Small Molecules Targeting H3K9 Methylation Prevent Silencing of Reactivated FMR1 Alleles in Fragile X Syndrome Patient Derived Cells

In fragile X syndrome (FXS), expansion of a CGG repeat tract in the 5′-untranslated region of the FMR1 gene to >200 repeats causes transcriptional silencing by inducing heterochromatin formation. Understanding the mechanism of FMR1 silencing is important as gene reactivation is a potential treatment approach for FXS. To date, only the DNA demethylating drug 5-azadeoxycytidine (AZA) has proved effective at gene reactivation; however, this drug is toxic. The repressive H3K9 methylation mark is enriched on the FMR1 gene in FXS patient cells and is thus a potential druggable target. However, its contribution to the silencing process is unclear. Here, we studied the effect of small molecule inhibitors of H3K9 methylation on FMR1 expression in FXS patient cells. Chaetocin showed a small effect on FMR1 gene reactivation and a synergistic effect on FMR1 mRNA levels when used in combination with AZA. Additionally, chaetocin, BIX01294 and 3-Deazaneplanocin A (DZNep) were able to significantly delay the re-silencing of AZA-reactivated FMR1 alleles. These data are consistent with the idea that H3K9 methylation precedes DNA methylation and that removal of DNA methylation is necessary to see the optimal effect of histone methyl-transferase (HMT) inhibitors on FMR1 gene expression. Nonetheless, our data also show that drugs targeting repressive H3K9 methylation marks are able to produce sustained reactivation of the FMR1 gene after a single dose of AZA.

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DNA Methylation, Mechanisms of FMR1 Inactivation and Therapeutic Perspectives for Fragile X Syndrome

Biomolecules ◽

10.3390/biom11020296 ◽

2021 ◽

Vol 11 (2) ◽

pp. 296

Author(s):

Veronica Nobile ◽

Cecilia Pucci ◽

Pietro Chiurazzi ◽

Giovanni Neri ◽

Elisabetta Tabolacci

Keyword(s):

Dna Methylation ◽

Fragile X Syndrome ◽

Intellectual Development ◽

Molecular Mechanisms ◽

Current Knowledge ◽

Fragile X ◽

Fmr1 Gene ◽

Critical Question ◽

Cgg Repeat

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.

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Faculty Opinions recommendation of Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732704934.793544461 ◽

2018 ◽

Author(s):

Laurent Villard

Keyword(s):

Dna Methylation ◽

Fragile X Syndrome ◽

Fragile X ◽

Fmr1 Gene

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Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells

10.1101/286732 ◽

2018 ◽

Author(s):

Jill M. Haenfler ◽

Geena Skariah ◽

Caitlin M. Rodriguez ◽

Andre Monteiro da Rocha ◽

Jack M. Parent ◽

...

Keyword(s):

Mrna Expression ◽

Fragile X Syndrome ◽

Fragile X ◽

Embryonic Stem ◽

Transcriptional Silencing ◽

Hesc Line ◽

Human Patient ◽

Cgg Repeat ◽

Heterochromatin Formation ◽

Fmr1 Mrna

ABSTRACTFragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5’ untranslated region of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing, and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRP’s pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the CRISPR/dCAS9 system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCAS9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 mRNA expression despite promoter and repeat methylation. These studies demonstrate that FMR1 mRNA expression can be selectively reactivated in human patient cells, creating a pathway forward for therapeutic development in Fragile X Syndrome.

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DNA Methylation at Birth Predicts Intellectual Functioning and Autism Features in Children with Fragile X Syndrome

International Journal of Molecular Sciences ◽

10.3390/ijms21207735 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7735

Author(s):

Claudine M Kraan ◽

Emma K Baker ◽

Marta Arpone ◽

Minh Bui ◽

Ling Ling ◽

...

Keyword(s):

Dna Methylation ◽

Fragile X Syndrome ◽

Fragile X ◽

Single Gene ◽

Intellectual Functioning ◽

Dried Blood Spots ◽

Reference Ranges ◽

Mrna Levels ◽

Blood Spots ◽

Fmr1 Mrna

Fragile X syndrome (FXS) is a leading single-gene cause of intellectual disability (ID) with autism features. This study analysed diagnostic and prognostic utility of the Fragile X-Related Epigenetic Element 2 DNA methylation (FREE2m) assessed by Methylation Specific-Quantitative Melt Analysis and the EpiTYPER system, in retrospectively retrieved newborn blood spots (NBS) and newly created dried blood spots (DBS) from 65 children with FXS (~2–17 years). A further 168 NBS from infants from the general population were used to establish control reference ranges, in both sexes. FREE2m analysis showed sensitivity and specificity approaching 100%. In FXS males, NBS FREE2m strongly correlated with intellectual functioning and autism features, however associations were not as strong for FXS females. Fragile X mental retardation 1 gene (FMR1) mRNA levels in blood were correlated with FREE2m in both NBS and DBS, for both sexes. In females, DNAm was significantly increased at birth with a decrease in childhood. The findings support the use of FREE2m analysis in newborns for screening, diagnostic and prognostic testing in FXS.

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A Novel Polymorphism in the FMR1 Gene: Implications for Clinical Testing of Fragile X Syndrome

Archives of Pathology & Laboratory Medicine ◽

10.5858/2008-132-95-anpitf ◽

2008 ◽

Vol 132 (1) ◽

pp. 95-98

Author(s):

Bharat Thyagarajan ◽

Matthew Bower ◽

Michael Berger ◽

Sidney Jones ◽

Michelle Dolan ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Mental Retardation ◽

Fragile X Syndrome ◽

Southern Blot ◽

Trinucleotide Repeat ◽

Fragile X ◽

Fmr1 Gene ◽

Chain Reaction ◽

Cgg Repeat ◽

Polymerase Chain

Abstract Fragile X syndrome is the most common cause of inherited mental retardation among males. In most cases, the molecular basis of fragile X syndrome is the expansion and subsequent methylation of a CGG trinucleotide repeat in the 5′ untranslated region of the fragile X mental retardation 1 (FMR1) gene. Laboratory diagnosis usually relies on a combination of Southern blot and polymerase chain reaction analyses. In this case report we describe an unusual Southern blot result in a patient who presented with developmental delay and had a normal CGG repeat number by polymerase chain reaction analysis. Further investigation revealed a novel G3310C transversion in the FMR1 gene resulting in a new recognition site for the BssHII restriction enzyme. This novel restriction site could potentially mimic a partial deletion of the FMR1 gene on Southern blot analysis and thus represents a possible pitfall in the diagnosis of fragile X syndrome.

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Reversion to Normal of FMR1 Expanded Alleles: A Rare Event in Two Independent Fragile X Syndrome Families

Genes ◽

10.3390/genes11030248 ◽

2020 ◽

Vol 11 (3) ◽

pp. 248

Author(s):

Elisabetta Tabolacci ◽

Roberta Pietrobono ◽

Giulia Maneri ◽

Laura Remondini ◽

Veronica Nobile ◽

...

Keyword(s):

Fragile X Syndrome ◽

Binding Sites ◽

Molecular Mechanisms ◽

Fragile X ◽

Rare Event ◽

The Other ◽

Fmr1 Gene ◽

Full Mutation ◽

Cgg Repeat ◽

Agg Interruptions

Fragile X syndrome (FXS) is mostly due to the expansion and subsequent methylation of a polymorphic CGG repeat in the 5’ UTR of the FMR1 gene. Full mutation alleles (FM) have more than 200 repeats and result in FMR1 gene silencing and FXS. FMs arise from maternal premutations (PM) that have 56–200 CGGs; contractions of a maternal PM or FM are rare. Here, we describe two unaffected boys in two independent FXS families who inherited a non-mosaic allele in the normal and intermediate range, respectively, from their mothers who are carriers of an expanded CGG allele. The first boy inherited a 51 CGG allele (without AGG interruptions) from his mother, who carries a PM allele with 72 CGGs. The other boy inherited from his FM mother an unusual allele with 19 CGGs resulting from a deletion, removing 85 bp upstream of the CGG repeat. Given that transcription of the deleted allele was found to be preserved, we assume that the binding sites for FMR1 transcription factors are excluded from the deletion. Such unusual cases resulting in non-mosaic reduction of maternal CGG expansions may help to clarify the molecular mechanisms underlying the instability of the FMR1 gene.

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