Figure 1.
Three possible types of mosaicism for cytosine methylation at FMR1.
(A) Mosaicism among cells, with either unmethylated (open squares) or hypermethylated (filled squares) epialleles; (B) mosaicism among CpG sites; and (C) mosaicism between the two strands of an individual DNA molecule. For the latter two possibilities, each square represents a CpG on a single strand of DNA; filled boxes depict methylated CpGs and open boxes depict unmethylated CpGs. Arrows represent possible transcriptional activity under each methylation-mosaicism scenario.
Figure 2.
Hairpin–bisulfite PCR at FMR1 (after Miner et al. [20]).
The genomic sequence from both strands of the FMR1 promoter, including the first five of the analyzed analyzed CpG sites, is shown. A hairpin linker, here illustrated for hairpin I, is ligated to both strands of an individual DNA molecule prior to bisulfite conversion and PCR amplification. The bisulfite-conversion reaction converts cytosine, but not 5-methylcytosine (“me”), to uracil, thus preserving information on methylation patterns in genomic DNA. After PCR amplification, uracil residues will appear as thymine; a cytosine detected in the sequence of a PCR product therefore indicates methylation of that base in the genomic DNA.
Figure 3.
Three separate hairpin–bisulfite PCR assays for the FMR1 promoter.
Three hairpins were developed to analyze a total of 52 CpG dyads representing 104 CpGs on both strands of the FMR1 promoter. Small vertical lines depict the distribution of CpG dyads present within the core region of the human FMR1 promoter and the adjacent track of CGG repeats. Horizontal gray bars above the CpG plot indicate the location of binding sites for transcription factors and the position of the CGG repeat. Arrows depict three initiation sites for transcription of FMR1.
Table 1.
Summary of FMR1-promoter methylation in samples from males with FXS and from female controls.
Figure 4.
CpG site-specific methylation frequencies among hypermethylated epialleles.
CpG site-specific methylation frequencies among hypermethylated epialleles as observed in samples from males with FXS who express (open circles), or do not express (filled circles) FMR1. Data were pooled across hairpin assays I, II, and III. Vertical lines indicate the boundaries of the three different hairpins.
Figure 5.
Assessment of methylation mosaicism between the two strands of individual DNA molecules in samples from males with fragile X syndrome.
Data are presented separately for samples from FMR1 mRNA-expressing males (A), and from non-expressing males (B). Weighted linear regression was used to account for differences in the numbers of CpG sites captured by the three different hairpins. The different icons of this “sunflower” plot reflect the number of observed epialleles with a given density of methylation on the top and bottom strands. A single epiallele is represented as a dot, two epialleles with identical methylation densities as a line, three epialleles as a “three-petal” flower, and so on up to the maximum in our data set of nine molecules.