RESUMEN
The cell type-specific expression of a gene is dependent on developmentally regulated modifications in chromatin structure that allow accessibility of basal and inducible transcription factors. In this study, we demonstrate that a cis-acting element in the second intron of the murine IL-4 gene has a dual function in regulating transcription in mast cells as well as chromatin accessibility of the IL-4 gene locus through its influence on the methylation state of the gene. Previous studies have shown that mast cell-restricted transcription factors GATA-1/2 and PU.1 associate with the intron element and regulate its activity. In this study, we use DNase I footprinting and mutational analyses to identify two additional sites that contribute to the element's ability to enhance transcription. One of these sites associates preferentially with STAT5a and STAT5b. We also demonstrate that deletion of the element or mutation of the GATA binding site in the context of a stably integrated IL-4 genomic construct prevents maintenance of a demethylated locus in IL-4-producing mast cells. These data indicate that, analogous to Ig and TCR intron regulatory elements, the intron enhancer has an essential role in maintaining developmentally regulated demethylation at the IL-4 gene locus. In addition, they indicate that members of the GATA family of transcription factors likely play an important role in these processes.
Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Interleucina-4/genética , Intrones/fisiología , Mastocitos/inmunología , Mastocitos/metabolismo , Factores de Transcripción/genética , Animales , Antígenos/biosíntesis , Secuencia de Bases , Sitios de Unión/genética , Sitios de Unión/inmunología , Línea Celular , Huella de ADN , Metilación de ADN , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Elementos de Facilitación Genéticos/inmunología , Marcadores Genéticos/inmunología , Interleucina-4/metabolismo , Intrones/inmunología , Ratones , Datos de Secuencia Molecular , Unión Proteica/genética , Unión Proteica/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Transactivadores , Factor de Transcripción Pit-1 , Factores de Transcripción/biosíntesis , Factores de Transcripción/inmunología , Factores de Transcripción/fisiologíaRESUMEN
Significant homology between dihydropteridine reductase (DHPR) from rat and human sources has been established by the ability of polyclonal antibodies raised to the rat-liver enzyme to detect the human protein in Western blots. The antibody also reacted with a single protein in bovine, dog and porcine kidney extracts, however, only trace reactivity was detected in rabbit. Quantitation of Western blots by soft laser densitometry showed that the response was proportional to total protein present in analyses of both pure rat-liver enzyme samples and crude extracts of rat and human liver. The DHPR contents of human blood cells were analysed by this method and the results compared to levels determined in enzymatic assays. Extracts of platelets and lymphocytes showed good correlation between these two methods, however, granulocytes exhibited high apparent enzyme activity but no DHPR protein detectable in blots. Erythrocyte extracts showed approximately 50% lower DHPR protein levels than predicted by activity measurements. These results are discussed in relation to the accuracy of detecting DHPR deficiencies in humans by enzymatic assay of whole blood samples.
Asunto(s)
Células Sanguíneas/enzimología , Dihidropteridina Reductasa/sangre , NADH NADPH Oxidorreductasas/sangre , Animales , Anticuerpos , Electroforesis , Humanos , Hígado/enzimología , Conejos , RatasRESUMEN
The cleavage of reductively alkylated rat liver dihydropteridine reductase with cyanogen bromide afforded a mixture of peptides, six of which (CB-1 to CB-6) were isolated and purified by C8 reverse-phase high performance liquid chromatography. Portions of peptides CB-1, CB-4, and CB-6 were sequenced by automated Edman degradation and high performance liquid chromatography and the carboxyl-terminal region by conventional procedures. Further proteolytic digestion of CB-6 and isolation of the products afforded a seven-amino acid peptide. A low degeneracy probe comprising 20 nucleotides was synthesized from the sequence of this peptide and was used to screen a rat liver cDNA expression library constructed in the vector lambda gt 10. Positive clones were isolated, and detailed examination of five of these by restriction endonucleases and dideoxy sequence analyses allowed identification of the entire coding region for dihydropteridine reductase. The gene was found to code for a protein of 240 amino acids (excluding the methionine initiator) of Mr = 25,420. Each of the sequences corresponding to the peptides CB-1, CB-4, CB-6, and the carboxyl terminus were identified in the deduced protein sequence. The rat enzyme is highly homologous to the human dihydropteridine reductase; the two proteins differ in only 10 amino acids, and all are conservative substitutions. In contrast, the sequence shows little homology with that of mammalian dihydrofolate reductase: reduced pyridine nucleotide-requiring enzymes with superficial mechanistic similarities.
Asunto(s)
Clonación Molecular , ADN/análisis , Dihidropteridina Reductasa/genética , Hígado/enzimología , NADH NADPH Oxidorreductasas/genética , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Secuencia de Bases , ADN/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , RatasRESUMEN
Purified rat-liver dihydropteridine reductase is homogeneous by gel filtration (Mr approximately 51,000), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 25,500), and native polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two identical subunits. However, analysis by isoelectric focusing has revealed three enzyme forms with approximate isoelectric points of 6.5, 5.9, and 5.7 (designated forms, I, II, and III, respectively). The three forms, isolated in 65% yield by preparative chromatofocusing, are stable in 0.05 M phosphate buffer, pH 6.8, containing 1 mM beta-mercaptoethanol and exhibit similar kinetic constants when the catalytic activities of the isolated forms are compared with quinonoid dihydrobiopterin as substrate. All forms generate complexes with the enzymatic cofactor NADH which are also detectable by IEF. When examined further by IEF under denaturing conditions in 6 M urea the enzyme demonstrates a differing subunit composition for its three forms. Two distinct subunits, designated alpha and beta, can be identified, and additional evidence suggests that the native enzyme forms I, II, and III represent the three differing dimeric combinations alpha alpha (form I), alpha beta (form II), and beta beta (form III).