Identification of novel genes involved in petunia flower development using transcript profiling and reverse genetics

[ENG] Petals are a key element on plant life cycle as, in many species, they attract pollinators, thus aiding to reproduction. Furthermore, they have economic importance in ornamental crops. In the present study, petal transcriptional patterns were compared within the ower organs in Arabidopsis thaliana. It was found that catalytic molecular functions were overrepresented in petals. A shortlist comprising the top ten di_erentially expressed genes in petals were mapped to the model species with industrial value Petunia hybrida, and further downregulated by RNAi. The silencing phenotypes found permitted to assign functions in petal development to seven novel genes: when silenced, they triggered alterations on ower size and shape (PhCYP76, PhNPH3, PhFeSOD, PhXTH, PhCYP96 and PhWAK), petal smoothness (PhPRA), color (PhNPH3 and PhWAK) and symmetry (PhCYP76). Pleiotropic phenotypes were found, such as changes in root morphology and leaf color (PhCYP76), ower number, capsule and seed morphology (PhCYP96) and plant height (PhCYP76 and PhCYP96). To accomplish the experimental design, three methods were developed. First, the \pESTle" management system that assembles, annotates, stores and serves expressed sequence tag data. Second, a reference gene selection for real time PCR experiments that includes a new method for stability estimation based on rank aggregation of published algorithms, and concludes that a normalization factor with two members of EF1_, SAND, CYP or RAN1 is stable enough under most conditions. And third, a PCR e_ciency estimator based on amplicon characteristics which allows e_ciency-driven primer design in a Web tool. [SPA] La corola es un elemento básico en el ciclo de vida vegetal puesto que, en muchos casos, atrae a polinizadores que intervienen en su propagación. Además, posee un valor económico en especies ornamentales. En el presente trabajo se realizó un análisis comparativo contrastando el transcriptoma de pétalos frente al resto de órganos orales de Arabidopsis thaliana. Como resultado se observó la preponderancia de genes involucrados en funciones catalíticas. Aún de dilucidar el rol de estos genes, se escogieron aquellos nueve con mayor expresión diferencial y se silenciaron de forma estable mediante ARN de interferencia. De esta forma, el análisis fenotípico ha permitido asignar un papel a siete nuevos genes: al ser silenciados, provocan alteraciones en la forma y tamaño (PhCYP76, PhNPH3, PhFeSOD, PhXTH, PhCYP96 y PhWAK), textura (PhPRA), color (PhNPH3 y Ph- WAK) y simetría (PhCYP76) de los pétalos; así como el color de las hojas y la morfología radicular (PhCYP76), el número de ores y la altura de la planta (PhCYP76 y PhCYP96). Aún de respaldar el diseño experimental se desarrollaron tres métodos. En primer lugar, el gestor _pESTle_, que aúna el ensamblaje, anotación, almacenamiento y difusión de ESTs. En segundo lugar, la selección de genes de referencia para PCR en tiempo real, que reúne un nuevo método para su evaluación basado en la agregación de rangos; y la descripción de la pertinencia de emplear un factor de normalización con dos genes de entre EF1_, SAND, CYP y RAN1 en la mayor parte de los casos. Finalmente, se produjo el estimador _pcrE_ciency_, que proporciona un entorno de diseño de cebadores que simultáneamente predice su eficiencia.Universidad Politécnica de Cartagen

Wanderer, an interactive viewer to explore DNA methylation and gene expression data in human cancer

BACKGROUND: The Cancer Genome Atlas (TCGA) offers a multilayered view of genomics and epigenomics data of many human cancer types. However, the retrieval of expression and methylation data from TCGA is a cumbersome and time-consuming task.RESULTS:Wanderer is an intuitive Web tool allowing real time access and visualization of gene expression and DNA methylation profiles from TCGA. Given a gene query and selection of a TCGA dataset (e.g., colon adenocarcinomas), the Web resource provides the expression profile, at the single exon level, and the DNA methylation levels of HumanMethylation450 BeadChip loci inside or in the vicinity of the queried gene. Graphic and table outputs include individual and summary data as well as statistical tests, allowing the comparison of tumor and normal profiles and the exploration along the genomic locus and across tumor collections. CONCLUSIONS: Wanderer offers a simple interface to straightforward access to TCGA data, amenable to experimentalists and clinicians without bioinformatics expertise. Wanderer may be accessed at http://maplab.catwanderer

Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida

<p>Abstract</p> <p>Background</p> <p>Identification of genes with invariant levels of gene expression is a prerequisite for validating transcriptomic changes accompanying development. Ideally expression of these genes should be independent of the morphogenetic process or environmental condition tested as well as the methods used for RNA purification and analysis.</p> <p>Results</p> <p>In an effort to identify endogenous genes meeting these criteria nine reference genes (RG) were tested in two Petunia lines (Mitchell and V30). Growth conditions differed in Mitchell and V30, and different methods were used for RNA isolation and analysis. Four different software tools were employed to analyze the data. We merged the four outputs by means of a non-weighted unsupervised rank aggregation method. The genes identified as optimal for transcriptomic analysis of Mitchell and V30 were <it>EF1α </it>in Mitchell and <it>CYP </it>in V30, whereas the least suitable gene was <it>GAPDH </it>in both lines.</p> <p>Conclusions</p> <p>The least adequate gene turned out to be <it>GAPDH </it>indicating that it should be rejected as reference gene in Petunia. The absence of correspondence of the best-suited genes suggests that assessing reference gene stability is needed when performing normalization of data from transcriptomic analysis of flower and leaf development.</p

The epigenetic landscape of Alu repeats delineates the structural and functional genomic architecture of colon cancer cells

Cancer cells exhibit multiple epigenetic changes with prominent local DNA hypermethylation and widespread hypomethylation affecting large chromosomal domains. Epigenome studies often disregard the study of repeat elements owing to technical complexity and their undefined role in genome regulation. We have developed NSUMA (Next-generation Sequencing of UnMethylated Alu), a cost-effective approach allowing the unambiguous interrogation of DNA methylation in more than 130,000 individual Alu elements, the most abundant retrotransposon in the human genome. DNA methylation profiles of Alu repeats have been analyzed in colon cancers and normal tissues using NSUMA and whole-genome bisulfite sequencing. Normal cells show a low proportion of unmethylated Alu (1%-4%) that may increase up to 10-fold in cancer cells. In normal cells, unmethylated Alu elements tend to locate in the vicinity of functionally rich regions and display epigenetic features consistent with a direct impact on genome regulation. In cancer cells, Alu repeats are more resistant to hypomethylation than other retroelements. Genome segmentation based on high/low rates of Alu hypomethylation allows the identification of genomic compartments with differential genetic, epigenetic, and transcriptomic features. Alu hypomethylated regions show low transcriptional activity, late DNA replication, and its extent is associated with higher chromosomal instability. Our analysis demonstrates that Alu retroelements contribute to define the epigenetic landscape of normal and cancer cells and provides a unique resource on the epigenetic dynamics of a principal, but largely unexplored, component of the primate genome

Long-term self-renewing stem cells in the adult mouse hippocampus identified by intravital imaging.

Neural stem cells (NSCs) generate neurons throughout life in the mammalian hippocampus. However, the potential for long-term self-renewal of individual NSCs within the adult brain remains unclear. We used two-photon microscopy and followed NSCs that were genetically labeled through conditional recombination driven by the regulatory elements of the stem cell-expressed genes GLI family zinc finger 1 (Gli1) or achaete-scute homolog 1 (Ascl1). Through intravital imaging of NSCs and their progeny, we identify a population of Gli1-targeted NSCs showing long-term self-renewal in the adult hippocampus. In contrast, once activated, Ascl1-targeted NSCs undergo limited proliferative activity before they become exhausted. Using single-cell RNA sequencing, we show that Gli1- and Ascl1-targeted cells have highly similar yet distinct transcriptional profiles, supporting the existence of heterogeneous NSC populations with diverse behavioral properties. Thus, we here identify long-term self-renewing NSCs that contribute to the generation of new neurons in the adult hippocampus.Wellcome Trus

pcrEfficiency: a Web tool for PCR amplification efficiency prediction

<p>Abstract</p> <p>Background</p> <p>Relative calculation of differential gene expression in quantitative PCR reactions requires comparison between amplification experiments that include reference genes and genes under study. Ignoring the differences between their efficiencies may lead to miscalculation of gene expression even with the same starting amount of template. Although there are several tools performing PCR primer design, there is no tool available that predicts PCR efficiency for a given amplicon and primer pair.</p> <p>Results</p> <p>We have used a statistical approach based on 90 primer pair combinations amplifying templates from bacteria, yeast, plants and humans, ranging in size between 74 and 907 bp to identify the parameters that affect PCR efficiency. We developed a generalized additive model fitting the data and constructed an open source Web interface that allows the obtention of oligonucleotides optimized for PCR with predicted amplification efficiencies starting from a given sequence.</p> <p>Conclusions</p> <p>pcrEfficiency provides an easy-to-use web interface allowing the prediction of PCR efficiencies prior to web lab experiments thus easing quantitative real-time PCR set-up. A web-based service as well the source code are provided freely at <url>http://srvgen.upct.es/efficiency.html</url> under the GPL v2 license.</p

Meta-analysis of (single-cell method) benchmarks reveals the need for extensibility and interoperability

Computational methods represent the lifeblood of modern molecular biology. Benchmarking is important for all methods, but with a focus here on computational methods, benchmarking is critical to dissect important steps of analysis pipelines, formally assess performance across common situations as well as edge cases, and ultimately guide users on what tools to use. Benchmarking can also be important for community building and advancing methods in a principled way. We conducted a meta-analysis of recent single-cell benchmarks to summarize the scope, extensibility, and neutrality, as well as technical features and whether best practices in open data and reproducible research were followed. The results highlight that while benchmarks often make code available and are in principle reproducible, they remain difficult to extend, for example, as new methods and new ways to assess methods emerge. In addition, embracing containerization and workflow systems would enhance reusability of intermediate benchmarking results, thus also driving wider adoption

Quantification of Unmethylated Alu (QUAlu): a tool to assess global hypomethylation in routine clinical samples

Hypomethylation of DNA is a hallmark of cancer and its analysis as tumor biomarker has been proposed, but its determination in clinical settings is hampered by lack of standardized methodologies. Here, we present QUAlu (Quantification of Unmethylated Alu), a new technique to estimate the Percentage of UnMethylated Alu (PUMA) as a surrogate for global hypomethylation. QUAlu consists in the measurement by qPCR of Alu repeats after digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation. QUAlu performance has been evaluated for reproducibility, trueness and specificity, and validated by deep sequencing. As a proof of use, QUAlu has been applied to a broad variety of pathological examination specimens covering five cancer types. Major findings of the preliminary application of QUAlu to clinical samples include: (1) all normal tissues displayed similar PUMA; (2) tumors showed variable PUMA with the highest levels in lung and colon and the lowest in thyroid cancer; (3) stools from colon cancer patients presented higher PUMA than those from control individuals; (4) lung squamous cell carcinomas showed higher PUMA than lung adenocarcinomas, and an increasing hypomethylation trend associated with smoking habits. In conclusion, QUAlu is a simple and robust method to determine Alu hypomethylation in human biospecimens and may be easily implemented in research and clinical settings.RB was supported by a FPI fellowship from Ministerio de Economía y Competitividad. AD-V was supported in part by a contract PTC2011-1091 from Ministerio de Economía y Competitividad. This work was supported by grants from FEDER, the Ministerio de Economía y Competitividad (SAF2011/23638 to MAP), the Instituto de Salud Carlos III (FIS PI11/02421 to JR, FIS PI11/01359 and FIS PI14/00240 to MR, FIS PI14/00308 to MJ, FIS PI12/00511 to MP), and Fundació Olga Torres (to MJ)

Quantification of unmethylated Alu (QUAlu): a tool to assess global hypomethylation in routine clinical samples

Hypomethylation of DNA is a hallmark of cancer and its analysis as tumor biomarker has been proposed, but its determination in clinical settings is hampered by lack of standardized methodologies. Here, we present QUAlu (Quantification of Unmethylated Alu), a new technique to estimate the Percentage of UnMethylated Alu (PUMA) as a surrogate for global hypomethylation. QUAlu consists in the measurement by qPCR of Alu repeats after digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation. QUAlu performance has been evaluated for reproducibility, trueness and specificity, and validated by deep sequencing. As a proof of use, QUAlu has been applied to a broad variety of pathological examination specimens covering five cancer types. Major findings of the preliminary application of QUAlu to clinical samples include: (1) all normal tissues displayed similar PUMA; (2) tumors showed variable PUMA with the highest levels in lung and colon and the lowest in thyroid cancer; (3) stools from colon cancer patients presented higher PUMA than those from control individuals; (4) lung squamous cell carcinomas showed higher PUMA than lung adenocarcinomas, and an increasing hypomethylation trend associated with smoking habits. In conclusion, QUAlu is a simple and robust method to determine Alu hypomethylation in human biospecimens and may be easily implemented in research and clinical settings

TDP-43 oligomerization and RNA binding are codependent but their loss elicits distinct pathologies

Aggregation of the RNA-binding protein TDP-43 is the main common neuropathological feature of TDP-43 proteinopathies. In physiological conditions, TDP-43 is predominantly nuclear and contained in biomolecular condensates formed via liquid-liquid phase separation (LLPS). However, in disease, TDP-43 is depleted from these compartments and forms cytoplasmic or, sometimes, intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Here, we show that self-oligomerization and RNA binding cooperatively govern TDP-43 stability, functionality, LLPS and cellular localization. Importantly, our data reveal that TDP-43 oligomerization is connected to, and conformationally modulated by, RNA binding. Mimicking the impaired proteasomal activity observed in patients, we found that TDP-43 forms nuclear aggregates via LLPS and cytoplasmic aggregates via aggresome formation. The favored aggregation pathway depended on the TDP-43 state –monomeric/oligomeric, RNA-bound/-unbound– and the subcellular environment –nucleus/cytoplasm. Our work unravels the origins of heterogeneous pathological species occurring in TDP-43 proteinopathies