CpG methylation of a silent controlling element in the murine A ^vy allele is incomplete and unresponsive to methyl donor supplementation

Background: The viable yellow allele of agouti (A^vy) is remarkable for its unstable and partially heritable epigenetic state, which produces wide variation in phenotypes of isogenic mice. In the A^vy allele an inserted intracisternal A particle (IAP) acts as a controlling element which deregulates expression of agouti by transcription from the LTR of the IAP; the phenotypic state has been linked to CpG methylation of the LTR. Phenotypic variation between A^vy mice indicates that the epigenetic state of the IAP is unstable in the germline. Principal Findings: We have made a detailed examination of somatic methylation of the IAP using bisulphite allelic sequencing, and find that the promoter is incompletely methylated even when it is transcriptionally silent. In utero exposure to supplementary methyl donors, which alters the spectrum of A^vy phenotypes, does not increase the density of CpG methylation in the silent LTR. Conclusions: Our findings suggest that, contrary to previous supposition, methyl donor supplementation acts through an indirect mechanism to silence A^vy. The incomplete cytosine methylation we observe at the somatically silent A^vy allele may reflect its unstable germline state, and the influence of epigenetic modifications underlying CpG methylation.