title: Role of CTCF protein in regulating FMR1 locus transcription.
abstract: Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, is caused by epigenetic silencing of the FMR1 gene, through expansion and methylation of a CGG triplet repeat (methylated full mutation). An antisense transcript (FMR1-AS1), starting from both promoter and intron 2 of the FMR1 gene, was demonstrated in transcriptionally active alleles, but not in silent FXS alleles. Moreover, a DNA methylation boundary, which is lost in FXS, was recently identified upstream of the FMR1 gene. Several nuclear proteins bind to this region, like the insulator protein CTCF. Here we demonstrate for the first time that rare unmethylated full mutation (UFM) alleles present the same boundary described in wild type (WT) alleles and that CTCF binds to this region, as well as to the FMR1 gene promoter, exon 1 and intron 2 binding sites. Contrariwise, DNA methylation prevents CTCF binding to FXS alleles. Drug-induced CpGs demethylation does not restore this binding. CTCF knock-down experiments clearly established that CTCF does not act as insulator at the active FMR1 locus, despite the presence of a CGG expansion. CTCF depletion induces heterochromatinic histone configuration of the FMR1 locus and results in reduction of FMR1 transcription, which however is not accompanied by spreading of DNA methylation towards the FMR1 promoter. CTCF depletion is also associated with FMR1-AS1 mRNA reduction. Antisense RNA, like sense transcript, is upregulated in UFM and absent in FXS cells and its splicing is correlated to that of the FMR1-mRNA. We conclude that CTCF has a complex role in regulating FMR1 expression, probably through the organization of chromatin loops between sense/antisense transcriptional regulatory regions, as suggested by bioinformatics analysis.
authors.affiliation: Istituto di Genetica Medica, Università Cattolica del S. Cuore, Rome, Italy.
authors.forename: Stella
authors.initials: S
authors.lastname: Lanni
authors.forename: Martina
authors.initials: M
authors.lastname: Goracci
authors.forename: Loredana
authors.initials: L
authors.lastname: Borrelli
authors.forename: Giorgia
authors.initials: G
authors.lastname: Mancano
authors.forename: Pietro
authors.initials: P
authors.lastname: Chiurazzi
authors.forename: Umberto
authors.initials: U
authors.lastname: Moscato
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Ferrè
authors.forename: Manuela
authors.initials: M
authors.lastname: Helmer-Citterich
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Tabolacci
authors.forename: Giovanni
authors.initials: G
authors.lastname: Neri
mesh_terms: D001665:Binding Sites; D000076246:CCCTC-Binding Factor; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D044127:Epigenesis, Genetic; D005091:Exons / Q000235:genetics; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics* / Q000378:metabolism; D005600:Fragile X Syndrome / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D007438:Introns / Q000235:genetics; D009154:Mutation; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D000076246:CCCTC-Binding Factor; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D051860:Fragile X Messenger Ribonucleoprotein 1; D012097:Repressor Proteins; C524946:CTCF protein, Drosophila; C000619697:CTCF protein, human; C467328:FMR1 protein, Drosophila