Enhancer-associated H3K4 methylation safeguards in vitro germline competence

Tore Bleckwehl,Michaela Bartusel,Patricia Respuela,Kaitlin Schaaf,Kristel M Dorighi,Joanna Wysocka,Giuliano Crispatzu,Stephen J Clark,Antonio Barral,Álvaro Rada-Iglesias,Wolf Reik,Laura Benson,Magdalena Laugsch,Miguel Manzanares,Wilfred F J Van Ijcken

doi:10.1038/s41467-021-26065-6

Abstract

Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.

Highlights

Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC)
Day 2 and Day 4 embryoid bodies (EB) showed cellular heterogeneity and formed distinct subclusters (Fig. 1b, c). One of these subclusters was identified as PGC-like cells (PGCLC) based on the high expression of major PGC markers (e.g. Prdm[14], Prdm[1], Tfap2c, Dppa3), while the additional subpopulations within d4 EB were annotated based on the expression of cell identity markers identified by single-cell transcriptional profiling of E8.25 mouse embryos[42] (Fig. 1c, Supplementary Fig. 1a, b)
Our analyses indicate that the decommissioning of a subset of PGCLC enhancers gets exacerbated in dCD epiblast like-cells (EpiLC) compared to their WT counterparts, resulting in a chromatin state similar to the one observed in WT Epiblast stem cells (EpiSC)

Summary

Introduction

Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that confer germline competence on the formative but not the primed epiblast cells remain obscure[8,9], partly due to the limited cell numbers that can be obtained in vivo from mouse peri-implantation embryos (E4.5–E6.5) These limitations were mitigated by a robust in vitro differentiation system whereby mouse embryonic stem cells (ESC) grown under 2i conditions (naïve pluripotency) can be sequentially differentiated into EpiLC and PGCLC that resemble the formative epiblast and E9.5 PGC, respectively[10]. It is currently unknown whether, during development, H3K4me[1] persistence once enhancers become decommissioned can facilitate their eventual re-activation[21]

Methods

Results

Conclusion