The E3 Ubiquitin Ligase Activity of Trip12 Is Essential for Mouse Embryogenesis

Abstract

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions [1], [2], [3], [4]. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1 [5], [6]. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12mt/mt) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12mt/mt embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16 [7], [8], [9], [10]. In contrast, Trip12mt/mt ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12mt/mt ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.

About the Authors:

Masashi Kajiro

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Mai Tsuchiya

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Yoh-ichi Kawabe

Affiliation: Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Ryohei Furumai

Affiliation: Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Naoya Iwasaki

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Yuki Hayashi

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Miyuki Katano

Affiliation: Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Yuka Nakajima

Affiliations Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan, Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Natsuka Goto

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Tatsuya Watanabe

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Akiko Murayama

Affiliations Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan, Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Hisashi Oishi

Affiliation: Department of Anatomy and Embryology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoudai, Tsukuba, Japan

Masatsugu Ema

Affiliation: Department of Anatomy and Embryology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoudai, Tsukuba, Japan

Satoru Takahashi

Affiliation: Department of Anatomy and Embryology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoudai, Tsukuba, Japan

Hiroyuki Kishimoto

Affiliations Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan, Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Junn Yanagisawa

* E-mail: [email protected]

Affiliations Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan, Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan

Introduction

Protein ubiquitination plays an important role in various cellular events [1], [2], [3], [4]. Ubiquitination occurs through sequential steps that are catalyzed by activating (E1), conjugating (E2), and ligase (E3) enzymes. In this reaction, the specificity for distinct substrates is determined by the ubiquitin ligase enzymes. From a mechanistic standpoint, ubiquitin ligases fall into two groups: HECT domain E3s and RING domain E3s [11], [12], [13]. The HECT family contains a diverse N-terminal region and a conserved C-terminal HECT domain, which has a reactive Cys residue. HECT-type ubiquitin ligases form a thioester intermediate with ubiquitin at the conserved Cys residue before transferring ubiquitin to the target proteins [12], [13].

Trip12 encodes a HECT domain containing an E3 ubiquitin ligase, which was originally identified as a thyroid hormone receptor-interacting protein in a yeast two-hybrid screen [14]. Recent reports indicate that Trip12 ubiquitinates ARF and APP-BP1 and regulates BAF57 turnover [5], [6], [15]. In this study, we showed that the ubiquitin ligase activity of Trip12 is essential for mouse development. We generated mice in which the coding region of the HECT domain within Trip12 was mutated (Trip12mt/mt mice). Targeted inactivation of Trip12 resulted in abnormal p16 induction and embryonic lethality in mice. We also found that Trip12mt/mt ES cells were viable but had decreased cell cycle progression. Trip12mt/mt ES cells had accumulated levels of the BAF57 protein and different gene expression patterns. Our data suggest that Trip12 is involved in global gene expression and is essential for mouse development.

Results

Generation of Trip12 mutant mice

To study the function of Trip12 in vivo, we generated Trip12 mutant mice by replacing exon 33 with FLAG and HA epitope tag sequences in frame and a neomycin resistant gene. This mutant Trip12 lacks the catalytic core for the ubiquitin ligase activity and has FLAG and HA epitope sequences at the C-terminus (Fig. 1A). The targeting vector was linearized, electroporated into ES cells and ES cells were grown under G418 selection. Selected ES cell clones were verified by Southern blot analysis and several clones were correctly targeted (Fig. 1B). We also detected the mutant Trip12 proteins in the targeted clones by Western blotting using an anti-HA antibody (Fig. 1C). Heterozygous Trip12+/mt mice were viable and fertile and did not exhibit any overt phenotype. To elucidate the Trip12 expression pattern in mouse embryos, anti-HA staining was performed. As shown in Fig. 1D, Trip12 is ubiquitously expressed in the embryo at E9.5 (Fig. 1D).

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Figure 1. Generation of Trip12 mutant mice.

(A) Schematic representation of the Trip12 gene targeting construct. Exon 33 of Trip12 genomic DNA containing the Cys residue that is required for ubiquitination was replaced in frame with FLAG and HA epitope tag sequences. Thus, the Trip12 mutant protein has FLAG and HA epitope tags instead of ubiquitination activity. Solid rectangles represent the exons of the Trip12 gene. (B) Southern blot analysis of the targeted allele in heterozygote Trip12 mutant ES cells. (C) Western blot analysis of Trip12 mutant protein expression in ES cells. (D) Whole mount immunohistochemistry of wild-type and heterozygous Trip12 embryos using an anti-HA antibody.

https://doi.org/10.1371/journal.pone.0025871.g001

TRIP12 ubiquitin ligase activity is essential for mouse embryogenesis

Trip12+/mt mice were intercrossed in order to produce Trip12mt/mt mice. However, no Trip12mt/mt mice were generated, suggesting that the ubiquitin ligase activity of Trip12 is essential for mouse development. To characterize the timing of this embryonic lethality, embryos derived from the Trip12+/mt intercross were genotyped at different stages of gestation. Trip12+/+, Trip12+/mt, and Trip12mt/mt embryos were born at the expected Mendelian ratio and were viable up to E8.5, while no Trip12mt/mt embryos were observed between E11.5 and E13.5 (Table 1). From these results, we concluded that Trip12 plays an important role in mouse embryogenesis.

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Table 1. Genotypes of the offspring from Trip12+/mt intercrosses.

https://doi.org/10.1371/journal.pone.0025871.t001

Phenotypes of Trip12mt/mt embryos

To characterize the nature of this embryonic lethality, we studied the morphology of embryos from timed Trip12+/mt intercrosses at different stages of gestation. An examination of these embryos at various developmental stages revealed that the development of Trip12mt/mt embryos appeared to be delayed at E8.5 (Fig. 2A). Despite this developmental delay, there were no gross morphological abnormalities in Trip12mt/mt embryos. We also confirmed that the number of somites was decreased in Trip12mt/mt embryos compared to wild-type embryos at E9.5 (Fig. 2B).

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Figure 2. Analysis of wild-type (+/+) and Trip12 mutant (mt/mt) embryos.

(A) Different developmental stages (E8.5 to E10.5) of wild-type (+/+) and Trip12 mutant (mt/mt) embryos. (×magnification) (B) The somite number of Trip12+/+, Trip12+/mt, and Trip12mt/mt embryos. The somite number of each embryo at E9.5 was scored by stereo microscopy. **, p<0.001; N.S., not significant. (C) Hematoxylin and eosin staining of sections of wild-type (+/+) and Trip12 mutant (mt/mt) embryos at different developmental stages (E7.5 to E9.5). Placental tissues at E9.5 are enlarged in the right panel. Scale bars represent 1 mm. (D) The expression levels of negative cell cycle regulators in wild-type (+/+) and Trip12 mutant (mt/mt) embryos. Total RNA was isolated from embryos, and the gene expression profiles were analyzed using real-time RT-PCR. Data are presented as the mean+s.d., n = 3. **, p<0.001; N.S., not significant.

https://doi.org/10.1371/journal.pone.0025871.g002

The Trip12mt/mt embryos were further characterized histologically at different developmental stages. In addition to a developmental delay, Trip12mt/mt embryos had abnormal placenta formation (Fig. 2C). Normal labyrinth formation was not observed and a number of erythrocytes accumulated in the placenta of Trip12mt/mt embryos (Fig. 2C right panel). This phenotype might be caused by impaired development of the allantois [16].

Given Trip12 enzymatic activity which is involved in ARF regulation (4), we examined whether mutant show altered expression of ARF and related proteins which could explain the developmental arrest of Trip12mt/mt embryos. Therefore, we examined the expression levels of several cell cycle-related genes and found that p16 and ARF expression levels were increased in Trip12mt/mt embryos (Fig. 2D). p16 is a cyclin-dependent kinase inhibitor and ARF increases p53 protein stability and promotes p53-dependent cell cycle arrest [7], [8], [9], [10]. However, the expression level of p21, a p53 target gene, was virtually unchanged. In addition, mutant embryo didn't show increased apoptosis compared to wild type embryo at corresponding developmental stage (Fig. S1). These data indicate that the embryonic lethality of Trip12mt/mt mice may be caused by abnormal p16 induction rather than p53 activation.

Generation and characterization of Trip12mt/mt ES cells

To further investigate the function of Trip12, we generated two Trip12mt/mt ES cell clones [17 and #2] (Fig. 3A, 3B, and 3C). We examined the ability of Trip12mt/mt ES cells to self-renew and differentiate. The expression levels of Nanog and Oct-4 were comparable in the two Trip12mt/mt ES cell clones and wild-type cell cultures with or without LIF (Fig. 3D). To assess whether ES cell differentiation was affected, we compared embryoid body (EB) formation on days 0 and 10. Trip12mt/mt ES cells displayed the same EB morphology (data not shown), and the expression patterns of endodermal (AFP)-, mesodermal (Flk-1)-, and ectodermal (NCAM)-specific genes were also similar between wild-type and Trip12mt/mt ES cells (Fig. 3E). These results suggest that Trip12 has a minor role in ES cell differentiation.

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Figure 3. Generation and characterization of Trip12 mutant (mt/mt) ES cells.

(A) Schematic representation of the Trip12 gene targeting construct (Targeted allele II). Solid rectangles represent the exons of the Trip12 gene. (B) Southern blot analysis of the targeted allele in Trip12 mutant (mt/mt) ES cells. (C) Western blot analysis of Trip12 mutant protein expression in ES cells. (D) Expression levels of Nanog and Oct-4 in Trip12 mutant (mt/mt) ES cells with or without LIF. The cells were cultured without LIF and harvested at the indicated time points. Total RNA was isolated from ES cells, and the gene expression profiles were analyzed using real-time RT-PCR. Data are presented as the mean+s.d., n = 3. (E) Embryoid body (EB) formation of wild-type (+/+) or Trip12 mutant (mt/mt) ES cells. The cells were cultured in suspension in ES cell medium without LIF in 10-mm dishes for 10 days. Scale bars: 100 µm. Total RNA was isolated from the cells, and the gene expression profiles were analyzed using real-time RT-PCR. Data are presented as the mean+s.d., n = 3.

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Trip12mt/mt ES cells exhibit cell cycle defects

Next, we tested whether the Trip12 mutation leads to a defect in cell proliferation. The MTT assay showed that Trip12mt/mt ES cells had decreased cell growth (Fig. 4A). Therefore, we analyzed cell cycle progression by flow cytometry, and found that the G2-M and SubG1 populations were increased in Trip12mt/mt ES cells (Fig. 4B). We further examined the expression levels of p16, ARF, and p21. Unexpectedly, the expression levels of these genes were virtually unchanged in the cells (Fig. 4C). It was previously reported that Trip12 ubiquitinates ARF and inhibits p53-dependent transcription [6]. However, there were no detectable alterations in p53-related protein levels in Trip12mt/mt ES cells (Fig. 4D).

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Figure 4. Cell cycle defects in Trip12 mutant (mt/mt) ES cells.

(A) Proliferation of Trip12 mutant (mt/mt) ES cells. The growth rate of wild-type or Trip12 mutant (mt/mt) ES cells was measured by the MTT assay. (B) Cell cycle progression in Trip12 mutant (mt/mt) ES cells was analyzed by flow cytometry. (C) The expression levels of negative cell cycle regulators in wild-type (+/+) or Trip12 mutant (mt/mt) ES cells. Total RNA was isolated from the cells, and the gene expression profiles were analyzed using real-time RT-PCR. Data are presented as the mean+s.d., n = 3. (D) Western blot analysis of proteins in the p53 pathway in wild-type (+/+) or Trip12 mutant (mt/mt) ES cells.

https://doi.org/10.1371/journal.pone.0025871.g004

Trip12 negatively regulates BAF57 protein stability and is involved in global gene expression in ES cells

Recent reports also suggest that Trip12 regulates BAF57 protein turnover [15]. Therefore, we examined whether inactivation of the Trip12 ubiquitin ligase activity affects the stability of BAF57. As shown in Fig. 5A, the endogenous BAF57 protein levels were increased in Trip12mt/mt ES cells (Fig. 5A). We also confirmed that endogenous BAF57 interacts with Trip12mt from targeted allele in ES cells (Fig. 5B). In addition, cycloheximide experiments showed that the turnover rate of endogenous BAF57 was decreased in Trip12mt/mt ES cells (t1/2≫6 h) compared to that in wild-type cells (t1/2≈4 h) (Fig. 5C). As shown in Fig. 5D, ectopically expressed wild type and mutant Trip12 were interacting with GST-BAF57 and mutant Trip12 showed stronger interaction than wild type suggesting that loss of catalytic activity of Trip12 resulted in decreased dissociation of BAF57 from Trip12. These data demonstrate that the ubiquitin ligase activity of Trip12 is required for BAF57 turnover in ES cells. BAF57 is a component of the SWI/SNF chromatin remodeling complex and involved in a variety of cellular processes [18], [19], [20], [21], [22], [23], [24], [25], [26]. Therefore, a gene expression study using microarray analysis was performed to elucidate the underlying mechanism that leads to cell proliferation defects in Trip12mt/mt ES cells. Microarray analysis of variance identified a total of 7635 genes that were significantly expressed in wild-type and Trip12mt/mt ES cells (p-values<0.05). To identify statistically significant functional categories by gene ontology, we selected genes that had greater than 2-fold changes in expression and placed these genes into DAVID [27]. As shown in Fig. 5D, both up- and down-regulated genes were enriched in the developmental process (Fig. 5D). These data suggest that Trip12 regulates gene expression by destabilizing BAF57 during mouse development.

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Figure 5. Loss of the ubiquitin ligase activity of Trip12 induces BAF57 protein accumulation in ES cells.

(A) The BAF57 expression levels in wild-type (+/+) or Trip12 mutant (mt/mt) ES cells. The BAF57 protein levels were determined by immunoblotting, and the signal intensity was quantified. (B) Co-immunoprecipitation of Trip12 with BAF57. Trip12mt was immunoprecipitated from Trip12mt/mt ES cell lysate with an anti-FLAG antibody, and the immunoprecipitates were immunoblotted with the indicated antibodies. (C) Trip12 regulates the turnover of BAF57. The cells were treated with 100 µg/ml cycloheximide and lysed at various time points. The protein lysates were separated by SDS-PAGE and detected by immunoblotting. (D) Wild type or mutant Trip12 was ectopically expressed with or without myc-tagged BAF57. Myc-BAF57 was immunoprecipitated with anti-myc antibody and immunoblotted with anti-Trip12 or anti-myc antibody. (E) A comparison of the mouse whole-genome RNA expression profile between wild-type and Trip12 mutant (mt/mt) ES cells. DAVID and the annotation sources GOTERM_BP2 (Biological process) were used to identify the functional categories.

https://doi.org/10.1371/journal.pone.0025871.g005

Discussion

In this report, we have determined that the E3 ubiquitin ligase activity of Trip12 is indispensable for mouse development. Previous reports indicated that Trip12 promotes the ubiquitination and degradation of ARF [6]. The down-regulation of Trip12 expression stabilizes ARF and enhances ARF-dependent, p53-mediated cell cycle arrest in normal human fibroblasts [6]. However, in our experiments, the p53 pathway was not activated in either Trip12mt/mt mice or Trip12mt/mt ES cells. Although Trip12mt/mt mice had increased ARF expression levels, the p21 (which is a well known p53 target gene) expression levels were unchanged in these mice. These findings suggest that the growth arrest phenotype of Trip12mt/mt mice was caused by p16 expression rather than ARF induction. Previous reports also showed that Trip12-mediated ARF ubiquitination is inhibited by c-Myc [6]. It is known that c-Myc expression levels are higher in mouse embryos and ES cells compared to differentiated cells [17], [28], [29], [30]. Considering these results, we speculate that Trip12 minimally contributes to ARF ubiquitination during mice embryogenesis because of elevated c-Myc expression. On one hand, our experiments showed that BAF57 protein stability was regulated by Trip12-mediated ubiquitination and that the expression profiles of several genes were changed between wild-type and Trip12mt/mt ES cells. This finding suggests that the ubiquitination activity of Trip12 is tightly regulated by individual target proteins. Further experiments using conditional knockout approaches and the Trip12 protein complex that is purified from different cell types may help elucidate the exact in vivo functions of the Trip12 protein.

Materials and Methods

Cell culture and embryoid body (EB) formation

E14 ES cells (ATCC) were maintained on mouse embryo fibroblast (MEF) feeder cell layers in Dulbecco-modified Eagle medium containing 14% KnockOut Serum Replacement (Invirtogen), 1% fetal bovine serum, 100 units/ml LIF, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and 100 µM 2-mercaptoethanol. For EB formation assays, 2×105 cells were seeded into 35 mm low attachment sterile cell plate in 2 ml ES cell medium without LIF. Fresh medium was provided every two days.

Targeting vectors and generation of Trip12 mutant mice

The targeting vectors for Trip12 were constructed by inserting FLAG and HA epitope tag sequences in frame and Neo or IRES-hyg cassette into the exon 33 of the mouse Trip12 genome. Correctly targeted ES cell clone were injected into C57BL/6 blastocysts, producing chimeric mice. Highly chimeric males were mated to females, and then the progeny were intercrossed. All experiments were performed in accordance with the Declaration of Helsinki and were approved by University of Tsukuba Ethics Committee for Animal Experiments (approval ID:040042).

Western Blot

Cells were lysed in TNE buffer [10 mM Tris-HCl (pH 7.8), 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 µM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin]. The proteins were separated by SDS-PAGE, transferred onto PVDF membranes (Millipore), immunoblotted with indicated antibodies. The antibodies employed in this study included mouse monoclonal antibodies specific for β-Actin (Sigma); rat monoclonal antibody for HA (Roche) and ARF (Calbiochem). Rabbit polyclonal antibody for BAF57 (GeneTex) and Trip12 (Bethyl Laboratories).

Real-time RT-PCR

Embryos and cells were homogenized in 1 ml of sepasol (nacalai tesque) and total RNA was extracted according to the instruction manual. cDNA was synthesized from total RNA using RevatraAce reverse transcriptase (Toyobo) and random primers. Real-time PCRs were performed to amplify fragments representing for the indicated mRNA expression. We used primers 5′- CATCTGGAGCAGCATGGAGTC-3′ and 5′-GGGTACGACCGAAAGAGTTCG-3′ for p16, 5′-GTTCTTGGTCACTGTGAGGATTCAG-3′ and 5′-CCATCATCATCACCTGGTCCAG-3′ for ARF, 5′-TTGCACTCTGGTGTCTGAGC-3′ and 5′-TGCGCTTGGAGTGATAGAAA-3′ for p21, 5′-TTGGGTCTCAGGCAAACTCT-3′ and 5′-CTGTTGGCCCTTTTGTTTTG-3′ for p27, 5′-AGTACCTCAGCCTCCAGCAGAT-3′ and 5′-GCTTGCACTTCATCCTTTGGTT-3′ for Nanog, 5′-CACGAGTGGAAAGCAACTCA-3′ and 5′-TTCATGTCCTGGGACTCCTC-3′ for Oct-4, 5′-CATGAACAGGTTCATCTATG-3′ and 5′-GTTGTCAGCTTTGCAGCATG-3′ for AFP, 5′-TGTCTATGTTCGAGATTACAG-3′ and 5′-CATTGAGGTTTGAAATCGAC-3′ for Flk-1, 5′-GGTCATTGTGAATGTACCAC-3′ and 5′-CCTTTGTCCAGCTCATGGTG-3′ for NCAM,

Cycloheximide treatment

Cells were treated with 100 µg/ml cycloheximide to prevent de novo protein synthesis, and lysed at various time points following cycloheximide treatment. The proteins were then separated by SDS-PAGE and detected by immunoblotting.

Whole mount immunostaining

Whole mount immunostaining of embryos were performed as described previously [31].

Microarray procedure and data analysis

The microarray procedure was performed as described in the Affymetrix GeneChip Expression Analysis manual (Affimetrix). Each array was analysed using GeneSpring (Agilent) program. Patterns were analysed to identify functional categories using Database for Annotation, Visualization and Integrated Discovery (DAVID) (http:// david.abcc.ncifcrf.gov) [27]. We used the functional annotation tool program and reported only GOTERM_BP (Biological process) that had p-values of <0.05.

Statistical analysis

All the data are representative of at least three different experiments. Statistical analysis was performed using t-test.

Supporting Information

[Figure omitted. See PDF.]

Figure S1.

TUNEL staining of wild type E8.5 embryo and Trip12mt/mt embryo at corresponding developmental stage.

https://doi.org/10.1371/journal.pone.0025871.s001

(TIF)

Author Contributions

Conceived and designed the experiments: M. Kajiro YK JY. Performed the experiments: M. Kajiro MT YK RF NI YH M. Katano HO ME ST HK JY. Analyzed the data: M. Kajiro MT YK HK JY. Contributed reagents/materials/analysis tools: YN NG TW AM. Wrote the paper: M. Kajiro HK JY.

Citation: Kajiro M, Tsuchiya M, Kawabe Y-i, Furumai R, Iwasaki N, Hayashi Y, et al. (2011) The E3 Ubiquitin Ligase Activity of Trip12 Is Essential for Mouse Embryogenesis. PLoS ONE6(10): e25871. https://doi.org/10.1371/journal.pone.0025871

References

1. Kitagawa K, Kotake Y, Kitagawa M (2009) Ubiquitin-mediated control of oncogene and tumor suppressor gene products. Cancer Sci 100: 1374–1381.K. KitagawaY. KotakeM. Kitagawa2009Ubiquitin-mediated control of oncogene and tumor suppressor gene products.Cancer Sci10013741381

2. Wertz IE, Dixit VM (2010) Signaling to NF-kappaB: regulation by ubiquitination. Cold Spring Harb Perspect Biol 2: a003350.IE WertzVM Dixit2010Signaling to NF-kappaB: regulation by ubiquitination.Cold Spring Harb Perspect Biol2a003350

3. Shabek N, Ciechanover A (2010) Degradation of ubiquitin: the fate of the cellular reaper. Cell Cycle 9: 523–530.N. ShabekA. Ciechanover2010Degradation of ubiquitin: the fate of the cellular reaper.Cell Cycle9523530

4. Pickart CM (2004) Back to the future with ubiquitin. Cell 116: 181–190.CM Pickart2004Back to the future with ubiquitin.Cell116181190

5. Park Y, Yoon SK, Yoon JB (2008) TRIP12 functions as an E3 ubiquitin ligase of APP-BP1. Biochem Biophys Res Commun 374: 294–298.Y. ParkSK YoonJB Yoon2008TRIP12 functions as an E3 ubiquitin ligase of APP-BP1.Biochem Biophys Res Commun374294298

6. Chen D, Shan J, Zhu WG, Qin J, Gu W (2010) Transcription-independent ARF regulation in oncogenic stress-mediated p53 responses. Nature 464: 624–627.D. ChenJ. ShanWG ZhuJ. QinW. Gu2010Transcription-independent ARF regulation in oncogenic stress-mediated p53 responses.Nature464624627

7. Gil J, Peters G (2006) Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 7: 667–677.J. GilG. Peters2006Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all.Nat Rev Mol Cell Biol7667677

8. Ruas M, Peters G (1998) The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta 1378: F115–177.M. RuasG. Peters1998The p16INK4a/CDKN2A tumor suppressor and its relatives.Biochim Biophys Acta1378F115177

9. Canepa ET, Scassa ME, Ceruti JM, Marazita MC, Carcagno AL, et al. (2007) INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions. IUBMB Life 59: 419–426.ET CanepaME ScassaJM CerutiMC MarazitaAL Carcagno2007INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions.IUBMB Life59419426

10. Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, et al. (1994) Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 368: 753–756.T. NoboriK. MiuraDJ WuA. LoisK. Takabayashi1994Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers.Nature368753756

11. Deshaies RJ, Joazeiro CA (2009) RING domain E3 ubiquitin ligases. Annu Rev Biochem 78: 399–434.RJ DeshaiesCA Joazeiro2009RING domain E3 ubiquitin ligases.Annu Rev Biochem78399434

12. Rotin D, Kumar S (2009) Physiological functions of the HECT family of ubiquitin ligases. Nat Rev Mol Cell Biol 10: 398–409.D. RotinS. Kumar2009Physiological functions of the HECT family of ubiquitin ligases.Nat Rev Mol Cell Biol10398409

13. Bernassola F, Karin M, Ciechanover A, Melino G (2008) The HECT family of E3 ubiquitin ligases: multiple players in cancer development. Cancer Cell 14: 10–21.F. BernassolaM. KarinA. CiechanoverG. Melino2008The HECT family of E3 ubiquitin ligases: multiple players in cancer development.Cancer Cell141021

14. Lee JW, Choi HS, Gyuris J, Brent R, Moore DD (1995) Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor. Mol Endocrinol 9: 243–254.JW LeeHS ChoiJ. GyurisR. BrentDD Moore1995Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor.Mol Endocrinol9243254

15. Keppler BR, Archer TK (2010) Ubiquitin-dependent and ubiquitin-independent control of subunit stoichiometry in the SWI/SNF complex. J Biol Chem 35665–35674.BR KepplerTK Archer2010Ubiquitin-dependent and ubiquitin-independent control of subunit stoichiometry in the SWI/SNF complex.J Biol Chem3566535674

16. Liu S, Wiggins JF, Sreenath T, Kulkarni AB, Ward JM, et al. (2006) Dph3, a small protein required for diphthamide biosynthesis, is essential in mouse development. Mol Cell Biol 26: 3835–3841.S. LiuJF WigginsT. SreenathAB KulkarniJM Ward2006Dph3, a small protein required for diphthamide biosynthesis, is essential in mouse development.Mol Cell Biol2638353841

17. Cartwright P, McLean C, Sheppard A, Rivett D, Jones K, et al. (2005) LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development 132: 885–896.P. CartwrightC. McLeanA. SheppardD. RivettK. Jones2005LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism.Development132885896

18. Link KA, Balasubramaniam S, Sharma A, Comstock CE, Godoy-Tundidor S, et al. (2008) Targeting the BAF57 SWI/SNF subunit in prostate cancer: a novel platform to control androgen receptor activity. Cancer Res 68: 4551–4558.KA LinkS. BalasubramaniamA. SharmaCE ComstockS. Godoy-Tundidor2008Targeting the BAF57 SWI/SNF subunit in prostate cancer: a novel platform to control androgen receptor activity.Cancer Res6845514558

19. Reisman D, Glaros S, Thompson EA (2009) The SWI/SNF complex and cancer. Oncogene 28: 1653–1668.D. ReismanS. GlarosEA Thompson2009The SWI/SNF complex and cancer.Oncogene2816531668

20. Garcia-Pedrero JM, Kiskinis E, Parker MG, Belandia B (2006) The SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen receptor function in breast cancer cells. J Biol Chem 281: 22656–22664.JM Garcia-PedreroE. KiskinisMG ParkerB. Belandia2006The SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen receptor function in breast cancer cells.J Biol Chem2812265622664

21. Hah N, Kolkman A, Ruhl DD, Pijnappel WW, Heck AJ, et al. (2010) A role for BAF57 in cell cycle-dependent transcriptional regulation by the SWI/SNF chromatin remodeling complex. Cancer Res 70: 4402–4411.N. HahA. KolkmanDD RuhlWW PijnappelAJ Heck2010A role for BAF57 in cell cycle-dependent transcriptional regulation by the SWI/SNF chromatin remodeling complex.Cancer Res7044024411

22. Heimeier RA, Hsia VS, Shi YB (2008) Participation of Brahma-related gene 1 (BRG1)-associated factor 57 and BRG1-containing chromatin remodeling complexes in thyroid hormone-dependent gene activation during vertebrate development. Mol Endocrinol 22: 1065–1077.RA HeimeierVS HsiaYB Shi2008Participation of Brahma-related gene 1 (BRG1)-associated factor 57 and BRG1-containing chromatin remodeling complexes in thyroid hormone-dependent gene activation during vertebrate development.Mol Endocrinol2210651077

23. Lai D, Wan M, Wu J, Preston-Hurlburt P, Kushwaha R, et al. (2009) Induction of TLR4-target genes entails calcium/calmodulin-dependent regulation of chromatin remodeling. Proc Natl Acad Sci U S A 106: 1169–1174.D. LaiM. WanJ. WuP. Preston-HurlburtR. Kushwaha2009Induction of TLR4-target genes entails calcium/calmodulin-dependent regulation of chromatin remodeling.Proc Natl Acad Sci U S A10611691174

24. Weissman B, Knudsen KE (2009) Hijacking the chromatin remodeling machinery: impact of SWI/SNF perturbations in cancer. Cancer Res 69: 8223–8230.B. WeissmanKE Knudsen2009Hijacking the chromatin remodeling machinery: impact of SWI/SNF perturbations in cancer.Cancer Res6982238230

25. Wang L, Baiocchi RA, Pal S, Mosialos G, Caligiuri M, et al. (2005) The BRG1- and hBRM-associated factor BAF57 induces apoptosis by stimulating expression of the cylindromatosis tumor suppressor gene. Mol Cell Biol 25: 7953–7965.L. WangRA BaiocchiS. PalG. MosialosM. Caligiuri2005The BRG1- and hBRM-associated factor BAF57 induces apoptosis by stimulating expression of the cylindromatosis tumor suppressor gene.Mol Cell Biol2579537965

26. Harte MT, O'Brien GJ, Ryan NM, Gorski JJ, Savage KI, et al. (2010) BRD7, a subunit of SWI/SNF complexes, binds directly to BRCA1 and regulates BRCA1-dependent transcription. Cancer Res 70: 2538–2547.MT HarteGJ O'BrienNM RyanJJ GorskiKI Savage2010BRD7, a subunit of SWI/SNF complexes, binds directly to BRCA1 and regulates BRCA1-dependent transcription.Cancer Res7025382547

27. Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4: 44–57.W. Huang daBT ShermanRA Lempicki2009Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat Protoc44457

28. Davis AC, Wims M, Spotts GD, Hann SR, Bradley A (1993) A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice. Genes Dev 7: 671–682.AC DavisM. WimsGD SpottsSR HannA. Bradley1993A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice.Genes Dev7671682

29. Downs KM, Martin GR, Bishop JM (1989) Contrasting patterns of myc and N-myc expression during gastrulation of the mouse embryo. Genes Dev 3: 860–869.KM DownsGR MartinJM Bishop1989Contrasting patterns of myc and N-myc expression during gastrulation of the mouse embryo.Genes Dev3860869

30. Harris LL, Talian JC, Zelenka PS (1992) Contrasting patterns of c-myc and N-myc expression in proliferating, quiescent, and differentiating cells of the embryonic chicken lens. Development 115: 813–820.LL HarrisJC TalianPS Zelenka1992Contrasting patterns of c-myc and N-myc expression in proliferating, quiescent, and differentiating cells of the embryonic chicken lens.Development115813820

31. Yokomizo T, Takahashi S, Mochizuki N, Kuroha T, Ema M, et al. (2007) Characterization of GATA-1(+) hemangioblastic cells in the mouse embryo. Embo J 26: 184–196.T. YokomizoS. TakahashiN. MochizukiT. KurohaM. Ema2007Characterization of GATA-1(+) hemangioblastic cells in the mouse embryo.Embo J26184196