Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine.
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.
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.
Roberta
R
Pietrobono
Maria Grazia
MG
Pomponi
Elisabetta
E
Tabolacci
Ben
B
Oostra
Pietro
P
Chiurazzi
Giovanni
G
Neri
D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D018899:CpG Islands / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D017422:Sequence Analysis, DNA
D001374:Azacitidine; D004247:DNA; D015254:DNA Modification Methylases; D000077209:Decitabine; D004791:Enzyme Inhibitors; D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C497683:FMR1 protein, human