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Human Molecular Genetics Advance Access originally published online on November 27, 2007
Human Molecular Genetics 2008 17(5):735-746; doi:10.1093/hmg/ddm346
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The Friedreich ataxia GAA repeat expansion mutation induces comparable epigenetic changes in human and transgenic mouse brain and heart tissues

Sahar Al-Mahdawi, Ricardo Mouro Pinto, Ozama Ismail, Dhaval Varshney, Stefania Lymperi, Chiranjeevi Sandi, Daniah Trabzuni and Mark Pook*

Hereditary Ataxia Group, Centre for Cell & Chromosome Biology and Brunel Institute of Cancer Genetics & Pharmacogenomics, Division of Biosciences, School of Health Sciences & Social Care, Brunel University, Uxbridge UB8 3PH, UK

* To whom correspondence should be addressed. Tel: +44 1895267243; Fax: +44 1895274348; Email: mark.pook{at}brunel.ac.uk

Received September 20, 2007; Revised November 17, 2007; Accepted November 25, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Friedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, leading to reduced expression of frataxin protein. Evidence suggests that the mutation may induce epigenetic changes and heterochromatin formation, thereby impeding gene transcription. In particular, studies using FRDA patient blood and lymphoblastoid cell lines have detected increased DNA methylation of specific CpG sites upstream of the GAA repeat and histone modifications in regions flanking the GAA repeat. In this report we show that such epigenetic changes are also present in FRDA patient brain, cerebellum and heart tissues, the primary affected systems of the disorder. Bisulfite sequence analysis of the FXN flanking GAA regions reveals a shift in the FRDA DNA methylation profile, with upstream CpG sites becoming consistently hypermethylated and downstream CpG sites becoming consistently hypomethylated. We also identify differential DNA methylation at three specific CpG sites within the FXN promoter and one CpG site within exon 1. Furthermore, we show by chromatin immunoprecipitation analysis that there is overall decreased histone H3K9 acetylation together with increased H3K9 methylation of FRDA brain tissue. Further studies of brain, cerebellum and heart tissues from our GAA repeat expansion-containing FRDA YAC transgenic mice reveal comparable epigenetic changes to those detected in FRDA patient tissue. We have thus developed a mouse model that will be a valuable resource for future therapeutic studies targeting epigenetic modifications of the FXN gene to increase frataxin expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
FRDA is an autosomal recessive neurodegenerative disorder that is predominantly caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene (1). Normal individuals have 5–30 GAA repeat sequences, whereas affected individuals have from approximately 70 to more than 1000 GAA triplets (2). The GAA repeat shows somatic instability, with progressive expansion throughout life, particularly in the cerebellum and dorsal root ganglia (DRG) (3–5). The effect of the GAA expansion mutation is to reduce the expression of frataxin (6), a mitochondrial protein that acts as an iron chaperone in iron–sulphur cluster and heme biosynthesis (7–9). Frataxin insufficiency leads to oxidative stress, mitochondrial iron accumulation and resultant cell death, with the primary site of pathology being in the large sensory neurons of the DRG and the dentate nucleus of the cerebellum (10). The outcome is progressive spinocerebellar neurodegeneration, causing symptoms of ataxia, dysarthria, muscle weakness and sensory loss, together with cardiomyopathy and diabetes. At present there is no effective treatment for FRDA, and affected individuals generally die in early adulthood from the associated heart disease.

Preclinical and clinical trials using antioxidants and iron chelators have demonstrated some limited success in alleviating FRDA heart pathology (11–16). However, a more effective overall therapeutic strategy may be to target the immediate effects of the GAA repeat expansion mutation to restore normal levels of frataxin expression. The exact mechanism by which the GAA repeat expansion leads to decreased frataxin expression is unknown, but several models have been put forward. First, it has been suggested that the GAA repeat expansion may adopt abnormal DNA or DNA/RNA hybrid structures that interfere with FXN gene transcription (17–20). Secondly, there is evidence that GAA repeat expansions produce a heterochromatin-mediated gene silencing effect (21). Epigenetic mechanisms, such as DNA methylation and the associated deacetylation and methylation of histones are known to affect gene expression by chromatin remodelling (22), and these epigenetic changes are likely to underpin any GAA repeat-induced heterochromatin-mediated gene silencing effects. In support of this hypothesis, research has recently shown increased DNA methylation of three specific CpG sites immediately upstream of the expanded GAA repeat sequence in FRDA patient lymphoblastoid cell lines and primary lymphocytes, and one of the three CpG sites was identified as an important enhancer of frataxin expression (23). Other studies have identified specific histone modifications that are associated with gene silencing within the GAA repeat expansion-flanking regions of the FXN intron 1 sequence in FRDA lymphoblastoid cell lines and primary lymphocytes (23,24). These changes include deacetylation of histone H3 and H4 lysine residues and increased di- and trimethylation of H3K9. Based on the hypothesis that the acetylation state of the core histones is responsible for gene silencing, novel histone deacetylase (HDAC) inhibitor compounds have been developed and have been shown to increase FXN transcription in FRDA lymphoblastoid cells and primary lymphocytes (24).

These previous epigenetic studies have provided valuable insights into the possible mechanism of GAA-induced transcription inhibition, but they do not address the issue of whether such epigenetic changes are actually present in the most clinically relevant FRDA tissues. Therefore, we decided to investigate epigenetic profiles of the FXN gene in FRDA patient autopsy brain, cerebellum and heart tissue. By bisulfite sequencing and ChIP analysis we now report changes in DNA methylation and histone modifications that are consistent with inhibition of FXN transcription. With a view to future epigenetic-based FRDA therapies, we also investigated the FXN epigenetic profiles within brain, cerebellum and heart tissue from our Y47, YG8 and YG22 FRDA YAC transgenic mouse models (25–27). We find that the GAA repeat expansion-containing FRDA mouse models (YG8 and YG22) exhibit comparable epigenetic changes to those detected in FRDA patient tissue. Therefore, these are excellent FRDA mouse models in which to investigate the therapeutic effects of epigenetically acting compounds, such as novel HDAC inhibitors or DNA methylation inhibitors.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
FXN gene DNA methylation profiles are distinctly altered in human FRDA brain and heart tissues
A previous investigation of the FXN gene in FRDA patient lymphoblastoid cell lines and blood samples has detected hypermethylation at three specific CpG sites immediately upstream of the expanded GAA repeat sequence. One of the three CpG sites was further identified as an important enhancer element for frataxin expression (23). This same study also reported a lack of any DNA methylation in the promoter region of either FRDA or unaffected cells.

However, cultured cells are known to often develop non-physiological DNA methylation profiles. Furthermore, FRDA is a systemic disorder that is known to have differentially affected tissues and cell types. Therefore, we chose to investigate the DNA methylation status in two of the primary affected tissues in FRDA, namely brain and heart. We obtained brain and heart autopsy tissues from two FRDA patients (GAA repeat sizes of 750/650 and 700/700) and two unaffected individuals, and we first determined the FXN transcription levels of the samples by quantitative RT–PCR. The FRDA brain and heart samples showed mean values of 23 and 65% FXN expression, respectively, compared with the unaffected samples (Fig. 1). We then analysed the DNA methylation status of the samples by performing bisulfite sequence analysis of three regions of the FXN gene: (i) a 475 bp sequence that encompasses part of the FXN promoter, exon 1 and start of intron 1, containing 59 CpG sites; (ii) a 286 bp sequence upstream of the GAA repeat, containing 8 CpG sites and (iii) a 275 bp sequence downstream of the GAA repeat, containing 12 CpG sites (Fig. 2). A comparison of the bisulfite sequences from the FRDA patient and control brain and heart tissues reveals a certain degree of DNA methylation in all eight of the upstream GAA CpG sites (Fig. 3C and D) and all 12 of the downstream GAA CpG sites (Fig. 3E and F). However, the data show a consistent shift in the DNA methylation pattern around the GAA repeat in both tissue types. The FRDA upstream GAA CpG sites are comparatively hypermethylated, whereas the FRDA downstream GAA CpG sites are comparatively hypomethylated (Fig. 3C–F). The greatest increases in DNA methylation within the upstream GAA region are seen at CpG sites 4, 5 and 6, the latter of which corresponds to the previously described E-box enhancer element (23). We observed 100% methylation at CpG site 6 in FRDA brain tissues (FXN mRNA level of 23%, Fig. 1) compared with a mean value of 90% methylation in heart tissue (FXN mRNA level of 65%, Fig. 1). Thus, the upstream GAA DNA methylation changes in both FRDA brain and heart are consistent with their proposed roles in inhibition of FXN transcription. However, the finding of decreased DNA methylation in the downstream GAA region (Fig. 3E and F) is somewhat unexpected, since all of the 12 CpG sites fall within an Alu repeat sequence and such sequences are usually repressed by heavy DNA methylation.


Figure 1
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  		Figure 1. Quantitative RT–PCR analysis of FXN mRNA isolated from brain and heart autopsy samples of two FRDA patients (750/650 and 700/700 GAA repeats) and two unaffected individuals. The mean values of FRDA patient tissue data are normalized to the mean FXN mRNA level of the unaffected individuals taken as 100%. Two individual cDNA samples were analysed for each tissue and each reaction was carried out in triplicate. Bars represent SEMs.

 


Figure 2
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  		Figure 2. Schematic representation of 2.2 kb at the 5' end of the FXN gene, indicating the promoter/exon 1 (Pro), upstream GAA (Up) and downstream GAA (Down) regions that were analysed by ChIP (black boxes) and bisulfite sequencing (hatched boxes). Numbers above indicate the position of CpG sites within the promoter and upstream GAA regions. The positions of the ATG translation start codon, exon 1 open reading frame and GAA repeat sequence within the Alu repeat sequence are shown. Numbers below indicate the chromosome 9 base pair numbering according to the 2006 build of the UCSC human DNA sequence database.

 


Figure 3
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  		Figure 3. DNA methylation analysis of the FXN promoter (A and B), upstream GAA (C and D) and downstream GAA (E and F) regions of human brain and heart tissues. In each case for the FXN promoter and upstream GAA regions the mean percentage (+SEM) of methylated CpG sites is shown as determined from the analysis of two FRDA patients and two unaffected individuals, with 7–12 independent cloned DNA sequences analysed for each. For the downstream region the percentage of methylated CpG sites has been determined from one FRDA patient and one unaffected individual (12 independent cloned DNA sequences analysed for each). Only eleven CpG sites are represented for the promoter region (A and B), as sites 11–22 and 24–59 did not show any methylation in either FRDA or unaffected samples in brain or heart. FRDA brain tissues and both FRDA and unaffected heart tissues did not show any DNA methylation at CpG site 23.

 
Another particularly interesting finding was the identification of differential DNA methylation at three specific CpG sites within the FXN promoter (sites 5, 7 and 8) and one CpG site within exon 1 (site 23) (Fig. 3A and B). All of the other 55 CpG sites in the total of 59 CpG sites analysed show complete lack of DNA methylation, as to be expected for a CpG island that is situated at the start of a gene. CpG sites 5, 7 and 8 show incomplete methylation in the unaffected heart, but complete methylation in the FRDA heart (Fig. 3B). Therefore, these CpG sites may be involved in reducing initiation of FXN gene transcription in FRDA heart. However, the DNA methylation pattern is different in brain tissue. Here we identified mean values of 10–35% DNA methylation at the four CpG sites in the unaffected tissues, but very little overall change of DNA methylation in FRDA tissues, or even loss of methylation at CpG sites 5 and 23 (Fig. 3A). Furthermore, the fact that we have identified some degree of DNA methylation at all in this region contrasts with the previous report that DNA methylation is absent in the FXN promoter region of both FRDA and unaffected lymphoblastoid cells (23). Therefore, we have shown that the influence of DNA methylation on FXN gene expression is likely to be complex, with some similarities (CpG site usage) but also some distinct differences (degree of CpG methylation) identified between different somatic tissues.

FXN gene histone modifications are altered in human FRDA brain tissue
Previous studies of the promoter, upstream GAA and downstream GAA regions of the FXN gene have identified specific histone modifications that are associated with gene silencing within the GAA repeat expansion-flanking regions of the FXN intron 1 sequence in FRDA lymphoblastoid cell lines and primary lymphocytes (23,24). We have now investigated acetylated histone H3 and H4 and methylated histone H3K9 modifications by ChIP analysis of the FXN promoter, upstream GAA and downstream GAA regions (Fig. 2) in autopsy brain tissues from two FRDA patients and two unaffected individuals. Our results show overall decreased histone H3 and H4 acetylation of FRDA brain tissue, particularly in the downstream GAA region (Fig. 4). All of the six acetylated histone residues that we have examined show a GAA-induced gradient of comparative acetylation that is highest in the FXN promoter and lowest in the downstream GAA region. The single most altered histone residue is H3K9, which exhibits progressive decreases in acetylation to comparative levels of 56, 32 and 11% in the FXN promoter, upstream GAA and downstream GAA regions, respectively. There is also a consistently increased H3K9 di- and tri-methylation of FRDA brain tissue in all three of the FXN gene regions (Fig. 4). These changes concur with the previous findings of increased H3K9 di- and tri-methylation in the upstream GAA region of other cell types (23,24). However, we have now extended these studies to show that in FRDA brain the H3K9 di- and tri-methylation spreads to both FXN promoter and downstream GAA regions.


Figure 4
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  		Figure 4. Analysis of histone modifications in human brain tissue. ChIP quantitative PCR results for the FXN promoter/exon1 (Pro), upstream GAA (Up) and downstream GAA (Down) amplified regions are represented as the relative amount of immunoprecipitated DNA compared with input DNA, having taken negligible –Ab control values into account. FXN values were normalized with human GAPDH and all values have been adjusted so that all of the Upstream GAA mean values from the unaffected individuals are 100%. In each case two individual ChIP samples from two FRDA patients and two unaffected controls were analysed in triplicate. The means and SEMs of these values are shown.

 
DNA methylation profiles of FXN transgenic mouse brain and heart tissues resemble the profiles of human tissue
Having determined the epigenetic profiles around the human FXN gene, we then investigated the epigenetic profiles of the FXN transgene in brain and heart tissue isolated from YG8 and YG22 GAA repeat expansion-containing FXN YAC transgenic mice (26) compared with Y47 normal-sized GAA repeat-containing FXN YAC transgenic mice (27). Initial determination of FXN transgene expression showed YG8 (90+190 GAA repeats) and YG22 (190 GAA repeats) to have mean decreased mRNA levels of ~26 and 35% in brain and 57 and 56% in heart compared with Y47 (Fig. 5). Thus, inhibition of FXN expression in transgenic mouse brain and heart was comparable with the mean values of 23 and 65% observed in the human FRDA brain and heart samples, respectively (Fig. 1). DNA methylation analysis was then performed on the GAA repeat expansion-containing YG8 and YG22 GAA repeat transgenic mouse tissue samples compared with the Y47 non-GAA repeat controls (3–4 individual mice for each group). As the mouse transgenes consist of entire human FXN gene sequence, we were able to investigate the DNA methylation profiles of exactly the same three regions of the FXN gene that we had previously analysed in human tissue (Fig. 2). Our data show that the DNA methylation profiles of upstream GAA regions of both YG8 and YG22 transgenic mouse brain and heart tissues closely resemble those found in human tissues (Fig. 6C and D). Namely, there is a consistent hypermethylation of the upstream GAA region induced by the GAA repeat expansion, with the most prominent hypermethylation at CpG sites 4, 5 and 6. However, the degree of DNA methylation at CpG sites 4 and 6 in YG8 and YG22 transgenic mouse brain tissue is less than that observed in FRDA human brain tissue, and indeed YG8 shows no difference at all at CpG site 6. The downstream GAA region differs from the human situation in that there is hypermethylation at all CpG sites, which is retained upon introduction of the GAA repeat expansion (Fig. 6E and F). Thus, there is no GAA-induced decrease in DNA methylation as detected in the human tissues. The promoter/exon 1 regions of the FXN transgenes in both mouse brain and heart tissues show a similarity to the human tissues in that DNA methylation is found at only four specific CpG sites: 5, 7, 8 and 23 (Fig. 6A and B). However, the changes in the DNA profiles of these four CpG sites upon introduction of the GAA repeat expansion differ markedly from those found in the human tissues. This time, the brain tissue shows either no change (CpG sites 5 and 7) or an increase (CpG sites 8 and 23) in DNA methylation, whereas the heart tissue shows an overall decrease in DNA methylation. Assessment of the entire mouse DNA methylation data indicates a similar overall DNA methylation profile around the start of the FXN gene that is consistent with inhibition of FXN transcription. However, there are also some specific differences, which may result from epigenetic-control or transcriptional-control variations between the human and the mouse that will require further investigation.


Figure 5
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  		Figure 5. Quantitative RT–PCR analysis of transgenic FXN mRNA isolated from Y47 (9 GAA), YG8 (190+90 GAA) and YG22 (190 GAA) mouse brain and heart tissues. Data are normalized to the mean FXN mRNA level found in the non-GAA transgenic control samples taken as 100%. Two individual cDNA samples were analysed for each tissue from two mice of each line and each reaction was carried out in triplicate. The means and SEMs of these values are shown.

 


Figure 6
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  		Figure 6. DNA methylation analysis of the FXN promoter (A and B), upstream GAA (C and D) and downstream GAA (E and F) regions of Y47 (black columns), YG8 (light grey columns) and YG22 (dark grey columns) transgenic mouse brain and heart tissues. In each case for the FXN promoter and upstream GAA regions, the mean percentage value (+SEM) of methylated CpG sites is shown as determined from the analysis of 7–12 independent cloned DNA sequences from each of 3–4 mice per group. For the downstream region the percentages of methylated CpG sites have been determined from one Y47, YG8 and YG22 mouse (12 independent cloned DNA sequences analysed for each). Only eleven CpG sites are represented for the promoter region, as sites 11–22 and 24–59 did not show any methylation in either FRDA or unaffected samples in brain or heart. Y47 brain tissues did not show any DNA methylation at CpG site 23, while YG22 heart tissues did not show any DNA methylation at any CpG site within the promoter region.

 
DNA methylation profiles of the upstream GAA region in FXN human and transgenic mouse cerebellar tissues are comparable and more severely altered than in other brain tissue
To investigate potential variation within distinct regions of the brain we further analysed the DNA methylation profiles within the upstream GAA region of cerebellar tissue from one FRDA patient compared with an unaffected control and two individual mice from each of the YG8 and YG22 lines compared with the Y47 control line. We chose to investigate the cerebellum because this structure is known to be involved in FRDA pathology (10) and we ourselves have observed increased GAA repeat instability primarily within the cerebellum of both human FRDA patient and FRDA transgenic mouse tissues (3,5,25). The results (shown in Fig. 7) reveal similar DNA methylation changes to those observed in both human and mouse brain tissues. However, the degree of hypermethylation change in the GAA repeat expansion-containing human and mouse cerebellar tissue is more severe, particularly at CpG sites 4, 5 and 6, compared with that seen in the brain tissues as a whole (Fig. 7A compared with Fig. 3C, and Fig. 7B compared with Fig. 6C). We have previously shown that frataxin expression in cerebellar tissue is reduced at both mRNA and protein levels compared with brain and brain stem tissue in our FXN transgenic mice (26). Therefore, the DNA hypermethylation patterns that we have now observed concur with the hypothesis that the upstream GAA region (CpG sites 4–6 in particular) is somehow involved in down-regulation of frataxin transcription.


Figure 7
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  		Figure 7. DNA methylation analysis of the upstream GAA regions of human (A) and transgenic mouse (B) cerebellar tissue. (A) In each case the mean percentage (+ SEM) of methylated CpG sites is shown as determined from the analysis of two FRDA patients (black columns) and two unaffected individuals (grey columns) with 10 independent cloned DNA sequences analysed for each. (B) Y47 (11 GAA) (black columns), YG8 (190+90 GAA) (light grey columns) and YG22 (190 GAA) (dark grey columns) transgenic mouse brain and heart tissues. In each case the mean percentage value (+SEM) of methylated CpG sites is shown as determined from the analysis of 7–12 independent cloned DNA sequences from each of two mice per group.

 
Histone modifications of FXN transgenic mouse brain tissue are comparable with histone modifications of human tissue
Acetylated histone H3 and H4 and di- and tri-methylated histone H3K9 modifications were detected by ChIP analysis of the three regions of the FXN transgene (Fig. 2) in brain tissue isolated from both YG8 and YG22 GAA repeat expansion-containing FXN YAC transgenic mice (26) and Y47 normal-sized GAA repeat-containing FXN YAC transgenic mice (27). Our results show overall GAA repeat-induced decreases in histone H3K9 acetylation and increases in H3K9 methylation for both YG8 and YG22 transgenic mice (Fig. 8), as we previously identified in human FRDA tissue (Fig. 4). However, the level of deacetylation in the transgenic mouse tissue was not as great as that seen in the human tissue, possibly as a consequence of the smaller transgenic GAA repeat expansion sizes (190+90 for YG8 mice and 190 for YG22 mice, compared with 750/650 and 700/700 for FRDA patients). Also, H4K16 acetylation is actually increased in all three FXN transgene regions of YG8 mouse tissue compared with Y47. Furthermore, H4K8 acetylation and H4K12 acetylation are increased in all three FXN transgene regions of YG22 mouse tissue compared with Y47, which is somewhat different to the finding in human tissues. The greatest consistent histone residue changes that we found between the non-GAA (Y47) and both of the GAA (YG8 and YG22) transgenic brain tissue samples were decreases in acetylated H3K9 and increases in di- and tri-methylated H3K9. The H4K12 residue also showed a significant degree of deacetylation, but only in the YG8 transgenic tissue. All of these major histone residue changes in mouse brain tissue reflect the GAA repeat-induced histone residue changes that we detected in human tissue. Furthermore, as with the human samples, we similarly identified a GAA repeat-induced gradient of decreased H3K9 acetylation in both YG8 and YG22 transgenic mouse tissues, with the highest comparative levels of acetylation in the FXN promoter and the lowest comparative levels in the downstream GAA region. The increases in H3K9 di- and tri-methylation were consistent throughout all of the three FXN gene regions in both YG8 and YG22 transgenic mice, once again agreeing with our findings in human FRDA tissue.


Figure 8
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  		Figure 8. Analysis of histone modifications in transgenic mouse brain tissues. ChIP quantitative PCR results for the transgenic FXN promoter/exon1 (Pro), upstream GAA (Up) and downstream GAA (Down) amplified regions are represented as the relative amount of immunoprecipitated DNA compared with input DNA, having taken negligible –Ab control values into account. FXN values were normalized with mouse GAPDH and all values have been adjusted so that all of the upstream GAA values from the non-GAA transgenic mouse tissues (Y47) are 100%. In each case two individual ChIP samples were analysed in triplicate from each of two mice per group. The means and SEMs of these values are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
For the consideration of future FRDA therapy, it is first essential to understand the mechanism of GAA-induced inhibition of FXN gene transcription. Previous studies of FRDA have implicated epigenetic changes, including the detection of increased DNA methylation of specific CpG sites upstream of the GAA repeat and histone modifications in regions flanking the GAA repeat that are both consistent with transcription inhibition (23,24). However, no DNA or histone methylation changes have previously been identified in the FXN promoter or downstream GAA regions, and clinically relevant FRDA brain and heart tissues have not previously been investigated. Different trinucleotide repeat expansion mutations have been shown to induce cis-acting epigenetic changes in several other human disorders (28,29). Thus, DNA methylation of the CGG repeat upstream of the FMR1 gene has been identified as a main epigenetic switch in Fragile X syndrome, with histone acetylation playing an ancillary role (30). Decreased Sp1 interaction associated with DNA hypermethylation upstream of the CTG repeat in the DMPK gene has also been reported for congenital myotonic dystrophy type 1 (29). Furthermore, both CTG and GAA repeat expansions have been shown to induce similar heterochromatin formation by position effect variegation studies of transgenic mice (21). However, it is still uncertain if different trinucleotide repeat sequences produce similar overall epigenetic effects or not.

Our investigations of the FXN gene in both FRDA human and transgenic mouse brain, cerebellum and heart tissues have now confirmed the presence of previously described DNA methylation changes (23) in the upstream GAA region of these clinically important tissues. Furthermore, our data have revealed an overall shift in the DNA methylation profile, moving from hypomethylation in the downstream GAA region towards hypermethylation in the upstream GAA region. This shift in DNA methylation profile could be explained by the known position of the GAA repeat within an Alu sequence, since Alu sequences have been shown to act as methylation centres leading to bi-directional spread of DNA methylation (31). Thus, the hypermethylation detected in the FRDA upstream GAA region may be due to the GAA repeat mutation enhancing the effect of a putative methylation centre at the 5' end of the Alu sequence. At the same time, the addition of the GAA repeat sequence would put extra distance between the methylation centre at the 5' end of the Alu sequence and the downstream GAA region. This may impede the spread of methylation to the downstream region when the distance is large enough (e.g. 2.25 kb for 750 human GAA repeats), but not when the distance is smaller (e.g. 600 bp for 190 transgenic mouse GAA repeats).

We have additionally identified differential DNA methylation at four specific CpG sites within the FXN promoter and exon 1 regions that have not previously been reported. The three CpG sites within the promoter region (sites 5, 7 and 8) are immediately upstream of the ATG translation start site, at nucleotide positions –27, –18 and –11, respectively. CpG sites 5 and 7 are also contained within Sp1 transcription factor binding sites (32). Interestingly, the region between –64 and the start of translation has previously been suggested to contain sequences important for positive regulation of frataxin production, although no candidate sequences were identified (33). Therefore, the three differentially methylated CpG sites that we have now uncovered in the FXN promoter, and in particular the two Sp1 recognition sites, are likely to represent these important regulatory sequences.

In comparison with other instances of trinucleotide repeat-induced DNA methylation changes that inhibit transcription (28,29), one would have predicted general hypermethylation to be associated with the FRDA GAA repeat expansion mutation. However, we actually identified three occurrences in human tissues (promoter and downstream GAA regions in brain, and downstream GAA region in heart) and one occurrence in mouse tissues (promoter region in heart) where there was in fact GAA repeat expansion-induced decrease in DNA methylation. This suggests the possible occurrence of demethylation and resultant active FXN gene expression, at least for some cells within the tissue. DNA demethylation has previously been shown to occur both passively due to DNA replication upon cell division (34) and actively in a process that may involve RNA (35), although the DNA demethylating activity has yet to be identified. DNA demethylation has also previously been associated with processes of DNA damage and repair. The formation of 8-OH-dG by oxidative DNA damage has been shown to affect the activity of human DNA methyltransferase and inhibit CpG methylation (36), and DNA demethylation has also been shown to occur as a result of homologous recombination repair of DNA damaged by double-strand breaks (37). However, DNA demethylation has not previously been considered for FRDA. A close inspection of our data reveals that potential DNA demethylation in the FXN promoter region occurs when the degree of DNA hypermethylation change in the upstream GAA region is greatest. Therefore, we now propose that the shutdown of transcription due to major epigenetic changes at the upstream GAA region may result in attempts to upregulate FXN transcription by Sp1 binding and subsequent DNA demethylation in the promoter region. In support of this proposal, Sp1 binding is known to occur independent of CpG methylation status (32), but at the same time has been shown to inhibit CpG methylation (38). Furthermore, DNA demethylation has previously been shown to occur when there are few methylated CpG sites within a CpG island, but not when all of the CpG sites are methylated (39), which is exactly the situation that we find for the FXN promoter region. However, the GAA repeat expansion-induced decreases in DNA methylation at the FXN promoter are not consistent throughout all human and mouse brain and heart tissues, suggesting the involvement of other factors. Such factors may include differential susceptibility of the brain and heart tissues to DNA damage and/or GAA repeat instability. Indeed, FRDA is a disorder that is known to involve both oxidative DNA damage (40) and somatic instability of GAA repeats (3–5). Therefore, cells that are initially methylated at the FXN promoter region may lose this methylation as part of the GAA repeat instability process, wherein demethylation subsequent to DNA damage repair (37) may be selected for due to beneficial effect of FXN expression and hence cell viability. The GAA repeat expansion-induced decreases in DNA methylation that we have observed in the downstream GAA region of human tissues, but not transgenic mouse tissues, are more likely due to differently sized GAA repeats within the Alu sequence, as we have previously discussed. However, potential DNA demethylation in this downstream GAA region could also indirectly lead to an increase in FXN transcription due to the removal of inhibitory effects on RNA polymerase II elongation.

Our investigations of histone modifications within FXN gene in both FRDA human and transgenic mouse brain tissues have now confirmed the changes previously reported for H3K9 deacetylation in the FXN promoter, upstream GAA and downstream GAA regions and H3K9 methylation in the upstream GAA region (23,24). Furthermore, we have extended the H3K9 methylation analysis to include the FXN promoter and downstream GAA regions that to our knowledge have not previously been reported for any FRDA tissue. Our findings from both human and transgenic mouse tissues indicate significant H3K9 deacetylation, which becomes more severe upon progression from the FXN promoter, through the upstream GAA region to the downstream GAA region. This correlates well with the results for both di- and tri-methylation of H3K9, which show a generally similar gradient of progressive increase from the FXN promoter, through the upstream GAA region, to the downstream GAA region. The only exception is the very high level of di-methylated H3K9 in the upstream GAA region of human FRDA brain tissue, which is higher than that in the downstream GAA region. All of the H3K9 changes correlate well with the DNA methylation changes in both human and transgenic mouse brain tissues. Thus, the patterns of progressively increasing H3K9 deacetylation and increasing H3K9 di- and tri-methylation in transgenic mouse brain correspond exactly to the pattern of increasing DNA methylation. Similarly, the patterns of progressively increasing H3K9 deacetylation and increasing H3K9 tri-methylation in human FRDA brain with a peak of H3K9 di-methylation in the upstream GAA region equate very well to the corresponding DNA methylation profiles. Therefore, our combined data thus far indicate major roles for DNA methylation, histone H3K9 deacetylation and histone H3K9 methylation in the inhibition of FXN transcription in brain and heart tissues, with a less prominent role for deacetylation of other histone residues. The more severe epigenetic changes within the FXN intron 1 region compared with the promoter region support a hypothesis of transcription inhibition due to interference with elongation rather than initiation. Further work will be required to determine the exact relationships between DNA methylation, histone acetylation and methylation, heterochromatin formation and transcription inhibition. However, our results are consistent with the generally described pathway for gene inactivation wherein initial histone H3K9 deacetylation leads to H3K9 methylation, recruitment of HP1, histone deacetylases, DNA methyltransferases and eventual long-term shut down of transcription by DNA methylation (41). However, this situation is not likely to be universal for all trinucleotide repeat disorders, as highlighted by research on the FMR1 gene which has shown both histone deacetylation and H3K9 methylation in the absence of DNA methylation without interfering in active gene transcription (42).

For now, the exact mechanism by which the GAA repeat mutation inhibits frataxin expression remains elusive. However, accumulating evidence, including the findings of this report, now highlights the importance of epigenetic changes that lead to heterochromatin formation. The epigenetic changes that we and others have now identified in FRDA do not in any way negate the importance of any abnormal DNA or DNA/RNA hybrid structures in the inhibition of frataxin expression, but rather suggest the involvement of several combined mechanisms. Indeed the existence of abnormal DNA structures may help to explain why the GAA repeat mutation induces epigenetic changes in the first place. Thus, there are reports that non-B DNA structures such as hairpins may induce DNA methylation (43,44), and GAA repeats have been shown to form hairpins (45). Alternatively, small double-stranded RNA (dsRNA) has also been shown to induce transcriptional gene silencing through a mechanism that involves DNA methylation (46,47). However, dsRNA has failed to induce DNA methylation in a study of mouse oocytes (48) and dsRNA targeted to the HD gene does not induce DNA methylation at the target huntingtin genomic locus in human cells (49). Thus, further studies are still required to identify any possible involvement of non-B DNA structures (such as GAA hairpins or triplex structures), DNA/RNA hybrids or dsRNA in the establishment of epigenetic changes and heterochromatin formation in FRDA.

In light of the epigenetic changes that we and others have identified in FRDA tissues and cells, several novel epigenetic-based therapeutic approaches can now be considered for FRDA. First, HDAC inhibitors can be used, and indeed these have already shown considerable promise by increasing acetylation of histones and thereby increasing FXN transcription in FRDA cells (24). Secondly, pharmacological approaches could be taken to decrease H3K9 methylation, as have recently been described for the combined use of mithramycin and cystamine in Huntington disease mice (50). Thirdly, therapies to decrease DNA methylation should now be considered for FRDA, as have previously been tried for other trinucleotide repeat disorders. In particular, 5-azadeoxycytidine (5-azadC) has been shown to remove DNA methylation of the CCG repeat expansion, increase H3 and H4 acetylation, decrease H3K9 methylation, increase H3K4 methylation and reactivate the FMR1 gene (30,51). Combined HDAC inhibitor and 5-azadC treatment has also been shown to synergistically increase FMR1 gene activity (52). Finally, short dsRNA molecules complementary to promoter sequences have recently been shown to induce gene activation (53,54), and such approaches may also prove effective in increasing FXN transcription. Our identification of a transgenic FRDA mouse model that shows comparable epigenetic changes to those seen in FRDA patients will now provide a valuable resource in the study of all such epigenetic-based FRDA therapies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Tissues
Human brain, cerebellum and heart tissue samples were obtained from autopsies of two FRDA patients (750/650 and 700/700 GAA repeats) and two non-FRDA individuals, in accordance with UK Human Tissue Authority ethical guidelines. Mouse brain and heart tissues were dissected from our previously reported FXN YAC transgenic mouse models: Y47 (two copies of nine GAA repeats); YG8 (2 copies of 90 and 190 GAA repeats) and YG22 (1 copy of 190 GAA repeats) (25,27).

mRNA expression analysis
Total RNA was isolated from frozen tissues by homogenization with Trizol (Invitrogen) and cDNA was then prepared by using AMV Reverse transcriptase (Invitrogen) with oligo-dT primers. Levels of human or mouse transgenic FXN mRNA expression were assessed by quantitative RT–PCR using an ABI7400 sequencer and SYBR® Green (Applied Biosystems) with the following primers: FxnRTF 5'-CAGAGGAAACGCTGGACTCT-3' and FxnRTR 5'-AGCCAGATTTGCTTGTTTGGC-3' (24). Human GAPDH or mouse Gapdh RT–PCR primers were used as control standards: human: GapdhhF 5'-GAAGGTGAAGGTCGGAGT-3' and GapdhhR 5'-GAAGATGGTGATGGGATTTC-3' mouse GapdhmF 5'-ACCCAGAAGACTGTGGATGG-3' and GapdhmR 5'-GGATGCAGGGATGATGTTCT-3'.

Bisulfite sequencing
Genomic DNA was isolated from frozen tissue by standard phenol/chloroform extraction and ethanol precipitation. Two micrograms of genomic DNA was digested with EcoRI prior to bisulfite treatment using the CpGenome kit (Calbiochem). Nested PCR was carried out on bisulfite-treated DNA to amplify three regions of the FXN gene using the following primers: Pro 1st primer pair: SL1F1 5'-TAGTTTTTAAGTTTTTTTTGTTTAG-3' and SL1R1 5'-CAAAACAAAATATCCCCTTTTC-3'; Pro 2nd primer pair: SL1F2 5'-GTTTTTTTATAGAAGAGTGTTTG-3' and SL1R2 5'-CAAAAACCAATATAAATACAACC-3'; Up 1st primer pair: F1G 5'-GAGGGATTTGTTTGGGTAAAG-3' and R1G 5'-ATACTAAATTTCACCATATTAACC-3'; Up 2nd primer pair: F2G 5'-GATTTGTTTGGGTAAAGGTTAG-3' and R2G 5'-CTCCCAAAATACTAAAATTATAAAC-3'; Down 1st primer pair: NH1F 5'-AAGAAGAAGAAGAAAATAAAGAAAAG-3' and SLGR2 5'-TCCTAAAAAAAATCTAAAAACCATC-3'; Down 2nd primer pair: NH2F 5'-AGAAGAAGAAAATAAAGAAAAGTTAG-3' and SLGR1 5'-AAAACCATCATAACCACACTTAC-3'. PCR products were then resolved on agarose gels, purified with Geneclean (BIO101) and cloned into pCR4.0 (Invitrogen) prior to DNA sequencing. A minimum of seven clones were sequenced for each tissue sample.

ChIP analysis
Histone modifications at the three FXN gene regions were detected by ChIP analysis of FRDA human and mouse tissues. This procedure involved initial cross-linking of DNA and protein by formaldehyde treatment of homogenized frozen tissue samples. DNA was then sheared by sonication, followed by immunoprecipitation with commercially available anti-histone and anti-acetylated histone H3 and H4 antibodies: H3K9ac, H3K14ac, H4K5ac, H4K8ac, H4K12ac, H4K16ac and H3K9me2 (Upstate), and H3K9me3 (Diagenode). For each experiment, normal rabbit serum (SIGMA) was used as a minus antibody immunoprecipitation control. After reversal of cross-linking, quantitative RT–PCR amplification of the resultant co-immunoprecipitated DNA was carried out with SYBR® Green in an ABI7400 sequencer (Applied Biosystems) using three sets of FXN primers (Pro, Up and Down) and human GAPDH control for the human samples as previously described (24). For the analysis of transgenic mouse samples, the same three sets of FXN primers were used together with the following mouse Gapdh control primers: GapdhMF, 5'-TGACAAGAGGGCGAGCG-3' and GapdhMR, 5'-GGAAGCCGAAGTCAGGAAC-3'. Each tissue sample was subjected to two independent ChIP procedures, followed by triplicate quantitative PCR analysis.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
This research has been supported by the Friedreich's Ataxia Research Alliance (FARA), the National Ataxia Foundation (NAF), Ataxia UK, GoFAR and the King Faisal Specialist Hospital and Research Center, KSA.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

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