EPIGENETIC VARIATION ILLUSTRATED BY DNA METHYLATION PATTERNS OF THE FRAGILE-X GENE FMR1

Genomic methylation patterns of mammals can vary among individuals and are subject to dynamic changes during development. In order to gain a better understanding of this variation, we have analyzed patterns of cytosine methylation within a 200 bp region at the CpG island of the h uman FMR1 gene from leukocyte DNA, FMR1 is normally methylated during inactivation of the X chromosome in females and it is also methylated and inactivated upon expansion of CGG repeats in fragile-X syndrome. P atterns of methylation (epigenotypes) were determined by the sequencin g of bisulfite-treated alleles from normal males and females and allel es from a family of five brothers who are methylation mosaics and are affected to various degrees by the fragile-X syndrome. Our data indica te that: (i) methylation of individual CpG cytosines is strikingly var iable in hypermethylated epigenotypes obtained from a single individua l, suggesting that maintenance of cytosine methylation is a dynamic pr ocess; (ii) methylation of non-CpG cytosines in the region studied may occur but is rare; (iii) mosaicism of methylation in the analyzed fra gile-X males is remarkably similar to that found for the active X and inactive X alleles in normal females, suggesting that the methylation mosaicism of some fragile-X males reflects similar on and off states o f FMR1 expression that exist in normal females; (iv) hypermethylation is slightly more pronounced on fragile-X alleles than on normal inacti ve X alleles of females; (v) the general dichotomy of hypo-and hyperme thylated alleles persisted over the 5 year period that separated sampl ings of the fragile-X males; (vi) methylation variability was most pro nounced at a consensus binding sequence for the alpha-PAL transcriptio n factor, a sequence that may play a role in regulating expression of FMR1.