Relationship between DNA methylation states and transcription of individual isoforms encoded by the protocadherin-alpha gene cluster.

The protocadherin-alpha (Pcdh-alpha) gene encodes diverse transmembrane proteins that are differentially expressed in individual neurons in the vertebrate central nervous system. The Pcdh-alpha genomic structure contains variable first exons, each regulated by its own promoter. Here, we investigated the effect of DNA methylation on gene regulation in the Pcdh-alpha gene cluster. We studied two mouse cell lines, C1300 and M3, that expressed different combinations of Pcdh-alpha isoforms and found that 1) the transcription of specific Pcdh-alpha isoforms correlated significantly with the methylation state of the promoter and the 5' (but not the 3') region of the first exon and 2) mosaic or mixed methylation states of the promoters were associated with both active and inactive transcription. Demethylation of C1300 cells up-regulated all of the Pcdh-alpha isoforms, and, in a promoter assay, hypermethylation of the promoters repressed their transcriptional activity. Cell lines subcloned from the demethylated C1300 cells transcribed different combinations of Pcdh-alpha isoforms than the parental, nondemethylated cells, and the promoters showed differential mosaic or mixed methylation patterns. In vivo, the promoter and 5'-regions of the Pcdh-alphaC1 and alphaC2 exons, which are transcribed in all neurons, were extensively hypomethylated. In contrast, the promoters of the Pcdh-alpha1 to -alpha12 isoforms, which are transcribed differentially by individual Purkinje cells, exhibited mosaic methylation patterns. Overall, our results demonstrated that mosaic or mixed DNA methylation states in the promoter and 5'-region of the first exon may help regulate differential Pcdh-alpha transcription and that hypermethylation is sufficient to repress transcription.