In vivo footprinting analysis of the FMR1 gene: Proposals concerning gene regulation in high-functioning males

Schwemmle, Sabine

doi:10.1002/(sici)1096-8628(19990528)84:3<266::aid-ajmg19>3.0.co;2-g

Cited by 5 publications

(2 citation statements)

References 8 publications

(5 reference statements)

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“…These individuals have no global impairment of DNA methylation (16) and are intellectually normal or`high functioning' males. Schwemmle (29) con®rmed the presence of footprints in one such individual, suggesting normal transcription. Probably the unmethylated CGG expansion does not impede transcription per se, neither in the premutation (15) nor in the full mutation range (30,31).…”

Section: Discussionmentioning

confidence: 95%

Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine

Pietrobono¹,

Pomponi²,

Tabolacci³

et al. 2002

Nucleic Acids Research

107

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In fragile X syndrome, hypermethylation of the expanded CGG repeat and of the upstream promoter leads to transcriptional silencing of the FMR1 gene. Absence of the FMR1 protein results in mental retardation. We previously proved that treatment with 5-azadeoxycytidine (5-azadC) of fragile X cell lines results in reactivation of the FMR1 gene. We now show that this treatment causes passive demethylation of the FMR1 gene promoter. We employed the bisulfite-sequencing technique to detect the methylation status of individual CpG sites in the entire promoter region, upstream of the CGG repeat. Lymphoblastoid cell lines of fragile X males with full mutations of different sizes were tested before and after treatment with 5-azadC at various time points. We observed that individual cells are either completely unmethylated or not, with few relevant exceptions. We also investigated the extent of methylation in the full mutation (CGG repeat) itself by Southern blot analysis after digestion with methylation-sensitive enzymes Fnu4HI and McrBC and found that the CGG repeat remains at least partially methylated in many cells with a demethylated promoter. This may explain the quantitative discrepancy between the large extent of promoter demethylation and the limited levels of FMR1 transcriptional reactivation estimated by quantitative real-time fluorescent RT-PCR analysis.

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Section: Discussionmentioning

confidence: 95%

Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine

Pietrobono¹,

Pomponi²,

Tabolacci³

et al. 2002

Nucleic Acids Research

107

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“…DNA footprinting studies have identified four footprints in the FMR1 gene promoter that correspond to the consensus binding site of four transcription factors, α-PAL/NRF1, Sp1, H4TF1/Sp1-like, and c-myc. These footprints are found in FMR1 in several different cell types derived from normal individuals but absent in FMR1 of cells derived from FXS individuals (Drouin et al, 1997; Schwemmle, 1999; Schwemmle et al, 1997). Therefore FMR1 gene repression is correlated with the absence of transcription factor binding.…”

Section: Fxs Is Caused By a Human Specific Mutationmentioning

confidence: 90%