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In tumors with WT p53, alternate mechanisms of p53 inactivation are reported. Here, we have identified a long noncoding RNA, PITAR ( p 53 I nactivating T RIM28 A ssociated R NA), as an inhibitor of p53. PITAR is an oncogenic Cancer/testis lncRNA and is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSC). We establish that TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase, is a direct target of PITAR. PITAR interaction with TRIM28 RNA stabilized TRIM28 mRNA, which resulted in increased TRIM28 protein levels and reduced p53 steady-state levels due to enhanced p53 ubiquitination. DNA damage activated PITAR , in addition to p53, in a p53-independent manner, thus creating an incoherent feedforward loop to inhibit the DNA damage response by p53. While PITAR silencing inhibited the growth of WT p53 containing GSCs in vitro and reduced glioma tumor growth in vivo, its overexpression enhanced the tumor growth in a TRIM28 -dependent manner and promoted resistance to Temozolomide. Thus, we establish an alternate way of p53 inactivation by PITAR , which maintains low p53 levels in normal cells and attenuates the DNA damage response by p53. Finally, we propose PITAR as a potential GBM therapeutic target., Competing Interests: SJ, MM, SM, BG, KP, LK, NJ, AC, VS, CK No competing interests declared, KS Reviewing editor, eLife, (© 2023, Jana et al.)

Background: Patients with peritoneal metastasis from colorectal cancer (PM-CRC) have inferior prognosis and respond particularly poorly to chemotherapy. This study aims to identify the molecular explanation for the observed clinical behavior and suggest novel treatment strategies in PM-CRC., Methods: Tumor samples (230) from a Norwegian national cohort undergoing surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C (MMC) for PM-CRC were subjected to targeted DNA sequencing, and associations with clinical data were analyzed. mRNA sequencing was conducted on a subset of 30 samples to compare gene expression in tumors harboring BRAF or KRAS mutations and wild-type tumors., Results: BRAF mutations were detected in 27% of the patients, and the BRAF-mutated subgroup had inferior overall survival compared to wild-type cases (median 16 vs 36 months, respectively, p < 0.001). BRAF mutations were associated with RNF43/RSPO aberrations and low expression of negative Wnt regulators (ligand-dependent Wnt activation). Furthermore, BRAF mutations were associated with gene expression changes in transport solute carrier proteins (specifically SLC7A6) and drug metabolism enzymes (CES1 and CYP3A4) that could influence the efficacy of MMC and irinotecan, respectively. BRAF-mutated tumors additionally exhibited increased expression of members of the novel butyrophilin subfamily of immune checkpoint molecules (BTN1A1 and BTNL9)., Conclusions: BRAF mutations were frequently detected and were associated with particularly poor survival in this cohort, possibly related to ligand-dependent Wnt activation and altered drug transport and metabolism that could confer resistance to MMC and irinotecan. Drugs that target ligand-dependent Wnt activation or the BTN immune checkpoints could represent two novel therapy approaches., (© 2024. The Author(s).)

In recent years, lactate has been recognized as an important circulating energy substrate rather than only a dead-end metabolic waste product generated during glucose oxidation at low levels of oxygen. The term "aerobic glycolysis" has been coined to denote increased glucose uptake and lactate production despite normal oxygen levels and functional mitochondria. Hence, in "aerobic glycolysis," lactate production is a metabolic choice, whereas in "anaerobic glycolysis," it is a metabolic necessity based on inadequate levels of oxygen. Interestingly, lactate can be taken up by cells and oxidized to pyruvate and thus constitutes a source of pyruvate that is independent of insulin. Here, we show that the transcription factor Foxp1 regulates glucose uptake and lactate production in adipocytes and myocytes. Overexpression of Foxp1 leads to increased glucose uptake and lactate production. In addition, protein levels of several enzymes in the glycolytic pathway are upregulated, such as hexokinase 2, phosphofructokinase, aldolase, and lactate dehydrogenase. Using chromatin immunoprecipitation and real-time quantitative PCR assays, we demonstrate that Foxp1 directly interacts with promoter consensus cis-elements that regulate expression of several of these target genes. Conversely, knockdown of Foxp1 suppresses these enzyme levels and lowers glucose uptake and lactate production. Moreover, mice with a targeted deletion of Foxp1 in muscle display systemic glucose intolerance with decreased muscle glucose uptake. In primary human adipocytes with induced expression of Foxp1, we find increased glycolysis and glycolytic capacity. Our results indicate Foxp1 may play an important role as a regulator of aerobic glycolysis in adipose tissue and muscle., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease., (© 2023. Springer Nature Limited.)

Background: Machine learning (ML) has gained significant attention for classifying immune states in adaptive immune receptor repertoires (AIRRs) to support the advancement of immunodiagnostics and therapeutics. Simulated data are crucial for the rigorous benchmarking of AIRR-ML methods. Existing approaches to generating synthetic benchmarking datasets result in the generation of naive repertoires missing the key feature of many shared receptor sequences (selected for common antigens) found in antigen-experienced repertoires., Results: We demonstrate that a common approach to generating simulated AIRR benchmark datasets can introduce biases, which may be exploited for undesired shortcut learning by certain ML methods. To mitigate undesirable access to true signals in simulated AIRR datasets, we devised a simulation strategy (simAIRR) that constructs antigen-experienced-like repertoires with a realistic overlap of receptor sequences. simAIRR can be used for constructing AIRR-level benchmarks based on a range of assumptions (or experimental data sources) for what constitutes receptor-level immune signals. This includes the possibility of making or not making any prior assumptions regarding the similarity or commonality of immune state-associated sequences that will be used as true signals. We demonstrate the real-world realism of our proposed simulation approach by showing that basic ML strategies perform similarly on simAIRR-generated and real-world experimental AIRR datasets., Conclusions: This study sheds light on the potential shortcut learning opportunities for ML methods that can arise with the state-of-the-art way of simulating AIRR datasets. simAIRR is available as a Python package: https://github.com/KanduriC/simAIRR., (© The Author(s) 2023. Published by Oxford University Press GigaScience.)

The regulatory nature of long non-coding RNAs (lncRNAs) has been well established in various processes of cellular growth, development, and differentiation. Therefore, it is vital to examine their contribution to cancer development. There are ample examples of lncRNAs whose cellular levels are significantly associated with clinical outcomes. However, whether these non-coding molecules can work as either key drivers or barriers to cancer development remains unknown. The current review aims to discuss some well-characterised lncRNAs in the process of oncogenesis and extrapolate the extent of their decisive contribution to tumour development. We ask if these lncRNAs can independently initiate neoplastic lesions or they always need the modulation of well characterized oncogenes or tumour suppressors to exert their functional properties. Finally, we discuss the emerging genetic approaches and appropriate animal and humanised models that can significantly contribute to the functional dissection of lncRNAs in cancer development and progression.

Although antisense transcription is a widespread event in the mammalian genome, double-stranded RNA (dsRNA) formation between sense and antisense transcripts is very rare and mechanisms that control dsRNA remain unknown. By characterizing the FGF-2 regulated transcriptome in normal and cancer cells, we identified sense and antisense transcripts IER3 and IER3-AS1 that play a critical role in FGF-2 controlled oncogenic pathways. We show that IER3 and IER3-AS1 regulate each other's transcription through HnRNPK-mediated post-transcriptional regulation. HnRNPK controls the mRNA stability and colocalization of IER3 and IER3-AS1. HnRNPK interaction with IER3 and IER3-AS1 determines their oncogenic functions by maintaining them in a single-stranded form. hnRNPK depletion neutralizes their oncogenic functions through promoting dsRNA formation and cytoplasmic accumulation. Intriguingly, hnRNPK loss-of-function and gain-of-function experiments reveal its role in maintaining global single- and double-stranded RNA. Thus, our data unveil the critical role of HnRNPK in maintaining single-stranded RNAs and their physiological functions by blocking RNA-RNA interactions., (© 2022. The Author(s).)

Background: Machine learning (ML) methodology development for the classification of immune states in adaptive immune receptor repertoires (AIRRs) has seen a recent surge of interest. However, so far, there does not exist a systematic evaluation of scenarios where classical ML methods (such as penalized logistic regression) already perform adequately for AIRR classification. This hinders investigative reorientation to those scenarios where method development of more sophisticated ML approaches may be required., Results: To identify those scenarios where a baseline ML method is able to perform well for AIRR classification, we generated a collection of synthetic AIRR benchmark data sets encompassing a wide range of data set architecture-associated and immune state-associated sequence patterns (signal) complexity. We trained ≈1,700 ML models with varying assumptions regarding immune signal on ≈1,000 data sets with a total of ≈250,000 AIRRs containing ≈46 billion TCRβ CDR3 amino acid sequences, thereby surpassing the sample sizes of current state-of-the-art AIRR-ML setups by two orders of magnitude. We found that L1-penalized logistic regression achieved high prediction accuracy even when the immune signal occurs only in 1 out of 50,000 AIR sequences., Conclusions: We provide a reference benchmark to guide new AIRR-ML classification methodology by (i) identifying those scenarios characterized by immune signal and data set complexity, where baseline methods already achieve high prediction accuracy, and (ii) facilitating realistic expectations of the performance of AIRR-ML models given training data set properties and assumptions. Our study serves as a template for defining specialized AIRR benchmark data sets for comprehensive benchmarking of AIRR-ML methods., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Background: The actin-binding protein filamin A (FLNA) regulates oncogenic signal transduction important for tumor growth, but the role of FLNA in the progression of neuroblastoma (NB) has not been explored., Methods: We analyzed FLNA mRNA expression in the R2 NB-database and FLNA protein expression in human NB tumors. We then silenced FLNA expression in human SKNBE2 and IMR32 NB cells by lentiviral vector encoding shRNA FLNA and assayed the cells for proliferation, migration, colony, spheroid formation, and apoptosis. SKNBE2 xenografts expressing or lacking FLNA in BALB/c nude mice were analyzed by both routine histopathology and immunohistochemistry., Results: We observed shorter patient survival with higher expression of FLNA mRNA than patients with lower FLNA mRNA expression, and high-risk NB tumors expressed higher FLNA levels. Overexpression of FLNA increased proliferation of SH-SY5 NB cells. NB cell lines transfected with siRNA FLNA proliferated and migrated less, expressed lower levels of phosphorylated AKT and ERK1/2, formed smaller colonies and spheroids, as well as increased apoptosis. After inoculation of SKNBE2 cells infected with lentivirus expressing shRNA FLNA , size of NB tumors and number of proliferating cells were decreased. Furthermore, we identified STAT3 as an interacting partner of FLNA. Silencing FLNA mRNA reduced levels of NF-κB, STAT3 and MYCN, and increased levels of p53 and cleaved caspase 3., Conclusion: Inhibition of FLNA impaired NB cell signaling and function and reduced NB tumor size in vivo , suggesting that drugs targeting either FLNA or its interaction with STAT3 may be useful in the treatment of NB., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Adaptive immune receptor repertoires (AIRR) are key targets for biomedical research as they record past and ongoing adaptive immune responses. The capacity of machine learning (ML) to identify complex discriminative sequence patterns renders it an ideal approach for AIRR-based diagnostic and therapeutic discovery. To date, widespread adoption of AIRR ML has been inhibited by a lack of reproducibility, transparency, and interoperability. immuneML (immuneml.uio.no) addresses these concerns by implementing each step of the AIRR ML process in an extensible, open-source software ecosystem that is based on fully specified and shareable workflows. To facilitate widespread user adoption, immuneML is available as a command-line tool and through an intuitive Galaxy web interface, and extensive documentation of workflows is provided. We demonstrate the broad applicability of immuneML by (i) reproducing a large-scale study on immune state prediction, (ii) developing, integrating, and applying a novel deep learning method for antigen specificity prediction, and (iii) showcasing streamlined interpretability-focused benchmarking of AIRR ML., Competing Interests: Competing Interests V.G. declares advisory board positions in aiNET GmbH and Enpicom B.V. VG is a consultant for Roche/Genentech.

Background: MYCN has been an attractive therapeutic target in neuroblastoma considering the widespread amplification of the MYCN locus in neuroblastoma, and its established role in neuroblastoma development and progression. Thus, understanding neuroblastoma-specific control of MYCN expression at the transcriptional and post-transcriptional level would lead to identification of novel MYCN -dependent oncogenic pathways and potential therapeutic strategies., Methods: By performing loss- and gain-of-function experiments of the neuroblastoma hotspot locus 6p22.3 derived lncRNAs CASC15-003 and NBAT1, together with coimmunoprecipitation and immunoblotting of MYCN, we have shown that both lncRNAs post-translationally control the expression of MYCN through regulating a deubiquitinase enzyme USP36. USP36 oncogenic properties were investigated using cancer cell lines and in vivo models. RNA-seq analysis of loss-of-function experiments of CASC15-003/NBAT1/MYCN/USP36 and JQ1-treated neuroblastoma cells uncovered MYCN -dependent oncogenic pathways., Results: We show that NBAT1/CASC15-003 control the stability of MYCN protein through their common interacting protein partner USP36. USP36 harbors oncogenic properties and its higher expression in neuroblastoma patients correlates with poor prognosis, and its downregulation significantly reduces tumor growth in neuroblastoma cell lines and xenograft models. Unbiased integration of RNA-seq data from CASC15-003 , NBAT1 , USP36 , and MYCN knockdowns and neuroblastoma cells treated with MYCN inhibitor JQ1, identified genes that are jointly regulated by the NBAT1 / CASC15-003 / USP3 6/ MYCN pathway. Functional experiments on one of the target genes, COL18A1 , revealed its role in the NBAT1 / CASC15-003 -dependent cell adhesion feature in neuroblastoma cells., Conclusion: Our data show post-translational regulation of MYCN by NBAT1 / CASC15-003 /USP36, which represents a new regulatory layer in the complex multilayered gene regulatory network that controls MYCN expression., (© The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Many long noncoding RNAs (lncRNAs) are enriched in pancreatic islets and several lncRNAs are linked to type 2 diabetes (T2D). Although they have emerged as potential players in β-cell biology and T2D, little is known about their functions and mechanisms in human β-cells. We identified an islet-enriched lncRNA, TUNAR (TCL1 upstream neural differentiation-associated RNA), which was upregulated in β-cells of patients with T2D and promoted human β-cell proliferation via fine-tuning of the Wnt pathway. TUNAR was upregulated following Wnt agonism by a glycogen synthase kinase-3 (GSK3) inhibitor in human β-cells. Reciprocally, TUNAR repressed a Wnt antagonist Dickkopf-related protein 3 (DKK3) and stimulated Wnt pathway signaling. DKK3 was aberrantly expressed in β-cells of patients with T2D and displayed a synchronized regulatory pattern with TUNAR at the single cell level. Mechanistically, DKK3 expression was suppressed by the repressive histone modifier enhancer of zeste homolog 2 (EZH2). TUNAR interacted with EZH2 in β-cells and facilitated EZH2-mediated suppression of DKK3 . These findings reveal a novel cell-specific epigenetic mechanism via islet-enriched lncRNA that fine-tunes the Wnt pathway and subsequently human β-cell proliferation. NEW & NOTEWORTHY The discovery that long noncoding RNA TUNAR regulates β-cell proliferation may be important in designing new treatments for diabetes.

Recent advances in genomics unraveled several actionable mutational drivers in lung cancer, leading to promising therapies such as tyrosine kinase inhibitors and immune checkpoint inhibitors. However, the tumors' acquired resistance to the newly-developed as well as existing therapies restricts life quality improvements. Therefore, we investigated the noncoding portion of the human transcriptome in search of alternative actionable targets. We identified an antisense transcript, LY6K-AS, with elevated expression in lung adenocarcinoma (LUAD) patients, and its higher expression in LUAD patients predicts poor survival outcomes. LY6K-AS abrogation interfered with the mitotic progression of lung cancer cells resulting in unfaithful chromosomal segregation. LY6K-AS interacts with and stabilizes 14-3-3 proteins to regulate the transcription of kinetochore and mitotic checkpoint proteins. We also show that LY6K-AS regulates the levels of histone H3 lysine 4 trimethylation (H3K4me3) at the promoters of kinetochore members. Cisplatin treatment and LY6K-AS silencing affect many common pathways enriched in cell cycle-related functions. LY6K-AS silencing affects the growth of xenografts derived from wildtype and cisplatin-resistant lung cancer cells. Collectively, these data indicate that LY6K-AS silencing is a promising therapeutic option for LUAD that inhibits oncogenic mitotic progression.

Neuroblastoma has a low mutation rate for the p53 gene. Alternative ways of p53 inactivation have been proposed in neuroblastoma, such as abnormal cytoplasmic accumulation of wild-type p53. However, mechanisms leading to p53 inactivation via cytoplasmic accumulation are not well investigated. Here we show that the neuroblastoma risk-associated locus 6p22.3-derived tumor suppressor NBAT1 is a p53-responsive lncRNA that regulates p53 subcellular levels. Low expression of NBAT1 provided resistance to genotoxic drugs by promoting p53 accumulation in cytoplasm and loss from mitochondrial and nuclear compartments. Depletion of NBAT1 altered CRM1 function and contributed to the loss of p53-dependent nuclear gene expression during genotoxic drug treatment. CRM1 inhibition rescued p53-dependent nuclear functions and sensitized NBAT1 -depleted cells to genotoxic drugs. Combined inhibition of CRM1 and MDM2 was even more effective in sensitizing aggressive neuroblastoma cells with p53 cytoplasmic accumulation. Thus, our mechanistic studies uncover an NBAT1 -dependent CRM1/MDM2-based potential combination therapy for patients with high-risk neuroblastoma. SIGNIFICANCE: This study shows how a p53-responsive lncRNA mediates chemotherapeutic response by modulating nuclear p53 pathways and identifies a potential treatment strategy for patients with high-risk neuroblastoma., (©2020 American Association for Cancer Research.)

Despite the rapid improvements in unveiling the importance of lncRNAs in all aspects of cancer biology, there is still a void in mechanistic understanding of their role in the DNA damage response. Here we explored the potential role of the oncogenic lncRNA SCAT7 (ELF3-AS1) in the maintenance of genome integrity. We show that SCAT7 is upregulated in response to DNA-damaging drugs like cisplatin and camptothecin, where SCAT7 expression is required to promote cell survival. SCAT7 silencing leads to decreased proliferation of cisplatin-resistant cells in vitro and in vivo through interfering with cell cycle checkpoints and DNA repair molecular pathways. SCAT7 regulates ATR signaling, promoting homologous recombination. Importantly, SCAT7 also takes part in proteasome-mediated topoisomerase I (TOP1) degradation, and its depletion causes an accumulation of TOP1-cc structures responsible for the high levels of intrinsic DNA damage. Thus, our data demonstrate that SCAT7 is an important constituent of the DNA damage response pathway and serves as a potential therapeutic target for hard-to-treat drug resistant cancers., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Cancer.)

From high-throughput DNA and RNA sequencing technologies, it is evident that more than two-thirds of the mammalian genome is transcribed and nearly 98% of the transcriptional output in humans constitute noncoding RNA, comprising tens of thousands of small and long noncoding RNAs. These observations have put the study of RNA expression levels at the center of molecular biology research. The transcriptional output of cells changes temporally throughout different cell cycle phases, or in response to a large panel of stimuli. In such instances, the measure of induced RNA transcripts might be obscured by the presence of steady-state RNA levels in the total transcriptome. With this protocol, we provide a method for labeling and purification of the nascent RNAs transcribed over short periods of time in cultured cells. The supplementation of cell culture medium with a chemically modified analog of uridine, ethynyl-uridine, allows for the subsequent biotinylation of ethynyl-uridine residues with a click-chemistry reaction. The labeled RNA is then purified on streptavidin beads and eluted. The purified RNA is suitable for use in RT-qPCR assays as well as in deep sequencing applications.

With the rapid revolution in RNA/DNA sequencing technologies, it is evident that mammalian genomes express tens of thousands of long noncoding RNAs (lncRNAs). Since a large majority of lncRNAs have been functionally implicated in cancer development and progression, there is an increasing appreciation for the use of antisense oligonucleotide (ASO)-based therapies targeting lncRNAs in several cancers. Despite their great potential in therapeutic applications, their use is still limited due to cellular toxicity and shortcomings in achieving required stability in biological fluids and tissue uptake. To overcome these limitations, major changes in ASO chemistry have been introduced to generate second and third generation ASOs, including locked nucleic acids (LNA) technology. Here we describe two different LNA-ASO delivery approaches, a peritumoral administration and a systemic delivery in xenograft models of lung adenocarcinoma, that significantly reduced tumor growth without inducing toxicity.

Cancer is characterized by genomic instability leading to deletion or amplification of oncogenes or tumor suppressors. However, most of the altered regions are devoid of known cancer drivers. Here, we identify lncRNAs frequently lost or amplified in cancer. Among them, we found amplified lncRNA associated with lung cancer-1 (ALAL-1) as frequently amplified in lung adenocarcinomas. ALAL-1 is also overexpressed in additional tumor types, such as lung squamous carcinoma. The RNA product of ALAL-1 is able to promote the proliferation and tumorigenicity of lung cancer cells. ALAL-1 is a TNFα- and NF-κB-induced cytoplasmic lncRNA that specifically interacts with SART3, regulating the subcellular localization of the protein deubiquitinase USP4 and, in turn, its function in the cell. Interestingly, ALAL-1 expression inversely correlates with the immune infiltration of lung squamous tumors, while tumors with ALAL-1 amplification show lower infiltration of several types of immune cells. We have thus unveiled a pro-oncogenic lncRNA that mediates cancer immune evasion, pointing to a new target for immune potentiation., (© 2020 Athie et al.)

Importance of sperm-derived transcripts and chromatin imprints in organismal development is poorly investigated. Here using an integrative approach, we show that human sperm transcripts are equally important as oocyte. Sperm-specific and sperm-oocyte common transcripts carry distinct chromatin structures at their promoters correlating with corresponding transcript levels in sperm. Interestingly, sperm-specific H3K4me3 patterns at the lincRNA promoters are not maintained in the germ layers and somatic tissues. However, bivalent chromatin at the sperm-specific protein-coding gene promoters is maintained throughout the development. Sperm-specific transcripts reach their peak expression during zygotic genome activation, whereas sperm-oocyte common transcripts are present during early preimplantation development but decline at the onset of zygotic genome activation. Additionally, there is an inverse correlation between sperm-specific and sperm-oocyte lincRNAs throughout the development. Sperm-lincRNAs also show aberrant activation in tumors. Overall, our observations indicate that sperm transcripts carrying chromatin imprints may play an important role in human development and cancer., Competing Interests: Declaration of Interests A patent application has been filed based on the findings presented in this study., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Autoantibodies to transglutaminase 2 (TG2) are hallmarks of celiac disease. To address B cell tolerance and autoantibody formation to TG2, we generated immunoglobulin knock-in (Ig KI) mice that express a prototypical celiac patient-derived anti-TG2 B cell receptor equally reactive to human and mouse TG2. We studied B cell development in the presence/absence of autoantigen by crossing the Ig KI mice to Tgm2-/- mice. Autoreactive B cells in Tgm2+/+ mice were indistinguishable from their naive counterparts in Tgm2-/- mice with no signs of clonal deletion, receptor editing, or B cell anergy. The autoreactive B cells appeared ignorant to their antigen, and they produced autoantibodies when provided T cell help. The findings lend credence to a model of celiac disease where gluten-reactive T cells provide help to autoreactive TG2-specific B cells by involvement of gluten-TG2 complexes, and they outline a general mechanism of autoimmunity with autoantibodies being produced by ignorant B cells on provision of T cell help., (© 2019 du Pré et al.)

Cerebral cavernous malformations (CCMs) are low-flow vascular malformations in the brain associated with recurrent hemorrhage and seizures. The current treatment of CCMs relies solely on surgical intervention. Henceforth, alternative non-invasive therapies are urgently needed to help prevent subsequent hemorrhagic episodes. Long non-coding RNAs (lncRNAs) belong to the class of non-coding RNAs and are known to regulate gene transcription and involved in chromatin remodeling via various mechanism. Despite accumulating evidence demonstrating the role of lncRNAs in cerebrovascular disorders, their identification in CCMs pathology remains unknown. The objective of the current study was to identify lncRNAs associated with CCMs pathogenesis using patient cohorts having 10 CCM patients and 4 controls from brain. Executing next generation sequencing, we performed whole transcriptome sequencing (RNA-seq) analysis and identified 1,967 lncRNAs and 4,928 protein coding genes (PCGs) to be differentially expressed in CCMs patients. Among these, we selected top 6 differentially expressed lncRNAs each having significant correlative expression with more than 100 differentially expressed PCGs. The differential expression status of the top lncRNAs, SMIM25 and LBX2-AS1 in CCMs was further confirmed by qRT-PCR analysis. Additionally, gene set enrichment analysis of correlated PCGs revealed critical pathways related to vascular signaling and important biological processes relevant to CCMs pathophysiology. Here, by transcriptome-wide approach we demonstrate that lncRNAs are prevalent in CCMs disease and are likely to play critical roles in regulating important signaling pathways involved in the disease progression. We believe, that detailed future investigations on this set of identified lncRNAs can provide useful insights into the biology and, ultimately, contribute in preventing this debilitating disease.

With the evolution of technologies that deal with global detection of RNAs to probing of lncRNA-chromatin interactions and lncRNA-chromatin structure regulation, we have been updated with a comprehensive repertoire of chromatin interacting lncRNAs, their genome-wide chromatin binding regions and mode of action. Evidence from these new technologies emphasize that chromatin targeting of lncRNAs is a prominent mechanism and that these chromatin targeted lncRNAs exert their functionality by fine tuning chromatin architecture resulting in an altered transcriptional readout. Currently, there are no unifying principles that define chromatin association of lncRNAs, however, evidence from a few chromatin-associated lncRNAs show presence of a short common sequence for chromatin targeting. In this article, we review how technological advancements contributed in characterizing chromatin associated lncRNAs, and discuss the potential mechanisms by which chromatin associated lncRNAs execute their functions., Competing Interests: The authors declare no conflict of interest.

Background: Plasma cells (PCs) are terminally differentiated B-lymphocytes producing antibodies. In coeliac disease (CeD) there is increased density of PCs in the small-intestinal lesion. Many of these PCs produce disease-specific autoantibodies targeting transglutaminase 2 (TG2)., Objective: The plasmacytosis of CeD motivated us to study the transcriptional programme of PCs from coeliac gut lesions., Methods: RNA-seq was performed on the PCs of CeD patients and disease controls, being specific or non-specific for TG2., Results: Being antibody-producing cells, 67% of the PCs' transcript was aligned to immunoglobulin genes. Strikingly, genes encoding ligands and receptors of chemokines and cytokines were abundant. Higher transcript levels of genes associated with cell activation and immune responses were observed in PCs of CeD patients compared to controls. TG2-specific compared to non-TG2 specific PCs expressed increased levels of CXCR3, CXCL10 and interleukin-15; factors that have been implicated in the pathogenesis of CeD yet with production attributed to other cells than PCs. The presence of transcripts of HLA class II and T-cell co-stimulatory molecules suggests that PCs may serve as antigen-presenting cells for CD4 + helper T cells., Conclusions: Our findings shed new light on the biology of intestinal PCs, implicating functions that go beyond the production of immunoglobulins., (© Author(s) 2019.)

EZH2 is overexpressed in poor-prognostic chronic lymphocytic leukaemia (CLL) cases, acting as an oncogene; however, thus far, the EZH2 target genes in CLL have not been disclosed. In this study, using ChIP-sequencing, we identified EZH2 and H3K27me3 target genes in two prognostic subgroups of CLL with distinct prognosis and outcome, i.e., cases with unmutated (U-CLL, n = 6) or mutated IGHV genes (M-CLL, n = 6). While the majority of oncogenic pathways were equally enriched for EZH2 target genes in both prognostic subgroups, PI3K pathway genes were differentially bound by EZH2 in U-CLL versus M-CLL. The occupancy of EZH2 for selected PI3K pathway target genes was validated in additional CLL samples (n = 16) and CLL cell lines using siRNA-mediated EZH2 downregulation and ChIP assays. Intriguingly, we found that EZH2 directly binds to the IGF1R promoter along with MYC and upregulates IGF1R expression in U-CLL, leading to downstream PI3K activation. By investigating an independent CLL cohort (n = 96), a positive correlation was observed between EZH2 and IGF1R expression with higher levels in U-CLL compared to M-CLL. Accordingly, siRNA-mediated downregulation of either EZH2, MYC or IGF1R and treatment with EZH2 and MYC pharmacological inhibitors in the HG3 CLL cell line induced a significant reduction in PI3K pathway activation. In conclusion, we characterize for the first time EZH2 target genes in CLL revealing a hitherto unknown implication of EZH2 in modulating the PI3K pathway in a non-canonical, PRC2-independent way, with potential therapeutic implications considering that PI3K inhibitors are effective therapeutic agents for CLL.

For tumors to progress efficiently, cancer cells must overcome barriers of oxidative stress. Although dietary antioxidant supplementation or activation of endogenous antioxidants by NRF2 reduces oxidative stress and promotes early lung tumor progression, little is known about its effect on lung cancer metastasis. Here, we show that long-term supplementation with the antioxidants N-acetylcysteine and vitamin E promotes KRAS-driven lung cancer metastasis. The antioxidants stimulate metastasis by reducing levels of free heme and stabilizing the transcription factor BACH1. BACH1 activates transcription of Hexokinase 2 and Gapdh and increases glucose uptake, glycolysis rates, and lactate secretion, thereby stimulating glycolysis-dependent metastasis of mouse and human lung cancer cells. Targeting BACH1 normalized glycolysis and prevented antioxidant-induced metastasis, while increasing endogenous BACH1 expression stimulated glycolysis and promoted metastasis, also in the absence of antioxidants. We conclude that BACH1 stimulates glycolysis-dependent lung cancer metastasis and that BACH1 is activated under conditions of reduced oxidative stress., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Combining HLA-DQ-gluten tetramers with mass cytometry and RNA sequencing analysis, we find that gluten-specific CD4 + T cells in the blood and intestines of patients with celiac disease display a surprisingly rare phenotype. Cells with this phenotype are also elevated in patients with systemic sclerosis and systemic lupus erythematosus, suggesting a way to characterize CD4 + T cells specific for disease-driving antigens in multiple autoimmune conditions.

Motivation: Many high-throughput methods produce sets of genomic regions as one of their main outputs. Scientists often use genomic colocalization analysis to interpret such region sets, for example to identify interesting enrichments and to understand the interplay between the underlying biological processes. Although widely used, there is little standardization in how these analyses are performed. Different practices can substantially affect the conclusions of colocalization analyses., Results: Here, we describe the different approaches and provide recommendations for performing genomic colocalization analysis, while also discussing common methodological challenges that may influence the conclusions. As illustrated by concrete example cases, careful attention to analysis details is needed in order to meet these challenges and to obtain a robust and biologically meaningful interpretation of genomic region set data., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press.)

Long non-coding RNAs (lncRNAs) can act as scaffolds that promote the interaction of proteins, RNA, and DNA. There is increasing evidence of sequence-specific interactions of lncRNAs with DNA via triple-helix (triplex) formation. This process allows lncRNAs to recruit protein complexes to specific genomic regions and regulate gene expression. Here we propose a computational method called Triplex Domain Finder (TDF) to detect triplexes and characterize DNA-binding domains and DNA targets statistically. Case studies showed that this approach can detect the known domains of lncRNAs Fendrr, HOTAIR and MEG3. Moreover, we validated a novel DNA-binding domain in MEG3 by a genome-wide sequencing method. We used TDF to perform a systematic analysis of the triplex-forming potential of lncRNAs relevant to human cardiac differentiation. We demonstrated that the lncRNA with the highest triplex-forming potential, GATA6-AS, forms triple helices in the promoter of genes relevant to cardiac development. Moreover, down-regulation of GATA6-AS impairs GATA6 expression and cardiac development. These data indicate the unique ability of our computational tool to identify novel triplex-forming lncRNAs and their target genes., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs are preferentially encoded from the genomic loci of key developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns in vivo in mouse and in human embryos. Signifying their developmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, which localizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncT depletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Adaptation to the environment and extraction of energy are essential for survival. Some species have found niches and specialized in using a particular source of energy, whereas others-including humans and several other mammals-have developed a high degree of flexibility 1 . A lot is known about the general metabolic fates of different substrates but we still lack a detailed mechanistic understanding of how cells adapt in their use of basic nutrients 2 . Here we show that the closely related fasting/starvation-induced forkhead transcription factors FOXK1 and FOXK2 induce aerobic glycolysis by upregulating the enzymatic machinery required for this (for example, hexokinase-2, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase), while at the same time suppressing further oxidation of pyruvate in the mitochondria by increasing the activity of pyruvate dehydrogenase kinases 1 and 4. Together with suppression of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 this leads to increased phosphorylation of the E1α regulatory subunit of the pyruvate dehydrogenase complex, which in turn inhibits further oxidation of pyruvate in the mitochondria-instead, pyruvate is reduced to lactate. Suppression of FOXK1 and FOXK2 induce the opposite phenotype. Both in vitro and in vivo experiments, including studies of primary human cells, show how FOXK1 and/or FOXK2 are likely to act as important regulators that reprogram cellular metabolism to induce aerobic glycolysis.

Background: Chronic lymphocytic leukemia (CLL) has been a good model system to understand the functional role of 5-methylcytosine (5-mC) in cancer progression. More recently, an oxidized form of 5-mC, 5-hydroxymethylcytosine (5-hmC) has gained lot of attention as a regulatory epigenetic modification with prognostic and diagnostic implications for several cancers. However, there is no global study exploring the role of 5-hydroxymethylcytosine (5-hmC) levels in CLL. Herein, using mass spectrometry and hMeDIP-sequencing, we analysed the dynamics of 5-hmC during B cell maturation and CLL pathogenesis., Results: We show that naïve B-cells had higher levels of 5-hmC and 5-mC compared to non-class switched and class-switched memory B-cells. We found a significant decrease in global 5-mC levels in CLL patients (n = 15) compared to naïve and memory B cells, with no changes detected between the CLL prognostic groups. On the other hand, global 5-hmC levels of CLL patients were similar to memory B cells and reduced compared to naïve B cells. Interestingly, 5-hmC levels were increased at regulatory regions such as gene-body, CpG island shores and shelves and 5-hmC distribution over the gene-body positively correlated with degree of transcriptional activity. Importantly, CLL samples showed aberrant 5-hmC and 5-mC pattern over gene-body compared to well-defined patterns in normal B-cells. Integrated analysis of 5-hmC and RNA-sequencing from CLL datasets identified three novel oncogenic drivers that could have potential roles in CLL development and progression., Conclusions: Thus, our study suggests that the global loss of 5-hmC, accompanied by its significant increase at the gene regulatory regions, constitute a novel hallmark of CLL pathogenesis. Our combined analysis of 5-mC and 5-hmC sequencing provided insights into the potential role of 5-hmC in modulating gene expression changes during CLL pathogenesis.

Trait associated single nucleotide polymorphisms often overlap with noncoding transcripts but their contribution to disease phenotype is poorly investigated. Our study on neuroblastoma risk associated 6p22.3 locus derived long noncoding RNAs (lncRNAs) demonstrates that functional co-operation between sense-antisense CASC15 and NBAT1 lncRNAs control neuroblastoma progression via regulating SOX9-CHD7-USP36 regulatory axis.