Expanded GAA repeats impede transcription elongation through the FXN gene and induce transcriptional silencing that is restricted to the FXN locus

Abstract

Friedreich's ataxia (FRDA) is a severe neurodegenerative disease caused by homozygous expansion of the guanine-adenine-adenine (GAA) repeats in intron 1 of the FXN gene leading to transcriptional repression of frataxin expression. Post-translational histone modifications that typify heterochromatin are enriched in the vicinity of the repeats, whereas active chromatin marks in this region are underrepresented in FRDA samples. Yet, the immediate effect of the expanded repeats on transcription progression through FXN and their long-range effect on the surrounding genomic context are two critical questions that remain unanswered in the molecular pathogenesis of FRDA. To address these questions, we conducted next-generation RNA sequencing of a large cohort of FRDA and control primary fibroblasts. This comprehensive analysis revealed that the GAA-induced silencing effect does not influence expression of neighboring genes upstream or downstream of FXN. Furthermore, no long-range silencing effects were detected across a large portion of chromosome 9. Additionally, results of chromatin immunoprecipitation studies confirmed that histone modifications associated with repressed transcription are confined to the FXN locus. Finally, deep sequencing of FXN pre-mRNA molecules revealed a pronounced defect in the transcription elongation rate in FRDA cells when compared with controls. These results indicate that approaches aimed to reactivate frataxin expression should simultaneously address deficits in transcription initiation and elongation at the FXN locus.

Figure 1.

Characterization of the FRDA fibroblast lines. (A) Determination of the number of GAA repeats using PCR; CR-GAA repeat expansion carrier harboring one expanded and one short GAA allele. (B) Analysis of the GAA interruption status using MboII digestion. (C and D) Correlation between the length of the shorter of the two expanded alleles (GAA1) and frataxin deficiency. Relative FXN mRNA and protein levels determined in 18 fibroblast lines using qRT-PCR (TaqMan) (C) and western blot (D) are plotted against the length of GAA1. Expression of the FXN transcript and frataxin was calculated relative to the average expression of FXN in 17 control fibroblast lines listed in the Supplementary Material, Table S1. The correlation coefficient value (r) is indicated in each graph.

Figure 2.

Transcriptional silencing induced by expanded GAA repeats is restricted to the FXN gene. (A) RNA-seq data from the analyses of two control and two FRDA fibroblast lines were aligned to GRCh37/hg19 and visualized in UCSC Genome Browser (http://genome.ucsc.edu). A representative snapshot was taken of ∼0.5 Mbp of chromosome 9 encompassing the PIP5K1B, FAM122A, FXN and TJP2 genes. No expression of the PIP5K1B and PRKACG mRNAs was detected in FRDA or control lines. (B) The expression level of FXN mRNA in 17 control (black bars) and 18 FRDA (white bars) fibroblast lines was determined using RNA-seq. Quantitative analysis was conducted using DESeq method. (C) Correlation between length of the GAA1 and FXN mRNA expression as determined using RNA-seq. (D, E, F) A cumulative analysis of FAM122 (D), FXN (E) and TPJ2 (F) gene expression in 17 control and 18 FRDA fibroblast lines. The FDR is shown.

Figure 3.

Epigenetic changes induced by expanded GAAs in FRDA cells are restricted to the FXN locus. Experiments were conducted in three control fibroblast lines (GM08399, GM01650 and GM02169; black bars) and three FRDA fibroblasts (4497, 281 and 203; white bars). (A–C) ChIP data for the indicated histone marks at the PIP5K1B, FAM122A and TJP2 genes in the vicinity of their transcription start sites and upstream of the GAA repeat region for the FXN locus. Statistically significant differences are denoted by asterisks (P < 0.05).

Figure 4.

Expanded GAA repeats impede transcription elongation rate at intron 1 of the FXN gene. (A) A combined landscape representation of RNA-seq tags for all 18 FRDA and 17 control samples mapped to the exon 1/intron 1 region of the FXN locus. Exon 1 and intron 1 are labeled below the plots, and the GAA repeat region is indicated by a dashed line. The nucleotide position on chromosome 9 is indicated above the plots, along with a 2-kb scale bar. RNA-seq tags were mapped to the human reference sequence GRCh37/hg19 lacking the expanded GAAs tract. Both landscapes are presented in the same scale (left of plots). (B) The ratio of RNA-seq signal measured upstream of the GAA repeats versus downstream of the GAAs was calculated for the control (black bar) and FRDA (white bar) cohorts. (C) RNA-seq intron gradient relative to 3′ splice site region (3′ss) for FXN, ZNF169 and TMEM38A. The RNA-seq read count was averaged for samples in the control group (left panel) and FRDA group (right panel). The RNA-seq reads are plotted in 200-bp windows and normalized to the number of reads at the 3′ end of the first intron to compensate for expression differences between mRNAs. The linear regression equation is shown for each plot.

Figure 5.

Histone H4K20me1 is decreased downstream of the expanded GAA repeats in FRDA cells. ChIP analyses of histone H4K20me1, H4K20me3 and H4K5ac enrichment in control and FRDA fibroblasts (black and white bars, respectively) upstream (UP) and downstream (DN) of the GAA repeats. The data are expressed as the mean ± SEM.