p53 Regulates Cell Cycle and MicroRNAs to Promote Differentiation of Human Embryonic Stem Cells

Abstract

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. Most cell types in an organism are generated from embryonic stem cells (ESCs), which are able to proliferate an unlimited number of times and have the potential to produce any kind of cell of that organism (this ability is called pluripotency). In order to maintain these properties, ESCs have to remain in a proliferate state, which is achieved by the collaboration of several factors. Expressing combinations of these factors in differentiated cells can result in ESC-like qualities; these induced pluripotent stem cells (iPSCs) can then function like ESCs. Previous studies suggested that p53, generally known for its roles in maintaining genomic integrity by regulating cell cycle and cell death pathways, also acts as a barrier to reprogramming adult cells during the creation of iPSCs; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays a significant role in actively promoting differentiation of human ESCs (hESCs). We find that, prior to differentiation, p53 is expressed at very low levels in hESCs, held in check by two negative regulators, HDM2 and TRIM24, that trigger p53 degradation. Upon induction of differentiation, lysine 373 of p53 is acetylated, and this disrupts the existing interactions with negative regulators, thus allowing stabilization and activation of p53. Active p53 in turn promotes expression of the cell cycle regulator p21 to slow down the hESC cell cycle; cells in the gap (G₁) phase of the cell cycle accumulate, preventing division. In parallel, p53 activates specific microRNAs, miR-34a and miR-145, that inhibit the expression of several stem cell factors and prevent differentiated cells from backsliding to pluripotency. Our results highlight a novel function of p53 in determining the human stem cell state.