Natural proteome diversity links aneuploidy tolerance to protein turnover.

Accessing the natural genetic diversity of species unveils hidden genetic traits, clarifies gene functions and allows the generalizability of laboratory findings to be assessed. One notable discovery made in natural isolates of Saccharomyces cerevisiae is that aneuploidy-an imbalance in chromosome copy numbers-is frequent1,2 (around 20%), which seems to contradict the substantial fitness costs and transient nature of aneuploidy when it is engineered in the laboratory3-5. Here we generate a proteomic resource and merge it with genomic1 and transcriptomic6 data for 796 euploid and aneuploid natural isolates. We find that natural and lab-generated aneuploids differ specifically at the proteome. In lab-generated aneuploids, some proteins-especially subunits of protein complexes-show reduced expression, but the overall protein levels correspond to the aneuploid gene dosage. By contrast, in natural isolates, more than 70% of proteins encoded on aneuploid chromosomes are dosage compensated, and average protein levels are shifted towards the euploid state chromosome-wide. At the molecular level, we detect an induction of structural components of the proteasome, increased levels of ubiquitination, and reveal an interdependency of protein turnover rates and attenuation. Our study thus highlights the role of protein turnover in mediating aneuploidy tolerance, and shows the utility of exploiting the natural diversity of species to attain generalizable molecular insights into complex biological processes.

A yeast living ancestor reveals the origin of genomic introgressions.

Genome introgressions drive evolution across the animal1, plant2 and fungal3 kingdoms. Introgressions initiate from archaic admixtures followed by repeated backcrossing to one parental species. However, how introgressions arise in reproductively isolated species, such as yeast4, has remained unclear. Here we identify a clonal descendant of the ancestral yeast hybrid that founded the extant Saccharomyces cerevisiae Alpechin lineage5, which carries abundant Saccharomyces paradoxus introgressions. We show that this clonal descendant, hereafter defined as a 'living ancestor', retained the ancestral genome structure of the first-generation hybrid with contiguous S. cerevisiae and S. paradoxus subgenomes. The ancestral first-generation hybrid underwent catastrophic genomic instability through more than a hundred mitotic recombination events, mainly manifesting as homozygous genome blocks generated by loss of heterozygosity. These homozygous sequence blocks rescue hybrid fertility by restoring meiotic recombination and are the direct origins of the introgressions present in the Alpechin lineage. We suggest a plausible route for introgression evolution through the reconstruction of extinct stages and propose that genome instability allows hybrids to overcome reproductive isolation and enables introgressions to emerge.

UTRdb 2.0: a comprehensive, expert curated catalog of eukaryotic mRNAs untranslated regions.

The 5' and 3' untranslated regions of eukaryotic mRNAs (UTRs) play crucial roles in the post-transcriptional regulation of gene expression through the modulation of nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization, and message stability. Since 1996, we have developed and maintained UTRdb, a specialized database of UTR sequences. Here we present UTRdb 2.0, a major update of UTRdb featuring an extensive collection of eukaryotic 5' and 3' UTR sequences, including over 26 million entries from over 6 million genes and 573 species, enriched with a curated set of functional annotations. Annotations include CAGE tags and polyA signals to label the completeness of 5' and 3'UTRs, respectively. In addition, uORFs and IRES are annotated in 5'UTRs as well as experimentally validated miRNA targets in 3'UTRs. Further annotations include evolutionarily conserved blocks, Rfam motifs, ADAR-mediated RNA editing events, and m6A modifications. A web interface allowing a flexible selection and retrieval of specific subsets of UTRs, selected according to a combination of criteria, has been implemented which also provides comprehensive download facilities. UTRdb 2.0 is accessible at http://utrdb.cloud.ba.infn.it/utrdb/.

Rice florigens control a common set of genes at the shoot apical meristem including the F-BOX BROADER TILLER ANGLE 1 that regulates tiller angle and spikelet development.

Rice flowering is triggered by transcriptional reprogramming at the shoot apical meristem (SAM) mediated by florigenic proteins produced in leaves in response to changes in photoperiod. Florigens are more rapidly expressed under short days (SDs) compared to long days (LDs) and include the HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) phosphatidylethanolamine binding proteins. Hd3a and RFT1 are largely redundant at converting the SAM into an inflorescence, but whether they activate the same target genes and convey all photoperiodic information that modifies gene expression at the SAM is currently unclear. We uncoupled the contribution of Hd3a and RFT1 to transcriptome reprogramming at the SAM by RNA sequencing of dexamethasone-inducible over-expressors of single florigens and wild-type plants exposed to photoperiodic induction. Fifteen highly differentially expressed genes common to Hd3a, RFT1, and SDs were retrieved, 10 of which still uncharacterized. Detailed functional studies on some candidates revealed a role for LOC_Os04g13150 in determining tiller angle and spikelet development and the gene was renamed BROADER TILLER ANGLE 1 (BRT1). We identified a core set of genes controlled by florigen-mediated photoperiodic induction and defined the function of a novel florigen target controlling tiller angle and spikelet development.

Genome evolution across 1,011 Saccharomyces cerevisiae isolates.

Large-scale population genomic surveys are essential to explore the phenotypic diversity of natural populations. Here we report the whole-genome sequencing and phenotyping of 1,011 Saccharomyces cerevisiae isolates, which together provide an accurate evolutionary picture of the genomic variants that shape the species-wide phenotypic landscape of this yeast. Genomic analyses support a single 'out-of-China' origin for this species, followed by several independent domestication events. Although domesticated isolates exhibit high variation in ploidy, aneuploidy and genome content, genome evolution in wild isolates is mainly driven by the accumulation of single nucleotide polymorphisms. A common feature is the extensive loss of heterozygosity, which represents an essential source of inter-individual variation in this mainly asexual species. Most of the single nucleotide polymorphisms, including experimentally identified functional polymorphisms, are present at very low frequencies. The largest numbers of variants identified by genome-wide association are copy-number changes, which have a greater phenotypic effect than do single nucleotide polymorphisms. This resource will guide future population genomics and genotype-phenotype studies in this classic model system.

Next generation sequencing of SARS-CoV-2 genomes: challenges, applications and opportunities.

Various next generation sequencing (NGS) based strategies have been successfully used in the recent past for tracing origins and understanding the evolution of infectious agents, investigating the spread and transmission chains of outbreaks, as well as facilitating the development of effective and rapid molecular diagnostic tests and contributing to the hunt for treatments and vaccines. The ongoing COVID-19 pandemic poses one of the greatest global threats in modern history and has already caused severe social and economic costs. The development of efficient and rapid sequencing methods to reconstruct the genomic sequence of SARS-CoV-2, the etiological agent of COVID-19, has been fundamental for the design of diagnostic molecular tests and to devise effective measures and strategies to mitigate the diffusion of the pandemic. Diverse approaches and sequencing methods can, as testified by the number of available sequences, be applied to SARS-CoV-2 genomes. However, each technology and sequencing approach has its own advantages and limitations. In the current review, we will provide a brief, but hopefully comprehensive, account of currently available platforms and methodological approaches for the sequencing of SARS-CoV-2 genomes. We also present an outline of current repositories and databases that provide access to SARS-CoV-2 genomic data and associated metadata. Finally, we offer general advice and guidelines for the appropriate sharing and deposition of SARS-CoV-2 data and metadata, and suggest that more efficient and standardized integration of current and future SARS-CoV-2-related data would greatly facilitate the struggle against this new pathogen. We hope that our 'vademecum' for the production and handling of SARS-CoV-2-related sequencing data, will contribute to this objective.

ViruClust: direct comparison of SARS-CoV-2 genomes and genetic variants in space and time.

MOTIVATION: The ongoing evolution of SARS-CoV-2 and the rapid emergence of variants of concern at distinct geographic locations have relevant implications for the implementation of strategies for controlling the COVID-19 pandemic. Combining the growing body of data and the evidence on potential functional implications of SARS-CoV-2 mutations can suggest highly effective methods for the prioritization of novel variants of potential concern, e.g. increasing in frequency locally and/or globally. However, these analyses may be complex, requiring the integration of different data and resources. We claim the need for a streamlined access to up-to-date and high-quality genome sequencing data from different geographic regions/countries, and the current lack of a robust and consistent framework for the evaluation/comparison of the results. RESULTS: To overcome these limitations, we developed ViruClust, a novel tool for the comparison of SARS-CoV-2 genomic sequences and lineages in space and time. ViruClust is made available through a powerful and intuitive web-based user interface. Sophisticated large-scale analyses can be executed with a few clicks, even by users without any computational background. To demonstrate potential applications of our method, we applied ViruClust to conduct a thorough study of the evolution of the most prevalent lineage of the Delta SARS-CoV-2 variant, and derived relevant observations. By allowing the seamless integration of different types of functional annotations and the direct comparison of viral genomes and genetic variants in space and time, ViruClust represents a highly valuable resource for monitoring the evolution of SARS-CoV-2, facilitating the identification of variants and/or mutations of potential concern. AVAILABILITY AND IMPLEMENTATION: ViruClust is openly available at http://gmql.eu/viruclust/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Deconvolution of intergenic polymorphisms determining high expression of Factor H binding protein in meningococcus and their association with invasive disease.

Neisseria meningitidis is a strictly human pathogen and is the major cause of septicemia and meningitis worldwide. Factor H binding protein (fHbp) is a meningococcal surface-exposed lipoprotein that binds the human Complement factor H allowing the bacterium to evade the host innate immune response. FHbp is also a key antigen in two vaccines against N. meningitidis serogroup B. Although the fHbp gene is present in most circulating meningococcal strains, level of fHbp expression varies among isolates and has been correlated to differences in promoter sequences upstream of the gene. Here we elucidated the sequence determinants that control fHbp expression in globally circulating strains. We analyzed the upstream fHbp intergenic region (fIR) of more than 5800 strains representative of the UK circulating isolates and we identified eleven fIR sequence alleles which represent 88% of meningococcal strains. By engineering isogenic recombinant strains where fHbp expression was under the control of each of the eleven fIR alleles, we confirmed that the fIR sequence determines a specific and distinct level of expression. Moreover, we identified the molecular basis for variation in expression through polymorphisms within key regulatory regions that are known to affect fHbp expression. We experimentally established three expression groups, high-medium-low, that correlated directly with the susceptibility to killing mediated by anti-fHbp antibodies and the ability of the meningococcal strain to survive within human serum. By using this sequence classification and information about the variant, we predicted fHbp expression in the panel of UK strains and we observed that strains with higher expressing fIR alleles are more likely associated with invasive disease. Overall, our findings can contribute to understand and predict vaccine coverage mediated by fHbp as well as to shed light on the role of this virulence factor in determining an invasive phenotype.

Lack of ApoA-I in ApoEKO Mice Causes Skin Xanthomas, Worsening of Inflammation, and Increased Coronary Atherosclerosis in the Absence of Hyperlipidemia.

BACKGROUND: HDL (high-density lipoprotein) and its major protein component, apoA-I (apolipoprotein A-I), play a unique role in cholesterol homeostasis and immunity. ApoA-I deficiency in hyperlipidemic, atheroprone mice was shown to drive cholesterol accumulation and inflammatory cell activation/proliferation. The present study was aimed at investigating the impact of apoA-I deficiency on lipid deposition and local/systemic inflammation in normolipidemic conditions. METHODS: ApoE deficient mice, apoE/apoA-I double deficient (DKO) mice, DKO mice overexpressing human apoA-I, and C57Bl/6J control mice were fed normal laboratory diet until 30 weeks of age. Plasma lipids were quantified, atherosclerosis development at the aortic sinus and coronary arteries was measured, skin ultrastructure was evaluated by electron microscopy. Blood and lymphoid organs were characterized through histological, immunocytofluorimetric, and whole transcriptome analyses. RESULTS: DKO were characterized by almost complete HDL deficiency and by plasma total cholesterol levels comparable to control mice. Only DKO showed xanthoma formation and severe inflammation in the skin-draining lymph nodes, whose transcriptome analysis revealed a dramatic impairment in energy metabolism and fatty acid oxidation pathways. An increased presence of CD4+ T effector memory cells was detected in blood, spleen, and skin-draining lymph nodes of DKO. A worsening of atherosclerosis at the aortic sinus and coronary arteries was also observed in DKO versus apoE deficient. Human apoA-I overexpression in the DKO background was able to rescue the skin phenotype and halt atherosclerosis development. CONCLUSIONS: HDL deficiency, in the absence of hyperlipidemia, is associated with severe alterations of skin morphology, aortic and coronary atherosclerosis, local and systemic inflammation.

Single nucleotide polymorphisms associated with wine fermentation and adaptation to nitrogen limitation in wild and domesticated yeast strains.

For more than 20 years, Saccharomyces cerevisiae has served as a model organism for genetic studies and molecular biology, as well as a platform for biotechnology (e.g., wine production). One of the important ecological niches of this yeast that has been extensively studied is wine fermentation, a complex microbiological process in which S. cerevisiae faces various stresses such as limited availability of nitrogen. Nitrogen deficiencies in grape juice impair fermentation rate and yeast biomass production, leading to sluggish or stuck fermentations, resulting in considerable economic losses for the wine industry. In the present work, we took advantage of the "1002 Yeast Genomes Project" population, the most complete catalogue of the genetic variation in the species and a powerful resource for genotype-phenotype correlations, to study the adaptation to nitrogen limitation in wild and domesticated yeast strains in the context of wine fermentation. We found that wild and domesticated yeast strains have different adaptations to nitrogen limitation, corroborating their different evolutionary trajectories. Using a combination of state-of-the-art bioinformatic (GWAS) and molecular biology (CRISPR-Cas9) methodologies, we validated that PNP1, RRT5 and PDR12 are implicated in wine fermentation, where RRT5 and PDR12 are also involved in yeast adaptation to nitrogen limitation. In addition, we validated SNPs in these genes leading to differences in fermentative capacities and adaptation to nitrogen limitation. Altogether, the mapped genetic variants have potential applications for the genetic improvement of industrial yeast strains.

VID22 counteracts G-quadruplex-induced genome instability.

Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism.

An Out-of-Patagonia migration explains the worldwide diversity and distribution of Saccharomyces eubayanus lineages.

Population-level sampling and whole-genome sequences of different individuals allow one to identify signatures of hybridization, gene flow and potential molecular mechanisms of environmental responses. Here, we report the isolation of 160 Saccharomyces eubayanus strains, the cryotolerant ancestor of lager yeast, from ten sampling sites in Patagonia along 2,000 km of Nothofagus forests. Frequency of S. eubayanus isolates was higher towards southern and colder regions, demonstrating the cryotolerant nature of the species. We sequenced the genome of 82 strains and, together with 23 available genomes, performed a comprehensive phylogenetic analysis. Our results revealed the presence of five different lineages together with dozens of admixed strains. Various analytical methods reveal evidence of gene flow and historical admixture between lineages from Patagonia and Holarctic regions, suggesting the co-occurrence of these ancestral populations. Analysis of the genetic contribution to the admixed genomes revealed a Patagonian genetic origin of the admixed strains, even for those located in the North Hemisphere. Overall, the Patagonian lineages, particularly the southern populations, showed a greater global genetic diversity compared to Holarctic and Chinese lineages, in agreement with a higher abundance in Patagonia. Thus, our results are consistent with a likely colonization of the species from peripheral glacial refugia from South Patagonia. Furthermore, fermentative capacity and maltose consumption resulted negatively correlated with latitude, indicating better fermentative performance in northern populations. Our genome analysis, together with previous reports in the sister species S. uvarum suggests that a S. eubayanus ancestor was adapted to the harsh environmental conditions of Patagonia, a region that provides the ecological conditions for the diversification of these ancestral lineages.

Whole-Exome and Transcriptome Sequencing Expands the Genotype of Majewski Osteodysplastic Primordial Dwarfism Type II.

Microcephalic Osteodysplastic Primordial Dwarfism type II (MOPDII) represents the most common form of primordial dwarfism. MOPD clinical features include severe prenatal and postnatal growth retardation, postnatal severe microcephaly, hypotonia, and an increased risk for cerebrovascular disease and insulin resistance. Autosomal recessive biallelic loss-of-function genomic variants in the centrosomal pericentrin (PCNT) gene on chromosome 21q22 cause MOPDII. Over the past decade, exome sequencing (ES) and massive RNA sequencing have been effectively employed for both the discovery of novel disease genes and to expand the genotypes of well-known diseases. In this paper we report the results both the RNA sequencing and ES of three patients affected by MOPDII with the aim of exploring whether differentially expressed genes and previously uncharacterized gene variants, in addition to PCNT pathogenic variants, could be associated with the complex phenotype of this disease. We discovered a downregulation of key factors involved in growth, such as IGF1R, IGF2R, and RAF1, in all three investigated patients. Moreover, ES identified a shortlist of genes associated with deleterious, rare variants in MOPDII patients. Our results suggest that Next Generation Sequencing (NGS) technologies can be successfully applied for the molecular characterization of the complex genotypic background of MOPDII.

Comparative Genomics Reveals Early Emergence and Biased Spatiotemporal Distribution of SARS-CoV-2.

Effective systems for the analysis of molecular data are fundamental for monitoring the spread of infectious diseases and studying pathogen evolution. The rapid identification of emerging viral strains, and/or genetic variants potentially associated with novel phenotypic features is one of the most important objectives of genomic surveillance of human pathogens and represents one of the first lines of defense for the control of their spread. During the COVID 19 pandemic, several taxonomic frameworks have been proposed for the classification of SARS-Cov-2 isolates. These systems, which are typically based on phylogenetic approaches, represent essential tools for epidemiological studies as well as contributing to the study of the origin of the outbreak. Here, we propose an alternative, reproducible, and transparent phenetic method to study changes in SARS-CoV-2 genomic diversity over time. We suggest that our approach can complement other systems and facilitate the identification of biologically relevant variants in the viral genome. To demonstrate the validity of our approach, we present comparative genomic analyses of more than 175,000 genomes. Our method delineates 22 distinct SARS-CoV-2 haplogroups, which, based on the distribution of high-frequency genetic variants, fall into four major macrohaplogroups. We highlight biased spatiotemporal distributions of SARS-CoV-2 genetic profiles and show that seven of the 22 haplogroups (and of all of the four haplogroup clusters) showed a broad geographic distribution within China by the time the outbreak was widely recognized-suggesting early emergence and widespread cryptic circulation of the virus well before its isolation in January 2020. General patterns of genomic variability are remarkably similar within all major SARS-CoV-2 haplogroups, with UTRs consistently exhibiting the greatest variability, with s2m, a conserved secondary structure element of unknown function in the 3'-UTR of the viral genome showing evidence of a functional shift. Although several polymorphic sites that are specific to one or more haplogroups were predicted to be under positive or negative selection, overall our analyses suggest that the emergence of novel types is unlikely to be driven by convergent evolution and independent fixation of advantageous substitutions, or by selection of recombined strains. In the absence of extensive clinical metadata for most available genome sequences, and in the context of extensive geographic and temporal biases in the sampling, many questions regarding the evolution and clinical characteristics of SARS-CoV-2 isolates remain open. However, our data indicate that the approach outlined here can be usefully employed in the identification of candidate SARS-CoV-2 genetic variants of clinical and epidemiological importance.

GIGANTEA Is a Negative Regulator of Abscisic Acid Transcriptional Responses and Sensitivity in Arabidopsis.

Transcriptional reprogramming plays a key role in drought stress responses, preceding the onset of morphological and physiological acclimation. The best-characterized signal regulating gene expression in response to drought is the phytohormone abscisic acid (ABA). ABA-regulated gene expression, biosynthesis and signaling are highly organized in a diurnal cycle, so that ABA-regulated physiological traits occur at the appropriate time of day. The mechanisms that underpin such diel oscillations in ABA signals are poorly characterized. Here we uncover GIGANTEA (GI) as a key gatekeeper of ABA-regulated transcriptional and physiological responses. Time-resolved gene expression profiling by RNA sequencing under different irrigation scenarios indicates that gi mutants produce an exaggerated ABA response, despite accumulating wild-type levels of ABA. Comparisons with ABA-deficient mutants confirm the role of GI in controlling ABA-regulated genes, and the analysis of leaf temperature, a read-out for transpiration, supports a role for GI in the control of ABA-regulated physiological processes. Promoter regions of GI/ABA-regulated transcripts are directly targeted by different classes of transcription factors (TFs), especially PHYTOCHROME-INTERACTING FACTOR and -BINDING FACTOR, together with GI itself. We propose a model whereby diel changes in GI control oscillations in ABA responses. Peak GI accumulation at midday contributes to establishing a phase of reduced ABA sensitivity and related physiological responses, by gating DNA binding or function of different classes of TFs that cooperate or compete with GI at target regions.

Critical assessment of bioinformatics methods for the characterization of pathological repeat expansions with single-molecule sequencing data.

A number of studies have reported the successful application of single-molecule sequencing technologies to the determination of the size and sequence of pathological expanded microsatellite repeats over the last 5 years. However, different custom bioinformatics pipelines were employed in each study, preventing meaningful comparisons and somewhat limiting the reproducibility of the results. In this review, we provide a brief summary of state-of-the-art methods for the characterization of expanded repeats alleles, along with a detailed comparison of bioinformatics tools for the determination of repeat length and sequence, using both real and simulated data. Our reanalysis of publicly available human genome sequencing data suggests a modest, but statistically significant, increase of the error rate of single-molecule sequencing technologies at genomic regions containing short tandem repeats. However, we observe that all the methods herein tested, irrespective of the strategy used for the analysis of the data (either based on the alignment or assembly of the reads), show high levels of sensitivity in both the detection of expanded tandem repeats and the estimation of the expansion size, suggesting that approaches based on single-molecule sequencing technologies are highly effective for the detection and quantification of tandem repeat expansions and contractions.

CorGAT: a tool for the functional annotation of SARS-CoV-2 genomes.

SUMMARY: While over 200 000 genomic sequences are currently available through dedicated repositories, ad hoc methods for the functional annotation of SARS-CoV-2 genomes do not harness all currently available resources for the annotation of functionally relevant genomic sites. Here, we present CorGAT, a novel tool for the functional annotation of SARS-CoV-2 genomic variants. By comparisons with other state of the art methods we demonstrate that, by providing a more comprehensive and rich annotation, our method can facilitate the identification of evolutionary patterns in the genome of SARS-CoV-2. AVAILABILITYAND IMPLEMENTATION: Galaxy.http://corgat.cloud.ba.infn.it/galaxy; software: https://github.com/matteo14c/CorGAT/tree/Revision_V1; docker: https://hub.docker.com/r/laniakeacloud/galaxy_corgat. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

VINYL: Variant prIoritizatioN bY survivaL analysis.

MOTIVATION: Clinical applications of genome re-sequencing technologies typically generate large amounts of data that need to be carefully annotated and interpreted to identify genetic variants potentially associated with pathological conditions. In this context, accurate and reproducible methods for the functional annotation and prioritization of genetic variants are of fundamental importance. RESULTS: In this article, we present VINYL, a flexible and fully automated system for the functional annotation and prioritization of genetic variants. Extensive analyses of both real and simulated datasets suggest that VINYL can identify clinically relevant genetic variants in a more accurate manner compared to equivalent state of the art methods, allowing a more rapid and effective prioritization of genetic variants in different experimental settings. As such we believe that VINYL can establish itself as a valuable tool to assist healthcare operators and researchers in clinical genomics investigations. AVAILABILITY AND IMPLEMENTATION: VINYL is available at http://beaconlab.it/VINYL and https://github.com/matteo14c/VINYL. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network.

The Sox2 transcription factor is necessary for the long-term self-renewal of neural stem cells (NSCs). Its mechanism of action is still poorly defined. To identify molecules regulated by Sox2, and acting in mouse NSC maintenance, we transduced, into Sox2-deleted NSC, genes whose expression is strongly downregulated following Sox2 loss (Fos, Jun, Egr2), individually or in combination. Fos alone rescued long-term proliferation, as shown by in vitro cell growth and clonal analysis. Furthermore, pharmacological inhibition by T-5224 of FOS/JUN AP1 complex binding to its targets decreased cell proliferation and expression of the putative target Suppressor of cytokine signaling 3 (Socs3). Additionally, Fos requirement for efficient long-term proliferation was demonstrated by the reduction of NSC clones capable of long-term expansion following CRISPR/Cas9-mediated Fos inactivation. Previous work showed that the Socs3 gene is strongly downregulated following Sox2 deletion, and its re-expression by lentiviral transduction rescues long-term NSC proliferation. Fos appears to be an upstream regulator of Socs3, possibly together with Jun and Egr2; indeed, Sox2 re-expression in Sox2-deleted NSC progressively activates both Fos and Socs3 expression; in turn, Fos transduction activates Socs3 expression. Based on available SOX2 ChIPseq and ChIA-PET data, we propose a model whereby Sox2 is a direct activator of both Socs3 and Fos, as well as possibly Jun and Egr2; furthermore, we provide direct evidence for FOS and JUN binding on Socs3 promoter, suggesting direct transcriptional regulation. These results provide the basis for developing a model of a network of interactions, regulating critical effectors of NSC proliferation and long-term maintenance.

Aortic Gene Expression Profiles Show How ApoA-I Levels Modulate Inflammation, Lysosomal Activity, and Sphingolipid Metabolism in Murine Atherosclerosis.

OBJECTIVE: HDL (high-density lipoprotein) particles are known to possess several antiatherogenic properties that include the removal of excess cholesterol from peripheral tissues, the maintenance of endothelial integrity, antioxidant, and anti-inflammatory activities. ApoA-I overexpression in apoE-deficient (EKO) mice has been shown to increase HDL levels and to strongly reduce atherosclerosis development. The aim of the study was to investigate gene expression patterns associated with atherosclerosis development in the aorta of EKO mice and how HDL plasma levels relate to gene expression patterns at different stages of atherosclerosis development and with different dietary treatments. Approach and Results: Eight-week-old EKO mice, EKO mice overexpressing human apoA-I, and wild-type mice as controls were fed either normal laboratory or Western diet for 6 or 22 weeks. Cholesterol distribution among lipoproteins was evaluated, and atherosclerosis of the aorta was quantified. High-throughput sequencing technologies were used to analyze the transcriptome of the aorta of the 3 genotypes in each experimental condition. In addition to the well-known activation of inflammation and immune response, the impairment of sphingolipid metabolism, phagosome-lysosome system, and osteoclast differentiation emerged as relevant players in atherosclerosis development. The reduced atherosclerotic burden in the aorta of EKO mice expressing high levels of apoA-I was accompanied by a reduced activation of immune system markers, as well as reduced perturbation of lysosomal activity and a better regulation of the sphingolipid synthesis pathway. CONCLUSIONS: ApoA-I modulates atherosclerosis development in the aorta of EKO mice affecting the expression of pathways additional to those associated with inflammation and immune response.