Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells

Abstract

Expanded triplet repeats have been identified as the genetic basis for a growing number of neurological and skeletal disorders. To examine the contribution of double-strand break repair to CAG⅐CTG repeat instability in mammalian systems, we developed zinc finger nucleases (ZFNs) that recognize and cleave CAG repeat sequences. Engineered ZFNs use a tandem array of zinc fingers, fused to the FokI DNA cleavage domain, to direct double-strand breaks (DSBs) in a site-specific manner. We first determined that the ZFNs cleave CAG repeats in vitro. Then, using our previously described tissue culture assay for identifying modifiers of CAG repeat instability, we found that transfection of ZFN-expression vectors induced up to a 15-fold increase in changes to the CAG repeat in human and rodent cell lines, and that longer repeats were much more sensitive to cleavage than shorter ones. Analysis of individual colonies arising after treatment revealed a spectrum of events consistent with ZFN-induced DSBs and dominated by repeat contractions. We also found that expressing a dominant-negative form of RAD51 in combination with a ZFN, dramatically reduced the effect of the nuclease, suggesting that DSB-induced repeat instability is mediated, in part, through homology directed repair. These studies identify a ZFN as a useful reagent for characterizing the effects of DSBs on CAG repeats in cells.DNA repair ͉ gene targeting ͉ triplet repeat instability ͉ zinc finger nucleases

Convergent Transcription through a Long CAG Tract Destabilizes Repeats and Induces Apoptosis

“…In the absence of NER, other repair processes that enhance repeat instability may become involved in resolving the bubble. For example, nicks in the two strands of the bubble would generate a double-strand break, whose repair we have previously shown is accompanied by a substantial increase in CAG repeat contractions (48). The persistence of this or some other abnormal structure is consistent with the idea that convergent transcription stresses the cell in a way that can lead to cell death.…”

Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice

“…Because zinc fingers can be readily reengineered to bind different DNA sequences (20)(21)(22)(23)(24)(25)(26)(27)(28)(29), including CAG repeats (30), they could, in principle, be used to target the HTT gene at a transcriptional level. Furthermore, zinc fingers can be easily concatenated into long chains, and different linker designs can alter the interaction kinetics substantially (31).…”

Hsp90 modulates CAG repeat instability in human cells

“…To test for a possible connection between Hsp90 function and the stability of repeat tracts, we employed a selection assay in human FLAH25 cells, which we have previously used to identify modifiers of CAG repeat stability (Gorbunova et al 2003(Gorbunova et al , 2004Lin et al 2006Lin et al , 2010Lin and Wilson 2007;Mittelman et al 2009). FLAH25 cells carry an integrated HPRT minigene that contains a single intron interrupted by 95 copies of a CAG repeat (Fig.…”

“…In both yeast and mammalian cells, CAG repeat tracts have been shown to be naturally prone to DSBs (Jankowski et al 2000;Meservy et al 2003). We previously developed CAG-specific zincfinger nucleases that introduce DSBs within CAG repeats and showed that they destabilize CAG repeats in a lengthdependent manner in human and rodent cells, triggering a substantial induction of large repeat contractions (Mittelman et al 2009). The susceptibility of CAG repeats to DSBs and the ability of Hsp90 inhibition to interfere with DSB repair led us to evaluate the possible role of Hsp90 in nucleotide repeat instability.…”