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-todate 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.

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure.

Endogenous retroviruses (ERVs) are an inherited part of the eukaryotic genomes, and represent ∼400 000 loci in the human genome. Human endogenous retroviruses (HERVs) can be divided into distinct families, composed of phylogenetically related but structurally heterogeneous elements. The majority of HERVs are silent in most physiological contexts, whereas a significant expression is observed in pathological contexts, such as cancers. Owing to their repetitive nature, few of the active HERV elements have been accurately identified. In addition, there are no criteria defining the active promoters among HERV long-terminal repeats (LTRs). Hence, it is difficult to understand the HERV (de)regulation mechanisms and their implication on the physiopathology of the host. We developed a microarray to specifically detect the LTR-containing transcripts from the HERV-H, HERV-E, HERV-W and HERV-K(HML-2) families. HERV transcriptome was analyzed in the placenta and seven normal/tumoral match-pair samples. We identified six HERV-W loci overexpressed in testicular cancer, including a usually placenta-restricted transcript of ERVWE1. For each locus, specific overexpression was confirmed by quantitative RT-PCR, and comparison of the activity of U3 versus U5 regions suggested a U3-promoted transcription coupled with 5′R initiation. The analysis of DNA from tumoral versus normal tissue revealed that hypomethylation of U3 promoters in tumors is a prerequisite for their activation.

Human endogenous retroviruses (HERVs) are globally silent in somatic cells. However, some HERVs display high transcription in physiological conditions. In particular, ERVWE1, ERVFRDE1 and ERV3, three proviruses of distinct families, are highly transcribed in placenta and produce envelope proteins associated with placenta development. As silencing of repeated elements is thought to occur mainly by DNA methylation, we compared the methylation of ERVWE1 and related HERVs to appreciate whether HERV methylation relies upon the family, the integration site, the tissue, the long terminal repeat (LTR) function or the associated gene function. CpG methylation of HERV-W LTRs in placenta-associated tissues was heterogeneous but a joint epigenetic control was found for ERVWE1 5′LTR and its juxtaposed enhancer, a mammalian apparent LTR retrotransposon. Additionally, ERVWE1, ERVFRDE1 and ERV3 5′LTRs were all essentially hypomethylated in cytotrophoblasts during pregnancy, but showed distinct and stage-dependent methylation profiles. In non-cytotrophoblastic cells, they also exhibited different methylation profiles, compatible with their respective transcriptional activities. Comparative analyses of transcriptional activity and LTR methylation in cell lines further sustained a role for methylation in the control of functional LTRs. These results suggest that HERV methylation might not be family related but copy-specific, and related to the LTR function and the tissue. In particular, ERVWE1 and ERV3 could be developmentally epigenetically regulated HERVs.

Human endogenous retroviruses (HERVs) are spread throughout the genome and their long terminal repeats (LTRs) constitute a wide collection of putative regulatory sequences. Phylogenetic similarities and the profusion of integration sites, two inherent characteristics of transposable elements, make it difficult to study individual locus expression in a large-scale approach, and historically apart from some placental and testis-regulated elements, it was generally accepted that HERVs are silent due to epigenetic control. Herein, we have introduced a generic method aiming to optimally characterize individual loci associated with 25-mer probes by minimizing cross-hybridization risks. We therefore set up a microarray dedicated to a collection of 5,573 HERVs that can reasonably be assigned to a unique genomic position. We obtained a first view of the HERV transcriptome by using a composite panel of 40 normal and 39 tumor samples. The experiment showed that almost one third of the HERV repertoire is indeed transcribed. The HERV transcriptome follows tropism rules, is sensitive to the state of differentiation and, unexpectedly, seems not to correlate with the age of the HERV families. The probeset definition within the U3 and U5 regions was used to assign a function to some LTRs (i.e. promoter or polyA) and revealed that (i) autonomous active LTRs are broadly subjected to operational determinism (ii) the cellular gene density is substantially higher in the surrounding environment of active LTRs compared to silent LTRs and (iii) the configuration of neighboring cellular genes differs between active and silent LTRs, showing an approximately 8 kb zone upstream of promoter LTRs characterized by a drastic reduction in sense cellular genes. These gathered observations are discussed in terms of virus/host adaptive strategies, and together with the methods and tools developed for this purpose, this work paves the way for further HERV transcriptome projects.

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodelling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed. However, they still present some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared to existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and emphasizes the role of transcription in the establishment of chromatin structure.RADICL-seq reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency. Application of RADICL-seq to mouse embryonic stem cells (mESCs) and mouse oligodendrocyte progenitor cells (mOPCs) reveals distinct genome occupancy patterns for specific classes of transcripts and uncovers cell-type specific RNA-chromatin interactions.Furthermore, our results highlight the role of transcription in the establishment of the threedimensional (3D) structure of chromatin. Results RADICL-seq technologyWe developed RADICL-seq by using R08, a Mus musculus male embryonic stem cell line with a deeply characterized transcriptome 11 , to identify genome-wide RNA-chromatin (or RNA-DNA) interactions in preserved nuclei (Fig. 1a). We crosslinked cells with two different formaldehyde (FA) concentrations (1% and 2%) to test whether captured interactions were dependent on the amount of crosslinking agent. After crosslinking we isolated the nuclei, partially digested the genomic DNA with DNase I and ends-prepared the chromatin. During technical development of RADICL-seq we evaluated different enzymes that specifically act on RNA to generate a 3'-hydroxyl end, compatible with RNA ligation (Supplementary Fig. 1a). Sequencing data of test RADICL-seq libraries showed that RNase H treatment increased the percentage of uniquely mapped RNA-chromatin interactions by decreasing the ribosomal RNA (rRNA) content, when compared to nuclease S1, RNase V1 or absence of treatment. RNase H enzymatic treatment is known to target RNA-DNA hybrids and, therefore, it could potentially digest nascent RNA bound to its transcription locus, including the highly transcribed rRNA. Indeed, we observed a 2.5-fold reduction in the number of RNA-DNA interactions occurring at a distance below 1 kb between RNAse H-treated and untreated samples (Supplementary Fig. 1b).After enzymatic treatment of the RNA, we introduced a bridge adapter to specifically ligate proximal RNA and DNA (Supplementary Fig. 1c). The adapter is a 5'...

Long non-coding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes and yet, their functions remain largely unknown. We systematically knockdown 285 lncRNAs expression 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 lncRNA 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 dataset with molecular phenotyping (over 1,000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

PRC2-mediated epigenetic function involves the interaction with long non-coding RNAs (lncRNAs). Although the identity of some of these RNAs has been elucidated in the context of developmental programs, their counterparts in postmitotic adult tissue homeostasis remain uncharacterized. To this aim, we used terminally differentiated postmitotic skeletal muscle cells in which oxidative stress induces the dynamic activation of PRC2-Ezh1 through Embryonic Ectoderm Develpment (EED) shuttling to the nucleus. We identify lncRNA Malat-1 as a necessary partner for PRC2-Ezh1-dependent response to oxidative stress. We show that in this pathway, PRC2-EZH1 dynamic assembly, and in turn stress induced skeletal muscle targeted genes repression, depends specifically on Malat-1. Our study reports about PRC2–RNA interactions in the physiological context of adaptive oxidative stress response and identifies the first lncRNA involved in PRC2-Ezh1 function.