Supporting data for “Deciphering the molecular underpinnings for the superior performance of engineered SOX17 as an inducer and maintainer of stemness”
An engineered SOX17 (eSOX) with three amino acid mutations inside the DNA binding domain (HMG box) has been previously created by our lab by directed evolution of reprogramming factors by cell selection and sequencing. This eSOX was termed SOX17FNV and has a much higher efficiency in inducing pluripotency than the wildly used Yamanaka factor SOX2 with unknown molecular bases. Thus, this project targets to uncover the mechanisms under the high potency of SOX17FNV, which will help to improve the transcription factor engineering.
We showed that SOX17FNV could mediate the highly potent mouse pluripotency reprogramming and induced neural stem cell (iNSC) reprogramming with a DOX-inducible lentivirus system. Next, we showed that SOX17FNV could functionally substitute the endogenous SOX2 to maintain pluripotency with similar transcriptomes, chromatin organizations, and interactomes to SOX2 by RNA-sequencing, ATAC-sequencing, and CO-IP Mass spectrometry. RT-qPCR was used to individually check the gene expressions.
Furthermore, we showed that the perturbation of the Wnt/beta-catenin signaling did not affect the SOX17FNV-induced cellular reprogramming using the DOX-inducible lentivirus system. We further showed that SOX17FNV and SOX17 could form liquid-liquid phase separation (LLPS) Mediator 1 more efficiently than SOX2 both in vitro and in cells. The EMSA results showed that the DNA preference of SOX17FNV was changed with a higher affinity to POU factors than SOX2 on canonical SoxOct motifs. Furthermore, the N and C terminus of SOX17 were subjected to a detailed sequence-structure-function analysis. We found the C-terminus of SOX17 was the key to highly efficient reprogramming and only several motifs were essential. By removing the nonessential regions, we thus defined a minimum SOX17FNV (miniSOX), which retains the potential but has only 70% length of the full-length protein. The miniSOX acted as SOX17FNV in pluripotency induction, maintenance, and self-organization examined by reprogramming assays, RNA-sequencing, RT-qPCR, and LLPS.
