Friedreich’s ataxia (FRDA) is known to be provoked by an abnormal GAA-repeat
expansion located in the first intron of the FXN gene. As a result of the GAA
expansion, patients exhibit low levels of FXN mRNA, leading to FRDA. Here, via
chromatin immunoprecipitation (ChIP), the presence of a RNA pol II transcriptional
pausing site at exon 1 of the FXN gene was demonstrated. At this site, FRDA EBVcell
lines exhibited elevated levels of the negative elongation factor NELF-E
depending on the presence of a GAA repeat expansion compared to controls. This
site may represent a rate-limiting step for FXN transcription and consequently
provide a means to modify transcription levels in FRDA. Moreover, RNA pol II
pausing site binding factors, such as NELF-E, were influenced by Nicotinamide
treatment, a HDAC class III inhibitor. Therefore, factors sensitive to chromatin
changes may influence the regulation of RNA pol II pausing and also balance
otherwise positive chromatin changes. This new finding could explain the relatively
minor effects of different drug approaches to up-regulate this gene. Furthermore,
CTCF and the histone demethylase LSD1 were also found to be located at the FXN
pausing site. Results suggest a function for LSD1 in demethylating H3K4me2 at the
pausing site and potentially also in demethylating H3K9me3 in the case of frequently
transcribed expanded GAA repeats. Therefore, LSD1 might play a crucial role in
preventing heterochromatinisation of a euchromatic gene. Using primary transcript
RNA-FISH, a delay in RNA pol II release from the pausing site and furthermore a
dramatic loss of RNA pol II elongation in the presence of expanded GAA repeats was
seen. The identified and characterised transcriptional pausing site at FXN is likely to
play a repressive role and participates in the pathogenesis of FRDA.