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.Imperial Users onl
