SMCHD1 is a non-canonical member of the structural maintenance of chromosomes (SMC) protein family, that has been established as an epigenetic repressor with roles in X chromosome inactivation, autosomal gene silencing and genomic imprinting. However, SMCHD1’s recruitment to chromatin and its mechanism of function, as well as high-resolution structure remain to be elucidated. Importantly, it has been established that SMCHD1 mutations contribute to the pathogenesis of the muscular dystrophy FSHD, and more recently discovered, a rare congenital condition termed Arhinia. Many of these mutations are located in SMCHD1’s ATPase-containing N-terminal region in both disease cohorts, highlighting a functional importance of this region in SMCHD1’s overall function.
In my Honours year, I chose ten SMCHD1 mutations identified in the two disease cohorts and generated these by site-directed mutagenesis, using the Mus musculus equivalent N-terminal region comprising of amino acids 111-702. Recombinant proteins were expressed using the Bac-to-Bac baculovirus expression system. I analyzed the activity of purified and stable recombinant proteins via an in vitro ATPase assay. Smchd1 found in FSHD patients all displayed a reduction in ATPase activity, correlating with SMCHD1’s established loss-of-function phenotype in these patients. Follow-up SAXS analysis of two FSHD mutants suggested an increase in flexibility was introduced in the ATPase-containing N-terminal end, potentially affecting ATP hydrolysis. In contrast, Smchd1 mutants found in Arhinia patients displayed varied changes in ATPase activity, implying a more complex disease mechanism. These findings suggest the well-established loss-of-function phenotype of SMCHD1 in FSHD patients correlates with loss of ATPase activity, and thus provide a framework for therapeutic treatment via activating molecules.