Differential epigenetic modifications in the FMR1 gene of the fragile X syndrome after reactivating pharmacological treatments.
The fragile X syndrome is caused by a >200 CGG repeat expansion within the FMR1 gene promoter, with consequent DNA hypermethylation and inactivation of its expression. To further clarify the mechanisms that suppress the activity of the mutant gene and the conditions that may permit its reactivation, we investigated the acetylation and methylation status of three different regions of the FMR1 gene (promoter, exon 1 and exon 16) of three fragile X cell lines, using a chromatin immunoprecipitation (ChIP) assay with antibodies against acetylated-H3/H4 histones and against dimethylated lysine residues K4 and K9 of histone H3 (H3-K4 and H3-K9). We then coupled the ChIP assay with real-time PCR, obtaining absolute quantification of immunoprecipitated chromatin. Basal levels of histone acetylation and H3-K4 methylation were much higher in transcriptionally active wild-type controls than in inactive fragile X cell lines. Treatment of fragile X cell lines with the DNA demethylating drug 5-aza-2-deoxycytidine (5-azadC), known to reactivate the FMR1 gene, induced a decrease of H3-K9 methylation, an increase of H3 and H4 acetylation and an increase of H3-K4 methylation. Treatment with acetyl-L-carnitine (ALC), a compound that reduces the in vitro expression of the FRAXA fragile site without affecting DNA methylation, caused an increase of H3 and H4 acetylation. However, H3-K4 methylation remained extremely low, in accordance with the observation that ALC alone does not reactivate the FMR1 gene. Our experiments indicate that H3-K4 methylation and DNA demethylation are the main epigenetic switches activating the expression of the FMR1 gene, with histone acetylation playing an ancillary role.
Tabolacci
Elisabetta
E
Istituto di Genetica Medica, Università Cattolica, Rome, Italy.
Pietrobono
Roberta
R
Moscato
Umberto
U
Oostra
Ben A
BA
Chiurazzi
Pietro
P
Neri
Giovanni
G
D000107:Acetylation; D000108:Acetylcarnitine / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D044127:Epigenesis, Genetic / Q000187:drug effects; D005091:Exons / Q000187:drug effects; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics*; D006657:Histones / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008297:Male; D009419:Nerve Tissue Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic / Q000187:drug effects; D016601:RNA-Binding Proteins / Q000235:genetics*
D000108:Acetylcarnitine; D001374:Azacitidine; D000077209:Decitabine; D051860:Fragile X Messenger Ribonucleoprotein 1; D006657:Histones; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; C497683:FMR1 protein, human