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

Tan, D. S., Chen, Y., Gao, Y., Bednarz, A., Wei, Y., Malik, V., et al. (2021). Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering. MOLECULAR BIOLOGY AND EVOLUTION, 38(7), 2854-2868. doi:10.1093/molbev/msab075.

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 Creators:
Tan, Daisylyn Senna, Author
Chen, Yanpu1, Author           
Gao, Ya, Author
Bednarz, Anastasia, Author
Wei, Yuanjie, Author
Malik, Vikas, Author
Ho, Derek Hoi-Hang, Author
Weng, Mingxi, Author
Ho, Sik Yin, Author
Srivastava, Yogesh, Author
Velychko, Sergiy, Author
Yang, Xiaoxiao, Author
Fan, Ligang, Author
Kim, Johnny1, Author           
Graumann, Johannes2, Author           
Stormo, Gary D., Author
Braun, Thomas1, Author           
Yan, Jian, Author
Schoeler, Hans R., Author
Jauch, Ralf, Author
Affiliations:
1Cardiac Development and Remodeling, Max Planck Institute for Heart and Lung Research, Max Planck Society, ou_2591695              
2Biomolecular Mass Spectrometry, Max Planck Institute for Heart and Lung Research, Max Planck Society, ou_2591705              

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Free keywords: TRANSCRIPTION FACTOR-BINDING; PLURIPOTENT STEM-CELLS; SOMATIC-CELLS; MOUSE; OCT4; EXPRESSION; DYNAMICS; PHOSPHORYLATION; IDENTIFICATION; STABILITYBiochemistry & Molecular Biology; Evolutionary Biology; Genetics & Heredity; reprogramming; protein engineering; POU; cell fate conversion; molecular evolution; transcription factor;
 Abstract: 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 genomewide 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.

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Language(s): eng - English
 Dates: 2021-03-152021-07
 Publication Status: Published in print
 Pages: 15
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: ISI: 000671060500014
DOI: 10.1093/molbev/msab075
PMID: 33720298
 Degree: -

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Title: MOLECULAR BIOLOGY AND EVOLUTION
Source Genre: Journal
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Publ. Info: GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND : OXFORD UNIV PRESS
Pages: - Volume / Issue: 38 (7) Sequence Number: - Start / End Page: 2854 - 2868 Identifier: ISSN: 0737-4038