In mammals, the formation of gamete precursors, the primordial germ cells (PGCs), leads to the repression of the somatic program and the expression of germline-specific genes, accompanied by a profound epigenetic remodeling. The reprogramming includes: genome-wide demethylation, important histone modification redistribution, erasure of the genomic imprints, and X-chromosome reactivation in females.

The re-expression of the inactive X chromosome (Xi), at the onset of sex gonadal differentiation, leads to a transient excess of X-linked gene products in females compared to males. X-reactivation involves the loss of the long non-coding RNA Xist, loss of repressive histone and DNA marks and the re-expression of X-linked genes from the Xi. Despite X-chromosome inactivation has been deeply studied, how X-chromosome reactivation is regulated in PGCs and its biological impact remains largely unknown.

In the lab, we aim to dissect the mechanisms behind Xi reactivation in female germ cells and its potential sex-specific reprogramming. We use a unique culture-based PGC-like cell system, carrying dual fluorescent reporters for both X-chromosome dynamics and PGC fate, an inducible XCI system and allele-specific information. These reporters are knock-in in mice embryonic stem cells (mESC) using CRISPR-Cas9 technology. We will determine the main contributors to X-chromosome reactivation during PGCLC specification by combining genome-wide CRISPR-knock-out screening and low-input genomic and transcriptomic profiling of our new reporter female cell lines.

This novel model allows us to analyze the sex-specific germline differentiation, advancing on the understanding of epigenetic mechanisms, as well as stem cell biology in reproduction.

Keywords: X-chromosome inactivation, primordial germ cell-like cells, genomic engineering, genome-wide CRISPR-KO screen, epigenetics