UNMETHYLATED THYROGLOBULIN PROMOTER MAY BE REPRESSED BY METHYLATION OF FLANKING DNA-SEQUENCES

The thyroglobulin gene, like many other tissue-specific genes, appears to be specifically less methylated in the differentiated cell type wh ere it is transcribed. The thyroglobulin gene promoter elements themse lves are highly CG-deficient and do not contain any HpaII/MspI sites. In this study, using DNA constructs that were methylated in vitro with HpaII or MspI methylases, we show that DNA methylation of vector sequ ences is sufficient to repress the activity of the thyroglobulin gene promoter in transient transfection experiments. Reporter-gene expressi on from a plasmid containing only the proximal thyroglobulin gene prom oter is sensitive to DNA methylation even in fully differentiated thyr ocytes. Transcription from methylated plasmids containing the thyroglo bulin gene enhancer and proximal promoter is also clearly reduced when the transfected cells are maintained under less-differentiated condit ions. These results indicate that DNA methylation can influence, from a distance, the activity of an unmodified promoter. Our results also a gree with the view that loss of DNA methylation does not constitute a prerequisite for thyroglobulin gene expression in differentiated thyro cytes, where the thyroglobulin gene enhancer and promoter are activate d. However, the production of thyroglobulin transcripts could be sever ely impaired when this activation is not maximal, as is the case in le ss-differentiated cells or when the enhancer element is lacking. We su ggest that DNA methylation helps to maintain the thyroglobulin gene in an inactive state unless all of the conditions required for its expre ssion are fulfilled, and that the thyroid-specific demethylation event s are a consequence of the activation state of the gene.