Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells

Background: Fragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5\u2032 UTR of the FMR1 gene and subsequent DNA methylation of both the expanded sequence and the CpGs of the promoter region. This epigenetic change causes transcriptional silencing of the gene. We have previously demonstrated that 5-aza-2-deoxycytidine (5-azadC) treatment of FXS lymphoblastoid cell lines reactivates the FMR1 gene, concomitant with CpG sites demethylation, increased acetylation of histones H3 and H4 and methylation of lysine 4 on histone 3. Results: In order to check the specificity of the 5-azadC-induced DNA demethylation, now we performed bisulphite sequencing of the entire methylation boundary upstream the FMR1 promoter region, which is preserved in control wild-type cells. We did not observe any modification of the methylation boundary after treatment. Furthermore, methylation analysis by MS-MLPA of PWS/AS and BWS/SRS loci demonstrated that 5-azadC treatment has no demethylating effect on these regions. Genome-wide methylation analysis through Infinium 450K (Illumina) showed no significant enrichment of specific GO terms in differentially methylated regions after 5-azadC treatment. We also observed that reactivation of FMR1 transcription lasts up to a month after a 7-day treatment and that maximum levels of transcription are reached at 10-15 days after last administration of 5-azadC. Conclusions: Taken together, these data demonstrate that the demethylating effect of 5-azadC on genomic DNA is not random, but rather restricted to specific regions, if not exclusively to the FMR1 promoter. Moreover, we showed that 5-azadC has a long-lasting reactivating effect on the mutant FMR1 gene

FAN1, a DNA Repair Nuclease, as a Modifier of Repeat Expansion Disorders

FAN1 encodes a DNA repair nuclease. Genetic deficiencies, copy number variants, and single nucleotide variants of FAN1 have been linked to karyomegalic interstitial nephritis, 15q13.3 microdeletion/microduplication syndrome (autism, schizophrenia, and epilepsy), cancer, and most recently repeat expansion diseases. For seven CAG repeat expansion diseases (Huntington’s disease (HD) and certain spinocerebellar ataxias), modification of age of onset is linked to variants of specific DNA repair proteins. FAN1 variants are the strongest modifiers. Non-coding disease-delaying FAN1 variants and coding disease-hastening variants (p.R507H and p.R377W) are known, where the former may lead to increased FAN1 levels and the latter have unknown effects upon FAN1 functions. Current thoughts are that ongoing repeat expansions in disease-vulnerable tissues, as individuals age, promote disease onset. Fan1 is required to suppress against high levels of ongoing somatic CAG and CGG repeat expansions in tissues of HD and FMR1 transgenic mice respectively, in addition to participating in DNA interstrand crosslink repair. FAN1 is also a modifier of autism, schizophrenia, and epilepsy. Coupled with the association of these diseases with repeat expansions, this suggests a common mechanism, by which FAN1 modifies repeat diseases. Yet how any of the FAN1 variants modify disease is unknown. Here, we review FAN1 variants, associated clinical effects, protein structure, and the enzyme’s attributed functional roles. We highlight how variants may alter its activities in DNA damage response and/or repeat instability. A thorough awareness of the FAN1 gene and FAN1 protein functions will reveal if and how it may be targeted for clinical benefit

ulcera peptica

<div><p>Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, is caused by epigenetic silencing of the <i>FMR1</i> gene, through expansion and methylation of a CGG triplet repeat (methylated full mutation). An antisense transcript (<i>FMR1</i>-<i>AS1</i>), starting from both promoter and intron 2 of the <i>FMR1</i> 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 <i>FMR1</i> 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 <i>FMR1</i> 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. <i>CTCF</i> knock-down experiments clearly established that CTCF does not act as insulator at the active <i>FMR1</i> locus, despite the presence of a CGG expansion. <i>CTCF</i> depletion induces heterochromatinic histone configuration of the <i>FMR1</i> locus and results in reduction of <i>FMR1</i> transcription, which however is not accompanied by spreading of DNA methylation towards the <i>FMR1</i> promoter. <i>CTCF</i> depletion is also associated with <i>FMR1-AS1</i> 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 <i>FMR1</i>-mRNA. We conclude that CTCF has a complex role in regulating <i>FMR1</i> expression, probably through the organization of chromatin loops between sense/antisense transcriptional regulatory regions, as suggested by bioinformatics analysis.</p></div

CTCF binding analysis on <i>FMR1</i> gene after <i>CTCF</i> knock-down.

<p>ChIP assay demonstrates the decrease of CTCF binding on <i>FMR1</i> promoter and exon 1 in WT fibroblasts after <i>CTCF</i> knock-down and <i>FMR1</i> reduction. Box-plots indicate the mean of at least three independent experiments, the corresponding standard error and standard deviation (thin lines). For both regions analyzed the level of CTCF binding in untreated WT (UT) is significantly higher respect to cells treated with siRNA against <i>CTCF</i> (siRNA) (p = 0.0003 for promoter region; p = 0.0001 for exon1). Note that the amount of IP-DNA (ng) is indicated on a logarithmic scale. ChIP experiments included negative controls performed by IgG immunoprecipitation and no template control (not shown).</p

Methylation analysis of <i>FMR1</i> locus after <i>CTCF</i> knock-down.

<p>Bisulfite sequencing of 82 CpGs within the 5′-UTR of <i>FMR1</i> gene after <i>CTCF</i> knock-down in WT (<b>A</b>) and UFM (<b>B</b>) fibroblasts. Every line corresponds to bisulfite sequencing of an individual cell. Black and white squares correspond to methylated and unmethylated CpG sites, respectively. In this experiment the <i>FMR1</i> transcriptional reduction was around 30% with a residual 20% of <i>CTCF</i> transcript. In spite of <i>FMR1</i> transcriptional reduction (indicated as siRNA), there was no methylation spreading towards active <i>FMR1</i> promoter, that remained unmethylated as in an untreated control (UT). Note that CpG pairs between 45 and 54 are within the promoter region. Black bars indicate CTCF binding sites in the MB and in the promoter region.</p

CTCF binding on <i>FMR1</i> locus through ChIP analysis after pharmacological demethylation.

<p>ChIP assay of CTCF binding to <i>FMR1</i> methylation boundary, promoter and exon 1 region after a 7-day treatment with [1 µM] 5-azadC on the S1 (FXS) lymphoblastoid cell line with 450 CGGs. After the pharmacological treatment we observed 25% of <i>FMR1</i> transcriptional reactivation. Box plots represent the amount of DNA bound by CTCF in untreated WT lymphoblasts, untreated FXS (FXSut) and FXS treated with 5-azadC (FXSazadC) cell lines. Note that the amount of IP-DNA (ng) is indicated in logarithmic scale. ChIP experiments included negative controls performed by IgG immunoprecipitation and no template control (not shown).</p

ChIP results after <i>CTCF</i> knock-down without <i>FMR1</i> transcript reduction.

<p>CTCF binding levels and H3-K4/H3-K9 methylation in WT and UFM fibroblasts before (UT) and after <i>CTCF</i> depletion (siRNA) not followed by <i>FMR1</i> transcript reduction for the promoter and exon 1 regions, respectively. All values correspond to the mean amount of IP-DNA (ng)±standard deviation.</p>*<p>indicated statistically significant values (p<0.05).</p

Histone H3 methylation analysis after <i>CTCF</i> knock-down.

<p>ChIP analysis of H3-K4 (<b>A</b>) and H3-K9 (<b>B</b>) methylation in WT fibroblasts after <i>CTCF</i> knock-down with <i>FMR1</i> reduction. Each box-plot depicts the amount (ng) of IP-DNA in promoter and exon 1 regions in control (UT) and siRNA transfected (siRNA) cells. The levels of H3-K4 methylation were significantly reduced in both regions (p<0.05), while those of H3-K9 were increased particularly in the promoter. Box-plots indicate the mean of at least three independent experiments, the corresponding standard error and standard deviation (thin lines). ChIP experiments included negative controls performed by IgG immunoprecipitation and no template control (not shown).</p

<i>CTCF</i> overexpression in WT, UFM and FXS fibroblasts.

<p><i>CTCF</i> overexpression in WT, UFM and FXS cell lines after transfection with a commercial vector containing the open reading frame of variant 1 of <i>CTCF</i>. Quantitative RT-PCR showed a strong increase of <i>CTCF</i> mRNA after 48 and 120 hours from transfection (<b>A</b>), while levels of <i>FMR1</i> transcription remained substantially unchanged (<b>B</b>). The levels of <i>CTCF</i> transcription in the untreated cells were arbitrarily set at 1 as well as those of <i>FMR1</i> transcript in WT and UFM fibroblasts, while those of <i>FMR1</i>-mRNA in FXS cells were set at 0. Bars indicate standard deviation.</p