RESUMO
Comparative cross-species genomic analysis has served as a powerful tool to discover novel noncoding regulatory regions that influence gene expression in several cytokine loci. In this study, we have identified several evolutionarily conserved regions (ECRs) that are shared between human, rhesus monkey, dog, and horse and that are upstream of the promoter regions that have been previously shown to play a role in regulating CCL2 gene expression. Of these, an ECR that was ~16.5 kb (-16.5 ECR) upstream of its coding sequence contained a highly conserved NF-κB site. The region encompassing the -16.5 ECR conferred TNF-α responsiveness to homologous and heterologous promoters. In vivo footprinting demonstrated that specific nucleotide residues in the -16.5 ECR were protected or became hypersensitive after TNF-α treatment. The footprinted regions were found to bind NF-κB subunits in vitro and in vivo. Mutation/deletion of the conserved NF-κB binding site in the -16.5 ECR led to loss of TNF-α responsiveness. After TNF-α stimulation, the -16.5 ECR showed increased sensitivity to nuclease digestion and loss of histone signatures that are characteristic of a repressive chromatin. Chromosome conformation capture assays indicated that -16.5 ECR physically interacts with the CCL2 proximal promoter after TNF-α stimulation. Taken together, these results suggest that the -16.5 ECR may play a critical role in the regulation of CCL2.
Assuntos
Quimiocina CCL2/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Fator de Necrose Tumoral alfa/genética , Animais , Sítios de Ligação , Sequência Conservada , Pegada de DNA , Evolução Molecular , Humanos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Especificidade da EspécieRESUMO
We investigated the association of polymorphisms in CCR5, the major human immunodeficiency virus (HIV)-1 coreceptor, and copy number of its potent ligand CCL3L1 with tuberculosis in 298 individuals from Colombia. The CCR5-HHD haplotype, a known genetic determinant of increased susceptibility to HIV-AIDS, and a high copy number of CCL3L1, a known genetic determinant of enhanced CCL3/CCL3L1 chemokine expression, each associated with presence of tuberculosis. Furthermore, CCR5-HHD was associated with higher CCR5 gene and surface expression. These results substantiate the strong link between the pro-inflammatory effects of CCR5 and its ligands with active tuberculosis and suggest that chemokine-chemokine receptor genetic determinants may influence tuberculosis in addition to HIV/AIDS.
Assuntos
Quimiocinas CC/genética , Receptores CCR5/genética , Tuberculose/genética , Adulto , Estudos de Casos e Controles , Colômbia/epidemiologia , Feminino , Dosagem de Genes , Haplótipos , Humanos , Desequilíbrio de Ligação , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Tuberculose/epidemiologiaRESUMO
Ligands of CCR5, the major coreceptor of HIV-1, costimulate T lymphocyte activation. However, the full impact of CCR5 expression on T cell responses remains unknown. Here, we show that compared with CCR5(+/+), T cells from CCR5(-/-) mice secrete lower amounts of IL-2, and a similar phenotype is observed in humans who lack CCR5 expression (CCR5-Delta32/Delta32 homozygotes) as well as after Ab-mediated blockade of CCR5 in human T cells genetically intact for CCR5 expression. Conversely, overexpression of CCR5 in human T cells results in enhanced IL-2 production. CCR5 surface levels correlate positively with IL-2 protein and mRNA abundance, suggesting that CCR5 affects IL-2 gene regulation. Signaling via CCR5 resulted in NFAT transactivation in T cells that was blocked by Abs against CCR5 agonists, suggesting a link between CCR5 and downstream pathways that influence IL-2 expression. Furthermore, murine T cells lacking CCR5 had reduced levels of intranuclear NFAT following activation. Accordingly, CCR5 expression also promoted IL-2-dependent events, including CD25 expression, STAT5 phosphorylation, and T cell proliferation. We therefore suggest that by influencing a NFAT-mediated pathway that regulates IL-2 production and IL-2-dependent events, CCR5 may play a critical role in T cell responses. In accord with our prior inferences from genetic-epidemiologic studies, such CCR5-dependent responses might constitute a viral entry-independent mechanism by which CCR5 may influence HIV-AIDS pathogenesis.
Assuntos
Interleucina-2/biossíntese , Ativação Linfocitária/imunologia , Fatores de Transcrição NFATC/metabolismo , Receptores CCR5/biossíntese , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Adulto , Animais , Linhagem Celular Tumoral , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Células Cultivadas , Humanos , Imunidade Celular/genética , Interleucina-2/fisiologia , Células Jurkat , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/fisiologia , Receptores CCR5/deficiência , Receptores CCR5/genética , Receptores CCR5/fisiologia , Transdução de Sinais/genéticaRESUMO
ABSTRACT Karyotypes of two Colocasia oresbia botanical varieties from Bangladesh were analyzed and compared with orcein, chromomycin A3 (CMA) and 4´-6 diamidino-2-phenylindole (DAPI). Both varieties had 2n=2x=26 chromosomes (karyotypic formula: 20m+6sm) and a pair of satellites each. Total chromosome length was 144.18 ± 2.45 μm in C. oresbia var. oresbia and 133.02 ± 2.75 μm in C. oresbia var. stolonifera. The karyotype of Colocasia oresbia var. oresbia is 2A whereas that of C. oresbia var. stolonifera is 1A. Six CMA and four DAPI bands were observed in C. oresbia var. oresbia and eight CMA and six DAPI bands in C. oresbia var. stolonifera. However, in these two morphologically distinct C. oresbia varieties of two different ecological zones, the same somatic chromosome number, diversification in various karyotypic parameters and CMA/DAPI-banding patterns were observed. In addition to taxonomic characters, the studied karyotype features will contribute to the characterization of these two C. oresbia varieties and to establish a base for future research.
RESUMEN Se analizaron y compararon los cariotipos de dos variedades botánicas de Colocasia oresbia de Bangladesh con orceína, chromomicina A3 (CMA) y 4-6 diamidino-2-phenilindol (DAPI). Ambas variedades presentaron 2n=2x=26 cromosomas (fórmula cariotípica: 20m+6sm) y un par de satélites cada una. La longitud total de cromosomas fue 144,18 ± 2,45 μm en C. oresbia var. oresbia y 133.02 ± 2.75 μm en C. oresbia var. stolonifera. El cariotipo de Colocasia oresbia var. oresbia es 2ª, y 1ª el de C. oresbia var. stolonifera. Se observaron seis bandas CMA y cuatro DAPI en C. oresbia var. oresbia y ocho bandas CMA y seis DAPI en C. oresbia var. stolonifera. Sin embargo, en estas dos variedades morfológicamente distintivas de C. oresbia de dos zonas ecológicas diferentes se observó el mismo número cromosómico somático, diversificación en varios parámetros cariotípicos y en patrones de bandeo CMA/DAPI. En adición a los caracteres taxonómicos, las características de los cariotipos estudiados contribuirán a la caracterización de estas dos variedades de C. oresbia y a establecer una base para futuras investigaciones.