Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering.
POU
cell fate conversion
molecular evolution
protein engineering
reprogramming
transcription factor
Journal
Molecular biology and evolution
ISSN: 1537-1719
Titre abrégé: Mol Biol Evol
Pays: United States
ID NLM: 8501455
Informations de publication
Date de publication:
25 06 2021
25 06 2021
Historique:
pubmed:
16
3
2021
medline:
22
9
2021
entrez:
15
3
2021
Statut:
ppublish
Résumé
Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and 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, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses 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 remain unchanged in ePOU. Compared with 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 enable 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.
Identifiants
pubmed: 33720298
pii: 6171154
doi: 10.1093/molbev/msab075
pmc: PMC8233511
doi:
Substances chimiques
KLF4 protein, human
0
Klf4 protein, mouse
0
Kruppel-Like Factor 4
0
POU Domain Factors
0
Types de publication
Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2854-2868Informations de copyright
© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
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