p 53 Regulates Cell Cycle and MicroRNAs to Promote Differentiation of Human Embryonic Stem Cells

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G1 phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state. Citation: Jain AK, Allton K, Iacovino M, Mahen E, Milczarek RJ, et al. (2012) p53 Regulates Cell Cycle and MicroRNAs to Promote Differentiation of Human Embryonic Stem Cells. PLoS Biol 10(2): e1001268. doi:10.1371/journal.pbio. 1001268 Academic Editor: Connie J. Eaves, B.C. Cancer Agency, Canada Received November 15, 2011; Accepted January 10, 2012; Published February 28, 2012 Copyright: 2012 Jain et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funds from NIH GM081627 to MCB, TPZ, and MK, and the NCI Cancer Center Support Grant to the University of Texas MD Anderson Cancer Center. AKJ is an Odyssey Fellow supported by the Odyssey Program and The Laura and John Arnold Foundation at The University of Texas MD Anderson Cancer Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: Adr, adriamycin; AP, alkaline phosphatase; ChIP, chromatin immunoprecipitation; Dox, doxycycline; CM, conditioned medium; ESC, embryonic stem cell; FGF, fibroblast growth factor; hESC, human embryonic stem cell; iPSC, induced pluripotent stem cell; MEF, mouse embryonic fibroblast; mESC, mouse embryonic stem cell; miRNA, microRNA; PI, propidium iodide; p53RE, p53 response element; p53WT, wild-type p53; qRT-PCR, quantitative real-time PCR; RA, retinoic acid; RT, room temperature; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEM, standard error of the mean; siRNA, small interfering RNA * E-mail: mbarton@mdanderson.org