C02 FAN1 controls cag repeat expansion in huntington’s disease by dual functions, MLH1 retention and nuclease activity

Abstract

<h3>Background</h3> Human genetic studies have shown that, after CAG repeat length, DNA maintenance is the main process influencing Huntington’s disease (HD) pathogenesis. This likely acts via somatic expansion - a process in which the pathogenic CAG repeat in HTT exon 1 expands throughout life. Genetic modifiers of HD in the DNA damage response include the interstrand crosslink repair nuclease FAN1 and several mismatch repair (MMR) factors, including MLH1, PMS2 and MSH3. FAN1 has shown to suppress repeat expansion, however, the mechanism of this remains unclear. <h3>Aims</h3> Explore the functional significance of the previously noted FAN1–MLH1 protein interaction in an HD context. Establish which regions or functions of FAN1 are required for CAG repeat stabilisation. <h3>Methods/Techniques</h3> In the FAN1 -/- U2OS cell system, we express FAN1 variants and assess protein interactions via co-immunoprecipitation. We measure somatic expansion in a co-expressed exogenous 118 CAG HTT exon 1 construct using fragment analysis, involving estimation of CAG repeat length by capillary electrophoresis. Functional assays such as 6-thioguanine and mitomycin C examine DNA repair integrity – mismatch repair and FAN1 nuclease activity, respectively. <h3>Results/Outcome</h3> We have elucidated FAN1’s protective function in HD and the molecular nature of its interaction with mismatch repair. In HD models (in vitro and in vivo), we confirm that FAN1 binds MLH1. We demonstrate that FAN1 directly competes with MSH3 for MLH1 through an evolutionary conserved N-terminal domain, 126SPYF129, reducing the activity of mismatch repair and also CAG repeat expansion. Additionally, FAN1’s canonical nuclease domain accounts for residual suppression of CAG repeat expansion. <h3>Conclusions</h3> The FAN1-MLH1 interaction regulates somatic expansion by attenuating mismatch repair activity, providing a novel therapeutic opportunity for HD and potentially other repeat expansion diseases.