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1.
EMBO Rep ; 24(10): e57369, 2023 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-37501563

RESUMO

Nutritional immunity includes sequestration of transition metals from invading pathogens. Yersinia pestis overcomes nutritional immunity by secreting yersiniabactin to acquire iron and zinc during infection. While the mechanisms for yersiniabactin synthesis and import are well-defined, those responsible for yersiniabactin secretion are unknown. Identification of this mechanism has been difficult because conventional mutagenesis approaches are unable to inhibit trans-complementation by secreted factors between mutants. To overcome this obstacle, we utilized a technique called droplet Tn-seq (dTn-seq), which uses microfluidics to isolate individual transposon mutants in oil droplets, eliminating trans-complementation between bacteria. Using this approach, we first demonstrated the applicability of dTn-seq to identify genes with secreted functions. We then applied dTn-seq to identify an AcrAB efflux system as required for growth in metal-limited conditions. Finally, we showed this efflux system is the primary yersiniabactin secretion mechanism and required for virulence during bubonic and pneumonic plague. Together, these studies have revealed the yersiniabactin secretion mechanism that has eluded researchers for over 30 years and identified a potential therapeutic target for bacteria that use yersiniabactin for metal acquisition.


Assuntos
Peste , Yersinia pestis , Humanos , Yersinia pestis/genética , Peste/genética , Peste/microbiologia , Fenóis , Tiazóis/farmacologia , Metais , Proteínas de Bactérias/genética
2.
Microb Genom ; 8(4)2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35416147

RESUMO

Streptococcus pneumoniae is a major human pathogen that can cause severe invasive diseases such as pneumonia, septicaemia and meningitis. Young children are at a particularly high risk, with an estimated 3-4 million cases of severe disease and between 300 000 and 500 000 deaths attributable to pneumococcal disease each year. The haemolytic toxin pneumolysin (Ply) is a primary virulence factor for this bacterium, yet despite its key role in pathogenesis, immune evasion and transmission, the regulation of Ply production is not well defined. Using a genome-wide association approach, we identified a large number of potential affectors of Ply activity, including a gene acquired horizontally on the antibiotic resistance-conferring Integrative and Conjugative Element (ICE) ICESp23FST81. This gene encodes a novel modular protein, ZomB, which has an N-terminal UvrD-like helicase domain followed by two Cas4-like domains with potent ATP-dependent nuclease activity. We found the regulatory effect of ZomB to be specific for the ply operon, potentially mediated by its high affinity for the BOX repeats encoded therein. Using a murine model of pneumococcal colonization, we further demonstrate that a ZomB mutant strain colonizes both the upper respiratory tract and lungs at higher levels when compared to the wild-type strain. While the antibiotic resistance-conferring aspects of ICESp23FST81 are often credited with contributing to the success of the S. pneumoniae lineages that acquire it, its ability to control the expression of a major virulence factor implicated in bacterial transmission is also likely to have played an important role.


Assuntos
Estudo de Associação Genômica Ampla , Streptococcus pneumoniae , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequências Repetitivas Dispersas/genética , Camundongos , Streptococcus pneumoniae/genética , Estreptolisinas , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
3.
Am J Respir Cell Mol Biol ; 66(6): 661-670, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35353673

RESUMO

The genome-wide association study (GWAS)-identified asthma susceptibility risk alleles on chromosome 17q21 increase the expression of ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) in lung tissue. Given the importance of epithelial integrity in asthma, we hypothesized that ORMDL3 directly impacted bronchial epithelial function. To determine whether and how ORMDL3 expression impacts the bronchial epithelium, in studies using both primary human bronchial epithelial cells and human bronchial epithelial cell line, 16HBE (16HBE14o-), we assessed the impact of ORMDL3 on autophagy. Studies included: autophagosome detection by electron microscopy, RFP-GFP-LC3B to assess autophagic activity, and Western blot analysis of autophagy-related proteins. Mechanistic assessments included immunoprecipitation assays, intracellular calcium mobilization assessments, and cell viability assays. Coexpression of ORMDL3 and autophagy-related genes was measured in primary human bronchial epithelial cells derived from 44 subjects. Overexpressing ORMDL3 demonstrated increased numbers of autophagosomes and increased levels of autophagy-related proteins LC3B, ATG3, ATG7, and ATG16L1. ORMDL3 overexpression promotes autophagy and subsequent cell death by impairing intracellular calcium mobilization through interacting with SERCA2. Strong correlation was observed between expression of ORMDL3 and autophagy-related genes in patient-derived bronchial epithelial cells. Increased ORMDL3 expression induces autophagy, possibly through interacting with SERCA2, thereby inhibiting intracellular calcium influx, and induces cell death, impairing bronchial epithelial function in asthma.


Assuntos
Asma , Proteínas de Membrana , Asma/genética , Asma/metabolismo , Asma/patologia , Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Cálcio/metabolismo , Epitélio/metabolismo , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
4.
Elife ; 92020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32543373

RESUMO

Within deep tissue sites, extracellular bacterial pathogens often replicate in clusters that are surrounded by immune cells. Disease is modulated by interbacterial interactions as well as bacterial-host cell interactions resulting in microbial growth, phagocytic attack and secretion of host antimicrobial factors. To overcome the limited ability to manipulate these infection sites, we established a system for Yersinia pseudotuberculosis (Yptb) growth in microfluidics-driven microdroplets that regenerates microbial social behavior in tissues. Chemical generation of nitric oxide (NO) in the absence of immune cells was sufficient to reconstruct microbial social behavior, as witnessed by expression of the NO-inactivating protein Hmp on the extreme periphery of microcolonies, mimicking spatial regulation in tissues. Similarly, activated macrophages that expressed inducible NO synthase (iNOS) drove peripheral expression of Hmp, allowing regeneration of social behavior observed in tissues. These results argue that topologically correct microbial tissue growth and associated social behavior can be reconstructed in culture.


Assuntos
Dispositivos Lab-On-A-Chip , Macrófagos/microbiologia , Interações Microbianas , Óxido Nítrico/metabolismo , Yersinia pseudotuberculosis/fisiologia , Interações Hospedeiro-Patógeno , Modelos Biológicos , Comportamento Social
5.
Nat Commun ; 10(1): 5729, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844066

RESUMO

While Tn-Seq is a powerful tool to determine genome-wide bacterial fitness in high-throughput, culturing transposon-mutant libraries in pools can mask community or other complex single-cell phenotypes. Droplet Tn-Seq (dTn-Seq) solves this problem by microfluidics facilitated encapsulation of individual transposon mutants into growth medium-in-oil droplets, thereby enabling isolated growth, free from the influence of the population. Here we describe and validate microfluidic chip design, production, encapsulation, and dTn-Seq sample preparation. We determine that 1-3% of mutants in Streptococcus pneumoniae have a different fitness when grown in isolation and show how dTn-Seq can help identify leads for gene function, including those involved in hyper-competence, processing of alpha-1-acid glycoprotein, sensitivity against the human leukocyte elastase and microcolony formation. Additionally, we show dTn-Seq compatibility with microscopy, FACS and investigations of bacterial cell-to-cell and bacteria-host cell interactions. dTn-Seq reduces costs and retains the advantages of Tn-Seq, while expanding the method's original applicability.


Assuntos
Elementos de DNA Transponíveis/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas Analíticas Microfluídicas/métodos , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , DNA Bacteriano/genética , Biblioteca Gênica , Genes Bacterianos/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Mutação , Análise de Célula Única/instrumentação , Streptococcus pneumoniae/genética
6.
Sci Rep ; 8(1): 12750, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30143676

RESUMO

Cigarette smoke (CS) is one of the major risk factors for many pulmonary diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. The first line of defense for CS exposure is the bronchial epithelial cells. Elucidation of the epigenetic changes during CS exposure is key to gaining a mechanistic understanding into how mature and differentiated bronchial epithelial cells respond to CS. Therefore, we performed epigenomic profiling in conjunction with transcriptional profiling in well-differentiated human bronchial epithelial (HBE) cells cultured in air-liquid interface (ALI) exposed to the vapor phase of CS. The genome-wide enrichment of histone 3 lysine 27 acetylation was detected by chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) in HBE cells and suggested the plausible binding of specific transcription factors related to CS exposure. Additionally, interrogation of ChIP-Seq data with gene expression profiling of HBE cells after CS exposure for different durations (3 hours, 2 days, 4 days) suggested that earlier epigenetic changes (3 hours after CS exposure) may be associated with later gene expression changes induced by CS exposure (4 days). The integration of epigenetics and gene expression data revealed signaling pathways related to CS-induced epigenetic changes in HBE cells that may identify novel regulatory pathways related to CS-induced COPD.


Assuntos
Brônquios/patologia , Diferenciação Celular , Epigenômica , Células Epiteliais/patologia , Fumar/genética , Acetilação , Células Cultivadas , Montagem e Desmontagem da Cromatina , Células Epiteliais/metabolismo , Epitélio/patologia , Histonas/metabolismo , Humanos , Lisina/metabolismo
7.
Am J Pathol ; 186(7): 1754-1761, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27157989

RESUMO

Traffic of activated monocytes into the dorsal root ganglia (DRG) is critical for pathology in HIV peripheral neuropathy. We have shown that accumulation of recently recruited (bromodeoxyuridine(+) MAC387(+)) monocytes is associated with severe DRG pathology and loss of intraepidermal nerve fibers in SIV-infected macaques. Herein, we blocked leukocyte traffic by treating animals with natalizumab, which binds to α4-integrins. SIV-infected CD8-depleted macaques treated with natalizumab either early (the day of infection) or late (28 days after infection) were compared with untreated SIV-infected animals sacrificed at similar times. Histopathology showed diminished DRG pathology with natalizumab treatment, including decreased inflammation, neuronophagia, and Nageotte nodules. Natalizumab treatment resulted in a decrease in the number of bromodeoxyuridine(+) (early), MAC387(+) (late), CD68(+) (early and late), and SIVp28(+) (late) macrophages in DRG tissues. The number of CD3(+) T lymphocytes in DRGs was not affected by natalizumab treatment. Vascular cell adhesion molecule 1, an adhesion molecule that mediates leukocyte traffic, was diminished in DRGs of all natalizumab-treated animals. These data show that blocking monocyte, but not T lymphocyte, traffic to the DRG results in decreased inflammation and pathology, supporting a role for monocyte traffic and activation in HIV peripheral neuropathy.


Assuntos
Gânglios Espinais/patologia , Integrina alfa4/metabolismo , Doenças do Sistema Nervoso Periférico/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/complicações , Molécula 1 de Adesão de Célula Vascular/biossíntese , Animais , Quimiotaxia de Leucócito/efeitos dos fármacos , Infecções por HIV , Imuno-Histoquímica , Fatores Imunológicos/farmacologia , Macaca mulatta , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Monócitos/imunologia , Monócitos/metabolismo , Natalizumab/farmacologia , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo
8.
Am J Respir Cell Mol Biol ; 54(1): 34-40, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26051534

RESUMO

Recurrent, rapidly growing nasal polyps are hallmarks of aspirin-exacerbated respiratory disease (AERD), although the mechanisms of polyp growth have not been identified. Fibroblasts are intimately involved in tissue remodeling, and the growth of fibroblasts is suppressed by prostaglandin E2 (PGE2), which elicits antiproliferative effects mediated through the E prostanoid (EP)2 receptor. We now report that cultured fibroblasts from the nasal polyps of subjects with AERD resist this antiproliferative effect. Fibroblasts from polyps of subjects with AERD resisted the antiproliferative actions of PGE2 and a selective EP2 agonist (P < 0.0001 at 1 µM) compared with nasal fibroblasts from aspirin-tolerant control subjects undergoing polypectomy or from healthy control subjects undergoing concha bullosa resections. Cell surface expression of the EP2 receptor protein was lower in fibroblasts from subjects with AERD than in fibroblasts from healthy control subjects and aspirin-tolerant subjects (P < 0.01 for both). Treatment of the fibroblasts with trichostatin A, a histone deacetylase inhibitor, significantly increased EP2 receptor mRNA in fibroblasts from AERD and aspirin-tolerant subjects but had no effect on cyclooxygenase-2, EP4, and microsomal PGE synthase 1 (mPGES-1) mRNA levels. Histone acetylation (H3K27ac) at the EP2 promoter correlated strongly with baseline EP2 mRNA (r = 0.80; P < 0.01). These studies suggest that the EP2 promotor is under epigenetic control, and one explanation for PGE2 resistance in AERD is an epigenetically mediated reduction of EP2 receptor expression, which could contribute to the refractory nasal polyposis typically observed in this syndrome.


Assuntos
Asma Induzida por Aspirina/metabolismo , Dinoprostona/farmacologia , Fibroblastos/efeitos dos fármacos , Pólipos Nasais/metabolismo , Receptores de Prostaglandina E Subtipo EP2/agonistas , Acetilação , Adulto , Asma Induzida por Aspirina/genética , Asma Induzida por Aspirina/patologia , Boston , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , Dinoprostona/análogos & derivados , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Epigênese Genética/efeitos dos fármacos , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/genética , Pólipos Nasais/patologia , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transdução de Sinais , Virginia
9.
BMC Plant Biol ; 15: 157, 2015 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-26105742

RESUMO

BACKGROUND: Ethylene plays critical roles in plant growth and development, including the regulation of cell expansion, senescence, and the response to biotic and abiotic stresses. Elements of the initial signal transduction pathway have been determined, but we are still defining regulatory mechanisms by which the sensitivity of plants to ethylene is modulated. RESULTS: We report here that members of the ARGOS gene family of Arabidopsis, previously implicated in the regulation of plant growth and biomass, function as negative feedback regulators of ethylene signaling. Expression of all four members of the ARGOS family is induced by ethylene, but this induction is blocked in ethylene-insensitive mutants. The dose dependence for ethylene induction varies among the ARGOS family members, suggesting that they could modulate responses across a range of ethylene concentrations. GFP-fusions of ARGOS and ARL localize to the endoplasmic reticulum, the same subcellular location as the ethylene receptors and other initial components of the ethylene signaling pathway. Seedlings with increased expression of ARGOS family members exhibit reduced ethylene sensitivity based on physiological and molecular responses. CONCLUSIONS: These results support a model in which the ARGOS gene family functions as part of a negative feedback circuit to desensitize the plant to ethylene, thereby expanding the range of ethylene concentrations to which the plant can respond. These results also indicate that the effects of the ARGOS gene family on plant growth and biomass are mediated through effects on ethylene signal transduction.


Assuntos
Arabidopsis/genética , Etilenos/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Família Multigênica , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Divisão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Cinética , Modelos Biológicos , Mutação/genética , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Plântula/metabolismo
10.
Hum Mol Genet ; 24(11): 3005-20, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25586491

RESUMO

Recent advances in genetics have spurred rapid progress towards the systematic identification of genes involved in complex diseases. Still, the detailed understanding of the molecular and physiological mechanisms through which these genes affect disease phenotypes remains a major challenge. Here, we identify the asthma disease module, i.e. the local neighborhood of the interactome whose perturbation is associated with asthma, and validate it for functional and pathophysiological relevance, using both computational and experimental approaches. We find that the asthma disease module is enriched with modest GWAS P-values against the background of random variation, and with differentially expressed genes from normal and asthmatic fibroblast cells treated with an asthma-specific drug. The asthma module also contains immune response mechanisms that are shared with other immune-related disease modules. Further, using diverse omics (genomics, gene-expression, drug response) data, we identify the GAB1 signaling pathway as an important novel modulator in asthma. The wiring diagram of the uncovered asthma module suggests a relatively close link between GAB1 and glucocorticoids (GCs), which we experimentally validate, observing an increase in the level of GAB1 after GC treatment in BEAS-2B bronchial epithelial cells. The siRNA knockdown of GAB1 in the BEAS-2B cell line resulted in a decrease in the NFkB level, suggesting a novel regulatory path of the pro-inflammatory factor NFkB by GAB1 in asthma.


Assuntos
Antiasmáticos/farmacologia , Asma/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Asma/metabolismo , Sequência de Bases , Relação Dose-Resposta a Droga , Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Inflamação/genética , Inflamação/metabolismo , Modelos Genéticos , NF-kappa B/genética , NF-kappa B/metabolismo , Mapeamento de Interação de Proteínas , Transdução de Sinais
11.
J Allergy Clin Immunol ; 134(5): 1153-62, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24934276

RESUMO

BACKGROUND: Genome-wide association studies have yet to identify the majority of genetic variants involved in asthma. We hypothesized that expression quantitative trait locus (eQTL) mapping can identify novel asthma genes by enabling prioritization of putative functional variants for association testing. OBJECTIVE: We evaluated 6706 cis-acting expression-associated variants (eSNPs) identified through a genome-wide eQTL survey of CD4(+) lymphocytes for association with asthma. METHODS: eSNPs were tested for association with asthma in 359 asthmatic patients and 846 control subjects from the Childhood Asthma Management Program, with verification by using family-based testing. Significant associations were tested for replication in 579 parent-child trios with asthma from Costa Rica. Further functional validation was performed by using formaldehyde-assisted isolation of regulatory elements (FAIRE) quantitative PCR and chromatin immunoprecipitation PCR in lung-derived epithelial cell lines (Beas-2B and A549) and Jurkat cells, a leukemia cell line derived from T lymphocytes. RESULTS: Cis-acting eSNPs demonstrated associations with asthma in both cohorts. We confirmed the previously reported association of ORMDL3/GSDMB variants with asthma (combined P = 2.9 × 10(-8)). Reproducible associations were also observed for eSNPs in 3 additional genes: fatty acid desaturase 2 (FADS2; P = .002), N-acetyl-α-D-galactosaminidase (NAGA; P = .0002), and Factor XIII, A1 (F13A1; P = .0001). Subsequently, we demonstrated that FADS2 mRNA is increased in CD4(+) lymphocytes in asthmatic patients and that the associated eSNPs reside within DNA segments with histone modifications that denote open chromatin status and confer enhancer activity. CONCLUSIONS: Our results demonstrate the utility of eQTL mapping in the identification of novel asthma genes and provide evidence for the importance of FADS2, NAGA, and F13A1 in the pathogenesis of asthma.


Assuntos
Asma , Linfócitos T CD4-Positivos/imunologia , Ácidos Graxos Dessaturases , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , alfa-N-Acetilgalactosaminidase , Asma/epidemiologia , Asma/genética , Asma/imunologia , Asma/patologia , Linfócitos T CD4-Positivos/patologia , Criança , Pré-Escolar , Costa Rica , Método Duplo-Cego , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/imunologia , Feminino , Humanos , Masculino , alfa-N-Acetilgalactosaminidase/genética , alfa-N-Acetilgalactosaminidase/imunologia
12.
Genomics ; 101(5): 263-72, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23459001

RESUMO

Hedgehog interacting protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.


Assuntos
Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Glicoproteínas de Membrana/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Transcriptoma , Brônquios/metabolismo , Brônquios/patologia , Proteínas de Transporte/genética , Estudos de Casos e Controles , Linhagem Celular , Proliferação de Células , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Humanos , Pulmão/metabolismo , Pulmão/patologia , Glicoproteínas de Membrana/genética , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Doença Pulmonar Obstrutiva Crônica/genética , RNA Interferente Pequeno/genética , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Transdução de Sinais , Regulação para Cima
13.
Hum Mol Genet ; 21(6): 1325-35, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22140090

RESUMO

Multiple intergenic single-nucleotide polymorphisms (SNPs) near hedgehog interacting protein (HHIP) on chromosome 4q31 have been strongly associated with pulmonary function levels and moderate-to-severe chronic obstructive pulmonary disease (COPD). However, whether the effects of variants in this region are related to HHIP or another gene has not been proven. We confirmed genetic association of SNPs in the 4q31 COPD genome-wide association study (GWAS) region in a Polish cohort containing severe COPD cases and healthy smoking controls (P = 0.001 to 0.002). We found that HHIP expression at both mRNA and protein levels is reduced in COPD lung tissues. We identified a genomic region located ∼85 kb upstream of HHIP which contains a subset of associated SNPs, interacts with the HHIP promoter through a chromatin loop and functions as an HHIP enhancer. The COPD risk haplotype of two SNPs within this enhancer region (rs6537296A and rs1542725C) was associated with statistically significant reductions in HHIP promoter activity. Moreover, rs1542725 demonstrates differential binding to the transcription factor Sp3; the COPD-associated allele exhibits increased Sp3 binding, which is consistent with Sp3's usual function as a transcriptional repressor. Thus, increased Sp3 binding at a functional SNP within the chromosome 4q31 COPD GWAS locus leads to reduced HHIP expression and increased susceptibility to COPD through distal transcriptional regulation. Together, our findings reveal one mechanism through which SNPs upstream of the HHIP gene modulate the expression of HHIP and functionally implicate reduced HHIP gene expression in the pathogenesis of COPD.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Elementos Facilitadores Genéticos/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Western Blotting , Brônquios/citologia , Brônquios/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Cromossomos Humanos Par 4/genética , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Predisposição Genética para Doença , Genótipo , Haplótipos/genética , Humanos , Pulmão/citologia , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Regiões Promotoras Genéticas/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fumar/genética , Fator de Transcrição Sp3/metabolismo
14.
Plant Cell ; 20(8): 2102-16, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18723577

RESUMO

The type B Arabidopsis Response Regulators (ARRs) of Arabidopsis thaliana are transcription factors that act as positive regulators in the two-component cytokinin signaling pathway. We employed a mutant-based approach to perform a detailed characterization of the roles of ARR1, ARR10, and ARR12 in plant growth and development. The most pronounced phenotype was found in the arr1-3 arr10-5 arr12-1 triple loss-of-function mutant, which showed almost complete insensitivity to high levels of exogenously applied cytokinins. The triple mutant exhibited reduced stature due to decreased cell division in the shoot, enhanced seed size, increased sensitivity to light, altered chlorophyll and anthocyanin concentrations, and an aborted primary root with protoxylem but no metaxylem. Microarray analysis revealed that expression of the majority of cytokinin-regulated genes requires the function of ARR1, ARR10, and ARR12. Characterization of double mutants revealed differing contributions of the type B ARRs to mutant phenotypes. Our results support a model in which cytokinin regulates a wide array of downstream responses through the action of a multistep phosphorelay that culminates in transcriptional regulation by ARR1, ARR10, and ARR12.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Citocininas/metabolismo , Transdução de Sinais/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citocininas/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Modelos Genéticos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
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