Optimizing data integration improves gene regulatory network inference in Arabidopsis thaliana.

MOTIVATIONS: Gene regulatory networks (GRNs) are traditionally inferred from gene expression profiles monitoring a specific condition or treatment. In the last decade, integrative strategies have successfully emerged to guide GRN inference from gene expression with complementary prior data. However, datasets used as prior information and validation gold standards are often related and limited to a subset of genes. This lack of complete and independent evaluation calls for new criteria to robustly estimate the optimal intensity of prior data integration in the inference process. RESULTS: We address this issue for two regression-based GRN inference models, a weighted random forest (weigthedRF) and a generalized linear model estimated under a weighted LASSO penalty with stability selection (weightedLASSO). These approaches are applied to data from the root response to nitrate induction in Arabidopsis thaliana. For each gene, we measure how the integration of transcription factor binding motifs influences model prediction. We propose a new approach, DIOgene, that uses model prediction error and a simulated null hypothesis in order to optimize data integration strength in a hypothesis-driven, gene-specific manner. This integration scheme reveals a strong diversity of optimal integration intensities between genes, and offers good performance in minimizing prediction error as well as retrieving experimental interactions. Experimental results show that DIOgene compares favorably against state-of-the-art approaches and allows to recover master regulators of nitrate induction. AVAILABILITY AND IMPLEMENTATION: The R code and notebooks demonstrating the use of the proposed approaches are available in the repository https://github.com/OceaneCsn/integrative_GRN_N_induction.

Short tandem repeats are important contributors to silencer elements in T cells.

The action of cis-regulatory elements with either activation or repression functions underpins the precise regulation of gene expression during normal development and cell differentiation. Gene activation by the combined activities of promoters and distal enhancers has been extensively studied in normal and pathological contexts. In sharp contrast, gene repression by cis-acting silencers, defined as genetic elements that negatively regulate gene transcription in a position-independent fashion, is less well understood. Here, we repurpose the STARR-seq approach as a novel high-throughput reporter strategy to quantitatively assess silencer activity in mammals. We assessed silencer activity from DNase hypersensitive I sites in a mouse T cell line. Identified silencers were associated with either repressive or active chromatin marks and enriched for binding motifs of known transcriptional repressors. CRISPR-mediated genomic deletions validated the repressive function of distinct silencers involved in the repression of non-T cell genes and genes regulated during T cell differentiation. Finally, we unravel an association of silencer activity with short tandem repeats, highlighting the role of repetitive elements in silencer activity. Our results provide a general strategy for genome-wide identification and characterization of silencer elements.

HT-smFISH: a cost-effective and flexible workflow for high-throughput single-molecule RNA imaging.

The ability to visualize RNA in its native subcellular environment by using single-molecule fluorescence in situ hybridization (smFISH) has reshaped our understanding of gene expression and cellular functions. A major hindrance of smFISH is the difficulty to perform systematic experiments in medium- or high-throughput formats, principally because of the high cost of generating the individual fluorescent probe sets. Here, we present high-throughput smFISH (HT-smFISH), a simple and cost-efficient method for imaging hundreds to thousands of single endogenous RNA molecules in 96-well plates. HT-smFISH uses RNA probes transcribed in vitro from a large pool of unlabeled oligonucleotides. This allows the generation of individual probes for many RNA species, replacing commercial DNA probe sets. HT-smFISH thus reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design. We provide a protocol that combines oligo pool design, probe set generation, optimized hybridization conditions and guidelines for image acquisition and analysis. The pipeline requires knowledge of standard molecular biology tools, cell culture and fluorescence microscopy. It is achievable in ~20 d. In brief, HT-smFISH is tailored for medium- to high-throughput screens that image RNAs at single-molecule sensitivity.

Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network.

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.

Identification of long regulatory elements in the genome of Plasmodium falciparum and other eukaryotes.

Long regulatory elements (LREs), such as CpG islands, polydA:dT tracts or AU-rich elements, are thought to play key roles in gene regulation but, as opposed to conventional binding sites of transcription factors, few methods have been proposed to formally and automatically characterize them. We present here a computational approach named DExTER (Domain Exploration To Explain gene Regulation) dedicated to the identification of candidate LREs (cLREs) and apply it to the analysis of the genomes of P. falciparum and other eukaryotes. Our analyses show that all tested genomes contain several cLREs that are somewhat conserved along evolution, and that gene expression can be predicted with surprising accuracy on the basis of these long regions only. Regulation by cLREs exhibits very different behaviours depending on species and conditions. In P. falciparum and other Apicomplexan organisms as well as in Dictyostelium discoideum, the process appears highly dynamic, with different cLREs involved at different phases of the life cycle. For multicellular organisms, the same cLREs are involved in all tissues, but a dynamic behavior is observed along embryonic development stages. In P. falciparum, whose genome is known to be strongly depleted of transcription factors, cLREs are predictive of expression with an accuracy above 70%, and our analyses show that they are associated with both transcriptional and post-transcriptional regulation signals. Moreover, we assessed the biological relevance of one LRE discovered by DExTER in P. falciparum using an in vivo reporter assay. The source code (python) of DExTER is available at https://gite.lirmm.fr/menichelli/DExTER.

A choreography of centrosomal mRNAs reveals a conserved localization mechanism involving active polysome transport.

Local translation allows for a spatial control of gene expression. Here, we use high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging reveals active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysome transport mediated by nascent proteins.

Fra-1 regulates its target genes via binding to remote enhancers without exerting major control on chromatin architecture in triple negative breast cancers.

The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate individually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1.

Probing transcription factor combinatorics in different promoter classes and in enhancers.

BACKGROUND: In eukaryotic cells, transcription factors (TFs) are thought to act in a combinatorial way, by competing and collaborating to regulate common target genes. However, several questions remain regarding the conservation of these combinations among different gene classes, regulatory regions and cell types. RESULTS: We propose a new approach named TFcoop to infer the TF combinations involved in the binding of a target TF in a particular cell type. TFcoop aims to predict the binding sites of the target TF upon the nucleotide content of the sequences and of the binding affinity of all identified cooperating TFs. The set of cooperating TFs and model parameters are learned from ChIP-seq data of the target TF. We used TFcoop to investigate the TF combinations involved in the binding of 106 TFs on 41 cell types and in four regulatory regions: promoters of mRNAs, lncRNAs and pri-miRNAs, and enhancers. We first assess that TFcoop is accurate and outperforms simple PWM methods for predicting TF binding sites. Next, analysis of the learned models sheds light on important properties of TF combinations in different promoter classes and in enhancers. First, we show that combinations governing TF binding on enhancers are more cell-type specific than that governing binding in promoters. Second, for a given TF and cell type, we observe that TF combinations are different between promoters and enhancers, but similar for promoters of mRNAs, lncRNAs and pri-miRNAs. Analysis of the TFs cooperating with the different targets show over-representation of pioneer TFs and a clear preference for TFs with binding motif composition similar to that of the target. Lastly, our models accurately distinguish promoters associated with specific biological processes. CONCLUSIONS: TFcoop appears as an accurate approach for studying TF combinations. Its use on ENCODE and FANTOM data allowed us to discover important properties of human TF combinations in different promoter classes and in enhancers. The R code for learning a TFcoop model and for reproducing the main experiments described in the paper is available in an R Markdown file at address https://gite.lirmm.fr/brehelin/TFcoop .

miRNA-30 Family Members Inhibit Breast Cancer Invasion, Osteomimicry, and Bone Destruction by Directly Targeting Multiple Bone Metastasis-Associated Genes.

miRNAs are master regulators of gene expression that play key roles in cancer metastasis. During bone metastasis, metastatic tumor cells must rewire their biology and express genes that are normally expressed by bone cells (a process called osteomimicry), which endow tumor cells with full competence for outgrowth in the bone marrow. Here, we establish miR-30 family members miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e as suppressors of breast cancer bone metastasis that regulate multiple pathways, including osteomimicry. Low expression of miR-30 in primary tumors from patients with breast cancer were associated with poor relapse-free survival. In addition, estrogen receptor (ER)-negative/progesterone receptor (PR)-negative breast cancer cells expressed lower miR-30 levels than their ER/PR-positive counterparts. Overexpression of miR-30 in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivoIn vitro, miR-30 did not affect tumor cell proliferation, but did inhibit tumor cell invasion. Furthermore, overexpression of miR-30 restored bone homeostasis by reversing the effects of tumor cell-conditioned medium on osteoclastogenesis and osteoblastogenesis. A number of genes associated with osteoclastogenesis stimulation (IL8, IL11), osteoblastogenesis inhibition (DKK-1), tumor cell osteomimicry (RUNX2, CDH11), and invasiveness (CTGF, ITGA5, ITGB3) were identified as targets for repression by miR-30. Among these genes, silencing CDH11 or ITGA5 in ER-/PR-negative breast cancer cells recapitulated inhibitory effects of miR-30 on skeletal tumor burden in vivo Overall, our findings provide evidence that miR-30 family members employ multiple mechanisms to impede breast cancer bone metastasis and may represent attractive targets for therapeutic intervention.Significance: These findings suggest miR-30 family members may serve as an effective means to therapeutically attenuate metastasis in triple-negative breast cancer. Cancer Res; 78(18); 5259-73. ©2018 AACR.

Human Enhancers Harboring Specific Sequence Composition, Activity, and Genome Organization Are Linked to the Immune Response.

The FANTOM5 consortium recently characterized 65,423 human enhancers from 1829 cell and tissue samples using the Cap Analysis of Gene Expression technology. We showed that the guanine and cytosine content at enhancer regions distinguishes two classes of enhancers harboring distinct DNA structural properties at flanking regions. A functional analysis of their predicted gene targets highlighted one class of enhancers as significantly enriched for associations with immune response genes. Moreover, these enhancers were specifically enriched for regulatory motifs recognized by transcription factors involved in immune response. We observed that enhancers enriched for links to immune response genes were more cell-type specific, preferentially activated upon bacterial infection, and with specific response activity. Looking at chromatin capture data, we found that the two classes of enhancers were lying in distinct topologically associating domains and chromatin loops. Our results suggest that specific nucleotide compositions encode for classes of enhancers that are functionally distinct and specifically organized in the human genome.

Mitochondrial Complex I activity signals antioxidant response through ERK5.

Oxidative phosphorylation (OXPHOS) generates ROS as a byproduct of mitochondrial complex I activity. ROS-detoxifying enzymes are made available through the activation of their antioxidant response elements (ARE) in their gene promoters. NRF2 binds to AREs and induces this anti-oxidant response. We show that cells from multiple origins performing OXPHOS induced NRF2 expression and its transcriptional activity. The NRF2 promoter contains MEF2 binding sites and the MAPK ERK5 induced MEF2-dependent NRF2 expression. Blocking OXPHOS in a mouse model decreased Erk5 and Nrf2 expression. Furthermore, fibroblasts derived from patients with mitochondrial disorders also showed low expression of ERK5 and NRF2 mRNAs. Notably, in cells lacking functional mitochondrial complex I activity OXPHOS did not induce ERK5 expression and failed to generate this anti-oxidant response. Complex I activity induces ERK5 expression through fumarate accumulation. Eukaryotic cells have evolved a genetic program to prevent oxidative stress directly linked to OXPHOS and not requiring ROS.

Differential long non-coding RNA expression profiles in human oocytes and cumulus cells.

Progress in assisted reproductive technologies strongly relies on understanding the regulation of the dialogue between oocyte and cumulus cells (CCs). Little is known about the role of long non-coding RNAs (lncRNAs) in the human cumulus-oocyte complex (COC). To this aim, publicly available RNA-sequencing data were analyzed to identify lncRNAs that were abundant in metaphase II (MII) oocytes (BCAR4, C3orf56, TUNAR, OOEP-AS1, CASC18, and LINC01118) and CCs (NEAT1, MALAT1, ANXA2P2, MEG3, IL6STP1, and VIM-AS1). These data were validated by RT-qPCR analysis using independent oocytes and CC samples. The functions of the identified lncRNAs were then predicted by constructing lncRNA-mRNA co-expression networks. This analysis suggested that MII oocyte lncRNAs could be involved in chromatin remodeling, cell pluripotency and in driving early embryonic development. CC lncRNAs were co-expressed with genes involved in apoptosis and extracellular matrix-related functions. A bioinformatic analysis of RNA-sequencing data to identify CC lncRNAs that are affected by maternal age showed that lncRNAs with age-related altered expression in CCs are essential for oocyte growth. This comprehensive analysis of lncRNAs expressed in human MII oocytes and CCs could provide biomarkers of oocyte quality for the development of non-invasive tests to identify embryos with high developmental potential.

Probing instructions for expression regulation in gene nucleotide compositions.

Gene expression is orchestrated by distinct regulatory regions to ensure a wide variety of cell types and functions. A challenge is to identify which regulatory regions are active, what are their associated features and how they work together in each cell type. Several approaches have tackled this problem by modeling gene expression based on epigenetic marks, with the ultimate goal of identifying driving regions and associated genomic variations that are clinically relevant in particular in precision medicine. However, these models rely on experimental data, which are limited to specific samples (even often to cell lines) and cannot be generated for all regulators and all patients. In addition, we show here that, although these approaches are accurate in predicting gene expression, inference of TF combinations from this type of models is not straightforward. Furthermore these methods are not designed to capture regulation instructions present at the sequence level, before the binding of regulators or the opening of the chromatin. Here, we probe sequence-level instructions for gene expression and develop a method to explain mRNA levels based solely on nucleotide features. Our method positions nucleotide composition as a critical component of gene expression. Moreover, our approach, able to rank regulatory regions according to their contribution, unveils a strong influence of the gene body sequence, in particular introns. We further provide evidence that the contribution of nucleotide content can be linked to co-regulations associated with genome 3D architecture and to associations of genes within topologically associated domains.

The PDK1 Inhibitor Dichloroacetate Controls Cholesterol Homeostasis Through the ERK5/MEF2 Pathway.

Controlling cholesterol levels is a major challenge in human health, since hypercholesterolemia can lead to serious cardiovascular disease. Drugs that target carbohydrate metabolism can also modify lipid metabolism and hence cholesterol plasma levels. In this sense, dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, augments usage of the glycolysis-produced pyruvate in the mitochondria increasing oxidative phosphorylation (OXPHOS). In several animal models, DCA decreases plasma cholesterol and triglycerides. Thus, DCA was used in the 70 s to treat diabetes mellitus, hyperlipoproteinemia and hypercholesterolemia with satisfactory results. However, the mechanism of action remained unknown and we describe it here. DCA increases LDLR mRNA and protein levels as well as LDL intake in several cell lines, primary human hepatocytes and two different mouse models. This effect is mediated by transcriptional activation as evidenced by H3 acetylation on lysine 27 on the LDLR promoter. DCA induces expression of the MAPK ERK5 that turns on the transcription factor MEF2. Inhibition of this ERK5/MEF2 pathway by genetic or pharmacological means decreases LDLR expression and LDL intake. In summary, our results indicate that DCA, by inducing OXPHOS, promotes ERK5/MEF2 activation leading to LDLR expression. The ERK5/MEF2 pathway offers an interesting pharmacological target for drug development.

An integrated expression atlas of miRNAs and their promoters in human and mouse.

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.

miR-125b controls monocyte adaptation to inflammation through mitochondrial metabolism and dynamics.

Metabolic changes drive monocyte differentiation and fate. Although abnormal mitochondria metabolism and innate immune responses participate in the pathogenesis of many inflammatory disorders, molecular events regulating mitochondrial activity to control life and death in monocytes remain poorly understood. We show here that, in human monocytes, microRNA-125b (miR-125b) attenuates the mitochondrial respiration through the silencing of the BH3-only proapoptotic protein BIK and promotes the elongation of the mitochondrial network through the targeting of the mitochondrial fission process 1 protein MTP18, leading to apoptosis. Proinflammatory activation of monocyte-derived macrophages is associated with a concomitant increase in miR-125b expression and decrease in BIK and MTP18 expression, which lead to reduced oxidative phosphorylation and enhanced mitochondrial fusion. In a chronic inflammatory systemic disorder, CD14+ blood monocytes display reduced miR-125b expression as compared with healthy controls, inversely correlated with BIK and MTP18 messenger RNA expression. Our findings not only identify BIK and MTP18 as novel targets for miR-125b that control mitochondrial metabolism and dynamics, respectively, but also reveal a novel function for miR-125b in regulating metabolic adaptation of monocytes to inflammation. Together, these data unravel new molecular mechanisms for a proapoptotic role of miR-125b in monocytes and identify potential targets for interfering with excessive inflammatory activation of monocytes in inflammatory disorders.

Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS) Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS) Production.

Tumor cell metabolism is altered during leukemogenesis. Cells performing oxidative phosphorylation (OXPHOS) generate reactive oxygen species (ROS) through mitochondrial activity. To limit the deleterious effects of excess ROS, certain gene promoters contain antioxidant response elements (ARE), e.g. the genes NQO-1 and HO-1. ROS induces conformational changes in KEAP1 and releases NRF2, which activates AREs. We show in vitro and in vivo that OXPHOS induces, both in primary leukemic cells and cell lines, de novo expression of NQO-1 and HO-1 and also the MAPK ERK5 and decreases KEAP1 mRNA. ERK5 activates the transcription factor MEF2, which binds to the promoter of the miR-23a-27a-24-2 cluster. Newly generated miR-23a destabilizes KEAP1 mRNA by binding to its 3'UTR. Lower KEAP1 levels increase the basal expression of the NRF2-dependent genes NQO-1 and HO-1. Hence, leukemic cells performing OXPHOS, independently of de novo ROS production, generate an antioxidant response to protect themselves from ROS.

microRNAs and Personalized Medicine: Evaluating Their Potential as Cancer Biomarkers.

microRNA deregulations are often, if not invariably, associated with human malignancies, including cancers. Though most of these deregulations may not be functionally implicated in tumorigenesis, the fact that microRNA expression can be monitored in a variety of human specimens, including biological fluids, supports studies aimed at characterizing microRNA signatures able to detect various cancers (diagnosis), predict their outcome (prognosis), monitor their treatment (theranosis), and adapt therapy to a patient (precision medicine). Here, we review and discuss pros and cons of microRNA-based approaches that can support their exploitation as cancer biomarkers.

Female aging alters expression of human cumulus cells genes that are essential for oocyte quality.

Impact of female aging is an important issue in human reproduction. There was a need for an extensive analysis of age impact on transcriptome profile of cumulus cells (CCs) to link oocyte quality and developmental potential with patient's age. CCs from patients of three age groups were analyzed individually using microarrays. RT-qPCR validation was performed on independent CC cohorts. We focused here on pathways affected by aging in CCs that may explain the decline of oocyte quality with age. In CCs collected from patients >37 years, angiogenic genes including ANGPTL4, LEPR, TGFBR3, and FGF2 were significantly overexpressed compared to patients of the two younger groups. In contrast genes implicated in TGF-ß signaling pathway such as AMH, TGFB1, inhibin, and activin receptor were underexpressed. CCs from patients whose ages are between 31 and 36 years showed an overexpression of genes related to insulin signaling pathway such as IGFBP3, PIK3R1, and IGFBP5. A bioinformatic analysis was performed to identify the microRNAs that are potential regulators of the differentially expressed genes of the study. It revealed that the pathways impacted by age were potential targets of specific miRNAs previously identified in our CCs small RNAs sequencing.