Associate Professor The University of Hong Kong Hong Kong, Hong Kong
Abstract: KLF4 is a pioneer transcription factor directing reprogramming towards pluripotent and multipotent stem cells. This activity is tied to its capacity to bind silenced chromatin, such as methylated CpG and nucleosome core particles. To dissect the basis for KLF4’s reprogramming activity and to enhance its function, we designed saturation mutagenesis libraries by randomizing positions that are critical to binding epigenetically modified DNA. Using pooled screens in mouse pluripotency reprogramming, we identified several evolved KLF4 (eKLF4) variants that change the reprogramming activity of wild-type KLF4. Notably, two double mutants in KLF4 support iPSC generation without the otherwise essential SOX2/OCT4 duo. The iPSCs derived from eKLF4 and c-MYC (eKM) exhibit all the molecular, cellular, and functional characteristics of embryonic stem cells. The eKM iPSCs display robust developmental potential and capacity for germline transmission. Mechanistically, eKLF4 differentially activates mesenchymal-to-epithelial transition, early pluripotency marker genes at the onset of eKM reprogramming and opens up chromatin regions that are less accessible to wild-type KLF4, potentially facilitating the induction of pluripotency. In vitro, eKLF4 shows a high affinity for nucleosome core particles and methylated DNA binding elements compared to the wild-type protein. Optimized pioneer factors are poised to transform personalized regenerative medicine, amplify the potential of stem cells, and facilitate rejuvenation by partial reprogramming.
