A Distinct DNA-Methylation Boundary in the 5′- Upstream Sequence of the FMR1 Promoter Binds Nuclear Proteins and Is Lost in Fragile X Syndrome
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PROVIDER: S-EPMC2801757 | biostudies-literature | 2010 Jan
REPOSITORIES: biostudies-literature
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Project description:We have discovered a distinct DNA-methylation boundary at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene. This boundary, identified by bisulfite sequencing, is present in all human cell lines and cell types, irrespective of age, gender, and developmental stage. The same boundary is found also in different mouse tissues, although sequence homology between human and mouse in this region is only 46.7%. This boundary sequence, in both the unmethylated and the CpG-methylated modes, binds specifically to nuclear proteins from human cells. We interpret this boundary as carrying a specific chromatin structure that delineates a hypermethylated area in the genome from the unmethylated FMR1 promoter and protecting it from the spreading of DNA methylation. In individuals with the fragile X syndrome (FRAXA), the methylation boundary is lost; methylation has penetrated into the FMR1 promoter and inactivated the FMR1 gene. In one FRAXA genome, the upstream terminus of the methylation boundary region exhibits decreased methylation as compared to that of healthy individuals. This finding suggests changes in nucleotide sequence and chromatin structure in the boundary region of this FRAXA individual. In the completely de novo methylated FMR1 promoter, there are isolated unmethylated CpG dinucleotides that are, however, not found when the FMR1 promoter and upstream sequences are methylated in vitro with the bacterial M-SssI DNA methyltransferase. They may arise during de novo methylation only in DNA that is organized in chromatin and be due to the binding of specific proteins.
| S-EPMC2775827 | biostudies-literature
Project description:This study characterizes the DNA methylation patterns specific to fragile X syndrome (FXS) with a full mutation (FM > 200 CGGs), premutation (PM 55-199 CGGs), and X inactivation in blood and brain tissues at the 3' boundary of the FMR1 promoter. Blood was analyzed from 95 controls and 462 individuals (32% males) with FM and PM alleles. Brain tissues (62% males) were analyzed from 12 controls and 4 with FXS. There was a significant increase in intron 1 methylation, extending to a newly defined 3' epigenetic boundary in the FM compared with that in the control and PM groups (p < 0.0001), and this was consistent between the blood and brain tissues. A distinct intron 2 site showed a significant decrease in methylation for the FXS groups compared with the controls in both sexes (p < 0.01). In all female groups, most intron 1 (but not intron 2 sites) were sensitive to X inactivation. In all PM groups, methylation at the 3' epigenetic boundary and the proximal sites was significantly decreased compared with that in the control and FM groups (p < 0.0001). In conclusion, abnormal FMR1 intron 1 and 2 methylation that was sensitive to X inactivation in the blood and brain tissues provided a novel avenue for the detection of PM and FM alleles through DNA methylation analysis.
| S-EPMC10342127 | biostudies-literature
Project description:Variability among individuals in the severity of fragile X syndrome (FXS) is influenced by epigenetic methylation mosaicism, which may also be common in other complex disorders. The epigenetic signal of dense promoter DNA methylation is usually associated with gene silencing, as was initially reported for FMR1 alleles in individuals with FXS. A paradox arose when significant levels of FMR1 mRNA were reported for some males with FXS who had been reported to have predominately methylated alleles. We have used hairpin-bisufite PCR, validated with molecular batch-stamps and barcodes, to collect and assess double-stranded DNA methylation patterns from these previously studied males. These patterns enable us to distinguish among three possible forms of methylation mosaicism, any one of which could explain FMR1 expression in these males. Our data indicate that cryptic inter-cell mosaicism in DNA methylation can account for the presence of FMR1 mRNA in some individuals with FXS.
| S-EPMC3166088 | biostudies-literature
Project description:Fragile X syndrome (FXS) results from a repeat expansion mutation near the FMR1 gene promoter and is the most common form of heritable intellectual disability and autism. Full mutations larger than 200 CGG repeats trigger FMR1 heterochromatinization and loss of gene expression, which is primarily responsible for the pathological features of FXS . In contrast, smaller pre-mutations of 55–200 CGG are associated with FMR1 overexpression and Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative condition. While the role of 5-methylcytosine (5mC) in FMR1 gene silencing has been studied extensively, the role of 5-hydroxymethylation (5hmC), a newly discovered epigenetic mark produced through active DNA demethylation, has not been previously investigated in FXS neurons. Here, we used two complementary epigenetic assays, 5hmC sensitive restriction digest and ten-eleven translocation-assisted bisulfite pyrosequencing, to quantify FMR1 5mC and 5hmC levels. We observed increased levels of 5hmC at the FMR1 promoter in FXS patient brains with full-mutations relative to pre-mutation carriers and unaffected controls. In addition, we found that 5hmC enrichment at the FMR1 locus in FXS cells is specific to neurons by utilizing a nuclei sorting technique to separate neuronal and glial DNA fractions from post-mortem brain tissues. This FMR1 5hmC enrichment was not present in cellular models of FXS including fibroblasts, lymphocytes and reprogrammed neurons, indicating they do not fully recapitulate this epigenetic feature of disease. Future studies could investigate the potential to leverage this epigenetic pathway to restore FMR1 expression and discern whether levels of 5hmC correlate with phenotypic severity.
| S-EPMC6078593 | biostudies-literature
Project description:Among the inherited causes of intellectual disability and autism, Fragile X syndrome (FXS) is the most frequent form, for which there is currently no cure. In most FXS patients, the FMR1 gene is epigenetically inactivated following the expansion over 200 triplets of a CGG repeat (FM: full mutation). FMR1 encodes the Fragile X Mental Retardation Protein (FMRP), which binds several mRNAs, mainly in the brain. When the FM becomes methylated at 10-12 weeks of gestation, the FMR1 gene is transcriptionally silent. The molecular mechanisms involved in the epigenetic silencing are not fully elucidated. Among FXS families, there is a rare occurrence of males carrying a FM, which remains active because it is not methylated, thus ensuring enough FMRPs to allow for an intellectual development within normal range. Which mechanisms are responsible for sparing these individuals from being affected by FXS? In order to answer this critical question, which may have possible implications for FXS therapy, several potential epigenetic mechanisms have been described. Here, we focus on current knowledge about the role of DNA methylation and other epigenetic modifications in FMR1 gene silencing.
| S-EPMC7920310 | biostudies-literature
Project description:Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.
| S-EPMC6375087 | biostudies-literature
Project description:Fragile X syndrome (FXS) is a common form of inherited intellectual disability caused by an expansion of CGG repeats located in the 5' untranslated region (UTR) of the FMR1 gene, which leads to hypermethylation and silencing of this locus. Although a dramatic increase in DNA methylation of the FMR1 full mutation allele is well documented, the extent to which these changes affect DNA methylation throughout the rest of the genome has gone unexplored.Here we examined genome-wide methylation in both peripheral blood (N = 62) and induced pluripotent stem cells (iPSCs; N = 10) from FXS individuals and controls.We not only found the expected significant DNA methylation differences in the FMR1 promoter and 5' UTR, we also saw that these changes inverse in the FMR1 gene body. Importantly, we found no other differentially methylated loci throughout the remainder of the genome, indicating the aberrant methylation of FMR1 in FXS is locus-specific.This study provides a comprehensive methylation profile of FXS and helps refine our understanding of the mechanisms behind FMR1 silencing.
| S-EPMC3599197 | biostudies-literature
Project description:ObjectiveAlmost all patients with Fragile X Syndrome (FXS) exhibit a CGG repeat expansion (full mutation) in the Fragile Mental Retardation 1 gene (FMR1). Here, the authors report five unrelated males with FXS harboring a somatic full mutation/deletion mosaicism.MethodsMutational profiles were only elucidated by using a combination of molecular approaches (CGG-based PCR, Sanger sequencing, MS-MLPA, Southern blot and mPCR).ResultsFour patients exhibited small deletions encompassing the CGG repeats tract and flanking regions, whereas the remaining had a larger deletion comprising at least exon 1 and part of intron 1 of FMR1 gene. The presence of a 2-3 base pairs microhomology in proximal and distal non-recurrent breakpoints without scars supports the involvement of microhomology mediated induced repair (MMBIR) mechanism in three small deletions.ConclusionThe authors data highlights the importance of using different research methods to elucidate atypical FXS mutational profiles, which are clinically undistinguishable and may have been underestimated.
| S-EPMC4956488 | biostudies-literature
Project description:Fragile X syndrome, the most common form of inherited mental retardation in males, arises when the normally stable 5 to 50 CGG repeats in the 5' untranslated region of the fragile X mental retardation protein 1 (FMR1) gene expand to over 200, leading to DNA methylation and silencing of the FMR1 promoter. Although the events that trigger local CGG expansion remain unknown, the stability of trinucleotide repeat tracts is affected by their position relative to an origin of DNA replication in model systems. Origins of DNA replication in the FMR1 locus have not yet been described. Here, we report an origin of replication adjacent to the FMR1 promoter and CGG repeats that was identified by scanning a 35-kb region. Prereplication proteins Orc3p and Mcm4p bind to chromatin in the FMR1 initiation region in vivo. The position of the FMR1 origin relative to the CGG repeats is consistent with a role in repeat maintenance. The FMR1 origin is active in transformed cell lines, fibroblasts from healthy individuals, fibroblasts from patients with fragile X syndrome, and fetal cells as early as 8 weeks old. The potential role of the FMR1 origin in CGG tract instability is discussed.
| S-EPMC1800797 | biostudies-literature
Project description:PurposeFragile X syndrome is associated with the expansion of CGG trinucleotide repeats and subsequent methylation of the FMR1 gene. Molecular diagnosis of fragile X currently requires Southern blot analysis to assess methylation. This study describes the evaluation of a polymerase chain reaction-only workflow for the determination of methylation status across a broad range of FMR1 genotypes in male and female specimens.MethodsWe evaluated a novel method that combines allele-specific methylation polymerase chain reaction and capillary electrophoresis with eight cell line and 80 clinical samples, including 39 full mutations. Methylation status was determined using a three-step workflow: (1) differential treatment of genomic DNA using a methylation-sensitive restriction enzyme; (2) polymerase chain reaction with two sets of dye-tagged primers; and (3) amplicon sizing by capillary electrophoresis. All samples were analyzed by both methylation polymerase chain reaction and Southern blot analysis.ResultsFMR1 methylation status and CGG repeat sizing were accurately and reproducibly determined in a set of methylation controls and genomic DNA samples representing a spectrum of CGG repeat lengths and methylation states. Moreover, methylation polymerase chain reaction revealed allele-specific methylation patterns in premutation alleles that were unobtainable using Southern blot analysis.ConclusionsMethylation polymerase chain reaction enabled high throughput, high resolution, and semiquantitative methylation assessments of FMR1 alleles, as well as determinations of CGG repeat length. Results for all samples were concordant with corresponding Southern blot analyses. As a result, this study presents a polymerase chain reaction-based method for comprehensive FMR1 analysis. In addition, the identification of novel methylation mosaic patterns revealed after polymerase chain reaction and capillary electrophoresis may be relevant to several FMR1 disorders.
| S-EPMC4043840 | biostudies-literature