GFP-Based Fluorescence Assay for CAG Repeat Instability in Cultured Human Cells

Abstract

Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries.

Environmental stress induces trinucleotide repeat mutagenesis in human cells

“…We found that induction of transcription, which efficiently destabilizes CAG repeat tracts in human cells (30,31,33,34), is not required for stress-induced production of GFP + cells. Heat, cold, hypoxic, and oxidative stress induced the same four-to fivefold increase in GFP + cells regardless of whether transcription of the GFP gene was induced by doxycycline before stress and occurred throughout the 3-d recovery period (Fig.…”

“…However, the mutations to the CAG repeat tracts in the GFP + cells-46% contractions and 54% indels-offer a clue. In most of our previous characterizations of CAG repeat instability, using the GFP-based assay or our HPRT selection system, we observed primarily simple contractions of the repeat tract; only about 5% were indels (30,33). Only when we introduced DSBs into the CAG repeat tract, using zinc-finger nucleases (42), did we observe a high frequency of indels (44%).…”

“…To establish a link between environmental stresses and CAG repeat instability, we used a GFP-based fluorescence assay to detect repeat contractions (30). As discussed previously, repeat contractions are a good indicator of repeat instability, including expansions (31).…”

“…Construction and characterization of the GFP(CAG) 89 and GFP(CAG) 0 HEK293 cell lines are described elsewhere (30). Briefly, the GFP(CAG) 89 cell line carries a GFP minigene, which is under the control of the inducible CMV/ TetO 2 hybrid promoter (the cytomegalovirus immediate early promoter plus two tetracycline operator 2 sites).…”

“…To detect TNR mutagenesis, we chose to use a long CAG repeat with heightened mutability and a sensitive GFP-based fluorescence assay to detect changes to the repeat tract (30). We showed that cold, heat, hypoxic, and oxidative stresses increased the frequency of TNR mutations in a way that depended on SRFs.…”