Functional annotation of human long noncoding RNAs via molecular phenotyping.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

FANTOM enters 20th year: expansion of transcriptomic atlases and functional annotation of non-coding RNAs.

The Functional ANnoTation Of the Mammalian genome (FANTOM) Consortium has continued to provide extensive resources in the pursuit of understanding the transcriptome, and transcriptional regulation, of mammalian genomes for the last 20 years. To share these resources with the research community, the FANTOM web-interfaces and databases are being regularly updated, enhanced and expanded with new data types. In recent years, the FANTOM Consortium's efforts have been mainly focused on creating new non-coding RNA datasets and resources. The existing FANTOM5 human and mouse miRNA atlas was supplemented with rat, dog, and chicken datasets. The sixth (latest) edition of the FANTOM project was launched to assess the function of human long non-coding RNAs (lncRNAs). From its creation until 2020, FANTOM6 has contributed to the research community a large dataset generated from the knock-down of 285 lncRNAs in human dermal fibroblasts; this is followed with extensive expression profiling and cellular phenotyping. Other updates to the FANTOM resource includes the reprocessing of the miRNA and promoter atlases of human, mouse and chicken with the latest reference genome assemblies. To facilitate the use and accessibility of all above resources we further enhanced FANTOM data viewers and web interfaces. The updated FANTOM web resource is publicly available at https://fantom.gsc.riken.jp/.

Meet-U: Educating through research immersion.

We present a new educational initiative called Meet-U that aims to train students for collaborative work in computational biology and to bridge the gap between education and research. Meet-U mimics the setup of collaborative research projects and takes advantage of the most popular tools for collaborative work and of cloud computing. Students are grouped in teams of 4-5 people and have to realize a project from A to Z that answers a challenging question in biology. Meet-U promotes "coopetition," as the students collaborate within and across the teams and are also in competition with each other to develop the best final product. Meet-U fosters interactions between different actors of education and research through the organization of a meeting day, open to everyone, where the students present their work to a jury of researchers and jury members give research seminars. This very unique combination of education and research is strongly motivating for the students and provides a formidable opportunity for a scientific community to unite and increase its visibility. We report on our experience with Meet-U in two French universities with master's students in bioinformatics and modeling, with protein-protein docking as the subject of the course. Meet-U is easy to implement and can be straightforwardly transferred to other fields and/or universities. All the information and data are available at www.meet-u.org.

SkewC: Identifying cells with skewed gene body coverage in single-cell RNA sequencing data.

The analysis and interpretation of single-cell RNA sequencing (scRNA-seq) experiments are compromised by the presence of poor-quality cells. For meaningful analyses, such poor-quality cells should be excluded as they introduce noise in the data. We introduce SkewC, a quality-assessment tool, to identify skewed cells in scRNA-seq experiments. The tool's methodology is based on the assessment of gene coverage for each cell, and its skewness as a quality measure; the gene body coverage is a unique characteristic for each protocol, and different protocols yield highly different coverage profiles. This tool is designed to avoid misclustering or false clusters by identifying, isolating, and removing cells with skewed gene body coverage profiles. SkewC is capable of processing any type of scRNA-seq dataset, regardless of the protocol. We envision SkewC as a distinctive QC method to be incorporated into scRNA-seq QC processing to preclude the possibility of scRNA-seq data misinterpretation.

Automatic identification of small molecules that promote cell conversion and reprogramming.

Controlling cell fate has great potential for regenerative medicine, drug discovery, and basic research. Although transcription factors are able to promote cell reprogramming and transdifferentiation, methods based on their upregulation often show low efficiency. Small molecules that can facilitate conversion between cell types can ameliorate this problem working through safe, rapid, and reversible mechanisms. Here, we present DECCODE, an unbiased computational method for identification of such molecules based on transcriptional data. DECCODE matches a large collection of drug-induced profiles for drug treatments against a large dataset of primary cell transcriptional profiles to identify drugs that either alone or in combination enhance cell reprogramming and cell conversion. Extensive validation in the context of human induced pluripotent stem cells shows that DECCODE is able to prioritize drugs and drug combinations enhancing cell reprogramming. We also provide predictions for cell conversion with single drugs and drug combinations for 145 different cell types.

miR200-regulated CXCL12ß promotes fibroblast heterogeneity and immunosuppression in ovarian cancers.

High-grade serous ovarian cancers (HGSOC) have been subdivided into molecular subtypes. The mesenchymal HGSOC subgroup, defined by stromal-related gene signatures, is invariably associated with poor patient survival. We demonstrate that stroma exerts a key function in mesenchymal HGSOC. We highlight stromal heterogeneity in HGSOC by identifying four subsets of carcinoma-associated fibroblasts (CAF-S1-4). Mesenchymal HGSOC show high content in CAF-S1 fibroblasts, which exhibit immunosuppressive functions by increasing attraction, survival, and differentiation of CD25+FOXP3+ T lymphocytes. The beta isoform of the CXCL12 chemokine (CXCL12ß) specifically accumulates in the immunosuppressive CAF-S1 subset through a miR-141/200a dependent-mechanism. Moreover, CXCL12ß expression in CAF-S1 cells plays a crucial role in CAF-S1 immunosuppressive activity and is a reliable prognosis factor in HGSOC, in contrast to CXCL12α. Thus, our data highlight the differential regulation of the CXCL12α and CXCL12ß isoforms in HGSOC, and reveal a CXCL12ß-associated stromal heterogeneity and immunosuppressive environment in mesenchymal HGSOC.

Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer.

Carcinoma-associated fibroblasts (CAF) are key players in the tumor microenvironment. Here, we characterize four CAF subsets in breast cancer with distinct properties and levels of activation. Two myofibroblastic subsets (CAF-S1, CAF-S4) accumulate differentially in triple-negative breast cancers (TNBC). CAF-S1 fibroblasts promote an immunosuppressive environment through a multi-step mechanism. By secreting CXCL12, CAF-S1 attracts CD4+CD25+ T lymphocytes and retains them by OX40L, PD-L2, and JAM2. Moreover, CAF-S1 increases T lymphocyte survival and promotes their differentiation into CD25HighFOXP3High, through B7H3, CD73, and DPP4. Finally, in contrast to CAF-S4, CAF-S1 enhances the regulatory T cell capacity to inhibit T effector proliferation. These data are consistent with FOXP3+ T lymphocyte accumulation in CAF-S1-enriched TNBC and show how a CAF subset contributes to immunosuppression.

Chronic oxidative stress promotes H2AX protein degradation and enhances chemosensitivity in breast cancer patients.

Anti-cancer drugs often increase reactive oxygen species (ROS) and cause DNA damage. Here, we highlight a new cross talk between chronic oxidative stress and the histone variant H2AX, a key player in DNA repair. We observe that persistent accumulation of ROS, due to a deficient JunD-/Nrf2-antioxidant response, reduces H2AX protein levels. This effect is mediated by an enhanced interaction of H2AX with the E3 ubiquitin ligase RNF168, which is associated with H2AX poly-ubiquitination and promotes its degradation by the proteasome. ROS-mediated H2AX decrease plays a crucial role in chemosensitivity. Indeed, cycles of chemotherapy that sustainably increase ROS reduce H2AX protein levels in Triple-Negative breast cancer (TNBC) patients. H2AX decrease by such treatment is associated with an impaired NRF2-antioxidant response and is indicative of the therapeutic efficiency and survival of TNBC patients. Thus, our data describe a novel ROS-mediated regulation of H2AX turnover, which provides new insights into genetic instability and treatment efficacy in TNBC patients.

miR-141 and miR-200a act on ovarian tumorigenesis by controlling oxidative stress response.

Although there is evidence that redox regulation has an essential role in malignancies, its impact on tumor prognosis remains unclear. Here we show crosstalk between oxidative stress and the miR-200 family of microRNAs that affects tumorigenesis and chemosensitivity. miR-141 and miR-200a target p38α and modulate the oxidative stress response. Enhanced expression of these microRNAs mimics p38α deficiency and increases tumor growth in mouse models, but it also improves the response to chemotherapeutic agents. High-grade human ovarian adenocarcinomas that accumulate miR-200a have low concentrations of p38α and an associated oxidative stress signature. The miR200a-dependent stress signature correlates with improved survival of patients in response to treatment. Therefore, the role of miR-200a in stress could be a predictive marker for clinical outcome in ovarian cancer. In addition, although oxidative stress promotes tumor growth, it also sensitizes tumors to treatment, which could account for the limited success of antioxidants in clinical trials.

Oxidative stress promotes myofibroblast differentiation and tumour spreading.

JunD regulates genes involved in antioxidant defence. We took advantage of the chronic oxidative stress resulting from junD deletion to examine the role of reactive oxygen species (ROS) in tumour development. In a model of mammary carcinogenesis, junD inactivation increased tumour incidence and revealed an associated reactive stroma. junD-inactivation in the stroma was sufficient to shorten tumour-free survival rate and enhance metastatic spread. ROS promoted conversion of fibroblasts into highly migrating myofibroblasts through accumulation of the hypoxia-inducible factor (HIF)-1alpha transcription factor and the CXCL12 chemokine. Accordingly, treatment with an antioxidant reduced the levels of HIF and CXCL12 and numerous myofibroblast features. CXCL12 accumulated in the stroma of HER2-human breast adenocarcinomas. Moreover, HER2 tumours exhibited a high proportion of myofibroblasts, which was significantly correlated to nodal metastases. Interestingly, this subset of tumours exhibited a significant nuclear exclusion of JunD and revealed an associated oxido-reduction signature, further demonstrating the relevance of our findings in human cancers. Collectively, our data uncover a new mechanism by which oxidative stress increases the migratory properties of stromal fibroblasts, which in turn potentiate tumour dissemination.