Primers for genotyping gene edited mESCs.

<div><p>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, <i>Klf4</i>. 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 <i>Klf4</i> enhancers contain suboptimal OCT4-SOX2 composite binding sites. Substitution with a high-affinity OCT4-SOX2 binding site in <i>Klf4</i> enhancer E2 rescued enhancer function and <i>Klf4</i> 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 <i>Klf4</i> enhancer E2. Altogether, our work suggests that the affinity of OCT4-SOX2 binding sites could facilitate enhancer functions in specific states of pluripotency.</p></div>