Developmental changes in the methylation status of regulatory elements in the murine alpha 1(I) collagen gene

Regulatory elements contributing to the tissue-specific regulation of the murine alpha 1(I) collagen (Colla1) gene have previously been identified in its promoter region and first intron. Because several lines of evidence indicate that DNA methylation may be involved in the tissue-specific regulation of Colla1 gene expression, we have analyzed the methylation status of the 5' region of the gene by restriction analysis and a methylation-dependent PCR assay. All sites tested were unmethylated in sperm DNA. The region surrounding the start site of transcription was partially or completely methylated in undifferentiated embryonal cell lines, suggesting that it may be marked by de novo methylation during early embryonic development. In differentiated cells and adult tissues, the Colla1 promoter was completely demethylated in collagen-producing and some nonproducing cells, and partially methylated in other nonproducing cells. The first intron was unmethylated in collagen-producing as well as nonproducing cells. Only sites in the first exon showed an inverse correlation with transcriptional activity, i.e., they were unmethylated in cells that express the gene, but predominantly methylated in cells that do not. Our results indicate that the methylation status of a small area (less than 1 kb) downstream of the Colla1 promoter, but not of the promoter itself or the first intron, may be critical for transcriptional activity of the promoter, presumably through an indirect mechanism.