title: Sustained expression of FMR1 mRNA from reactivated fragile X syndrome alleles after treatment with small molecules that prevent trimethylation of H3K27.
abstract: Expansion of a CGG-repeat tract in the 5'-untranslated region of the FMR1 gene to >200 repeats results in epigenetic silencing of the gene by a mechanism that is still unknown. FMR1 gene silencing results in fragile X syndrome (FXS), the most common heritable cause of intellectual disability. We have previously shown that reactivation of the FMR1 gene in FXS cells with 5-azadeoxycytidine (AZA) leads to the transient recruitment of EZH2, the polycomb repressive complex 2 (PRC2) component responsible for H3K27 trimethylation, and that this recruitment depends on the presence of the FMR1 transcript. However, whether H3K27 trimethylation was essential for FMR1 re-silencing was not known. We show here that EZH2 inhibitors increased FMR1 expression and significantly delayed re-silencing of the FMR1 gene in AZA-treated FXS cells. This delay occurred despite the fact that EZH2 inhibition did not prevent the return of DNA methylation. Treatment with compound 1a, a small molecule that targets CGG-repeats in the FMR1 mRNA, also resulted in sustained expression of the FMR1 gene in AZA-treated cells. This effect of 1a was also associated with a decrease in the levels of H3K27 trimethylation but not DNA methylation. Thus, our data show that EZH2 plays a critical role in the FMR1 gene silencing process and that its inhibition can prolong expression of the FMR1 gene even in the presence of its transcript.
authors.affiliation: Section on Genomic Structure and Function, Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA damank@helix.nih.gov.
authors.forename: Daman
authors.initials: D
authors.lastname: Kumari
authors.affiliation: Section on Genomic Structure and Function, Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Usdin
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D019175:DNA Methylation; D000077209:Decitabine; D000071221:Enhancer of Zeste Homolog 2 Protein / Q000378:metabolism*; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008297:Male; D008745:Methylation / Q000187:drug effects; D018895:Microsatellite Repeats / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology*; D015854:Up-Regulation
chemical_list: D001374:Azacitidine; D000077209:Decitabine; D000071221:Enhancer of Zeste Homolog 2 Protein; D051860:Fragile X Messenger Ribonucleoprotein 1; D006657:Histones; D054852:Small Molecule Libraries; C497683:FMR1 protein, human; C498409:EZH2 protein, human