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Huntington’s disease (HD) is one of a large group of human disorders that are caused by expanded DNA repeats. These repeat expansion disorders can have repeat units of different size and sequence that can be located in any part of the gene and, while the pathological consequences of the expansion can differ widely, there is evidence to suggest that the underlying mutational mechanism may be similar. In the case of HD, the expanded repeat unit is a CAG trinucleotide located in exon 1 of the huntingtin (HTT) gene, resulting in an expanded polyglutamine tract in the huntingtin protein. Expansion results in neuronal cell death, particularly in the striatum. Emerging evidence suggests that somatic CAG expansion, specifically expansion occurring in the brain during the lifetime of an individual, contributes to an earlier disease onset and increased severity. In this review we will discuss mouse models of two non-CAG repeat expansion diseases, specifically the Fragile X-related disorders (FXDs) and Friedreich ataxia (FRDA). We will compare and contrast these models with mouse and patient-derived cell models of various other repeat expansion disorders and the relevance of these findings for somatic expansion in HD. We will also describe additional genetic factors and pathways that modify somatic expansion in the FXD mouse model for which no comparable data yet exists in HD mice or humans. These additional factors expand the potential druggable space for diseases like HD where somatic expansion is a significant contributor to disease impact.

Heavy metal elements in particulate matter can cause adverse effects on human health, and the smaller the particle size, the greater the harm. A total of 16 heavy metal elements (Al, Si, K, Ca, V, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Ba, Pb, and Cd) in PM

Fragile X-related disorders (FXDs), also known as FMR1 disorders, are examples of repeat expansion diseases (REDs), clinical conditions that arise from an increase in the number of repeats in a disease-specific microsatellite. In the case of FXDs, the repeat unit is CGG/CCG and the repeat tract is located in the 5′ UTR of the X-linked FMR1 gene. Expansion can result in neurodegeneration, ovarian dysfunction, or intellectual disability depending on the number of repeats in the expanded allele. A growing body of evidence suggests that the mutational mechanisms responsible for many REDs share several common features. It is also increasingly apparent that in some of these diseases the pathologic consequences of expansion may arise in similar ways. It has long been known that many of the disease-associated repeats form unusual DNA and RNA structures. This review will focus on what is known about these structures, the proteins with which they interact, and how they may be related to the causative mutation and disease pathology in the FMR1 disorders.

Analysis of individual gametes has a number of applications in the study of the mechanism of repeat expansion in mouse models of the Fragile X-related disorders, as well as in mouse models of other Repeat Expansion Diseases. This chapter describes the techniques required to isolate oocytes and male gametes of different stages of maturity, along with the techniques required to accurately determine the repeat number in these gametes.

Expansion of a CGG-repeat tract in the 5’ untranslated region of the FMR1 gene causes the fragile X-related disorders (FXDs; aka the FMR1 disorders). The expansion mechanism is likely shared by the 35+ other diseases resulting from expansion of a disease-specific microsatellite, but many steps in this process are unknown. We have shown previously that expansion is dependent upon functional mismatch repair proteins, including an absolute requirement for MutLγ, one of the three MutL heterodimeric complexes found in mammalian cells. We demonstrate here that both MutLα and MutLβ, the two other MutL complexes present in mammalian cells, are also required for most, if not all, expansions in a mouse embryonic stem cell model of the FXDs. A role for MutLα and MutLβ is consistent with human GWA studies implicating these complexes as modifiers of expansion risk in other Repeat Expansion Diseases. The requirement for all three complexes suggests a novel model in which these complexes co-operate to generate expansions. It also suggests that the PMS1 subunit of MutLβ may be a reasonable therapeutic target in those diseases in which somatic expansion is an important disease modifier., Author summary Repeat Expansion Diseases, including the fragile X-related disorders, are a large group of human genetic disorders caused by a mutation in a disease-specific tandem repeat or microsatellite. This mutation increases the number of repeats in that microsatellite. Unusual features of this mutation include its high frequency and its absolute requirement for proteins involved in Mismatch Repair, some of the very proteins that normally protect against classical microsatellite instability. Proteins known to be essential for this mutation include MLH3, the MLH1 binding partner in MutLγ, one of the three mammalian MutL complexes. Here we show that PMS2 and PMS1, MLH1-binding partners in the remaining two MutL complexes, MutLα and MutLβ respectively, are also required for expansion in a cell-based model of the fragile X-related disorders. This has interesting implications for the mechanism of repeat expansion. It also identifies another potential therapeutic target for reducing the mutation responsible for these diseases.

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.

The Fragile X-associated disorders (FXDs) and Friedreich ataxia (FRDA) are genetic conditions resulting from expansion of a trinucleotide repeat in a region of the affected gene that is transcribed but not translated. In the case of the FXDs, pathology results from expansion of CGG•CCG-repeat tract in the 5' UTR of the FMR1 gene, while pathology in FRDA results from expansion of a GAA•TTC-repeat in intron 1 of the FXN gene. Expansion occurs during gametogenesis or early embryogenesis by a mechanism that is not well understood. Associated Expansion then produces disease pathology in various ways that are not completely understood either. In the case of the FXDs, alleles with 55-200 repeats express higher than normal levels of a transcript that is thought to be toxic, while alleles with200 repeats are silenced. In addition, alleles with200 repeats are associated with a cytogenetic abnormality known as a fragile site, which is apparent as a constriction or gap in the chromatin that is seen when cells are grown in presence of inhibitors of thymidylate synthase. FRDA alleles show a deficit of the FXN transcript. This review will address the role of repeat-mediated chromatin changes in these aspects of FXD and FRDA disease pathology. This article is part of a Special Issue entitled: Chromatin in time and space.

We have previously reported that the human ACAT1 gene produces a chimeric mRNA through the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG(1397-1399) and GGC(1274-1276) as translation initiation codons to produce normal 50-kDa ACAT1 and a novel enzymatically active 56-kDa isoform, respectively, with the latter being authentically present in human cells, including human monocyte-derived macrophages. In this work, we report that RNA secondary structures located in the vicinity of the GGC(1274-1276) codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops (nt 1255-1268, 1286-1342 and 1355-1384) were tested individually by transfecting expression plasmids into cells that contained the wild-type, deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame (ORF) or to the ORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop(1255-1268) from chromosome 7 and downstream stem-loop(1286-1342) from chromosome 1 were needed for production of the 56-kDa isoform, whereas the last stem-loop(1355-1384) from Chromosome 1 was dispensable. The results of experiments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC(1274-1276) codon was mediated by an internal ribosome entry site (IRES). Further experiments revealed that translation initiation from the GGC(1274-1276) codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACAT1 transcript.

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., Author Summary The repeat expansion diseases are a group of human genetic disorders that are caused by expansion of a specific microsatellite in a single affected gene. How this expansion occurs is unknown, but previous work in various models for different diseases in the group, including the fragile X-related disorders (FXDs), has implicated the mismatch repair complex MutSβ in the process. With the exception of somatic expansion in Friedreich ataxia, MutSα has not been reported to contribute to generation of expansions in other disease models. Here we show that MutSα does in fact play a role in both germ line and somatic expansions in a mouse model of the FXDs since the expansion frequency is significantly reduced in Msh6-/- mice. However, since we have previously shown that loss of MutSβ eliminates almost all expansions, MutSα is apparently not able to fully substitute for MutSβ in the expansion process. We also show here that MutSα increases the stability of the structures formed by the fragile X repeats that are thought to be the substrates for expansion and promotes binding of MutSβ to the repeats. This, together with our genetic data, suggests possible models for how MutSα and MutSβ, could co-operate to generate repeat expansions in the FXDs.

The Fragile X-related disorders are X-linked disorders resulting from the inheritance of FMR1 alleles with >54 CGG/CCG repeats in their 5' UTR. The repeats expand both somatically and on intergenerational transmission and increased repeat numbers are associated with increased risk of disease and increased risk of further expansion. The mechanism responsible for expansion is unknown. Here, we show in a knockin mouse model of these disorders that somatic expansion is much less common in females than in males. We show that this is due in large part to the fact that expansions occur only when the repeat is on the active X chromosome. However, even when this is taken into account, expansions in females are still less common than expected. This additional gender effect is not due to a protective effect of estrogen, a deleterious effect of testosterone or to differences in the expression of the Fmr1 gene or a variety of X-linked and autosomal DNA repair genes. However, our data do suggest that a higher level of expression of genes that protect against oxidative damage in females may contribute to their lower levels of expansion. Whatever the basis, our data suggest that the risk for somatic expansion may be lower in women than it is in men. This could help explain the reduced penetrance of some aspects of disease pathology in women. The fact that expansion only occurs when the Fmr1 allele is on the active X chromosome has important implications for the mechanism of repeat expansion.

The liver plays a central role in cholesterol homeostasis. It exclusively receives and metabolizes oxysterols, which are important metabolites of cholesterol and are more cytotoxic than free cholesterol, from all extrahepatic tissues. Hepatocellular carcinomas (HCCs) impair certain liver functions and cause pathological alterations in many processes including cholesterol metabolism. However, the link between an altered cholesterol metabolism and HCC development is unclear. Human ACAT2 is abundantly expressed in intestine and fetal liver. Our previous studies have shown that ACAT2 is induced in certain HCC tissues. Here, by investigating tissue samples from HCC patients and HCC cell lines, we report that a specific cholesterol metabolic pathway, involving induction of ACAT2 and esterification of excess oxysterols for secretion to avoid cytotoxicity, is established in a subset of HCCs for tumor growth. Inhibiting ACAT2 leads to the intracellular accumulation of unesterified oxysterols and suppresses the growth of both HCC cell lines and their xenograft tumors. Further mechanistic studies reveal that HCC-linked promoter hypomethylation is essential for the induction of ACAT2 gene expression. We postulate that specifically blocking this HCC-established cholesterol metabolic pathway may have potential therapeutic applications for HCC patients.

Trans-splicing, a process involving the cleavage and joining of two separate transcripts, can expand the transcriptome and proteome in eukaryotes. Chimeric RNAs generated by trans-splicing are increasingly described in literatures. The widespread presence of antibiotic resistance genes in natural environments and human intestines is becoming an important challenge for public health. Certain antibiotic resistance genes, such as ampicillin resistance gene (Amp(r)), are frequently used in recombinant plasmids. Until now, trans-splicing involving recombinant plasmid-derived exogenous transcripts and endogenous cellular RNAs has not been reported. Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a key enzyme involved in cellular cholesterol homeostasis. The 4.3-kb human ACAT1 chimeric mRNA can produce 50-kDa and 56-kDa isoforms with different enzymatic activities. Here, we show that human ACAT1 56-kDa isoform is produced from an mRNA species generated through the trans-splicing of an exogenous transcript encoded by the antisense strand of Amp(r) (asAmp) present in common Amp(r)-plasmids and the 4.3-kb endogenous ACAT1 chimeric mRNA, which is presumably processed through a prior event of interchromosomal trans-splicing. Strikingly, DNA fragments containing the asAmp with an upstream recombined cryptic promoter and the corresponding exogenous asAmp transcripts have been detected in human cells. Our findings shed lights on the mechanism of human ACAT1 56-kDa isoform production, reveal an exogenous-endogenous trans-splicing system, in which recombinant plasmid-derived exogenous transcripts are linked with endogenous cellular RNAs in human cells, and suggest that exogenous DNA might affect human gene expression at both DNA and RNA levels.

We have previously reported that human ACAT1 mRNAs produce the 50 kDa protein using the AUG(11397-1399) initiation codon, and also a minor 56 kDa isoform using the upstream in-frame GGC(1274-1276) initiation codon. The GGC(1274-1276) codon is located at the optional long 5'-untranslated region (5'-UTR, nt 1-1396) of the mRNAs. The DNA sequences corresponding to this 5'-UTR are located in two different chromosomes, 7 and 1. In the current work, we report that the optional long 5'-UTR significantly impairs the production of human ACAT1 protein initiated from the AUG(1397-1399)codon, mainly by promoting its mRNA decay. The western blot analyses indicated that the optional long 5'-UTR potently impaired the production of different proteins initiated from the AUG(1397-1399) codon, meaning that this impairing effect was not influenced by the 3'-UTR or the coding sequence of ACAT1 mRNA. The results of reverse transcription-quantitative polymerase chain reaction demonstrated that this 5'- UTR dramatically reduced the contents of its linked mRNAs. Analyses of the protein to mRNA ratios showed that this 5'-UTR mainly decreased its mRNA stability rather than altering its translational efficiency. We next performed the plasmid transfection experiments and used actinomycin D to inhibit transcription. The results showed that this 5'-UTR promoted its mRNA decay. Additional transfection and nucleofection experiments using RNAs prepared in vitro illustrated that, in both the cytoplasm and the nucleus of cells, the optional long 5'-UTR-linked mRNAs decayed faster than those without the link. Overall, our study brings new insight to the regulation of the human ACAT1 gene expression at the post-transcription level.

Humans express two ACAT (acyl-CoA:cholesterol acyltransferase) genes, ACAT1 and ACAT2. ACAT1 is ubiquitously expressed, whereas ACAT2 is primarily expressed in intestinal mucosa and plays an important role in intestinal cholesterol absorption. To investigate the molecular mechanism(s) responsible for the tissue-specific expression of ACAT2, we identified five cis-elements within the human ACAT2 promoter, four for the intestinal-specific transcription factor CDX2 (caudal type homeobox transcription factor 2), and one for the transcription factor HNF1alpha (hepatocyte nuclear factor 1alpha). Results of luciferase reporter and electrophoretic mobility shift assays show that CDX2 and HNF1alpha exert a synergistic effect, enhancing the ACAT2 promoter activity through binding to these cis-elements. In undifferentiated Caco-2 cells, the ACAT2 expression is increased when exogenous CDX2 and/or HNF1alpha are expressed by co-transfection. In differentiated Caco-2 cells, the ACAT2 expression significantly decreases when the endogenous CDX2 or HNF1alpha expression is suppressed by using RNAi (RNA interference) technology. The expression levels of CDX2, HNF1alpha, and ACAT2 are all greatly increased when the Caco-2 cells differentiate to become intestinal-like cells. These results provide a molecular mechanism for the tissue-specific expression of ACAT2 in intestine. In normal adult human liver, CDX2 expression is not detectable and the ACAT2 expression is very low. In the hepatoma cell line HepG2 the CDX2 expression is elevated, accounting for its elevated ACAT2 expression. A high percentage (seven of fourteen) of liver samples from patients affected with hepatocellular carcinoma exhibited elevated ACAT2 expression. Thus, the elevated ACAT2 expression may serve as a new biomarker for certain form(s) of hepatocellular carcinoma.