Enhancer-associated H3K4 methylation safeguards in vitro germline competence [scRNA-seq]

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. ChIP-seq, ATAC-seq, genome-wide bisulftie sequencing, 4C-seq, RNA-seq and single cell RNA-seq from different stages of the PGCLC differentiation.

哺乳动物的生殖系特化通过诱导过程完成：植入后上胚层中的感受态细胞可分化为原始生殖细胞（primordial germ cell, PGC）。目前尚不明确赋予上胚层细胞响应生殖系诱导信号能力的内在因子。对体外类原始生殖细胞（PGC-like cell, PGCLC）分化系统的多阶段单细胞RNA测序结果显示，初始表达于初始多能态（naïve pluripotent stage）的PGCLC相关基因，在生殖系感受态类上胚层细胞（epiblast-like cell, EpiLC）中会被均匀地沉默。与之相对，与这些生殖系基因相关的增强子的失活过程并不完全。具体而言，这类增强子中的一部分会部分保留组蛋白H3赖氨酸4单甲基化（H3K4me1）修饰，所积累的异染色质标记更少，且仍保持染色质开放状态并可响应转录激活因子。随后，当体外培养体系中的生殖系感受态特性丧失时，这些增强子会进一步失活，并失去对转录激活因子的响应能力。值得注意的是，通过构建H3K4me1缺陷型细胞，我们证实该组蛋白修饰的缺失会降低EpiLC的生殖系感受态能力，并削弱PGCLC的分化效率。本研究表明，尽管H3K4me1可能并非增强子行使功能所必需，但它能够在特定发育转变过程中，促进增强子的（再）激活以及基因表达程序的建立。本数据集涵盖PGCLC分化不同阶段的染色质免疫共沉淀测序（ChIP-seq）、转座酶可及性测序（ATAC-seq）、全基因组亚硫酸氢盐测序、4C测序、RNA测序以及单细胞RNA测序数据。