NF-E2-related factor 2 (Nrf2) regulates expression and coordinated induction of a battery of chemoprotective genes in response to oxidative and electrophilic stress. This leads to protection against oxidative stress and neoplastic diseases. Nuclear import and export of Nrf2 play a significant role in control of nuclear levels of Nrf2 and thus the expression of Nrf2 downstream genes. Tyrosine kinase Fyn phosphorylates tyrosine 568 of Nrf2 that leads to the nuclear export of Nrf2. In this study, we investigated the upstream factor(s) in regulation of Fyn and Fynmediated nuclear export of Nrf2. The investigations shed light on a novel mechanism of Nrf2 regulation in response to oxidative stress. We demonstrate that GSK-3␤ acts upstream of Fyn kinase in control of nuclear export of Nrf2. Chemical and short interfering RNA-mediated inhibition of GSK-3␤ led to nuclear accumulation of Nrf2 and transcriptional activation of the Nrf2 downstream gene nqo1. Chemical and short interfering RNA inhibition of GSK-3␤ and Fyn individually and in combination revealed that both kinases follow the same pathway to regulate nuclear export of Nrf2. We further demonstrate that hydrogen peroxide phosphorylates tyrosine 216 of GSK-3␤. This leads to activation of GSK-3␤. The activated GSK-3␤ phosphorylates Fyn at threonine residue(s). Phosphorylated Fyn accumulates in the nucleus and phosphorylates Nrf2 at tyrosine 568. This leads to nuclear export, ubiquitination, and degradation of Nrf2.NF-E2-related factor (Nrf2) 2 is a nuclear transcription factor that binds to antioxidant-response element (ARE) and regulates expression and coordinated induction of a battery of chemoprotective genes in response to antioxidants, oxidants, and radiations (1). This induction involves a mechanism essential for cellular protection against oxidative and electrophilic stress and neoplastic diseases (1). Nrf2-null mice are born normal and viable, indicating that Nrf2 is not required for development and growth of mice (2). Nrf2-null mice express significantly lower levels and no induction of chemoprotective proteins that include NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase Ya subunit (GST-Ya), ␥-glutamylcysteinyl synthetase, and heme oxygenase (HO-1) (1). These mice demonstrate slower wound healing and pulmonary emphysema in response to exposure to tobacco smoke (3, 4).A cytosolic inhibitor of Nrf2, INrf2 (inhibitor of Nrf2) or Keap1, retains Nrf2 in the cytoplasm (5, 6). The INrf2-Nrf2 complex serves as cellular sensor of oxidative and electrophilic stress (1). The cellular exposure to oxidants, antioxidants, and radiations antagonizes this interaction and leads to the release of Nrf2 from INrf2 (1). Nrf2 translocates into the nucleus and induces the expression of chemoprotective proteins. Electrophilic adduction of selected cysteines in INrf2 and PKC phosphorylation of Nrf2 are known to contribute to the release of Nrf2 from INrf2 (1). Disruption of INrf2 in mice leads to postnatal death, probably from malnutrition resulting from hyperk...

The antioxidant response element (ARE) and Nrf2 are known to regulate the expression and coordinated induction of genes encoding detoxifying enzymes including NAD(P)H:quinone oxidoreductase1 (NQO1) in response to antioxidants. In this

Numerous studies focus on the tumor suppressor p53 as a protector of genomic stability, mediator of cell cycle arrest and apoptosis, and target of mutation in 50% of all human cancers. The vast majority of information on p53, its protein-interaction partners and regulation, comes from studies of tumor-derived, cultured cells where p53 and its regulatory controls may be mutated or dysfunctional. To address regulation of endogenous p53 in normal cells, we created a mouse and stem cell model by knock-in (KI) of a tandem-affinity-purification (TAP) epitope at the endogenous Trp-53 locus. Mass spectrometry of TAP-purified p53-complexes from embryonic stem cells revealed Tripartite-motif protein 24 (Trim24), a previously unknown partner of p53. Mutation of TRIM24 homolog, bonus, in Drosophila led to apoptosis, which could be rescued by p53-depletion. These in vivo analyses establish TRIM24/bonus as a pathway that negatively regulates p53 in Drosophila. The Trim24-p53 link is evolutionarily conserved, as TRIM24 depletion in human breast cancer cells caused p53-dependent, spontaneous apoptosis. We found that Trim24 ubiquitylates and negatively regulates p53 levels, suggesting Trim24 as a therapeutic target to restore tumor suppression by p53.apoptosis ͉ bonus ͉ TAP ͉ ubiquitylation

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.

SUMMARY Recent evidence suggests that lncRNAs play an integral regulatory role in numerous functions, including determination of cellular identity. We determined global expression (RNA-seq) and genome wide profiles (ChIP-seq) of histone post-translational modifications and p53 binding in human embryonic stem cells (hESCs) undergoing differentiation to define a high-confidence set of 40 lncRNAs, which are p53 transcriptional targets. We focused on lncRNAs highly expressed in pluripotent hESCs and repressed by p53 during differentiation to identify lncPRESS1 as a p53-regulated transcript that maintains hESC pluripotency in concert with core pluripotency factors. RNA-seq of hESCs depleted of lncPRESS1 revealed that lncPRESS1 controls a gene network that promotes pluripotency. Further, we found that lncPRESS1 physically interacts with SIRT6 and prevents SIRT6 chromatin localization, which maintains high levels of histone H3K56 and H3K9 acetylation at promoters of pluripotency genes. In summary, we describe a p53-regulated, pluripotency-specific lncRNA that safeguards the hESC state by disrupting SIRT6 activity.