Expansions of CTG/CAG repeats in specific human genes cause numerous neurological diseases, including myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. These diseases arise when the number of triplet repeats increases beyond a threshold of about 25-35 repeats to a length that has pathologic consequences. This application focuses on the basis for CTG/CAG repeat instability. Studies in model systems have shown that processes that expose single strands of DMA?replication, recombination, repair, transcription?are capable of destabilizing triplet repeats. CTG and CAG repeats in single strands form hairpins and slipped-strand structures, which are the key intermediates in instability. These structures either interfere with normal repair or trigger aberrant repair, which changes the length of the repeat tract. In no case, however, has the mechanism for triplet repeat instability in humans been defined. Using a novel selection assay for CAG repeat contraction, we have identified two processes that greatly destabilize triplet repeats in mammalian cells: genome-wide demethylation and transcription. The two documented periods of repeat instability in development?early embryogenesis and germline differentiation?correspond to the two waves of epigenetic reprogramming that occur in mammals. Thus, demethylation-induced repeat instability is likely to be directly relevant to the germline events that lead to the progressive worsening of the disease phenotype in subsequent generations?the clinical phenomenon of anticipation. Ongoing, age-dependent repeat instability occurs in affected patient tissues such as neurons, which do not divide, and may exacerbate the disease pathology. Because the disease genes are transcribed in these tissues, transcription-induced instability may be a major contributor to this ongoing, non-replication-dependent instability. We propose to define the specific DNA repair proteins responsible for demethylation- and transcription-induced instability, and in that way define the molecular mechanisms underlying CTG/CAG repeat instability. We will use siRNA knockdowns in selection assays in human cells to identify specific components required for repeat instability. We will extend our studies to mice to examine the effects of altered genomic methylation and transcription on repeat stability in germline and somatic tissues. Finally, we propose to use our selection assay to screen insertion vector libraries, siRNA libraries, and chemical libraries to identify genes that alter repeat stability. Our goal is to delineate those processes that are responsible for both the germline and somatic CTG/CAG repeat instability that characterizes myotonic dystrophy and other neurological diseases.
