http://orcid.org/0000-0002-3956-6576
Figures
Abstract
PIERCE1, p53 induced expression 1 in Rb null cells, is a novel p53 target involved in the DNA damage response and cell cycle in mice. These facts prompted us to study the function of PIERCE1 with respect to p53-associated pathophysiology of cancer in humans. Unexpectedly, PIERCE1 did not respond to overexpression and activation of p53 in humans. In this study, we swapped p53 protein expression in human and mouse cells to find the clue of this difference between species. Human p53 expression in mouse cells upregulated PIERCE1 expression, suggesting that p53-responsive elements on the PIERCE1 promoter are crucial, but not the p53 protein itself. Indeed, in silico analyses of PIERCE1 promoters revealed that p53-responsive elements identified in mice are not conserved in humans. Consistently, chromatin immunoprecipitation-sequencing (ChIP-seq) analyses confirmed p53 enrichment against the PIERCE1 promoter region in mice, not in human cells. To complement the p53 study in mice, further promoter analyses suggested that the human PIERCE1 promoter is more similar to guinea pigs, lemurs, and dogs than to rodents. Taken together, our results confirm the differential responsiveness of PIERCE1 expression to p53 due to species differences in PIERCE1 promoters. The results also show partial dissimilarity after p53 induction between mice and humans.
Citation: Kim HJ, Lee SE, Na H, Roe J-S, Roh J-i, Lee H-W (2020) Divergence of the PIERCE1 expression between mice and humans as a p53 target gene. PLoS ONE 15(8): e0236881. https://doi.org/10.1371/journal.pone.0236881
Editor: Klaus Roemer, Universitat des Saarlandes, GERMANY
Received: February 4, 2020; Accepted: July 16, 2020; Published: August 3, 2020
Copyright: © 2020 Kim 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.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by grants from the National Research Foundation of Korea (NRF; https://www.nrf.re.kr/eng/index) funded by the Korean government (MEST; 2018R1A2A1A05022746 and 2017R1A4A1015328) to Han-Woong Lee. 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.
Introduction
PIERCE1, p53-induced expression 1 in retinoblastoma (RB)-null cells also known as C9orf116 and RbEst47, was first shown to be upregulated in Rb-deficient mouse embryonic fibroblasts (MEFs) [1]. PIERCE1 is highly induced by p53 overexpression and activation because it consists of three p53-responsive elements at the region adjacent to the transcription start site [2]. As a result, genotoxic stresses such as ultraviolet C (UVC) light can activate PIERCE1 expression [2]. PIERCE1 expression fluctuates during the cell cycle, and its protein stability is highly affected by the proteasome system as it harbors the PEST domain at the N-terminus [1]. PIERCE1 expression is restricted in normal human tissues such as the brain, kidney, and lung [1]. In vivo analyses of PIERCE1 knockout (KO) mice revealed that approximately half of PIERCE1 homozygous KO mice exhibit embryonic lethality due to developmental defects, and 40% of survivors have situs inversus totalis, which reverses all organs from their normal positions [3].
p53, as an essential tumor suppressor gene, is a guardian of the genome that is the most frequently mutated or deleted in approximately 50% of all human cancers [4–7]. Activated p53 under stress conditions regulates target gene expression, resulting in the alteration of multiple physiologies including senescence, apoptosis, metabolism, and the cell cycle [8–11]. p53 is a sequence-specific pleiotropic transcription factor that binds specifically to a palindromic consensus sequence, RRRCWWGYYY (N0-13) RRRCWWGYYY [12] with at least one mismatch. Mutation hotspots in p53 are mainly found in the DNA binding region across tumor types, suggesting that transcriptional activity is critical for its tumor suppressor function [13–15].
Since PIERCE1 is significantly induced after p53 overexpression and activation in mice, we examined tumor related PIERCE1 function under p53 activation and mutation conditions in humans. Unexpectedly, PIERCE1 expression was not affected by p53 overexpression and activation in normal and cancerous human cell types, while mouse cells successfully induced Pierce1 expression regardless of p53 protein species origins. At the molecular level, the human PIERCE1 promoter did not harbor the p53-responsive element near the transcription start site, but mice have three responsive elements. Though mice have been used as a typical model organism due to several genetic and physiological advantages and similarities to humans [16], recent reports suggested that mouse models are less reliable as representative human disease models [17]. Thus, as a part of p53 targets are differ between two species, molecular and biochemical data that comparing mice and humans need to be accounted more carefully especially in aspect to p53.
Materials & methods
Mice
FVB/NTac mice and p53 KO (Trp53tm1Tyj) mice were purchased from Taconic Biosciences and Jackson Laboratory, respectively [18]. p53 heterozygous KO mice were backcrossed onto the FVB/N for more than 20 generations. All mice were maintained in the specific pathogen-free (SPF) facility of the Yonsei Animal Research Center [19–21]. Animal suffering was minimized, and all experiments were performed in accordance with the Korean Food and Drug Administration (KFDA) guidelines for animal research. The experimental protocols for generating the genetically engineered mouse models were approved by the Institutional Animal Care and Use Committee (IACUC-A-201709-359-03) at Yonsei University.
Cell culture
B16F1, H1299, and HCT-116 cells were purchased from the Korean Cell Line Bank (KCLB). MCF-7, A549, and BJ cells were purchased from the American Type Culture Collection (ATCC). Mouse embryonic fibroblasts (MEFs) were prepared and cultured as previously described [1]. Briefly, MEFs were isolated from 13.5-day embryos of wild-type (WT) and p53 KO mice. Lung cancer cells were isolated from tumor nodules of the KRASLa2 mouse model [22]. AC-16, Beas-2b, and 293A cells were provided by WJ Park (Gwangju Institute of Science and Technology) and HW Park (Yonsei University, Department of Biochemistry). All cells were cultured and maintained in RPMI 1640 (Gibco) or Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Sigma) and 1% penicillin/streptomycin (Gibco) at 37°C in a humidified chamber containing 5% CO2 [23].
Transfection, RNA isolation, and real-time quantitative PCR
RNA was isolated and used for quantitative PCR as previously described [24]. In brief, total RNAs from cells were prepared with TRIsure reagent (Bioline) after treating for 12 hours with cisplatin (Sigma) or transfection. Cell extracts were harvested after 24 hours of transfection with Lipofectamine 3000 reagent (Invitrogen) or 36 hours of transfection with RNAiMAX reagent (Thermo Fisher Scientific). One microgram of total RNA was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific), and quantitative PCR was performed using SYBR Green (Bioline) and CFX Connect Real-Time System (Bio Rad) [25]. Experiments were conducted in triplicate, and signals were normalized to mouse and human GAPDH. All primers and siRNA sequences are listed in S1 and S2 Tables.
Reporter gene constructs and luciferase assays
Reporter genes were constructed by subcloning the genomic DNA fragment containing the PIERCE1 promoter region into the pGL3 Basic vector (Promega) [2]. p53 response elements were predicted by directly comparing DNA sequences with the consensus sequence [2, 12]. The mutant PIERCE1 promoter constructs were generated using PCR-based mutagenesis [26] with the following primers: 5’- AAC AGG ACG CCG CCT TGC CGC AGC AGG CAC AGA CTT GAT CGC TTC TCC TCC AGG CAC AAT GT-3’ and 5’- GAG GAG AAG CGA TCA AGT CTG TGC CTG CTG CGG CAA GGC GGC GTC CTG TTG CCA AGC GAC GG-3’ for BS1; 5’- GGC AGG TTC CAG ACT TGC CTA CAG CTA GCT GCC CGG CCC ACG CGC GGC GCC TT-3’ and 5’- GTG GGC CGG GCA GCT AGC TGT AGG CAA GTC TGG AAC CTG CCG GGC GAC TCC CCA AG-3’ for BS2 and were verified by sequencing. Reporter genes and effector constructs pcDNA3-p53 were transfected with Lipofectamine 3000 reagent (Invitrogen). Luciferase assays were conducted as previously described [27].
Chromatin immunoprecipitation-sequencing (ChIP-seq) enrichment peak analysis
The enrichment peaks were analyzed as previously described [28]. In brief, human and mouse p53 ChIP-seq data were aligned to the human and mouse reference genome assemblies hg19 and mm9 [29, 30], respectively. The peaks were re-calculated by normalizing to read depth of 10 million. The Gene Expression Omnibus accession number for the acquisition of publicly available ChIP-Seq data reported in this paper is GSE55727 (p53 ChIP-Seq in MEF) and GSE86164 (p53 ChIP-Seq in HCT116 and MCF7).
Results
Differential regulation of PIERCE1 expression by p53 in mice and humans
PIERCE1 is a novel p53 target involved in the DNA damage response in mice [1, 2]. Since mice are predicted to have similar genetic and physiological aspects to humans [31, 32], these data prompted us to examine whether PIERCE1 expression is affected by p53 and apply the previous results to humans to investigate p53-related and tumor regulating functions of PIERCE1. Consistent with previous data, ectopic p53 expression significantly upregulated Pierce1 expression approximately 2.5- and 10-fold in mouse primary fibroblasts and cancer cells, respectively (Fig 1) [2]. However, unlike p53-dependent regulation in mouse cells, the transcript level of PIERCE1 was not affected by p53 overexpression in p53-deficient HCT-116 cells, even though induction of other p53 targets was confirmed (Fig 2A and 2B). Additionally, transient p53 overexpression and knockdown could not alter the PIERCE1 expression in six different cell lines originating from humans (Fig 2C–2G), suggesting that PIERCE1 is not the downstream target of p53 in humans.
(A–C) Relative p53 and Pierce1 mRNA level determined using qPCR in cells 24 hours after transient transfection with empty vector (yellow bars) or p53 (dark blue bars) in indicated mouse cell lines. p53-deficient mouse embryonic fibroblasts (MEFs, A) and cancer cells (B, melanoma; C, lung cancer) were used for the analyses. GAPDH was used for normalization. Data are presented as mean ± SD (Student’s t-test, **P<0.01, ***P<0.001).
(A-B) RT-PCR analyses of p53 (A) and its target (B) mRNA levels 24 hours after transient transfection of empty vector (yellow bars) and p53 (red bars) in p53 KO HCT-116 cells. (C-F) RT-PCR analyses of p53 and PIERCE1 transcripts 24 hours after transient transfection of control (yellow bars) and p53 (red bars) in Beas-2b (C), MCF-7 (D), A549 (E), and H1299 (F) cell lines. (G) Relative p53 and PIERCE1 mRNA levels of no transfection (NT, white bars) or 36 hours after transient transfection of siRNA against control (siCTRL, gray bars) or p53 (sip53, black bars) in AC16 cells. The level of each gene was normalized to GAPDH. ns indicates no significant difference. Data are presented as mean ± SD (Student’s t-test, ***P<0.001).
Since genotoxic stress such as platinum treatment can promote p53 activity followed by induction of its downstream targets [6], genotoxic stresses were applied to examine gene expression alteration of p53 targets. Consistent with the previous report [2], significant upregulation of both p21 and Pierce1 was detected with cisplatin treatment conditions in MEFs (S1 Fig). However, unlike mice, the PIERCE1 mRNA level was not changed by cisplatin in human primary fibroblast and cancer cell lines, but the p21 mRNA level increased (Fig 3). These data implied that genotoxic stress-mediated p53 activation is not sufficient to induce human PIERCE1.
(A–E) Relative expression of p21 (green bars) and PIERCE1 (red bars) transcripts after 12 hours of cisplatin treatment at the indicated doses in BJ (A), HFF (B), p53 KO HCT-116 (C), MCF-7 (D), and A549 (E) cell lines. GAPDH was used for normalization. Data are presented as mean ± SD (Student’s t-test, triplicate).
Poor transcriptional activation is not due to p53 protein, but low conservation of p53 responsive elements in human PIERCE1 promoter
The differential responsiveness of PIERCE1 expression to p53 can be due to either the p53 protein or PIERCE1 promoter. To validate the factor that determines the species difference, the cDNA encoding human p53 was introduced into mouse cells and vice versa. Pierce1 transcript was significantly upregulated up to 23.5-fold when human p53 was overexpressed in MEFs, the response which is similar to that of mouse p53 (Figs 1A and 4A), whereas overexpression of mouse p53 did not affect PIERCE1 expression in p53 KO HCT-116 cells (Fig 4B and 4C). These results indicated that the PIERCE1 promoter, rather than the p53 protein, is crucial for determining the species differences.
(A) Relative mRNA level of p53 and Pierce1 24 hours after transient transfection of empty vector (yellow) or human p53 (red) in p53 KO MEF. (B-C) Relative mRNA level of p53 (B) and its targets (C) 24 hours after transient expression of empty vector (yellow bars), human p53 (red bars), and mouse p53 (dark blue bars) in p53 KO HCT-116 cells. GAPDH was used for normalization. Data are presented as mean ± SD (Student’s t-test, ***P<0.001; ns, no significance).
Since the PIERCE1 promoter is predicted to differ between mice and humans, in silico analyses of the PIERCE1 promoter were conducted to examine which element varies between them. Based on the previous reports [2, 12], the mouse Pierce1 promoter was compared to the human promoter and promoters in other species, especially in the regions of p53-responsive elements (Fig 5A and S2 Fig). By re-analyzing publicly available mouse and human p53 ChIP-Seq, we confirmed that only mouse PIERCE1 promoter selectively accumulates p53 while the promoter of known p53 target (i.e. CDKN1A) is occupied regardless of mouse and human cells (Fig 5B and 5C and S3 Fig). We measured p53 occupancy at the PIERCE1 promoter and compared it to the well-known p53 target gene, CDKN1A, in MEFs, HCT-116, and MCF-7 cell lines. Given that the p53 activation status is based on CDKN1A peaks, significant binding affinity at Pierce1 promoter was identified in MEFs. No remarkable interaction was seen in HCT-116, and MCF-7 cells (Fig 5B and 5C). These data indicated that p53 binding sites (BSs) in PIERCE1 promoter are not conserved in humans.
(A) A schematic view of PIERCE1 promoter region with p53-responsive elements (BS, shown in blue) in the mouse Pierce1 locus (upper panel) and predicted BS sites in the PIERCE1 locus of human and rat (human locus is shown in the bottom panel). Predicted BS sites are shown in blue, and two RRRCWWGYYY sequences, are highlighted in yellow. Mismatches with the p53 consensus sequence are shown in green. R, purine; Y, pyrimidine; W, adenine, or thymine. Arrows indicate the transcription start region. (B-C) ChIP-sequencing profiles and identified peaks of direct p53 binding to PIERCE1 in mouse cell, MEFs, (B) and human cell lines HCT-116, and MCF-7 (C). A schematic view below the profiles shows the PIERCE1 gene and promoter region. Exons are shown as blue boxes, and introns are marked by blue lines. (D) A conceptual diagram of WT and mutant PIERCE1 promoters with putative p53 BSs and relative luciferase activity of these promoters 24 hours after transfection of empty vector (yellow bars) or p53 (red bars) in 293A cells. Each mutant PIERCE1 constructs contains mouse p53 binding site as described with ovals. Gray bars, human PIERCE1 promoter; White ovals, WT; Black ovals, p53 BS sequence of mouse Pierce1. Data are presented as mean ± SD (Student’s t-test, triplicate).
To investigate whether alteration of putative p53 BSs from the human to mouse sequences can affect p53 responsiveness, we used mutant forms of human PIERCE1 promoter constructs called BS1mut, BS2mut, and BS1/2mut, which contain either mouse p53 binding site 1 or 2 (BS1 and 2), or both in human PIERCE1 promoter, respectively (Fig 5A and 5D). As expected, luciferase analyses revealed no notable activation of human PIERCE1 promoter by p53. However, mutant promoters, especially the constructs containing the BS1 mutation, showed significant upregulation of promoter activity in response to p53 (Fig 5D). Consistent with previous data [2], BS2mut did not respond to p53. These data suggest that p53-response elements are the determining factors of PIERCE1 expression across the species.
Proposal of alternative experimental animal models for p53 study
Although the mouse is the most frequently used model to examine p53 function, our data suggested that there is some inconsistency between mouse data and human cases. Thus, we investigated the PIERCE1 promoter in the representative animal models to suggest representatives with more similarity to humans. Sequence analyses in the PIERCE1 promoter region revealed that rat has high p53 BS consensus sequence homology with mouse, but these sites are poorly conserved in primates such as chimpanzees and humans (Fig 5A, the data for chimpanzee are not shown). Furthermore, the PIERCE1 promoter phylogenetic tree analyses suggested that zebrafish, dog, pika, and guinea pig have similar PIERCE1 promoter structure to humans (S2 Fig). Additionally, previous reports indicated that Lpin1 and Cpt1c genes are differentially controlled between mice and humans [33]. We also found that p53-responsive elements in these genes are preserved in rats, partially in guinea pigs and lemurs, but not in dogs and primates (S4 Fig). Furthermore, this tendency was stronger in the more closely related species. Collectively, these data indicate that mouse p53 is not fully representative of humans. On the contrary, guinea pig, lemur, and dog models might show a better correlation with human p53 function.
Discussion
Here, we identified PIERCE1 as a differentially-regulated gene across species due to its promoter region variation. These dissimilarities inhibit the responsiveness of PIERCE1 to human p53. In a proof-of-concept, mutagenesis of the human PIERCE1 promoter at p53-responsive elements to the mouse allele successfully enabled transcriptional activation of the human PIERCE1 by p53 or genotoxic stress conditions.
Analyses of PIERCE1 promoter sequences in several species suggested that zebrafish, dogs, guinea pigs, and lemurs were predicted to be similar to humans in expression patterns and p53 or genotoxic stress responsiveness. On the contrary, transcriptional activity and responsiveness to p53 in the mouse Pierce1 promoter might be similar to rats, frogs, squirrels, and tree shrews. The transcriptional responses to p53 are not restricted in PIERCE1, but approximately 1010 genes including POLK [34], LPIN1 [35], and CPT1C [36] also show species variation in their expression [33]. Gene ontology (GO) classification of these genes suggested that they are related to the DNA damage response, DNA metabolism, and cell cycle [33]. p53-induced DNA damage response and cell cycle alteration could vary between mice and humans due to the dissimilar expression of p53-dependent downstream target genes. Therefore, the use of animal models showing differential p53 responsiveness might interrupt proper p53 function interpretation.
Supporting information
S1 Fig. Pierce1 expression is responsible for cisplatin-induced p53 activation in mice.
Relative mRNA level of Pierce1 12 hours after cisplatin treatment at the indicated doses in MEFs. GAPDH was used for normalization. Data are presented as mean ± SD (Student’s t-test, triplicate).
https://doi.org/10.1371/journal.pone.0236881.s001
(TIF)
S2 Fig. Phylogenetic tree of the p53 binding sites of the PIERCE1 promoter region in the indicated species.
Deep blue lines show that no binding site exists. Light blue and yellow lines are predicted to harbor only BS2 or BS1, respectively. Red lines indicate that both two binding sites exist. (BS, binding site; O, exist; X, does not exist).
https://doi.org/10.1371/journal.pone.0236881.s002
(TIF)
S3 Fig. Identification of direct p53 binding sites.
ChIP-seq profiles and identified peaks of direct p53 binding to CDKN1A in mouse cells (MEFs) (A) and human HCT-116 and MCF-7 cell lines (B). The CDKN1A gene schematic is shown below. Exons are shown as blue boxes, and introns are marked in blue lines.
https://doi.org/10.1371/journal.pone.0236881.s003
(TIF)
S4 Fig. p53 responsiveness prediction based on promoter sequence analysis in several species.
p53 response sequence alignment of the Lpin1 (A) and Cpt1c (B) in mice, and its consensus regions in rats, guinea pigs, lemurs, dogs, and humans. Predicted response elements are shown in blue, and two RRRCWWGYYY sequences separated by 0 to 13 bp of spacer DNA are highlighted in yellow. Mismatches with the p53 consensus sequences are shown in green. R, purine; Y, pyrimidine; W, adenine, or thymine.
https://doi.org/10.1371/journal.pone.0236881.s004
(TIF)
References
- 1. Sung YH, Kim HJ, Lee HW. Identification of a novel Rb-regulated gene associated with the cell cycle. Mol Cells. 2007;24(3):409–15. Epub 2008/01/10. pmid:18182857.
- 2. Sung YH, Kim HJ, Devkota S, Roh J, Lee J, Rhee K, et al. Pierce1, a novel p53 target gene contributing to the ultraviolet-induced DNA damage response. Cancer Res. 2010;70(24):10454–63. Epub 2010/12/17. pmid:21159655.
- 3. Sung YH, Baek IJ, Kim YH, Gho YS, Oh SP, Lee YJ, et al. PIERCE1 is critical for specification of left-right asymmetry in mice. Sci Rep. 2016;6:27932. Epub 2016/06/17. pmid:27305836; PubMed Central PMCID: PMC4917697.
- 4. Liu ZG, Baskaran R, Lea-Chou ET, Wood LD, Chen Y, Karin M, et al. Three distinct signalling responses by murine fibroblasts to genotoxic stress. Nature. 1996;384(6606):273–6. Epub 1996/11/21. pmid:8918879.
- 5. Beroud C, Soussi T. p53 gene mutation: software and database. Nucleic Acids Res. 1998;26(1):200–4. Epub 1998/02/21. pmid:9399836; PubMed Central PMCID: PMC147206.
- 6. Colman MS, Afshari CA, Barrett JC. Regulation of p53 stability and activity in response to genotoxic stress. Mutat Res. 2000;462(2–3):179–88. Epub 2000/04/18. pmid:10767629.
- 7. Nam TW, Park SY, Lee JH, Roh JI, Lee HW. Effect of EI24 expression on the tumorigenesis of Apc(Min/+) colorectal cancer mouse model. Biochem Biophys Res Commun. 2019;514(4):1087–92. Epub 2019/05/18. pmid:31097220.
- 8. Horvath MM, Wang X, Resnick MA, Bell DA. Divergent evolution of human p53 binding sites: cell cycle versus apoptosis. PLoS Genet. 2007;3(7):e127. Epub 2007/08/07. pmid:17677004; PubMed Central PMCID: PMC1934401.
- 9. Harms K, Nozell S, Chen X. The common and distinct target genes of the p53 family transcription factors. Cell Mol Life Sci. 2004;61(7–8):822–42. Epub 2004/04/20. pmid:15095006.
- 10. Wong J, Li PX, Klamut HJ. A novel p53 transcriptional repressor element (p53TRE) and the asymmetrical contribution of two p53 binding sites modulate the response of the placental transforming growth factor-beta promoter to p53. J Biol Chem. 2002;277(29):26699–707. Epub 2002/05/16. pmid:12011055.
- 11. Lee TK, Park YE, Park CW, Kim B, Lee JC, Park JH, et al. Age-dependent changes of p53 and p63 immunoreactivities in the mouse hippocampus. Lab Anim Res. 2019;35:20. Epub 2020/04/08. pmid:32257908; PubMed Central PMCID: PMC7081572.
- 12. el-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B. Definition of a consensus binding site for p53. Nat Genet. 1992;1(1):45–9. Epub 1992/04/01. pmid:1301998.
- 13. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997;88(3):323–31. Epub 1997/02/07. pmid:9039259.
- 14. Ko LJ, Prives C. p53: puzzle and paradigm. Genes Dev. 1996;10(9):1054–72. Epub 1996/05/01. pmid:8654922.
- 15. Harris CC. p53: at the crossroads of molecular carcinogenesis and risk assessment. Science. 1993;262(5142):1980–1. Epub 1993/12/24. pmid:8266092.
- 16. Jonas AM. The mouse in biomedical research. Physiologist. 1984;27(5):330–46. pmid:6393160.
- 17. Perlman RL. Mouse models of human disease: An evolutionary perspective. Evol Med Public Health. 2016;2016(1):170–6. pmid:27121451; PubMed Central PMCID: PMC4875775.
- 18. Jacks T, Remington L, Williams BO, Schmitt EM, Halachmi S, Bronson RT, et al. Tumor spectrum analysis in p53-mutant mice. Curr Biol. 1994;4(1):1–7. Epub 1994/01/01. pmid:7922305.
- 19. Kim H, Jeong H, Cho Y, Lee J, Nam KT, Lee HW. Disruption of the Tff1 gene in mice using CRISPR/Cas9 promotes body weight reduction and gastric tumorigenesis. Lab Anim Res. 2018;34(4):257–63. pmid:30671113; PubMed Central PMCID: PMC6333602.
- 20. Park BM, Roh JI, Lee J, Lee HW. Generation of knockout mouse models of cyclin-dependent kinase inhibitors by engineered nuclease-mediated genome editing. Lab Anim Res. 2018;34(4):264–9. Epub 2019/01/24. pmid:30671114; PubMed Central PMCID: PMC6333600.
- 21. Lee H, Kim JI, Park JS, Roh JI, Lee J, Kang BC, et al. CRISPR/Cas9-mediated generation of a Plac8 knockout mouse model. Lab Anim Res. 2018;34(4):279–87. Epub 2019/01/24. pmid:30671116; PubMed Central PMCID: PMC6333607.
- 22. Johnson L, Mercer K, Greenbaum D, Bronson RT, Crowley D, Tuveson DA, et al. Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature. 2001;410(6832):1111–6. Epub 2001/04/27. pmid:11323676.
- 23. Jin Y, You L, Kim HJ, Lee HW. Telomerase Reverse Transcriptase Contains a BH3-Like Motif and Interacts with BCL-2 Family Members. Mol Cells. 2018;41(7):684–94. Epub 2018/06/26. pmid:29937479; PubMed Central PMCID: PMC6078858.
- 24. Roh JI, Kim Y, Oh J, Kim Y, Lee J, Lee J, et al. Hexokinase 2 is a molecular bridge linking telomerase and autophagy. PLoS One. 2018;13(2):e0193182. pmid:29462198; PubMed Central PMCID: PMC5819818.
- 25. Roh JI, Lee J, Park SU, Kang YS, Lee J, Oh AR, et al. CRISPR-Cas9-mediated generation of obese and diabetic mouse models. Exp Anim. 2018;67(2):229–37. Epub 2018/01/19. pmid:29343656; PubMed Central PMCID: PMC5955754.
- 26. Roh JI, Cheong C, Sung YH, Lee J, Oh J, Lee BS, et al. Perturbation of NCOA6 leads to dilated cardiomyopathy. Cell Rep. 2014;8(4):991–8. Epub 2014/08/19. pmid:25131203; PubMed Central PMCID: PMC4150217.
- 27. Oh W, Lee EW, Sung YH, Yang MR, Ghim J, Lee HW, et al. Jab1 induces the cytoplasmic localization and degradation of p53 in coordination with Hdm2. J Biol Chem. 2006;281(25):17457–65. Epub 2006/04/21. pmid:16624822.
- 28. Roe JS, Hwang CI, Somerville TDD, Milazzo JP, Lee EJ, Da Silva B, et al. Enhancer Reprogramming Promotes Pancreatic Cancer Metastasis. Cell. 2017;170(5):875–88 e20. pmid:28757253; PubMed Central PMCID: PMC5726277.
- 29. Andrysik Z, Galbraith MD, Guarnieri AL, Zaccara S, Sullivan KD, Pandey A, et al. Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity. Genome Res. 2017;27(10):1645–57. Epub 2017/09/15. pmid:28904012; PubMed Central PMCID: PMC5630028.
- 30. Kenzelmann Broz D, Spano Mello S, Bieging KT, Jiang D, Dusek RL, Brady CA, et al. Global genomic profiling reveals an extensive p53-regulated autophagy program contributing to key p53 responses. Genes Dev. 2013;27(9):1016–31. Epub 2013/05/09. pmid:23651856; PubMed Central PMCID: PMC3656320.
- 31. Bedell MA, Jenkins NA, Copeland NG. Mouse models of human disease. Part I: techniques and resources for genetic analysis in mice. Genes Dev. 1997;11(1):1–10. pmid:9000047.
- 32. Bedell MA, Largaespada DA, Jenkins NA, Copeland NG. Mouse models of human disease. Part II: recent progress and future directions. Genes Dev. 1997;11(1):11–43. pmid:9000048.
- 33. Fischer M. Conservation and divergence of the p53 gene regulatory network between mice and humans. Oncogene. 2019;38(21):4095–109. Epub 2019/02/03. pmid:30710145; PubMed Central PMCID: PMC6755996.
- 34. Velasco-Miguel S, Richardson JA, Gerlach VL, Lai WC, Gao T, Russell LD, et al. Constitutive and regulated expression of the mouse Dinb (Polkappa) gene encoding DNA polymerase kappa. DNA Repair (Amst). 2003;2(1):91–106. Epub 2003/01/02. pmid:12509270.
- 35. Assaily W, Rubinger DA, Wheaton K, Lin Y, Ma W, Xuan W, et al. ROS-mediated p53 induction of Lpin1 regulates fatty acid oxidation in response to nutritional stress. Mol Cell. 2011;44(3):491–501. Epub 2011/11/08. pmid:22055193.
- 36. Sanchez-Macedo N, Feng J, Faubert B, Chang N, Elia A, Rushing EJ, et al. Depletion of the novel p53-target gene carnitine palmitoyltransferase 1C delays tumor growth in the neurofibromatosis type I tumor model. Cell Death Differ. 2013;20(4):659–68. Epub 2013/02/16. pmid:23412344; PubMed Central PMCID: PMC3595492.