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. Overall design: 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.