Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering

Abstract

Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming require efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, ePOU can induce pluripotency with Sox2 alone and in the absence of Sox2 in three factor - ePOU/Klf4/c-Myc cocktails. Biochemical assays combined with genome-wide analyses show that ePOU acquires a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins are not changed in ePOU. Compared to Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: (1) differentially activates several genes hitherto not implicated in reprogramming, (2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and (3) binds a distinct class of retrotransposons. Collectively, these features enabled ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.

The functional diversity of the POUV-class proteins across vertebrates

“…For example, it was proposed that the cooperation between the HMG-containing Dichaete and the POU protein vvl occurs during Drosophila neurogenesis [49][50][51]. On the other hand, in vivo data (ChIP-seq) show that DNA-dependent formation of the Sox-Oct dimer is more typical for POUV-class proteins, while other POU factors prefer binding to DNA as homodimers [52][53][54][55].…”

Enhancing Sox/Oct cooperativity induces higher-grade developmental reset

“…Perhaps the apparent barrier is merely a testimony to the inadequacy of the wild-type factor-based reprogramming machinery, which we currently employ. We and others have reported engineering enhanced reprogramming factors that increased the efficiency of mouse reprogramming 31,106–108 but none of the studies showed a substantial gain of efficiency and faithfulness of iPSC generation beyond the mouse model. All alternative cocktails that improved the developmental potential of mouse iPSCs also decreased the efficiency of reprogramming, making them impractical even if they did work for humans 22,66,67 .…”

“…Many studies addressed uniqueness of Oct4 among POU factors by domain swapping and mutagenesis 20,39,48,106,113–116 . We and others determined the importance of the Oct4-POU S , POU HD linker, and CTD domains.…”

“…Our engineered super-SOX harvests the beneficial naturally evolved structural elements of two major development regulators, Sox2 and Sox17. It is likely that even more efficient reprogramming factors could be built by means of rational engineering and directed evolution 106,107 . Our data suggest that enhancing cooperativity between key co-factors should be one of the goals of future designers.…”

Evolutionary origin of vertebrate OCT4/POU5 functions in supporting pluripotency

“…10 ), such as the POU-S domain residues D159 (POU29) required for the mOct4-mSox2 interaction and iPSC formation 53 , V166 (POU36) required for optimal reprogramming 49 and a gain-of-function mutation (T152R (POU22) ) identified in an enhanced POU (ePOU) 63 . In addition, multiple positions in the first helix of the linker region have been identified as important for reprogramming 49 , including positions N206 (POU76) , N207 (POU77) , N209 (POU79) , L210 (POU80) and Q211 (POU81) and another gain-of-function mutation (E208P (POU78) ) 63 . Simultaneous mutation of N206 (POU76) , N207 (POU77) , N209 (POU79) and L210 (POU80) abolishes OCT4-rescue activity 49 .…”