Nucleic Acids Research, 2002, Vol. 30, No. 14 3278-3285
© 2002 Oxford University Press
Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine
Istituto di Genetica Medica, Università Cattolica, and Centro Ricerche per la Disabilità Mentale e Motoria, Associazione Anni Verdi, Largo F. Vito 1, 00168 Rome, Italy, 1 Department of Clinical Genetics and Center for Biomedical Genetics, Erasmus University, PO Box 1738, DR 3000 Rotterdam, The Netherlands and 2 Dipartimento di Scienze Pediatriche Mediche e Chirurgiche, Policlinico Universitario, Messina, Italy
*To whom correspondence should be addressed. Tel: +39 06 305 4449; Fax: +39 06 305 0031; Email: gneri{at}rm.unicatt.it
In fragile X syndrome, hypermethylation of the expanded CGG repeat and of the upstream promoter leads to transcriptional silencing of the FMR1 gene. Absence of the FMR1 protein results in mental retardation. We previously proved that treatment with 5-azadeoxycytidine (5-azadC) of fragile X cell lines results in reactivation of the FMR1 gene. We now show that this treatment causes passive demethylation of the FMR1 gene promoter. We employed the bisulfite-sequencing technique to detect the methylation status of individual CpG sites in the entire promoter region, upstream of the CGG repeat. Lymphoblastoid cell lines of fragile X males with full mutations of different sizes were tested before and after treatment with 5-azadC at various time points. We observed that individual cells are either completely unmethylated or not, with few relevant exceptions. We also investigated the extent of methylation in the full mutation (CGG repeat) itself by Southern blot analysis after digestion with methylation-sensitive enzymes Fnu4HI and McrBC and found that the CGG repeat remains at least partially methylated in many cells with a demethylated promoter. This may explain the quantitative discrepancy between the large extent of promoter demethylation and the limited levels of FMR1 transcriptional reactivation estimated by quantitative real-time fluorescent RT–PCR analysis.
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