Friedreich ataxia (FRDA), the most common hereditary ataxia, is caused by mutations in the frataxin (FXN) gene. The vast majority of FRDA mutations involve expansion of a GAA•TTC-repeat tract in intron 1, which leads to an FXN mRNA deficit. Bisulfite mapping demonstrates that the region adjacent to the repeat was methylated in both unaffected and affected individuals. However, methylation was more extensive in patients. Additionally, three residues were almost completely methylation-free in unaffected individuals but almost always methylated in those with FRDA. One of these residues is located within an E-box whose deletion caused a significant drop in promoter activity in reporter assays. Elevated levels of histone H3 dimethylated on lysine 9 were seen in FRDA cells consistent with a more repressive chromatin organization. Such chromatin is known to reduce transcription elongation. This may be one way in which the expanded repeats contribute to the frataxin deficit in FRDA. Our data also suggest that repeat-mediated chromatin changes may also affect transcription initiation by blocking binding of factors that increase frataxin promoter activity. Our results also raise the possibility that the repeat-mediated increases in DNA methylation in the FXN gene in FRDA patients are secondary to the chromatin changes.

Repeat Expansion Diseases result from expansion of a specific tandem repeat. The three Fragile X-related disorders (FXDs) arise from germline expansions of a CGG•CCG repeat tract in the 5′ UTR of the FMR1 gene. We show here that in addition to germline expansion, expansion also occurs in the somatic cells of both mice and humans carriers of premutation alleles. Expansion in mice primarily affects brain, testis and liver with very little expansion in heart or blood. Our data would be consistent with a simple two-factor model for the organ specificity. Somatic expansion in humans may contribute to the mosaicism often seen in individuals with one of the FXDs. Since expansion risk and disease severity are related to repeat number, somatic expansion may exacerbate disease severity and contribute to the age-related increased risk of expansion seen on paternal transmission in humans. Since little somatic expansion occurs in murine lymphocytes, our data also raise the possibility that there may be discordance in humans between repeat numbers measured in blood and that present in brain. This could explain, at least in part, the variable penetrance seen in some of these disorders.

Expansion of a GAA⅐TTC repeat in the first intron of the frataxin (FXN) gene causes an mRNA deficit that results in Friedreich ataxia (FRDA). The region flanking the repeat on FRDA alleles is associated with more extensive DNA methylation than is seen on normal alleles and histone modifications typical of repressed genes. However, whether these changes are responsible for the mRNA deficit is controversial. Using chromatin immunoprecipitation and cell lines from affected and unaffected individuals, we show that certain marks of active chromatin are also reduced in the promoter region of the FXN gene in patient cells. Thus, the promoter chromatin may be less permissive for transcription initiation than it is on normal alleles. Furthermore, we show that the initiating form of RNA polymerase II and histone H3 trimethylated on lysine 4, a chromatin mark tightly linked to transcription initiation, are both present at lower levels on FRDA alleles. In addition, a mark of transcription elongation, trimethylated H3K36, shows a reduced rate of accumulation downstream of the repeat. Our data thus suggest that repeat expansion reduces both transcription initiation and elongation in FRDA cells. Our findings may have implications for understanding the mechanism responsible for FRDA as well as for therapeutic approaches to reverse the transcription deficit.Friedreich ataxia (FRDA), 3 an autosomal recessive disorder with early onset, is the most common inherited ataxia (1). In addition to ataxia, diabetes and hypertrophic cardiomyopathy are also commonly seen, with congestive heart failure being a frequent cause of early mortality. The disorder results from mutations in the frataxin (FXN) gene. Most affected individuals have large expansions of a GAA⅐TTC repeat in intron 1 of both FXN alleles (1). Expanded alleles produce lower than normal amounts of FXN mRNA, resulting in a deficiency of frataxin, a protein essential for mitochondrial iron metabolism (1). In general, the repeat number is directly related to the age of onset and the severity of symptoms (2).Brain, peripheral blood leukocytes, and lymphoblastoid cells from individuals with FRDA all show a similar FXN mRNA deficit, and the region flanking the repeat in these cells is aberrantly methylated and enriched for histone modifications characteristic of genes that are transcriptionally repressed (3-7). Because the repeat is relatively close to the promoter, the repeat-mediated chromatin changes could affect transcription initiation if they were to spread to the promoter. This could produce a promoter chromatin configuration that was less permissive for transcription initiation (8). Because intragenic DNA methylation can reduce transcription elongation (8), repeat expansion may also affect the ability of RNA polymerase II (RNAPII) to transcribe through the intron.An alternative explanation for the FXN mRNA deficit in FRDA has been suggested based on the observation that long GAA⅐TTC repeats block transcription elongation on unchromatinized templates in vitro or when propaga...