Methylation of Expanded CCG Triplet Repeat DNA from Fragile X Syndrome Patients Enhances Nucleosome Exclusion

Long tracts of CCG trinucleotide or CCGNN pentanucleotide repeats in DNA have previously been shown to resist assembly into nucleosomes. This may provide a molecular explanation for the nature of certain rare, folate-sensitive fragile sites in human chromosomes that contain expanded CCG triplet tracts. Further, it is known that methylation of CpG dinucleotides at or near these fragile sites enhances the fragile phenotype. Here DNAs containing 76 tandem CCG triplets or 48 CCGNN pentanucleotide repeats were methylated with SssI methylase at three different levels of methylation. Using competitive nucleosome reconstitution/gel shift assays, the ability of these DNAs and a mixed sequence DNA from the pUC19 plasmid were compared in their ability to assemble into nucleosomes. DNA methylation had no significant effect on nucleosome formation over the pUC 19 fragment. However, the highly methylated DNAs containing 76 CCG triplets or 48 CCGNN pentanucleotide repeats were 2.0 +/- 0. 2-fold and 2.1 +/- 0.3-fold less efficient in nucleosome assembly than the respective unmethylated forms, and 4.4 +/- 0.4-fold and 12. 6 +/- 1.6-fold less efficient than a pUC19 fragment of similar length.