Claire Rougeulle’s group studies the function of long non-coding RNAs (lncRNAs) in controlling gene expression programs in stem cells, differentiation and development and their contribution to the plasticity of epigenetic regulation in evolution. Our group focuses on X-chromosome inactivation in mammals as a paradigm for epigenetic processes that involves lncRNAs and displays substantial variability between species in the underlying regulatory mechanisms. Our research also aims to investigate the involvement of lncRNAs in pathological contexts.
We couple cellular and animal models to the latest technologies of genome engineering to study, ex vivo and in vivo, the function of lncRNAs in various species. In particular we use mouse and human pluripotent stem cells and their differentiated derivatives. Our experimental strategy combines classic molecular approaches with single cell analysis and large scale transcriptomic and epigenomic techniques.
Key words: Non-coding RNAs; X chromosome inactivation; stem cells; development; epigenetics
Long non-coding RNAs (lncRNAs) represent a special class of transcripts: they are abundant in complex organisms such as vertebrates and have important roles in diverse cellular processes, yet they are subject to low selection pressure during evolution. LncRNAs play central roles in the epigenetic control of gene expression and they contribute to the establishment and/or maintenance of cellular identity. LncRNAs are also frequently found associated with diseases, in particular cancers as well as neurodevelopmental disorders and psychiatric diseases.
Our main question is to understand how lncRNAs regulate gene expression and contribute to cellular identities, in physiological and pathological contexts. Our projects are also devoted to multi-species analyses to investigate the contribution of lncRNAs to the plasticity of epigenetic regulation in evolution. We study X-chromosome inactivation as one of the most striking example of developmentally regulated epigenetic processes involving lncRNAs (Fig. 1).
X-inactivation is a fascinating process which implies the differential treatment of two chromosome homologues within the same nucleus. X-inactivation is tightly regulated during embryonic development and is linked to cellular state and in particular to differentiation; instability of X-inactivation is observed in various poorly differentiated contexts including human pluripotent stem cells and cancers. Furthermore, the mechanisms underlying X-inactivation display remarkable variation between mammalian species and the question arises as to which extent lncRNAs contribute to this variation.
Our main goals are:
– To identify and characterize lncRNAs controlling X-chromosome inactivation in mouse and human
– To probe how lncRNAs contribute to the variation in X-inactivation strategies between species
– To address the link between lncRNA, pluripotency, differentiation and development
– To investigate on a larger scale the contribution of lncRNAs and epigenetic alteration to pathological states
LncRNAs controlling X-inactivation in the mouse
X-inactivation in eutherians is triggered by the accumulation of the XIST lncRNA on one of the two X chromosome in female individuals. Deciphering the network that control the monoallelic expression of XIST in a developmental manner is thus critical to understand the mechanisms of X-inactivation regulation. Our lab has participated in the identification of the transcriptional network ensuring XIST repression and its activation in appropriate developmental contexts in the mouse. Several additional non-coding genes located in the vicinity of XIST, within a region called the X-inactivation center are also contributing to the regulation of XIST and of X-inactivation (Fig. 1). We are focusing on FTX, a complex locus giving rise to multiple ncRNA isoforms and the only gene of the inactivation center to host micro-RNAs. We are interrogating the function of FTX in X-inactivation and in differentiation using complementary in vivo and ex vivo approaches (Fig. 2).
Fig. 2: Principle of functional analysis of lncRNAs through CRISPR/Cas9-mediated genome editing
Regulation of X-inactivation in humans, and contribution of lncRNAs to the variation in X-inactivation strategies between species
Although X-inactivation takes place in every mammals, the strategies employed display quite unexpected variations between species. Our research focuses on the differences between mouse and human X-inactivation and we use human embryonic stem cells to address the regulation of X-inactivation in humans (Fig. 3). We identified XACT, a human specific lncRNA that shows the unique property of coating active X chromosomes in pluripotent contexts. Using state of the art genome engineering approaches such as CRISPR/Cas9 based editing, we are studying the function of XACT in controlling the activity status of the X chromosome in humans (Fig.2). We are also interested in understanding the extent to which conserved lncRNAs of the X inactivation center, such as FTX, play similar function in different species.
Fig. 3: RNA-FISH analysis of XIST (in green) and XACT (in red) expression in human embryonic stem cells
LncRNAs in pathological contexts
As lncRNAs are increasingly recognized as central elements in many biological processes, it is not surprising to observe that deregulation of the lncRNA circuitry is an important factor in various pathologies, including cancers. We are investigating the contribution of lncRNA to pathologies, and cancer in particular, through gene-centered as well as genome-wide approaches (Fig. 4).
Fig. 4: Genome-wide analysis of chromatin marks and gene expression by ChIP-seq and RNA-seq
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