Investigation on stem cell-associated transcription factors : artificial evolution of KLF4 and natural evolution of SOX

Abstract

The generation of induced pluripotent stem cells (iPSCs) via ectopic expression of OCT4, SOX2, KLF4, and c-MYC has revolutionized the field of regenerative medicine. Among these factors, SOX2 and KLF4 are the functional core whilst OCT4 and c-MYC can be omitted. However, additional investigations are necessary for elucidating mechanisms of cell fate control, decoding the molecular basis for their ability to act as pioneer transcription factors and applying these factors for inducing next-generation stem cells for translational applications.

In the first part of this thesis, directed molecular evolution was employed to re-engineer how reprogramming factors read and write the epigenome to facilitate cell fate transitions. In the second part, the investigation was undertaken to ascertain the evolutionary origin of the transcription factor toolkit of stem cells.

Firstly, the aim was to develop new cell fate engineering paradigms by artificially evolving and enhancing the function of KLF4. From screens with three site-saturation libraries, engineered versions of KLF4 were identified, referred to as eKLF4. Two related eKLF4 variants possess correlated double mutations that enhance the affinity to DNA elements with methylated CpG sites. When combined with the other three factors, eKLF4 enables more rapid and efficient iPSC generation. Surprisingly, eKLF4 compensates for the function of the otherwise essential SOX2/OCT4 duo, allowing for reprogramming using a minimalistic two-factor cocktail alongside c-MYC. iPSCs derived from eKLF4 and c-MYC (eKM) exhibit similar characteristics to mouse embryonic stem cells in vitro and in vivo. Mechanistically, eKLF4 triggers cell-autonomous innate immunity, boosts mesenchymal to epithelial transition at the onset of reprogramming, and specifically upregulates trophectoderm genes. Chromatin accessibility profiling indicates that eKLF4 turned into a super-pioneer factor as it opens up pluripotency-associated chromatin regions otherwise requiring the presence of SOX2/OCT4. Our findings hold great promise not only for iPSC reprogramming but also for lineage reprogramming, induction of totipotency and the reprogramming of cells types and species that are challenging to turn into stem cells.

Traditionally, the Sox and POU factors have been considered specific to animals. However, we discovered that POU and Sox sequences are present in unicellular relatives of animals. Staggeringly, Sox factors from unicellular species without stem cells can replace Sox2 to induce pluripotency in mice. Likewise, resurrected Sox factors that presumably existed 700 million years ago showed that Sox genes from all the ancestral nodes leading to the Sox2 lineage have the capacity to induce pluripotency. Overall, these findings provide new insights into the evolutionary origins of stemness-associated factors and suggest that the evolution of stem cells in multicellular animals utilized a pre-existing set of transcription factors.

Taken together, in this thesis, it is demonstrated that the molecular toolkit of pluripotency predates the emergence of stem cells themselves. Through the utilization of directed molecular evolution techniques in mammalian cells, it is discovered that this toolkit can be further adapted to enable cell fate transition paradigms that were previously considered impossible.