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

Nakatani R, Nakamori M, Fujimura H, Mochizuki H, Takahashi MP - Sci Rep (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Osaka University Graduate School of Medicine.

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.

No MeSH data available.


Related in: MedlinePlus

Effects of sustained trans-factor knockdown on CTG•CAG repeat instability in HT1080-800R cells.Repeat instability was analyzed by small-pool PCR followed by Southern blotting. Histograms show the repeat-length distributions in the HT1080-800R cells treated with each siRNA. The frequency distribution of unstable alleles is indicated as gray bars. The frequency of stable alleles is indicated as black bars. Allele lengths are grouped in bins spanning 50 repeats. More than 50 alleles were sized per group.
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f2: Effects of sustained trans-factor knockdown on CTG•CAG repeat instability in HT1080-800R cells.Repeat instability was analyzed by small-pool PCR followed by Southern blotting. Histograms show the repeat-length distributions in the HT1080-800R cells treated with each siRNA. The frequency distribution of unstable alleles is indicated as gray bars. The frequency of stable alleles is indicated as black bars. Allele lengths are grouped in bins spanning 50 repeats. More than 50 alleles were sized per group.

Mentions: MMR proteins have been extensively studied to elucidate the mechanism of repeat instability. MSH2 was especially suggested as a potential promoter of repeat instability both in vitro and in vivo1. In our study, sustained MSH2 knockdown significantly reduced both the expansion and contraction modes of repeat instability and eliminated “big jumps” involving expansions of several hundred repeats (Fig. 2 and Supplemental Figure S2). The cumulative frequency of unstable alleles in siMSH2-treated cells was 27.1% (6.8% expansions and 20.3% contractions), whereas that in control-treated cells was 65.4% (21.8% expansions and 43.6% contractions; Table 1). Similarly, knockdown of MSH3, which forms MutSβ with MSH2, also reduced repeat instability, especially expansions (6.5% expansion versus 54.5% unchanged versus 39.0% contraction for all alleles; Fig. 2 and Table 1). Previous cell model-based studies reported that knockdown of MSH6, which forms MutSα with MSH2, did not affect repeat instability81218. However, in our study, MSH6 knockdown strongly enhanced repeat instability with a bias toward expansion (41.8% expansion versus 25.5% unchanged versus 32.7% contraction for all alleles; Fig. 2 and Table 1). Many alleles exhibited changes of >200 repeats and some alleles gained >1000-CTG repeat expansions (average change in the repeat size: +21 CTG repeats versus −90 in the control). In contrast, knockdown of MLH1 or PMS2, which form the MutLα heterodimer and act downstream of MutS homologue mismatch recognition, did not affect repeat instability.


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

Nakatani R, Nakamori M, Fujimura H, Mochizuki H, Takahashi MP - Sci Rep (2015)

Effects of sustained trans-factor knockdown on CTG•CAG repeat instability in HT1080-800R cells.Repeat instability was analyzed by small-pool PCR followed by Southern blotting. Histograms show the repeat-length distributions in the HT1080-800R cells treated with each siRNA. The frequency distribution of unstable alleles is indicated as gray bars. The frequency of stable alleles is indicated as black bars. Allele lengths are grouped in bins spanning 50 repeats. More than 50 alleles were sized per group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4457148&req=5

f2: Effects of sustained trans-factor knockdown on CTG•CAG repeat instability in HT1080-800R cells.Repeat instability was analyzed by small-pool PCR followed by Southern blotting. Histograms show the repeat-length distributions in the HT1080-800R cells treated with each siRNA. The frequency distribution of unstable alleles is indicated as gray bars. The frequency of stable alleles is indicated as black bars. Allele lengths are grouped in bins spanning 50 repeats. More than 50 alleles were sized per group.
Mentions: MMR proteins have been extensively studied to elucidate the mechanism of repeat instability. MSH2 was especially suggested as a potential promoter of repeat instability both in vitro and in vivo1. In our study, sustained MSH2 knockdown significantly reduced both the expansion and contraction modes of repeat instability and eliminated “big jumps” involving expansions of several hundred repeats (Fig. 2 and Supplemental Figure S2). The cumulative frequency of unstable alleles in siMSH2-treated cells was 27.1% (6.8% expansions and 20.3% contractions), whereas that in control-treated cells was 65.4% (21.8% expansions and 43.6% contractions; Table 1). Similarly, knockdown of MSH3, which forms MutSβ with MSH2, also reduced repeat instability, especially expansions (6.5% expansion versus 54.5% unchanged versus 39.0% contraction for all alleles; Fig. 2 and Table 1). Previous cell model-based studies reported that knockdown of MSH6, which forms MutSα with MSH2, did not affect repeat instability81218. However, in our study, MSH6 knockdown strongly enhanced repeat instability with a bias toward expansion (41.8% expansion versus 25.5% unchanged versus 32.7% contraction for all alleles; Fig. 2 and Table 1). Many alleles exhibited changes of >200 repeats and some alleles gained >1000-CTG repeat expansions (average change in the repeat size: +21 CTG repeats versus −90 in the control). In contrast, knockdown of MLH1 or PMS2, which form the MutLα heterodimer and act downstream of MutS homologue mismatch recognition, did not affect repeat instability.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Osaka University Graduate School of Medicine.

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.

No MeSH data available.


Related in: MedlinePlus