Evolution of X-inactivation and regulatory lncRNA in primates – PrimateXCI

PRIMATE XCI exploits pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells) from the three chosen primate species to model early development, when critical steps of X chromosome regulation occurs. The activity status of the X chromosome will be characterized at various stages. We will identify, in each species and through candidate and systematic approaches, noncoding regulators and we will characterize their mechanisms of action. We will also determine how these regulators couple, at the molecular level, X-inactivation to cellular state, and thus to development. PRIMATE XCI relies on state of the art CRISPR/Cas9 technologies and derivatives for robust and efficient functional investigation of noncoding elements, as well as on innovative development for mimicking in vitro various status of pluripotency.

We have first collected material and built tools for characterization and functional investigation in non-human primates. We have determined the activity status of the X chromosomes in females rhesus and chimpanzee pluripotent stem cells. We have moreover built on RNA-seq and Hi-C data to characterize the transcriptional and 3D landscapes of the X-inactivation center in primates. These unbiased approaches already revealed differences across species, the functional consequences of which remains to be determined.
We have also obtained the first evidence toward a role for candidate noncoding regulators in controlling X chromosome inactivation in non-human primates.

This project will bring novel insights into the X chromosome inactivation process in primates, for which data are mostly inexistent. This is all the more important as rodents, which served as the main model system thus far, are now thought to be an exception in how X-inactivation is established.
This original combination of biological systems, creative approaches and tools will moreover allow us to unravel the phenotypic and mechanistic diversity of X-inactivation across primates and the contribution of the noncoding genome to general principles in the regulation of X-inactivation in primates as well as to species-specific mechanisms.

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The noncoding genome carries key regulatory function and participates to the establishment of gene expression programs, and, as such, to cell identity and fate. It encompasses a vast repertoire of long noncoding RNA genes (LRG), a fraction of which derives from endogenous retroviruses, and whose function may be conveyed by the RNA itself, but also by the genomic locus, the act of transcription, or any downstream smaller RNA by-products. LRG are subject to rapid evolutionary turnover -even among mammals- and orthologous LRG display a high rate of sequence divergence. The extent to which LRG contribute to phenotypic variation, notably among closely related species, is however poorly understood.
X-chromosome inactivation (XCI) is a process of choice to address this question, as it involves a large number of LRG and displays displays marked variability in the underlying mechanisms across species. Previous work from Partner 1 has demonstrated major differences in the involvement of LRG in human XCI compared to the mouse, which has been so far the reference model for the study of XCI. However, these two species belong to different orders of eutherians, primates and rodents respectively, which diverged 90 million years ago and display different developmental programs and pluripotency networks, which might account, at least in part, for these differences. Probing the link between XCI and early cell fate commitment across more closely related species constitutes an innovative paradigm to understand how the noncoding genome allows for rapid adaptation and species-specification on a short evolutionary timescale.
The PRIMATE XCI project aims at deciphering phenotypic and mechanistic variability of XCI across primates, through the study of 3 model species (human, chimp and macaque) and in the context of naïve and primed pluripotent stem cells. We will identify, in each species and through candidate and systematic approaches, noncoding regulators and we will characterize their mechanisms of action. We will also determine how these regulators couple, at the molecular level, XCI to cellular state, and thus to development. PRIMATE XCI relies on state of the art CRISPR/Cas9 technologies and derivatives for robust and efficient functional investigation in primates PSC, as well as on innovative development for resetting primed into naïve pluripotency. This original combination of biological systems, creative approaches and tools will allow us to define how LRG contribute to general principles in the regulation of XCI in primates as well as to species-specific mechanisms. This project is at the crossroads of several competitive research fields, namely stem cells, LRG, epigenetics and evolution, and will likely impact multiple scientific communities.