RESUMO
TCRD V segments rearrange in an ordered fashion during human and murine thymic development. Recombination requires the accessibility of substrate gene segments, and transcriptional enhancers and promoters have been shown to regulate the accessible chromatin configuration. We therefore investigated the regulation of TCRD V rearrangements by characterizing the promoter of the first TCRD V segment to be rearranged, DV101S1, under the influence of its own enhancer. Sequences required for full promoter activity were identified by transient transfections of normal and mutated promoters into a human gammadelta lymphoma, and necessary elements fall between -86 and +66 nt, relative to the major transcription start site. They include a cAMP responsive element (CRE) at -62, an Ets site at -39, a TATA box at -26, the major transcriptional start site sequence (-8 to -5 and -2 to +11), and a downstream sequence (+12 to +33). Gel shift analyses and in vitro DNase I footprinting showed that nuclear proteins bind to the functionally relevant CRE, Ets, +1 to +10 sequence, and the +17 to +21 sequence. Nuclear proteins also bind to an E box at -52, and GATA-3 binds to a GATA motif at -5, as shown by Ab ablation-supershift experiments, but mutations that abrogated protein binding to these sites failed to affect DV101S1 promoter activity. We conclude that not all protein-binding sites within the DV101S1 minimal promoter are important for enhancer driven TCRD gene transcription. Further, the possibility remains that the GATA and E box sites function in enhancer independent DV101S1 germline transcription.
Assuntos
Regiões Promotoras Genéticas/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Sequências Reguladoras de Ácido Nucleico/imunologia , Animais , Sequência de Bases , Extratos Celulares/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA3 , Rearranjo Gênico da Cadeia delta dos Receptores de Antígenos dos Linfócitos T/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/imunologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Iniciação Traducional da Cadeia Peptídica/genética , Iniciação Traducional da Cadeia Peptídica/imunologia , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas/imunologia , TATA Box/imunologia , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica/imunologia , Células Tumorais CultivadasRESUMO
In a previous study, we showed that the immunoglobulin heavy-chain (IgH) enhancer (IgHe) is near or in an initiation zone of chromosomal DNA replication, which is preferentially active in B cells (K. Ariizumi, Z. Wang, and P. W. Tucker, Proc. Natl. Acad. Sci. USA 90:3695-3699, 1993). This suggests the existence of a functional relationship between IgHe-mediated transcription and DNA replication. To test this theory, we utilized simian virus 40 (SV40) DNA replication as a model of chromosomal replication. IgHe or its operationally divisible domains (5'-En, core, and 3'-En) were introduced into SV40 minichromosomes (IgHe-SV40). Results of replication assays with IgHe-SV40 replicons indicated that the 5'-En and 3'-En activated or suppressed SV40 DNA replication regardless of the presence of SV40 enhancers or promoters in these replicons. The activity did not reside in IgHe core sequences. The results suggested that the 5'- and 3'-En regulated SV40 replication through direct interaction with the origin, not through suppression at the SV40 enhancer and/or promoter. In an effort to identify elements within the 5'-En motif that contributed to this effect, we found that the E site, but not microE5 and microE2 boxes, upregulated DNA replication. Our results provide another possible regulatory function for the 5'-En and 3'-En domains besides transcriptional suppression of IgHe.
Assuntos
Replicação do DNA , Elementos Facilitadores Genéticos , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Vírus 40 dos Símios/genética , Animais , Composição de Bases , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Técnicas In Vitro , Dados de Sequência Molecular , Matriz Nuclear/metabolismo , Oligodesoxirribonucleotídeos/química , Sequências Reguladoras de Ácido NucleicoRESUMO
By generating phosphorylcholine (PC)-specific, wild-type (mu), and chimeric (mu-I-A alpha) antigen receptor transfectants of mature B cells, we have shown that the COOH terminus of the mu heavy chain is essential for three major functions: immediate signal transduction (measured as changes in intracellular Ca2+), antigen presentation, and induction of immunoglobulin M secretion. A more detailed analysis of structural requirements of the COOH-terminal domains contributing to these functions was achieved by systematically replacing the spacer, cytoplasmic, and transmembranal domains of the mu-I-A alpha chimeric chain with those of mu. Using this rescue approach, we show that the carboxyl two-thirds of the transmembranal domain (proximal to the cytoplasmic domain) is required for induction of intracellular Ca2+, whereas the complete transmembranal domain is required for the function of antigen presentation but is dispensable for induction of antibody secretion.
