The transcription factor Oct4 is a core component of molecular cocktails inducing pluripotent stem cells (iPSCs), while other members of the POU family cannot replace Oct4 with comparable efficiency. Rather, group III POU factors such as Oct6 induce neural lineages. Here, we sought to identify molecular features determining the differential DNA‐binding and reprogramming activity of Oct4 and Oct6. In enhancers of pluripotency genes, Oct4 cooperates with Sox2 on heterodimeric SoxOct elements. By re‐analyzing ChIP‐Seq data and performing dimerization assays, we found that Oct6 homodimerizes on palindromic OctOct more cooperatively and more stably than Oct4. Using structural and biochemical analyses, we identified a single amino acid directing binding to the respective DNA elements. A change in this amino acid decreases the ability of Oct4 to generate iPSCs, while the reverse mutation in Oct6 does not augment its reprogramming activity. Yet, with two additional amino acid exchanges, Oct6 acquires the ability to generate iPSCs and maintain pluripotency. Together, we demonstrate that cell type‐specific POU factor function is determined by select residues that affect DNA‐dependent dimerization.

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