The effect of treatment with 5-aza-dC, splitomicin and TSA on the DNA methylation of the promoter of a FXS allele.

<p>Lymphoblastoid cells from a FXS patient were treated with the indicated compounds as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000017#s4" target="_blank">Materials and Methods</a>. Genomic DNA isolated from cells with and without treatment was tested for DNA methylation at the <i>FMR1</i> promoter. The derivative of the dissociation curve of the bisulfite modified PCR fragment obtained from this procedure (dRFU/dT) was plotted as a function of temperature. The point of inflection corresponds to the Tm of the PCR product. Note that 2 peaks in the 5-aza-dC-treated samples are seen, one corresponding to completely demethylated alleles and a much smaller one, indicated by an asterisk, reflecting residual partially methylated alleles. RFU: relative fluorescent units.</p

Splitomicin and 5-aza-dC-induced chromatin changes at the 5′ end of the <i>FMR1</i> gene in affected and unaffected individuals.

<p>Lymphoblastoid cells from an unaffected (GM06865) and affected individual (GM03200B) were treated with 700 µM splitomicin and 10 µM 5-aza-dC as before. ChIP was performed using antibodies to H3K9Ac, H4K16Ac and H4Kme2. Real time PCR was carried out on the immunoprecipitated material and the results expressed as the percentage of input DNA and normalized to GAPDH. Panels A depicts the chromatin modifications occurring in untreated and treated cells in the promoter region. Panel B depicts the chromatin modifications occurring in untreated and treated cells in exon 1.</p

Model for the effect of 5-aza-dC and splitomicin on reactivation of <i>FMR1</i> full mutation alleles.

<p>Binding of a DNA methyl-binding protein (MeBP) to the methylated 5′ end allows SIRT1 to be recruited. This results in deacetylation of H4K16 and H3K9. The deacetylated H3K9 can now be methylated. A) Inhibition of DNA methylation prevents binding of the MeBP and thus the recruitment of SIRT1. This facilitates the acetylation of H4K16 by hMOF, which promotes chromatin opening and transcriptional activation. B) Inhibition of SIRT1, allows hMOF to acetylate H3K16 and thus to adopt a more open chromatin conformation without affecting DNA methylation.</p

Gene reactivation and FMRP production.

<p>(A) The effect of HDAC and DNA methylation inhibitors on <i>FMR1</i> gene expression in FXS cells. Lymphoblastoid cells from an unaffected (GM06895) and affected individual (GM03200B) were treated with 10 µM 5-aza-dC for 72 hr, or with 700 µM splitomicin (SPT) or 3 µM TSA for 24 hr. <i>FMR1</i> mRNA levels were measured by real time PCR and the <i>FMR1</i> expression in patient cells was plotted as a percentage of the <i>FMR1</i> mRNA produced from unaffected cells without any treatment. (B) Representative western blot with an anti-FMRP antibody showing the extent of FMRP production in lymphoblasts from unaffected and affected individuals with and without treatment with either 300 µM (GM06897) or 700 µM splitomicin. (C) Quantification of FMRP levels in untreated and splitomicin treated cells. FMRP levels were determined by densitometric analysis. After normalization to β-actin to control for differences in protein loading, the results were expressed as a fraction of the amount of FMRP in untreated cells from an unaffected individual (GM06895). The results shown represent the average of 3 independent experiments. The difference in FMRP levels in GM06897 cells with and without treatment was significant at p = 0.0151.</p

The effect of nicotinamide and splitomicin on <i>FMR1</i> gene expression in unaffected and FXS cell lines.

<p>(A). Lymphoblastoid cells from an unaffected individual (GM02168), individuals with FXS (GM06897 and GM03200B) treated with the indicated concentrations of nicotinamide. (B and C) Lymphoblastoid cells from an unaffected individual (GM02168), individuals with FXS (GM06897, GM03200B, GM09145 and GM04025) treated with the indicated concentrations of splitomicin. (D) FXS fibroblasts (GM05131 and GM05848) treated with 700 µM splitomicin. <i>FMR1</i> mRNA levels were measured by real time PCR using Taqman primer-probe mixes. The <i>FMR1</i> expression in patient cells was plotted as a percentage of the <i>FMR1</i> mRNA produced from unaffected cells without any treatment. The decrease in <i>FMR1</i> mRNA levels at higher nicotinamide and splitomicin concentrations seen in the normal cells (GM02168) was not significant by Students T-test. However, while the effect of 300 µM splitomicin on GM06897 was significant (p = 0.0016), some inhibition of <i>FMR1</i> mRNA levels was seen at 700 µM such that <i>FMR1</i> mRNA levels were not significantly different in untreated and splitomicin treated cells (p = 0.49). This inhibition was not seen with other cells and may reflect “off-target” effects of splitomicin on other genes/proteins in these cells.</p

The association of SIRT1 with the <i>FMR1</i> promoter in unaffected and affected cells.

<p>Fibroblasts were transfected with pCruzWTSIRT1-HA which expresses a SIRT-HA tag fusion protein. ChIP was carried out using anti-HA antibody. Real time PCR was carried out on the immunoprecipitated material and the fold change of the <i>FMR1</i> promoter and the first exon DNA were expressed relative to input DNA.</p

The effect of SIRT1 on <i>FMR1</i> gene expression.

<p>Vectors expressing either SIRT1 or a dominant negative version of SIRT1 (dnSIRT1) were transfected into fibroblasts from an unaffected individual and individuals with FXS. After 48 hrs <i>FMR1</i> mRNA levels were measured by real time PCR and plotted relative to the <i>FMR1</i> mRNA produced from cells transfected with empty vector. The results represent the average and standard deviations of 3 independent experiments.</p

Additional file 1: Figure S1. of CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons

Analysis of premutation iPSCs and full mutation ESCs. a Immunostaining for pluripotency markers in SC120 and HT14 iPSCs and WCMC37 ESCs. Cells were grown in 24-well plates, fixed and stained for the indicated pluripotency markers (red) as described in the supplemental experimental procedure. Nuclei were stained with DAPI (blue). Scale bar: 200 μm. b SC120 iPSCs were differentiated into neurons and stained for neuronal markers. SC120 neural progenitor cells (NPC) were stained at passage 2 with Nestin (green) and Sox1 (red). Scale bar: 200 μm. SC120 neurons were stained at 4 weeks of differentiation with Map2 (red) and TuJ1 (red). Nuclei were stained with DAPI (blue). Scale bar: 100 μm. c DNA methylation at the FMR1 promoter was analyzed by qMS-PCR in SC120 iPSCs, NPCs, and neurons at indicated weeks (wk) of differentiation. d Immunostaining for FMRP (red) was done in indicated cell lines. The percentage of FMRP-positive cells is indicated under each panel. In SC120 iPSCs, all the cells were positive for FMRP expression but the intensity was much reduced compared to the control H1 cells. Scale bar: 100 μm. e Western blot for FMRP levels in indicated cell lines. β-Actin is used as loading control. f Western blot analysis for MSH2, MSH3, and MSH6 proteins in stem cells and fibroblasts was done as described in the supplemental experimental procedures. β-Actin is used as loading control. (TIFF 11721 kb

PolB+/C mice show a reduced somatic instability index in testis and tail.

<p>The somatic instability index of different organs of three 16 month old <i>PolB+/+</i> and three 16 month old <i>PolB+/C</i> mice with ~140 repeats was determined as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005181#pgen.1005181.ref046" target="_blank">46</a>]. Tail 1 and tail 2 refer to tail samples taken at 3 weeks of age and tail samples taken at 16 months respectively. The error bars represent the standard deviations. The significance of the differences in the SII for different genotypes was determined using Student’s t-test. The tissues in which the SII was significantly lower in <i>PolB+/C</i> mice are indicated by asterisks. The SII for <i>PolB+/C</i> testis was significantly lower at p = 0.001 and the SII for the <i>PolB+/C</i> tail 2 sample was significantly lower at p = 0.013.</p

The effect of heterozygosity for the <i>PolBC</i> mutation on the distribution of repeat number changes seen in the gametes of 3-month-old male mice.

<p>The percentage of alleles with the indicated gains or losses in repeat number for <i>PolB+/+</i> and <i>PolB+/C</i> mice was plotted. The mean gain of repeats was 3.17 (SD = 2.52) for <i>PolB+/+</i> and 5.43 (SD = 4.48) for <i>PolB+/C</i>. This resulted in a distribution of expanded alleles that was significantly different in the two genotypes (p = 0.0001; <i>t</i> test). The mean loss of repeats was 10.83 (SD = 11.05) for <i>PolB+/+</i> and 19.68 (SD = 31.58) for <i>PolB+/C</i>. The very high standard deviations due to the presence of some very large contractions particularly in the <i>PolB+/C</i> mice resulted in a distribution of contracted alleles that was not significantly different in the two genotypes. Inset: <i>PolB+/C</i> mice have fewer small expansions and more large expansions than <i>PolB+/+</i> mice. The error bars represent the 95% confidence interval. Repeat size classes that are significantly different in <i>PolB+/C</i> mice are marked with an asterisk. The decrease in the number of alleles with 1–5 repeats was significant at p = 0.0001, and the increase in the number of alleles with >10 repeats was significant at p = 0.005.</p