DNA methylation in transcriptional repression of two differentially expressed X-linked genes, GPC3 and SYBL1

Methylation of CpG islands is an established transcriptional repressive mec hanism and is a feature of silencing in X chromosome inactivation, Housekee ping genes that are subject to X inactivation exhibit differential methylat ion of their CpG islands such that the inactive alleles are hypermethylated , In this report, we examine two contrasting X-linked genes with CpG island s for regulation by DNA methylation: SYBL1, a housekeeping gene in the Xq p seudoautosomal region, and GPC3, a tissue-specific gene in Xq26 that is imp licated in the etiology of the Simpson-Golabi-Behmel overgrowth syndrome. W e observed that in vitro methylation of either the SYBL1 or the GPC3 promot er resulted in repression of reporter constructs. In normal contexts, we fo und that both the Y and inactive X alleles of SYBL1 are repressed and hyper methylated, whereas the active X allele is expressed and unmethylated. Furt hermore, the Y and inactive X alleles of SYBL1 were derepressed by treatmen t with the demethylating agent azadeoxycytidine, GPC3 is also subject to X inactivation, and the active X allele is unmethylated in nonexpressing leuk ocytes as well as in an expressing cell line, suggesting that methylation i s not involved in the tissue-specific repression of this allele. The inacti ve X allele, however, is hypermethylated in leukocytes, presumably reflecti ng early X inactivation events that become important for gene dosage in exp ressing lineages. These and other data suggest that all CPG islands on Xq, including the pseudoautosomal region, are subject to X inactivation-induced meth ylation, Additionally, methylation of SYBL1 on Yq may derive from a p rocess related to X inactivation that targets large chromatin domains for t ranscriptional repression.