All three MutL complexes are required for repeat expansion in a human stem cell model of CAG-repeat expansion mediated glutaminase deficiency.

The Repeat Expansion Diseases (REDs) arise from the expansion of a disease-specific short tandem repeat (STR). Different REDs differ with respect to the repeat involved, the cells that are most expansion prone and the extent of expansion. Furthermore, whether these diseases share a common expansion mechanism is unclear. To date, expansion has only been studied in a limited number of REDs. Here we report the first studies of the expansion mechanism in induced pluripotent stem cells derived from a patient with a form of the glutaminase deficiency disorder known as Global Developmental Delay, Progressive Ataxia, And Elevated Glutamine (GDPAG; OMIM# 618412) caused by the expansion of a CAG-STR in the 5' UTR of the glutaminase (GLS) gene. We show that alleles with as few as ~ 120 repeats show detectable expansions in culture despite relatively low levels of R-loops formed at this locus. Additionally, using a CRISPR-Cas9 knockout approach we show that PMS2 and MLH3, the constituents of MutLα and MutLγ, the 2 mammalian MutL complexes known to be involved in mismatch repair (MMR), are essential for expansion. Furthermore, PMS1, a component of a less well understood MutL complex, MutLß, is also important, if not essential, for repeat expansion in these cells. Our results provide insights into the factors important for expansion and lend weight to the idea that, despite some differences, the same mechanism is responsible for expansion in many, if not all, REDs.

All three MutL complexes are required for repeat expansion in a human stem cell model of CAG-repeat expansion mediated glutaminase deficiency.

The Repeat Expansion Diseases (REDs) arise from the expansion of a disease-specific short tandem repeat (STR). Different REDs differ with respect to the repeat involved, the cells that are most expansion prone and the extent of expansion. Furthermore, whether these diseases share a common expansion mechanism is unclear. To date, expansion has only been studied in a limited number of REDs. Here we report the first studies of the expansion mechanism in induced pluripotent stem cells derived from a patient with a form of the glutaminase deficiency disorder known as Global Developmental Delay, Progressive Ataxia, And Elevated Glutamine (GDPAG; OMIM# 618412) caused by the expansion of a CAG-STR in the 5' UTR of the glutaminase (GLS) gene. We show that alleles with as few as ~120 repeats show detectable expansions in culture despite relatively low levels of R-loops formed at this locus. Additionally, using a CRISPR-Cas9 knockout approach we show that PMS2 and MLH3, the constituents of MutLα and MutLγ, the 2 mammalian MutL complexes known to be involved in mismatch repair (MMR), are essential for expansion. Furthermore, PMS1, a component of a less well understood MutL complex, MutLß, is also important, if not essential, for repeat expansion in these cells. Our results provide insights into the factors important for expansion and lend weight to the idea that, despite some differences, the same mechanism is responsible for expansion in many, if not all, REDs.

Sustained expression of FMR1 mRNA from reactivated fragile X syndrome alleles after treatment with small molecules that prevent trimethylation of H3K27.

Expansion of a CGG-repeat tract in the 5'-untranslated region of the FMR1 gene to >200 repeats results in epigenetic silencing of the gene by a mechanism that is still unknown. FMR1 gene silencing results in fragile X syndrome (FXS), the most common heritable cause of intellectual disability. We have previously shown that reactivation of the FMR1 gene in FXS cells with 5-azadeoxycytidine (AZA) leads to the transient recruitment of EZH2, the polycomb repressive complex 2 (PRC2) component responsible for H3K27 trimethylation, and that this recruitment depends on the presence of the FMR1 transcript. However, whether H3K27 trimethylation was essential for FMR1 re-silencing was not known. We show here that EZH2 inhibitors increased FMR1 expression and significantly delayed re-silencing of the FMR1 gene in AZA-treated FXS cells. This delay occurred despite the fact that EZH2 inhibition did not prevent the return of DNA methylation. Treatment with compound 1a, a small molecule that targets CGG-repeats in the FMR1 mRNA, also resulted in sustained expression of the FMR1 gene in AZA-treated cells. This effect of 1a was also associated with a decrease in the levels of H3K27 trimethylation but not DNA methylation. Thus, our data show that EZH2 plays a critical role in the FMR1 gene silencing process and that its inhibition can prolong expression of the FMR1 gene even in the presence of its transcript.

Mutsß generates both expansions and contractions in a mouse model of the Fragile X-associated disorders.

Fragile X-associated disorders are Repeat Expansion Diseases that result from expansion of a CGG/CCG-repeat in the FMR1 gene. Contractions of the repeat tract also occur, albeit at lower frequency. However, these contractions can potentially modulate disease symptoms or generate an allele with repeat numbers in the normal range. Little is known about the expansion mechanism and even less about contractions. We have previously demonstrated that the mismatch repair (MMR) protein MSH2 is required for expansions in a mouse model of these disorders. Here, we show that MSH3, the MSH2-binding partner in the MutSß complex, is required for 98% of germ line expansions and all somatic expansions in this model. In addition, we provide evidence for two different contraction mechanisms that operate in the mouse model, a MutSß-independent one that generates small contractions and a MutSß-dependent one that generates larger ones. We also show that MutSß complexes formed with the repeats have altered kinetics of ATP hydrolysis relative to complexes with bona fide MMR substrates and that MutSß increases the stability of the CCG-hairpins at physiological temperatures. These data may have important implications for our understanding of the mechanism(s) of repeat instability and for the role of MMR proteins in this process.

Somatic expansion in mouse and human carriers of fragile X premutation alleles.

Repeat expansion diseases result from expansion of a specific tandem repeat. The three fragile X-related disorders (FXDs) arise from germline expansions of a CGG•CCG repeat tract in the 5' UTR (untranslated region) of the fragile X mental retardation 1 (FMR1) gene. We show here that in addition to germline expansion, expansion also occurs in the somatic cells of both mice and humans carriers of premutation alleles. Expansion in mice primarily affects brain, testis, and liver with very little expansion in heart or blood. Our data would be consistent with a simple two-factor model for the organ specificity. Somatic expansion in humans may contribute to the mosaicism often seen in individuals with one of the FXDs. Because expansion risk and disease severity are related to repeat number, somatic expansion may exacerbate disease severity and contribute to the age-related increased risk of expansion seen on paternal transmission in humans. As little somatic expansion occurs in murine lymphocytes, our data also raise the possibility that there may be discordance in humans between repeat numbers measured in blood and that present in brain. This could explain, at least in part, the variable penetrance seen in some of these disorders.

The role of DNA damage response pathways in chromosome fragility in Fragile X syndrome.

FRAXA is one of a number of fragile sites in human chromosomes that are induced by agents like fluorodeoxyuridine (FdU) that affect intracellular thymidylate levels. FRAXA coincides with a >200 CGG*CCG repeat tract in the 5' UTR of the FMR1 gene, and alleles prone to fragility are associated with Fragile X (FX) syndrome, one of the leading genetic causes of intellectual disability. Using siRNA depletion, we show that ATR is involved in protecting the genome against FdU-induced chromosome fragility. We also show that FdU increases the number of gamma-H2AX foci seen in both normal and patient cells and increases the frequency with which the FMR1 gene colocalizes with these foci in patient cells. In the presence of FdU and KU55933, an ATM inhibitor, the incidence of chromosome fragility is reduced, suggesting that ATM contributes to FdU-induced chromosome fragility. Since both ATR and ATM are involved in preventing aphidicolin-sensitive fragile sites, our data suggest that the lesions responsible for aphidicolin-induced and FdU-induced fragile sites differ. FRAXA also displays a second form of chromosome fragility in absence of FdU, which our data suggest is normally prevented by an ATM-dependent process.