C011 Functions of FAN1 protecting against CAG repeat expansions

<h3>Background</h3> Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by an expanded trinucleotide CAG repeat in the huntingtin gene, HTT. Disease onset is primarily driven by HTT CAG repeat length, but DNA damage response (DDR) genes have emerged as major regulators of HD onset and progression. Ongoing expansion of the CAG tract in affected tissues, known as somatic expansion, correlates with an earlier age at onset and faster disease progression, suggesting that CAG repeat instability drives disease pathogenesis. DDR proteins MSH2, MSH3 and MLH1 are all required for expansions to occur, whilst FAN1 protects against repeat expansions. We identified a highly conserved motif at the N-terminus of FAN1 that binds to MLH1, 126SPYF129. This interaction limits the recruitment of MLH1 by MSH3, thus inhibiting the assembly of a functional MMR complex that would otherwise promote CAG repeat expansion. <h3>Methods</h3> Using an HD-iPSC line with 125 HTT CAG repeats we examine the functional properties of FAN1 that stabilise the expanded CAG repeat. We have used CRISPR/Cas9 to knock-out FAN1, disrupt FAN1-MLH1 binding domain at the SPYF motif in FAN1 (Y128A/F129A) or ablate FAN1 nuclease activity (D960A). <h3>Results</h3> Loss of FAN1 and FAN1 variants show accelerated HTT CAG repeat expansions in iPSCs and iPSC-derived neurons. We have explored the effect of FAN1 variants on protein-protein interactions and characterised their activity in interstrand crosslink repair and mismatch repair. <h3>Conclusions</h3> Our data suggests that the relationship between FAN1, MLH1 and MSH3 is crucial in modulation of HTT CAG repeat dynamics.