CHROMATIN STRUCTURE, DNA METHYLATION AND VITELLOGENIN GENE EXPRESSION.

摘要

The methylation patterns and the chromatin conformation of the Xenopus vitellogenin genes were examined to determine their relationship to the transcriptional state of the genes. The role of these two potential regulatory mechanisms was of particular interest because the estrogen induction of vitellogenin mRNA does not simply involve modulation of the rate at which a set of genes which are transcriptionally active in the absence of hormone is transcribed. Instead it involves the switching on of a set of transcriptionally inactive genes in a stable non-dividing cell type. The fact that the induction of vitellogenin is such an immense response made the study of the regulatory mechanisms responsible for this hormonal induction even more interesting.;The potential role of undermethylation of DNA in the activation of vitellogenin gene transcription was examined with methylation sensitive restriction enzymes. The finding that the majority of the potential methylation sites in the vitellogenin A1 gene are fully methylated in both induced hepatocyte DNA and red blood cell DNA indicates two important features of methylation in the vitellogenic response. First, the methylation of the vitellogenin gene in the induced hepatocyte DNA does not inhibit its transcription. All of the sites that were mapped to exon regions were fully methylated during rapid gene transcription. And second, no differences in methylation were found between the untranscribed and the extensively transcribed vitellogenin gene. The existence of some regions of partial undermethylation in the intron regions of the vitellogenin A1 gene in both blood cell DNA and induced hepatocyte DNA suggests that the methylation of Xenopus DNA may serve as a modulator in other cellular processes such as replication and recombination or in splicing enzyme recognition signals.;The relationship between chromatin conformation and transcription was determined by measuring the sensitivity of the vitellogenin genes to digestion by DNase 1 during the vitellogenic response. The chromatin conformation of the vitellogenin genes in uninduced liver was not preferentially sensitive to DNase digestion. However when the transcription of the genes was induced by administration of estrogen, the vitellogenin genes were preferentially digested by DNase 1. The change in the nuclease sensitivity of the genes in the uninduced and induced state indicates that the "actively" transcribed vitellogenin genes exist in a conformation that is different from the conformation of the uninduced, transcriptionally silent genes. When hormone withdrawn liver nuclei were digested with DNase 1, the genes were not preferentially digested. This result suggested that the change in chromatin conformation is a reversible phenomenon. This finding also suggested that the "memory effect" is not the consequence of the maintainance of the nuclease sensitive "active" conformation in withdrawn liver. Although these results do not exclude the possibility of another type of stable chromatin change such as the maintainance of a small region of hypersensitivity in the withdrawn state.