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

Research output: Journal Publications and ReviewsRGC 21 - Publication in refereed journalpeer-review

10 Scopus Citations
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Author(s)

  • Daisylyn Senna Tan
  • Yanpu Chen
  • Ya Gao
  • Anastasia Bednarz
  • Yuanjie Wei
  • Vikas Malik
  • Derek Hoi-Hang Ho
  • Mingxi Weng
  • Sik Yin Ho
  • Yogesh Srivastava
  • Sergiy Velychko
  • Xiaoxiao Yang
  • Ligang Fan
  • Johnny Kim
  • Johannes Graumann
  • Gary D. Stormo
  • Thomas Braun
  • Hans R. Schöler
  • Ralf Jauch

Related Research Unit(s)

Detail(s)

Original languageEnglish
Article numbermsab075
Pages (from-to)2854-2868
Number of pages15
Journal / PublicationMolecular Biology and Evolution
Volume38
Issue number7
Online published15 Mar 2021
Publication statusPublished - Jul 2021

Link(s)

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.

Research Area(s)

  • reprogramming, protein engineering, POU, cell fate conversion, molecular evolution, transcription factor

Citation Format(s)

Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering. / Tan, Daisylyn Senna; Chen, Yanpu; Gao, Ya et al.
In: Molecular Biology and Evolution, Vol. 38, No. 7, msab075, 07.2021, p. 2854-2868.

Research output: Journal Publications and ReviewsRGC 21 - Publication in refereed journalpeer-review

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