Compared to differentiated cells, ES cells are hypersensitive to DNA damage-induced apoptosis. It is thought that this hypersensitivity to DNA damage-induced apoptosis contributes to the low mutation burden of ES cells because ES cells with mutated DNA caused by DNA damage are removed by apoptosis. We have hypothesized that ES cell-specific factors contribute to this ES cell-specific hypersensitivity to DNA damage-induced apoptosis. During fiscal year (FY) of 2015, we have made significant progress of identifying these ES cell-specific factors that could potentially regulate the hypersensitivity of mES cells to DNA damage. We have previously mapped a global p53 signaling in ES cells (Li M, et al., Molecular Cell, 2012). Based on the datasets generated by this earlier study, we have identified ES cell-enriched factors that may be involved in p53 signaling in ES cells. We have decided to focus on one transcript called Apela for further study because Apela is repressed by p53 and encodes a putative secretory peptide, Apela. Apela peptide binds to its receptor, Aplnr, to regulate cell movement in differentiated cells. We found that Apela positive regulates p53-regulated apoptosis in mouse ES cells as knockdown of Apela compromised p53-mediated apoptosis in the cells. Surprisingly, the coding ability of Apela is dispensable for its role in p53-mediated apoptosis. Instead, Apela binds and antagonizes the function of heterogeneous nuclear ribonuclear protein L (hnRNPL). hnRNPL interacts with p53 and inhibits p53 activation. Therefore, we have discovered an Apela RNA-mediated negative feedback loop in mouse ES cells that regulates apoptosis. Given that Apela is specifically expressed in ES cells, our findings provide an explanation to the hypersensitivity of ES cells to DNA damage. Our study on Apela also establishes foundations for investigating two aspects of the regulation of p53 signaling in ES cells. First, under unstressed condition, p53 activity needs to be controlled to allow ES cells to proliferate. However, the factor(s) inhibits p53 activity under unstressed condition is(are) unknown. Here, we identified hnRNPL as one of such inhibitory factors that control p53 activities in ES cells. Interestingly, hnRNPL knockout embryos die at the blastocyst stage. We plan to test whether hnRNPL is the inhibitory factor for p53 activity in vivo. A second aspect of the regulation of p53 signaling concerns about the mechanisms underlying the enhancer interference by activated p53. We have previously found that p53 represses ES cell-specific genes, such as Nanog, Oct4, and Sox2, by interfering with their enhancer activities. However, the mechanisms of enhancer interference are unclear. We were not able to use Nanog, Oct4, or Sox2 as our model genes to study the mechanisms because prolonged down-regulation of either of these genes will lead to ES cell differentiation. Apela down-regulation, however, does not cause ES cell differentiation. Therefore, Apela serves as a good model gene to study enhancer interference. We plan to use Apela and CRISPR technology to investigate the molecular mechanism of p53-directed enhancer interference in ES cells.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Yan, Hualong; Shi, Yun-Bo; Huang, Jing (2017) iPSCs are safe! Cell Biosci 7:30
Li, Mangmang; Gou, Hongfeng; Tripathi, Brajendra K et al. (2015) An Apela RNA-Containing Negative Feedback Loop Regulates p53-Mediated Apoptosis in Embryonic Stem Cells. Cell Stem Cell 16:669-83
Shi, Jianxin; Marconett, Crystal N; Duan, Jubao et al. (2014) Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue. Nat Commun 5:3365
Zhang, Xinyue; He, Yunlong; Lee, Kyoung-Hwa et al. (2013) Rap2b, a novel p53 target, regulates p53-mediated pro-survival function. Cell Cycle 12:1279-91
Shin, Min Hwa; He, Yunlong; Huang, Jing (2013) Embryonic stem cells shed new light on the developmental roles of p53. Cell Biosci 3:42
Li, Mangmang; He, Yunlong; Dubois, Wendy et al. (2012) Distinct regulatory mechanisms and functions for p53-activated and p53-repressed DNA damage response genes in embryonic stem cells. Mol Cell 46:30-42
Li, Mangmang; He, Yunlong; Feng, Xi et al. (2012) Genome-wide studies of the transcriptional regulation by p53. Biochim Biophys Acta 1819:684-7
He, Yunlong; Korboukh, Ilia; Jin, Jian et al. (2012) Targeting protein lysine methylation and demethylation in cancers. Acta Biochim Biophys Sin (Shanghai) 44:70-9
Liu, Feng; Barsyte-Lovejoy, Dalia; Allali-Hassani, Abdellah et al. (2011) Optimization of cellular activity of G9a inhibitors 7-aminoalkoxy-quinazolines. J Med Chem 54:6139-50
Li, Mangmang; Huang, Jing (2010) A new puzzling role of p53 in mouse embryonic stem cells. Cell Cycle 9:1669-70

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