Expansions of CAG repeats in specific human genes cause numerous neurological diseases, including Huntington disease, myotonic dystrophy, and several spinocerebellar ataxias. These diseases cause immense human suffering, with few potential treatments, and no cures. Our long-term goal is to determine the basis for CAG repeat instability in human patients. Our approach is to combine the strengths of a novel selection assay in human cells with the power of mouse models to discover processes that are responsible for repeat instability in human disease. This application focuses on the role of transcription in triggering CAG repeat instability. Our overarching hypothesis is that transcription promotes formation of repeat-dependent secondary structures that call into play various repair proteins whose actions change the length of the repeat tract. Using siRNA knockdowns and chemical treatments in human cells, we showed that transcription-induced repeat instability was either enhanced or suppressed by several processes, including mismatch repair, transcription-coupled nucleotide excision repair (NER), single-strand break (SSB) repair, double-strand break (DSB) repair, proteasome degradation, transcriptional R-loops, antisense transcription, and DNA damage signaling pathways. In every single case, the effect of the interfering agent was manifest only when there was active transcription through the repeat tract. Critically, we showed that eliminating nucleotide excision repair in a SCA1 mouse model virtually eliminated CAG instability in striatum, in accord with results in human cells. In this application, we propose to concentrate on three striking aspects of our current work.
In Aim 1, we propose to investigate in human cells and mice our observation that convergent transcription through a CAG tract-simultaneous sense and antisense transcription-not only synergistically increases repeat instability, but also triggers apoptosis, providing a potential link to disease pathogenesis.
In Aim 2, we propose to test the inference that transcription drives repeat instability via NER and SSB repair in vitro, in cells, and in mice.
In Aim 3, we propose to exploit the CAG-specific zinc-finger nucleases we developed in the current grant period to investigate the roles of DSBs and strand-specific SSBs in CAG repeat instability in human cells and mouse models. Our overall goal is to delineate those processes that are responsible for both germline and somatic CAG repeat instability that characterizes several neurological diseases.
PROJECT NARRATIVE Expansions of CAG repeats in specific human genes cause numerous neurological diseases. Using human cells and mice, we propose to investigate the role of transcription, which destabilizes CAG repeats by engaging DNA repair processes that not only change the length of the repeat tract, but also can cause cell death. These studies will define basic mechanisms of repeat instability in germline and somatic tissues, test a novel mechanism of disease pathogenesis, and develop a reagent with potential therapeutic applications.
|Chatterjee, Nimrat; Lin, Yunfu; Wilson, John H (2016) Fanconi anemia pathway regulates convergent transcription-induced cell death at trinucleotide repeats in human cells. Postdoc J 4:46-54|
|Chatterjee, Nimrat; Lin, Yunfu; Yotnda, Patricia et al. (2016) Environmental Stress Induces Trinucleotide Repeat Mutagenesis in Human Cells by Alt-Nonhomologous End Joining Repair. J Mol Biol 428:2978-80|
|Chatterjee, Nimrat; Lin, Yunfu; Wilson, John H (2016) Mismatch repair enhances convergent transcription-induced cell death at trinucleotide repeats by activating ATR. DNA Repair (Amst) 42:26-32|
|Mittelman, David; Wilson, John H (2013) The fractured genome of HeLa cells. Genome Biol 14:111|
|Lin, William Y; Wilson, John H; Lin, Yunfu (2013) Repair of chromosomal double-strand breaks by precise ligation in human cells. DNA Repair (Amst) 12:480-7|
|Lin, Yunfu; Leng, Mei; Wan, Ma et al. (2010) Convergent transcription through a long CAG tract destabilizes repeats and induces apoptosis. Mol Cell Biol 30:4435-51|
|Dion, Vincent; Lin, Yunfu; Price, Brandee A et al. (2008) Genome-wide demethylation promotes triplet repeat instability independently of homologous recombination. DNA Repair (Amst) 7:313-20|