| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Synergistic effect of histone hyperacetylation and DNA demethylation in the reactivation of the FMR1 gene
Introduction
Results
Discussion
Materials And Methods
Cell culture and stock solutions
Treatments with histone hyperacetylating drugs
Treatment with the DNA demethylating drug
Combined treatments
RT-PCR analysis
Densitometric analysis
Acknowledgements
References
Synergistic effect of histone hyperacetylation and DNA demethylation in the reactivation of the FMR1 gene
Received July 21, 1999; Revised and Accepted September 1, 1999
Most fragile X syndrome patients have expansion of a (CGG)n sequence with >200 repeats (full mutation) in the FMR1 gene responsible for this condition. Hypermethylation of the expanded repeat and of the FMR1 promoter is almost always present and apparently suppresses transcription, resulting in absence of the FMR1 protein. We recently showed that transcriptional reactivation of FMR1 full mutations can be achieved by inducing DNA demethylation with 5-azadeoxycytidine (5-azadC). The level of histone acetylation is another important factor in regulating gene expression; therefore, we treated lymphoblastoid cell lines of non-mosaic full mutation patients with three drugs capable of inducing histone hyperacetylation. We observed a consistent, although modest, reactivation of the FMR1 gene with 4-phenylbutyrate, sodium butyrate and trichostatin A, as shown by RT-PCR. However, we report that combining these drugs with 5-azadC results in a 2- to 5-fold increase in FMR1 mRNA levels obtained with 5-azadC alone, thus showing a marked synergistic effect of histone hyperacetylation and DNA demethylation in the reactivation of FMR1 full mutations.
INTRODUCTION
Fragile X syndrome is a common cause of inherited mental retardation with minor physical manifestations, due to mutations in the FMR1 gene located in Xq27.3 (1). Most patients have a mutation in the 5[prime]-untranslated region of this gene, consisting of an expanded (CGG)n sequence with >200 repeats (full mutation). Hypermethylation of most cytosines in the CGG repeat and in the upstream CpG island is observed and this correlates with gene silencing and 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 (2-4). This clearly shows that absence of the FMR1 protein is the cause of the disease. Given this observation and knowing that the open reading frame of the FMR1 gene is intact in patients with a full mutation, we investigated whether gene activity could be restored in vitro by inducing DNA demethylation with 5-azadeoxycytidine (5-azadC) in fragile X lymphoblastoid cell lines. In fact, we found that transcriptional reactivation of FMR1 full mutations and restoration of protein production can be achieved with 5-azadC treatment (5).
It was shown recently that DNA methylation can cause transcriptional silencing of genes through local deacetylation of histone proteins (6-8). Actually, transcriptionally active chromatin appears to contain more acetylated core histones than tightly packaged heterochromatin (9-11) and treatment with inhibitors of histone deacetylases can increase gene expression (12). We had shown previously that these drugs inhibit cyto-genetic expression of the fragile site FRAXA in cultured lymphocytes of patients (13).
Therefore, we decided to test whether treatment with histone hyperacetylating drugs could also lead to transcriptional reactivation of the FMR1 gene in lymphoblastoid cell lines of fragile X patients. Since fragile X patients are often mosaics harbouring several amplified alleles of different lengths, as well as premutations which are still transcriptionally active, we only worked with cell lines harbouring fully expanded alleles. We used 4-phenylbutyrate (4-PBA) and sodium butyrate (BA), which are reversible inhibitors of histone deacetylases (HDACs) active at millimolar concentrations (14), and trichostatin A (TSA), which is also a reversible HDAC inhibitor but at micromolar concentrations (15,16). We also extended our initial observations on 5-azadC by employing different concentrations of the drug for variable periods of time. Most importantly, we tested whether combined treatment with 5-azadC and hyperacetylating drugs would result in a synergistic or simply additive effect.
RESULTS
Lymphoblastoid cell lines were established from peripheral blood lymphocytes of several male fragile X patients and the size of the (CGG)n expansion was determined by Southern blot analysis after restriction with HindIII and EagI (data not shown). Only cell lines harbouring a full mutation (i.e. with >200 CGG repeats) and without constitutive FMR1 expression were used in these experiments. More precisely, cell line E3 contains ~270-370 CGG repeats, line E4 contains 370-520 repeats, while lines E6 and E7 have between 590 and 710 CGG repeats. All the full mutations were completely methylated at the EagI site when tested by Southern blotting. Reverse transcription-polymerase chain reaction (RT-PCR), employing primers for FMR1 mRNA and HPRT mRNA as an internal control, was performed in order to confirm silencing of the FMR1 gene in the untreated patient cell lines.
Lymphoblasts were cultured and treated as described below with either a histone deacetylase inhibitor alone or in combination with the demethylating agent 5-azadC. After RNA extraction, its concentration was measured and the same amount was used in an optimized semi-quantitative RT-PCR assay to estimate the relative abundance of the FMR1 and HPRT transcripts. It should be noted immediately that although the same amount of total RNA was employed in each RT reaction, the absolute RNA yield and the number of harvested cells decreased with increasing concentrations of the drugs used in the treatment. This effect on cell viability was much more pronounced with TSA (up to 90-95% decrease) than with either BA or 4-PBA, depending on the drug concentration (data not shown). Only a slight decrease was observed with 5-azadC alone (up to 50%), as reported previously (5). A typical result obtained after performing a radioactive RT-PCR is presented in Figure 1. The 195 bp band corresponding to the FMR1 mRNA is visible in the normal (male) control line (lane 11) and in the treated patients' cell lines (lanes 2-5 and 7-10) but not in the untreated ones (lanes 1 and 6). The 386 bp band specific for the HPRT transcript serves as an internal control for the quantification of input RNA. After densitometric analysis the results were normalized to the intensity of the HPRT band and ratios were calculated in order to express FMR1 reactivation as a fraction of FMR1 mRNA in the control cell line.
Figure 1. Typical RT-PCR experiment illustrating the results of a combined treatment with 5-azadC and 4-PBA. The upper band (386 bp) corresponds to the transcript of the HPRT housekeeping gene used as internal control. The lower band (195 bp) is specific for the FMR1 mRNA and is absent in both patients' untreated cell lines (lanes 1 and 6). U, untreated; 0.2 and 1.0, treated with either 0.2 or 1.0 µM 5-azadC; 0.2+ and 1.0+, treated with either 0.2 or 1.0 µM 5-azadC and 5 mM 4-PBA; WT, normal control sample.
A modest but consistent reactivation of the FMR1 gene was obtained by treating the cell lines for 48 h with various concentrations of 4-PBA (Fig. 2a) and TSA (Fig. 2b) in the millimolar and micromolar ranges, respectively. Similar results were obtained when the same cell lines were treated with BA in place of 4-PBA (data not shown). 4-PBA was effective at and above 5 mM concentration, while TSA-induced reactivation peaked at 300-500 nM and decreased above 1 µM, possibly because of its adverse effect on cell viability. As noted above, the number of cells harvested sharply decreased after treatment with HDAC inhibitors, which are known to have powerful growth-arresting effects (17,18). Therefore, we repeated the treatment with 4-PBA over a 48 or 96 h period, but washing the cells free of the drug for either 4 or 8 h every day. Although the FMR1:HPRT ratio did not change, we observed an increase in cell number and viability (data not shown). Figure 2c illustrates the results of a similar experiment performed over a 72 h interval with different concentrations of 5-azadC alone, which shows that 5-azadC has a much greater effect (sometimes up to 10-fold) on FMR1 expression, compared with the small reactivation obtained with HDAC inhibitors. It is also clear that the effect becomes apparent above 200 nM and reaches a plateau above 2 µM concentration. Furthermore, a different reactivation response was observed between the two patient cell lines employed in this experiment, which suggests that some full mutations may be reactivated more easily than others.
Figure 2. Relative levels of FMR1 reactivation after treatment with various concentrations of 4-PBA (a), TSA (b) or 5-azadC (c). A single dose of 4-PBA and TSA was added for 48 h, while a daily dose of 5-azadC was added for 72 h before cell harvesting. Open circles and diamonds correspond to the cell lines of patients E4 and E3, respectively. Reactivation levels are indicated as a percentage of wild-type expression. The experiments were replicated with minimal differences for each cell line.
In order to estimate an optimal incubation time for drug action, the same cell lines were treated with either 4-PBA or 5-azadC for variable time intervals of between 12 h and 8 days (Fig. 3). With 4-PBA treatment (5 mM), adding the drug every 48 h after the medium had been refreshed, the FMR1:HPRT ratio started to increase after 24 h and tended to plateau during the last 4 days (Fig. 3a). The effect of daily addition of 5-azadC was tested at 1 or 0.2 µM concentration (Fig. 3b). 5-azadC at 1 µM again had a much stronger effect than 4-PBA (5 mM) and the FMR1:HPRT ratio clearly increased after 48 h up to the end of the treatment. The lower concentration (0.2 µM) was suboptimal at all time points.
Figure 3. Relative levels of FMR1 reactivation after treatment with 5 mM 4-PBA (a) and 1 µM (open symbols) or 0.2 µM (filled symbols) 5-azadC (b) for 12, 24, 48, 96 and 192 h, respectively. Circles and diamonds correspond to the cell lines of patients E4 and E3, respectively. 4-PBA was added every 48 h and 5-azadC daily, while cell culture medium was changed every 48 h. Reactivation levels are indicated as a percentage of wild-type expression. The experiments were replicated with minimal differences for each cell line.
Combined treatments with 5-azadC and HDAC inhibitors were then performed in order to test whether the respective effects would be simply additive or possibly synergistic. 5-azadC was added daily for a total of 72 h at two concentrations (0.2 and 1 µM) with the aim of facilitating the detection of any synergy of treatment when using the suboptimal dose of 0.2 µM. Optimal concentrations of 4-PBA (5 mM), BA (5 mM) and TSA (0.3 µM) were then added for the last 24 h to one culture while another received a mock treatment [with adequate volumes of phosphate-buffered saline or dimethyl sulfoxide (DMSO)]. Figure 1 visually depicts the results for two of the cell lines corresponding to patients E3 and E6 treated with 5-azadC and 4-PBA. The complete results of the combined treatments are presented in graphical form in Figure 4. It is readily appreciated that a synergistic increase (2- to 5-fold) in the levels of FMR1 reactivation was obtained when 4-PBA or BA were added. Again there was some variation in the level of FMR1 reactivation between the different cell lines treated with either 5-azadC alone (open bars) or in combination with the deacetylase inhibitors (filled bars); nevertheless, the effect of the combined treatment with 4-PBA and BA was always more than additive. Although TSA did slightly reactivate the FMR1 gene when used alone (Fig. 2b), it is not clear whether the FMR1:HPRT ratio increased when TSA was added to the 5-azadC-treated cells for the last 24 h (data not shown). In fact, the extremely low yield of total RNA from the cell cultures treated with TSA did not allow a sufficiently reliable RT-PCR to be performed.
Figure 4. Relative levels of FMR1 reactivation after combined treatment with 5-azadC (0.2 or 1.0 µM) for a total of 72 h and 4-PBA or BA (5 mM) for the last 24 h. Open bars correspond to cell cultures treated with 5-azadC alone, while filled bars represent the cell lines which received the combined treatment. The cell line name and the secondary drug employed (4-PBA or BA) are indicated in bold beneath the 5-azadC concentration (0.2 or 1 µM). Reactivation levels are indicated as a percentage of wild-type expression.
DISCUSSION
Though it has been known for a long time that cytosine methylation in the promoter region of genes often correlates with their transcriptional silencing, it is not clear whether just the binding of transcription factors is affected by this epigenetic modification or whether the chromatin structure is first altered and then transcription is inhibited (19). Although the first possibility has not been excluded, it was reported recently that binding of proteins such as MeCP2 to methylated DNA can recruit a multiprotein complex, including HDACs, which determines increased packaging of chromatin (6-8). Therefore, it appears that DNA methylation effectively modifies the local chromatin structure and consequently reduces the potential access of transcription factors to gene promoter regions (20,21).
The N-terminal tails of histones H4, H3 and H2B extend beyond the nucleosomal core (22) and acetylation of lysines K5, K8, K12 and K16 of histone H4, which neutralizes its positive charge, possibly weakens the interaction with DNA and destabilizes internucleosomal binding, thereby facilitating the unfolding of chromatin fibres and access of the transcriptional machinery (10). HDACs remove precisely those acetyl groups which are added by specific histone acetyltransferases (HATs) to the lysine residues in the N-termini of core histones H3 and H4. Antibodies against specific acetylation sites in histone H4 have been used to show that potentially active euchromatin can be modified at all acetylable lysines, whereas H4 is hypoacetylated in heterochromatin (23,24).
It is worth remembering that the action of HATs and HDACs is directly modulated by a variety of transcriptional activators or repressors interacting in multiprotein complexes, including the Mad-Max heterodimers and members of the nuclear receptor superfamily (11,18,25). DNA methylation seems to be but one of the many triggers of local histone deacetylation and chromatin inactivation; in fact, a selective distribution of acetylated histones is also observed in organisms such as yeast (26) and Drosophila (27), both of which lack DNA methylation.
As far as the FMR1 gene is concerned, we have shown that DNA hypermethylation of its promoter associated with a large (CGG)n expansion (full mutation) actually causes transcriptional silencing of the gene, because demethylation with 5-azadC can reactivate its expression (5). We have now extended our previous observations on the effects of 5-azadC and found that concentrations between 1 and 10 µM are most effective, although the extent of FMR1 reactivation is quite variable in different patients' cell lines (Fig. 2c). It is obvious that the reactivation obtained with 5-azadC is only partial and we have shown, by immunocytochemistry, that it correlates with the proportion of cells (10-20%) actually re-expressing the protein (5). In fact, although individual reactivated cells have almost normal levels of FMR1 protein, many cells do not respond to the treatment, either because of a low efficiency of 5-azadC incorporation and subsequent passive DNA demethylation or because of the toxicity of the drug (above 10 µM), which causes formation of DNA adducts by irreversibly binding to the catalytic core of DNA methyltransferases (28). It is tempting to speculate that the difference between individual cell lines in FMR1 reactivation levels after 5-azadC treatment may correlate with the extent of their promoter hypermethylation. To elucidate this point, further investigations such as those of Stöger et al. (29), assessing the methylation of individual CpG sites in the FMR1 promoter before and after various drug treatments, will be required.
A schematic representation of the chromatin modification possibly caused by FMR1 promoter hypermethylation is shown in Figure 5, which adapts for the fragile X locus the model proposed recently by several groups (6-8). It is clear from the diagram that inhibition of HDACs should allow unfolding of the chromatin fibre and repositioning of nucleosomes, possibly determining reappearance of the DNase I hypersensitive site which is normally present in the promoter region (30). In fact, we have observed reactivation of FMR1 expression after treatment with 4-PBA, BA and TSA alone, although at levels <2% of those observed in normal cells (Fig. 2a and b). This effect is presumably due to a global hyperacetylation of histones H4 and H3 that may increase the transcription of several genes, including the fully mutated FMR1. Coffee et al. (31) confirmed that the 5[prime]-region of the FMR1 gene is associated with acetylated histones H3 and H4 in cells from normal individuals, but acetylation is reduced in cells from fragile X patients. Treatment of fragile X lymphoblasts with 5-azadC resulted in reassociation of acetylated histones H3 and H4 with the FMR1 promoter and transcriptional reactivation. However, treatment with TSA (0.3 µM for 24 h), which led to almost complete acetylation of histone H4 but little acetylation of histone H3, did not cause any reactivation of FMR1 expression (31). Different experimental and cell culture conditions may explain why we were able to detect FMR1 reactivation after TSA treatment, although the extreme anti-proliferative effect of TSA in particular may severely interfere with FMR1 expression, as discussed below.
Figure 5. Schematic representation of the chromatin structure at the FMR1 promoter. (a) In the normal situation the active gene has an open chromatin with spaced nucleosomes and acetylated histone tails. The promoter is free of nucleosomes and the DNase I hypersensitive site is shown. (b) When the CGG repeat is expanded (shaded in grey), it becomes hypermethylated and then the binding of MeCP2 and recruitment of a multiprotein histone deacetylating complex rapidly leads to a more packaged and less accessible chromatin structure (c), causing inactivation of the FMR1 gene. DNA hypermethylation could also lead to chromatin inactivation without directly affecting histone acetylation, possibly through a chromatin remodelling complex (CRC) interacting with the adaptor protein mSin3A, with MeCP2 (open arrowheads) or with other proteins binding methylated DNA.
There could be several explanations for the modest level of FMR1 reactivation by HDAC inhibitors, the first being the persistence of DNA methylation which triggers the chromatin remodelling cascade shown in Figure 5. Whereas treatment with 5-azadC actually prevents assembly of the multisubunit repressor complex, HDAC inhibitors block the chromatin inactivating machinery at a later step. This possibility is in line with the results of Cameron et al. (32), who combined 5-azadC and TSA treatment in cancer cell lines with aberrant promoter hypermethylation of some tumour suppressor genes and concluded that DNA methylation is dominant over histone acetylation in determining silencing at those loci.
Secondly, 4-PBA, BA and especially TSA arrest the cell cycle at multiple points (17,33,34) and induce cell differentiation (35), whereas FMR1 expression is increased in dividing cells (36), with the notable exception of neurons. Therefore, potential FMR1 reactivation may be counteracted by the growth-arresting properties of these drugs. In fact, it appears that a pulsedtreatment with 4-PBA is more effective in our in vitro model (data not shown) and also in vivo when boosting the levels of [gamma]-globin chain expression (37).
Finally, it now seems likely that chromatin structure and nucleosome positioning can be modified by several redundant chromatin remodelling complexes (38), some of which do not affect histone acetylation and harbour an SWI2/SNF2 ATPase family protein (39). If promoter hypermethylation could increase chromatin packaging and cause FMR1 gene silencing without directly acting on histone acetylation (21,40,41), as indicated by the open arrows in Figure 5, then the HDAC inhibitors would only partially relieve the silencing effects of DNA methylation.
The time curve experiment illustrated in Figure 3 demonstrates that a continued treatment over several days can increase the effect of 5-azadC, possibly by allowing more complete DNA demethylation. This result retrospectively supports our previous choice of a 7 day treatment with 5-azadC (5) and suggests that a passive demethylation process is taking place in the treated cells. On the other hand, 4-PBA action is already apparent after 24 h, whereas in the last 96 h the growth arrest induced by treatment may counteract FMR1 reactivation, as discussed above. TSA has been shown by others (32) to be active within 6 h after its addition and its anti-proliferative effect is apparent after 12 h.
The combined effect of 5-azadC and 4-PBA or BA treatment is clearly synergistic and not simply additive (Fig. 4). The fact that the effects of the DNA demethylating and histone hyperacetylating treatments reinforce each other confirms that DNA methylation and histone deacetylation occur in sequence in the same pathway which leads to silencing of the fully mutated FMR1 gene. It is worth pointing out that the extent of FMR1 reactivation seems to correlate with the size of the full mutation; cell lines E6 and E7 harbour ~600-700 CGG repeats and respond less to the treatments than lines E3 (270-370 repeats) and E4 (370-530 repeats). A larger CGG expansion will contain more methylated cytosines triggering local inactivation of the chromatin and a longer time might be required to achieve thorough demethylation with 5-azadC treatment.
Our results are also in agreement with the recent observation of a synergistic interaction of 5-azadC and TSA in regulating the re-expression of silenced tumor suppressor genes (32). Although we were not able to unequivocally prove that TSA can also reinforce reactivation of the FMR1 gene induced by 5-azadC, it is possible that exposing the cell cultures for shorter times (<24 h) to this potent drug may eventually uncover its synergic effect. However, McCaffrey et al. (42) have noted that TSA was less effective than arginine butyrate in increasing expression of the [gamma]-globingene in vitro, probably because of its pronounced cytotoxic effect. In fact, TSA does not appear to be a suitable drug for in vivo applications because of its extremely strong side effects which confine its use to in vitro experiments.
On the other hand, both BA and 4-PBA have been used in vivo to boost [gamma]-globin expression in patients with either sickle cell disease or [beta]-thalassemia (37,43). 4-PBA, which is a new FDA approved drug, was also employed to increase expression of the variant [Delta]F508 CFTR protein in patients with cystic fibrosis (44) and was effective in reactivating an analogue of the ALD gene in knock-out mice (45).
The synergistic effect of 5-azadC and 4-PBA or BA suggests the possibility of using lower dosages of both drugs in order to obtain appreciable FMR1 reactivation levels in vivo. 5-azadC has already been employed in vivo (46), in order to increase the expression of [gamma]-globin gene expression in [beta]-thalassemia patients, although its potential mutagenic effects limit the duration of therapeutic application. It remains to be established whether after a short-term treatment with 5-azadC leading to partial demethylation and reactivation of the FMR1 gene the patients' full mutation can remain stably active, as happens for the rare cases of normal males with an unmethylated full mutation (3), or whether the abnormal (CGG)n expansion would inevi-tably cause remethylation of the promoter and gene silencing. Further in vitro investigations will hopefully indicate whether long-term treatment with histone hyperacetylating drugs such as 4-PBA may help not only in obtaining initial FMR1 reactivation but also in maintaining such an active status.
MATERIALS AND METHODS
Cell culture and stock solutions
Lymphoblastoid cell lines were established by Epstein-Barr virus (EBV) transformation of peripheral blood lymphocytes from male fragile X patients and normal male controls. Cells were grown in RPMI 1640 medium with 10% fetal calf serum and penicillin/streptomycin at 37°C in closed flasks without CO2. BA (Sigma, St Louis, MO) and 4-PBA (Fluka, St Louis, MO) were resuspended in sterile water to a concentration of 500 mM and stored at -80°C in aliquots. TSA (Sigma) was resuspended in DMSO to a concentration of 10 mM and stored at -20°C in small aliquots and a working solution of 1 mM was prepared just before use. Finally, a 10 mM stock solution of 5-azadC (Sigma) was prepared in sterile water and stored at -80°C in aliquots.
Treatments with histone hyperacetylating drugs
Cells were counted, split and seeded at an initial concentration of 2.5-3 × 105 cells/ml in a total volume of 10 ml per flask. A single dose of either 4-PBA, BA or TSA was added to the flasks and was thoroughly resuspended. A control flask for each patient cell line was left untreated or received a mock treatment with a comparable volume of DMSO in the case of the TSA treatment. Cells were harvested after 48 h and the RNA extracted. When a time curve was performed for 4-PBA, the optimal concentration of the drug (5 mM) was added to the cells and RNA was extracted from a treated flask at each time point (12, 24, 48, 96 and 192 h, respectively). Cells were washed and resuspended in fresh medium with the appropriate drug concentration every 48 h.
Treatment with the DNA demethylating drug
Cells were counted, split and seeded at an initial concentration of 2.5-3 × 105 cells/ml in a total volume of 10 ml per flask. A daily dose of 5-azadC was added to the flasks and was thoroughly resuspended, while a control flask was left untreated. Cells were harvested after 72 h treatment when various micromolar concentrations were tested, while the time curve was performed by adding 1 µM of 5-azadC every day and extracting the RNA at the same time points used with 4-PBA. The medium was changed every 48 h.
Combined treatments
Cells were counted and seeded as described above. 5-azadC (200 nM or 1 µM) was added daily for 72 h, while an optimal dose of either 4-PBA (5 mM), BA (5 mM) or TSA (300 nM) was added during the last 24 h. Control cultures were grown with either 5-azadC alone or without any treatment for the total duration of the experiment. After 72 h, RNA was extracted from all flasks at the same time.
RT-PCR analysis
Total RNA was extracted with the single-step acid phenol method, using RNAzol B (Tel-Test, Friendswood, TX). cDNA synthesis was carried out at 37°C for 120 min in a total volume of 40 µl with 180 U MoMLV-RT in the supplied buffer (Gibco BRL, Life Technologies, Rockville, MD), 1 mM DTT, 10 U RNase inhibitor (Promega, Madison, WI) and 0.8 mM each dNTP, and preincubating 5 µg of total RNA with 0.6 µg of random hexamers (Pharmacia, Uppsala, Sweden) at 65°C for 10 min. Expression of FMR1-specific mRNA was determined by RT-PCR using primers K9 (GTA TGG TAC CAT TTG TTT TTG TG, exon 3) and K6 (CAT CAT CAG TCA CAT AGC TTT TTT C, exon 4), which yield a 195 bp product. As an internal control for amplification and to normalize the quantity of input RNA, we employed primers 244 (AAT TAT GGA CAG GAC TGA ACG TC, exons 2 and 3) and 243 (CGT GGG TCC TTT TCA CCA GCA AG, exon 7) of the housekeeping gene HPRT, which yield a 386 bp product. Test reactions were initially performed with 18, 21 and 24 cycles and also with variable amounts of input cDNA from a control cell line to ensure that the PCR reaction would be still in the linear range (data not shown). Twenty-four cycles of amplification were further employed (94°C for 1 min; 55°C for 1 min; 72°C for 2 min) in a total volume of 10 µl with 1 mM MgCl2, 250 µM each dNTP, 0.5 mM spermidine, 0.75 U Taq polymerase (Gibco BRL), 0.1 µl of [[alpha]-32P]dCTP (10 mCi/ml; Amersham Pharmacia, Uppsala, Sweden) and 0.7 µl of cDNA. A large excess of primers (5 pmol each) was added to the reaction in order to maintain it in the exponential phase at 24 cycles. PCR products were separated on a denaturing 6% polyacrylamide gel with 7 M urea run at 70 W for 2 h on an S2 apparatus (Gibco BRL). The gel was fixed with 10% methanol/10% acetic acid solution, driedfor at least 40 min at 80°C with a vacuum pump and exposed to autoradiographic film New RX (Fuji, Tokyo, Japan) for a variable time interval of 3 h to 6 days, in order to have several exposures to scan for densitometric analysis.
Densitometric analysis
Autoradiographic films were scanned at high resolution (300 d.p.i.) and densitometric analysis was carried out with the Windows version of the NIH Image software available from Scion (beta v.3b, July 1998). Band intensity of the FMR1 transcript was normalized to the intensity of the HPRT signal in order to account for differences in input RNA or RT-PCR efficiency. Ratios between the intensity of the FMR1-specific signals in the treated and control cell lines were then calculated in order to express the reactivation levels as a fraction of wild-type expression.
ACKNOWLEDGEMENTS
P.C. is the recipient of Telethon fellowship 283B. This work was supported by a grant from the FRAXA Research Foundation and by Telethon grant E.245 to G.N.
REFERENCES
+To whom correspondence should be addressed. G.N.-Tel: +39 06 305 4449; Fax: +39 06 305 0031; Email: gneri{at}rm.unicatt.it B.A.O.-Tel: +31 10 408 7198; Fax: +31 10 408 9489; Email: oostra{at}kgen.fgg.eur.nl
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S. Fujii, R. Z. Luo, J. Yuan, M. Kadota, M. Oshimura, S. R. Dent, Y. Kondo, J.-P. J. Issa, R. C. Bast Jr, and Y. Yu Reactivation of the silenced and imprinted alleles of ARHI is associated with increased histone H3 acetylation and decreased histone H3 lysine 9 methylation Hum. Mol. Genet., August 1, 2003; 12(15): 1791 - 1800. [Abstract] [Full Text] [PDF]
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P. Weisman-Shomer, E. Cohen, I. Hershco, S. Khateb, O. Wolfovitz-Barchad, L. H. Hurley, and M. Fry The cationic porphyrin TMPyP4 destabilizes the tetraplex form of the fragile X syndrome expanded sequence d(CGG)n Nucleic Acids Res., July 15, 2003; 31(14): 3963 - 3970. [Abstract] [Full Text] [PDF]
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 E Pascale, E Battiloro, G C. Reale, R Pietrobono, M G Pomponi, P Chiurazzi, R Nicolai, M Calvani, G Neri, and E D'Ambrosio Modulation of methylation in the FMR1 promoter region after long term treatment with L-carnitine and acetyl-L-carnitine J. Med. Genet., June 1, 2003; 40(6): e76 - 76. [Full Text] [PDF]
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R. Pietrobono, M. G. Pomponi, E. Tabolacci, B. Oostra, P. Chiurazzi, and G. Neri Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine Nucleic Acids Res., July 15, 2002; 30(14): 3278 - 3285. [Abstract] [Full Text] [PDF]
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J. Worm, A. F. Kirkin, K. N. Dzhandzhugazyan, and P. Guldberg Methylation-dependent Silencing of the Reduced Folate Carrier Gene in Inherently Methotrexate-resistant Human Breast Cancer Cells J. Biol. Chem., October 19, 2001; 276(43): 39990 - 40000. [Abstract] [Full Text] [PDF]
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D. Benjamin and J.-P. Jost Reversal of methylation-mediated repression with short-chain fatty acids: evidence for an additional mechanism to histone deacetylation Nucleic Acids Res., September 1, 2001; 29(17): 3603 - 3610. [Abstract] [Full Text] [PDF]
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 G. Csankovszki, A. Nagy, and R. Jaenisch Synergism of Xist Rna, DNA Methylation, and Histone Hypoacetylation in Maintaining X Chromosome Inactivation J. Cell Biol., May 14, 2001; 153(4): 773 - 784. [Abstract] [Full Text] [PDF]
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S. B. Baylin, M. Esteller, M. R. Rountree, K. E. Bachman, K. Schuebel, and J. G. Herman Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer Hum. Mol. Genet., April 1, 2001; 10(7): 687 - 692. [Abstract] [Full Text] [PDF]
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S. B. Fulmer-Smentek and U. Francke Association of acetylated histones with paternally expressed genes in the Prader-Willi deletion region Hum. Mol. Genet., March 1, 2001; 10(6): 645 - 652. [Abstract] [Full Text] [PDF]
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X. Yang, A. T. Ferguson, S. J. Nass, D. L. Phillips, K. A. Butash, S. M. Wang, J. G. Herman, and N. E. Davidson Transcriptional Activation of Estrogen Receptor {{alpha}} in Human Breast Cancer Cells by Histone Deacetylase Inhibition Cancer Res., December 1, 2000; 60(24): 6890 - 6894. [Abstract] [Full Text]
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P. Jin and S. T. Warren Understanding the molecular basis of fragile X syndrome Hum. Mol. Genet., April 1, 2000; 9(6): 901 - 908. [Abstract] [Full Text] [PDF]
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D. Kumari and K. Usdin Interaction of the Transcription Factors USF1, USF2, and alpha -Pal/Nrf-1 with the FMR1 Promoter. IMPLICATIONS FOR FRAGILE X MENTAL RETARDATION SYNDROME J. Biol. Chem., February 2, 2001; 276(6): 4357 - 4364. [Abstract] [Full Text] [PDF]
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 H.-L. Kang, S. Benzer, and K.-T. Min Life extension in Drosophila by feeding a drug PNAS, January 22, 2002; 99(2): 838 - 843. [Abstract] [Full Text] [PDF]
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