Genome-wide demethylation promotes triplet repeat instability independently of homologous recombination.
Trinucleotide repeat instability is intrinsic to a family of human neurodegenerative diseases. The mechanism leading to repeat length variation is unclear. We previously showed that treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) dramatically increases triplet repeat instability in mammalian cells. Based on previous reports that demethylation increases homologous recombination (HR), and our own observations that HR destabilizes triplet repeats, we hypothesized that demethylation alters repeat stability by stimulating HR. Here, we test that hypothesis at the adenosine phosphoribosyl transferase (Aprt) locus in CHO cells, where CpG demethylation and HR have both been shown to increase CAG repeat instability. We find that the rate of HR at the Aprt locus is not altered by demethylation. The spectrum of recombinants, however, was shifted from the usual 6:1 ratio of conversions to crossovers to more equal proportions in 5-aza-CdR-treated cells. The subtle influences of demethylation on HR at the Aprt locus are not sufficient to account for its dramatic effects on repeat instability. We conclude that 5-aza-CdR promotes triplet repeat instability independently of HR.
Dion
Vincent
V
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Lin
Yunfu
Y
Price
Brandee A
BA
Fyffe
Sharyl L
SL
Seluanov
Andrei
A
Gorbunova
Vera
V
Wilson
John H
JH
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics; D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019175:DNA Methylation; D017931:DNA Primers / Q000235:genetics; D000077209:Decitabine; D042822:Genomic Instability / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018911:Trinucleotide Repeats / Q000235:genetics*
D017931:DNA Primers; D000077209:Decitabine; D000228:Adenine Phosphoribosyltransferase; D001374:Azacitidine