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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 17, 2004 as doi:10.1096/fj.04-2499fje. |
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Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands
1Corresponding author: André T. Hoogeveen, PhD, Department of Clinical Genetics, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: a.hoogeveen{at}erasmusmc.nl
SPECIFIC AIMS
In fragile X syndrome the coding sequence of the FMR1 gene is intact; when a CGG trinucleotide repeat expands above 200 repeat units, the CGG repeat itself and the upstream CpG island of the promoter become hypermethylated and transcriptional silencing of the gene occurs. Rare male individuals have been described that carry a FMR1 gene with an expanded repeat >200 units, but the FMR1 promoter is not methylated and FMRP is produced. These high-functioning males (HFM) are intellectually and physically unaffected, an indication that methylation of the FMR1 promoter, not the expanded CGG repeat itself, is crucial for silencing the gene and that this hypermethylation is reversible.
The aim of the present study was to test the influence of different genetic backgrounds in fibroblasts from individuals with fragile X syndrome (FX) and from control males, as characterization of the mechanism involved in the unique epigenetic silencing of the FMR1 promoter may provide important clues for understanding the silencing pathway in general.
PRINCIPAL FINDINGS
1. Fibroblasts from highly functional males and control individuals have a mechanism to repair aberrant DNA methylation
We used primary fibroblasts from three unrelated individuals with fragile X syndrome. These cells were labeled, cocultivated, and fused with HFM or control cells. The heterokaryons containing nuclei from both parental cell lines were sorted out and DNA was analyzed. Using polymorphic markers, we showed that the DNA analyzed after cell fusion derives from both parental cell lines and that no preferential loss of one of the X chromosomes occurred. By bisulfite genomic sequencing, we analyzed methylation of the 52 CpG sites in the FMR1 promoter (Fig. 1
A).
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Figure 1C
shows the results from fusions of FX cells from two individuals with fragile X syndrome (FX1 and FX2) with cells from the HFM. All 34 clones analyzed were found almost completely unmethylated. When FX cells (FX1) were fused with control cells (C1), only demethylated sequences of the FMR1 promoter were identified (Fig. 1D
, upper panel). The same approach was used with FX2 and FX3. FX2 was fused with C1 as well as C2, and FX3 was fused with C2. Again, only demethylated clones of the FMR1 promoter were found (Fig. 1D
, lower panel).
These cell complementation studies showed that the FMR1 promoter in FX cells was specifically demethylated 24 h after cell fusion.
Alternatively, we analyzed DNA from cocultivated and fused cells (FX1 and C1) by Southern hybridization. We applied total DNA from the entire cell culture after fusion without cell sorting. In addition to bands corresponding to normal, not methylated, allele from C1 (2.8 kb) and the full mutation methylated allele from FX1 (6.5 kb), a third band of 
4.0 kb was detected corresponding to the demethylated allele with a long CGG repeat belonging to FX1 cells, which were fused with C1 cells (Fig. 2
E, lane d).
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These experiments demonstrated that the FMR1 promoter in FX cells could be demethylated when the cells are fused with control or HFM cells.
2. Cell fusion is not inducing demethylation
To test whether the fusion procedure itself is influencing the methylated status of the FMR1 gene, we analyzed FX cells fused with each other. Only methylated copies of the FMR1 promoter were found in the DNA analyzed (Fig. 1F
, upper and middle panels), confirming that the demethylation found in the first series of experiments was not due to the cell fusion procedure itself but rather to factors apparently present in the control/HFM cell lines that can operate in trans (promoter of the FMR1 gene in the nucleus of the FX cell).
3. Cells from individuals with fragile X syndrome have lost the factor(s) to demethylate their own FMR1 promoter
When cells from unrelated individuals with fragile X syndrome were fused, only methylated clones were found, indicating FX cells of different genetic background cannot complement each other (Fig. 1F
, lower panel).
4. The demethylation does not affect all promoters
Additional control experiments indicated no change in methylation status of the SNRPN CpG island; therefore, demethylation of the FMR1 promoter seems to be specific.
5. The hypermethylated FMR1 promoter can be specifically demethylated without DNA replication
Tests for DNA replication after cell fusion indicated that in the ensuing 24 h <20% of the DNA in the heterokaryons was replicated. Since we observed complete demethylation of the FMR1 promoter in 100% of the DNA, we conclude that the latter occurs within a short time frame and does not necessarily involve DNA replication.
CONCLUSIONS AND SIGNIFICANCE
The extended CGG repeat and the FMR1 promoter in FX cells are hypermethylated. Rare cases of HFM who have a full mutation CGG repeat, but without methylation, indicate that in some conditions the cell can "escape" from this aberrant methylation. In laboratory conditions, demethylation of the FMR1 gene even for alleles with a repeat as long as 800 CGG units has been achieved using 5-azadeoxycytidine.
Our observation that fibroblasts from normal individuals are able to demethylate the FMR1 promoter as effectively as the HFM fibroblasts suggests that, for the demethylation observed, no additional factors are present in HFM vs. control cells. However, when fibroblasts from different individuals with fragile X syndrome were fused, no demethylation was observed in the heterokaryons, indicating that FX cells are missing the necessary factor(s) for active demethylation of the FMR1 gene.
Several mechanisms for demethylation have been described. The demethylation we observed is fast and does not require DNA replication, so we believe it represents an active mechanism.
Microinjection of a human X chromosome with a methylated FMR1 allele in mouse embryonic cells led to demethylation and reactivation of the FMR1 gene. However, the same allele remained unchanged when introduced into immortalized rodent cells (A9). In such human-rodent hybrids, human trans- and sometimes even cis-acting factors that influence the methylation of the FMR1 promoter could not play a role; these studies are different from our experimental approach. We expose the FMR1 gene to the whole cellular machinery without depriving the FMR1 promoter from the influence of possible cis- and trans-acting factors. The fine-tuning of gene regulation involves the simultaneous binding of activating and repressing complexes to the promoters of genes. The balance between these complexes competing for binding sites is regulated by cellular signals, and this balance could be changed after the cell fusion.
We hypothesize that by fusion of fibroblasts from individuals with fragile X syndrome with fibroblasts from HFM or normal individuals, we provide FX fibroblasts with the necessary repair/demethylating factor(s) already operating and active in the normal cells. These factors seem to be specifically targeted to the hypermethylated FMR1 promoter.
We were not able to analyze the possible restoration of expression of the FMR1 gene in the FX cells. The finding that no demethylation of the FMR1 promoter is observed in heterokaryons of FX cells (lacking FMRP), together with published data for the role of FMRP on the regulation of expression of a large number of genes, suggests that FMRP may be (in)directly involved in the demethylation/repair pathway of its own promoter.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2499fje;
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6.4–6.5 kb); b, DNA from C1 cells (2.8 kb); c, DNA from a premutation patient (99 CGG repeats, not methylated) (