LATS1 controls CTCF chromatin occupancy and hormonal response of 3D-grown breast cancer cells.

The cancer epigenome has been studied in cells cultured in two-dimensional (2D) monolayers, but recent studies highlight the impact of the extracellular matrix and the three-dimensional (3D) environment on multiple cellular functions. Here, we report the physical, biochemical, and genomic differences between T47D breast cancer cells cultured in 2D and as 3D spheroids. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, as well as altered expression of ~2000 genes, the majority of which become repressed. Hi-C analysis reveals that cells in 3D are enriched for regions belonging to the B compartment, have decreased chromatin-bound CTCF and increased fusion of topologically associating domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, thereby likely causing the observed TAD fusions. 3D cells show higher chromatin binding of progesterone receptor (PR), leading to an increase in the number of hormone-regulated genes. This effect is in part mediated by LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.

LCOR mediates interferon-independent tumor immunogenicity and responsiveness to immune-checkpoint blockade in triple-negative breast cancer.

Ligand-dependent corepressor (LCOR) mediates normal and malignant breast stem cell differentiation. Cancer stem cells (CSCs) generate phenotypic heterogeneity and drive therapy resistance, yet their role in immunotherapy is poorly understood. Here we show that immune-checkpoint blockade (ICB) therapy selects for LCORlow CSCs with reduced antigen processing/presentation machinery (APM) driving immune escape and ICB resistance in triple-negative breast cancer (TNBC). We unveil an unexpected function of LCOR as a master transcriptional activator of APM genes binding to IFN-stimulated response elements (ISREs) in an IFN signaling-independent manner. Through genetic modification of LCOR expression, we demonstrate its central role in modulation of tumor immunogenicity and ICB responsiveness. In TNBC, LCOR associates with ICB clinical response. Importantly, extracellular vesicle (EV) Lcor-messenger RNA therapy in combination with anti-PD-L1 overcame resistance and eradicated breast cancer metastasis in preclinical models. Collectively, these data support LCOR as a promising target for enhancement of ICB efficacy in TNBC, by boosting of tumor APM independently of IFN.

Coordinated post-transcriptional control of oncogene-induced senescence by UNR/CSDE1.

Oncogene-induced senescence (OIS) is a form of stable cell-cycle arrest arising in response to oncogenic stimulation. OIS must be bypassed for transformation, but the mechanisms of OIS establishment and bypass remain poorly understood, especially at the post-transcriptional level. Here, we show that the RNA-binding protein UNR/CSDE1 enables OIS in primary mouse keratinocytes. Depletion of CSDE1 leads to senescence bypass, cell immortalization, and tumor formation, indicating that CSDE1 behaves as a tumor suppressor. Unbiased high-throughput analyses uncovered that CSDE1 promotes OIS by two independent molecular mechanisms: enhancement of the stability of senescence-associated secretory phenotype (SASP) factor mRNAs and repression of Ybx1 mRNA translation. Importantly, depletion of YBX1 from immortal keratinocytes rescues senescence and uncouples proliferation arrest from the SASP, revealing multilayered mechanisms exerted by CSDE1 to coordinate senescence. Our data highlight the relevance of post-transcriptional control in the regulation of senescence.

Distinct roles for PARP-1 and PARP-2 in c-Myc-driven B-cell lymphoma in mice.

Dysregulation of the c-Myc oncogene occurs in a wide variety of hematologic malignancies, and its overexpression has been linked with aggressive tumor progression. Here, we show that poly (ADP-ribose) polymerase 1 (PARP-1) and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphoma. PARP-1 and PARP-2 catalyze the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc-driven B-cell lymphoma, whereas PARP-1 deficiency accelerates lymphomagenesis in the Eµ-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in preleukemic Eµ-Myc B cells, resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc-driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1 deficiency induces a proinflammatory response and an increase in regulatory T cells, likely contributing to immune escape of B-cell lymphoma, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc-driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centered therapeutic strategies, with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc-driven tumors.

Environmental Enrichment Induces Epigenomic and Genome Organization Changes Relevant for Cognition.

In early development, the environment triggers mnemonic epigenomic programs resulting in memory and learning experiences to confer cognitive phenotypes into adulthood. To uncover how environmental stimulation impacts the epigenome and genome organization, we used the paradigm of environmental enrichment (EE) in young mice constantly receiving novel stimulation. We profiled epigenome and chromatin architecture in whole cortex and sorted neurons by deep-sequencing techniques. Specifically, we studied chromatin accessibility, gene and protein regulation, and 3D genome conformation, combined with predicted enhancer and chromatin interactions. We identified increased chromatin accessibility, transcription factor binding including CTCF-mediated insulation, differential occupancy of H3K36me3 and H3K79me2, and changes in transcriptional programs required for neuronal development. EE stimuli led to local genome re-organization by inducing increased contacts between chromosomes 7 and 17 (inter-chromosomal). Our findings support the notion that EE-induced learning and memory processes are directly associated with the epigenome and genome organization.

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

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

Transcriptomic microRNA Profiling of Dendritic Cells in Response to Gut Microbiota-Secreted Vesicles.

The interconnection between nutrients, metabolites and microbes is a key factor governing the healthy/pathological status of an individual. Thus, microbiota-based research is essential in order to better understand human health and nutrition. Gut bacteria release membrane vesicles (MVs) as an intercellular communication mechanism that allows the direct delivery of factors that prime the host's innate immune system. We have previously shown that MVs from intestinal E. coli activate dendritic cells (DCs) in a strain-specific manner. To gain insights into the regulatory mechanisms involved, here, we have used an RNA deep sequencing approach to identify differentially expressed miRNAs (microRNAs) in DCs which are challenged by the MVs of the probiotic Nissle 1917 (EcN) or the commensal ECOR12. MicroRNAs are post-transcriptional regulatory mediators that permit the fine tuning of signaling pathways. This approach allowed the identification of a common set of miRNAs which are modulated by MVs from both strains and miRNAs which are differentially expressed in response to EcN or ECOR12 MVs. Based on the differential expression of the target genes and subsequent validation experiments, we correlated some of the selected miRNAs with the reported cytokine profile and specific T cell responses. As far as we know, this is the first study to analyze the regulation of miRNAs in DCs by MVs released by gut microbiota.

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

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

Genome Sequence of the Yeast Saprochaete ingens CBS 517.90.

Chromosome-scale genome assembly of the yeast Saprochaete ingens CBS 517.90 was determined by a combination of technologies producing short (HiSeq X; Illumina) and long (MinION; Oxford Nanopore Technologies) reads. The 21.2-Mbp genome sequence has a GC content of 36.9% and codes for 6,475 predicted proteins.

EV-associated miRNAs from pleural lavage as potential diagnostic biomarkers in lung cancer.

Lung cancer is the leading cause of cancer-related deaths among men and women in the world, accounting for the 25% of cancer mortality. Early diagnosis is an unmet clinical issue. In this work, we focused to develop a novel approach to identify highly sensitive and specific biomarkers by investigating the use of extracellular vesicles (EVs) isolated from the pleural lavage, a proximal fluid in lung cancer patients, as a source of potential biomarkers. We isolated EVs by ultracentrifuge method from 25 control pleural fluids and 21 pleural lavages from lung cancer patients. Analysis of the expression of EV-associated miRNAs was performed using Taqman OpenArray technology through which we could detect 288 out of the 754 miRNAs that were contained in the OpenArray. The differential expression analysis yielded a list of 14 miRNAs that were significantly dysregulated (adj. p-value < 0.05 and logFC lower or higher than 3). Using Machine Learning approach we discovered the lung cancer diagnostic biomarkers; miRNA-1-3p, miRNA-144-5p and miRNA-150-5p were found to be the best by accuracy. Accordance with our finding, these miRNAs have been related to cancer processes in previous studies. This results opens the avenue to the use of EV-associated miRNA of pleural fluids and lavages as an untapped source of biomarkers, and specifically, identifies miRNA-1-3p, miRNA-144-5p and miRNA 150-5p as promising biomarkers of lung cancer diagnosis.

EV-Associated miRNAs from Peritoneal Lavage are a Source of Biomarkers in Endometrial Cancer.

Endometrial cancer (EC) is the sixth most common cancer in women worldwide and is responsible for more than 89,000 deaths every year. Mortality is associated with presence of poor prognostic factors at diagnosis, i.e., diagnosis at an advanced stage, with a high grade and/or an aggressive histology. Development of novel approaches that would permit us to improve the clinical management of EC patients is an unmet need. In this study, we investigate a novel approach to identify highly sensitive and specific biomarkers of EC using extracellular vesicles (EVs) isolated from the peritoneal lavage of EC patients. EVs of peritoneal lavages of 25 EC patients were isolated and their miRNA content was compared with miRNAs of EVs isolated from the ascitic fluid of 25 control patients. Expression of the EV-associated miRNAs was measured using the Taqman OpenArray technology that allowed us to detect 371 miRNAs. The analysis showed that 114 miRNAs were significantly dysregulated in EC patients, among which eight miRNAs, miRNA-383-5p, miRNA-10b-5p, miRNA-34c-3p, miRNA-449b-5p, miRNA-34c-5p, miRNA-200b-3p, miRNA-2110, and miRNA-34b-3p, demonstrated a classification performance at area under the receiver operating characteristic curve (AUC) values above 0.9. This finding opens an avenue for the use of EV-associated miRNAs of peritoneal lavages as an untapped source of biomarkers for EC.

EV-associated miRNAs from peritoneal lavage as potential diagnostic biomarkers in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer-related mortality worldwide. Current systematic methods for diagnosing have inherent limitations so development of a minimally-invasive diagnosis, based on the identification of sensitive biomarkers in liquid biopsies could therefore facilitate screening among population at risk. METHODS: In this study, we aim to develop a novel approach to identify highly sensitive and specific biomarkers by investigating the use of extracellular vesicles (EVs) isolated from the peritoneal lavage as a source of potential miRNA diagnostic biomarkers. We isolated EVs by ultracentrifugation from 25 ascitic fluids and 25 peritoneal lavages from non-cancer and CRC patients, respectively. Analysis of the expression of EV-associated miRNAs was performed using Taqman OpenArray technology through which we could detect 371 miRNAs. RESULTS: 210 miRNAs were significantly dysregulated (adjusted p value < 0.05 and abs(logFC) ≥ 1). The top-10 miRNAs, which had the AUC value higher than 0.95, were miRNA-199b-5p, miRNA-150-5p, miRNA-29c-5p, miRNA-218-5p, miRNA-99a-3p, miRNA-383-5p, miRNA-199a-3p, miRNA-193a-5p, miRNA-10b-5p and miRNA-181c-5p. CONCLUSIONS: This finding opens the avenue to the use of EV-associated miRNA of peritoneal lavages as an untapped source of biomarkers for CRC.

Genome Sequence of Flavor-Producing Yeast Saprochaete suaveolens NRRL Y-17571.

Saprochaete suaveolens is an ascomycetous yeast that produces a range of fruity flavors and fragrances. Here, we report the high-contiguity genome sequence of the ex-holotype strain, NRRL Y-17571 (CBS 152.25). The nuclear genome sequence contains 24.4 Mbp and codes for 8,119 predicted proteins.

Wnt/ß-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) are pluripotent and can differentiate into cells belonging to the three germ layers of the embryo. However, mESC pluripotency and genome stability can be compromised in prolonged in vitro culture conditions. Several factors control mESC pluripotency, including Wnt/ß-catenin signaling pathway, which is essential for mESC differentiation and proliferation. Here we show that the activity of the Wnt/ß-catenin signaling pathway safeguards normal DNA methylation of mESCs. The activity of the pathway is progressively silenced during passages in culture and this results into a loss of the DNA methylation at many imprinting control regions (ICRs), loss of recruitment of chromatin repressors, and activation of retrotransposons, resulting into impaired mESC differentiation. Accordingly, sustained Wnt/ß-catenin signaling maintains normal ICR methylation and mESC homeostasis and is a key regulator of genome stability.

Publisher Correction: Purification of replicating pancreatic ß-cells for gene expression studies.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Purification of replicating pancreatic ß-cells for gene expression studies.

ß-cell proliferation is a rare event in adult pancreatic islets. To study the replication-related ß-cell biology we designed a replicating ß-cells sorting system for gene expression experiments. Replicating ß-cells were identified by EdU incorporation and purified by flow cytometry. For ß-cell separation islet cells were sorted by size, granularity and Newport Green fluorescence emission that was combined with emitted fluorescence for EdU-labelled replicating cells sorting. The purity of the resulting sorted populations was evaluated by insulin staining and EdU for ß-cell identification and for replicating cells, respectively. Total RNA was isolated from purified cell-sorted populations for gene expression analysis. Cell sorting of dispersed islet cells resulted in 96.2% purity for insulin positivity in the collected ß-cell fraction and 100% efficiency of the EdU-based cell separation. RNA integrity was similar between FACS-sorted replicating and quiescent ß-cells. Global transcriptome analysis of replicating vs quiescent ß-cells showed the expected enrichment of categories related to cell division and DNA replication. Indeed, key genes in the spindle check-point were the most upregulated genes in replicating ß-cells. This work provides a method that allows for the isolation of replicating ß-cells, a very scarce population in adult pancreatic islets.

Gene expression changes in blood RNA after swimming in a chlorinated pool.

Exposure to disinfection by-products (DBP) such as trihalomethanes (THM) in swimming pools has been linked to adverse health effects in humans, but their biological mechanisms are unclear. We evaluated short-term changes in blood gene expression of adult recreational swimmers after swimming in a chlorinated pool. Volunteers swam 40min in an indoor chlorinated pool. Blood samples were drawn and four THM (chloroform, bromodichloromethane, dibromochloromethane and bromoform) were measured in exhaled breath before and after swimming. Intensity of physical activity was measured as metabolic equivalents (METs). Gene expression in whole blood mRNA was evaluated using IlluminaHumanHT-12v3 Expression-BeadChip. Linear mixed models were used to evaluate the relationship between gene expression changes and THM exposure. Thirty-seven before-after pairs were analyzed. The median increase from baseline to after swimming were: 0.7 to 2.3 for MET, and 1.4 to 7.1µg/m3 for exhaled total THM (sum of the four THM). Exhaled THM increased on average 0.94µg/m3 per 1 MET. While 1643 probes were differentially expressed post-exposure. Of them, 189 were also associated with exhaled levels of individual/total THM or MET after False Discovery Rate. The observed associations with the exhaled THM were low to moderate (Log-fold change range: -0.17 to 0.15). In conclusion, we identified short-term gene expression changes associated with swimming in a pool that were minor in magnitude and their biological meaning was unspecific. The high collinearity between exhaled THM levels and intensity of physical activity precluded mutually adjusted models with both covariates. These exploratory results should be validated in future studies.

From PTEN loss of expression to RICTOR role in smooth muscle differentiation: complex involvement of the mTOR pathway in leiomyosarcomas and pleomorphic sarcomas.

Over the past decade, comprehensive genomic studies demonstrated that leiomyosarcomas and most of the tumors previously labeled as 'malignant fibrous histiocytomas' share complex karyotypes and genomic profiles, and can be referred to as 'sarcomas with complex genomics'. We recently reported a series of 160 sarcomas with complex genomics such as leiomyosarcomas, myxofibrosarcomas, pleomorphic liposarcomas/rhabdomyosarcomas and undifferentiated pleomorphic sarcomas. These tumors present with a frequent loss of chromosome 10 region encompassing the tumor suppressor gene PTEN. In the present study, we assessed PTEN genomic level and protein expression in this large series of sarcomas with complex genomics, as well as activation of downstream pathways. PTEN partial genomic loss was observed in only 46% of tumors, especially in well-differentiated leiomyosarcomas, whereas up to 68% of these tumors demonstrate a loss of protein expression on western blot analysis. Specific discrepancies in PTEN immunohistochemical results suggested bias in this latter technique. PTEN mutations were rare, with only 4 point mutations in the 65 samples studied. Subsequent activation of AKT and mTOR pathways was only observed in 2 out of 3 of PTEN-deleted tumors. On the other hand, RICTOR, a major component of the mTOR complex 2, was significantly overexpressed in well-differentiated leiomyosarcomas. These results, confirmed on tissue micro-array immunohistochemical analysis of 459 sarcomas, could suggest a link between RICTOR overexpression and leiomyosarcomas oncogenesis. As therapeutics directed against the mTOR pathway are assessed in sarcomas, RICTOR overexpression in sarcomas and its links to therapeutic response need to be assessed.

TEQC: an R package for quality control in target capture experiments.

UNLABELLED: TEQC is an R/Bioconductor package for quality assessment of target enrichment experiments. Quality measures comprise specificity and sensitivity of the capture, enrichment, per-target read coverage and its relation to hybridization probe characteristics, coverage uniformity and reproducibility, and read duplicate analysis. Several diagnostic plots allow visual inspection of the data quality. AVAILABILITY AND IMPLEMENTATION: TEQC is implemented in the R language (version >2.12.0) and is available as a Bioconductor package for Linux, Windows and MacOS from www.bioconductor.org.

New insights in sarcoma oncogenesis: a comprehensive analysis of a large series of 160 soft tissue sarcomas with complex genomics.

Adult soft tissue sarcomas (STS) are rare tumours of mesenchymal lineage. Based on cytogenetic and comparative genomic hybridization (CGH) data, they can be divided into 'STS with simple genomics', displaying a characteristic genetic alteration, and 'STS with complex genomics' (SCG), where multiple genomic alterations occur. This latter group is mostly composed of leiomyosarcomas (LMS) and pleiomorphic undifferentiated tumours previously labelled as 'malignant fibrous histiocytomas' (MFH), corresponding in fact to myxofibrosarcomas (MFS), pleiomorphic liposarcomas/rhabdomyosarcomas (P-LPS, P-RMS), and undifferentiated pleiomorphic sarcomas (UPS). Their pathobiology is still not well understood, leading to challenges in diagnosis and therapeutic management. We report here a comprehensive study encompassing array-CGH and transcriptome analysis data of a large series of 160 SCG. Non-supervised clustering of transcriptome data led to the identification of five groups of tumours, one of them (group A) corresponding to well-differentiated LMS and the other four (B-E) to 'MFH' and poorly differentiated LMS. Welch analysis of transcriptome data in these groups allowed us to retrieve several genes of potential interest. Among them, RB1 alteration is a constant thread in SCG, often associated with RBL2 loss. PTEN tumour suppressor deletion would also stand out as a major recurrent event, especially in groups A, C, and D. The WNT canonical pathway could be potentially involved, as demonstrated by up-regulation of one of its inhibitors, DKK1, in groups D and E, whereas DKK1 is significantly down-regulated in groups A, B, and C. These data suggest a very complex interplay between pathways downstream of PTEN and the WNT canonical pathway, providing new hints about SCG pathobiology and their potential therapeutic targets.