Biochemistry & Molecular Biology Program, Lewis & Clark College, Portland, Oregon, United States of America.
Biology Department, Lewis & Clark College, Portland, Oregon, United States of America.
PLoS One. 2024 Sep 30;19(9):e0311120. doi: 10.1371/journal.pone.0311120. eCollection 2024.
Enhancers have critical functions in the precise, spatiotemporal control of transcription during development. It is thought that enhancer grammar, or the characteristics and arrangements of transcription factor binding sites, underlie the specific functions of developmental enhancers. In this study, we sought to identify grammatical constraints that direct enhancer activity in the naïve state of pluripotency, focusing on the enhancers for the naïve-state specific gene, Klf4. Using a combination of biochemical tests, reporter assays, and endogenous mutations in mouse embryonic stem cells, we have studied the binding sites for the transcription factors OCT4 and SOX2. We have found that the three Klf4 enhancers contain suboptimal OCT4-SOX2 composite binding sites. Substitution with a high-affinity OCT4-SOX2 binding site in Klf4 enhancer E2 rescued enhancer function and Klf4 expression upon loss of the ESRRB and STAT3 binding sites. We also observed that the low-affinity of the OCT4-SOX2 binding site is crucial to drive the naïve-state specific activities of Klf4 enhancer E2. Altogether, our work suggests that the affinity of OCT4-SOX2 binding sites could facilitate enhancer functions in specific states of pluripotency.
增强子在发育过程中对转录的精确时空控制中具有关键功能。人们认为，增强子语法（即转录因子结合位点的特征和排列）是发育增强子特定功能的基础。在这项研究中，我们试图确定在多能性的原始状态下指导增强子活性的语法约束，重点是原始状态特异性基因 Klf4 的增强子。我们使用生化测试、报告基因测定和小鼠胚胎干细胞中的内源性突变的组合，研究了转录因子 OCT4 和 SOX2 的结合位点。我们发现，三个 Klf4 增强子包含非最佳的 OCT4-SOX2 复合结合位点。用 Klf4 增强子 E2 中的高亲和力 OCT4-SOX2 结合位点取代 ESRRB 和 STAT3 结合位点可挽救增强子功能和 Klf4 的表达。我们还观察到，OCT4-SOX2 结合位点的低亲和力对于驱动 Klf4 增强子 E2 的原始状态特异性活性至关重要。总之，我们的工作表明，OCT4-SOX2 结合位点的亲和力可以促进特定多能状态下增强子的功能。
