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

Yuh-Hwa Wang,Jack Griffith

doi:10.1074/jbc.271.38.22937

Abstract

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.

Highlights

In eukaryotic cells methylation of CpG dinucleotides by DNA methyltransferase can directly inhibit gene expression
The fragile X syndrome was named for the presence of a rare, folate-sensitive fragile site on the X chromosome (FRAXA) whose appearance correlates with the disease
Because the expression of the five fragile sites described above depends on the degree of methylation, it seemed important to examine the effects of DNA methylation on the ability of DNAs containing CCG or CCGNN repeats to assemble into chromatin

Summary

The abbreviations used are

MeCP-1, methyl-CpG-binding protein; FRAX, fragile X syndrome; bp, base pair(s); DHFR, dihydrofolate reductase. Because the expression of the five fragile sites described above depends on the degree of methylation, it seemed important to examine the effects of DNA methylation on the ability of DNAs containing CCG or CCGNN repeats to assemble into chromatin. We have used competitive nucleosome reconstitution/gel retardation assays to investigate this issue and show that methylation inhibits nucleosome formation 2-fold beyond the inherent exclusion due to the sequences themselves. This observation provides further support for a model in which nucleosome exclusion creates unstable chromatin at these fragile sites, which may further facilitate the binding of the MeCP-1 protein to inhibit transcription from this region

EXPERIMENTAL PROCEDURES

RESULTS

16 Ϯ 1 13 Ϯ 0

DISCUSSION