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. 2016 Oct 6:7:42.
doi: 10.1186/s13229-016-0105-9. eCollection 2016.

CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons

Yifan Zhou  1 , Daman Kumari  1 , Nicholas Sciascia  2 , Karen Usdin  1
Affiliations

CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons

Yifan Zhou et al. Mol Autism. .

Abstract

Background: Fragile X syndrome (FXS), a common cause of intellectual disability and autism, results from the expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene to >200 repeats. Such expanded alleles, known as full mutation (FM) alleles, are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP, a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial.

Methods: We monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing, and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs.

Results: We show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells (ESCs), some silenced alleles contract and when the repeat number drops below ~400, DNA methylation erodes, even when the repeat number remains >200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore, there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation.

Conclusions: Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo, (ii) large unmethylated alleles may be deleterious in stem cells, (iii) methylation can occur on alleles with >400 repeats very early in embryogenesis, and (iv) expansion and contraction may occur by different mechanisms. Our data also suggest that the threshold for stable methylation of FM alleles may be higher than previously thought. A higher threshold might explain why some carriers of FM alleles escape methylation. It may also provide a simple explanation for why silencing has not been observed in mouse models with >200 repeats.

Keywords: Fragile X syndrome; Repeat contractions; Repeat expansion mutation; Repeat-mediated gene silencing; Stem cells.

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Figures

Fig. 1
Fig. 1
CGG-repeat instability in iPSCs from PM carriers. CGG-repeat size analysis was done by RPT-PCR followed by capillary electrophoresis as described in the “Methods” section for the hiPSCs HT14 (a) and SC120 iPSCs (b), individual lines derived from HT-14 iPSCs (c) and neuronal cells derived from SC120 iPSC (d). See Additional file 1: Figure S1a for pluripotency marker staining for these cells and Additional file 1: Figure S1b for neuronal differentiation of SC120 cells
Fig. 2
Fig. 2
CGG-repeat instability in FX ESCs. a CGG-repeat size and methylation analysis for the WCMC37 ESCs and the individual lineages derived from it was done by MS_RPT-PCR followed by agarose gel electrophoresis as described in the “Methods” section. The “+” and “−“ signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. b Pyrosequencing analysis of DNA methylation in the FMR1 promoter of WCMC37 and 37A cells at passage 44 and 48, respectively. c qMS-PCR analysis of DNA methylation in the FMR1 promoter of the samples analyzed in a. The extent of methylation was determined by the ∆∆Ct method and the individual technical replicates varied by <0.3 Ct. d The abundance of total histone H3, H3K9me2, H3K9me3, H3K27me3, and H4K20me3 in the FMR1 exon1 region is shown relative to GAPDH. Data shown are an average of two independent experiments and error bars represent standard deviation. The 37A line at passage 59 (p59) showed the presence of mostly active FMR1 alleles with only 15 % DNA methylation, consistent with the low levels of repressive histone marks on the FMR1 exon1 compared to WCMC37 cells that carry a silenced FMR1 allele. The passage number of the cells used in panels a and c are as follows: WCMC37 p44, 37A p48, 37B p53, 37C p55, 37D p55, 37 F p55, 37E p55. See Additional file 1: Figure S1a for pluripotency marker staining in WCMC37 cells
Fig. 3
Fig. 3
Selective growth advantage of cells carrying methylated FMR1 alleles with large CGG repeats. a–c The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “Methods” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. a, b Data for 37D and 37A lineages that were maintained in culture for extended periods of time. The DNA methylation status is indicated by the grey line and symbols in the right hand panel, and the mRNA level is indicated by the black line and symbols. c Growth of methylated 37A and unmethylated 37D cells. Late passage 37A cells that were completely methylated and late passage 37D cells that were unmethylated were either grown separately (i) or in a ~1:1 mixture (ii) for ~20 passages. S refers to the cells at the start of the experiment and E to the cells at the end of the experiment. Data for the mixed cultures are shown from two independent experiments (Rep 1 and Rep 2). Panel (iii) shows the DNA methylation for each set of cultures at the start and end of the experiment as an average from two experiments and the error bars indicate standard deviation
Fig. 4
Fig. 4
Unmethylated FM alleles do not become silenced on differentiation into neurons. The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “Methods” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. A late passage culture of 37D ESCs containing little, if any methylated alleles, was differentiated into neurons as described in the “Methods” section. Methylation levels were measured by qMS-PCR on the indicated number of days after the initiation of neuronal differentiation. See Additional file 3: Figure S3 for representative images during neuronal differentiation of 37D cells
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