TRIM25 Has a Dual Function in the P53/Mdm2 Circuit

ORIGINAL ARTICLE TRIM25 has a dual function in the p53/Mdm2 circuit

P Zhang1,2, S Elabd1, S Hammer3, V Solozobova1,HYan1,2, F Bartel3, S Inoue4, T Henrich5, J Wittbrodt6, F Loosli1, G Davidson1 and C Blattner1

P53 is an important tumor suppressor that, upon activation, induces growth arrest and cell death. Control of p53 is thus of prime importance for proliferating cells, but also for cancer therapy, where p53 activity contributes to the eradication of tumors. Mdm2 functionally inhibits p53 and targets the tumor suppressor protein for degradation. In a genetic screen, we identiﬁed TRIM25 as a novel regulator of p53 and Mdm2. TRIM25 increased p53 and Mdm2 abundance by inhibiting their ubiquitination and degradation in 26 S proteasomes. TRIM25 co-precipitated with p53 and Mdm2 and interfered with the association of p300 and Mdm2, a critical step for p53 polyubiquitination. Despite the increase in p53 levels, p53 activity was inhibited in the presence of TRIM25. Downregulation of TRIM25 resulted in an increased acetylation of p53 and p53-dependent cell death in HCT116 cells. Upon genotoxic insults, TRIM25 dampened the p53-dependent DNA damage response. The downregulation of TRIM25 furthermore resulted in massive apoptosis during early embryogenesis of medaka, which was rescued by the concomitant downregulation of p53, demonstrating the functional relevance of the regulation of p53 by TRIM25 in an organismal context.

Oncogene (2015) 34, 5729–5738; doi:10.1038/onc.2015.21; published online 2 March 2015

INTRODUCTION RESULTS The p53 protein is a transcription factor that mainly controls the TRIM25 increases the abundance of p53 and Mdm2 by protein transcription of cell cycle control and cell death genes.1 As an anti- stabilization proliferative protein, p53 is tightly controlled. The major regulator We identiﬁed TRIM25, a protein belonging to the family of TRIMs, in a for p53 is Mdm2, which binds to the transactivation domain of p53 cell culture-based expression screen that we performed in order to and mediates its ubiquitination and degradation.2 As mdm2 is identify novel regulators of p53 and Mdm2. The overexpression of also a target gene of p53, both proteins function in a negative TRIM25 increased p53 and Mdm2 abundance in a dose-dependent feed-back loop. Several other proteins impinge on this negative manner (Figure 1a; Supplementary Figure S1A), whereas the feed-back loop and affect the abundance and activity of p53 and/or downregulation of TRIM25 reduced p53 and Mdm2 levels 4 Mdm2.2 (Figure 1b). As TRIM25 levels can be increased by β-estradiol, we TRIM25 belongs to the Tripartite motif protein (TRIM) family, tested the effect of this steroid hormone on the induction of p53 and which contains an N-terminal RING (really interesting new gene)- Mdm2 in order to further investigate the physiologic relevance. domain, one or two B-boxes and a coiled-coil region.3 TRIM25 is When we treated MCF7 cells with β-estradiol, we observed a ﬁ induced by estrogen and is therefore particularly abundant in signi cant upregulation of both p53 and Mdm2 that accompanied β placenta and uterus. TRIM25 mRNA expression is also high in the -estradiol-mediated upregulation of TRIM25 (Figure 1c, thyroid gland, aorta and spleen, but low in most other tissues.4,5 Supplementary Figure S1B). The downregulation of TRIM25 by two different small interfering RNAs strongly reduced or blocked The absence of TRIM25 leads to underdeveloped uteri in female β-estradiol- mediated induction of p53 and Mdm2, respectively mice and reduces estrogen-responsiveness,6 whereas its over- (Figure 1c, Supplementary Figure S1B). This indicated that the expression has been observed in several ovarian and breast induction of p53 and Mdm2 by β-estradiol is (at least partly) tumors and is associated with advanced disease and poor 7 mediated by TRIM25 and that the regulation of p53 and Mdm2 by prognosis. Similar to other RING-domain proteins, TRIM25 TRIM25 occurs under physiologically relevant conditions. possesses E3 ligase activity and is able to transfer ubiquitin We next investigated the mechanism leading to increased 8 and ISG15 to target proteins. abundance of p53 and Mdm2 by TRIM25. Since p53 and Mdm2 are We show that TRIM25 enhances p53 and Mdm2 abundance connected by a negative feed-back loop,2 we asked whether by preventing their proteasomal degradation. At the same TRIM25 can increase p53 abundance in the absence of over- time, TRIM25 inhibits p53’s transcriptional activity and dampens expressed Mdm2 (and Mdm2 abundance in the absence of p53). the response to DNA damage. TRIM25 is essential for medaka We therefore co-transfected increasing amounts of TRIM25 into development and this dependence is rescued by silencing H1299 cells together with p53 but without Mdm2 (and together of p53. with Mdm2 but without p53). Figure 2a shows that the

1Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany; 2Faculty of Biosciences, University of Heidelberg, Heidelberg, Germany; 3Institute of Pathology, Faculty of Medicine, University of Halle-Wittenberg, Halle, Germany; 4Department of Anti-Aging Medicine, University of Tokyo, Tokyo, Japan; 5Chemistry/Biology Combined Major Program, International College, Osaka University, Osaka, Japan and 6Center for Organismal Studies, Developmental Biology and Physiology, Heidelberg University, Heidelberg, Germany. Correspondence: Dr C Blattner or Dr G Davidson, Institute of Toxicology and Genetics, Karlsruher Institute of Technology, PO Box 3640, Karlsruhe 76021, Baden-Württemberg, Germany. E-mail: [email protected] or [email protected] The authors declare no conflict of interest. Received 26 May 2014; revised 17 December 2014; accepted 19 December 2014; published online 2 March 2015 TRIM25 regulates p53 and Mdm2 P Zhang et al 5730 upregulation of p53 by TRIM25 only occurred when Mdm2 was co- P53 can be ubiquitinated by several ubiquitin ligases and these transfected (Figure 2a, lanes 1–4), whereas a TRIM25-mediated ubiquitin ligases usually need to interact directly with p53.2 As increase in Mdm2 levels did not require p53 (Figure 2a, lanes 5–8). TRIM25 reduced ubiquitination of p53 after the overexpression of Considering that p53 and Mdm2 are mainly regulated by Mdm2, we wondered whether this might be caused by a protein stability,2 we next asked whether TRIM25 increases p53 reduction of the interaction of p53 and Mdm2. To test this, we and Mdm2 abundance by inhibiting their degradation. We overexpressed p53 and Mdm2 in H1299 cells in the presence and therefore used cycloheximide to block de novo protein synthesis absence of TRIM25, immunoprecipitated p53 and monitored the after the overexpression of p53, Mdm2 and TRIM25 in H1299 cells abundance of Mdm2 by western blotting. In contrast to our and monitored the decay of p53 and Mdm2 by western blotting. expectation, TRIM25 did not reduce the association of p53 and Without co-transfection of TRIM25, p53 and Mdm2 showed a half- Mdm2. In fact, it slightly enhanced their association (Figure 2d, life of ~ 20-25 min, however, this was strongly increased after the compare lanes 2 and 3), probably owing to an increase in the overexpression of TRIM25 (Figure 2b). TRIM25 did not affect the abundance of p53 and Mdm2 in the presence of TRIM25. levels of p53 and Mdm2 mRNA (Supplementary Figure S2A), A further requirement for p53 polyubiquitination is the supporting the idea that the regulation of p53 and Mdm2 interaction of Mdm2 with p300.9 We therefore tested whether abundance by TRIM25 occurs by protecting the proteins from TRIM25 interferes with the binding of p300 to Mdm2. We degradation. In order to see whether this principle also applies downregulated TRIM25 in HCT116 cells, precipitated p300 and under more physiologically relevant conditions, we treated MCF7 4 monitored the associated Mdm2 by western blotting. As shown in cells with estrogen, which increases TRIM25 abundance. In line Figure 2e, only a minor amount of Mdm2 was associated with with the results obtained after the overexpression of TRIM25, p300 under normal growth conditions. Downregulation of TRIM25, treatment with estrogen increased the half-life of p53 and Mdm2 however, resulted in a strong increase in the amount of Mdm2 (Supplementary Figure S2B). that was bound to p300, whereas the amount of p53 that was Most proteins that are degraded by 26 S proteasomes require bound to p300 was not affected (Figure 2e). This result suggests covalent attachment of polyubiquitin chains. As TRIM25 stabilized that TRIM25 reduces p53 polyubiquitination by hindering the the p53 and Mdm2 protein, we asked whether TRIM25 affects their binding of p300 to Mdm2. polyubiquitination. We therefore transfected H1299 cells with His- tagged ubiquitin together with Mdm2 and/or p53 and TRIM25, purified ubiquitinated proteins by using metal affinity and TRIM25 associates with p53 and Mdm2 monitored the abundance of p53 and Mdm2 by western blotting. As TRIM25 interfered with p53 and Mdm2 polyubiquitination, we As shown in Figure 2c, the overexpression of TRIM25 strongly predicted that TRIM25 would associate with p53 and Mdm2. To reduced the polyubiquitination of both p53 and Mdm2 (Figure 2c). investigate this, we precipitated endogenous TRIM25 from MCF7

Figure 1. TRIM25 increases the abundance of p53 and Mdm2. (a) H1299 cells were transfected with increasing amounts of V5-tagged human TRIM25 and with p53 and Mdm2. Twenty-four hours after transfection, the amounts of p53, Mdm2 and V5-tagged TRIM25 were monitored by western blotting. Hybridization with an antibody targeted against PCNA (proliferating cell nuclear antigen) was performed for loading control. (b) HCT116 cells were transfected with two different TRIM25 siRNAs (small interfering RNAs) or with control siRNA. Seventy-eight hours after transfection, cells were harvested and the abundance of p53, Mdm2 and TRIM25 was monitored by western blotting. (c) MCF7 cells were transfected with 100 nM of a TRIM25 siRNA or a control siRNA. Forty-eight hours after transfection, cells were treated with 10 nM β-estradiol. Twenty-four hours after β-estradiol addition, the abundance of p53, Mdm2 and TRIM25 was monitored by western blotting.

Figure 2. TRIM25 increases p53 and Mdm2 abundance by protein stabilization (a) H1299 cells were transfected with increasing amounts of a plasmid encoding V5-tagged hTRIM25 together with p53 or Mdm2. Twenty-four hours after transfection, the amount of p53, Mdm2 and V5- tagged TRIM25 was monitored by western blotting. (b) H1299 cells were co-transfected with p53, Mdm2 and TRIM25 or with control DNA. Twenty-four hours after transfection, cycloheximid (CHX) was added. Cells were harvested at the indicated times after CHX addition and the amounts of p53, Mdm2 and TRIM25 were monitored by western blotting. Signals for p53, Mdm2 and PCNA (proliferating cell nuclear antigen) were quantiﬁed densitometrically and the relative amounts of p53 and Mdm2 were assessed. Mean values and s.d. of three independent experiments were calculated and plotted. Relative abundance of p53 and Mdm2 protein at the time of CHX addition was set to 100%. P-values were calculated by two-tailed t-test (*Po0.05; **Po0.01). (c) H1299 cells were co-transfected with Mdm2, His-tagged ubiquitin (His-ubi) p53 and TRIM25 in the indicated combinations. Twenty-four hours after transfection, the cells were treated with MG132 for 8 h before harvesting. An aliquot of the cells was analyzed for the expression of p53, Mdm2 and TRIM25 (input). The remaining cells were lysed in guanidinium buffer. Ubiquitinated proteins were collected by adsorption to Ni2+-agarose, eluted and analyzed by western blotting for the presence of p53 and Mdm2. (d) H1299 cells were co-transfected with plasmids encoding Mdm2 and p53 or TRIM25 or both. Twenty-four hours after transfection, MG132 was added to a ﬁnal concentration of 10 μM. Four hours after MG132 addition, the cells were lysed. The cellular lysate (5%) was used to monitor the expression of p53, Mdm2 and TRIM25 by western blotting (input). From the remaining 95% of the cellular lysate p53 was immunoprecipitated. Precipitation with IgG was performed for control. The precipitates were resolved by gel electrophoresis. Immunoprecipitated p53 and associated Mdm2 were detected by western blotting. (e) HCT116 cells were transfected with siRNA targeted against TRIM25. Forty-eight hours after transfection the cells were lysed. An aliquot of the cells was analyzed for the abundance of Mdm2, p53 and TRIM25 (input). From the remaining cells, p300 was immunoprecipitated and associated p53 and Mdm2 were detected by western blotting.

Oncogene (2015) 5729 – 5738 © 2015 Macmillan Publishers Limited TRIM25 regulates p53 and Mdm2 P Zhang et al 5731 cells and monitored the associated p53 and Mdm2 by western co-precipitated with p53 (Figure 3b) and TRIM25 and p53 co- blotting. In agreement with our prediction, Mdm2 and p53 precipitated with Mdm2 (Figure 3c). In order to get further co-precipitated with TRIM25 (Figure 3a). Furthermore, in reciprocal evidence that these interactions are indeed physiologic, we co-immunoprecipitation experiments, TRIM25 and Mdm2 performed sucrose density centrifugation. With this approach, it

Figure 3. TRIM25 interacts with Mdm2 and p53. (a) MCF7 cells were lysed. The cellular lysate (5%) was used to monitor the expression of p53, Mdm2 and TRIM25 by western blotting (input). From the remaining cellular lysate, TRIM25 was immunoprecipitated and associated p53 and Mdm2 were detected by western blotting. Precipitation with IgG was performed for control. (b) MCF7 cells were lysed. The cellular lysate (5%) was used to monitor the expression of p53, Mdm2 and TRIM25 by western blotting (input). From the remaining cellular lysate, p53 was immunoprecipitated and processed as described in part A. (c) MCF7 cells were lysed. The cellular lysate (5%) was used to monitor the expression of p53, Mdm2 and TRIM25 by western blotting (input). From the remaining cellular lysate, Mdm2 was immunoprecipitated and processed as described in part A.

is possible to separate higher-order complexes under native (Supplementary Figure S4A). Interestingly, the abundance of conditions. Although the majority of p53 eluted in fractions that Mdm2 RNA was not significantly changed under these conditions. contained protein complexes of a molecular weight 4500 kD, One function of p53 is to induce apoptosis.2 Indeed, when whereas TRIM25 eluted in fractions that contained proteins and TRIM25 was downregulated in HCT116 cells by siRNA, a strong protein complexes with a molecular weight below o500 kD, both increase in cell death was observed (Supplementary Figure S4B) proteins were detected in fractions 20–26. Of note, the majority of together with the cleavage of PARP and Caspase 3 (Figure 4c; Mdm2 that is well known to form a complex with p532 also eluted Supplementary Figure S4C). Importantly, cell death did not occur in -/- in fractions with a molecular weight o500 kD. Importantly, the HCT116 p53 cells, demonstrating that TRIM25 regulates cell death majority of Mdm2 eluted in fraction 20–26, which also contained in a p53-dependent manner (Figure 4c, Supplementary Figure S4B). p53 and TRIM25 (Supplementary Figure S3A). p53 and Mdm2 are It should be noted that in p53-negative HCT116 cells, we also primarily nuclear proteins.10 In order to determine whether all the observed some reduction in cell number upon downregulation of fi three proteins are in the same cellular compartment we TRIM25, however, this decrease was signi cantly weaker than in performed cell fractionation with subsequent western blotting. HCT116 cells with wild-type p53 (Supplementary Figure S4D). β-Actin (GAPDH in Supplementary Figure S3B.II) and p300 were One condition that would result in increased transcriptional used as markers for cytoplasmic and nuclear fractions, respec- activity of p53 would be increased binding of p53 to promoters of tively. As expected, the majority of p53 and Mdm2 were detected target genes. However, in spite of the increase in p53 transcrip- in the nuclear compartment. A significant amount (almost 50%) of tional activity after RNAi-mediated downregulation of TRIM25, we TRIM25 was also nuclear (Supplementary Figure S3B.I), showing observed a decrease in the binding of p53 to its consensus DNA that a significant part of all the three proteins is present in the (Supplementary Figure S4E.I). The reduction in binding most likely mirrors the decrease in the overall abundance of p53 upon same cellular compartment. Moreover, as shown in supplementary reduction of TRIM25 levels (Supplementary Figure S4E.II). This Figure 3B.II, Mdm2 and p53 co-precipitated with TRIM25 result implies that it is not the absolute amount of p53 bound to irrespective of whether the cytoplasmic or nuclear lysate was promoter sites that determines the activity of p53 but that used (Supplementary Figure S3B.II). additional alterations, like post-translational modifications of the As TRIM25 associated with p53 and Mdm2, and p53 strongly bound p53, dictate the transcriptional response. binds to Mdm22, we next asked whether p53 could influence the fl As p53-dependent induction of cell death requires acetylation binding of Mdm2 to TRIM25 and whether Mdm2 could in uence of C-terminal lysines,11 we tested whether the downregulation of the binding of p53 to TRIM25. We therefore overexpressed TRIM25 TRIM25 affected p53 acetylation. We therefore probed the cell and Mdm2 in the presence or absence of p53 and TRIM25 and p53 lysates with an antibody against acetylated lysine 382 of p53. in the presence and absence of Mdm2. The overexpression of p53 Indeed, lysates from HCT116 cells treated with TRIM25 siRNA had only a minor effect on the association of Mdm2 with TRIM25 showed a strong increase in p53 acetylation (Figures 4c and e, (Supplementary Figure S3C); however, the overexpression of Supplementary Figure S4C). Also TRIM25-/- mouse embryonic Mdm2 strongly enhanced the association of p53 with TRIM25 fibroblasts showed enhanced acetylation of p53. This acetylation (Supplementary Figure S3D). was accompanied by a strong increase in the abundance of the histone acetyltransferase p300 (Figure 4d), a major acetyltransfer- TRIM25 reduces p53 transcriptional activity ase for p53.12,13 Inhibition of p300 with curcumin14 strongly Usually, increased abundance of p53 is accompanied by an reduced the acetylation of p53 after the downregulation of increase in p53’s transcriptional activity. To see whether this is also TRIM25 (Figure 4e), showing that TRIM25 indeed controls p300- the case for p53 in the presence of TRIM25, we transfected the mediated acetylation of p53. Downregulation of TRIM25 and p53-dependent reporter PG13, which contains 13 repeats of the acetylation of p53 furthermore resulted in the induction of the p53-consensus site fused to a minimal promoter into MCF7 cells, p53 target gene P21 in MCF7 cells. When we concomitantly together with increasing amounts of TRIM25 and monitored the downregulated p300, acetylation and P21 induction were reporter activity. Surprisingly, the overexpression of TRIM25 did clearly reduced (Supplementary Figure S4F). Whether p300 is not increase but actually decreased p53-dependent transcriptional the only acetylase that is involved in this process remains to be activity (Figure 4a). In agreement with this result, the down- determined. regulation of TRIM25 resulted in an increased p53 reporter activity (Figure 4b). Consistently, the downregulation of TRIM25 increased TRIM25 reduces p53 activity in response to DNA damage mRNA levels of p21 and 14-3-3σ, whereas it decreased the mRNA P53 activity is most important during the DNA damage response. abundance of CDC25c, whose expression is repressed by p53 Here p53 accumulates to high levels and its activity is enhanced.2

Figure 4. TRIM25 reduces p53’s activity. (a) MCF7 cells were transfected with the p53-dependent reporter PG13 (Firefly luciferase) and Renilla luciferase, together with increasing amounts of TRIM25. 48 h after transfection, Firefly and Renilla luciferase activity was determined. Relative Firefly activity was calculated by normalization with the readings for Renilla luciferase. Relative Firefly activity of cells without TRIM25 transfection was set to 100%. The graph shows mean values and s.d. of three independent experiments. P-values were calculated by two- tailed t-test. *Po0.05; **Po0.01. (b) MCF7 cells were transfected with the p53-dependent reporter PG13 and Renilla luciferase together with a TRIM25 siRNA or a control siRNA. Forty-eight hours after transfection, cells were harvested and processed as described in the legend to part A. (c) HCT116 wild-type (wt) and HCT p53-/- cells were transfected with TRIM25 siRNA or with a control siRNA. Forty-eight hours after transfection, cells were analyzed for expression of p53, acetylated p53 (ac-p53), Mdm2, PARP, cleaved Caspase 3 and TRIM25. (d) Fibroblasts were prepared from murine embryos that possess two, one or no allele of TRIM25. Cells were harvested and analyzed for the expression of p300, p53, acetylated p53, Mdm2 and TRIM25. (e) HCT116 wt cells were transfected with two different siRNAs (small interfering RNA) targeted against TRIM25 or with a control siRNA. Forty-eight hours after transfection, the cells were treated with 25 or 40 μM (f.c.) curcumin. Twenty-four hours after curcumin addition, the cells were lysed and analyzed for acetylated p53, Mdm2 and TRIM25.

As TRIM25 inhibits p53 transcriptional activity, this raises the transcription. To investigate this possibility, we measured question whether p53 is released from this TRIM25-mediated p21-dependent reporter activity in response to DNA damage repression during the DNA damage response. One possibility by with or without overexpressed TRIM25. In the absence of DNA which this could be achieved is via a reduction in the amount of damage, the overexpression of TRIM25 reduced p21-dependent TRIM25 after DNA damage. Treatment of cells with γ-rays or reporter activity (Figure 5b) as it did with PG13-dependent etoposide, however, resulted in a slight but reproducible increase reporter activity (Figure 4a). As expected, in cells treated with in TRIM25 levels (Figure 5a). Alternatively, TRIM25 could be etoposide an increase in p21-dependent reporter activity was inhibited and thus no longer be able to repress p53-dependent detected (Figure 5b). Overexpression of TRIM25 reduced this

© 2015 Macmillan Publishers Limited Oncogene (2015) 5729 – 5738 TRIM25 regulates p53 and Mdm2 P Zhang et al 5734 p21-dependent reporter activity in response to DNA damage Developmental defects in medaka embryos induced by knocking (Figure 5b). Likewise, the overexpression of TRIM25 reduced the down TRIM25 are rescued by the silencing of p53 induction of endogenous P21 protein in response to DNA damage A TRIM25-like gene has been reported to be essential for early (Supplementary Figure S5A). Conversely, when we downregulated zebra-fish development.15 To investigate whether this is also the TRIM25, P21 and Bax levels were increased and this increase was case for medaka, we performed a functional analysis of these raised further in response to DNA damage (Figure 5c, proteins in medaka embryos during early development. Owing Supplementary Figure S5B). Likewise, mice with a genetic deletion to teleost-specific gene duplications, medaka possesses two of TRIM25 showed higher levels of the p53 targets P21 and 14-3- homologs of TRIM25 (Supplementary Figure S6A). Both homologs 3σ and these higher levels were further increased after DNA contain the RING-domain, the two B-boxes and the coiled-coil damage (Figure 5d), showing that TRIM25 reduces p53 activity domain (Supplementary Figure S6B) and both homologs of also in response to DNA damage. Interestingly, transcription of medaka TRIM25 are capable of increasing p53 and Mdm2 Mdm2 was again rather insensitive to the alterations in TRIM25 abundance (Supplementary Figure S6C). Although homolog-2 abundance (Figure 5d). was more efficient in regulating p53 and Mdm2 abundance than

Figure 5. TRIM25 reduces the activation of p53 in response to DNA damage. (a) U2OS cells were irradiated with γ-rays (5 Gy) or treated with etoposide (Eto; 50 μM) for the indicated time. Abundance of TRIM25, p53, Mdm2, P21 and PCNA (proliferating cell nuclear antigen; for loading control) was determined by western blotting. (b) U2OS cells were transfected with the P21-reporter (Firefly luciferase) and Renilla luciferase together with vector DNA or hTRIM25. Forty hours after transfection, Eto or the carrier were added. Sixteen hours after Eto addition, cells were harvested and the Firefly and Renilla luciferase activity was determined. Relative p21-promoter activity was calculated by normalization with the readings for Renilla luciferase. The value of cells treated with carrier was set to 100%. The graph shows mean values and s.d. of three independent experiments. P-values were calculated by two-tailed t-test. *Po0.05; **Po0.01. (c) HCT116 cells were transfected with TRIM25 siRNA or a control siRNA. Forty hours after transfection, cells were irradiated with γ-rays (5 Gy), treated with etoposide (Eto: 50 μM f.c.) or left untreated for control (C). Eight hours after DNA damage, the cells were lysed and the abundance of TRIM25, P21 and Bax was determined by western blotting. (d) Primary fibroblasts from mice with a genetic deletion of TRIM25 or from the corresponding wild-type mice were treated with etoposide (Eto) or left untreated for control (untd). Eight hours after treatment, the cells were harvested and the total RNA was prepared. The relative amount of P21, 14-3-3σ and Mdm2, mRNA was determined by qRT (quantitative reverse transcription)-PCR. Mean values and s.d. were calculated from the 2^dCT values of three independent experiments and blotted. Values of mRNA in cells transfected with control siRNA were set to 1. P-values were calculated by the two-tailed t-test (**Po0.01; *Po0.05; n.s, not significant).

Oncogene (2015) 5729 – 5738 © 2015 Macmillan Publishers Limited TRIM25 regulates p53 and Mdm2 P Zhang et al 5735 homolog 1, we cannot exclude that this difference is caused by was rather small, these data indicate that TRIM25 also controls p53 different expression levels, due to the lack of antibodies. activity in mammals. A prerequisite for the regulation of p53 by TRIM25 during development is that both proteins are temporally and spatially co- TRIM25 is frequently overexpressed in human ovarian tumors expressed. By whole-mount in situ hybridization we found that The p53/Mdm2 loop is an important hub for tumor development, p53, mdm2 and trim25 were all ubiquitously expressed in medaka which is displayed by the fact that ~ 50% of human tumors have (Supplementary Figure S6D). Expression of p53 and trim25 was mutated p53. We investigated the expression level of TRIM25 in a fi also constant during the rst 6 days of development, whereas the cohort of ovarian tumors with known p53 status.16 In agreement expression of mdm2 was reduced from day 1 to day 2 post with previous reports, we found that TRIM25 was frequently fertilization but stayed constant during the following days overexpressed in human tumors.17 In fact, we found a high (Supplementary Figure S6E). expression of TRIM25 in 11 out of 24 tumors. Three out of seven In order to investigate whether TRIM25 is required for medaka tumors with wild-type p53 and five out of eleven tumors with a development, we injected antisense-morpholino-oligonucleotides missense mutation in the p53 gene showed high expression of (MO) targeting both homologs of medaka TRIM25 at the one-cell TRIM25. Strikingly, in tumors with a nonsense mutation resulting stage. This led to a dose-dependent shortening of the embryonic in a shortened p53 protein, TRIM25 was overexpressed in three axis and retarded development (Figure 6a, Supplementary out of four cases (Table 1, Supplementary Figure S7B). Figure S6F). The phenotype induced by TRIM25-MOs was rescued to a significant part by the co-injection of TRIM25 mRNA (Supplementary Figure S6G), demonstrating that the develop- DISCUSSION mental defect upon injection of TRIM25-MOs is specific to the We identified the Tripartitite-Motif-protein TRIM25 as a novel absence of TRIM25 and not due to off-target effects. regulator for p53 and Mdm2 by a cell culture-based over- Importantly, co-injection of p53-MOs together with TRIM25- expression screen using a complementary DNA library.18 Increased MOs reduced the number of embryos with an abnormal abundance of TRIM25 either by its direct overexpression or via phenotype from 70 to 20%, whereas the injection of p53-MOs estrogen-dependent TRIM25-induction resulted in a higher alone had no effect (Figure 6a). As the phenotype of TRIM25-MO- abundance of p53 and Mdm2. injected embryos was reminiscent of apoptosis, we performed Increased abundance of p53 and Mdm2 after estrogen TUNEL staining and found that injection of TRIM25-MOs indeed treatment has been reported before,19–21 but the dependence induced apoptosis and that this induction of apoptosis was also on TRIM25 has not been investigated. Downregulation of TRIM25 reversed by p53-MOs (Figure 6b). These data show that TRIM25 completely blunted the induction of Mdm2 by estrogen, also controls p53 function in vivo. Likewise, a reduction in body demonstrating that the induction of TRIM25 is responsible for weight that was observed in TRIM25-/- mice was rescued when the increase. Induction of p53 was, however, not completely p53 was also deleted (Supplementary Figure S7A). Although the inhibited under these conditions. Earlier reports showed that phenotype in mice was not as strong as in medaka, and the cohort estrogen increases p53 mRNA levels 21 and protein stability, 19 and

Figure 6. Malformations caused by TRIM25 knockdown in medaka embryos are rescued by the silencing of p53. (a) The indicated MOs were injected into fertilized medaka eggs. One day after injection, the morphology of the embryos was monitored by microscopy. The graph shows the mean values and s.d. of three independent experiments with 28 to 46 analyzed embryos in each experiment. The total number of analyzed embryos was set to 100%. The P-value was calculated from the embryos injected with TRIM25-MOs and the embryos injected with TRIM25- and p53-MOs by two-tailed t-test. **Po0.01. (b) Fertilized medaka eggs were injected with the indicated MOs. One day after injection, the embryos were ﬁxed and apoptotic cells were labelled using the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) technique and detected by microscopy.

Figure 7. Regulation of p53 by TRIM25. Mdm2, as an E3 ligase, can ubiquitinate p53 and target it for proteasomal degradation. Efﬁcient polyubiquitination and degradation of p53, however, requires p300. P300 furthermore acetylates p53 and this modiﬁcation is essential for the transcriptional activation of most target genes of p53. TRIM25 inhibits these activities of p300 resulting in higher levels of p53, which is, however, usually transcriptionally inactive p53.

death of wt HCT116 cells, whereas HCT116 cells with a genetic Table 1. Relative expression of TRIM25 and p53 mutational status of deletion of both alleles of p53 (HCT p53-/-) showed no signs of 24 tumor samples from patients with ovarian carcinoma cell death, demonstrating the p53-dependence of this process. Nevertheless, HCT p53-/- cells also showed some, although much TRIM25 expression p53 Mutational status weaker, reduction in proliferation upon downregulation of Wt Missense Nonsense Not TRIM25. This reduction is most likely due to increased abundance mutation mutation determined of 14-3-3σ, a p53 target gene that is targeted for degradation by TRIM257,23 and this activity of TRIM25 does not depend on p53. Low or undetectable 4/7 (57%) 6/11 (55%) 1/4 (25%) 2/2 (100%) The observation that TRIM25 increases p53 abundance while at High 3/7 (43%) 5/11 (45%) 3/4 (75%) 0/2 (0%) the same time inhibiting its transcriptional activity appears Abbreviations: TRIM, tripartite motif protein; Wt, wild type. Tumor samples counter-intuitive. One possible explanation is that TRIM25 keeps with known p53 status from patients with ovarian carcinoma were p53 levels high to allow rapid p53-dependent responses but analyzed for TRIM25 and p53 abundance. The table shows the correlation inactive enough to ensure cell survival. When p53 activity is of high or low TRIM25 abundance with the expression of wild-type p53, required, the activity-suppressing function of TRIM25 could be with p53 possessing missense mutations or with p53 possessing mutations rapidly switched off, resulting in the immediate presence of high that result in premature termination of translation. levels of active p53. How the p53-inhibiting activity of TRIM25 is released remains to be determined. it is most likely that these mechanisms also contribute to the One major function of p53 is to inhibit cell proliferation in induction of p53 by estrogen. response to DNA damage.24 As TRIM25 inhibits p53 activity, the Mdm2 and p53 are short-lived proteins that are degraded in question arose whether TRIM25 is inactivated in response to DNA 26 S proteasomes upon prior ubiquitination. TRIM25 reduced p53 damage. Surprisingly, TRIM25 levels were even enhanced after and Mdm2 ubiquitination, resulting in increased stability of p53 DNA damage and also the activity of TRIM25 appeared not to be and Mdm2. Polyubiquitination and degradation of p53 requires restricted as we always observed reduced transcriptional activa- the association of Mdm2 with p300.9 This association was strongly tion of p53 in the presence of TRIM25, irrespectively if DNA lesions reduced in the presence of TRIM25 resulting in reduced were present or not. As the supervision of genomic integrity is one ubiquitination and degradation of p53. Whether the interaction of the main routes by which p53 suppresses carcinogenesis, of p300 with Mdm2 is also required for ubiquitination of Mdm2 abundance and activity of TRIM25 during the DNA damage remains to be determined. response is an important issue. Indeed, we found that TRIM25 is Most surprisingly, although p53 levels were elevated, p53’s over-expressed in ovarian tumors and its over-expression in other transcriptional activity was reduced by TRIM25. This result tumors has also been reported17,25 and this study. Moreover, suggests that TRIM25 has two separate activities with regard to downregulation of TRIM25 strongly reduced tumor growth in an p53, one is regulation of p53 abundance and the other is control experimental setting.7 Whether these observations are directly of its transcriptional activity. The increase in p53’s transcriptional linked to the regulation of p53 will, however, require a larger activity was accompanied by increased acetylation of p53, a cohort of tumors with known p53 status. Importantly, as DNA modification that is required for transcription particularly of damage is not only an important trigger for the onset of growth-arresting and pro-apoptotic target genes.22 The histone carcinogenesis but also frequently used for cancer treatment, acetyltransferase P300 contributes significantly to p53 which often involves activation of p53, the status of TRIM25 might acetylation.2 Interestingly, fibroblasts with a genetic deletion of be an important determinant for successful tumor therapy. TRIM25 showed much stronger expression of p300. The mechan- TRIM25 is essential for zebrafish development15 and as we show ism for this upregulation of p300 in the absence of TRIM25 is also for the development of medaka. The downregulation of unclear. Surprisingly, despite the increase in target gene activa- TRIM25 resulted in severe malformations and the embryos tion, the amount of p53 bound to DNA was reduced upon the eventually die by p53-induced apoptosis. These results show in downregulation of TRIM25. This reduction in DNA-bound p53 was a whole animal that the control of p53 activity by TRIM25 is of vital probably due to an overall reduction in p53 abundance after the importance. In contrast to fish, mice with a genetic deletion of downregulation of TRIM25. The increase in the amount of TRIM25 are viable. However the uterus of these mice is acetylated p53, however, could most likely compensate for this underdeveloped and the proliferation of fibroblasts derived from reduction, resulting in increased transcriptional activation despite these mice is strongly reduced.6,7 Furthermore, mice with a a reduction in the overall p53 levels. deletion of TRIM25 frequently showed reduced body weight, Increased activity of p53 after downregulation of TRIM25 which was not observed when we compared it with sex-matched resulted in the activation of Caspase 3, cleavage of PARP and siblings from the same litter. All these properties are consistent

Oncogene (2015) 5729 – 5738 © 2015 Macmillan Publishers Limited TRIM25 regulates p53 and Mdm2 P Zhang et al 5737 with an overly active p53 protein. The strong dependence of Ubiquitination assay teleost development on TRIM25-mediated control of p53 raises Ubiquitination assay was performed as described in Kulikov et al.26 the question why this dependence is not seen so robustly in the mouse. The most likely explanation is that in higher eukaryotes, Immunoprecipitation the abundance of TRIM25 might be too low in fetal tissues and fl Cells were lysed in lysis buffer (20 mM Tris pH 7.5, 1 mM EDTA, 100 mM NaCl, therefore the in uence of TRIM25 is limited. In teleosts, the impact 0.5% NP40, 10% glycerol and EDTA-free Protease Inhibitor Cocktail (Roche, of TRIM25 might be higher as the protein might be more Mannheim, Germany)) for 15 min on ice. The lysate was cleared by abundant. However, if TRIM25 expression is low in most tissues, centrifugation at 13 200 r.p.m. for 10 min at 4 °C. The protein lysate (5%) why then is p53 not overly active in these settings? Most likely, was analyzed by western blotting (input). The remaining lysate was added other proteins can replace TRIM25 in controlling p53 activity in to protein A and G sepharose precoupled with antibody and incubated for most tissues and this could have restricted the requirement for 4 h at 4 °C. The precipitates were washed with lysis buffer, suspended in TRIM25 to certain tissues such as the uterus where TRIM25 is sample buffer and separated by SDS–PAGE. present in higher amounts.6 Quantitative reverse transcription PCR MATERIALS AND METHODS Quantitative reverse transcription PCR. was performed as described in Solozobova et al.27 Cell lines and their treatments -/- H1299, U2OS, MCF7, HCT116 and HCT116 p53 were cultured in Luciferase assay Dulbecco's modified Eagle's medium containing 10% fetal bovine For luciferase assays, 1x104 cells per well were plated in 96-well plates. For serum and 1% penicillin/streptomycin according to standard conditions. TRIM25 overexpression studies, each well was transfected with 50 ng of the H1299 cells were transiently transfected by calcium-phosphate DNA co- p53-dependent reporter PG13 or p21 and 5 ng of a plasmid encoding precipitation26 or with PromoFectin (PromoKine, Heidelberg, Germany). Renilla luciferase together with a plasmid encoding TRIM25 or with vector U2OS and HCT116 cells were transfected with GeneJuice (Novagene, DNA. For TRIM25 downregulation studies, each well was transfected with Darmstadt, Germany). For transfection of siRNA, Ribojuice (Novagen) or 30 ng of PG13 or p21 and 3 ng of a plasmid encoding Renilla luciferase Viromer (Lipocalyx, Halle, Germany) was used. Sequences of small together with TRIM25 or control siRNA. Forty-eight hours after transfection, interfering RNAs are available on request. the cells were lysed and the luciferase activity was determined. β-estradiol was used at a final concentration of 10 nM in RPMI ( Roswell Park Memorial Institute) medium without phenol red, supplemented with 10% charcoal-stripped fetal bovine serum and 1% penicillin/streptomycin. Whole-mount in situ hybridization Cells were transferred to RPMI medium 2 days before estrogen treatment. Whole-mount in situ hybridization was performed as described in Loosli μ μ MG132 was used at 10 M (f.c.), cycloheximide at 60 g/ml (f.c.) and et al.28 etoposide at 50 μM (f.c.). γ-irradiation was performed in culture medium with 5 Gy at a dose rate of 0.5 Gy per minute using a 60cobalt-γ-source. Ethical issues Fish strains and housing Patient samples were used in an anonymized form. The local ethical committee has approved the study. The medaka inbred line Cab was maintained at 26° C. Embryos were raised at 28° C. ACKNOWLEDGEMENTS Plasmids We thank Germana Meroni (CBM S.c.r.l., Trieste) for the TRIM25 plasmid, Yi Su for help The plasmids encoding p53, Mdm2 and His-ubiquitin, have been described with the screening and Christina Bauer, Tanja Kuhn, Beate Heydel and Cathrin Herder previously.26 TRIM25 was amplified by PCR and cloned into pcDNA3 vector. for technical assistance. PZ was a CSC fellow. GD acknowledges the funding from the For cloning of V5-tagged TRIM25 the same strategy was used with DFG (FOR 1036) for the screening experiments. This work is supported by COST inclusion of the V5-tag sequence in the reverse primer. The plasmids for Action BM1307. mdTRIM25 homolog 1 and 2 were from the cDNA (complementary DNA) library. Sequences of cloning primers are available on request. REFERENCES Antibodies 1 Riley T, Sontag E, Chen P, Levine A. Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol 2008; 9: 402–412. 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