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.

Targeting senescence and inflammation in chronic destructive TNF-driven joint pathology.

We had shown that administration of the senolytic Dasatinib abolishes arthritis in the human TNF transgenic mouse model of chronic destructive arthritis when given in combination with a sub-therapeutic dose of the anti-TNF mAb Infliximab (1 mg/kg). Herein, we found that while the number of senescent chondrocytes (GL13+/Ki67-), assessed according to guideline algorithmic approaches, was not affected by either Dasatinib or sub-therapeutic Infliximab monotherapies, their combination reduced senescent chondrocytes by 50 %, which was comparable to levels observed with therapeutic Infliximab monotherapy (10 mg/kg). This combination therapy also reduced the expression of multiple factors of senescence-associated secretory phenotype in arthritic joints. Studies to elucidate the interplay of inflammation and senescence may help in optimizing treatment strategies also for age-related pathologies characterized by chronic low-grade joint inflammation.

Promyelocytic leukemia protein regulates angiogenesis and epithelial-mesenchymal transition to limit metastasis in MDA-MB-231 breast cancer cells.

Promyelocytic leukemia protein (PML) modulates diverse cell functions that contribute to both tumor suppressor and pro-oncogenic effects, depending on the cellular context. We show here that PML knockdown (KD) in MDA-MB-231, but not MCF7, breast cancer cells, prolonged stem-cell-like survival, and increased cell proliferation and migration, which is in line with gene-enrichment results from their RNA sequencing analysis. Of note, increased migration was accompanied by higher levels of the epithelial-mesenchymal transition (EMT) regulator Twist-related protein 2 (TWIST2). We showed here that PML binds to TWIST2 via its basic helix-loop-helix (bHLH) region and functionally interferes with the suppression of the epithelial target of TWIST2, CD24. In addition, PML ablation in MDA-MB-231 cells led to higher protein levels of hypoxia-inducible factor 1-alpha (HIF1a), resulting in a higher cell hypoxic response. Functionally, PML directly suppressed the induction of the HIF1a target gene vascular endothelial growth factor A (VEGFa). In line with these results, tumor xenografts of MDA-MB-231 PML-KD cells had enhanced aggressive properties, including higher microvessel density, faster local growth, and higher metastatic ability, with a preference for lung. Collectively, PML suppresses the cancer aggressive behavior by multiple mechanisms that impede both the HIF-hypoxia-angiogenic and EMT pathways.

The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency.

Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

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.

Distinct chromosomal "niches" in the genome of Saccharomyces cerevisiae provide the background for genomic innovation and shape the fate of gene duplicates.

Nearly one third of Saccharomyces cerevisiae protein coding sequences correspond to duplicate genes, equally split between small-scale duplicates (SSD) and whole-genome duplicates (WGD). While duplicate genes have distinct properties compared to singletons, to date, there has been no systematic analysis of their positional preferences. In this work, we show that SSD and WGD genes are organized in distinct gene clusters that occupy different genomic regions, with SSD being more peripheral and WGD more centrally positioned close to centromeric chromatin. Duplicate gene clusters differ from the rest of the genome in terms of gene size and spacing, gene expression variability and regulatory complexity, properties that are also shared by singleton genes residing within them. Singletons within duplicate gene clusters have longer promoters, more complex structure and a higher number of protein-protein interactions. Particular chromatin architectures appear to be important for gene evolution, as we find SSD gene-pair co-expression to be strongly associated with the similarity of nucleosome positioning patterns. We propose that specific regions of the yeast genome provide a favourable environment for the generation and maintenance of small-scale gene duplicates, segregating them from WGD-enriched genomic domains. Our findings provide a valuable framework linking genomic innovation with positional genomic preferences.

The 3D enhancer network of the developing T cell genome is shaped by SATB1.

Mechanisms of tissue-specific gene expression regulation via 3D genome organization are poorly understood. Here we uncover the regulatory chromatin network of developing T cells and identify SATB1, a tissue-specific genome organizer, enriched at the anchors of promoter-enhancer loops. We have generated a T-cell specific Satb1 conditional knockout mouse which allows us to infer the molecular mechanisms responsible for the deregulation of its immune system. H3K27ac HiChIP and Hi-C experiments indicate that SATB1-dependent promoter-enhancer loops regulate expression of master regulator genes (such as Bcl6), the T cell receptor locus and adhesion molecule genes, collectively being critical for cell lineage specification and immune system homeostasis. SATB1-dependent regulatory chromatin loops represent a more refined layer of genome organization built upon a high-order scaffold provided by CTCF and other factors. Overall, our findings unravel the function of a tissue-specific factor that controls transcription programs, via spatial chromatin arrangements complementary to the chromatin structure imposed by ubiquitously expressed genome organizers.

Revisiting the Non-Coding Nature of Pospiviroids.

Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study.

Circulating miRNAs as Potential Biomarkers in Prostate Cancer Patients Undergoing Radiotherapy.

INTRODUCTION: Disease recurrence is a major concern in patients with localized prostate cancer (PCa) following treatment with radiotherapy (RT), and few studies have evaluated the clinical relevance of microRNAs (miRNAs) prior and post-RT. PURPOSE: We aimed to investigate the significance of miRNAs in the outcomes of prostate cancer patients undergoing radiotherapy and to identify the related pathways through bioinformatics analysis. MATERIALS AND METHODS: The expression levels of miR-21, miR-106b, miR-141 and miR-375 involved in the response to radiotherapy were assessed by RT-qPCR in the serum of PCa patients (n=56) prior- and post-RT. RESULTS: Low expression levels of miR-106b prior-RT were associated with extracapsular extension and seminal vesicles invasion by the tumor (p=0.031 and 0.044, respectively). In the high-risk subgroup (n=47), post-RT expression levels of miR-21 were higher in patients with biochemical relapse (BR) compared to non-relapse (p=0.043). Also, in the salvage treatment subgroup (post-operative BR; n=20), post-RT expression levels of miR-21 and miR-106b were higher in patients with BR compared to non-relapse (p=0.043 and p=0.032, respectively). In the whole group of patients, high expression levels of miR-21 prior-RT and of miR-106b post-RT were associated with significantly shorter overall survival (OS; p=0.049 and p=0.050, respectively). No associations were observed among miR-141 and miR-375 expression levels with clinicopathological features or treatment outcome. Bioinformatics analysis revealed significant enrichment in DNA damage response pathways. CONCLUSION: Circulating miRNAs prior or post-RT may hold prognostic implications in patients with PCa.

Extensive Changes in Transcription Dynamics Reflected on Alternative Splicing Events in Systemic Lupus Erythematosus Patients.

In addition to increasing the complexity of the transcriptional output, alternative RNA splicing can lead to the reduction of mRNA translation or the production of non-functional or malfunctional proteins, thus representing a vital component of the gene regulation process. Herein, we set out to detect and characterize alternative splicing events that occur in whole-blood samples of patients with Systemic Lupus Erythematosus (SLE) as compared to healthy counterparts. Through the implementation of a computational pipeline on published RNA-sequencing data, we identified extensive changes in the transcription dynamics affecting a large number of genes. We found a predominance of intron retention events, with the majority introducing premature stop codons, suggestive of gene repression, in both inactive and active SLE patient samples. Alternative splicing affected a distinct set of genes from the ones detected as differentially expressed in the same comparisons, while alternatively spliced genes tended to reside in genome areas associated with increased gene co-expression. Functional analysis of genes affected by alternative splicing pointed towards particular functions related to metabolism and histone acetylation as of potential interest. Together, our findings underline the importance of incorporating alternative splicing analyses in the context of molecular characterization of complex diseases such as SLE.

MicroRNAs Regulating Tumor and Immune Cell Interactions in the Prediction of Relapse in Early Stage Breast Cancer.

MicroRNAs (miRNAs) are involved in the regulation of immune response and hold an important role in tumor immune escape. We investigated the differential expression of the immunomodulatory miR-10b, miR-19a, miR-20a, miR-126, and miR-155 in the plasma of healthy women and patients with early stage breast cancer and interrogated their role in the prediction of patients' relapse. Blood samples were obtained from healthy women (n = 20) and patients with early stage breast cancer (n = 140) before adjuvant chemotherapy. Plasma miRNA expression levels were assessed by RT-qPCR. Relapse predicting models were developed using binary logistic regression and receiver operating curves (ROC) were constructed to determine miRNA sensitivity and specificity. Only miR-155 expression was lower in patients compared with healthy women (p = 0.023), whereas miR-155 and miR-10b were lower in patients who relapsed compared with healthy women (p = 0.039 and p = 0.002, respectively). MiR-155 expression combined with axillary lymph node infiltration and tumor grade demonstrated increased capability in distinguishing relapsed from non-relapsed patients [(area under the curve, (AUC = 0.861; p < 0.001)]. Combined miR-19a and miR-20a expression had the highest performance in discriminating patients with early relapse (AUC = 0.816; p < 0.001). Finally, miR-10b in combination with lymph node status and grade had the highest accuracy to discriminate patients with late relapse (AUC = 0.971; p < 0.001). The robustness of the relapse predicting models was further confirmed in a 10-fold cross validation. Deregulation of circulating miRNAs involved in tumor-immune interactions may predict relapse in early stage breast cancer. Their successful clinical integration could potentially address the significance challenge of treatment escalation or de-escalation according to the risk of recurrence.

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.

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.

Extensive fragmentation and re-organization of transcription in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is the prototype of autoimmune diseases, characterized by extensive gene expression perturbations in peripheral blood immune cells. Circumstantial evidence suggests that these perturbations may be due to altered epigenetic profiles and chromatin accessibility but the relationship between transcriptional deregulation and genome organization remains largely unstudied. In this work we propose a genomic approach that leverages patterns of gene coexpression from genome-wide transcriptome profiles in order to identify statistically robust Domains of Co-ordinated gene Expression (DCEs). Application of this method on a large transcriptome profiling dataset of 148 SLE patients and 52 healthy individuals enabled the identification of significant disease-associated alterations in gene co-regulation patterns, which also correlate with SLE activity status. Low disease activity patient genomes are characterized by extensive fragmentation leading to overall fewer DCEs of smaller size. High disease activity genomes display extensive redistribution of co-expression domains with expanded and newly-appearing (emerged) DCEs. The dynamics of domain fragmentation and redistribution are associated with SLE clinical endophenotypes, with genes of the interferon pathway being highly enriched in DCEs that become disrupted and with functions associated to more generalized symptoms, being located in domains that emerge anew in high disease activity genomes. Our results suggest strong links between the SLE phenotype and the underlying genome structure and underline an important role for genome organization in shaping gene expression in SLE.

Circulating MicroRNAs Regulating DNA Damage Response and Responsiveness to Cisplatin in the Prognosis of Patients with Non-Small Cell Lung Cancer Treated with First-Line Platinum Chemotherapy.

The expression of microRNA (miR)-21, miR-128, miR-155, and miR-181a involved in DNA damage response (DDR) and tumor responsiveness to platinum was assessed by RT-qPCR in the plasma of patients with non-small cell lung cancer (NSCLC; n = 128) obtained prior to initiation of first-line platinum chemotherapy. U6 small nuclear RNA (snRNA) was used for normalization, and fold change of each miRNA expression relative to the expression in healthy controls was calculated by the 2-ΔΔCt method. MicroRNA expression levels were correlated with patients' outcomes. Integrated function and pathway enrichment analysis was performed to identify putative target genes. MiR-128, miR-155, and miR-181a expressions were higher in patients compared to healthy donors. MiRNA expression was not associated with response to treatment. High miR-128 and miR-155 were correlated with shorter overall survival (OS), whereas performance status (PS) 2 and high miR-128 independently predicted for decreased OS. In the squamous (SqCC) subgroup (n = 41), besides miR-128 and miR-155, high miR-21 and miR-181a expressions were also associated with worse survival and high miR-155 independently predicted for shorter OS. No associations of miRNA expression with clinical outcomes were observed in patients with non-SqCC (n = 87). Integrated function and pathway analysis on miRNA targets revealed significant enrichments in hypoxia-related pathways. Our study shows for the first time that plasma miR-128 and miR-155 hold independent prognostic implications in NSCLC patients treated with platinum-based chemotherapy possibly related to their involvement in tumor response to hypoxia. Further studies are needed to investigate the potential functional role of these miRNAs in an effort to exploit their therapeutic potential.

Monitoring the prolonged Tnf stimulation in space and time with topological-functional networks.

Genes in linear proximity often share regulatory inputs, expression and evolutionary patterns, even in complex eukaryote genomes with extensive intergenic sequences. Gene regulation, on the other hand, is effected through the co-ordinated activation (or suppression) of genes participating in common biological pathways, which are often transcribed from distant loci. Existing approaches for the study of gene expression focus on the functional aspect, taking positional constraints into account only marginally. In this work we propose a novel concept for the study of gene expression, through the combination of topological and functional information into bipartite networks. Starting from genome-wide expression profiles, we define extended chromosomal regions with consistent patterns of differential gene expression and then associate these domains with enriched functional pathways. By analyzing the resulting networks in terms of size, connectivity and modularity we can draw conclusions on the way genome organization may underlie the gene regulation program. Implementation of this approach in a detailed RNASeq profiling of sustained Tnf stimulation of mouse synovial fibroblasts, allowed us to identify unexpected regulatory changes taking place in the cells after 24 h of stimulation. Bipartite network analysis suggests that the cytokine response set by Tnf, progresses through two distinct transitions. An early generalization of the inflammatory response, that is followed by a late shutdown of immune-related functions and the redistribution of expression to developmental and cell adhesion pathways and distinct chromosomal regions. We show that the incorporation of topological information may provide additional insights in the complex propagation of Tnf activation.