VlincRNAs controlled by retroviral elements are a hallmark of pluripotency and cancer

Background The function of the non-coding portion of the human genome remains one of the most important questions of our time. Its vast complexity is exemplified by the recent identification of an unusual and notable component of the transcriptome - very long intergenic non-coding RNAs, termed vlincRNAs. Results Here we identify 2,147 vlincRNAs covering 10 percent of our genome. We show they are present not only in cancerous cells, but also in primary cells and normal human tissues, and are controlled by canonical promoters. Furthermore, vlincRNA promoters frequently originate from within endogenous retroviral sequences. Strikingly, the number of vlincRNAs expressed from endogenous retroviral promoters strongly correlates with pluripotency or the degree of malignant transformation. These results suggest a previously unknown connection between the pluripotent state and cancer via retroviral repeat-driven expression of vlincRNAs. Finally, we show that vlincRNAs can be syntenically conserved in humans and mouse and their depletion using RNAi can cause apoptosis in cancerous cells. Conclusions These intriguing observations suggest that vlincRNAs could create a framework that combines many existing short ESTs and lincRNAs into a landscape of very long transcripts functioning in the regulation of gene expression in the nucleus. Certain types of vlincRNAs participate at specific stages of normal development and, based on analysis of a limited set of cancerous and primary cell lines, they appear to be co-opted by cancer-associated transcriptional programs. This provides additional understanding of transcriptome regulation during the malignant state, and could lead to additional targets and options for its reversal

Identification de très longs ARN non codants ou vlincRNA régulés au cours de la sénescence et caractérisation d'un vlincRNA nommé VAD requis pour le maintien de la sénescence

La sénescence est un mécanisme anti-tumoral qui conduit à un arrêt stable et irréversible de la prolifération cellulaire. Ce processus est caractérisé par des changements majeurs de la structure chromatinienne, avec l'apparition de foyers d'hétérochromatine, appelés SAHF (Senescence Associated Heterochromatin Foci) dans lesquels les gènes prolifératifs sont réprimés. Les ARN non codants (ARNnc) sont des acteurs majeurs de la structure chromatinienne, notamment au cours de l'assemblage de l'hétérochromatine péricentrique chez les mammifères. Notre hypothèse de travail est que les ARNnc pourraient être impliqués dans la mise en place des SAHF et plus généralement dans le processus de sénescence. Afin de caractériser le rôle des ARNnc dans la sénescence, nous avons analysé les modifications du transcriptome dans un modèle de sénéscence induite par l'oncogène RAF1. Une analyse à grande échelle a été réalisée de façon brin spécifique (par tiling array) sur les chromosomes 1 & 6 dans les cellules proliférantes et sénescentes. La comparaison des profils obtenus a permis d'identifier un certain nombre de transcrits différentiellement exprimés au cours de la sénescence. Étonnamment, alors que les ARN différentiellement exprimés lors de l'induction de la sénescence sont majoritairement réprimés, les ARNnc appartenant à la classe récemment décrite des vlincRNA (very long intergenic non-coding (>50kb)) sont principalement activés, suggérant qu'ils puissent être impliqués dans la mise en place du processus de sénescence. Au cours de ma thèse, je me suis intéressée à l'un de ces très long ARNnc, partiellement antisens au gène DDAH1, jusque là inconnu. J'ai caractérisé cet ARN en montrant qu'il s'agit d'un ARN issu d'une seule unité de transcription de plus 200 kb, à priori non codant et très faiblement poly-adénylé. Cet ARN possède toutes les caractéristiques de très longs ARN récemment décrits comme des vlincRNA, aux fonctions inconnues à l'heure actuelle. Ainsi, nous l'avons nommé VAD (VlincRNA Antisens de DDAH1). J'ai montré que VAD est requis pour l'arrêt stable de la prolifération et dans la mise en place des SAHF. De plus, VAD intervient dans la régulation de l'expression des gènes suppresseurs de tumeurs (p15INK4b, p16 INK4a, p14ARF et p21) impliqués dans le contrôle du cycle cellulaire et dans la mise en place de la sénescence. J'ai également montré que VAD possède une action chromatinienne répressive en cis; alors qu'en trans VAD possède une action chromatinienne activatrice au niveau du locus INK4 (qui code pour p15INK4b, p16 INK4a et p14ARF), acteur majeur de l'entrée en sénescence. Ces expériences établissent ainsi la première démonstration du rôle d'un vlincRNA dans le processus de sénescence, dont l'action est de moduler la structure chromatinienne en cis et en trans. Afin d'analyser plus extensivement les vlincRNA en sénescence, nous avons réalisé un séquençage brin spécifique des ARN totaux des cellules proliférantes versus sénescentes. Cette étude a été corrélée avec l'analyse du paysage chromatinien de H2AZ grâce à des expériences de séquençage de ChIP. Ces expériences ont déjà permis l'identification d'autres vlincRNA et d'en étudier la régulation pendant la sénescence, et devraient augmenter notre compréhension des mécanismes de régulation des vlincRNA et de l'importance de ces derniers dans la mise en place du contrôle épigénétique au cours de la sénescence. Ces approches plus globales devraient permettre une meilleure compréhension des mécanismes de régulation de processus cellulaires par des ARN non codants.Senescence is an anti-tumor mechanism which leads to a stable and irreversible arrest of cell proliferation. During this process there are major changes in chromatin structure, characteristic of senescent cells, called SAHFs (Senescence Associated Heterochromatin Foci) in which proliferative genes are repressed. Non-coding RNAs (ncRNAs) are known to be major actors of the formation of chromatin domains, e.g. assembly of pericentric heterochromatin in mammals. Our working hypothesis is that ncRNAs may be involved in the formation of SAHFs and more generally in senescence process. To characterize the role of ncRNAs in senescence, we analyzed the transcriptomic changes in a model of RAF1 oncogene-induced human senescence. A large-scale strand-specific analysis, by tiling array, was performed on chromosomes 1 & 6 in proliferative and senescent cells. Comparison between the obtained profiles identified a number of transcripts differentially expressed during senescence. Surprisingly, while RNAs differentially expressed during the induction of senescence are largely repressed; ncRNAs, belonging to the recently described vlincRNA (very long intergenic non-coding (>50kb)) class, are mainly activated. This suggests that vlincRNAs may be involved in the establishment of senescence. During my PhD, I focused on one of these long ncRNAs, partially antisense to DDAH1 gene, hitherto unknown. I could show that this RNA is transcribed from a single transcription unit longer than 200 kb, and is weakly polyadenylated and most probably non-coding. This RNA has all the characteristics of very long RNAs recently described as vlincRNA, without any known functions up to date. We term this RNA VAD (VlincRNA Antisense to DDAH1). I showed that VAD is required for stable arrest of proliferation and the establishment of SAHFs. Moreover, VAD is involved in the regulation of the expression of tumor suppressor genes (p15INK4b, p16INK4a, p14ARF and p21) that control the cell cycle and the establishment of senescence. I also showed that VAD has an epigenetic repressive action in cis; whereas in trans, VAD has an epigenetic activating action on INK4 locus (encoding p15 INK4b, p16 INK4a and p14ARF). These data represent the first demonstration of the role of a vlincRNA in senescence. In order to analyze more extensively vlincRNA involvement in senescence, we performed strand-specific sequencing of total RNAs from proliferative cells compared to senescent cells. This was correlated with the analysis of chromatin landscape through ChIPseq experiments. These approaches led to identify other vlincRNAs and to analyze their regulation during senescence. These experiments should provide a better understanding of the mechanisms regulating vlincRNAs and their importance in the epigenetic control during senescence. These broader approaches would provide insights into mechanisms regulating cellular processes by non-coding RNAs

Control of Gene Expression in Senescence through Transcriptional Read-Through of Convergent Protein-Coding Genes

Antisense RNAs are non-coding RNAs that can regulate their corresponding sense RNAs and are generally produced from specific promoters. We uncover here a family of antisense RNAs, named START RNAs, produced during cellular senescence by transcriptional read-through at convergent protein-coding genes. Importantly, START RNAs repress the expression of their corresponding sense RNAs. In proliferative cells, we found that the Pol II elongation rate is limited downstream of TTS at START loci, allowing transcription termination to occur before Pol II reaches the convergent genes, thus preventing antisense RNA production and interference with the expression of the convergent genes. START RNAs are repressed by H2A.Z histone variant, whose local occupancy decreases in senescence. Our results thus uncover a mechanism of gene expression regulation relying on read-through antisense transcript production at convergent genes, underlining the functional importance of chromatin regulation in the control of RNA pol II elongation rate at intergenic regions

A vlincRNA participates in senescence maintenance by relieving H2AZ-mediated repression at the INK4 locus.

International audienceNon-coding RNAs (ncRNAs) play major roles in proper chromatin organization and function. Senescence, a strong anti-proliferative process and a major anticancer barrier, is associated with dramatic chromatin reorganization in heterochromatin foci. Here we analyze strand-specific transcriptome changes during oncogene-induced human senescence. Strikingly, while differentially expressed RNAs are mostly repressed during senescence, ncRNAs belonging to the recently described vlincRNA (very long intergenic ncRNA) class are mainly activated. We show that VAD, a novel antisense vlincRNA strongly induced during senescence, is required for the maintenance of senescence features. VAD modulates chromatin structure in cis and activates gene expression in trans at the INK4 locus, which encodes cell cycle inhibitors important for senescence-associated cell proliferation arrest. Importantly, VAD inhibits the incorporation of the repressive histone variant H2A.Z at INK4 gene promoters in senescent cells. Our data underline the importance of vlincRNAs as sensors of cellular environment changes and as mediators of the correct transcriptional response