RESUMEN
We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Silenciador del Gen , Mamíferos/genética , Animales , Marcadores Genéticos , Globinas/genética , Proteínas Fluorescentes Verdes/genética , Histonas/genética , Metilación , Ratones , Ratones Transgénicos , Recombinación GenéticaRESUMEN
Using recombinase-mediated cassette exchange to test multiple transgenes at the same site of integration, we demonstrate a novel chromatin context-dependent silencer activity of the beta-globin locus control region (LCR). This silencer activity requires DNase I hypersensitive sites HS2 and HS3 but not HS4. After silencing, the silenced cassettes adopt a typical closed chromatin conformation (histone H3 and H4 deacetylation, histone H3-K4 methylation, DNA methylation, and replication in late S phase). In the absence of the LCR at the same site of integration, the chromatin remains decondensed. We demonstrate that the LCR is necessary but not sufficient to trigger these chromatin changes. We also provide evidence that this novel silencing activity is caused by transcriptional interference triggered by activation of transcription in the flanking sequences by the LCR.
Asunto(s)
Silenciador del Gen , Globinas/genética , Región de Control de Posición/genética , Activación Transcripcional/genética , Animales , Línea Celular , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Cromosomas de los Mamíferos/genética , Metilación de ADN , Replicación del ADN/genética , ADN Intergénico/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Modelos Genéticos , Regiones Promotoras Genéticas/genética , Transcripción Genética/genética , Transgenes/genéticaRESUMEN
Protein disulfide-isomerase has been isolated from human liver. The preparative procedure involved heat treatment, (NH(4))(2)SO(4) precipitation, CM-Sephadex C50 and DEAE-fast flow chromatography. The enzyme was homogenous and had a molecular mass of 60 kD or 120 kD as determined by sodium dodecy1 sulphate electro-phoresis and gel filtration respectively, indicating that the enzyme was a 120 kD dimmer with a subunit with molecular mass of 60 kD. The enzyme activity was as high as 830 U/g.protein as measured by the reactivation of "scrambled" ribonuclease. The antiserum of high titer was prepared by immunizing New Zealand rabbit with a mixture of the protein disulfide-isomerase and adjuvant.