title: CGG-repeat dynamics andFMR1gene silencing in fragile X syndrome stem cells and stem cell-derived neurons.
abstract: Fragile X syndrome (FXS), a common cause of intellectual disability and autism, results from the expansion of a CGG-repeat tract in the 5' untranslated region of theFMR1gene to >200 repeats. Such expanded alleles, known as full mutation (FM) alleles, are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP, a protein important for learning and memory. The timing of repeat expansion andFMR1gene silencing is controversial.
  
  
  
  We monitored the repeat size and methylation status ofFMR1alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing, and a quantitative methylation-specific PCR for the analysis ofFMR1promoter methylation. TheFMR1mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with theFMR1gene in FXS ESCs.
  
  
  
  We show here that whileFMR1gene silencing can be seen in FXS embryonic stem cells (ESCs), some silenced alleles contract and when the repeat number drops below ~400, DNA methylation erodes, even when the repeat number remains >200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore, there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation.
  
  
  
  Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo, (ii) large unmethylated alleles may be deleterious in stem cells, (iii) methylation can occur on alleles with >400 repeats very early in embryogenesis, and (iv) expansion and contraction may occur by different mechanisms. Our data also suggest that the threshold for stable methylation of FM alleles may be higher than previously thought. A higher threshold might explain why some carriers of FM alleles escape methylation. It may also provide a simple explanation for why silencing has not been observed in mouse models with >200 repeats.
authors.affiliation: Section on Gene Structure and Disease, Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD USA.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Section on Gene Structure and Disease, Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD USA.
authors.forename: Daman
authors.identifier: 0000-0001-6508-4530
authors.initials: D
authors.lastname: Kumari
authors.affiliation: Section on Gene Structure and Disease, Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD USA ; Present Address: Laboratory of Genome Integrity, National Cancer Institute, Bethesda, MD USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Sciascia
authors.affiliation: Section on Gene Structure and Disease, Laboratory of Molecular and Cellular 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: D020121:5' Untranslated Regions; D000483:Alleles; D002454:Cell Differentiation; D002460:Cell Line; D019175:DNA Methylation; D053595:Embryonic Stem Cells / Q000378:metabolism* / Q000473:pathology; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics* / Q000378:metabolism; D005600:Fragile X Syndrome / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020868:Gene Silencing; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism / Q000473:pathology; D008297:Male; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D061251:Primary Cell Culture; D013997:Time Factors; D019680:Trinucleotide Repeat Expansion
chemical_list: D020121:5' Untranslated Regions; D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human
keywords: Fragile X syndrome; Repeat contractions; Repeat expansion mutation; Repeat-mediated gene silencing; Stem cells