Spatio-temporal X-linked gene reactivation in the mouse germline reveals site-specific retention of epigenetic silencing [RNA-seq]

Random X-chromosome inactivation (XCI) is a hallmark of female mammalian somatic cells. This epigenetic mechanism, mediated by the long non-coding RNA Xist, occurs in the epiblast and is stably maintained to ensure proper dosage compensation of X-linked genes during life. However, this silencing is lost during primordial germ cell (PGC) development. Using a combination of single-cell allele-specific RNA sequencing and low-input chromatin profiling on in vivo developing mouse PGC, we provide unprecedented detailed maps of gene reactivation. We demonstrate that PGC still carry a fully silent X chromosome at embryonic day (E) 9.5, despite the loss of Xist expression. X-linked genes are then gradually reactivated outside the Xist first-bound regions. At E12.5, a significant part of the inactive X chromosome (Xi) still resists reactivation, carrying an epigenetic memory of its silencing. Late-reactivated genes are enriched in repressive chromatin marks, including DNA methylation and H3K27me3 marks. Our results define the timing of reactivation of the silent X chromosome a key event in female PGC reprogramming with direct implications for reproduction. After dissection of embryos at the location of the PGC, according to embryonic stage, and sexing of the embryos, samples were resuspended in 200 µL of 0.25 % trypsin and incubated at 37 °C for 3 min. Trypsin was inactivated with serum and a single-cell solution was obtained by vigorous up-and-down. For scRNA-seq experiments from E10.5, cells were manually picked based on their GFP and size and washed in PBS-acetylated BSA. For the earlier developmental stages, cells were collected by FACS and processed quickly for cDNA amplification. Poly(A)+ mRNA extracted from each single cell was reverse-transcribed from 3’-UTRs and amplified according to Tang et al, 2010 and Borensztein et al, 2018.