatic steatosis and insulin resistance. We show here that hepatic PPARγ expression in Tg mice is increased by 2-fold compared to wild type (WT) mice. Consequently, PPARγ target genes Plin2 and Cide A/C are overexpressed, leading to increased lipid droplets formation. Expression of the fatty acid transporter CD36, another PPARgamma target, is also increased in Tg mice associated with elevated fatty acid uptake as measured in primary hepatocytes. Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme. Fatty acid synthase expression is also induced but the hepatic lipogenesis measured in vivo is not significantly different between WT and Tg mice. In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated. Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA), the main apoD ligand, increases the transcriptional activity of PPARγ. Supporting the role of apoD in AA transport, we observed enrichment in hepatic AA and a decrease in plasmatic AA concentration. Taken together, our results demonstrate that the hepatic steatosis observed in apoD Tg mice is a consequence of increased PPARγ transcriptional activity by AA leading to increased fatty acid uptake by the liver.


Assuntos
Apolipoproteínas D/genética , Ácidos Graxos/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , PPAR gama/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Ácido Araquidônico/sangue , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Antígenos CD36/metabolismo , Ácido Graxo Sintases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Lipogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , PPAR gama/genética , Perilipina-2 , Transporte Proteico/efeitos dos fármacos , Proteínas/metabolismo
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