Assuntos
Ebolavirus/isolamento & purificação , Reações Falso-Negativas , Doença pelo Vírus Ebola/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ebolavirus/genética , Guiné , Humanos , Plasma/virologiaRESUMO
Activation of the pDbeta1 promoter at the TCRbeta locus requires a functional distal enhancer, Ebeta. Here, we have analyzed the mechanism of promoter activation in thymocytes from mice containing or lacking Ebeta. We found that pDbeta1 shows a complex profile of transcription factor and chromatin remodeling complex occupancy even at Ebeta(-) alleles. The presence of Ebeta, however, results in a few specific changes in factor occupancy at the promoter. These differences correlate with localized alterations in histone modifications and in the recruitment of the basal transcriptional machinery. In addition, Ebeta is also bound by CBP and Pol II, suggesting a mechanism for delivery of a holoenzyme complex to the pDbeta1 promoter. These results illustrate a specialized, long-range function of an enhancer in the hierarchical events that regulate assembly of a cell type-specific promoter.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Regiões Promotoras Genéticas , Células 3T3 , Animais , Sítios de Ligação , Cromatina/genética , Cromatina/imunologia , Cromatina/ultraestrutura , Mapeamento Cromossômico , Pegada de DNA , Proteínas de Ligação a DNA/metabolismo , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleossomos/genética , Reação em Cadeia da PolimeraseRESUMO
To assess the role of the T cell receptor (TCR) beta gene enhancer (Ebeta) in regulating the processing of VDJ recombinase-generated coding ends, we assayed TCRbeta rearrangement of Ebeta-deleted (DeltaEbeta) thymocytes in which cell death is inhibited via expression of a Bcl-2 transgene. Compared with DeltaEbeta, DeltaEbeta Bcl-2 thymocytes show a small accumulation of TCRbeta standard recombination products, including coding ends, that involves the proximal Dbeta-Jbeta and Vbeta14 loci but not the distal 5' Vbeta genes. These effects are detectable in double negative pro-T cells, predominate in double positive pre-T cells, and correlate with regional changes in chromosomal structure during double negative-to-double positive differentiation. We propose that Ebeta, by driving long range nucleoprotein interactions and the control of locus expression and chromatin structure, indirectly contributes to the stabilization of coding ends within the recombination processing complexes. The results also illustrate Ebeta-dependent and -independent changes in chromosomal structure, suggesting distinct modes of regulation of TCRbeta allelic exclusion depending on the position within the locus.