MSH3 polymorphisms and protein levels affect CAG repeat instability in huntington's disease mice

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)~100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases

CTCF cis-Regulates Trinucleotide Repeat Instability in an Epigenetic Manner: A Novel Basis for Mutational Hot Spot Determination

At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting “instability elements,” and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF—a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability

A novel approach to investigate tissue-specific trinucleotide repeat instability

Abstract Background In Huntington's disease (HD), an expanded CAG repeat produces characteristic striatal neurodegeneration. Interestingly, the HD CAG repeat, whose length determines age at onset, undergoes tissue-specific somatic instability, predominant in the striatum, suggesting that tissue-specific CAG length changes could modify the disease process. Therefore, understanding the mechanisms underlying the tissue specificity of somatic instability may provide novel routes to therapies. However progress in this area has been hampered by the lack of sensitive high-throughput instability quantification methods and global approaches to identify the underlying factors. Results Here we describe a novel approach to gain insight into the factors responsible for the tissue specificity of somatic instability. Using accurate genetic knock-in mouse models of HD, we developed a reliable, high-throughput method to quantify tissue HD CAG repeat instability and integrated this with genome-wide bioinformatic approaches. Using tissue instability quantified in 16 tissues as a phenotype and tissue microarray gene expression as a predictor, we built a mathematical model and identified a gene expression signature that accurately predicted tissue instability. Using the predictive ability of this signature we found that somatic instability was not a consequence of pathogenesis. In support of this, genetic crosses with models of accelerated neuropathology failed to induce somatic instability. In addition, we searched for genes and pathways that correlated with tissue instability. We found that expression levels of DNA repair genes did not explain the tissue specificity of somatic instability. Instead, our data implicate other pathways, particularly cell cycle, metabolism and neurotransmitter pathways, acting in combination to generate tissue-specific patterns of instability. Conclusion Our study clearly demonstrates that multiple tissue factors reflect the level of somatic instability in different tissues. In addition, our quantitative, genome-wide approach is readily applicable to high-throughput assays and opens the door to widespread applications with the potential to accelerate the discovery of drugs that alter tissue instability

Continuous and Periodic Expansion of CAG Repeats in Huntington's Disease R6/1 Mice

Huntington's disease (HD) is one of several neurodegenerative disorders caused by expansion of CAG repeats in a coding gene. Somatic CAG expansion rates in HD vary between organs, and the greatest instability is observed in the brain, correlating with neuropathology. The fundamental mechanisms of somatic CAG repeat instability are poorly understood, but locally formed secondary DNA structures generated during replication and/or repair are believed to underlie triplet repeat expansion. Recent studies in HD mice have demonstrated that mismatch repair (MMR) and base excision repair (BER) proteins are expansion inducing components in brain tissues. This study was designed to simultaneously investigate the rates and modes of expansion in different tissues of HD R6/1 mice in order to further understand the expansion mechanisms in vivo. We demonstrate continuous small expansions in most somatic tissues (exemplified by tail), which bear the signature of many short, probably single-repeat expansions and contractions occurring over time. In contrast, striatum and cortex display a dramatic—and apparently irreversible—periodic expansion. Expansion profiles displaying this kind of periodicity in the expansion process have not previously been reported. These in vivo findings imply that mechanistically distinct expansion processes occur in different tissues

Histone Deacetylase Complexes Promote Trinucleotide Repeat Expansions

Genetic analysis in budding yeast and in cultured human astrocytes reveals that specific histone deacetylase complexes accelerate expansion mutations in DNA triplet repeats

Onset and Progression of Behavioral and Molecular Phenotypes in a Novel Congenic R6/2 Line Exhibiting Intergenerational CAG Repeat Stability

In the present study we report on the use of speed congenics to generate a C57BL/6J congenic line of HD-model R6/2 mice carrying 110 CAG repeats, which uniquely exhibits minimal intergenerational instability. We also report the first identification of the R6/2 transgene insertion site. The relatively stable line of 110 CAG R6/2 mice was characterized for the onset of behavioral impairments in motor, cognitive and psychiatric-related phenotypes as well as the progression of disease-related impairments from 4 to 10 weeks of age. 110Q mice exhibited many of the phenotypes commonly associated with the R6/2 model including reduced activity and impairments in rotarod performance. The onset of many of the phenotypes occurred around 6 weeks and was progressive across age. In addition, some phenotypes were observed in mice as early as 4 weeks of age. The present study also reports the onset and progression of changes in several molecular phenotypes in the novel R6/2 mice and the association of these changes with behavioral symptom onset and progression. Data from TR-FRET suggest an association of mutant protein state changes (soluble versus aggregated) in disease onset and progression

Breast Cancer Stem-Like Cells Are Inhibited by a Non-Toxic Aryl Hydrocarbon Receptor Agonist

Cancer stem cells (CSCs) have increased resistance to cancer chemotherapy. They can be enriched as drug-surviving CSCs (D-CSCs) by growth with chemotherapeutic drugs, and/or by sorting of cells expressing CSC markers such as aldehyde dehydrogenase-1 (ALDH). CSCs form colonies in agar, mammospheres in low-adherence cultures, and tumors following xenotransplantation in Scid mice. We hypothesized that tranilast, a non-toxic orally active drug with anti-cancer activities, would inhibit breast CSCs.We examined breast cancer cell lines or D-CSCs generated by growth of these cells with mitoxantrone. Tranilast inhibited colony formation, mammosphere formation and stem cell marker expression. Mitoxantrone-selected cells were enriched for CSCs expressing stem cell markers ALDH, c-kit, Oct-4, and ABCG2, and efficient at forming mammospheres. Tranilast markedly inhibited mammosphere formation by D-CSCs and dissociated formed mammospheres, at pharmacologically relevant concentrations. It was effective against D-CSCs of both HER-2+ and triple-negative cell lines. Tranilast was also effective in vivo, since it prevented lung metastasis in mice injected i.v. with triple-negative (MDA-MB-231) mitoxantrone-selected cells. The molecular targets of tranilast in cancer have been unknown, but here we demonstrate it is an aryl hydrocarbon receptor (AHR) agonist and this plays a key role. AHR is a transcription factor activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatic hydrocarbons and other ligands. Tranilast induced translocation of the AHR to the nucleus and stimulated CYP1A1 expression (a marker of AHR activation). It inhibited binding of the AHR to CDK4, which has been linked to cell-cycle arrest. D-CSCs expressed higher levels of the AHR than other cells. Knockdown of the AHR with siRNA, or blockade with an AHR antagonist, entirely abrogated the anti-proliferative and anti-mammosphere activity of tranilast. Thus, the anti-cancer effects of tranilast are AHR dependent.We show that tranilast is an AHR agonist with inhibitory effects on breast CSCs. It is effective against CSCs of triple-negative breast cancer cells selected for anti-cancer drug resistance. These results suggest it might find applications in the treatment of breast cancer

The GAA triplet-repeat is unstable in the context of the human FXN locus and displays age-dependent expansions in cerebellum and DRG in a transgenic mouse model

Friedreich ataxia (FRDA) is caused by homozygosity for FXN alleles containing an expanded GAA triplet-repeat (GAA-TR) sequence. This expanded GAA-TR sequence is unstable in somatic cells of FRDA patients, showing age-dependent expansions in dorsal root ganglia (DRG), the tissue where pathology occurs earliest and is most significant. This is thought to be the basis for the progressive, tissue-specific pathology seen in FRDA, but the mechanism(s) for this somatic instability is unknown. We show that transgenic mice containing the expanded GAA-TR sequence (190 or 82 triplets) in the context of the human FXN locus show tissue-specific and age-dependent somatic instability that mimics the human condition. Small pool PCR analysis, which allows quantitative analysis of instability by assaying individual transgenes in vivo, showed age-dependent expansions specifically in the cerebellum and DRG. The (GAA)190 allele showed some instability by 2 months, progressed at about 0.3 – 0.4 triplets/week, resulting in a significant number of expansions by 12 months. Repeat length determined the age of onset of somatic instability, and the rate and magnitude of expansion. Whereas the GAA-TR was unstable in the context of the human FXN locus, pure GAATR sequences at other genetic loci in the human and murine genomes showed no instability. These data indicate that somatic instability of the GAA-TR sequence in the human FXN gene is determined by a combination of unique cis and trans-acting factors. This mouse model will serve as a useful tool to delineate the mechanism(s) of diseasespecific somatic instability in FRDA

Progressive GAA·TTC Repeat Expansion in Human Cell Lines

Trinucleotide repeat expansion is the genetic basis for a sizeable group of inherited neurological and neuromuscular disorders. Friedreich ataxia (FRDA) is a relentlessly progressive neurodegenerative disorder caused by GAA·TTC repeat expansion in the first intron of the FXN gene. The expanded repeat reduces FXN mRNA expression and the length of the repeat tract is proportional to disease severity. Somatic expansion of the GAA·TTC repeat sequence in disease-relevant tissues is thought to contribute to the progression of disease severity during patient aging. Previous models of GAA·TTC instability have not been able to produce substantial levels of expansion within an experimentally useful time frame, which has limited our understanding of the molecular basis for this expansion. Here, we present a novel model for studying GAA·TTC expansion in human cells. In our model system, uninterrupted GAA·TTC repeat sequences display high levels of genomic instability, with an overall tendency towards progressive expansion. Using this model, we characterize the relationship between repeat length and expansion. We identify the interval between 88 and 176 repeats as being an important length threshold where expansion rates dramatically increase. We show that expansion levels are affected by both the purity and orientation of the repeat tract within the genomic context. We further demonstrate that GAA·TTC expansion in our model is independent of cell division. Using unique reporter constructs, we identify transcription through the repeat tract as a major contributor to GAA·TTC expansion. Our findings provide novel insight into the mechanisms responsible for GAA·TTC expansion in human cells