Controlled X-chromosome dynamics defines meiotic potential of female mouse in vitro germ cells.

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X-chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X-inactivation and reactivation dynamics using a tailor-made in vitro system of primordial germ cell-like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X-inactivation in PGCLCs in vitro and in germ cell-competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X-inactivation is followed by step-wise X-reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X-inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine-tuned X-chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.

NFAT5 Amplifies Antipathogen Responses by Enhancing Chromatin Accessibility, H3K27 Demethylation, and Transcription Factor Recruitment.

The ability of innate immune cells to respond to pathogen-associated molecular patterns across a wide range of intensities is fundamental to limit the spreading of infections. Studies on transcription responses to pathogen-activated TLRs have often used relatively high TLR ligand concentrations, and less is known about their regulation under mild stimulatory conditions. We had shown that the transcription factor NFAT5 facilitates expression of antipathogen genes under TLR stimulation conditions corresponding to low pathogen loads. In this study, we analyze how NFAT5 optimizes TLR-activated responses in mouse macrophages. We show that NFAT5 was required for effective recruitment of central effectors p65/NF-κB and c-Fos to specific proinflammatory target genes, such as Nos2, Il6, and Tnf in primary macrophages responding to low doses of the TLR4 ligand LPS. By contrast, NFAT5 was not required for p65/NF-κB recruitment in response to high LPS doses. Using the transposase-accessible chromatin with high-throughput sequencing assay, we show that NFAT5 facilitated chromatin accessibility mainly at promoter regions of multiple TLR4-responsive genes. Analysis of various histone marks that regulate gene expression in response to pathogens identified H3K27me3 demethylation as an early NFAT5-dependent mechanism that facilitates p65 recruitment to promoters of various TLR4-induced genes. Altogether, these results advance our understanding about specific mechanisms that optimize antipathogen responses to limit infections.

Selective cleavage of ncRNA and antiviral activity by RNase2/EDN in THP1-induced macrophages.

RNase2 is the member of the RNaseA family most abundant in macrophages. Here, we knocked out RNase2 in THP-1 cells and analysed the response to Respiratory Syncytial Virus (RSV). RSV induced RNase2 expression, which significantly enhanced cell survival. Next, by cP-RNAseq sequencing, which amplifies the cyclic-phosphate endonuclease products, we analysed the ncRNA population. Among the ncRNAs accumulated in WT vs KO cells, we found mostly tRNA-derived fragments (tRFs) and second miRNAs. Differential sequence coverage identified tRFs from only few parental tRNAs, revealing a predominant cleavage at anticodon and D-loops at U/C (B1) and A (B2) sites. Selective tRNA cleavage was confirmed in vitro using the recombinant protein. Likewise, only few miRNAs were significantly more abundant in WT vs RNase2-KO cells. Complementarily, by screening of a tRF & tiRNA array, we identified an enriched population associated to RNase2 expression and RSV exposure. The results confirm the protein antiviral action and provide the first evidence of its cleavage selectivity on ncRNAs.

Determination of primary microRNA processing in clinical samples by targeted pri-miR-sequencing.

MicroRNA expression is important for gene regulation and deregulated microRNA expression is often observed in diseases such as cancer. The processing of primary microRNA transcripts is an important regulatory step in microRNA biogenesis. Due to low expression level and association with chromatin, primary microRNAs are challenging to study in clinical samples where input material is limited. Here, we present a high-sensitivity targeted method to determine processing efficiency of several hundred primary microRNAs from total RNA that requires relatively few RNA sequencing reads. We validate the method using RNA from HeLa cells and show the applicability to clinical samples by analyzing RNA from normal liver and hepatocellular carcinoma. We identify 24 primary microRNAs with significant changes in processing efficiency from normal liver to hepatocellular carcinoma, among those the highly expressed miRNA-122 and miRNA-21, demonstrating that differential processing of primary microRNAs is occurring and could be involved in disease. With our method presented here we provide means to study pri-miRNA processing in disease from clinical samples.

Genome-wide transcriptomic changes reveal the genetic pathways involved in insect migration.

Insects are capable of extraordinary feats of long-distance movement that have profound impacts on the function of terrestrial ecosystems. The ability to undertake these movements arose multiple times through the evolution of a suite of traits that make up the migratory syndrome, however the underlying genetic pathways involved remain poorly understood. Migratory hoverflies (Diptera: Syrphidae) are an emerging model group for studies of migration. They undertake seasonal movements in huge numbers across large parts of the globe and are important pollinators, biological control agents and decomposers. Here, we assembled a high-quality draft genome of the marmalade hoverfly (Episyrphus balteatus). We leveraged this genomic resource to undertake a genome-wide transcriptomic comparison of actively migrating Episyrphus, captured from a high mountain pass as they flew south to overwinter, with the transcriptomes of summer forms which were non-migratory. We identified 1543 genes with very strong evidence for differential expression. Interrogation of this gene set reveals a remarkable range of roles in metabolism, muscle structure and function, hormonal regulation, immunity, stress resistance, flight and feeding behaviour, longevity, reproductive diapause and sensory perception. These features of the migrant phenotype have arisen by the integration and modification of pathways such as insulin signalling for diapause and longevity, JAK/SAT for immunity, and those leading to octopamine production and fuelling to boost flight capabilities. Our results provide a powerful genomic resource for future research, and paint a comprehensive picture of global expression changes in an actively migrating insect, identifying key genomic components involved in this important life-history strategy.

Zrf1 is required to establish and maintain neural progenitor identity.

The molecular mechanisms underlying specification from embryonic stem cells (ESCs) and maintenance of neural progenitor cells (NPCs) are largely unknown. Recently, we reported that the Zuotin-related factor 1 (Zrf1) is necessary for chromatin displacement of the Polycomb-repressive complex 1 (PRC1). We found that Zrf1 is required for NPC specification from ESCs and that it promotes the expression of NPC markers, including the key regulator Pax6. Moreover, Zrf1 is essential to establish and maintain Wnt ligand expression levels, which are necessary for NPC self-renewal. Reactivation of proper Wnt signaling in Zrf1-depleted NPCs restores Pax6 expression and the self-renewal capacity. ESC-derived NPCs in vitro resemble most of the characteristics of the self-renewing NPCs located in the developing embryonic cortex, which are termed radial glial cells (RGCs). Depletion of Zrf1 in vivo impairs the expression of key self-renewal regulators and Wnt ligand genes in RGCs. Thus, we demonstrate that Zrf1 plays an essential role in NPC generation and maintenance.

QCloud2: An Improved Cloud-based Quality-Control System for Mass-Spectrometry-based Proteomics Laboratories.

QCloud is a cloud-based system to support proteomics laboratories in daily quality assessment using a user-friendly interface, easy setup, and automated data processing. Since its release, QCloud has facilitated automated quality control for proteomics experiments in many laboratories. QCloud provides a quick and effortless evaluation of instrument performance that helps to overcome many analytical challenges derived from clinical and translational research. Here we present an improved version of the system, QCloud2. This new version includes enhancements in the scalability and reproducibility of the quality-control pipelines, and it features an improved front end for data visualization, user management, and chart annotation. The QCloud2 system also includes programmatic access and a standalone local version.

An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms.

Alternative splicing (AS) generates remarkable regulatory and proteomic complexity in metazoans. However, the functions of most AS events are not known, and programs of regulated splicing remain to be identified. To address these challenges, we describe the Vertebrate Alternative Splicing and Transcription Database (VastDB), the largest resource of genome-wide, quantitative profiles of AS events assembled to date. VastDB provides readily accessible quantitative information on the inclusion levels and functional associations of AS events detected in RNA-seq data from diverse vertebrate cell and tissue types, as well as developmental stages. The VastDB profiles reveal extensive new intergenic and intragenic regulatory relationships among different classes of AS and previously unknown and conserved landscapes of tissue-regulated exons. Contrary to recent reports concluding that nearly all human genes express a single major isoform, VastDB provides evidence that at least 48% of multiexonic protein-coding genes express multiple splice variants that are highly regulated in a cell/tissue-specific manner, and that >18% of genes simultaneously express multiple major isoforms across diverse cell and tissue types. Isoforms encoded by the latter set of genes are generally coexpressed in the same cells and are often engaged by translating ribosomes. Moreover, they are encoded by genes that are significantly enriched in functions associated with transcriptional control, implying they may have an important and wide-ranging role in controlling cellular activities. VastDB thus provides an unprecedented resource for investigations of AS function and regulation.

Fine tuning the extracellular environment accelerates the derivation of kidney organoids from human pluripotent stem cells.

The generation of organoids is one of the biggest scientific advances in regenerative medicine. Here, by lengthening the time that human pluripotent stem cells (hPSCs) were exposed to a three-dimensional microenvironment, and by applying defined renal inductive signals, we generated kidney organoids that transcriptomically matched second-trimester human fetal kidneys. We validated these results using ex vivo and in vitro assays that model renal development. Furthermore, we developed a transplantation method that utilizes the chick chorioallantoic membrane. This approach created a soft in vivo microenvironment that promoted the growth and differentiation of implanted kidney organoids, as well as providing a vascular component. The stiffness of the in ovo chorioallantoic membrane microenvironment was recapitulated in vitro by fabricating compliant hydrogels. These biomaterials promoted the efficient generation of renal vesicles and nephron structures, demonstrating that a soft environment accelerates the differentiation of hPSC-derived kidney organoids.

The penile microbiota of Black South African men: relationship with human papillomavirus and HIV infection.

BACKGROUND: To date, the microbiota of the human penis has been studied mostly in connection with circumcision, HIV risk and female partner bacterial vaginosis (BV). These studies have shown that male circumcision reduces penile anaerobic bacteria, that greater abundance of penile anaerobic bacteria is correlated with increased cytokine levels and greater risk of HIV infection, and that the penile microbiota is an important harbour for BV-associated bacteria. While circumcision has been shown to significantly reduce the risk of acquiring human papillomavirus (HPV) infection, the relationship of the penile microbiota with HPV is still unknown. In this study, we examined the penile microbiota of HPV-infected men as well as the impact of HIV status. RESULTS: The penile skin microbiota of 238 men from Cape Town (South Africa) were profiled using Illumina sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene. Corynebacterium and Prevotella were found to be the most abundant genera. Six distinct community state types (CSTs) were identified. CST-1, dominated by Corynebacterium, corresponded to less infections with high-risk HPV (HR-HPV) relative to CSTs 2-6. Men in CST-5 had greater relative abundances of Prevotella, Clostridiales, and Porphyromonas and a lower relative abundance of Corynebacterium. Moreover, they were significantly more likely to have HPV or HR-HPV infections than men in CST-1. Using a machine learning approach, we identified greater relative abundances of the anaerobic BV-associated bacteria (Prevotella, Peptinophilus, and Dialister) and lower relative abundance of Corynebacterium in HR-HPV-infected men compared to HR-HPV-uninfected men. No association was observed between HIV and CST, although the penile microbiota of HIV-infected men had greater relative abundances of Staphylococcus compared to HIV-uninfected men. CONCLUSIONS: We found significant differences in the penile microbiota composition of men with and without HPV and HIV infections. HIV and HR-HPV infections were strongly associated with greater relative abundances of Staphylococcus and BV-associated bacterial taxa (notably Prevotella, Peptinophilus and Dialister), respectively. It is possible that these taxa could increase susceptibility to HIV and HR-HPV acquisition, in addition to creating conditions in which infections persist. Further longitudinal studies are required to establish causal relationships and to determine the extent of the effect.

Age-associated inflammation inhibits epidermal stem cell function.

Altered stem cell homeostasis is linked to organismal aging. However, the mechanisms involved remain poorly understood. Here we report novel alterations in hair follicle stem cells during skin aging, including increased numbers, decreased function, and an inability to tolerate stress. Performing high-throughput RNA sequencing on aging stem cells, cytokine arrays, and functional assays, we identify an age-associated imbalance in epidermal Jak-Stat signaling that inhibits stem cell function. Collectively, this study reveals a role for the aging epidermis in the disruption of cytokine and stem cell homeostasis, suggesting that stem cell decline during aging may be part of broader tumor-suppressive mechanisms.

Prp4 Kinase Grants the License to Splice: Control of Weak Splice Sites during Spliceosome Activation.

The genome of the fission yeast Schizosaccharomyces pombe encodes 17 kinases that are essential for cell growth. These include the cell-cycle regulator Cdc2, as well as several kinases that coordinate cell growth, polarity, and morphogenesis during the cell cycle. In this study, we further characterized another of these essential kinases, Prp4, and showed that the splicing of many introns is dependent on Prp4 kinase activity. For detailed characterization, we chose the genes res1 and ppk8, each of which contains one intron of typical size and position. Splicing of the res1 intron was dependent on Prp4 kinase activity, whereas splicing of the ppk8 intron was not. Extensive mutational analyses of the 5' splice site of both genes revealed that proper transient interaction with the 5' end of snRNA U1 governs the dependence of splicing on Prp4 kinase activity. Proper transient interaction between the branch sequence and snRNA U2 was also important. Therefore, the Prp4 kinase is required for recognition and efficient splicing of introns displaying weak exon1/5' splice sites and weak branch sequences.

DPY30 regulates pathways in cellular senescence through ID protein expression.

Cellular senescence is an intrinsic defense mechanism to various cellular stresses: while still metabolically active, senescent cells stop dividing and enter a proliferation arrest. Here, we identify DPY30, a member of all mammalian histone H3K4 histone methyltransferases (HMTases), as a key regulator of the proliferation potential of human primary cells. Following depletion of DPY30, cells show a severe proliferation defect and display a senescent phenotype, including a flattened and enlarged morphology, elevated level of reactive oxygen species (ROS), increased SA-ß-galactosidase activity, and formation of senescence-associated heterochromatin foci (SAHFs). While DPY30 depletion leads to a reduced level of H3K4me3-marked active chromatin, we observed a concomitant activation of CDK inhibitors, including p16INK4a, independent of H3K4me3. ChIP experiments show that key regulators of cell-cycle progression, including ID proteins, are under direct control of DPY30. Because ID proteins are negative regulators of the transcription factors ETS1/2, depletion of DPY30 leads to the transcriptional activation of p16INK4a by ETS1/2 and thus to a senescent-like phenotype. Ectoptic re-introduction of ID protein expression can partially rescue the senescence-like phenotype induced by DPY30 depletion. Thus, our data indicate that DPY30 controls proliferation by regulating ID proteins expression, which in turn lead to senescence bypass.

MyMpn: a database for the systems biology model organism Mycoplasma pneumoniae.

MyMpn (http://mympn.crg.eu) is an online resource devoted to studying the human pathogen Mycoplasma pneumoniae, a minimal bacterium causing lower respiratory tract infections. Due to its small size, its ability to grow in vitro, and the amount of data produced over the past decades, M. pneumoniae is an interesting model organisms for the development of systems biology approaches for unicellular organisms. Our database hosts a wealth of omics-scale datasets generated by hundreds of experimental and computational analyses. These include data obtained from gene expression profiling experiments, gene essentiality studies, protein abundance profiling, protein complex analysis, metabolic reactions and network modeling, cell growth experiments, comparative genomics and 3D tomography. In addition, the intuitive web interface provides access to several visualization and analysis tools as well as to different data search options. The availability and--even more relevant--the accessibility of properly structured and organized data are of up-most importance when aiming to understand the biology of an organism on a global scale. Therefore, MyMpn constitutes a unique and valuable new resource for the large systems biology and microbiology community.

The genome of melon (Cucumis melo L.).

We report the genome sequence of melon, an important horticultural crop worldwide. We assembled 375 Mb of the double-haploid line DHL92, representing 83.3% of the estimated melon genome. We predicted 27,427 protein-coding genes, which we analyzed by reconstructing 22,218 phylogenetic trees, allowing mapping of the orthology and paralogy relationships of sequenced plant genomes. We observed the absence of recent whole-genome duplications in the melon lineage since the ancient eudicot triplication, and our data suggest that transposon amplification may in part explain the increased size of the melon genome compared with the close relative cucumber. A low number of nucleotide-binding site-leucine-rich repeat disease resistance genes were annotated, suggesting the existence of specific defense mechanisms in this species. The DHL92 genome was compared with that of its parental lines allowing the quantification of sequence variability in the species. The use of the genome sequence in future investigations will facilitate the understanding of evolution of cucurbits and the improvement of breeding strategies.

Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern.

BACKGROUND: Novosphingobium sp. strain PP1Y is a marine α-proteobacterium adapted to grow at the water/fuel oil interface. It exploits the aromatic fraction of fuel oils as a carbon and energy source. PP1Y is able to grow on a wide range of mono-, poly- and heterocyclic aromatic hydrocarbons. Here, we report the complete functional annotation of the whole Novosphingobium genome. RESULTS: PP1Y genome analysis and its comparison with other Sphingomonadal genomes has yielded novel insights into the molecular basis of PP1Y's phenotypic traits, such as its peculiar ability to encapsulate and degrade the aromatic fraction of fuel oils. In particular, we have identified and dissected several highly specialized metabolic pathways involved in: (i) aromatic hydrocarbon degradation; (ii) resistance to toxic compounds; and (iii) the quorum sensing mechanism. CONCLUSIONS: In summary, the unraveling of the entire PP1Y genome sequence has provided important insight into PP1Y metabolism and, most importantly, has opened new perspectives about the possibility of its manipulation for bioremediation purposes.

Comparative transcriptomics of early dipteran development.

BACKGROUND: Modern sequencing technologies have massively increased the amount of data available for comparative genomics. Whole-transcriptome shotgun sequencing (RNA-seq) provides a powerful basis for comparative studies. In particular, this approach holds great promise for emerging model species in fields such as evolutionary developmental biology (evo-devo). RESULTS: We have sequenced early embryonic transcriptomes of two non-drosophilid dipteran species: the moth midge Clogmia albipunctata, and the scuttle fly Megaselia abdita. Our analysis includes a third, published, transcriptome for the hoverfly Episyrphus balteatus. These emerging models for comparative developmental studies close an important phylogenetic gap between Drosophila melanogaster and other insect model systems. In this paper, we provide a comparative analysis of early embryonic transcriptomes across species, and use our data for a phylogenomic re-evaluation of dipteran phylogenetic relationships. CONCLUSIONS: We show how comparative transcriptomics can be used to create useful resources for evo-devo, and to investigate phylogenetic relationships. Our results demonstrate that de novo assembly of short (Illumina) reads yields high-quality, high-coverage transcriptomic data sets. We use these data to investigate deep dipteran phylogenetic relationships. Our results, based on a concatenation of 160 orthologous genes, provide support for the traditional view of Clogmia being the sister group of Brachycera (Megaselia, Episyrphus, Drosophila), rather than that of Culicomorpha (which includes mosquitoes and blackflies).

Nanopore Direct RNA Sequencing Data Processing and Analysis Using MasterOfPores.

This chapter describes MasterOfPores v.2 (MoP2), an open-source suite of pipelines for processing and analyzing direct RNA Oxford Nanopore sequencing data. The MoP2 relies on the Nextflow DSL2 framework and Linux containers, thus enabling reproducible data analysis in transcriptomic and epitranscriptomic studies. We introduce the key concepts of MoP2 and provide a step-by-step fully reproducible and complete example of how to use the workflow for the analysis of S. cerevisiae total RNA samples sequenced using MinION flowcells. The workflow starts with the pre-processing of raw FAST5 files, which includes basecalling, read quality control, demultiplexing, filtering, mapping, estimation of per-gene/transcript abundances, and transcriptome assembly, with support of the GPU computing for the basecalling and read demultiplexing steps. The secondary analyses of the workflow focus on the estimation of RNA poly(A) tail lengths and the identification of RNA modifications. The MoP2 code is available at https://github.com/biocorecrg/MOP2 and is distributed under the MIT license.

microRNA sequencing for biomarker detection in the diagnosis, classification and prognosis of Diffuse Large B Cell Lymphoma.

Despite being considered a single disease, Diffuse Large B Cell Lymphoma (DLBCL) presents with variable backgrounds, which results in heterogeneous outcomes among patients, with 40% of them still having primary refractory disease or relapse. Thus, novel biomarkers are needed. In addition, multiple factors regarding its pathogenesis remain unclear. In this context, recent investigations point to the relevance of microRNAs (miRNAs) in cancer. However, regarding DLBCL, there is inconsistency in the data reported. Therefore, in this work, the main goals were to determine a miRNA set with utility as biomarkers for DLBCL diagnosis, classification, prognosis and treatment response, as well as to decipher the mechanism of action of deregulated miRNAs in the origin of the disease. We analyzed miRNA expression in a cohort of 78 DLBCL patients and 17 controls using small RNA sequencing and performed a miRNA-mRNA interaction network analysis. This way, we were able to define new miRNA expression signatures for diagnosis, classification, treatment response and prognosis, and we identified plausible mechanisms of action by which deregulated miRNAs could be involved in DLBCL pathogenesis. In summary, our study remarks that miRNAs could play an important role in DLBCL.

SET-PP2A complex as a new therapeutic target in KMT2A (MLL) rearranged AML.

KMT2A-rearranged (KMT2A-R) is an aggressive and chemo-refractory acute leukemia which mostly affects children. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of survival and drug resistance in KMT2A-R leukemia. In contrast, the contribution and regulation of phosphatases is unknown. In this study we uncover the essential role and underlying mechanisms of SET, the endogenous inhibitor of Ser/Thr phosphatase PP2A, in KMT2A-R-leukemia. Investigation of SET expression in acute myeloid leukemia (AML) samples demonstrated that SET is overexpressed, and elevated expression of SET is correlated with poor prognosis and with the expression of MEIS and HOXA genes in AML patients. Silencing SET specifically abolished the clonogenic ability of KMT2A-R leukemic cells and the transcription of KMT2A targets genes HOXA9 and HOXA10. Subsequent mechanistic investigations showed that SET interacts with both KMT2A wild type and fusion proteins, and it is recruited to the HOXA10 promoter. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3ß, AURB and PLK1 and led to suppression of MYC, supporting the hypothesis of a feedback loop among PP2A, AURB, PLK1, MYC, and SET. Our findings illustrate that SET is a novel player in KMT2A-R leukemia and they provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia.