SCA8 CAG/CTG Expansions, a Tale of Two TOXICities: A Unique or Common Case?

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Polyglutamine Expansion Accelerates the Dynamics of Ataxin-1 and Does Not Result in Aggregate Formation

Polyglutamine expansion disorders are caused by an expansion of the polyglutamine (polyQ) tract in the disease related protein, leading to severe neurodegeneration. All polyQ disorders are hallmarked by the presence of intracellular aggregates containing the expanded protein in affected neurons. The polyQ disorder SpinoCerebellar Ataxia 1 (SCA1) is caused by a polyQ-expansion in the ataxin-1 protein, which is thought to lead to nuclear aggregates.Using advanced live cell fluorescence microscopy and a filter retardation assay we show that nuclear accumulations formed by polyQ-expanded ataxin-1 do not resemble aggregates of other polyQ-expanded proteins. Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1. During mitosis, ataxin-1 accumulations redistributed equally among daughter cells, in contrast to polyQ aggregates. Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before.These results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1. The unexpected findings for a polyQ-expanded protein and their consequences for ongoing SCA1 research are discussed

Endogenous myoglobin in human breast cancer is a hallmark of luminal cancer phenotype

BACKGROUND: We aimed to clarify the incidence and the clinicopathological value of non-muscle myoglobin (Mb) in a large cohort of non-invasive and invasive breast cancer cases. METHODS: Matched pairs of breast tissues from 10 patients plus 17 breast cell lines were screened by quantitative PCR for Mb mRNA. In addition, 917 invasive and 155 non-invasive breast cancer cases were analysed by immunohistochemistry for Mb expression and correlated to clinicopathological parameters and basal molecular characteristics including oestrogen receptor-alpha (ERalpha)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1alpha (HIF-1alpha), HIF-2alpha, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERalpha or FASN was followed up by double immunofluorescence. Finally, the effects of estradiol treatment and FASN inhibition on Mb expression in breast cancer cells were analysed. RESULTS: Myoglobin mRNA was found in a subset of breast cancer cell lines; in microdissected tumours Mb transcript was markedly upregulated. In all, 71% of tumours displayed Mb protein expression in significant correlation with a positive hormone receptor status and better prognosis. In silico data mining confirmed higher Mb levels in luminal-type breast cancer. Myoglobin was also correlated to FASN, HIF-2alpha and CAIX, but not to HIF-1alpha or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERalpha or FASN and Mb. In addition, Mb levels were modulated on estradiol treatment and FASN inhibition in a cell model. CONCLUSION: We conclude that in breast cancer, Mb is co-expressed with ERalpha and co-regulated by oestrogen signalling and can be considered a hallmark of luminal breast cancer phenotype. This and its possible new role in fatty acid metabolism may have fundamental implications for our understanding of Mb in solid tumours

A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis

DNA methylation is an indispensible epigenetic modification required for regulating the expression of mammalian genomes. Immunoprecipitation-based methods for DNA methylome analysis are rapidly shifting the bottleneck in this field from data generation to data analysis, necessitating the development of better analytical tools. In particular, an inability to estimate absolute methylation levels remains a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling. To address this issue, we developed a cross-platform algorithm - Bayesian tool for methylation analysis (Batman) - for analyzing methylated DNA immunoprecipitation (MeDIP) profiles generated using oligonucleotide arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). We developed the latter approach to provide a high-resolution whole-genome DNA methylation profile (DNA methylome) of a mammalian genome. Strong correlation of our data, obtained using mature human spermatozoa, with those obtained using bisulfite sequencing suggest that combining MeDIP-seq or MeDIP-chip with Batman provides a robust, quantitative and cost-effective functional genomic strategy for elucidating the function of DNA methylation. © 2008 Nature Publishing Group.Fil: Down, Thomas A.. Wellcome Trust Sanger Institute; Reino UnidoFil: Rakyan, Vardhman K.. Institute of Cell and Molecular Science; Reino UnidoFil: Turner, Daniel J.. Wellcome Trust Sanger Institute; Reino UnidoFil: Flicek, Paul. European Bioinformatics Institute; Reino UnidoFil: Li, Heng. Wellcome Trust Sanger Institute; Reino UnidoFil: Kulesha, Eugene. European Bioinformatics Institute; Reino UnidoFil: Gräf, Stefan. European Bioinformatics Institute; Reino UnidoFil: Johnson, Nathan. European Bioinformatics Institute; Reino UnidoFil: Herrero, Javier. European Bioinformatics Institute; Reino UnidoFil: Tomazou, Eleni M.. Wellcome Trust Sanger Institute; Reino UnidoFil: Thorne, Natalie P.. University of Cambridge; Reino UnidoFil: Bäckdahl, Liselotte. University College London; Reino UnidoFil: Herberth, Marlis. University of Cambridge; Reino UnidoFil: Howe, Kevin L.. University of Cambridge; Reino UnidoFil: Jackson, David K.. Wellcome Trust Sanger Institute; Reino UnidoFil: Miretti, Marcos Mateo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; Argentina. Wellcome Trust Sanger Institute; Reino UnidoFil: Marioni, John C.. University of Cambridge; Reino UnidoFil: Birney, Ewan. European Bioinformatics Institute; Reino UnidoFil: Hubbard, Tim J. P.. Wellcome Trust Sanger Institute; Reino UnidoFil: Durbin, Richard. Wellcome Trust Sanger Institute; Reino UnidoFil: Tavaré, Simon. University of Cambridge; Reino UnidoFil: Beck, Stephan G.. University College London; Reino Unid

Discovery of Novel Hypermethylated Genes in Prostate Cancer Using Genomic CpG Island Microarrays

BACKGROUND: Promoter and 5' end methylation regulation of tumour suppressor genes is a common feature of many cancers. Such occurrences often lead to the silencing of these key genes and thus they may contribute to the development of cancer, including prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify methylation changes in prostate cancer, we performed a genome-wide analysis of DNA methylation using Agilent human CpG island arrays. Using computational and gene-specific validation approaches we have identified a large number of potential epigenetic biomarkers of prostate cancer. Further validation of candidate genes on a separate cohort of low and high grade prostate cancers by quantitative MethyLight analysis has allowed us to confirm DNA hypermethylation of HOXD3 and BMP7, two genes that may play a role in the development of high grade tumours. We also show that promoter hypermethylation is responsible for downregulated expression of these genes in the DU-145 PCa cell line. CONCLUSIONS/SIGNIFICANCE: This study identifies novel epigenetic biomarkers of prostate cancer and prostate cancer progression, and provides a global assessment of DNA methylation in prostate cancer

DNA Demethylation and USF Regulate the Meiosis-Specific Expression of the Mouse Miwi

Miwi, a member of the Argonaute family, is required for initiating spermiogenesis; however, the mechanisms that regulate the expression of the Miwi gene remain unknown. By mutation analysis and transgenic models, we identified a 303 bp proximal promoter region of the mouse Miwi gene, which controls specific expression from midpachytene spermatocytes to round spermatids during meiosis. We characterized the binding sites of transcription factors NF-Y (Nuclear Factor Y) and USF (Upstream Stimulatory Factor) within the core promoter and found that both factors specifically bind to and activate the Miwi promoter. Methylation profiling of three CpG islands within the proximal promoter reveals a markedly inverse correlation between the methylation status of the CpG islands and germ cell type–specific expression of Miwi. CpG methylation at the USF–binding site within the E2 box in the promoter inhibits the binding of USF. Transgenic Miwi-EGFP and endogenous Miwi reveal a subcellular co-localization pattern in the germ cells of the Miwi-EGFP transgenic mouse. Furthermore, the DNA methylation profile of the Miwi promoter–driven transgene is consistent with that of the endogenous Miwi promoter, indicating that Miwi transgene is epigenetically modified through methylation in vivo to ensure its spatio-temporal expression. Our findings suggest that USF controls Miwi expression from midpachytene spermatocytes to round spermatids through methylation-mediated regulation. This work identifies an epigenetic regulation mechanism for the spatio-temporal expression of mouse Miwi during spermatogenesis

Congo Basin peatlands: threats and conservation priorities

The recent publication of the first spatially explicit map of peatlands in the Cuvette Centrale, central Congo Basin, reveals it to be the most extensive tropical peatland complex, at ca. 145,500 km2. With an estimated 30.6 Pg of carbon stored in these peatlands, there are now questions about whether these carbon stocks are under threat and, if so, what can be done to protect them. Here, we analyse the potential threats to Congo Basin peat carbon stocks and identify knowledge gaps in relation to these threats, and to how the peatland systems might respond. Climate change emerges as a particularly pressing concern, given its potential to destabilise carbon stocks across the whole area. Socio-economic developments are increasing across central Africa and, whilst much of the peatland area is protected on paper by some form of conservation designation, the potential exists for hydrocarbon exploration, logging, plantations and other forms of disturbance to significantly damage the peatland ecosystems. The low level of human intervention at present suggests that the opportunity still exists to protect the peatlands in a largely intact state, possibly drawing on climate change mitigation funding, which can be used not only to protect the peat carbon pool but also to improve the livelihoods of people living in and around these peatlands

Correlation of Inter-Locus Polyglutamine Toxicity with CAG•CTG Triplet Repeat Expandability and Flanking Genomic DNA GC Content

Dynamic expansions of toxic polyglutamine (polyQ)-encoding CAG repeats in ubiquitously expressed, but otherwise unrelated, genes cause a number of late-onset progressive neurodegenerative disorders, including Huntington disease and the spinocerebellar ataxias. As polyQ toxicity in these disorders increases with repeat length, the intergenerational expansion of unstable CAG repeats leads to anticipation, an earlier age-at-onset in successive generations. Crucially, disease associated alleles are also somatically unstable and continue to expand throughout the lifetime of the individual. Interestingly, the inherited polyQ length mediating a specific age-at-onset of symptoms varies markedly between disorders. It is widely assumed that these inter-locus differences in polyQ toxicity are mediated by protein context effects. Previously, we demonstrated that the tendency of expanded CAG•CTG repeats to undergo further intergenerational expansion (their ‘expandability’) also differs between disorders and these effects are strongly correlated with the GC content of the genomic flanking DNA. Here we show that the inter-locus toxicity of the expanded polyQ tracts of these disorders also correlates with both the expandability of the underlying CAG repeat and the GC content of the genomic DNA flanking sequences. Inter-locus polyQ toxicity does not correlate with properties of the mRNA or protein sequences, with polyQ location within the gene or protein, or steady state transcript levels in the brain. These data suggest that the observed inter-locus differences in polyQ toxicity are not mediated solely by protein context effects, but that genomic context is also important, an effect that may be mediated by modifying the rate at which somatic expansion of the DNA delivers proteins to their cytotoxic state

Formation and Toxicity of Soluble Polyglutamine Oligomers in Living Cells

Aggregation and cytotoxicity of mutant proteins containing an expanded number of polyglutamine (polyQ) repeats is a hallmark of several diseases, including Huntington's disease (HD). Within cells, mutant Huntingtin (mHtt) and other polyglutamine expansion mutant proteins exist as monomers, soluble oligomers, and insoluble inclusion bodies (IBs). Determining which of these forms constitute a toxic species has proven difficult. Recent studies support a role for IBs as a cellular coping mechanism to sequester levels of potentially toxic soluble monomeric and oligomeric species of mHtt.When fused to a fluorescent reporter (GFP) and expressed in cells, the soluble monomeric and oligomeric polyglutamine species are visually indistinguishable. Here, we describe two complementary biophysical fluorescence microscopy techniques to directly detect soluble polyglutamine oligomers (using Htt exon 1 or Htt(ex1)) and monitor their fates in live cells. Photobleaching analyses revealed a significant reduction in the mobilities of mHtt(ex1) variants consistent with their incorporation into soluble microcomplexes. Similarly, when fused to split-GFP constructs, both wildtype and mHtt(ex1) formed oligomers, as evidenced by the formation of a fluorescent reporter. Only the mHtt(ex1) split-GFP oligomers assembled into IBs. Both FRAP and split-GFP approaches confirmed the ability of mHtt(ex1) to bind and incorporate wildtype Htt into soluble oligomers. We exploited the irreversible binding of split-GFP fragments to forcibly increase levels of soluble oligomeric mHtt(ex1). A corresponding increase in the rate of IBs formation and the number formed was observed. Importantly, higher levels of soluble mHtt(ex1) oligomers significantly correlated with increased mutant cytotoxicity, independent of the presence of IBs.Our study describes powerful and sensitive tools for investigating soluble oligomeric forms of expanded polyglutamine proteins, and their impact on cell viability. Moreover, these methods should be applicable for the detection of soluble oligomers of a wide variety of aggregation prone proteins