The emergence of the stem cell-associated Sox and POU transcription factors predates animal origins (Spec-Seq)
Pluripotent stem cells are a hallmark of animal multicellularity. Sox and POU family transcription factors are pivotal for stemness and were believed to be animal innovations as they were reported absent from the genomes of their unicellular relatives. Here we describe new unicellular holozoan orthologues to Sox and POU families, indicating that they emerged before the appearance of animals. We show that choanoflagellate and filasterean Sox genes have DNA binding specificity similar to Sox2. Choanoflagellate Sox can partner with the POU member Oct4 on DNA elements found in pluripotency enhancers. Consistently, choanoflagellate – but not filasterean – Sox genes can replace Sox2 to reprogram mouse somatic cells into induced pluripotent stem cells (iPSC). In contrast, choanoflagellate POU harbors a unique DNA-binding profile that differs from Oct4 and cannot generate iPSCs. Pluripotency reprogramming with reconstructed ancestral Sox genes shows that their molecular ability to induce stemness was already present in the last common ancestor of animals and their unicellular relatives. Our findings imply that the evolution of stem cells exploited a pre-existing set of transcription factors, where the critical innovation involved an initial change in DNA specificity of POU and the exaptation of the ancestral capacity to interact with Sox transcription factors. Overall design: To investigate the binding specificity of Sox and POU proteins found in unicellular organisms such as Salpingoeca helianthica (Salhel) and Pigoraptor chileana (Pchi), the DNA binding domains (DBD) of Sox proteins from Salhel and Pchi, as well as the POU protein from Salhel, were purified. These proteins along with DBDs of mouse Sox2 and Oct4 proteins were then subjected to Specificity by sequencing (Spec-seq) experiments using  libraries of degenerate sequences of Sox motif (CATTGTT) and Octamer motif (ATGCTAAT), respectively.