title: Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells.
abstract: BACKGROUND
  Fragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5' UTR of the FMR1 gene and subsequent DNA methylation of both the expanded sequence and the CpGs of the promoter region. This epigenetic change causes transcriptional silencing of the gene. We have previously demonstrated that 5-aza-2-deoxycytidine (5-azadC) treatment of FXS lymphoblastoid cell lines reactivates the FMR1 gene, concomitant with CpG sites demethylation, increased acetylation of histones H3 and H4 and methylation of lysine 4 on histone 3.
  
  
  RESULTS
  In order to check the specificity of the 5-azadC-induced DNA demethylation, now we performed bisulphite sequencing of the entire methylation boundary upstream the FMR1 promoter region, which is preserved in control wild-type cells. We did not observe any modification of the methylation boundary after treatment. Furthermore, methylation analysis by MS-MLPA of PWS/AS and BWS/SRS loci demonstrated that 5-azadC treatment has no demethylating effect on these regions. Genome-wide methylation analysis through Infinium 450K (Illumina) showed no significant enrichment of specific GO terms in differentially methylated regions after 5-azadC treatment. We also observed that reactivation of FMR1 transcription lasts up to a month after a 7-day treatment and that maximum levels of transcription are reached at 10-15 days after last administration of 5-azadC.
  
  
  CONCLUSIONS
  Taken together, these data demonstrate that the demethylating effect of 5-azadC on genomic DNA is not random, but rather restricted to specific regions, if not exclusively to the FMR1 promoter. Moreover, we showed that 5-azadC has a long-lasting reactivating effect on the mutant FMR1 gene.
authors.affiliation: Istituto di Medicina Genomica, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Tabolacci
authors.affiliation: Istituto di Medicina Genomica, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy.
authors.forename: Giorgia
authors.initials: G
authors.lastname: Mancano
authors.affiliation: Istituto di Medicina Genomica, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy.
authors.forename: Stella
authors.initials: S
authors.lastname: Lanni
authors.affiliation: Istituto di Medicina Genomica, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Palumbo
authors.affiliation: Istituto di Medicina Genomica, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Goracci
authors.affiliation: Department of Biology, Centre for Molecular Bioinformatics (CBM), University of Rome Tor Vergata, Rome, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Ferrè
authors.affiliation: Department of Biology, Centre for Molecular Bioinformatics (CBM), University of Rome Tor Vergata, Rome, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Helmer-Citterich
authors.affiliation: Istituto di Medicina Genomica, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Neri
keywords: 5-aza-2-deoxycytidine; DNA methylation; Epigenetic modifications; FMR1 gene; Fragile X syndrome; In vitro pharmacological demethylation; Whole methylation analysis