Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China.
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
Cell Prolif. 2020 Nov;53(11):e12914. doi: 10.1111/cpr.12914. Epub 2020 Sep 29.
Mouse embryonic stem cell (mESC) culture contains various heterogeneous populations, which serve as excellent models to study gene regulation in early embryo development. The heterogeneity is typically defined by transcriptional activities, for example, the expression of Nanog or Rex1 mRNA. Our objectives were to identify mESC heterogeneity that are caused by mechanisms other than transcriptional control.
Klf3 mRNA and protein were analysed by RT-qPCR, Western blotting or immunofluorescence in mESCs, C2C12 cells, early mouse embryos and various mouse tissues. An ESC reporter line expressing KLF3-GFP fusion protein was made to study heterogeneity of KLF3 protein expression in ESCs. GFP-positive mESCs were sorted for further analysis including RT-qPCR and RNA-seq.
In the majority of mESCs, KLF3 protein is actively degraded due to its proline-rich sequence and highly disordered structure. Interestingly, KLF3 protein is stabilized in a small subset of mESCs. Transcriptome analysis indicates that KLF3-positive mESCs upregulate genes that are initially activated in 8-cell embryos. Consistently, KLF3 protein but not mRNA is dramatically increased in 8-cell embryos. Forced expression of KLF3 protein in mESCs promotes the expression of 8-cell-embryo activated genes.
Our study identifies previously unrecognized heterogeneity due to KLF3 protein expression in mESCs.
小鼠胚胎干细胞（mESC）培养物包含各种异质群体，这些群体是研究早期胚胎发育中基因调控的极佳模型。异质性通常通过转录活性来定义，例如 Nanog 或 Rex1 mRNA 的表达。我们的目标是确定除转录控制以外的机制引起的 mESC 异质性。
通过 RT-qPCR、Western blot 或免疫荧光分析 mESCs、C2C12 细胞、早期小鼠胚胎和各种小鼠组织中的 Klf3 mRNA 和蛋白质。构建了表达 KLF3-GFP 融合蛋白的 ESC 报告系，以研究 ESC 中 KLF3 蛋白表达的异质性。对 GFP 阳性 mESCs 进行分选，用于进一步分析，包括 RT-qPCR 和 RNA-seq。
在大多数 mESCs 中，由于其脯氨酸丰富的序列和高度无序的结构，KLF3 蛋白被积极降解。有趣的是，一小部分 mESCs 中 KLF3 蛋白稳定。转录组分析表明，KLF3 阳性 mESCs 上调了在 8 细胞胚胎中最初激活的基因。一致地，KLF3 蛋白而不是 mRNA 在 8 细胞胚胎中显著增加。在 mESCs 中强制表达 KLF3 蛋白可促进 8 细胞胚胎激活基因的表达。
我们的研究确定了 mESCs 中由于 KLF3 蛋白表达而导致的先前未被识别的异质性。
