Methylation of CpGs as a determinant of transcriptional activation at alternative promoters for transforming growth factor-beta 3

The human transforming growth factor-beta 3 (TGF-beta 3) gene has a typical CpG island, the core of which is centered just upstream of its principle p romoter. Activation of an alternative downstream promoter, leading to the p roduction of a truncated mRNA lacking the portion of the 5' noncoding regio n responsible for translational inhibition of TGF-beta 3 mRNA, is only evid ent in breast cancer cells. We compared the methylation status of genomic D NA isolated from a panel of breast (SKBR-3 and T47-D) and non-breast cancer (HT-1080, A673, and A375) cell lines by sequencing sodium metabisulfite-tr eated DNA, In all cell lines, the core of the TGF-beta 3 CpG island was pre dominantly unmethylated? irrespective of promoter usage associated with tha t cell line, In contrast, we observed a marked difference in methylation at 19 CpG sites immediately flanking and downstream of the alternative promot er's transcription initiation site. Specifically, the non-breast cancer cel l lines exhibited nearly complete methylation of these CpG, sites, whereas in the breast cancer cell lines, these CpGs vr ere predominantly unmethylat ed, Our data support the hypothesis that methylation of a limited number of CpGs at the periphery of an otherwise unmethylated CpG island underlies th e transcriptional repression of the downstream promoter in non-breast cance r cells, thereby serving to regulate the use of alternative promoters for T GF-beta 3.