Repeat-induced epigenetic changes in intron 1 of the frataxin gene and its consequences in Friedreich ataxia

Abstract

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.

Figure 1.

The 5′ end of the frataxin gene. (A) Schematic representation of the 5′ end of the human FXN gene showing the location of the FXN repeat relative to the other repeated DNA elements and to the FXN promoter. The 5′ end of the region depicted here corresponds to base 68 878 851 of chromosome 9 in the 2004 build of the UCSC human DNA sequence database. The beginning of the FXN repeat corresponds to base 68 881 758 of this sequence. The numbers 3, 6 and 13 refer to those CpG residues that are protected from methylation in cells from unaffected individuals but that are methylated in FRDA cells. These residues correspond to the Cs marked with asterisks in panel B. (B) The sequence of region of intron 1 analyzed by bisulfite mapping showing an alignment of the human with chimpanzee and macaque FXN genes. The bold numbers 1–15 indicate the 15 methylatable C residues that were analyzed. The boxed sequences indicate interspersed repeated DNA elements. The dotted gray line above the sequence indicates the core muscle-specific Mt-binding site. The solid gray line below the sequence marks the core E-box site.

Figure 2.

Methylation status of 15 CpG residues in the region 5′ of the FXN repeat in cells from unaffected and affected individuals. DNA from lymphoblastoid cell lines from four unaffected individuals and four individuals with FRDA was bisulphite modified and cloned as described in the Materials and Methods section. The numbers below the bars indicate the residue number using the same numbering convention used in Figure 1. The gray arrows indicate the three residues that are frequently methylated in affected individuals but that are very infrequently methylated, if at all, in the unaffected population.

Figure 3.

The effect of different lengths of intron 1 sequence on the activity of the FXN promoter. (A–C). Luciferase activities of constructs containing different amounts of intron 1. Constructs containing the promoter, exon 1 and different lengths of intron 1 sequence were transfected into mouse myoblasts as described in the Materials and Methods section. The dotted lines demarcate deleted regions. The suffix ‘Me’ in the construct FXN81658Me indicates that the FXN81658 construct was specifically methylated at the E-box/Mt-binding site. The luciferase levels from the FXN reporter constructs were normalized to luciferase levels from the pRLNull vector and expressed as a percentage of the normalized luciferase activity of the full-length construct. The data are an average of three independent transfection experiments. (D) Actinomycin D treatment of cells transfected with the FXN81658, FXN81423, FXNE-box/Mt+ and FXNE-box/Mt- constructs. The values for the luciferase protein and mRNA after exposure to Actinomycin D for 8 h are shown expressed as a percentage of the values at 0 h. The data shown represent the average of three independent experiments.

Figure 4.

Electrophoretic gel mobility shift analysis (EMSA) of proteins binding to FXN intron 1. EMSA was carried in the presence of molar excesses of E-boxcon, a canonical E-box sequence, FXN E-box which contains the FXN E-box together with the muscle-specific Mt-binding site, and FXN E-BoxΔ which contains the 3′ end of the E-box together with the full Mt-binding site.

Figure 5.

FXN histone H3 K9 dimethylation and mRNA levels in unaffected and affected individuals. (A) The amount of FXN DNA immunoprecipitated by antibodies to histone H3-K9Me2 in each lymphoblastoid cell line was determined by real-time PCR as described in the Materials and Methods section. The data shown is an average of three independent ChIP experiments. In all instances P < 0.002. When the data from all the patient samples was combined and compared to the combined data from the unaffected individuals P = 0.0001. (B) FXN RNA levels were determined in the indicated cell lines using real-time PCR and expressed relative to the amount of GUS mRNA in the same cells.

Figure 6.

Model for chromatin organization in the region containing residue 13 in normal and FRDA alleles.