Acute expression of human APOBEC3B in mice results in RNA editing and lethality.

BACKGROUND: RNA editing has been described as promoting genetic heterogeneity, leading to the development of multiple disorders, including cancer. The cytosine deaminase APOBEC3B is implicated in tumor evolution through DNA mutation, but whether it also functions as an RNA editing enzyme has not been studied. RESULTS: Here, we engineer a novel doxycycline-inducible mouse model of human APOBEC3B-overexpression to understand the impact of this enzyme in tissue homeostasis and address a potential role in C-to-U RNA editing. Elevated and sustained levels of APOBEC3B lead to rapid alteration of cellular fitness, major organ dysfunction, and ultimately lethality in mice. Importantly, RNA-sequencing of mouse tissues expressing high levels of APOBEC3B identifies frequent UCC-to-UUC RNA editing events that are not evident in the corresponding genomic DNA. CONCLUSIONS: This work identifies, for the first time, a new deaminase-dependent function for APOBEC3B in RNA editing and presents a preclinical tool to help understand the emerging role of APOBEC3B as a driver of carcinogenesis.

Frequent aneuploidy in primary human T cells after CRISPR-Cas9 cleavage.

Multiple clinical trials of allogeneic T cell therapy use site-specific nucleases to disrupt T cell receptor (TCR) and other genes1-6. In this study, using single-cell RNA sequencing, we investigated genome editing outcomes in primary human T cells transfected with CRISPR-Cas9 and guide RNAs targeting genes for TCR chains and programmed cell death protein 1. Four days after transfection, we found a loss of chromosome 14, harboring the TCRα locus, in up to 9% of the cells and a chromosome 14 gain in up to 1.4% of the cells. Chromosome 7, harboring the TCRß locus, was truncated in 9.9% of the cells. Aberrations were validated using fluorescence in situ hybridization and digital droplet PCR. Aneuploidy was associated with reduced proliferation, induced p53 activation and cell death. However, at 11 days after transfection, 0.9% of T cells still had a chromosome 14 loss. Aneuploidy and chromosomal truncations are, thus, frequent outcomes of CRISPR-Cas9 cleavage that should be monitored and minimized in clinical protocols.

Gene network analysis reveals a role for striatal glutamatergic receptors in dysregulated risk-assessment behavior of autism mouse models.

Autism spectrum disorder (ASD) presents a wide, and often varied, behavioral phenotype. Improper assessment of risks has been reported among individuals diagnosed with ASD. Improper assessment of risks may lead to increased accidents and self-injury, also reported among individuals diagnosed with ASD. However, there is little knowledge of the molecular underpinnings of the impaired risk-assessment phenotype. In this study, we have identified impaired risk-assessment activity in multiple male ASD mouse models. By performing network-based analysis of striatal whole transcriptome data from each of these ASD models, we have identified a cluster of glutamate receptor-associated genes that correlate with the risk-assessment phenotype. Furthermore, pharmacological inhibition of striatal glutamatergic receptors was able to mimic the dysregulation in risk-assessment. Therefore, this study has identified a molecular mechanism that may underlie risk-assessment dysregulation in ASD.

Genome-wide localization of the polyphenol quercetin in human monocytes.

BACKGROUND: Quercetin is a polyphenol of great interest given its antioxidant activity and involvement in the immune response. Although quercetin has been well studied at the molecular level as a gene regulator and an activator of specific cellular pathways, not much attention has been given to its mechanism of action at the genome-wide level. The present study aims to characterize quercetin's interaction with cellular DNA and to show its subsequent effect on downstream transcription. RESULTS: Two massive parallel DNA-sequencing technologies were used: Chem-seq and RNA-seq. We demonstrate that upon binding to DNA or genome-associated proteins, quercetin acts as a cis-regulatory transcription factor for the expression of genes that are involved in the cell cycle, differentiation and development. CONCLUSIONS: Such findings could provide new and important insights into the mechanisms by which the dietary polyphenol quercetin influences cellular functions.

Antidepressants affect gut microbiota and Ruminococcus flavefaciens is able to abolish their effects on depressive-like behavior.

Accumulating evidence demonstrates that the gut microbiota affects brain function and behavior, including depressive behavior. Antidepressants are the main drugs used for treatment of depression. We hypothesized that antidepressant treatment could modify gut microbiota which can partially mediate their antidepressant effects. Mice were chronically treated with one of five antidepressants (fluoxetine, escitalopram, venlafaxine, duloxetine or desipramine), and gut microbiota was analyzed, using 16s rRNA gene sequencing. After characterization of differences in the microbiota, chosen bacterial species were supplemented to vehicle and antidepressant-treated mice, and depressive-like behavior was assessed to determine bacterial effects. RNA-seq analysis was performed to determine effects of bacterial treatment in the brain. Antidepressants reduced richness and increased beta diversity of gut bacteria, compared to controls. At the genus level, antidepressants reduced abundances of Ruminococcus, Adlercreutzia, and an unclassified Alphaproteobacteria. To examine implications of the dysregulated bacteria, we chose one of antidepressants (duloxetine) and investigated if its antidepressive effects can be attenuated by simultaneous treatment with Ruminococcus flavefaciens or Adlercreutzia equolifaciens. Supplementation with R. flavefaciens diminished duloxetine-induced decrease in depressive-like behavior, while A. equolifaciens had no such effect. R. flavefaciens treatment induced changes in cortical gene expression, up-regulating genes involved in mitochondrial oxidative phosphorylation, while down-regulating genes involved in neuronal plasticity. Our results demonstrate that various types of antidepressants alter gut microbiota composition, and further implicate a role for R. flavefaciens in alleviating depressive-like behavior. Moreover, R. flavefaciens affects gene networks in the brain, suggesting a mechanism for microbial regulation of antidepressant treatment efficiency.

Differential contribution of cis and trans gene transcription regulatory mechanisms in amygdala and prefrontal cortex and modulation by social stress.

While both individual transcription factors and cis-acting sites have been studied in relation to psychiatric disorders, there is little knowledge of the relative contribution of trans-acting and cis-acting factors to gene transcription in the brain. Using an RNA-seq approach in mice bred from two evolutionary-distinct mice strains, we determined the contribution of cis and trans factors to gene expression in the prefrontal cortex and amygdala, two regions of the brain relevant to the stress response, and the contribution of cis and trans factors in the prefrontal cortex after Chronic Social Defeat (CSD) in mice. More genes were regulated by cis-regulatory factors in both brain regions, underlying the importance of cis-acting gene regulation in the brain. However, there was an increase in genes regulated by trans-regulatory mechanisms in the amygdala, compared to the prefrontal cortex. These genes were involved in synaptic functions, and were enriched for binding sites for transcription factors, including Egr1. CSD induced an increase in genes regulated by trans-regulatory mechanisms in the prefrontal cortex, and induced a pattern similar to the unstressed amygdala. Overall, we show brain site-specific patterns in cis and trans regulatory mechanisms, and show that these patterns can be modified by a psychological trigger.

Dysregulation of Cortical Neuron DNA Methylation Profile in Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a complex neuropsychiatric syndrome whose etiology includes genetic and environmental components. Since epigenetic marks are sensitive to environmental insult, they may be involved in the development of ASD. Initial brain studies have suggested a dysregulation of epigenetic marks in ASD. However, due to cellular heterogeneity in the brain, these studies have not determined if there is a true change in the neuronal epigenetic signature. Here, we report a genome-wide methylation study on fluorescence-activated cell sorting-sorted neuronal nuclei from the frontal cortex of 16 male ASD and 15 male control subjects. Using the 450 K BeadArray, we identified 58 differentially methylated regions (DMRs) that included loci associated to GABAergic system genes, particularly ABAT and GABBR1, and brain-specific MicroRNAs. Selected DMRs were validated by targeted Next Generation Bisulfite Sequencing. Weighted gene correlation network analysis detected 3 co-methylation modules which are significantly correlated to ASD that were enriched for genomic regions underlying neuronal, GABAergic, and immune system genes. Finally, we determined an overlap of the 58 ASD-related DMRs with neurodevelopment associated DMRs. This investigation identifies alterations in the DNA methylation pattern in ASD cortical neurons, providing further evidence that epigenetic alterations in disorder-relevant tissues may be involved in the biology of ASD.

Virtual screening for potential inhibitors of Mcl-1 conformations sampled by normal modes, molecular dynamics, and nuclear magnetic resonance.

Myeloid cell leukemia-1 (Mcl-1) is often overexpressed in human cancer and is an important target for developing antineoplastic drugs. In this study, a data set containing 2.3 million lead-like molecules and a data set of all the US Food and Drug Administration (FDA)-approved drugs are virtually screened for potential Mcl-1 ligands using Protein Data Bank (PDB) ID 2MHS. The potential Mcl-1 ligands are evaluated and computationally docked on to three conformation ensembles generated by normal mode analysis (NMA), molecular dynamics (MD), and nuclear magnetic resonance (NMR), respectively. The evaluated potential Mcl-1 ligands are then compared with their clinical use. Remarkably, half of the top 30 potential drugs are used clinically to treat cancer, thus partially validating our virtual screen. The partial validation also favors the idea that the other half of the top 30 potential drugs could be used in the treatment of cancer. The normal mode-, MD-, and NMR-based conformation greatly expand the conformational sampling used herein for in silico identification of potential Mcl-1 inhibitors.

Sequence variation in PPP1R13L results in a novel form of cardio-cutaneous syndrome.

Dilated cardiomyopathy (DCM) is a life-threatening disorder whose genetic basis is heterogeneous and mostly unknown. Five Arab Christian infants, aged 4-30 months from four families, were diagnosed with DCM associated with mild skin, teeth, and hair abnormalities. All passed away before age 3. A homozygous sequence variation creating a premature stop codon at PPP1R13L encoding the iASPP protein was identified in three infants and in the mother of the other two. Patients' fibroblasts and PPP1R13L-knocked down human fibroblasts presented higher expression levels of pro-inflammatory cytokine genes in response to lipopolysaccharide, as well as Ppp1r13l-knocked down murine cardiomyocytes and hearts of Ppp1r13l-deficient mice. The hypersensitivity to lipopolysaccharide was NF-κB-dependent, and its inducible binding activity to promoters of pro-inflammatory cytokine genes was elevated in patients' fibroblasts. RNA sequencing of Ppp1r13l-knocked down murine cardiomyocytes and of hearts derived from different stages of DCM development in Ppp1r13l-deficient mice revealed the crucial role of iASPP in dampening cardiac inflammatory response. Our results determined PPP1R13L as the gene underlying a novel autosomal-recessive cardio-cutaneous syndrome in humans and strongly suggest that the fatal DCM during infancy is a consequence of failure to regulate transcriptional pathways necessary for tuning cardiac threshold response to common inflammatory stressors.

Multidimensional patterns of metabolic response in abiotic stress-induced growth of Arabidopsis thaliana.

KEY MESSAGE: Contextualization of specific transcriptional responses of Arabidopsis within the stress-tissue-time perspective provides a simplified representation of the cellular transcriptional response pathways to abiotic stress, while reducing the dimensions in gene-oriented response description. Crops resistant to abiotic stresses are a long-term goal of many research programs, thus understanding the progression of stress responses is of great interest. We reanalyzed the AtGenExpress transcription dataset to go beyond gene-level characterization, and to contextualize the discrete information into (1) a process-level signature of stress-specific, time-specific, and tissue-specific responses and (2) identify patterns of response progression across a time axis. To gain a functional perspective, ∼1000 pathways associated with the differentially-expressed genes were characterized across all experiments. We find that the global response of pathways to stress is multi-dimensional and does not obviously cluster according to stress, time or tissue. The early response to abiotic stress typically involves induction of genes involved in transcription, hormone synthesis and signaling modules; a later response typically involves metabolism of amino acids and secondary metabolites. By linking specific primary and secondary response pathways, we outline possible stress-associated routes of response progression. The contextualization of specific processes within stress-tissue-time perspective provides a simplified representation of cellular response while reducing the dimensions in gene-oriented response description. Such simplified representation allows finding stress-specific markers based on process-combinations pointing whether a stress-specific response was invoked as well as provide a reference point for the conductance of comparative inter-plant study of stress response, bypassing the need in detailed orthologous mapping.

Systems approach for exploring the intricate associations between sweetness, color and aroma in melon fruits.

BACKGROUND: Melon (Cucumis melo) fruits exhibit phenotypic diversity in several key quality determinants such as taste, color and aroma. Sucrose, carotenoids and volatiles are recognized as the key compounds shaping the above corresponding traits yet the full network of biochemical events underlying their synthesis have not been comprehensively described. To delineate the cellular processes shaping fruit quality phenotypes, a population of recombinant inbred lines (RIL) was used as a source of phenotypic and genotypic variations. In parallel, ripe fruits were analyzed for both the quantified level of 77 metabolic traits directly associated with fruit quality and for RNA-seq based expression profiles generated for 27,000 unigenes. First, we explored inter-metabolite association patterns; then, we described metabolites versus gene association patterns; finally, we used the correlation-based associations for predicting uncharacterized synthesis pathways. RESULTS: Based on metabolite versus metabolite and metabolite versus gene association patterns, we divided metabolites into two key groups: a group including ethylene and aroma determining volatiles whose accumulation patterns are correlated with the expression of genes involved in the glycolysis and TCA cycle pathways; and a group including sucrose and color determining carotenoids whose accumulation levels are correlated with the expression of genes associated with plastid formation. CONCLUSIONS: The study integrates multiple processes into a genome scale perspective of cellular activity. This lays a foundation for deciphering the role of gene markers associated with the determination of fruit quality traits.

A Role of AREB in the Regulation of PACC-Dependent Acid-Expressed-Genes and Pathogenicity of Colletotrichum gloeosporioides.

Gene expression regulation by pH in filamentous fungi and yeasts is controlled by the PACC/RIM101 transcription factor. In Colletotrichum gloeosporioides, PACC is known to act as positive regulator of alkaline-expressed genes, and this regulation was shown to contribute to fungal pathogenicity. PACC is also a negative regulator of acid-expressed genes, however; the mechanism of downregulation of acid-expressed genes by PACC and their contribution to C. gloeosporioides pathogenicity is not well understood. RNA sequencing data analysis was employed to demonstrate that PACC transcription factor binding sites (TFBS) are significantly overrepresented in the promoter of PACC-upregulated, alkaline-expressed genes. In contrast, they are not overrepresented in the PACC-downregulated, acid-expressed genes. Instead, acid-expressed genes showed overrepresentation of AREB GATA TFBS in C. gloeosporioides and in homologs of five other ascomycetes genomes. The areB promoter contains PACC TFBS; its transcript was upregulated at pH 7 and repressed in ΔpacC. Furthermore, acid-expressed genes were found to be constitutively upregulated in ΔareB during alkalizing conditions. The areB mutants showed significantly reduced ammonia secretion and pathogenicity on tomato fruit. Present results indicate that PACC activates areB expression, thereby conditionally repressing acid-expressed genes and contributing critically to C. gloeosporioides pathogenicity.

PHI-DAC: protein homology database through dihedral angle conservation.

UNLABELLED: Finding related conformations in the Protein Data Bank is essential in many areas of bioscience. To assist this task, we designed a dihedral angle database for searching protein segment homologs. The search engine relies on encoding of the protein coordinates into text characters representing amino acid sequence, φ and ψ dihedral angles. The search engine is advantageous owing to its high speed and interactive nature and is expected to assist scientists in discovering conformation homologs and evolutionary kinship. The search engine is fast, with query times lasting a few seconds, and freely available at http://tarshish.md.biu.ac.il/∼samsona. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Global aspects of pacC regulation of pathogenicity genes in Colletotrichum gloeosporioides as revealed by transcriptome analysis.

Colletotrichum gloeosporioides alkalinizes its surroundings during colonization of host tissue. The transcription factor pacC is a regulator of pH-controlled genes and is essential for successful colonization. We present here the sequence assembly of the Colletotrichum fruit pathogen and use it to explore the global regulation of pathogenicity by ambient pH. The assembled genome size was 54 Mb, encoding 18,456 genes. Transcriptomes of the wild type and ΔpacC mutant were established by RNA-seq and explored for their global pH-dependent gene regulation. The analysis showed that pacC upregulates 478 genes and downregulates 483 genes, comprising 5% of the fungal genome, including transporters, antioxidants, and cell-wall-degrading enzymes. Interestingly, gene families with similar functionality are both up- and downregulated by pacC. Global analysis of secreted genes showed significant pacC activation of degradative enzymes at alkaline pH and during fruit infection. Select genes from alkalizing-type pathogen C. gloeosporioides and from acidifying-type pathogen Sclerotinia sclerotiorum were verified by quantitative reverse-transcription polymerase chain reaction analysis at different pH values. Knock out of several pacC-activated genes confirmed their involvement in pathogenic colonization of alkalinized surroundings. The results suggest a global regulation by pacC of key pathogenicity genes during pH change in alkalinizing and acidifying pathogens.

A novel multi-scale modeling approach to infer whole genome divergence.

We propose a novel and simple approach to elucidate genomic patterns of divergence using principal component analysis (PCA). We applied this methodology to the metric space generated by M. musculus genome-wide SNPs. Distance profiles were computed between M. musculus and its closely related species, M. spretus, which was used as external reference. While the speciation dynamics were apparent in the first principal component, the within M. musculus differentiation dimensions gave rise to three minor components. We were unable to obtain a clear divergence signature discriminating laboratory strains, suggesting a stronger effect of genetic drift. These results were at odds with wild strains which exhibit defined deterministic signals of divergence. Finally, we were able to rank novel and previously known genes according to their likelihood to be under selective pressure. In conclusion, we posit PCA as a robust methodology to unravel diverging DNA regions without any a priori forcing.

The genetic background effect on domesticated species: a mouse evolutionary perspective.

Laboratory mouse strains are known for their large phenotypic diversity and serve as a primary mammalian model in genotype-phenotype association studies. One possible attempt to understand the reason for this diversity could be addressed by careful investigation of the unique evolutionary history of their wild-derived founders and the consequence that it may have on the genetic makeup of the laboratory mouse strains during the history of human fancy breeding. This review will summarize recently published literature that endeavors to unravel the genetic background of laboratory mouse strains, as well as give new insights into novel evolutionary approaches. I will explain basic concepts of molecular evolution and the reason why it is important in order to infer function even among closely related wild and domesticated species. I will also discuss future frontiers in the field and how newly emerging sequencing technologies could help us to better understand the relationship between genotype and phenotype.

The consequence of natural selection on genetic variation in the mouse.

Laboratory mouse strains are known to have emerged from recent interbreeding between individuals of Mus musculus isolated populations. As a result of this breeding history, the collection of polymorphisms observed between laboratory mouse strains is likely to harbor the effects of natural selection between reproductively isolated populations. Until now no study has systematically investigated the consequences of this breeding history on gene evolution. Here we have used a novel, unbiased evolutionary approach to predict the founder origin of laboratory mouse strains and to assess the balance between ancient and newly emerged mutations in the founder subspecies. Our results confirm a contribution from at least four distinct subspecies. Additionally, our method allowed us to identify regions of relaxed selective constraint among laboratory mouse strains. This unique structure of variation is likely to have significant consequences on the use of mouse to find genes underlying phenotypic variation.

Phenostat: visualization and statistical tool for analysis of phenotyping data.

The effective extraction of information from multidimensional data sets derived from phenotyping experiments is a growing challenge in biology. Data visualization tools are important resources that can aid in exploratory data analysis of complex data sets. Phenotyping experiments of model organisms produce data sets in which a large number of phenotypic measures are collected for each individual in a group. A critical initial step in the analysis of such multidimensional data sets is the exploratory analysis of data distribution and correlation. To facilitate the rapid visualization and exploratory analysis of multidimensional complex trait data, we have developed a user-friendly, web-based software tool called Phenostat. Phenostat is composed of a dynamic graphical environment that allows the user to inspect the distribution of multiple variables in a data set simultaneously. Individuals can be selected by directly clicking on the graphs and thus displaying their identity, highlighting corresponding values in all graphs, allowing their inclusion or exclusion from the analysis. Statistical analysis is provided by R package functions. Phenostat is particularly suited for rapid distribution and correlation analysis of subsets of data. An analysis of behavioral and physiologic data stemming from a large mouse phenotyping experiment using Phenostat reveals previously unsuspected correlations. Phenostat is freely available to academic institutions and nonprofit organizations and can be used from our website at: (http://www.bioinfo.embl.it/phenostat/).

Mouse Phenotype Database Integration Consortium: integration [corrected] of mouse phenome data resources.

Understanding the functions encoded in the mouse genome will be central to an understanding of the genetic basis of human disease. To achieve this it will be essential to be able to characterize the phenotypic consequences of variation and alterations in individual genes. Data on the phenotypes of mouse strains are currently held in a number of different forms (detailed descriptions of mouse lines, first-line phenotyping data on novel mutations, data on the normal features of inbred lines) at many sites worldwide. For the most efficient use of these data sets, we have initiated a process to develop standards for the description of phenotypes (using ontologies) and file formats for the description of phenotyping protocols and phenotype data sets. This process is ongoing and needs to be supported by the wider mouse genetics and phenotyping communities to succeed. We invite interested parties to contact us as we develop this process further.