SRRM2 splicing factor modulates cell fate in early development.

Embryo development is an orchestrated process that relies on tight regulation of gene expression to guide cell differentiation and fate decisions. The Srrm2 splicing factor has recently been implicated in developmental disorders and diseases, but its role in early mammalian development remains unexplored. Here, we show that Srrm2 dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness, characterised by the coexistence of cells expressing naive and formative pluripotency markers, together with extensive changes in gene expression, including genes regulated by serum-response transcription factor (SRF) and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells shows that the earliest effects of Srrm2 heterozygosity are specific alternative splicing events on a small number of genes, followed by expression changes in metabolism and differentiation-related genes. Our findings unveil molecular and cellular roles of Srrm2 in stemness and lineage commitment, shedding light on the roles of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis.

Transcription readthrough is prevalent in healthy human tissues and associated with inherent genomic features.

Transcription termination is a crucial step in the production of conforming mRNAs and functional proteins. Under cellular stress conditions, the transcription machinery fails to identify the termination site and continues transcribing beyond gene boundaries, a phenomenon designated as transcription readthrough. However, the prevalence and impact of this phenomenon in healthy human tissues remain unexplored. Here, we assessed transcription readthrough in almost 3000 transcriptome profiles representing 23 human tissues and found that 34% of the expressed protein-coding genes produced readthrough transcripts. The production of readthrough transcripts was restricted in genomic regions with high transcriptional activity and was associated with inefficient splicing and increased chromatin accessibility in terminal regions. In addition, we showed that these transcripts contained several binding sites for the same miRNA, unravelling a potential role as miRNA sponges. Overall, this work provides evidence that transcription readthrough is pervasive and non-stochastic, not only in abnormal conditions but also in healthy tissues. This suggests a potential role for such transcripts in modulating normal cellular functions.

Identification of biomarkers predictive of metastasis development in early-stage colorectal cancer using network-based regularization.

Colorectal cancer (CRC) is the third most common cancer and the second most deathly worldwide. It is a very heterogeneous disease that can develop via distinct pathways where metastasis is the primary cause of death. Therefore, it is crucial to understand the molecular mechanisms underlying metastasis. RNA-sequencing is an essential tool used for studying the transcriptional landscape. However, the high-dimensionality of gene expression data makes selecting novel metastatic biomarkers problematic. To distinguish early-stage CRC patients at risk of developing metastasis from those that are not, three types of binary classification approaches were used: (1) classification methods (decision trees, linear and radial kernel support vector machines, logistic regression, and random forest) using differentially expressed genes (DEGs) as input features; (2) regularized logistic regression based on the Elastic Net penalty and the proposed iTwiner-a network-based regularizer accounting for gene correlation information; and (3) classification methods based on the genes pre-selected using regularized logistic regression. Classifiers using the DEGs as features showed similar results, with random forest showing the highest accuracy. Using regularized logistic regression on the full dataset yielded no improvement in the methods' accuracy. Further classification using the pre-selected genes found by different penalty factors, instead of the DEGs, significantly improved the accuracy of the binary classifiers. Moreover, the use of network-based correlation information (iTwiner) for gene selection produced the best classification results and the identification of more stable and robust gene sets. Some are known to be tumor suppressor genes (OPCML-IT2), to be related to resistance to cancer therapies (RAC1P3), or to be involved in several cancer processes such as genome stability (XRCC6P2), tumor growth and metastasis (MIR602) and regulation of gene transcription (NME2P2). We show that the classification of CRC patients based on pre-selected features by regularized logistic regression is a valuable alternative to using DEGs, significantly increasing the models' predictive performance. Moreover, the use of correlation-based penalization for biomarker selection stands as a promising strategy for predicting patients' groups based on RNA-seq data.

Genetic and microenvironmental intra-tumor heterogeneity impacts colorectal cancer evolution and metastatic development.

Colorectal cancer (CRC) is a highly diverse disease, where different genomic instability pathways shape genetic clonal diversity and tumor microenvironment. Although intra-tumor heterogeneity has been characterized in primary tumors, its origin and consequences in CRC outcome is not fully understood. Therefore, we assessed intra- and inter-tumor heterogeneity of a prospective cohort of 136 CRC samples. We demonstrate that CRC diversity is forged by asynchronous forms of molecular alterations, where mutational and chromosomal instability collectively boost CRC genetic and microenvironment intra-tumor heterogeneity. We were able to depict predictor signatures of cancer-related genes that can foresee heterogeneity levels across the different tumor consensus molecular subtypes (CMS) and primary tumor location. Finally, we show that high genetic and microenvironment heterogeneity are associated with lower metastatic potential, whereas late-emerging copy number variations favor metastasis development and polyclonal seeding. This study provides an exhaustive portrait of the interplay between genetic and microenvironment intra-tumor heterogeneity across CMS subtypes, depicting molecular events with predictive value of CRC progression and metastasis development.

Concerted Regulation of Glycosylation Factors Sustains Tissue Identity and Function.

Glycosylation is a fundamental cellular process affecting human development and health. Complex machinery establishes the glycan structures whose heterogeneity provides greater structural diversity than other post-translational modifications. Although known to present spatial and temporal diversity, the evolution of glycosylation and its role at the tissue-specific level is poorly understood. In this study, we combined genome and transcriptome profiles of healthy and diseased tissues to uncover novel insights into the complex role of glycosylation in humans. We constructed a catalogue of human glycosylation factors, including transferases, hydrolases and other genes directly involved in glycosylation. These were categorized as involved in N-, O- and lipid-linked glycosylation, glypiation, and glycosaminoglycan synthesis. Our data showed that these glycosylation factors constitute an ancient family of genes, where evolutionary constraints suppressed large gene duplications, except for genes involved in O-linked and lipid glycosylation. The transcriptome profiles of 30 healthy human tissues revealed tissue-specific expression patterns preserved across mammals. In addition, clusters of tightly co-expressed genes suggest a glycosylation code underlying tissue identity. Interestingly, several glycosylation factors showed tissue-specific profiles varying with age, suggesting a role in ageing-related disorders. In cancer, our analysis revealed that glycosylation factors are highly perturbed, at the genome and transcriptome levels, with a strong predominance of copy number alterations. Moreover, glycosylation factor dysregulation was associated with distinct cellular compositions of the tumor microenvironment, reinforcing the impact of glycosylation in modulating the immune system. Overall, this work provides genome-wide evidence that the glycosylation machinery is tightly regulated in healthy tissues and impaired in ageing and tumorigenesis, unveiling novel potential roles as prognostic biomarkers or therapeutic targets.

A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth.

Angiogenesis, the process by which endothelial cells (ECs) form new blood vessels from existing ones, is intimately linked to the tissue's metabolic milieu and often occurs at nutrient-deficient sites. However, ECs rely on sufficient metabolic resources to support growth and proliferation. How endothelial nutrient acquisition and usage are regulated is unknown. Here we show that these processes are instructed by Yes-associated protein 1 (YAP)/WW domain-containing transcription regulator 1 (WWTR1/TAZ)-transcriptional enhanced associate domain (TEAD): a transcriptional module whose function is highly responsive to changes in the tissue environment. ECs lacking YAP/TAZ or their transcriptional partners, TEAD1, 2 and 4 fail to divide, resulting in stunted vascular growth in mice. Conversely, activation of TAZ, the more abundant paralogue in ECs, boosts proliferation, leading to vascular hyperplasia. We find that YAP/TAZ promote angiogenesis by fuelling nutrient-dependent mTORC1 signalling. By orchestrating the transcription of a repertoire of cell-surface transporters, including the large neutral amino acid transporter SLC7A5, YAP/TAZ-TEAD stimulate the import of amino acids and other essential nutrients, thereby enabling mTORC1 activation. Dissociating mTORC1 from these nutrient inputs-elicited by the loss of Rag GTPases-inhibits mTORC1 activity and prevents YAP/TAZ-dependent vascular growth. Together, these findings define a pivotal role for YAP/TAZ-TEAD in controlling endothelial mTORC1 and illustrate the essentiality of coordinated nutrient fluxes in the vasculature.

Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops.

DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. Here, we show that the presence of 5hmC in the transcribed gene promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. Depletion of TET enzymes reduced global R-loops in the absence of gene expression changes, whereas CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. The genome-wide distribution of 5hmC and R-loops shows a positive correlation in mouse and human stem cells and overlap in half of all active genes. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, disclosing new mechanisms of gene expression regulation.

Comprehensive Detection of Pseudogenes Transcribed by Readthrough.

Transcription termination is a critical stage for the production of legitimate mRNAs, and consequently functional proteins. However, the transcription machinery can ignore the stop signs and continue elongating beyond gene boundaries, invading downstream neighboring genes. Such phenomenon, designated transcription readthrough, can trigger the expression of pseudogenes usually silenced or lacking the proper regulatory signals. Due to the sequence similarity to parental genes, readthrough transcribed pseudogenes can regulate relevant protein-coding genes and impact biological functions. Here, we describe a computational pipeline that employs already existent bioinformatic tools to detect readthrough transcribed pseudogenes from expression profiles. We also unveil that combining strand-specific transcriptome data and epigenetic profiles can enhance and corroborate the results. By applying such approach to renal cancer biopsies, we show that pseudogenes can be readthrough transcribed as part of unspliced transcripts or processed RNA chimeras. Overall, our pipeline allows us to scrutinize transcriptome profiles to detect a diversity of readthrough events leading to expression of pseudogenes.

Overexpression of wild-type IL-7Rα promotes T-cell acute lymphoblastic leukemia/lymphoma.

Tight regulation of IL-7Rα expression is essential for normal T-cell development. IL-7Rα gain-of-function mutations are known drivers of T-cell acute lymphoblastic leukemia (T-ALL). Although a subset of patients with T-ALL display high IL7R messenger RNA levels and cases with IL7R gains have been reported, the impact of IL-7Rα overexpression, rather than mutational activation, during leukemogenesis remains unclear. In this study, overexpressed IL-7Rα in tetracycline-inducible Il7r transgenic and Rosa26 IL7R knockin mice drove potential thymocyte self-renewal, and thymus hyperplasia related to increased proliferation of T-cell precursors, which subsequently infiltrated lymph nodes, spleen, and bone marrow, ultimately leading to fatal leukemia. The tumors mimicked key features of human T-ALL, including heterogeneity in immunophenotype and genetic subtype between cases, frequent hyperactivation of the PI3K/Akt pathway paralleled by downregulation of p27Kip1 and upregulation of Bcl-2, and gene expression signatures evidencing activation of JAK/STAT, PI3K/Akt/mTOR and Notch signaling. Notably, we also found that established tumors may no longer require high levels of IL-7R expression upon secondary transplantation and progressed in the absence of IL-7, but remain sensitive to inhibitors of IL-7R-mediated signaling ruxolitinib (Jak1), AZD1208 (Pim), dactolisib (PI3K/mTOR), palbociclib (Cdk4/6), and venetoclax (Bcl-2). The relevance of these findings for human disease are highlighted by the fact that samples from patients with T-ALL with high wild-type IL7R expression display a transcriptional signature resembling that of IL-7-stimulated pro-T cells and, critically, of IL7R-mutant cases of T-ALL. Overall, our study demonstrates that high expression of IL-7Rα can promote T-cell tumorigenesis, even in the absence of IL-7Rα mutational activation.

Control of endothelial quiescence by FOXO-regulated metabolites.

Endothelial cells (ECs) adapt their metabolism to enable the growth of new blood vessels, but little is known how ECs regulate metabolism to adopt a quiescent state. Here, we show that the metabolite S-2-hydroxyglutarate (S-2HG) plays a crucial role in the regulation of endothelial quiescence. We find that S-2HG is produced in ECs after activation of the transcription factor forkhead box O1 (FOXO1), where it limits cell cycle progression, metabolic activity and vascular expansion. FOXO1 stimulates S-2HG production by inhibiting the mitochondrial enzyme 2-oxoglutarate dehydrogenase. This inhibition relies on branched-chain amino acid catabolites such as 3-methyl-2-oxovalerate, which increase in ECs with activated FOXO1. Treatment of ECs with 3-methyl-2-oxovalerate elicits S-2HG production and suppresses proliferation, causing vascular rarefaction in mice. Our findings identify a metabolic programme that promotes the acquisition of a quiescent endothelial state and highlight the role of metabolites as signalling molecules in the endothelium.

Innate immune evasion revealed in a colorectal zebrafish xenograft model.

Cancer immunoediting is a dynamic process of crosstalk between tumor cells and the immune system. Herein, we explore the fast zebrafish xenograft model to investigate the innate immune contribution to this process. Using multiple breast and colorectal cancer cell lines and zAvatars, we find that some are cleared (regressors) while others engraft (progressors) in zebrafish xenografts. We focus on two human colorectal cancer cells derived from the same patient that show contrasting engraftment/clearance profiles. Using polyclonal xenografts to mimic intra-tumor heterogeneity, we demonstrate that SW620_progressors can block clearance of SW480_regressors. SW480_regressors recruit macrophages and neutrophils more efficiently than SW620_progressors; SW620_progressors however, modulate macrophages towards a pro-tumoral phenotype. Genetic and chemical suppression of myeloid cells indicates that macrophages and neutrophils play a crucial role in clearance. Single-cell-transcriptome analysis shows a fast subclonal selection, with clearance of regressor subclones associated with IFN/Notch signaling and escaper-expanded subclones with enrichment of IL10 pathway. Overall, our work opens the possibility of using zebrafish xenografts as living biomarkers of the tumor microenvironment.

New Insights into Immunological Involvement in Congenital Disorders of Glycosylation (CDG) from a People-Centric Approach.

Congenital disorders of glycosylation (CDG) are rare diseases with variable phenotypes and severity. Immunological involvement remains a largely uncharted topic in CDG, mainly due to lack of robust data. To better characterize immune-related manifestations' prevalence, relevance, and quality-of-life (QoL) impact, we developed electronic questionnaires targeting (1) CDG patients and (2) the general "healthy" population. Two-hundred and nine CDG patients/caregivers and 349 healthy participants were included in this study. PMM2-CDG was the most represented CDG (n = 122/209). About half of these participants (n = 65/122) described relevant infections with a noteworthy prevalence of those affecting the gastrointestinal tract (GI) (63.1%, n = 41/65). Infection burden and QoL impact were shown as infections correlated with more severe clinical phenotypes and with a set of relevant non-immune PMM2-CDG signs. Autoimmune diseases had only a marginal presence in PMM2-CDG (2.5%, n = 3/122), all being GI-related. Allergy prevalence was also low in PMM2-CDG (33%, n = 41/122) except for food allergies (26.8%, n = 11/41, of PMM2-CDG and 10.8%, n = 17/158, of controls). High vaccination compliance with greater perceived ineffectiveness (28.3%, n = 17/60) and more severe adverse reactions were described in PMM2-CDG. This people-centric approach not only confirmed literature findings, but created new insights into immunological involvement in CDG, namely by highlighting the possible link between the immune and GI systems in PMM2-CDG. Finally, our results emphasized the importance of patient/caregiver knowledge and raised several red flags about immunological management.

Analysis of Mammalian Native Elongating Transcript sequencing (mNET-seq) high-throughput data.

Mammalian Native Elongating Transcript sequencing (mNET-seq) is a recently developed technique that generates genome-wide profiles of nascent transcripts associated with RNA polymerase II (Pol II) elongation complexes. The ternary transcription complexes formed by Pol II, DNA template and nascent RNA are first isolated, without crosslinking, by immunoprecipitation with antibodies that specifically recognize the different phosphorylation states of the polymerase large subunit C-terminal domain (CTD). The coordinate of the 3' end of the RNA in the complexes is then identified by high-throughput sequencing. The main advantage of mNET-seq is that it provides global, bidirectional maps of Pol II CTD phosphorylation-specific nascent transcripts and coupled RNA processing at single nucleotide resolution. Here we describe the general pipeline to prepare and analyse high-throughput data from mNET-seq experiments.

A Systematic Pan-Cancer Analysis of Genetic Heterogeneity Reveals Associations with Epigenetic Modifiers.

Intratumor genetic heterogeneity (ITH) is the main obstacle to effective cancer treatment and a major mechanism of drug resistance. It results from the continuous evolution of different clones of a tumor over time. However, the molecular features underlying the emergence of genetically-distinct subclonal cell populations remain elusive. Here, we conducted an exhaustive characterization of ITH across 2807 tumor samples from 16 cancer types. Integration of ITH scores and somatic variants detected in each tumor sample revealed that mutations in epigenetic modifier genes are associated with higher ITH levels. In particular, genes that regulate genome-wide histone and DNA methylation emerged as being determinant of high ITH. Indeed, the knockout of histone methyltransferase SETD2 or DNA methyltransferase DNMT3A using the CRISPR/Cas9 system on cancer cells led to significant expansion of genetically-distinct clones and culminated in highly heterogeneous cell populations. The ITH scores observed in knockout cells recapitulated the heterogeneity levels observed in patient tumor samples and correlated with a better mitochondrial bioenergetic performance under stress conditions. Our work provides new insights into tumor development, and discloses new drivers of ITH, which may be useful as either predictive biomarkers or therapeutic targets to improve cancer treatment.

RNA Polymerase II Phosphorylated on CTD Serine 5 Interacts with the Spliceosome during Co-transcriptional Splicing.

The highly intronic nature of protein coding genes in mammals necessitates a co-transcriptional splicing mechanism as revealed by mNET-seq analysis. Immunoprecipitation of MNase-digested chromatin with antibodies against RNA polymerase II (Pol II) shows that active spliceosomes (both snRNA and proteins) are complexed to Pol II S5P CTD during elongation and co-transcriptional splicing. Notably, elongating Pol II-spliceosome complexes form strong interactions with nascent transcripts, resulting in footprints of approximately 60 nucleotides. Also, splicing intermediates formed by cleavage at the 5' splice site are associated with nearly all spliced exons. These spliceosome-bound intermediates are frequently ligated to upstream exons, implying a sequential, constitutive, and U12-dependent splicing process. Finally, lack of detectable spliced products connected to the Pol II active site in human HeLa or murine lymphoid cells suggests that splicing does not occur immediately following 3' splice site synthesis. Our results imply that most mammalian splicing requires exon definition for completion.

Nutrient sensing modulates malaria parasite virulence.

The lifestyle of intracellular pathogens, such as malaria parasites, is intimately connected to that of their host, primarily for nutrient supply. Nutrients act not only as primary sources of energy but also as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Canonical nutrient-sensing pathways are presumed to be absent from the causative agent of malaria, Plasmodium, thus raising the question of whether these parasites can sense and cope with fluctuations in host nutrient levels. Here we show that Plasmodium blood-stage parasites actively respond to host dietary calorie alterations through rearrangement of their transcriptome accompanied by substantial adjustment of their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status. KIN shares homology with SNF1/AMPKα, and yeast complementation studies suggest that it is part of a functionally conserved cellular energy-sensing pathway. Overall, these findings reveal a key parasite nutrient-sensing mechanism that is critical for modulating parasite replication and virulence.

Transcription Dynamics Prevent RNA-Mediated Genomic Instability through SRPK2-Dependent DDX23 Phosphorylation.

Genomic instability is frequently caused by nucleic acid structures termed R-loops that are formed during transcription. Despite their harmful potential, mechanisms that sense, signal, and suppress these structures remain elusive. Here, we report that oscillations in transcription dynamics are a major sensor of R-loops. We show that pausing of RNA polymerase II (RNA Pol II) initiates a signaling cascade whereby the serine/arginine protein kinase 2 (SRPK2) phosphorylates the DDX23 helicase, culminating in the suppression of R-loops. We show that in the absence of either SRPK2 or DDX23, accumulation of R-loops leads to massive genomic instability revealed by high levels of DNA double-strand breaks (DSBs). Importantly, we found DDX23 mutations in several cancers and detected homozygous deletions of the entire DDX23 locus in 10 (17%) adenoid cystic carcinoma (ACC) samples. Our results unravel molecular details of a link between transcription dynamics and RNA-mediated genomic instability that may play important roles in cancer development.

A pan-cancer analysis of MYC-PVT1 reveals CNV-unmediated deregulation and poor prognosis in renal carcinoma.

The PVT1 lncRNA has recently been involved in tumorigenesis by affecting the protein stability of the MYC proto-oncogene. Both MYC and PVT1 reside in a well-known cancer-risk locus and enhanced levels of their products have been reported in different human cancers. Nonetheless, the extension and relevance of the MYC-PVT1 deregulation in tumorigenesis has not yet been systematically addressed.Here we performed a pan-cancer analysis of matched copy number, transcriptomic, methylation, proteomic and clinicopathological profiles for almost 7000 patients from 17 different cancers represented in the TCGA cohorts. Among all cancers types, kidney renal clear cell carcinoma (KIRC) showed the strongest upregulation of PVT1 and increased levels of both MYC and PVT1 correlated with the clinical outcome. PVT1 misregulation in KIRC is mostly associated to promoter hypomethylation rather than locus amplification. Furthermore, we found an association between MYC levels and PVT1 expression, which impacted on MYC-target genes.Collectively, our study discloses the role of PVT1 as a novel prognostic factor and as a molecular target for novel therapeutic interventions in renal carcinoma.

Transcriptome profiling of two Iberian freshwater fish exposed to thermal stress.

The congeneric freshwater fish Squalius carolitertii and S. torgalensis inhabit different Iberian regions with distinct climates; Atlantic in the North and Mediterranean in the South, respectively. While northern regions present mild temperatures, fish in southern regions often experience harsh temperatures and droughts. Previous work with two hsp70 genes suggested that S. torgalensis is better adapted to harsher thermal conditions than S. carolitertii as a result of the different environmental conditions. We present a transcriptomic characterisation of these species' thermal stress responses. Through differential gene expression analysis of the recently available transcriptomes of these two endemic fish species, comprising 12 RNA-seq libraries from three tissues (skeletal muscle, liver and fins) of fish exposed to control (18 °C) and test (30 °C) conditions, we intend to lay the foundations for further studies on the effects of temperature given predicted climate changes. Results showed that S. carolitertii had more upregulated genes, many of which are involved in transcription regulation, whereas S. torgalensis had more downregulated genes, particularly those responsible for cell division and growth. However, both species displayed increased gene expression of many hsps genes, suggesting that they are able to deal with protein damage caused by heat, though with a greater response in S. torgalensis. Together, our results suggest that S. torgalensis may have an energy saving strategy during short periods of high temperatures, re-allocating resources from growth to stress response mechanisms. In contrast, S. carolitertii regulates its metabolism by increasing the expression of genes involved in transcription and promoting the stress response, probably to maintain homoeostasis. Additionally, we indicate a set of potential target genes for further studies that may be particularly suited to monitoring the responses of Cyprinidae to changing temperatures, particularly for species living in similar conditions in the Mediterranean Peninsulas.