MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome

Here we present MethylQuant, a novel method that allows accurate quantification of the methylation level of a specific cytosine within a complex genome. This method relies on the well-established treatment of genomic DNA with sodium bisulfite, which converts cytosine into uracil without modifying 5-methyl cytosine. The region of interest is then PCR-amplified and quantification of the methylation status of a specific cytosine is performed by methylation-specific real-time PCR with SYBR Green I using one of the primers whose 3′ end discriminates between the methylation states of this cytosine. The presence of a locked nucleic acid at the 3′ end of the discriminative primer provides the specificity necessary for accurate and sensitive quantification, even when one of the methylation states is present at a level as low as 1% of the overall population. We demonstrate that accurate quantification of the methylation status of specific cytosines can be achieved in biological samples. The method is high-throughput, cost-effective, relatively simple and does not require any specific equipment other than a real-time PCR instrument

Glucocorticoid-induced DNA demethylation and gene memory during development

Glucocorticoid hormones were found to regulate DNA demethylation within a key enhancer of the rat liver-specific tyrosine aminotransferase (Tat) gene. Genomic footprinting analysis shows that the glucocorticoid receptor uses local DNA demethylation as one of several steps to recruit transcription factors in hepatoma cells. Demethylation occurs within 2–3 days following rapid (<1 h) chromatin remodeling and recruitment of a first transcription factor, HNF-3. Upon demethylation, two additional transcription factors are recruited when chromatin is remodeled. In contrast to chromatin remodeling, the demethylation is stable following hormone withdrawal. As a stronger subsequent glucocorticoid response is observed, demethylation appears to provide memory of the first stimulation. During development, this demethylation occurs before birth, at a stage where the Tat gene is not yet inducible, and it could thus prepare the enhancer for subsequent stimulation by hypoglycemia at birth. In vitro cultures of fetal hepatocytes recapitulate the regulation analyzed in hepatoma cells. There fore, demethylation appears to contribute to the fine-tuning of the enhancer and to the memorization of a regulatory event during development

Identification of Gender- and Subtype-Specific Gene Expression Associated with Patient Survival in Low-Grade and Anaplastic Glioma in Connection with Steroid Signaling

Low-grade gliomas are rare primary brain tumors, which fatally evolve to anaplastic gliomas. The current treatment combines surgery, chemotherapy, and radiotherapy. If gender differences in the natural history of the disease were widely described, their underlying mechanisms remain to be determined for the identification of reliable markers of disease progression. We mined the transcriptomic and clinical data from the TCGA-LGG and CGGA databases to identify male-over-female differentially expressed genes and selected those associated with patient survival using univariate analysis, depending on molecular characteristics (IDH wild-type/mutated; 1p/19q codeleted/not) and grade. Then, the link between the expression levels (low or high) of the steroid biosynthesis enzyme or receptors of interest and survival was studied using the log-rank test. Finally, a functional analysis of gender-specific correlated genes was performed. HOX-related genes appeared to be differentially expressed between males and females in both grades, suggesting that a glioma could originate in perturbation of developmental signals. Moreover, aromatase, androgen, and estrogen receptor expressions were associated with patient survival and were mainly related to angiogenesis or immune response. Therefore, consideration of the tight control of steroid hormone production and signaling seems crucial for the understanding of glioma pathogenesis and emergence of future targeted therapies

Nature of the Accessible Chromatin at a Glucocorticoid-Responsive Enhancer

To gain a better understanding of the nature of active chromatin in mammals, we have characterized in living cells the various chromatin modification events triggered by the glucocorticoid receptor (GR) at the rat tyrosine aminotransferase gene. GR promotes a local remodeling at a glucocorticoid-responsive unit (GRU) located 2.5 kb upstream of the transcription start site, creating nuclease hypersensitivity that encompasses 450 bp of DNA. Nucleosomes at the GRU occupy multiple frames that are remodeled without nucleosome repositioning, showing that nucleosome positioning is not the key determinant of chromatin accessibility at this locus. Remodeling affects nucleosomes and adjacent linker sequences, enhancing accessibility at both regions. This is associated with decreased interaction of both the linker histone H1 and the core histone H3 with DNA. Thus, our results indicate that nucleosome and linker histone removal rather than nucleosome repositioning is associated with GR-triggered accessibility. Interestingly, GR induces hyperacetylation of histones H3 and H4, but this is not sufficient either for remodeling or for transcriptional activation. Finally, our data favor the coexistence of several chromatin states within the population, which may account for the previously encountered difficulties in characterizing unambiguously the active chromatin structure in living cells

Lack of miRNA Misregulation at Early Pathological Stages in Drosophila Neurodegenerative Disease Models

International audienceLate onset neurodegenerative diseases represent a major public health concern as the populationin many countries ages. Both frequent diseases such as Alzheimer disease (AD,14% incidence for 80–84 year-old Europeans) or Parkinson disease (PD, 1.4% prevalencefor >55 years old) share, with other low-incidence neurodegenerative pathologies suchas spinocerebellar ataxias (SCAs, 0.01% prevalence) and frontotemporal lobar degeneration(FTLD, 0.02% prevalence), a lack of efficient treatment in spite of important researchefforts. Besides significant progress, studies with animal models have revealed unexpectedcomplexities in the degenerative process, emphasizing a need to better understand theunderlying pathological mechanisms. Recently, microRNAs (miRNAs), a class of small regulatorynon-coding RNAs, have been implicated in some neurodegenerative diseases.Thecurrent data supporting a role of miRNAs in PD, tauopathies, dominant ataxias, and FTLDwill first be discussed to emphasize the different levels of the pathological processes whichmay be affected by miRNAs.To investigate a potential involvement of miRNA dysregulationin the early stages of these neurodegenerative diseases we have used Drosophila modelsfor seven diseases (PD, 3 FTLD, 3 dominant ataxias) that recapitulate many features of thehuman diseases.We performed deep sequencing of head small RNAs after 3 days of pathologicalprotein expression in the fly head neurons.We found no evidence for a statisticallysignificant difference in miRNA expression in this early stage of the pathological process.In addition, we could not identify small non-coding CAG repeat RNAs (sCAG) in polyQ diseasemodels. Thus our data suggest that transcriptional deregulation of miRNAs or sCAGis unlikely to play a significant role in the initial stages of neurodegenerative diseases

Single amino-acid mutation in a Drosoph ila melanogaster ribosomal protein: An insight in uL11 transcriptional activity.

The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs

Single amino-acid mutation in a Drosoph ila melanogaster ribosomal protein: An insight in uL11 transcriptional activity

International audienceThe ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes ( RPGs ). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes ( RiBis ) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D . melanogaster uL11 . Unexpectedly, the uL11 K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11 K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11 K3A but not in uL11 K3Y . Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs

AutomiG, a biosensor to detect alterations in miRNA biogenesis and in small RNA silencing guided by perfect target complementarity.

International audienceDefects in miRNA biogenesis or activity are associated to development abnormalities and diseases. In Drosophila, miRNAs are predominantly loaded in Argonaute-1, which they guide for silencing of target RNAs. The miRNA pathway overlaps the RNAi pathway in this organism, as miRNAs may also associate with Argonaute-2, the mediator of RNAi. We set up a gene construct in which a single inducible promoter directs the expression of the GFP protein as well as two miRNAs perfectly matching the GFP sequences. We show that self-silencing of the resulting automiG gene requires Drosha, Pasha, Dicer-1, Dicer-2 and Argonaute-2 loaded with the anti-GFP miRNAs. In contrast, self-silencing of the automiG gene does not involve Argonaute-1. Thus, automiG reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing, providing a powerful biosensor to identify situations where miRNA or siRNA pathways are impaired. As a proof of concept, we used automiG as a biosensor to screen a chemical library and identified 29 molecules that strongly inhibit miRNA silencing, out of which 5 also inhibit RNAi triggered by long double-stranded RNA. Finally, the automiG sensor is also self-silenced by the anti-GFP miRNAs in HeLa cells and might be easily used to identify factors involved in miRNA biogenesis and silencing guided by perfect target complementarity in mammals

Cyclin G and the Polycomb Repressive complexes PRC1 and PR-DUB cooperate for developmental stability

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81 compounds induce strong fluorescence of automiG cells.

<p>The primary screen identified 46 compounds that induced a fluorescence fold-change >3.9 and 57 compound for which more that 500 fluorescent spots were counted from well imaging (see materials and methods). The merging of these two sets yield 81 compounds whose effects on automiG cells are presented. Each panel correspond to one image taken by the automated Nikon TE2000 inverted microscope. Note that out of the 81 compounds, 17 were discarded as false positives because they induced by themselves fluorescence (see Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074296#pone.0074296.s002" target="_blank">Table S2</a>, shaded identifiers).</p