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

Abstract

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.

Figure 1. Phenotypic variation in A^vy mice, A^vy allele structure, and complex CpG methylation at the IAP.

A. Phenotypes of isogenic A^vy littermates range from pure yellow and obese (left) through mottled yellow/agouti to lean fully agouti, called pseudoagouti (right). B. Schematic of the A^vy locus, with the sequence of the amplified region, which includes portions of the 5′LTR and pseudoexon 1A (−240 to +92 relative to the cryptic promoter, which is marked by an arrow). The start point of A^vy transcription is marked by an arrow. CpG dinucleotides are displayed in red. C. Representative bisulphite allelic sequencing profiles of individual alleles from yellow and pseudoagouti mice. Each single row represents a single allele, and each box a CpG (white: unmethylated; black: methylated).

Figure 2. Incomplete CpG methylation of the silent A^vy IAP.

A. Bisulphite allelic sequencing profiles at the A^vy IAP and three other IAPs. Each line represents an allele, and each box a CpG dinucleotide (white: unmethylated; black: methylated). Each block represents sequences derived from a single mouse. B. Histograms of allelic CpG methylation density. Each histogram displays the frequency of alleles with a given number of CpGs methylated across the sequenced region. For IAP C, only 10 CpGs were sequenced. n = number of alleles sequenced; *p<0.001.

Figure 3. Disruption of cytosine methylation at the silent A^vy allele by methyl-donor supplementation.

The histograms display the frequency of alleles with a given number of CpGs methylated across the sequenced region in unsupplemented mice (top), mice exposed to methyl-donor supplementation in utero (middle) and unsupplemented offspring of supplemented mice (bottom). Each histogram represents combined bisulphite allelic sequencing data from three pseudoagouti mice. The red line shows the pattern of normal distribution. The average percent methylation over all sequenced CpGs for each mouse group is indicated. *p = 0.037.