Molecular determinants of CTG.CAG repeat expansions in Huntington's disease

Abstract

CTG•CAG repeat expansions are the underlying genetic cause for at least 12 inherited neurological disorders, including Huntington’s disease (HD) and myotonic dystrophy type I (DM1). Trinucleotide repeats (TNRs) are very unstable with a strong expansion bias between generations in affected families. High-frequency somatic expansions in affected tissues are also evident for some diseases. Expansions determine whether disease will ensue, at what age and the speed of disease progression. Inherited and somatic expansions require the DNA mismatch repair protein MutSb. Previous studies investigated MutSb abundance, MutSb ATPase function and naturally occurring polymorphisms of Msh2 and Msh3. These studies were carried out in several different experimental systems and in some studies the protein variants were expressed at lower levels than wild-type proteins which makes it difficult to draw firm conclusions about the mechanism of MutSb and expansions. I used CRISPR/Cas9 genome editing technology to mutate Msh3 in human SVG-A astrocytes to create a Msh3-/- cell line that is selectively deficient for MutSb. CTG•CAG repeat expansion frequencies were measured using the well-established SVG-A expansion assay. The Msh3-/- cell line was found to be severely defective for expansions with no effect on contractions. Over-expression of Msh3 showed a rescue of expansion phenotype and an increase in expansions in line with Msh3 expression, thereby indicating that MutSb abundance is limiting for CTG•CAG expansions in human cells. The Msh3-/- cell line provided me with a novel experimental platform for studying key Msh3 variants in a biologically relevant cell system by adding back clones expressing those variants to the Msh3-/- cells. I was able to identify cell lines that express the variant protein at near wild type levels, thereby allowing me to distinguish between the variant protein effect and any effect of protein abundance on expansions. Mutation of the Walker B motif of the Msh3 ATPase domain was shown to affect hydrolysis of ATP by Msh3 but is predicted to not affect binding of ATP. When assayed for expansions, there was a significant decrease in expansions to a similar level to the Msh3-/- cell line. Thus, the ATPase mutant appeared completely defective in driving expansions. I also investigated Msh3 polymorphisms in this system, previous studies had identified the mouse T321I and the human T1045A x viii Msh3 gene polymorphism as modifiers of genomic instability. Like the studies mentioned earlier, these polymorphic proteins were also found to be expressed at lower levels than wild-type. I tested the human equivalent to the T321I polymorphism, T363I and the T1045A polymorphism at near wild type levels and found no significant difference in expansion rates. This indicates that the effect on genome stability seen in the other studies was due to lower protein expression and not due to altered biochemical activities arising from the polymorphisms. I also describe the creation of a novel Msh35KR cell line which expresses a mutant Msh35KR protein with five known acetylation sites mutated from lysine to arginine residues. The Msh35KR protein is expressed at wild type level, mimics a deacetylated Msh3 protein and the cell line containing this mutation is active for CTG•CAG repeat expansions. This cell line is particularly useful for investigating the relationship between Msh3 and histone deacetylase 3 (HDAC3), another known promoter of CTG•CAG repeat expansions. I report my initial findings on the effect of HDAC3 inhibition on CTG•CAG repeat expansions in the Msh35KR and wild type cell lines and provide a solid framework for further investigation into the relationship between MutSb and HDAC3 and how they promote CTG• CAG repeat instability in human cells.