Large expansion of CTG•CAG repeats is exacerbated by MutSβ in human cells.

Rie Nakatani,Masanori P Takahashi,Harutoshi Fujimura,Hideki Mochizuki,Masayuki Nakamori

doi:10.1038/srep11020

Rie Nakatani, Masanori P Takahashi + Show 3 more

Open Access

https://doi.org/10.1038/srep11020

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Abstract

Trinucleotide repeat expansion disorders (TRED) are caused by genomic expansions of trinucleotide repeats, such as CTG and CAG. These expanded repeats are unstable in germline and somatic cells, with potential consequences for disease severity. Previous studies have demonstrated the involvement of DNA repair proteins in repeat instability, although the key factors affecting large repeat expansion and contraction are unclear. Here we investigated these factors in a human cell model harboring 800 CTG•CAG repeats by individually knocking down various DNA repair proteins using short interfering RNA. Knockdown of MSH2 and MSH3, which form the MutSβ heterodimer and function in mismatch repair, suppressed large repeat expansions, whereas knockdown of MSH6, which forms the MutSα heterodimer with MSH2, promoted large expansions exceeding 200 repeats by compensatory increases in MSH3 and the MutSβ complex. Knockdown of topoisomerase 1 (TOP1) and TDP1, which are involved in single-strand break repair, enhanced large repeat contractions. Furthermore, knockdown of senataxin, an RNA/DNA helicase which affects DNA:RNA hybrid formation and transcription-coupled nucleotide excision repair, exacerbated repeat instability in both directions. These results indicate that DNA repair factors, such as MutSβ play important roles in large repeat expansion and contraction, and can be an excellent therapeutic target for TRED.

Highlights

More than 20 human neurodegenerative diseases are caused by trinucleotide repeat expansions in genomic DNA, including Huntington disease (HD) and several forms of spinocerebellar ataxia (SCA; CAG expansions), as well as myotonic dystrophy type 1 (DM1; CTG expansions) [reviewed by1]
We demonstrated the importance of MutSβ with respect to large repeat expansion using a simple knockdown method in our human cell model
These results indicate that MutSβ enhances the repeat instability of both short and long expansion as well as different repeat motifs

Summary

Materials and methods

The expanded CTG repeats were sized by small-pool PCR followed by Southern blot as described previously[15]. RNA was prepared from human brain samples as described previously[17]. Proteins were extracted from human brain samples by mechanical homogenization in lysis buffer [0.125-M, Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate (SDS), 10% glycerol] containing Protease Inhibitor Cocktail (Sigma–Aldrich). Chromatin immunoprecipitation (ChIP) assays were performed using an EZ-ChIP kit (Merck Millipore) according to the manufacturer’s instructions. The quantitative PCR-based analysis with primers specific for the region downstream of the CTG repeats was performed as described previously[14]. Χ 2-tests were performed to compare the frequencies of expanded, unchanged, and contracted alleles in each set of experiments as reported previously[14]. Paired t-tests were performed for expression analysis of mRNA and protein

Result

Discussion