DNA METHYLATION INHIBITS TRANSCRIPTION OF PROCOLLAGEN ALPHA-2(I) PROMOTERS

Our previous studies have demonstrated that a 2-[N-(acetoxyacetyl)amino]fluorene-transformed rat epithelial-like cell line, W8, contains a transcriptionally inactive alpha-2(I) gene with a hypermethylated promoter/first-exon region. We have cloned the rat promoter/first-exon region (- 211 to + 207) from W8 cells and their parent cell line, K16, which expresses alpha-2(I) collagen. There were no sequence differences between the clones from the two cell lines, indicating that a mutation was not responsible for transcriptional inhibition. The alpha-2(I) rat promoters were cloned upstream of the chloramphenicol acetyltransferase gene. Both constructs were equally active in both cell lines, suggesting that trans-activating factors for alpha-2(I) transcription are present in W8 cells. Finally, methylation of plasmids at all CpG sites with SssI methylase completely inhibited transcription using alpha-2(I) promoters, but methylation did not inhibit simian-virus-40 promoter-driven transcription. Certain methylation sites partially inhibit promoter activity. An HhaI methylation site inhibited transcriptional activity of the alpha-2(I) promoter 8-fold, whereas methylation at the HpaII site in the rat alpha-2(I) promoter did not decrease transcriptional activity. This provides further evidence that methylation at specific sites in the collagen alpha-2(I) promoter is responsible for the inactivation of transcription in W8 cells.