Gene Regulation Studies of the Friedreich Ataxia Locus Using Genomic Reporter Assays
Friedreich ataxia (FA) is a progressive neurodegenerative disease caused by a trinucleotide repeat expansion in the first intron of the FRDA gene resulting in insufficiency of frataxin protein. As the coding sequence of the FRDA gene is unaltered, targeted upregulation of gene expression may restore cellular frataxin to therapeutic levels in patients. Understanding the mechanisms that regulate FRDA gene expression should enable a rational approach for the pharmacological restoration of frataxin levels and the therapy of FA. No information is currently available about the position of any long-range, cis-acting regulatory sequences that regulate human FRDA gene expression. We have established a system for the bioinformatic identification and experimental verification of regulatory mechanisms that direct the expression of the FRDA gene. Utilisation of data from the sequence assemblies of the human and other mammalian genomes for cross-species comparative genomics analysis has identified a number of conserved, non-coding regions surrounding the FRDA gene. The role of these sequences will be evaluated by deletions in the context of an FRDA-EGFP genomic reporter, consisting of the fusion of the EGFP gene to the entire normal genomic FRDA locus on a BAC clone. To facilitate studies in human cells we have developed a dual-reporter enhanced BAC (EBAC) vector. The EBAC can be stably maintained episomally and includes the FRDA-EGFP genomic reporter and a gene encoding DsRed-Express fluorescent protein. The EGFP/DsRed-Express ratio will provide a sensitive and specific assay for detecting the effects of deletions of regulatory regions of the gene while concurrently allowing for correction of differing transfection efficiencies.