Human PNPase causes RNA stabilization and accumulation of R-loops in the Escherichia coli model system.

Polyribonucleotide phosphorylase (PNPase) is a phosphorolytic RNA exonuclease highly conserved throughout evolution. In Escherichia coli, PNPase controls complex phenotypic traits like biofilm formation and growth at low temperature. In human cells, PNPase is located in mitochondria, where it is implicated in the RNA import from the cytoplasm, the mitochondrial RNA degradation and the processing of R-loops, namely stable RNA-DNA hybrids displacing a DNA strand. In this work, we show that the human PNPase (hPNPase) expressed in E. coli causes oxidative stress, SOS response activation and R-loops accumulation. Hundreds of E. coli RNAs are stabilized in presence of hPNPase, whereas only few transcripts are destabilized. Moreover, phenotypic traits typical of E. coli strains lacking PNPase are strengthened in presence of the human enzyme. We discuss the hypothesis that hPNPase expressed in E. coli may bind, but not degrade, the RNA, in agreement with previous in vitro data showing that phosphate concentrations in the range of those found in the bacterial cytoplasm and, more relevant, in the mitochondria, inhibit its activity.

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/.

The evolutionary history of the polyQ tract in huntingtin sheds light on its functional pro-neural activities.

Huntington's disease is caused by a pathologically long (>35) CAG repeat located in the first exon of the Huntingtin gene (HTT). While pathologically expanded CAG repeats are the focus of extensive investigations, non-pathogenic CAG tracts in protein-coding genes are less well characterized. Here, we investigated the function and evolution of the physiological CAG tract in the HTT gene. We show that the poly-glutamine (polyQ) tract encoded by CAGs in the huntingtin protein (HTT) is under purifying selection and subjected to stronger selective pressures than CAG-encoded polyQ tracts in other proteins. For natural selection to operate, the polyQ must perform a function. By combining genome-edited mouse embryonic stem cells and cell assays, we show that small variations in HTT polyQ lengths significantly correlate with cells' neurogenic potential and with changes in the gene transcription network governing neuronal function. We conclude that during evolution natural selection promotes the conservation and purity of the CAG-encoded polyQ tract and that small increases in its physiological length influence neural functions of HTT. We propose that these changes in HTT polyQ length contribute to evolutionary fitness including potentially to the development of a more complex nervous system.

FOS Rescues Neuronal Differentiation of Sox2-Deleted Neural Stem Cells by Genome-Wide Regulation of Common SOX2 and AP1(FOS-JUN) Target Genes.

The transcription factor SOX2 is important for brain development and for neural stem cells (NSC) maintenance. Sox2-deleted (Sox2-del) NSC from neonatal mouse brain are lost after few passages in culture. Two highly expressed genes, Fos and Socs3, are strongly downregulated in Sox2-del NSC; we previously showed that Fos or Socs3 overexpression by lentiviral transduction fully rescues NSC's long-term maintenance in culture. Sox2-del NSC are severely defective in neuronal production when induced to differentiate. NSC rescued by Sox2 reintroduction correctly differentiate into neurons. Similarly, Fos transduction rescues normal or even increased numbers of immature neurons expressing beta-tubulinIII, but not more differentiated markers (MAP2). Additionally, many cells with both beta-tubulinIII and GFAP expression appear, indicating that FOS stimulates the initial differentiation of a "mixed" neuronal/glial progenitor. The unexpected rescue by FOS suggested that FOS, a SOX2 transcriptional target, might act on neuronal genes, together with SOX2. CUT&RUN analysis to detect genome-wide binding of SOX2, FOS, and JUN (the AP1 complex) revealed that a high proportion of genes expressed in NSC are bound by both SOX2 and AP1. Downregulated genes in Sox2-del NSC are highly enriched in genes that are also expressed in neurons, and a high proportion of the "neuronal" genes are bound by both SOX2 and AP1.

Cleaning the Medicago Microarray Database to Improve Gene Function Analysis.

Transcriptomics studies have been facilitated by the development of microarray and RNA-Seq technologies, with thousands of expression datasets available for many species. However, the quality of data can be highly variable, making the combined analysis of different datasets difficult and unreliable. Most of the microarray data for Medicago truncatula, the barrel medic, have been stored and made publicly accessible on the web database Medicago truncatula Gene Expression atlas (MtGEA). The aim of this work is to ameliorate the quality of the MtGEA database through a general method based on logical and statistical relationships among parameters and conditions. The initial 716 columns available in the dataset were reduced to 607 by evaluating the quality of data through the sum of the expression levels over the entire transcriptome probes and Pearson correlation among hybridizations. The reduced dataset shows great improvements in the consistency of the data, with a reduction in both false positives and false negatives resulting from Pearson correlation and GO enrichment analysis among genes. The approach we used is of general validity and our intent is to extend the analysis to other plant microarray databases.

The coding and long noncoding single-cell atlas of the developing human fetal striatum.

Deciphering how the human striatum develops is necessary for understanding the diseases that affect this region. To decode the transcriptional modules that regulate this structure during development, we compiled a catalog of 1116 long intergenic noncoding RNAs (lincRNAs) identified de novo and then profiled 96,789 single cells from the early human fetal striatum. We found that D1 and D2 medium spiny neurons (D1- and D2-MSNs) arise from a common progenitor and that lineage commitment is established during the postmitotic transition, across a pre-MSN phase that exhibits a continuous spectrum of fate determinants. We then uncovered cell type-specific gene regulatory networks that we validated through in silico perturbation. Finally, we identified human-specific lincRNAs that contribute to the phylogenetic divergence of this structure in humans. This work delineates the cellular hierarchies governing MSN lineage commitment.

Using RNentropy to Detect Significant Variation in Gene Expression Across Multiple RNA-Seq or Single-Cell RNA-Seq Samples.

RNA-Seq has become the de facto standard technique for characterization and quantification of transcriptomes, and a large number of methods and tools have been proposed to model and detect differential gene expression based on the comparison of transcript abundances across different samples. However, state-of-the-art methods for this task are usually designed for pairwise comparisons, that is, can identify significant variation of expression only between two conditions or samples. We describe the use of RNentropy, a methodology based on information theory, devised to overcome this limitation. RNentropy can thus detect significant variations of gene expression in RNA-Seq data across any number of samples and conditions, and can be applied downstream of any analysis pipeline for the quantification of gene expression from raw sequencing data. RNentropy takes as input gene (or transcript) expression values, defined with any measure suitable for the comparison of transcript levels across samples and conditions. The output consists of genes (or transcripts) exhibiting significant variation of expression across the conditions studied, together with the samples in which they result to be over- or underexpressed. RNentropy is implemented as an R package and freely available from the CRAN repository. We provide a detailed guide to the functions and parameters of the package and usage examples to demonstrate the software capabilities, also showing how it can be applied to the analysis of single-cell RNA sequencing data.

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.

aScan: A Novel Method for the Study of Allele Specific Expression in Single Individuals.

In diploid organisms, two copies of each allele are normally inherited from parents. Paternal and maternal alleles can be regulated and expressed unequally, which is referred to as allele-specific expression (ASE). In this work, we present aScan, a novel method for the identification of ASE from the analysis of matched individual genomic and RNA sequencing data. By performing extensive analyses of both real and simulated data, we demonstrate that aScan can correctly identify ASE with high accuracy and sensitivity in different experimental settings. Additionally, by applying our method to a small cohort of individuals that are not included in publicly available databases of human genetic variation, we outline the value of possible applications of ASE analysis in single individuals for deriving a more accurate annotation of "private" low-frequency genetic variants associated with regulatory effects on transcription. All in all, we believe that aScan will represent a beneficial addition to the set of bioinformatics tools for the analysis of ASE. Finally, while our method was initially conceived for the analysis of RNA-seq data, it can in principle be applied to any quantitative NGS assay for which matched genotypic and expression data are available. AVAILABILITY: aScan is currently available in the form of an open source standalone software package at: https://github.com/Federico77z/aScan/. aScan version 1.0.3, available at https://github.com/Federico77z/aScan/releases/tag/1.0.3, has been used for all the analyses included in this manuscript. A Docker image of the tool has also been made available at https://github.com/pmandreoli/aScanDocker.

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.

On the NF-Y regulome as in ENCODE (2019).

NF-Y is a trimeric Transcription Factor -TF- which binds with high selectivity to the conserved CCAAT element. Individual ChIP-seq analysis as well as ENCODE have progressively identified locations shared by other TFs. Here, we have analyzed data introduced by ENCODE over the last five years in K562, HeLa-S3 and GM12878, including several chromatin features, as well RNA-seq profiling of HeLa cells after NF-Y inactivation. We double the number of sequence-specific TFs and co-factors reported. We catalogue them in 4 classes based on co-association criteria, infer target genes categorizations, identify positional bias of binding sites and gene expression changes. Larger and novel co-associations emerge, specifically concerning subunits of repressive complexes as well as RNA-binding proteins. On the one hand, these data better define NF-Y association with single members of major classes of TFs, on the other, they suggest that it might have a wider role in the control of mRNA production.

Targeting the scaffolding role of LSD1 (KDM1A) poises acute myeloid leukemia cells for retinoic acid-induced differentiation.

The histone demethylase LSD1 is deregulated in several tumors, including leukemias, providing the rationale for the clinical use of LSD1 inhibitors. In acute promyelocytic leukemia (APL), pharmacological doses of retinoic acid (RA) induce differentiation of APL cells, triggering degradation of the PML-RAR oncogene. APL cells are resistant to LSD1 inhibition or knockout, but targeting LSD1 sensitizes them to physiological doses of RA without altering of PML-RAR levels, and extends survival of leukemic mice upon RA treatment. The combination of RA with LSD1 inhibition (or knockout) is also effective in other non-APL, acute myeloid leukemia (AML) cells. Nonenzymatic activities of LSD1 are essential to block differentiation, while RA with targeting of LSD1 releases a differentiation gene expression program, not strictly dependent on changes in histone H3K4 methylation. Integration of proteomic/epigenomic/mutational studies showed that LSD1 inhibitors alter the recruitment of LSD1-containing complexes to chromatin, inhibiting the interaction between LSD1 and the transcription factor GFI1.

Integrating Peak Colocalization and Motif Enrichment Analysis for the Discovery of Genome-Wide Regulatory Modules and Transcription Factor Recruitment Rules.

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) has opened new avenues of research in the genome-wide characterization of regulatory DNA-protein interactions at the genetic and epigenetic level. As a consequence, it has become the de facto standard for studies on the regulation of transcription, and literally thousands of data sets for transcription factors and cofactors in different conditions and species are now available to the scientific community. However, while pipelines and best practices have been established for the analysis of a single experiment, there is still no consensus on the best way to perform an integrated analysis of multiple datasets in the same condition, in order to identify the most relevant and widespread regulatory modules composed by different transcription factors and cofactors. We present here a computational pipeline for this task, that integrates peak summit colocalization, a novel statistical framework for the evaluation of its significance, and motif enrichment analysis. We show examples of its application to ENCODE data, that led to the identification of relevant regulatory modules composed of different factors, as well as the organization on DNA of the binding motifs responsible for their recruitment.

Histone acetylation landscape in S. cerevisiae nhp6ab mutants reflects altered glucose metabolism.

BACKGROUND: The execution of many genetic programs, influenced by environmental conditions, is epigenetically controlled. Thus, small molecules of the intermediate metabolism being precursors of most of nutrition-deriving epigenetic modifications, sense the cell surrounding environment. METHODS: Here we describe histone H4K16 acetylation distribution in S. cerevisiae nhp6ab mutant, using ChIP-seq analysis; its transcription profile by RNA-seq and its metabolic features by studying the metabolome. We then intersected these three -omic approaches to unveil common crosspoints (if any). RESULTS: In the nhp6ab mutant, the glucose metabolism is switched to pathways leading to Acetyl-CoA synthesis. These enhanced pathways could lead to histone hyperacetylation altering RNA transcription, particularly of those metabolic genes that maintain high Acetyl-CoA availability. CONCLUSIONS: Thus, the absence of chromatin regulators like Nhp6 A and B, interferes with a regulative circular mechanism where histone modification, transcription and metabolism influence each other and contribute to clarify the more general phenomenon in which gene regulation feeds metabolic alterations on epigenetic basis. GENERAL SIGNIFICANCE: This study allowed us to identify, in these two factors, a common element of regulation in metabolism and chromatin acetylation state that could represent a powerful tool to find out relationships existing between metabolism and gene expression in more complex systems.

Epigenetic signatures of stress adaptation and flowering regulation in response to extended drought and recovery in Zea mays.

During their lifespan, plants respond to a multitude of stressful factors. Dynamic changes in chromatin and concomitant transcriptional variations control stress response and adaptation, with epigenetic memory mechanisms integrating environmental conditions and appropriate developmental programs over the time. Here we analyzed transcriptome and genome-wide histone modifications of maize plants subjected to a mild and prolonged drought stress just before the flowering transition. Stress was followed by a complete recovery period to evaluate drought memory mechanisms. Three categories of stress-memory genes were identified: i) "transcriptional memory" genes, with stable transcriptional changes persisting after the recovery; ii) "epigenetic memory candidate" genes in which stress-induced chromatin changes persist longer than the stimulus, in absence of transcriptional changes; iii) "delayed memory" genes, not immediately affected by the stress, but perceiving and storing stress signal for a delayed response. This last memory mechanism is described for the first time in drought response. In addition, applied drought stress altered floral patterning, possibly by affecting expression and chromatin of flowering regulatory genes. Altogether, we provided a genome-wide map of the coordination between genes and chromatin marks utilized by plants to adapt to a stressful environment, describing how this serves as a backbone for setting stress memory.

Mapping the Global Chromatin Connectivity Network for Sox2 Function in Neural Stem Cell Maintenance.

The SOX2 transcription factor is critical for neural stem cell (NSC) maintenance and brain development. Through chromatin immunoprecipitation (ChIP) and chromatin interaction analysis (ChIA-PET), we determined genome-wide SOX2-bound regions and Pol II-mediated long-range chromatin interactions in brain-derived NSCs. SOX2-bound DNA was highly enriched in distal chromatin regions interacting with promoters and carrying epigenetic enhancer marks. Sox2 deletion caused widespread reduction of Pol II-mediated long-range interactions and decreased gene expression. Genes showing reduced expression in Sox2-deleted cells were significantly enriched in interactions between promoters and SOX2-bound distal enhancers. Expression of one such gene, Suppressor of Cytokine Signaling 3 (Socs3), rescued the self-renewal defect of Sox2-ablated NSCs. Our work identifies SOX2 as a major regulator of gene expression through connections to the enhancer network in NSCs. Through the definition of such a connectivity network, our study shows the way to the identification of genes and enhancers involved in NSC maintenance and neurodevelopmental disorders.

hmSEEKER: Identification of hmSILAC Doublets in MaxQuant Output Data.

Heavy methyl Stable Isotope Labeling with Amino acids in Cell culture (hmSILAC) is a metabolic labeling strategy employed in proteomics to increase the confidence of global identification of methylated peptides by MS. However, to this day, the automatic and robust identification of heavy and light peak doublets from MS-raw data of hmSILAC experiments is a challenging task, for which the choice of computational methods is very limited. Here, hmSEEKER, a software designed to work downstream of a MaxQuant analysis for in-depth search of MS peak pairs that correspond to light and heavy methyl-peptide within MaxQuant-generated tables is described with good sensitivity and specificity. The software is written in Perl, and its code and user manual are freely available at Bitbucket (https://bit.ly/2scCT9u).

Microtubules play a role in trafficking prevacuolar compartments to vacuoles in tobacco pollen tubes.

Fine regulation of exocytosis and endocytosis plays a basic role in pollen tube growth. Excess plasma membrane secreted during pollen tube elongation is known to be retrieved by endocytosis and partially reused in secretory pathways through the Golgi apparatus. Dissection of endocytosis has enabled distinct degradation pathways to be identified in tobacco pollen tubes and has shown that microtubules influence the transport of plasma membrane internalized in the tip region to vacuoles. Here, we used different drugs affecting the polymerization state of microtubules together with SYP21, a marker of prevacuolar compartments, to characterize trafficking of prevacuolar compartments in Nicotiana tabacum pollen tubes. Ultrastructural and biochemical analysis showed that microtubules bind SYP21-positive microsomes. Transient transformation of pollen tubes with LAT52-YFP-SYP21 revealed that microtubules play a key role in the delivery of prevacuolar compartments to tubular vacuoles.

Sox2 conditional mutation in mouse causes ataxic symptoms, cerebellar vermis hypoplasia, and postnatal defects of Bergmann glia.

Sox2 is a transcription factor active in the nervous system, within different cell types, ranging from radial glia neural stem cells to a few specific types of differentiated glia and neurons. Mutations in the human SOX2 transcription factor gene cause various central nervous system (CNS) abnormalities, involving hippocampus and eye defects, as well as ataxia. Conditional Sox2 mutation in mouse, with different Cre transgenes, previously recapitulated different essential features of the disease, such as hippocampus and eye defects. In the cerebellum, Sox2 is active from early embryogenesis in the neural progenitors of the cerebellar primordium; Sox2 expression is maintained, postnatally, within Bergmann glia (BG), a differentiated cell type essential for Purkinje neurons functionality and correct motor control. By performing Sox2 Cre-mediated ablation in the developing and postnatal mouse cerebellum, we reproduced ataxia features. Embryonic Sox2 deletion (with Wnt1Cre) leads to reduction of the cerebellar vermis, known to be commonly related to ataxia, preceded by deregulation of Otx2 and Gbx2, critical regulators of vermis development. Postnatally, BG is progressively disorganized, mislocalized, and reduced in mutants. Sox2 postnatal deletion, specifically induced in glia (with GLAST-CreERT2), reproduces the BG defect, and causes (milder) ataxic features. Our results define a role for Sox2 in cerebellar function and development, and identify a functional requirement for Sox2 within postnatal BG, of potential relevance for ataxia in mouse mutants, and in human patients.

Birth, evolution, and transmission of satellite-free mammalian centromeric domains.

Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Equus caballus Chromosome 11. Here, we investigated the satellite-free centromeres of Equus asinus by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regions. A subset of these centromeres is associated with DNA amplification. The location of CENPA binding domains can vary in different individuals, giving rise to epialleles. The analysis of epiallele transmission in hybrids (three mules and one hinny) showed that centromeric domains are inherited as Mendelian traits, but their position can slide in one generation. Conversely, centromere location is stable during mitotic propagation of cultured cells. Our results demonstrate that the presence of more than half of centromeres void of satellite DNA is compatible with genome stability and species survival. The presence of amplified DNA at some centromeres suggests that these arrays may represent an intermediate stage toward satellite DNA formation during evolution. The fact that CENPA binding domains can move within relatively restricted regions (a few hundred kilobases) suggests that the centromeric function is physically limited by epigenetic boundaries.