Kang-Xuan Jin, a , b , 1 Rujuan Zuo, a , 1 Konstantinos Anastassiadis, c Arne Klungland, b , d Carsten Marr, e , 2 and Adam Filipczyk a , 2
"aLaboratory for Stem Cell Dynamics, Department of Microbiology, Division of Laboratory Medicine, Oslo University Hospital, Rikshospitalet, Oslo 4950, Norway;
bDepartment of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo 1072, Norway;
cStem Cell Engineering, Biotechnology Centre, Technische Universität Dresden, 01307 Dresden, Germany;
dLaboratory for Dynamic Gene Regulation, Department of Microbiology, Division of Laboratory Medicine, Oslo University Hospital, Rikshospitalet, Oslo 4950, Norway;
eInstitute of Computational Biology, Helmholtz Zentrum München–German Research Centre for Environmental Health, 85764 Neuherberg, Germany
2To whom correspondence may be addressed. Email: [email protected] or [email protected]
Edited by Frank McCormick, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA; received March 31, 2021; accepted November 02, 2021
Author contributions: A.F. designed and supervised research; K.-X.J. and R.Z. performed research; K.A. and C.M. contributed new reagents/analytic tools; K.A., A.K., C.M., and A.F. analyzed data; A.F. wrote the paper; and A.F. acquired funding.
1K.-X.J. and R.Z. contributed equally to this work."
"N6-methyladenosine (m6A) deposition on messenger RNA (mRNA) controls embryonic stem cell (ESC) fate by regulating the mRNA stabilities of pluripotency and lineage transcription factors (TFs) [P. J. Batista et al., Cell Stem Cell 15, 707–719 (2014); Y. Wang et al., Nat. Cell Biol. 16, 191–198 (2014); and S. Geula et al., Science 347, 1002–1006 (2015)]. If the mRNAs of these two TF groups become stabilized, it remains unclear how the pluripotency or lineage commitment decision is implemented. We performed noninvasive quantification of Nanog and Oct4 TF protein levels in reporter ESCs to define cell-state dynamics at single-cell resolution. Long-term single-cell tracking shows that immediate m6A depletion by Mettl3 knock-down in serum/leukemia inhibitory factor supports both pluripotency maintenance and its departure. This is mediated by differential and opposing signaling pathways. Increased FGF5 mRNA stability activates pErk, leading to Nanog down-regulation. FGF5-mediated coactivation of pAkt reenforces Nanog expression. In formative stem cells poised toward differentiation, m6A depletion activates both pErk and pAkt, increasing the propensity for mesendodermal lineage induction. Stable m6A depletion by Mettl3 knock-out also promotes pErk activation. Higher pErk counteracts the pluripotency exit delay exhibited by stably m6A-depleted cells upon differentiation. At single-cell resolution, we illustrate that decreasing m6A abundances activates pErk and pAkt-signaling, regulating pluripotency departure.
DOI: https://dx.doi.org/ 10.1073/pnas.2105192118
Imaging was conducted on previously mentioned wide-field microscope (Zeiss), with a pE-4000 CoolLED
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The pE-4000 Universal Illumination System offers 16 selectable wavelengths from 365 - 770 nm, making it a highly flexible illuminator covering a wide variety of fluorophores
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