SMAD1/5-Mediated Recruitment of the Histone Demethylase KDM1A Controls Cell Fate Programs in Embryonic Stem Cells [scRNA-seq]

Bone morphogenetic proteins (BMPs) play diverse roles in mouse embryonic stem cell (mESC) biology. Recent studies suggest that BMPs induce multiple cell fates and enhance mESC heterogeneity by cross-activating multiple signaling pathways. Although BMPs primarily signal through SMAD1 and SMAD5 in mESCs, their roles remain incompletely defined. Here, we investigated the SMAD signaling pathway using Smad1/5 double knockout (S1/5 dKO) mESCs. While SMAD1/5 depletion may influence mESC heterogeneity, single-cell RNA sequencing (scRNA-seq) revealed only minor differences between S1/5 dKO and WT cells, suggesting that the observed changes are not because of altered cell states. Chromatin immunoprecipitation sequencing (ChIP-seq) demonstrated that SMAD1/5 recruit the histone demethylase KDM1A/LSD1 to specific genomic regions, where it removes H3K4me1/2 marks associated with enhancers. Published scRNA-seq data from Kdm1a-deficient mESCs during embryoid body differentiation further supported this mechanism. This study reveals a transcriptional repression mechanism of SMAD1/5, involving KDM1A-dependent H3K4me1/2 depletion and the regulation of cell type–specific gene expression programs. scRNA-seq: parental E14 mouse ESCs (WT) and Smad1/5 double knockout cells (S1/5 dKO) were submitted for single cell RNA sequencing using the 10x Genomics platform. The experiments were performed without replicates.

骨形态发生蛋白（Bone morphogenetic proteins, BMPs）在小鼠胚胎干细胞（mouse embryonic stem cell, mESC）的生物学过程中发挥多样功能。近期研究表明，BMPs可通过交叉激活多条信号通路，诱导多种细胞命运并增强mESC的异质性。尽管在小鼠胚胎干细胞中，BMPs主要通过SMAD1与SMAD5传递信号，但二者的具体功能仍未完全阐明。本研究利用Smad1/5双基因敲除（double knockout, S1/5 dKO）小鼠胚胎干细胞对SMAD信号通路展开探究。虽然SMAD1/5的缺失可能会影响小鼠胚胎干细胞的异质性，但单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）结果显示，S1/5 dKO细胞与野生型（wild type, WT）细胞之间仅存在微小差异，这表明观测到的变化并非由细胞状态改变所致。染色质免疫共沉淀测序（Chromatin immunoprecipitation sequencing, ChIP-seq）实验证实，SMAD1/5可将组蛋白去甲基化酶KDM1A/LSD1招募至特定基因组区域，该酶可去除与增强子相关的H3K4me1/2修饰。已发表的Kdm1a缺陷型小鼠胚胎干细胞在拟胚体分化过程中的单细胞RNA测序数据进一步支持了这一机制。本研究揭示了SMAD1/5的转录抑制机制：该机制依赖KDM1A介导的H3K4me1/2去修饰，并调控细胞类型特异性基因表达程序。本次单细胞RNA测序的样本包括亲本E14小鼠ESC（野生型WT）与Smad1/5双基因敲除细胞（S1/5 dKO），采用10x Genomics平台完成测序，实验未设置生物学重复。