In vitro reactivation of the FMR1 gene involved in fragile X syndrome
In vitro reactivation of the FMR1 gene involved in fragile X syndromePietro Chiurazzi, M. Grazia Pomponi, Rob Willemsen1, Ben A. Oostra1 and Giovanni Neri*
Istituto di Genetica Medica, Università Cattolica, and Centro Ricerche per la Disabilità Mentale e Motoria, Associazione Anni Verdi, Largo F. Vito 1, 00168 Rome, Italy and 1Department of Clinical Genetics, Erasmus University, Rotterdam, The Netherlands
Received September 4, 1997;Revised and Accepted October 23, 1997
Fragile X syndrome is the most frequent cause of heritable mental retardation. Most patients have a mutation in the 5' untranslated region of the FMR1 gene, consisting of the amplification of a polymorphic (CGG)n repeat sequence, and cytogenetically express the folate-sensitive fragile site FRAXA in Xq27.3. Fragile X patients harbour an expanded sequence with >200 CGG repeats (full mutation), accompanied by methylation of most cytosines of the sequence itself and of the upstream CpG island. This abnormal hypermethylation of the promoter suppresses gene transcription, resulting in the absence of the FMR1 protein. Rare individuals of normal intelligence were shown to carry a completely or partially unmethylated full mutation and to express the FMR1 protein. Given this observation and knowing that the open reading frame of the mutated FMR1 gene is intact, we decided to investigate whether its activity could be restored in vitro by inducing DNA demethylation with 5-azadeoxycytidine (5-azadC) in fragile X patients' lymphoblastoid cells. We report that treatment with 5-azadC causes reactivation of fully mutated FMR1 genes with 300-800 repeats, as shown by the restoration of specific mRNA and protein production. This effect correlates with the extent of promoter demethylation, determined by restriction analysis with methylation-sensitive enzymes. These results confirm the critical role of FMR1 promoter hypermethylation in the pathogenesis of the fragile X syndrome, provide an additional explanation for the normal IQ of the rare males with unmethylated full mutations and pave the way to future attempts at pharmacologically restoring mutant FMR1 gene activity in vivo.
Fragile X syndrome is the most frequent cause of heritable mental retardation (18 ). Over 95% of patients have a mutation in exon 1 of the FMR1 gene, consisting of the amplification of a (CGG)n sequence (18 ). This sequence, which is polymorphic in the normal population with a mean copy number of 30 CGGs, is transcribed but not translated (18 ). It becomes meiotically and mitotically unstable in pre-mutation carriers with 50-200 CGG repeats. Patients with fragile X syndrome have an expansion of >200 repeats (full mutation), accompanied by methylation of most cytosines of the CGG stretch and of the upstream CpG island (18 ,18 ). It has been suggested that the abnormal hypermethylation of the FMR1 promoter suppresses gene transcription, resulting in the absence of specific mRNA and protein (18 -18 ), in spite of the fact that the open reading frame of the gene is intact. The observation of rare individuals of normal intelligence, carrying a completely or partially unmethylated full mutation and expressing the FMR1 protein (18 -18 ), also supports this hypothesis. However, since patients are often mosaics harbouring several amplified alleles of different lengths, as well as pre-mutations which are still transcriptionally active, genotype-phenotype correlations in vivo are of limited use.
Therefore, we decided to try to restore the activity of the FMR1 gene in vitro by inducing DNA demethylation with 5-azadeoxycytidine (5-azadC) in lymphoblastoid cell lines established from fragile X patients with a `pure' full mutation, which did not have any detectable level of FMR1 mRNA and protein expression.
Lymphoblastoid cell lines from five male fragile X patients and two male controls were used in the experiments. The size of the amplification in the patients' cell lines ranged from 300 to 800 CGG repeats. Both peripheral lymphocytes and Epstein-Barr virus (EBV)-transformed lymphoblasts of control individuals abundantly express FMR1 protein, while the patients' cell lines did not produce any detectable amounts of either specific mRNA or protein, with the partial exception of patient 5, as discussed below. All cell lines were cultured in duplicate as described in Materials and Methods, and one aliquot was treated with 5-azadC for 7 days while tissue culture medium was added as mock treatment to the other aliquot. Cell counts at the end of each experiment indicated that cell viability was not significantly different in treated and untreated cell lines.
The effect of 5-azadC on transcription was tested by RT-PCR (Fig. 1 ) using primers 4924-4925 for FMR1 (145 bp product) and, as an internal control, primers 243-244 for the housekeeping HPRT gene (370 bp product). No FMR1 product is seen in lanes 2, 4, 6, 8 and 10, corresponding to the untreated patients' samples (patients 1-5), while the specific band is clearly present in lanes 3, 5, 7, 9 and 11 that contain samples of the same patients treated with 5-azadC. Some FMR1 mRNA is actually detectable in the untreated sample from patient 5 (lane 10), in whom DNA methylation analysis showed that the full mutation was partially demethylated (data not shown). In the untreated and treated control males' cells, the FMR1 gene is fully expressed (lanes 12 and 13) and the 5-azadC treatment does not appear to affect the FMR1/HPRT ratio. A modified radioactive RT-PCR protocol with a reduced number of cycles allowed semi-quantitation of the FMR1 transcripts detectable in the treated patients' lines (Fig. 2 ), which range from 10 to >50% of the normal mean value, as evaluated by densitometric analysis of the autoradiograms.
DNA methylation analysis using methylation-sensitive enzymes showed loss of the usual hypermethylation of patients' full mutations. A representative experiment is shown in Figure 3 for patients 3 and 4, who harbour full mutations of 700 and 500 CGG repeats, respectively. Partial demethylation of the SstII (equivalent to SacII) site of the FMR1 promoter is clearly present in 5-azadC-treated cells (lanes 4 and 6), while hypermethylation is complete in the corresponding untreated samples (lanes 3 and 5). Again, there was no evidence of any pre-mutation or normal sized fragments after treatment. Methylation analysis was also performed with the enzymes EagI and BssHII (data not shown). However, these enzymes were not as effective as SstII, suggesting an uneven distribution of the demethylated sites across the FMR1 CpG island. It is worth noting that expression of the FMR1 gene has also been demonstrated in a tumour harbouring a large pre-mutation with both EagI and BssHII sites completely methylated (18 ). Thus it seems that the methylation density of the 5' CpG island as a whole may be more important than the methylation of specific sites in determining the transcriptional status of the FMR1 gene.
Reappearance of the FMR1 protein in the cytoplasm of the treated patients' cell lines was demonstrated by immunocytochemistry, as shown in Figure 4 . Cells from an untreated patient show no specific fuchsin red staining because no protein is recognized by the specific mouse monoclonal antibody (Fig. 4 a), as opposed to cells from a normal control (Fig. 4 c), whose cytoplasm stains positive. After 5-azadC treatment, a proportion of patients' cells show specific staining (Fig. 4 b), though sometimes of reduced intensity. The number of cells expressing FMR1 protein is in line with the observed level of promoter demethylation.
We have shown that mutant FMR1 genes with expansions of 300-800 CGG triplet repeats are able to resume transcription after 5-azadC treatment (Figs 1 and 2 ), concomitant with demethylation of the promoter (Fig. 3 ). The efficiency of transcription appears to correlate with the degree of demethylation, as also indirectly suggested by previous studies in which the activity of a recombinant construct containing the FMR1 promoter was inhibited by hypermethylation (9 ,10 ). The extent of promoter demethylation attained in our experiments is probably related to the fraction of cycling cells which incorporated 5-azadC in culture. Thus, it appears that the amount of specific FMR1 mRNA produced by the cells depends on the degree of demethylation of the promoter, while the amplification itself does not seem to block transcription of hypomethylated alleles. Similar effects of both 5-azadC and 5-azacytidine (5-azaC) have been reported on the transcriptional reactivation of several genes, like PGK1 and HPRT (19 -21 ), which are also normally silenced by the hypermethylation of their respective 5' CpG islands on the inactive X chromosome.
Moreover, we showed that mutant FMR1 transcripts with up to 800 CGGs can be translated into protein (Fig. 4 ), while it had been suggested that, above a threshold of 280 triplets, translation of abnormal mRNAs is completely abolished because of stalling of the repeated sequence on the small ribosomal subunit (22 ). In other words, we provide direct evidence that hypermethylation of the FMR1 promoter and not amplification of the CGG repeat is the major determinant in abolishing FMR1 protein production. The difference between our results and those of Feng et al. (22 ), who tested fibroblast-like cell lines, could be due to different cell specificity in the expression of the FMR1 protein. Our experiments are in line with the observations of Smeets et al. (11 ) on lymphoblastoid cell lines of two normal males with unmethylated full mutation, and we were able to induce a comparable situation with a DNA demethylation protocol. Thus, even if the abnormal length of the amplified FMR1 mRNA is likely to diminish the translational efficiency somewhat, fragile X full mutations can still be transcribed and translated when the promoter of the gene is demethylated. Reduced levels of the protein appear to be sufficient to prevent mental retardation in the few males with unmethylated full mutations (11 ). Putting this in perspective, we think that our experiments pave the way to future attempts at pharmacologically restoring gene activity in fragile X patients. End-stage patients with [beta]-thalassaemia were treated with intravenous azacytidine, which was able to boost [gamma]-globin expression up to therapeutic levels (23 ). While long-term treatment with such agents may have serious adverse effects, use of safer DNA demethylating drugs, after in vitro testing for efficacy and lack of toxicity, may represent a not so remote therapeutic possibility for fragile X patients. Eventually, one might even consider the possibility of a preventive treatment. In fact, there is evidence that methylation of the fully mutated FMR1 gene takes place only during a short time period before week 10 of fetal development (7 ). If this early methylation event could be either prevented or reversed, it might be expected that the gene will stay demethylated during further development and after birth.
EBV-transformed lymphoblastoid cell lines were established from blood samples of fragile X patients and normal controls using the procedure of Neitzel (24 ). Cells were grown in RPMI1640 medium with 10% fetal calf serum and penicillin/streptomycin at 37°C, in a 5% CO2 atmosphere. Cells were counted and seeded at the initial concentration of 3 × 105 /ml and synchronized with two 8 h blocks of 1 mM thymidine (Sigma) spaced by a 10 h interval. After each thymidine block, the cells were centrifuged and resuspended in fresh medium. Treatment with the DNA demethylating agent 5-azadC (Sigma, 0.5-1 µM) was started after the first thymidine block. Total cell culture volume was tripled by the end of the treatment (from 20 to 60 ml) and fresh 5-azadC was added every 24-48 h, to keep its concentration constant. A control flask for each cell line was also grown without addition of 5-azadC. At day 7, cell concentration was measured and both treated and untreated coltures were recovered and subdivided.
Total RNA was extracted with the single-step acid phenol method (25 ). cDNA synthesis was carried out with 200 U of Moloney murine leukaemia virus reverse transcriptase (Gibco/BRL) incubating 5 µg of total RNA and 1.2 µg of random hexamers (Pharmacia) at 37°C for 60-90 min. Expression of specific mRNA was determined by RT-PCR using primers 4925-4924 located in exons 3 and 4 of the FMR1 gene (6 ). As internal control of amplification, we employed primers 244-243 of the HPRT gene which is expressed constitutively in all cells (6 ). Thirty two PCR cycles (94°C 1 min, 55°C 1 min, 72°C 2 min) were employed to detect the amplification products with ethidium bromide after separation on a 1.2% agarose gel. Cycle number was lowered to 21 when [[alpha]-32P]dCTP was added, and PCR products were separated on a denaturing 6% polyacrylamide gel (7 M urea). Densitometric analysis of the autoradiographic film was carried out with the NIH Image software (version 1.61).
GenomicDNA was extracted according to a routine salt-chloroform protocol (26 ). Southern blots were prepared digesting 10 µg of genomic DNA with 24 U of methylation-sensitive enzyme SstII (Gibco/BRL), an isoschizomer of SacII, and 50 U of HindIII (MBI/Fermentas), and incubated overnight at 37°C in the HindIII buffer. Samples were run on a 0.8% agarose gel and blotted on Hybond N+ nylon membrane (Amersham). The DNA methylation status of the FMR1 promoter was evaluated by hybridization of probe Ox1.9 (27 ). Filters were hybridized overnight at 65°C in 0.5 M sodium phosphate/7% SDS buffer and subsequently washed for 30 min with 2× SSC/0.1% SDS at room temperature and for 10 min with 0.5× SSC/0.1% SDS at 65°C, then exposed for 3-7 days at -80°C to Kodak Ektamat G films with intensifying screens.
Cytospins of treated and untreated cell suspensions were prepared on microscope slides and the presence of the FMR1 protein was tested with a previously described antibody test (28 ) that employs the specific mouse monoclonal antibody 1A1 (Euromedex). Cells were fixed for 10 min with a drop of 4% paraformaldehyde and permeabilized with 100% methanol for 20 min. Slides were then washed in 0.1 M phosphate-buffered saline (PBS) + 0.15% glycine, 0.5% bovine serum albumin and incubated for 1 h at room temperature with 1:200 mouse monoclonal antibody. After washing and another 1 h incubation with 1:200 biotinylated goat anti-mouse immunoglobulins, a final 45 min incubation followed, with 1:100 streptavidin-biotinylated alkaline phosphatase (StreptABComplex/alkaline phosphatase, DAKO). Revelation was obtained with the New Fuchsin substrate-chromogen system (DAKO) for 15-30 min after extensive washing with 0.1 Tris. Nuclei were counterstained with Gill's haematoxylin and slides mounted.
P.C. wishes to thank Professor Hans Galjaard for hospitality at the Department of Clinical Genetics of the Erasmus University in Rotterdam. This work was supported by Telethon grant E.245 to G.N.
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*To whom correspondence should be addressed. Tel: +39 6 30154606; Fax: +39 6 3050031; Email: ibige@rm.unicatt.it
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