Intergenic Regions of Saccharomycotina Yeasts are Enriched in Potential to Encode Transmembrane Domains.

Intergenic genomic regions have essential regulatory and structural roles that impose constraints on their sequences. But regions that do not currently encode proteins also carry the potential to do so in the future. De novo gene emergence, the evolution of novel genes out of previously noncoding sequences has now been established as a potent force for genomic novelty. Recently, it was shown that intergenic regions in the genome of Saccharomyces cerevisiae harbor pervasive cryptic potential to, if theoretically translated, form transmembrane domains (TM domains) more frequently than expected by chance given their nucleotide composition, a property that we refer to as TM-forming enrichment. The source and biological relevance of this property is unknown. Here, we expand the investigation into the TM-forming potential of intergenic regions to the entire Saccharomycotina budding yeast subphylum, in an effort to explain this property and understand its importance. We find pervasive but variable enrichment in TM-forming potential across the subphylum regardless of the composition and average size of intergenic regions. This cryptic property is evenly spread across the genome, cannot be explained by the hydrophobic content of the sequence, and does not appear to localize to regions containing regulatory motifs. This TM-forming enrichment specifically, and not the actual TM-forming potential, is associated, across genomes, with more TM domains in evolutionarily young genes. Our findings shed light on this newly discovered feature of yeast genomes and constitute a first step toward understanding its evolutionary importance.

SEGCOND predicts putative transcriptional condensate-associated genomic regions by integrating multi-omics data.

MOTIVATION: The compartmentalization of biochemical reactions, involved in the activation of gene expression in the eukaryotic nucleus, leads to the formation of membraneless bodies through liquid-liquid phase separation. These formations, called transcriptional condensates, appear to play important roles in gene regulation as they are assembled through the association of multiple enhancer regions in 3D genomic space. To date, we are still lacking efficient computational methodologies to identify the regions responsible for the formation of such condensates, based on genomic and conformational data. RESULTS: In this work, we present SEGCOND, a computational framework aiming to highlight genomic regions involved in the formation of transcriptional condensates. SEGCOND is flexible in combining multiple genomic datasets related to enhancer activity and chromatin accessibility, to perform a genome segmentation. It then uses this segmentation for the detection of highly transcriptionally active regions of the genome. At a final step, and through the integration of Hi-C data, it identifies regions of putative transcriptional condensates (PTCs) as genomic domains where multiple enhancer elements coalesce in 3D space. SEGCOND identifies a subset of enhancer segments with increased transcriptional activity. PTCs are also found to significantly overlap highly interconnected enhancer elements and super enhancers obtained through two independent approaches. Application of SEGCOND on data from a well-defined system of B-cell to macrophage transdifferentiation leads to the identification of previously unreported genes with a likely role in the process. AVAILABILITY AND IMPLEMENTATION: Source code and details for the implementation of SEGCOND is available at https://github.com/AntonisK95/SEGCOND. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Dissecting Hes-centred transcriptional networks in neural stem cell maintenance and tumorigenesis in Drosophila.

Neural stem cells divide during embryogenesis and juvenile life to generate the entire complement of neurons and glia in the nervous system of vertebrates and invertebrates. Studies of the mechanisms controlling the fine balance between neural stem cells and more differentiated progenitors have shown that, in every asymmetric cell division, progenitors send a Delta-Notch signal to their sibling stem cells. Here, we show that excessive activation of Notch or overexpression of its direct targets of the Hes family causes stem-cell hyperplasias in the Drosophila larval central nervous system, which can progress to malignant tumours after allografting to adult hosts. We combined transcriptomic data from these hyperplasias with chromatin occupancy data for Dpn, a Hes transcription factor, to identify genes regulated by Hes factors in this process. We show that the Notch/Hes axis represses a cohort of transcription factor genes. These are excluded from the stem cells and promote early differentiation steps, most likely by preventing the reversion of immature progenitors to a stem-cell fate. We describe the impact of two of these 'anti-stemness' factors, Zfh1 and Gcm, on Notch/Hes-triggered tumorigenesis.

Combination of subtherapeutic anti-TNF dose with dasatinib restores clinical and molecular arthritogenic profiles better than standard anti-TNF treatment.

BACKGROUND: New medications for Rheumatoid Arthritis (RA) have emerged in the last decades, including Disease Modifying Antirheumatic Drugs (DMARDs) and biologics. However, there is no known cure, since a significant proportion of patients remain or become non-responders to current therapies. The development of new mode-of-action treatment schemes involving combination therapies could prove successful for the treatment of a greater number of RA patients. METHODS: We investigated the effect of the Tyrosine Kinase inhibitors (TKIs) dasatinib and bosutinib, on the human TNF-dependent Tg197 arthritis mouse model. The inhibitors were administered either as a monotherapy or in combination with a subtherapeutic dose of anti-hTNF biologics and their therapeutic effect was assessed clinically, histopathologically as well as via gene expression analysis and was compared to that of an efficient TNF monotherapy. RESULTS: Dasatinib and, to a lesser extent, bosutinib inhibited the production of TNF and proinflammatory chemokines from arthritogenic synovial fibroblasts. Dasatinib, but not bosutinib, also ameliorated significantly and in a dose-dependent manner both the clinical and histopathological signs of Tg197 arthritis. Combination of dasatinib with a subtherapeutic dose of anti-hTNF biologic agents, resulted in a synergistic inhibitory effect abolishing all arthritis symptoms. Gene expression analysis of whole joint tissue of Tg197 mice revealed that the combination of dasatinib with a low subtherapeutic dose of Infliximab most efficiently restores the pathogenic gene expression profile to that of the healthy state compared to either treatment administered as a monotherapy. CONCLUSION: Our findings show that dasatinib exhibits a therapeutic effect in TNF-driven arthritis and can act in synergy with a subtherapeutic anti-hTNF dose to effectively treat the clinical and histopathological signs of the pathology. The combination of dasatinib and anti-hTNF exhibits a distinct mode of action in restoring the arthritogenic gene signature to that of a healthy profile. Potential clinical applications of combination therapies with kinase inhibitors and anti-TNF agents may provide an interesting alternative to high-dose anti-hTNF monotherapy and increase the number of patients responding to treatment.

An integrative transcriptome analysis framework for drug efficacy and similarity reveals drug-specific signatures of anti-TNF treatment in a mouse model of inflammatory polyarthritis.

Anti-TNF agents have been in the first line of treatment of various inflammatory diseases such as Rheumatoid Arthritis and Crohn's Disease, with a number of different biologics being currently in use. A detailed analysis of their effect at transcriptome level has nevertheless been lacking. We herein present a concise analysis of an extended transcriptomics profiling of four different anti-TNF biologics upon treatment of the established hTNFTg (Tg197) mouse model of spontaneous inflammatory polyarthritis. We implement a series of computational analyses that include clustering of differentially expressed genes, functional analysis and random forest classification. Taking advantage of our detailed sample structure, we devise metrics of treatment efficiency that take into account changes in gene expression compared to both the healthy and the diseased state. Our results suggest considerable variability in the capacity of different biologics to modulate gene expression that can be attributed to treatment-specific functional pathways and differential preferences to restore over- or under-expressed genes. Early intervention appears to manage inflammation in a more efficient way but is accompanied by increased effects on a number of genes that are seemingly unrelated to the disease. Administration at an early stage is also lacking in capacity to restore healthy expression levels of under-expressed genes. We record quantifiable differences among anti-TNF biologics in their efficiency to modulate over-expressed genes related to immune and inflammatory pathways. More importantly, we find a subset of the tested substances to have quantitative advantages in addressing deregulation of under-expressed genes involved in pathways related to known RA comorbidities. Our study shows the potential of transcriptomic analyses to identify comprehensive and distinct treatment-specific gene signatures combining disease-related and unrelated genes and proposes a generalized framework for the assessment of drug efficacy, the search of biosimilars and the evaluation of the efficacy of TNF small molecule inhibitors.

Mosquitoes cloak their legs to resist insecticides.

Malaria incidence has halved since the year 2000, with 80% of the reduction attributable to the use of insecticides. However, insecticide resistance is now widespread, is rapidly increasing in spectrum and intensity across Africa, and may be contributing to the increase of malaria incidence in 2018. The role of detoxification enzymes and target site mutations has been documented in the major malaria vector Anopheles gambiae; however, the emergence of striking resistant phenotypes suggests the occurrence of additional mechanisms. By comparing legs, the most relevant insect tissue for insecticide uptake, we show that resistant mosquitoes largely remodel their leg cuticles via enhanced deposition of cuticular proteins and chitin, corroborating a leg-thickening phenotype. Moreover, we show that resistant female mosquitoes seal their leg cuticles with higher total and different relative amounts of cuticular hydrocarbons, compared with susceptible ones. The structural and functional alterations in Anopheles female mosquito legs are associated with a reduced uptake of insecticides, substantially contributing to the resistance phenotype.

Genome urbanization: clusters of topologically co-regulated genes delineate functional compartments in the genome of Saccharomyces cerevisiae.

The eukaryotic genome evolves under the dual constraint of maintaining coordinated gene transcription and performing effective DNA replication and cell division, the coupling of which brings about inevitable DNA topological tension. DNA supercoiling is resolved and, in some cases, even harnessed by the genome through the function of DNA topoisomerases, as has been shown in the concurrent transcriptional activation and suppression of genes upon transient deactivation of topoisomerase II (topoII). By analyzing a genome-wide transcription run-on experiment upon thermal inactivation of topoII in Saccharomyces cerevisiae we were able to define 116 gene clusters of consistent response (either positive or negative) to topological stress. A comprehensive analysis of these topologically co-regulated gene clusters reveals pronounced preferences regarding their functional, regulatory and structural attributes. Genes that negatively respond to topological stress, are positioned in gene-dense pericentromeric regions, are more conserved and associated to essential functions, while upregulated gene clusters are preferentially located in the gene-sparse nuclear periphery, associated with secondary functions and under complex regulatory control. We propose that genome architecture evolves with a core of essential genes occupying a compact genomic 'old town', whereas more recently acquired, condition-specific genes tend to be located in a more spacious 'suburban' genomic periphery.

Circulating microRNAs in the early prediction of disease recurrence in primary breast cancer.

BACKGROUND: In primary breast cancer metastases frequently arise from a state of dormancy that may persist for extended periods of time. We investigated the efficacy of plasma micro-RNA (miR)-21, miR-23b, miR-190, miR-200b and miR-200c, related to dormancy and metastasis, to predict the outcome of patients with early breast cancer. METHODS: miRNAs were evaluated by RT-qPCR in plasma obtained before adjuvant chemotherapy. miRNA expression, classified as high or low according to median values, correlated with relapse and survival. Receiver operating characteristic (ROC) curves were constructed to determine miRNA sensitivity and specificity. RESULTS: miR-21 (p < 0.001), miR-23b (p = 0.028) and miR-200c (p < 0.001) expression were higher and miR-190 was lower (p = 0.013) in relapsed (n = 49), compared to non-relapsed patients (n = 84). Interestingly, miR-190 was lower (p = 0.0032) in patients with early relapse (at < 3 years; n = 23) compared to those without early relapse (n = 110). On the other hand, miR-21 and miR-200c were higher (p = 0.015 and p < 0.001, respectively) in patients with late relapse (relapse at ≥ 5 years; n = 20) as compared to non-relapsed patients. High miR-200c was associated with shorter disease-free survival (DFS) (p = 0.005) and high miR-21 with both shorter DFS and overall survival (OS) (p < 0.001 and p = 0.033, respectively) compared to low expression. ROC curve analysis revealed that miR-21, miR-23b, miR-190 and miR-200c discriminated relapsed from non-relapsed patients. A combination of of miR-21, miR-23b and miR-190 showed higher sensitivity and specificity in ROC analyses compared to each miRNA alone; accuracy was further improved by adding lymph node infiltration and tumor grade to the panel of three miRs (AUC 0.873). Furthermore, the combination of miR-200c, lymph node infiltration, tumor grade and estrogen receptor predicted late relapse (AUC 0.890). CONCLUSIONS: Circulating miRNAs are differentially expressed among relapsed and non-relapsed patients with early breast cancer and predict recurrence many years before its clinical detection. Our results suggest that miRNAs represent potential circulating biomarkers in early breast cancer.

Invisible cities: segregated domains in the yeast genome with distinct structural and functional attributes.

Recent advances in our understanding of the three-dimensional organization of the eukaryotic nucleus have rendered the spatial distribution of genes increasingly relevant. In a recent work (Tsochatzidou et al., Nucleic Acids Res 45:5818-5828, 2017), we proposed the existence of a functional compartmentalization of the yeast genome according to which, genes occupying the chromosomal regions at the nuclear periphery have distinct structural, functional and evolutionary characteristics compared to their centromeric-proximal counterparts. Around the same time, it was also shown that the genome of Saccharomyces cerevisiae is organized in topologically associated domains (TADs), which are largely associated with the replication timing. In this work, we proceed to investigate whether such units of three-dimensional genomic organization can be linked to transcriptional activity as a driving force for the shaping of genomic architecture. Through the application of a simple boundary-calling criterion in genome-wide 3C data, we define ~100 TAD-like domains which can be clustered in six different classes with radically different nucleosomal organizations, significant variations in transcription factor binding and uneven chromosomal distribution. Approximately ~20% of the genome is found to be confined in regions with "closed" chromatin structure around gene promoters. Most interestingly, we find both "open" and "closed" regions to be segregated, in the sense that they tend to avoid inter-chromosomal interactions. Our data further enforce the notion of a marked compartmentalization of the yeast genome in isolated territories, with implications in its function and evolution.

Structure and Chromosomal Organization of Yeast Genes Regulated by Topoisomerase II.

Cellular DNA topoisomerases (topo I and topo II) are highly conserved enzymes that regulate the topology of DNA during normal genome transactions, such as DNA transcription and replication. In budding yeast, topo I is dispensable whereas topo II is essential, suggesting fundamental and exclusive roles for topo II, which might include the functions of the topo IIa and topo IIb isoforms found in mammalian cells. In this review, we discuss major findings of the structure and chromosomal organization of genes regulated by topo II in budding yeast. Experimental data was derived from short (10 min) and long term (120 min) responses to topo II inactivation in top-2 ts mutants. First, we discuss how short term responses reveal a subset of yeast genes that are regulated by topo II depending on their promoter architecture. These short term responses also uncovered topo II regulation of transcription across multi-gene clusters, plausibly by common DNA topology management. Finally, we examine the effects of deactivated topo II on the elongation of RNA transcripts. Each study provides an insight into the particular chromatin structure that interacts with the activity of topo II. These findings are of notable clinical interest as numerous anti-cancer therapies interfere with topo II activity.

Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis.

Tumor progression locus-2 (Tpl2) kinase is a major inflammatory mediator in immune cell types recently found to be genetically associated with inflammatory bowel diseases (IBDs). Here we show that Tpl2 may exert a dominant homeostatic rather than inflammatory function in the intestine mediated specifically by subepithelial intestinal myofibroblasts (IMFs). Mice with complete or IMF-specific Tpl2 ablation are highly susceptible to epithelial injury-induced colitis showing impaired compensatory proliferation in crypts and extensive ulcerations without significant changes in inflammatory responses. Following epithelial injury, IMFs sense innate or inflammatory signals and activate, via Tpl2, the cyclooxygenase-2 (Cox-2)-prostaglandin E2 (PGE2) pathway, which we show here to be essential for the epithelial homeostatic response. Exogenous PGE2 administration rescues mice with complete or IMF-specific Tpl2 ablation from defects in crypt function and susceptibility to colitis. We also show that Tpl2 expression is decreased in IMFs isolated from the inflamed ileum of IBD patients indicating that Tpl2 function in IMFs may be highly relevant to human disease. The IMF-mediated mechanism we propose also involves the IBD-associated genes IL1R1, MAPK1, and the PGE2 receptor-encoding PTGER4. Our results establish a previously unidentified myofibroblast-specific innate pathway that regulates intestinal homeostasis and may underlie IBD susceptibility in humans.

Inferring active regulatory networks from gene expression data using a combination of prior knowledge and enrichment analysis.

BACKGROUND: Under both physiological and pathological conditions gene expression programs are shaped through the interplay of regulatory proteins and their gene targets, interactions between which form intricate gene regulatory networks (GRN). While the assessment of genome-wide expression for the complete set of genes at a given condition has become rather straight-forward and is performed routinely, we are still far from being able to infer the topology of gene regulation simply by analyzing its "descendant" expression profile. In this work we are trying to overcome the existing limitations for the inference and study of such regulatory networks. We are combining our approach with state-of-the-art gene set enrichment analyses in order to create a tool, called Regulatory Network Enrichment Analysis (RNEA) that will prioritize regulatory and functional characteristics of a genome-wide expression experiment. RESULTS: RNEA combines prior knowledge, originating from manual literature curation and small-scale experimental data, to construct a reference network of interactions and then uses enrichment analysis coupled with a two-level hierarchical parsing of the network, to infer the most relevant subnetwork for a given experimental setting. It is implemented as an R package, currently supporting human and mouse datasets and was herein tested on one test case for each of the two organisms. In both cases, RNEA's gene set enrichment analysis was comparable to state-of-the-art methodologies. Moreover, through its distinguishing feature of regulatory subnetwork reconstruction, RNEA was able to define the key transcriptional regulators for the studied systems as supported from the literature. CONCLUSIONS: RNEA constitutes a novel computational approach to obtain regulatory interactions directly from a genome-wide expression profile. Its simple implementation, with minimal requirements from the user is coupled with easy-to-parse enrichment lists and a subnetwork file that may be readily visualized to reveal the most important components of the regulatory hierarchy. The combination of prior information and novel concept of a hierarchical reconstruction of regulatory interactions makes RNEA a very useful tool for a first-level interpretation of gene expression profiles.

Targeted Metabolic Profiling of the Tg197 Mouse Model Reveals Itaconic Acid as a Marker of Rheumatoid Arthritis.

Rheumatoid arthritis is a progressive, highly debilitating disease where early diagnosis, enabling rapid clinical intervention, would provide obvious benefits to patients, healthcare systems, and society. Novel biomarkers that enable noninvasive early diagnosis of the onset and progression of the disease provide one route to achieving this goal. Here a metabolic profiling method has been applied to investigate disease development in the Tg197 arthritis mouse model. Hind limb extract profiling demonstrated clear differences in metabolic phenotypes between control (wild type) and Tg197 transgenic mice and highlighted raised concentrations of itaconic acid as a potential marker of the disease. These changes in itaconic acid concentrations were moderated or indeed reversed when the Tg197 mice were treated with the anti-hTNF biologic infliximab (10 mg/kg twice weekly for 6 weeks). Further in vitro studies on synovial fibroblasts obtained from healthy wild-type, arthritic Tg197, and infliximab-treated Tg197 transgenic mice confirmed the association of itaconic acid with rheumatoid arthritis and disease-moderating drug effects. Preliminary indications of the potential value of itaconic acid as a translational biomarker were obtained when studies on K4IM human fibroblasts treated with hTNF showed an increase in the concentrations of this metabolite.

Rad9 interacts with Aft1 to facilitate genome surveillance in fragile genomic sites under non-DNA damage-inducing conditions in S. cerevisiae.

DNA damage response and repair proteins are centrally involved in genome maintenance pathways. Yet, little is known about their functional role under non-DNA damage-inducing conditions. Here we show that Rad9 checkpoint protein, known to mediate the damage signal from upstream to downstream essential kinases, interacts with Aft1 transcription factor in the budding yeast. Aft1 regulates iron homeostasis and is also involved in genome integrity having additional iron-independent functions. Using genome-wide expression and chromatin immunoprecipitation approaches, we found Rad9 to be recruited to 16% of the yeast genes, often related to cellular growth and metabolism, while affecting the transcription of ∼2% of the coding genome in the absence of exogenously induced DNA damage. Importantly, Rad9 is recruited to fragile genomic regions (transcriptionally active, GC rich, centromeres, meiotic recombination hotspots and retrotransposons) non-randomly and in an Aft1-dependent manner. Further analyses revealed substantial genome-wide parallels between Rad9 binding patterns to the genome and major activating histone marks, such as H3K36me, H3K79me and H3K4me. Thus, our findings suggest that Rad9 functions together with Aft1 on DNA damage-prone chromatin to facilitate genome surveillance, thereby ensuring rapid and effective response to possible DNA damage events.

Topoisomerase II regulates yeast genes with singular chromatin architectures.

Eukaryotic topoisomerase II (topo II) is the essential decatenase of newly replicated chromosomes and the main relaxase of nucleosomal DNA. Apart from these general tasks, topo II participates in more specialized functions. In mammals, topo IIα interacts with specific RNA polymerases and chromatin-remodeling complexes, whereas topo IIß regulates developmental genes in conjunction with chromatin remodeling and heterochromatin transitions. Here we show that in budding yeast, topo II regulates the expression of specific gene subsets. To uncover this, we carried out a genomic transcription run-on shortly after the thermal inactivation of topo II. We identified a modest number of genes not involved in the general stress response but strictly dependent on topo II. These genes present distinctive functional and structural traits in comparison with the genome average. Yeast topo II is a positive regulator of genes with well-defined promoter architecture that associates to chromatin remodeling complexes; it is a negative regulator of genes extremely hypo-acetylated with complex promoters and undefined nucleosome positioning, many of which are involved in polyamine transport. These findings indicate that yeast topo II operates on singular chromatin architectures to activate or repress DNA transcription and that this activity produces functional responses to ensure chromatin stability.

Nucleosomal DNA has topological memory.

One elusive aspect of the chromosome architecture is how it constrains the DNA topology. Nucleosomes stabilise negative DNA supercoils by restraining a DNA linking number difference (∆Lk) of about -1.26. However, whether this capacity is uniform across the genome is unknown. Here, we calculate the ∆Lk restrained by over 4000 nucleosomes in yeast cells. To achieve this, we insert each nucleosome in a circular minichromosome and perform Topo-seq, a high-throughput procedure to inspect the topology of circular DNA libraries in one gel electrophoresis. We show that nucleosomes inherently restrain distinct ∆Lk values depending on their genomic origin. Nucleosome DNA topologies differ at gene bodies (∆Lk = -1.29), intergenic regions (∆Lk = -1.23), rDNA genes (∆Lk = -1.24) and telomeric regions (∆Lk = -1.07). Nucleosomes near the transcription start and termination sites also exhibit singular DNA topologies. Our findings demonstrate that nucleosome DNA topology is imprinted by its native chromatin context and persists when the nucleosome is relocated.

Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor.

Cellular senescence is acknowledged as a key contributor to organismal ageing and late-life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro-inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent-like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity.

Extensive Alternative Splicing Patterns in Systemic Lupus Erythematosus Highlight Sexual Differences.

Substantial evidence highlights divergences in immune responses between men and women. Women are more susceptible to autoimmunity, whereas men suffer from the more severe presentation of autoimmune disorders. The molecular mechanism of this sexual dimorphism remains elusive. Herein, we conducted a comprehensive analysis of sex differences in whole-blood gene expression focusing on alternative splicing (AS) events in systemic lupus erythematosus (SLE), which is a prototype sex-biased disease. This study included 79 SLE patients with active disease and 58 matched healthy controls who underwent whole-blood RNA sequencing. Sex differences in splicing events were widespread, existent in both SLE and a healthy state. However, we observed distinct gene sets and molecular pathways targeted by sex-dependent AS in SLE patients as compared to healthy subjects, as well as a notable sex dissimilarity in intron retention events. Sexually differential spliced genes specific to SLE patients were enriched for dynamic cellular processes including chromatin remodeling, stress and inflammatory responses. Remarkably, the extent of sexual differences in AS in the SLE patients and healthy individuals exceeded those in gene expression. Overall, this study reveals an unprecedent variation in sex-dependent splicing events in SLE and the healthy state, with potential implications for understanding the molecular basis of sexual dimorphism in autoimmunity.