Xiao-Nan Zhao

The Mismatch Repair Protein MSH2 is Rate Limiting for Repeat Expansion in a Fragile X Premutation Mouse Model

Fragile X‐associated tremor and ataxia syndrome, Fragile X‐associated primary ovarian insufficiency, and Fragile X syndrome are Repeat Expansion Diseases caused by expansion of a CGG•CCG‐repeat microsatellite in the 5′ UTR of the FMR1 gene. To help understand the expansion mechanism responsible for these disorders, we have crossed mice containing ∼147 CGG•CCG repeats in the endogenous murine Fmr1 gene with mice containing a null mutation in the gene encoding the mismatch repair protein MSH2. MSH2 mutations are associated with elevated levels of generalized microsatellite instability. However, we show here for the first time that in the FX mouse model, all maternally and paternally transmitted expansions require Msh2. Even the loss of one Msh2 allele reduced the intergenerational expansion frequency significantly. Msh2 is also required for all somatic expansions and loss of even one functional Msh2 allele reduced the extent of somatic expansion in some organs. Tissues with lower levels of MSH2 were more sensitive to the loss of a single Msh2 allele. This suggests that MSH2 is rate limiting for expansion in this mouse model and that MSH2 levels may be a key factor that accounts for tissue‐specific differences in expansion risk.

Repeat-mediated genetic and epigenetic changes at the FMR1 locus in the Fragile X-related disorders

The Fragile X-related disorders are a group of genetic conditions that include the neurodegenerative disorder, Fragile X-associated tremor/ataxia syndrome (FXTAS), the fertility disorder, Fragile X-associated primary ovarian insufficiency (FXPOI) and the intellectual disability, Fragile X syndrome (FXS). The pathology in all these diseases is related to the number of CGG/CCG-repeats in the 5′ UTR of the Fragile X mental retardation 1 (FMR1) gene. The repeats are prone to continuous expansion and the increase in repeat number has paradoxical effects on gene expression increasing transcription on mid-sized alleles and decreasing it on longer ones. In some cases the repeats can simultaneously both increase FMR1 mRNA production and decrease the levels of the FMR1 gene product, Fragile X mental retardation 1 protein (FMRP). Since FXTAS and FXPOI result from the deleterious consequences of the expression of elevated levels of FMR1 mRNA and FXS is caused by an FMRP deficiency, the clinical picture is turning out to be more complex than once appreciated. Added complications result from the fact that increasing repeat numbers make the alleles somatically unstable. Thus many individuals have a complex mixture of different sized alleles in different cells. Furthermore, it has become apparent that the eponymous fragile site, once thought to be no more than a useful diagnostic criterion, may have clinical consequences for females who inherit chromosomes that express this site. This review will cover what is currently known about the mechanisms responsible for repeat instability, for the repeat-mediated epigenetic changes that affect expression of the FMR1 gene, and for chromosome fragility. It will also touch on what current and future options are for ameliorating some of these effects.

The Repeat Expansion Diseases: The dark side of DNA repair

DNA repair normally protects the genome against mutations that threaten genome integrity and thus cell viability. However, growing evidence suggests that in the case of the Repeat Expansion Diseases, disorders that result from an increase in the size of a disease-specific microsatellite, the disease-causing mutation is actually the result of aberrant DNA repair. A variety of proteins from different DNA repair pathways have thus far been implicated in this process. This review will summarize recent findings from patients and from mouse models of these diseases that shed light on how these pathways may interact to cause repeat expansion.

Gender and cell-type specific effects of the transcription coupled repair protein, ERCC6/CSB, on repeat expansion in a mouse model of the Fragile X-related disorders

The repeat expansion diseases are human genetic disorders that arise from the expansion of a tandem‐repeat tract. The Fragile X‐related disorders are members of this disease group in which the repeat unit is CGG/CCG and is located in the 5′ untranslated region of the FMR1 gene. Affected individuals often show mosaicism with respect to repeat number resulting from both expansion and contraction of the repeat tract; however, the mechanism responsible for these changes in repeat number is unknown. The work from a variety of model systems suggests that transcription‐coupled repair (TCR) may contribute to repeat instability in diseases resulting from CAG/CTG‐repeat expansion. To test whether TCR could contribute to repeat instability in the Fragile X‐related disorders, we tested the effect of mutations in Csb (Cockayne syndrome group B), a gene essential for TCR, in a knock‐in mouse model of these disorders. We found that the loss of CSB affects expansions in a gender and cell‐type‐specific manner. Our data also show an unanticipated gender difference in instability even in Csb+/+ animals that may have implications for our understanding of the mechanism of repeat expansion in the FX mouse model and perhaps for humans as well.

Mutsβ 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 MutSβ 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 MutSβ-independent one that generates small contractions and a MutSβ-dependent one that generates larger ones. We also show that MutSβ complexes formed with the repeats have altered kinetics of ATP hydrolysis relative to complexes with bona fide MMR substrates and that MutSβ 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.

A MutSβ-Dependent Contribution of MutSα to Repeat Expansions in Fragile X Premutation Mice?

The fragile X-related disorders result from expansion of a CGG/CCG microsatellite in the 5’ UTR of the FMR1 gene. We have previously demonstrated that the MSH2/MSH3 complex, MutSβ, that is important for mismatch repair, is essential for almost all expansions in a mouse model of these disorders. Here we show that the MSH2/MSH6 complex, MutSα also contributes to the production of both germ line and somatic expansions as evidenced by the reduction in the number of expansions observed in Msh6-/- mice. This effect is not mediated via an indirect effect of the loss of MSH6 on the level of MSH3. However, since MutSβ is required for 98% of germ line expansions and almost all somatic ones, MutSα is apparently not able to efficiently substitute for MutSβ in the expansion process. Using purified human proteins we demonstrate that MutSα, like MutSβ, binds to substrates with loop-outs of the repeats and increases the thermal stability of the structures that they form. We also show that MutSα facilitates binding of MutSβ to these loop-outs. These data suggest possible models for the contribution of MutSα to repeat expansion. In addition, we show that unlike MutSβ, MutSα may also act to protect against repeat contractions in the Fmr1 gene.

The transcription-coupled repair protein ERCC6/CSB also protects against repeat expansion in a mouse model of the fragile X premutation.

The fragile X‐related disorders (FXDs) are members of the group of diseases known as the repeat expansion diseases. The FXDs result from expansion of an unstable CGG/CCG repeat tract in the 5′ UTR of the FMR1 gene. Contractions are also seen, albeit at lower frequency. We have previously shown that ERCC6/CSB plays an auxiliary role in promoting germ line and somatic expansions in a mouse model of the FXDs. However, work in model systems of other repeat expansion diseases has suggested that CSB may protect against expansions by promoting contractions. Since FXD mice normally have such a high expansion frequency, it is possible that such a protective effect would have been masked. We thus examined the effect of the loss of CSB in an Msh2+/− background where the germ line expansion frequency is reduced and in an Msh2−/− background where expansions do not occur, but contractions do. Our data show that in addition to promoting repeat expansion, CSB does in fact protect the genome from germ line expansions in the FXD mouse model. However, it likely does so not by promoting contractions but by promoting an error‐free process that preserves the parental allele.

Ups and Downs: Mechanisms of Repeat Instability in the Fragile X-Related Disorders

The Fragile X-related disorders (FXDs) are a group of clinical conditions resulting from the expansion of a CGG/CCG-repeat tract in exon 1 of the Fragile X mental retardation 1 (FMR1) gene. While expansions of the repeat tract predominate, contractions are also seen with the net result being that individuals can show extensive repeat length heterogeneity in different tissues. The mechanisms responsible for expansion and contraction are still not well understood. This review will discuss what is known about these processes and current evidence that supports a model in which expansion arises from the interaction of components of the base excision repair, mismatch repair and transcription coupled repair pathways.

Timing of Expansion of Fragile X Premutation Alleles During Intergenerational Transmission in a Mouse Model of the Fragile X-Related Disorders

Fragile X syndrome (FXS) is caused by the maternal expansion of an unstable CGG-repeat tract located in the first exon of the FMR1 gene. Further changes in repeat number occur during embryogenesis resulting in individuals sometimes being highly mosaic. Here we show in a mouse model that, in males, expansions are already present in primary spermatocytes with no additional expansions occurring in later stages of gametogenesis. We also show that, in females, expansion occurs in the post-natal oocyte. Additional expansions and a high frequency of large contractions are seen in two-cell stage embryos. Expansion in oocytes, which are non-dividing, would be consistent with a mechanism involving aberrant DNA repair or recombination rather than a problem with chromosomal replication. Given the difficulty of replicating large CGG-repeat tracts, we speculate that very large expanded alleles may be prone to contract in the mitotically proliferating spermatagonial stem cells in men. However, expanded alleles may not be under such pressure in the non-dividing oocyte. The high degree of both expansions and contractions seen in early embryos may contribute to the high frequency of somatic mosaicism that is observed in humans. Our data thus suggest an explanation for the fact that FXS is exclusively maternally transmitted and lend support to models for repeat expansion that are based on problems arising during DNA repair.

MutLγ promotes repeat expansion in a Fragile X mouse model while EXO1 is protective

The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases resulting from an expansion of a CGG-repeat tract at the 5’ end of the FMR1 gene. The mechanism responsible for this unusual mutation is not fully understood. We have previously shown that mismatch repair (MMR) complexes, MSH2/MSH3 (MutSβ) and MSH2/MSH6 (MutSα), together with Polβ, a DNA polymerase important for base excision repair (BER), are important for expansions in a mouse model of these disorders. Here we show that MLH1/MLH3 (MutLγ), a protein complex that can act downstream of MutSβ in MMR, is also required for all germ line and somatic expansions. However, exonuclease I (EXO1), which acts downstream of MutL proteins in MMR, is not required. In fact, a null mutation in Exo1 results in more extensive germ line and somatic expansions than is seen in Exo1+/+ animals. Furthermore, mice homozygous for a point mutation (D173A) in Exo1 that eliminates its nuclease activity but retains its native conformation, shows a level of expansion that is intermediate between Exo1+/+ and Exo1-/- animals. Thus, our data suggests that expansion of the FX repeat in this mouse model occurs via a MutLγ-dependent, EXO1-independent pathway, with EXO1 protecting against expansion both in a nuclease-dependent and a nuclease-independent manner. Our data thus have implications for the expansion mechanism and add to our understanding of the genetic factors that may be modifiers of expansion risk in humans.