PI3Kß-regulated ß-catenin mediates EZH2 removal from promoters controlling primed human ESC stemness and primitive streak gene expression.

The mechanism governing the transition of human embryonic stem cells (hESCs) toward differentiated cells is only partially understood. To explore this transition, the activity and expression of the ubiquitous phosphatidylinositol 3-kinase (PI3Kα and PI3Kß) were modulated in primed hESCs. The study reports a pathway that dismantles the restraint imposed by the EZH2 polycomb repressor on an essential stemness gene, NODAL, and on transcription factors required to trigger primitive streak formation. The primitive streak is the site where gastrulation begins to give rise to the three embryonic cell layers from which all human tissues derive. The pathway involves a PI3Kß non-catalytic action that controls nuclear/active RAC1 levels, activation of JNK (Jun N-terminal kinase) and nuclear ß-catenin accumulation. ß-Catenin deposition at promoters triggers release of the EZH2 repressor, permitting stemness maintenance (through control of NODAL) and correct differentiation by allowing primitive streak master gene expression. PI3Kß epigenetic control of EZH2/ß-catenin might be modulated to direct stem cell differentiation.

sRNA Analysis Evidenced the Involvement of Different Plant Viruses in the Activation of RNA Silencing-Related Genes and the Defensive Response Against Plum pox virus of 'GF305' Peach Grafted with 'Garrigues' Almond.

Plum pox virus (PPV) causes sharka disease in Prunus trees. Peach (P. persica) trees are severely affected by PPV, and no definitive source of genetic resistance has been identified. However, previous results showed that PPV-resistant 'Garrigues' almond (P. dulcis) was able to transfer its resistance to 'GF305' peach through grafting, reducing symptoms and viral load in PPV-infected plants. A recent study tried to identify genes responsible for this effect by studying messenger RNA expression through RNA sequencing in peach and almond plants, before and after grafting and before and after PPV infection. In this work, we used the same peach and almond samples but focused the high-throughput analyses on small RNA (sRNA) expression. We studied massive sequencing data and found an interesting pattern of sRNA overexpression linked to antiviral defense genes that suggested activation of these genes followed by downregulation to basal levels. We also discovered that 'Garrigues' almond plants were infected by different plant viruses that were transferred to peach plants. The large amounts of viral sRNA found in grafted peaches indicated a strong RNA silencing antiviral response and led us to postulate that these plant viruses could be collaborating in the observed "Garrigues effect."

WeReview: CRISPR Tools-Live Repository of Computational Tools for Assisting CRISPR/Cas Experiments.

Computational tools are essential in the process of designing a CRISPR/Cas experiment for the targeted modification of an organism's genome. Among other functionalities, these tools facilitate the design of a guide-RNA (gRNA) for a given nuclease that maximizes its binding to the intended genomic site, while avoiding binding to undesired sites with similar sequences in the genome of the organism of interest (off-targets). Due to the popularity of this methodology and the rapid pace at which it evolves and changes, new computational tools show up constantly. This rapid turnover, together with the intrinsic high death-rate of bioinformatics tools, mean that many of the published tools become unavailable at some point. Consequently, the traditional ways to inform the community about the landscape of available tools, i.e., reviews in the scientific literature, are not adequate for this fast-moving field. To overcome these limitations, we have developed "WeReview: CRISPR Tools," a live, on-line, user-updatable repository of computational tools to assist researchers in designing CRISPR/Cas experiments. In its web site users can find an updated comprehensive list of tools and search for those fulfilling their specific needs, as well as proposing modifications to the data associated with the tools or the incorporation of new ones.

Automatic detection of genomic regions with informative epigenetic patterns.

BACKGROUND: Epigenetic phenomena are crucial for explaining the phenotypic plasticity seen in the cells of different tissues, developmental stages and diseases, all holding the same DNA sequence. As technology is allowing to retrieve epigenetic information in a genome-wide fashion, massive epigenomic datasets are being accumulated in public repositories. New approaches are required to mine those data to extract useful knowledge. We present here an automatic approach for detecting genomic regions with epigenetic variation patterns across samples related to a grouping of these samples, as a way of detecting regions functionally associated to the phenomenon behind the classification. RESULTS: We show that the regions automatically detected by the method in the whole human genome associated to three different classifications of a set of epigenomes (cancer vs. healthy, brain vs. other organs, and fetal vs. adult tissues) are enriched in genes associated to these processes. CONCLUSIONS: The method is fully automatic and can exhaustively scan the whole human genome at any resolution using large collections of epigenomes as input, although it also produces good results with small datasets. Consequently, it will be valuable for obtaining functional information from the incoming epigenomic information as it continues to accumulate.

Parallel Evolution of High-Level Aminoglycoside Resistance in Escherichia coli Under Low and High Mutation Supply Rates.

Antibiotic resistance is a major concern in public health worldwide, thus there is much interest in characterizing the mutational pathways through which susceptible bacteria evolve resistance. Here we use experimental evolution to explore the mutational pathways toward aminoglycoside resistance, using gentamicin as a model, under low and high mutation supply rates. Our results show that both normo and hypermutable strains of Escherichia coli are able to develop resistance to drug dosages > 1,000-fold higher than the minimal inhibitory concentration for their ancestors. Interestingly, such level of resistance was often associated with changes in susceptibility to other antibiotics, most prominently with increased resistance to fosfomycin. Whole-genome sequencing revealed that all resistant derivatives presented diverse mutations in five common genetic elements: fhuA, fusA and the atpIBEFHAGDC, cyoABCDE, and potABCD operons. Despite the large number of mutations acquired, hypermutable strains did not pay, apparently, fitness cost. In contrast to recent studies, we found that the mutation supply rate mainly affected the speed (tempo) but not the pattern (mode) of evolution: both backgrounds acquired the mutations in the same order, although the hypermutator strain did it faster. This observation is compatible with the adaptive landscape for high-level gentamicin resistance being relatively smooth, with few local maxima; which might be a common feature among antibiotics for which resistance involves multiple loci.

A Conserved Carbon Starvation Response Underlies Bud Dormancy in Woody and Herbaceous Species.

Plant shoot systems give rise to characteristic above-ground plant architectures. Shoots are formed from axillary meristems and buds, whose growth and development is modulated by systemic and local signals. These cues convey information about nutrient and water availability, light quality, sink/source organ activity and other variables that determine the timeliness and competence to maintain development of new shoots. This information is translated into a local response, in meristems and buds, of growth or quiescence. Although some key genes involved in the onset of bud latency have been identified, the gene regulatory networks (GRNs) controlled by these genes are not well defined. Moreover, it has not been determined whether bud dormancy induced by environmental cues, such as a low red-to-far-red light ratio, shares genetic mechanisms with bud latency induced by other causes, such as apical dominance or a short-day photoperiod. Furthermore, the evolution and conservation of these GRNs throughout angiosperms is not well established. We have reanalyzed public transcriptomic datasets that compare quiescent and active axillary buds of Arabidopsis, with datasets of axillary buds of the woody species Vitis vinifera (grapevine) and apical buds of Populus tremula x Populus alba (poplar) during the bud growth-to-dormancy transition. Our aim was to identify potentially common GRNs induced during the process that leads to bud para-, eco- and endodormancy. In Arabidopsis buds that are entering eco- or paradormancy, we have identified four induced interrelated GRNs that correspond to a carbon (C) starvation syndrome, typical of tissues undergoing low C supply. This response is also detectable in poplar and grapevine buds before and during the transition to dormancy. In all eukaryotes, C-limiting conditions are coupled to growth arrest and latency like that observed in dormant axillary buds. Bud dormancy might thus be partly a consequence of the underlying C starvation syndrome triggered by environmental and endogenous cues that anticipate or signal conditions unfavorable for sustained shoot growth.

Apoptosis, Toll-like, RIG-I-like and NOD-like Receptors Are Pathways Jointly Induced by Diverse Respiratory Bacterial and Viral Pathogens.

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here.

Transcriptional repressor DREAM regulates trigeminal noxious perception.

Expression of the downstream regulatory element antagonist modulator (DREAM) protein in dorsal root ganglia and spinal cord is related to endogenous control mechanisms of acute and chronic pain. In primary sensory trigeminal neurons, high levels of endogenous DREAM protein are preferentially localized in the nucleus, suggesting a major transcriptional role. Here, we show that transgenic mice expressing a dominant active mutant of DREAM in trigeminal neurons show increased responses following orofacial sensory stimulation, which correlates with a decreased expression of prodynorphin and brain-derived neurotrophic factor in trigeminal ganglia. Genome-wide analysis of trigeminal neurons in daDREAM transgenic mice identified cathepsin L and the monoglyceride lipase as two new DREAM transcriptional targets related to pain. Our results suggest a role for DREAM in the regulation of trigeminal nociception. This article is part of the special article series "Pain".

Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes.

The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5' or 3' and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request.

Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease.

Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD.

An unbiased genetic screen reveals the polygenic nature of the influenza virus anti-interferon response.

UNLABELLED: Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, interferon (IFN)-dependent upregulation of IFN-stimulated genes (ISGs), and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterized populations and selected mutants of wild-type viruses selected by passage through non-IFN-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that nonsynonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterization induced IFN and ISGs and were unable to counteract the effects of exogenous IFN, yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs but nonetheless replicated to high titers. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in non-IFN-responsive cells allows the virus to relax from these constraints and find a new genome consensus within its sequence space. IMPORTANCE: In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses, and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes to interferon evasion have not been well characterized. Here, we examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titers had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine-tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response.

DREAM controls the on/off switch of specific activity-dependent transcription pathways.

Changes in nuclear Ca(2+) homeostasis activate specific gene expression programs and are central to the acquisition and storage of information in the brain. DREAM (downstream regulatory element antagonist modulator), also known as calsenilin/KChIP-3 (K(+) channel interacting protein 3), is a Ca(2+)-binding protein that binds DNA and represses transcription in a Ca(2+)-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca(2+)-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Using genome-wide analysis, we show that DREAM regulates the expression of specific activity-dependent transcription factors in the hippocampus, including Npas4, Nr4a1, Mef2c, JunB, and c-Fos. Furthermore, DREAM regulates its own expression, establishing an autoinhibitory feedback loop to terminate activity-dependent transcription. Ablation of DREAM does not modify activity-dependent transcription because of gene compensation by the other KChIP family members. The expression of daDREAM in the forebrain resulted in a complex phenotype characterized by loss of recurrent inhibition and enhanced long-term potentiation (LTP) in the dentate gyrus and impaired learning and memory. Our results indicate that DREAM is a major master switch transcription factor that regulates the on/off status of specific activity-dependent gene expression programs that control synaptic plasticity, learning, and memory.

Analysis of genome-wide changes in the translatome of Arabidopsis seedlings subjected to heat stress.

Heat stress is one of the most prominent and deleterious environmental threats affecting plant growth and development. Upon high temperatures, plants launch specialized gene expression programs that promote stress protection and survival. These programs involve global and specific changes at the transcriptional and translational levels. However, the coordination of these processes and their specific role in the establishment of the heat stress response is not fully elucidated. We have carried out a genome-wide analysis to monitor the changes in the translation efficiency of individual mRNAs of Arabidopsis thaliana seedlings after the exposure to a heat shock stress. Our results demonstrate that translation exerts a wide but dual regulation of gene expression. For the majority of mRNAs, translation is severely repressed, causing a decreased of 50% in the association of the bulk of mRNAs to polysomes. However, some relevant mRNAs involved in different aspects of homeostasis maintenance follow a differential pattern of translation. Sequence analyses of the differentially translated mRNAs unravels that some features, such as the 5'UTR G+C content and the cDNA length, may take part in the discrimination mechanisms for mRNA polysome loading. Among the differentially translated genes, master regulators of the stress response stand out, highlighting the main role of translation in the early establishment of the physiological response of plants to elevated temperatures.

Distinct and conserved transcriptomic changes during nematode-induced giant cell development in tomato compared with Arabidopsis: a functional role for gene repression.

Root-knot nematodes (RKNs) induce giant cells (GCs) from root vascular cells inside the galls. Accompanying molecular changes as a function of infection time and across different species, and their functional impact, are still poorly understood. Thus, the transcriptomes of tomato galls and laser capture microdissected (LCM) GCs over the course of parasitism were compared with those of Arabidopsis, and functional analysis of a repressed gene was performed. Microarray hybridization with RNA from galls and LCM GCs, infection-reproduction tests and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) transcriptional profiles in susceptible and resistant (Mi-1) lines were performed in tomato. Tomato GC-induced genes include some possibly contributing to the epigenetic control of GC identity. GC-repressed genes are conserved between tomato and Arabidopsis, notably those involved in lignin deposition. However, genes related to the regulation of gene expression diverge, suggesting that diverse transcriptional regulators mediate common responses leading to GC formation in different plant species. TPX1, a cell wall peroxidase specifically involved in lignification, was strongly repressed in GCs/galls, but induced in a nearly isogenic Mi-1 resistant line on nematode infection. TPX1 overexpression in susceptible plants hindered nematode reproduction and GC expansion. Time-course and cross-species comparisons of gall and GC transcriptomes provide novel insights pointing to the relevance of gene repression during RKN establishment.

Neuronal Ca(2+) dyshomeostasis in Huntington disease.

The expansion of the N-terminal poly-glutamine tract of the huntingtin (Htt) protein is responsible for Huntington disease (HD). A large number of studies have explored the neuronal phenotype of HD, but the molecular aethiology of the disease is still very poorly understood. This has hampered the development of an appropriate therapeutical strategy to at least alleviate its symptoms. In this short review, we have focused our attention on the alteration of a specific cellular mechanism common to all HD models, either genetic or induced by treatment with 3-NPA, i.e. the cellular dyshomeostasis of Ca(2+). We have highlighted the direct and indirect (i.e. transcriptionally mediated) effects of mutated Htt on the maintenance of the intracellular Ca(2+) balance, the correct modulation of which is fundamental to cell survival and the disturbance of which plays a key role in the death of the cell.

Reduced Mid1 Expression and Delayed Neuromotor Development in daDREAM Transgenic Mice.

Downstream regulatory element antagonist modulator (DREAM) is a Ca(2+)-binding protein that binds DNA and represses transcription in a Ca(2+)-dependent manner. Previous work has shown a role for DREAM in cerebellar function regulating the expression of the sodium/calcium exchanger 3 (NCX3) in cerebellar granular neurons to control Ca(2+) homeostasis and survival of these neurons. To achieve a global view of the genes regulated by DREAM in the cerebellum, we performed a genome-wide analysis in transgenic cerebellum expressing a Ca(2+)-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Here we show that DREAM regulates the expression of the midline 1 (Mid1) gene early after birth. As a consequence, daDREAM mice exhibit a significant shortening of the rostro-caudal axis of the cerebellum and a delay in neuromotor development early after birth. Our results indicate a role for DREAM in cerebellar function.

Improved protein-binding microarrays for the identification of DNA-binding specificities of transcription factors.

Transcriptional regulation depends on the specificity of transcription factors (TFs) recognizing cis regulatory sequences in the promoters of target genes. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies, revealing DNA motifs bound by a TF with high affinity. These strategies are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. Here we report on the development of a protein-binding microarray (PBM11) containing all possible double-stranded 11-mers for the determination of DNA-binding specificities of TFs. The large number of sequences in the PBM11 allows accurate and high-throughput quantification of TF-binding sites, outperforming previous methods. We applied this tool to determine binding site specificities of two Arabidopsis TFs, MYC2 and ERF1, rendering the G-box and the GCC-box, respectively, as their highest-affinity binding sites. In addition, we identified variants of the G-box recognized by MYC2 with high and medium affinity, whereas ERF1 only recognized GCC variants with low affinity, indicating that ERF1 binding to DNA has stricter base requirements than MYC2. Analysis of transcriptomic data revealed that high- and medium-affinity binding sites have biological significance, probably representing relevant cis-acting elements in vivo. Comparison of promoter sequences with putative orthologs from closely related species demonstrated a high degree of conservation of all the identified DNA elements. The combination of PBM11, transcriptomic data and phylogenomic footprinting provides a straightforward method for the prediction of biologically active cis-elements, and thus for identification of in vivo DNA targets of TFs.

Increased B cell proliferation and reduced Ig production in DREAM transgenic mice.

DREAM/KChIP-3 is a calcium-dependent transcriptional repressor highly expressed in immune cells. Transgenic mice expressing a dominant active DREAM mutant show reduced serum Ig levels. In vitro assays show that reduced Ig secretion is an intrinsic defect of transgenic B cells that occurs without impairment in plasma cell differentiation, class switch recombination, or Ig transcription. Surprisingly, transgenic B cells show an accelerated entry in cell division. Transcriptomic analysis of transgenic B cells revealed that hyperproliferative B cell response could be correlated with a reduced expression of Klf9, a cell-cycle regulator. Pulse-chase experiments demonstrated that the defect in Ig production is associated with reduced translation rather than with increased protein degradation. Importantly, transgenic B cells showed reduced expression of the Eif4g3 gene, which encodes a protein related to protein translation. Our results disclose, to our knowledge, a novel function of DREAM in proliferation and Ig synthesis in B lymphocytes.

Genome-wide identification of small RNA targets based on target enrichment and microarray hybridizations.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two classes of abundant 21-24 nucleotide small RNAs (smRNAs) that control gene expression in plants, mainly by guiding cleavage and degradation of target transcripts. Target identification based on predictive algorithms for base-paired complementarity requires further experimental validation and often fails to recognize miRNA::target pairs that escape from stringent complementarity rules. Here, we report on a microarray-based methodology to identify target mRNAs of miRNAs and siRNAs at a genomic scale. This strategy takes advantage of the RNA ligase-mediated amplification of 5' cDNA ends (RLM-RACE) to isolate miRNA or siRNA cleavage products from biological samples. Cleaved transcripts are then subjected to T7 RNA polymerase-mediated amplification and microarray hybridizations. The use of suitable hybridization controls is what makes our strategy outperform previous analyses. We applied this method and identified more than 100 putative novel miRNA or siRNA target mRNAs that had not been previously predicted by computational or microarray-based methods. Our data expand the regulatory role of endogenous smRNAs to a wide range of cellular processes, with prevalence in the regulation of cellular solute homeostasis. The methodology described here is straightforward, avoids extensive computational analysis and allows simultaneous analyses of several biological replicates, thus reducing the biological variability inherent in genomic analysis. The application of this simple methodology offers a framework for systematic analysis of smRNA-guided cleaved transcriptomes in different plant tissues, genotypes or stress conditions, and should contribute to understanding of the physiological role of smRNAs in plants.

Bioinformatics methods for the analysis of expression arrays: data clustering and information extraction.

Expression arrays facilitate the monitoring of changes in the expression patterns of large collections of genes. The analysis of expression array data has become a computationally-intensive task that requires the development of bioinformatics technology for a number of key stages in the process, such as image analysis, database storage, gene clustering and information extraction. Here, we review the current trends in each of these areas, with particular emphasis on the development of the related technology being carried out within our groups.