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Vol. 18, Issue 5, 1701-1709, May 2007
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Departments of *Molecular Genetics and Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Osaka 537-8511, Japan; Division of Molecular Biology, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan; ||Kan Research Institute, Kyoto, Kyoto 600-8815, Japan; and ¶Department of Internal Medicine and McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical School, Dallas, TX 75390
Submitted August 25, 2006;
Revised February 9, 2007;
Accepted February 16, 2007
Monitoring Editor: A. Gregory Matera
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Chen et al., 2005). Tumor suppressor proteins p53 and pRB both act in pathways that regulate the cellular senescence induced by oncogenic signals (Serrano et al., 1997; Campisi, 2005). The activity of p53 is modulated mostly by stabilization through posttranslational modifications, including phosphorylation and acetylation (Bode and Dong, 2004). In addition to these posttranslational modifications, recruitment of p53 into promyelocytic leukemia (PML)-nuclear bodies (NBs) also plays an important role in its activation in response to genotoxic stress (Fogal et al., 2000; Pearson et al., 2000). PML-NBs are highly dynamic nuclear structures containing an essential component, PML, to which many proteins are recruited to regulate transcription or DNA repair (Borden, 2002). Expression of PML IV, one of the PML isoforms encoded by several alternative spliced transcripts, recruits p53 and CBP to PML-NBs, activates them and induces cellular senescence (Pearson et al., 2000). However, little is known about how p53 or other proteins are recruited into PML-NBs or other nuclear subdomains.
MORC3 (microrchidia3), also called KIAA0136, ZCWCC3, and NXP-2 (Kimura et al., 2002), is a member of the MORC protein family characterized by conserved structures consisting of a GHL (Gyrase B, Hsp90 and MutL)-ATPase domain (Dutta and Inouye, 2000) at the amino-terminus, a zinc finger type CW domain containing conserved four cysteines and two tryptophans (Perry and Zhao, 2003), a nuclear localization signal (NLS) and coiled-coil domains at the carboxy-terminus (Supplementary Figure 1). We previously identified one of MORC family proteins, mouse Morc1 (Inoue et al., 1999), whose deficiency causes arrest of spermatogenesis before the pachytene stage of meiosis prophase I (Watson et al., 1998). There are four predicted MORC family proteins (MORC1, MORC2 [KIAA0852, ZCWCC1], MORC3, and MORC4 [ZCWCC2]) in human and five (Morc1, Morc2a, Morc2b, Morc3, and Morc4) in mice (Supplementary Figure 1 and Supplementary Table 1). The MORC family proteins belong to CW-domain–containing subfamilies I (MORC1 and MORC2) or IX (MORC3 and MORC4) (Perry and Zhao, 2003). MORC1 is expressed specifically in male germ cells (Inoue et al., 1999), whereas MORC2 and MORC3 in human are ubiquitously expressed (The HUGE database, http://www.kazusa.or.jp/huge/index.html). Caenorhabditis elegans has only one MORC family member, ZC155.3, whose knockdown by siRNA is embryonic lethal (Wormbase, http://www.wormbase.org/).
Although MORC family proteins are conserved in higher eukaryotes, no molecular function of any MORC family protein has yet been clarified. Here we show that MORC3 is involved in recruiting p53 and Sp100 but not PML or CBP from the nucleoplasm to PML-NBs. In addition, MORC3 regulates p53 activation in a manner dependent on MORC3-ATPase activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). pMEPy-enhanced green fluorescent protein (EGFP)-tag vector was generated by introduction of an EGFP fragment in which the stop codon was replaced with SalI site. pME-I-P vector was constructed by subcloning the internal ribosomal entry site-puromycin resistance gene (I-P) fragment derived from pIRESpuro2 (Clontech, Mountain View, CA) into the pME vector. We replaced the initiation codon of human MORC3 with a SalI site and inserted the resulting construct into the expression vectors. MORC3 mutants were generated by the QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). p53, PML IV, and Maltose-binding protein (MBP) cDNA carrying a SalI site instead of the initiation codon were subcloned into the FLAG- or HA-tag vectors. A knockdown vector, pH1'-DsRed2-I-P and pH1'-SV40-puro was constructed by the introduction of HindIII-KpnI fragment of pME-DsRed2-I-P and a fragment of puromycin resistance gene transcribed from SV40 promoter, respectively, into pH1' vector (Hasuwa et al., 2002). MORC3 knockdown vectors, pH1'-shRNA732-DsRed2-I-P (shRNA732 [short hairpin RNA]) were designed to contain the following inserts: 5'-CCCGGGGTACAAGAAGCAGGAAAGTTCAAGAGACTTTCCTGCTTCTTGTACCCCTTTTTGGAAA-3' (the target sense and antisense sequences are underlined). As a negative control, we replaced shRNA732 with the following shRNA695 sequence to target the same region of mouse Morc3: 5'-CCCaGGGTACAAGAAaCAaGAAAGTTCAAGAGACTTTCtTGtTTCTTGTACCCtTTTTTGGAAA-3' (the mismatch nucleotides are lowercased). PML-knockdown vector pH1'-shRNA-PML1555 (shRNA-PML1555) was designed to contain the following inserts: 5'-CCC- GGGGTACAAGAAGCAGGAAAGTTCAAGAGACTTTCCTGCTTCTTGTA- CCCCTTTTTGGAAA-3'. Proper construction of all plasmids was confirmed by restriction digestions and sequencing. These plasmids and their sequences are available upon request.
Cell Culture and Transfection
Cells were cultured in DMEM (Sigma, St. Louis, MO) for HeLa and mouse embryonic fibroblasts (MEFs), McCoy's 5A (Sigma) for U-2 OS (ATCC, Manassas, VA) and Saos-2, RPMI 1640 (Sigma) for H1299 (ATCC) and NB4, and Minimum essential medium (Invitrogen, Carlsbad, CA) for WI38 (RIKEN cell bank) at 37°C under 5% CO2 atmosphere. All media were supplemented with 10% fetal calf serum (FCS; 15% FCS for Saos-2), 100U/ml penicillin, and 100 µg/ml streptomycin. Plasmids were transfected into cells with PolyFect (Qiagen, Chatsworth, CA) or FuGENE6 (Roche, Indianapolis, IN) for HeLa, FuGENE6 (Roche) for U-2 OS and Saos-2, and Lipofectamine 2000 (Invitrogen) for H1299, according to the manufacturers' instructions.
Retroviral Infection and Senescence Analysis
The retroviral vectors, pLNCX2-EGFP-MORC3-I-P and pLNCX2-FLAG-MORC3-I-P were constructed by inserting EGFP-MORC3-I-P and FLAG-MORC3-I-P fragments, respectively, between the EcoRI and NotI sites of pLNCX2 vector (Clontech). pLNCX2-EGFP-PML IV-I-P and pLNCX2-FLAG-PML IV-I-P were generated from pLNCX2-EGFP-MORC3-I-P and pLNCX2-FLAG-MORC3-I-P, respectively, by replacing their MORC3 fragments with PML IV fragment. pLNCX2-EGFP-I-P was generated from pLNCX2-EGFP-MORC3-I-P by replacing its EGFP-MORC3 fragment with EGFP fragment derived from pEGFP vector. Plat-E or Plat-A packaging cells were used for production of the retrovirus, as described previously (Morita et al., 2000). Infected cells were selected with puromycin (2 µg/ml for WI38 and 3 µg/ml for MEFs) for 4 d. Day 0 is the first day after the selection. At day –1, cells were plated in 15 wells of 12-well plates at 2 x 104 cells/well. At the indicated time points, the numbers of cells in three wells were counted using a Z1 particle counter (Beckman Coulter, Fullerton, CA). The population doublings were calculated as described previously (Bischof et al., 2002). When cells became semiconfluent, 25% of the cells were further subcultured. Senescence-associated 
-galactosidase (SA--Gal) activity was detected as previously described (Dimri et al., 1995).
Luciferase Assay
The pGL3-PG12S reporter vector derived from pGL3-Promoter (Promega, Madison, WI) contains 12 repeats of a p53-binding site (5'-CCTGCCTGGACTTGCCTGG-3'; el-Deiry et al., 1993) upstream of an SV40 promoter. As a negative control, the pGL3-MG14S vector was generated by replacing the p53-binding sites with 14 repeats of mutated p53-binding site (5'-CCTTAATGGACTTTAATGG-3'). Transfection efficiency was normalized using Renilla luciferase activity expressed from pGL3-Renilla, containing the Renilla luciferase gene derived from phRL-TK vector (Promega).
pCBG99-p21 and pCBG99-Bax reporter vectors were constructed by inserting the 2.4-kbp p21 promoter region (el-Deiry et al., 1993) and the Bax promoter (–690 base pairs to –317 base pairs; Miyashita and Reed, 1995), respectively, upstream of the green-emitting luciferase gene of pCBG99-Basic (Promega). An internal control vector, pCBR-SV40pro, was generated using an SV40 promoter derived from pGL3-Control (Promega). The total amount of transfected DNA was kept constant by adjusting with empty vectors or pMEPy-GST. One or two days after transfection, luciferase activities were measured using a Dual-Glo Luciferase Assay System (Promega) or a Chroma-Glo Luciferase Assay System (Promega). Means were calculated from the results of three independent experiments.
Generation of Morc3-Knockout Mice
We obtained Morc3 genomic BAC clones by screening the RPCI-22 129s6/SvEvTAC mouse BAC library (BACPAC Resources Center, Oakland, CA). A 3-kbp fragment containing Morc3 exon 3 and exon 4 (103 base pairs), hrGFP gene (Stratagene) fused in frame to Morc3, and a rabbit -globin polyadenylation signal was cloned into the SmaI site of the pBluescript-neo/Diphtheria toxin fragment A (DT-A)/loxP vector (Ikeda et al., 1999). A 4-kbp fragment containing Morc3 exon 6 was cloned into the EcoRV site of the same vector. Part of Morc3 exon 4 (112 base pairs) and exon 5 encoding GHL-ATPase motifs III and IV were replaced by the hrGFP gene and neomycin resistance gene flanked by loxP sites. RW4 Embryonic stem (ES) cells transfected with the NotI-linearized vector were selected with G418 and screened with a 1-kbp probe (see Figure 3A) using the ALKphos direct labeling and detection system with CDP-Star (GE Healthcare, Waukesha, WI). Generation of Morc3-knockout mice from two independent targeted ES clones was carried out as described previously (Ikeda et al., 1999). MEFs were generated by intercrossing Morc3+/– mice backcrossed to C57/BL6J one to three times. Animal experiments were conducted in accordance with our institutional guidelines.
Antibodies
The following antibodies and antisera were used: mouse anti-FLAG M2 (Sigma), rabbit polyclonal anti-FLAG (Sigma), rabbit polyclonal anti-HA (Sigma), mouse monoclonal anti-PML (PG-M3; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-PML (H-238; Santa Cruz), goat polyclonal anti-mouse Pml (E-15; Santa Cruz), rabbit polyclonal anti-Sp100 (Chemicon, Temecula, CA), mouse monoclonal anti-human p53 (DO-1; Santa Cruz), rabbit polyclonal anti-mouse p53 (CM5; Novocastra, Newcastle, United Kingdom), rabbit polyclonal anti-p21 (C-19; Santa Cruz), mouse monoclonal anti-p16 (DCS-50; Sigma), rabbit anti-CBP (A-22; Santa Cruz), and mouse monoclonal anti--tubulin (TUB 2.1; Sigma) antibodies. The following rabbit polyclonal antibodies were used to assay posttranslational modifications of p53: anti-phospho-p53 (Ser15) (9284; Cell Signaling, Beverly, MA), anti-phospho-p53 (Ser20) (9287; Cell Signaling), and anti-acetyl-p53 (Lys373, Lys382) (06–758; Upstate Biotechnology, Lake Placid, NY) antibodies. Anti-MORC3 rabbit polyclonal antiserum was raised against recombinant Histidine-tagged-MORC3 (His-MORC3) purified from Rosetta DE3 (Novagen, Madison, WI) carrying pET16b-His-MORC3. We established a hybridoma clone (17A9) from mice immunized with His-MORC3 and purified monoclonal anti-MORC3 antibody from it by standard methods.
Western Blotting
Proteins were transferred from SDS-PAGE gels to Hybond-P (GE Healthcare) or Immobilon-P (Millipore, Bedford, MA) membranes and visualized by incubation with various primary antibodies and peroxidase-conjugated secondary antibodies (Sigma), using ECL or ECL-Plus detection reagents (GE Healthcare).
Immunofluorescence
Immunofluorescence studies were performed using the standard procedure previously described (Watanabe et al., 1996). Briefly, cells grown on Lab-Tek II Chamber Slides (Nalge Nunc, Naperville, IL) were fixed and permeabilized. Cells were then incubated with the indicated antibodies and further incubated with appropriate Alexa Fluor-488– or Alexa Fluor-594–conjugated, cross-adsorbed secondary antibodies (Invitrogen). Confocal imaging was performed using a Radiance 2000 laser-scanning system (Bio-Rad, Richmond, CA) connected to a BX51 microscope (Olympus, Melville, NY). When cells were permeabilized before fixation, they were treated with CSK buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 3 mM MgCl2, 300 mM sucrose, and complete protease inhibitor cocktail [Complete Mini, EDTA free; Roche]) with 0.5% Triton X-100 for 5 min and fixed with 4% paraformaldehyde at 4°C overnight. The fixed cells were stained and observed as described above. NB4 cells were treated with 1 µM of all-trans-retinoic acid (ATRA, Sigma) or dimethyl sulfoxide (DMSO) as vehicle for 3 d, attached to slide glasses with Shandon Cytospin 4 (Thermo Electron, Pittsburgh, PA), and stained as described above.
ATP Depletion Analysis
ATP depletion analysis was performed as previously described (Platani et al., 2002). HeLa cells transiently transfected with pME-FLAG-MORC3-I-P were incubated in glucose-free medium containing 10 mM sodium azide (Wako, Richmond, VA) and 50 mM 2-deoxyglucose (Sigma) for 30 min and then fixed and stained with anti-FLAG antibodies and anti-PML antibodies as described above. To determine the time course of MORC3 localization, HeLa cells were transfected with pME-EGFP-MORC3-I-P and pME-FLAG-MORC3-I-P (1:8) to prevent EGFP aggregation. Live cells were imaged using an IX70 microscope (Olympus) and CoolSnap cf CCD camera (Roper Scientific, Tucson, AZ).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To clarify whether MORC3 induces premature senescence, infected cells were stained for SA--Gal activity at day 6 (Figure 1, B and C). The percentage of SA--Gal–positive cells infected with the MORC3-retrovirus vector (51.0 ± 5.1%) was significantly greater than that of controls infected with vector alone (2.3 ± 0.8%). MORC3 expression also strongly induced p16, another senescence marker (Figure 1D).
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), we next examined the effect of MORC3 expression in wild-type and p53-deficient MEFs (Gondo et al., 1994) (Supplementary Figure 2B). Wild-type MEFs (p53+/+) infected with the MORC3-retrovirus vector ceased proliferating (Figure 1E) and exhibited the same large flat morphology as MORC3-infected WI38 cells (Supplementary Figure 2C). However, MORC3 did not impair proliferation or induce the flat morphology of p53–/– MEFs derived from littermates of p53+/+ mice (Figure 1E and Supplementary Figure 2C).
Expression of MORC3 Activates p53
To examine whether MORC3 regulates p53 activity, we measured the effect of MORC3 expression on p53-mediated transactivation by luciferase reporter analyses. FLAG-MORC3 expression enhanced transcriptional activation by endogenous p53 of a reporter containing 12 p53-responsive elements (PG12S) in a dose-dependent manner, but not activation of a reporter containing 14 mutated responsive elements (MG14S) (Figure 2, A and B). In contrast, FLAG-MORC3-E35A, a mutant of MORC3 lacking ATPase activity but retaining ATP-binding activity generated by mutation of the conserved catalytic glutamic acid 35 in N-box of the GHL-ATPase domain to alanine (Supplementary Figure 1; Panaretou et al., 1998; Ban et al., 1999) showed no transcriptional enhancement (Figure 2A). To examine whether MORC3 also affects transactivation of the promoters of p53-inducible genes, MORC3 was expressed together with p53, and luciferase was transcribed from p21 and Bax promoters in p53-deficient H1299 cells. MORC3 enhanced the p53-dependent activation of p21 (2.5-fold, Figure 2C, left) and Bax (2.6-fold, Figure 2C, right) promoters. MORC3 but not MORC3-E35A also induced the expression of endogenous p21 in U-2 OS cells expressing wild-type p53 (Figure 2D).
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). Therefore, we examined whether overexpression of MORC3 induces p53 stabilization by the phosphorylation and acetylation of p53. In MORC3-overexpressed WI38 cells, p53 was stabilized and phosphorylated at serine 15 but barely acetylated at lysine 373 and 382 (Supplementary Figure 3).
Deficiency of Morc3 Reduces p21 Induction by Adriamycin in MEFs
To investigate whether p53 is activated by genotoxic signals in MORC3 deficient cells, we produced Morc3-knockout mice by homologous recombination in murine ES cells (Figure 3, A–C). We generated two lines of Morc3-targeted mice from independently targeted ES cell clones as described previously (Ikeda et al., 1999). All mice homozygous for the Morc3-disrupted allele (Morc3–/–) died at birth or within a day thereafter for unknown reasons (Yoshida, Takahashi, Mimura, Ishizaki, Tanaka-Okamoto, Miyoshi, Shime, and Inoue, unpublished results). Wild-type, heterozygous and homozygous embryos at 18.5 d postcoitum (dpc) were present at almost the expected Mendelian ratio. MEFs of Morc3+/+ and Morc3–/– established from littermate embryos at 14.5 dpc were morphologically indistinguishable.
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MORC3 Localizes on PML-NBs
In addition to posttranslational modification, recruitment of p53 into PML-NBs is important for its activation (Fogal et al., 2000; Pearson et al., 2000). The mechanism of this recruitment is poorly understood. Because p53 was stabilized but not activated in ADR-treated Morc3–/– cells, we hypothesized that MORC3 could be involved in p53 localization into PML-NBs.
To test this hypothesis, we first determined the subcellular localization of MORC3. An anti-MORC3 mAb specifically stained entire nuclei and a few small nuclear foci together with Pml, except for the nucleolus, in Morc3+/+ but not Morc3–/– MEFs (Figure 4A and Supplementary Figure 4, A and B). In human cell lines, Saos-2 (Figure 4B) and HeLa cells (Supplementary Figure 4C), endogenous MORC3 was detected primarily on nuclear foci, together with PML (Figure 4B, top images) and Sp100, which is constitutively localized on PML-NBs (Figure 4B, bottom images) and also in entire nuclei. These results indicate that MORC3 localizes to both PML-NBs and the nucleoplasm. Although we detected MORC3 nuclear foci in almost all PML-NBs of most human and mouse cells, the size and the number of these nuclear foci were different in cell lines. When FLAG-MORC3 was expressed in HeLa cells, endogenous PML was localized within almost all of the nuclear foci formed by overexpressed FLAG-MORC3 in all cells (Figure 4C, top images), and endogenous Sp100 was completely colocalized with FLAG-MORC3 in 44% of the FLAG-MORC3–expressing cells (Figure 4C, bottom images).
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(RAR) (Dyck et al., 1994; Weis et al., 1994). In these cells, PML-NB structure is disrupted but is restored by treatment with all-trans-retinoic acid (ATRA; Figure 4D). MORC3 was diffusely distributed in the entire nucleus of untreated NB4 cells, and nuclear foci were detected in only 2.86% of the cells (Figure 4D, top images). ATRA treatment induced PML-NB formation and recruitment of MORC3 (Figure 4D, bottom images) as well as Sp100 and CBP (Supplementary Figure 5, A and B) to the reorganized PML-NBs in 15.4% of the cells. The localization of MORC3 to nuclear foci was also suppressed in PML-knockdown cells (Figure 4E). Furthermore, in WI38 cells, overexpression of PML IV increased the localization of endogenous MORC3 to PML-NBs (Figure 4F).
Role of GHL-ATPase Domain in Localization of MORC3 into PML-NBs
Because expression of the ATPase-deficient MORC3-E35A mutant protein did not activate p53, we examined the effect of this mutation on MORC3 localization. FLAG-MORC3-E35A showed diffuse nuclear localization in HeLa cells (Figure 4G). Furthermore, depleting HeLa cells expressing wild-type FLAG-MORC3 of ATP by adding sodium azide and 2-deoxyglucose to the medium caused MORC3 to disperse from PML-NBs within 30 min in most cells, whereas endogenous PML remained localized to PML-NBs (Figure 4H). To study the time course of this dispersion in living cells, we expressed EGFP-MORC3 in HeLa cells. EGFP-MORC3 gradually dispersed after ATP depletion (Figure 4I).
MORC3 Induces Localization of p53 and Sp100 into PML-NBs
In premature senescence induced by expression of PML IV or oncogenic Ras, p53 and CBP are recruited into PML-NBs to form a ternary complex with PML (Pearson et al., 2000; Bischof et al., 2002). To test whether MORC3 mediates the recruitment of p53 and CBP into PML-NBs, we analyzed the effect of MORC3 overexpression on p53 and CBP localization. Overexpressed FLAG-MORC3 colocalized with endogenous p53 (Figure 5, A and B, top images) and CBP (Figure 5, A and B, bottom images) in U-2 OS cells (Figure 5A) and WI38 cells (Figure 5B), but p53 showed diffuse nuclear localization in nontransfected and FLAG-MORC3-E35A–transfected cells (Supplementary Figure 6). Furthermore, p53 and PML IV specifically formed a complex with MORC3 (Supplementary Figure 7). We then examined the effect of MORC3 repression on Sp100, p53, and CBP subnuclear localization. MORC3 was efficiently repressed by shRNA732 in HeLa cells (Figure 5C). Endogenous Sp100 was completely dispersed or localized to two or fewer nuclear foci in DsRed2-positive cells (41.3%) in which MORC3 was simultaneously repressed (Figure 5D). In contrast, fewer than 10% of cells transfected with a mutant shRNA695 (8.7%) or a control vector (5.3%) showed diffuse localization of Sp100 (Supplementary Figure 8A). Notably, subcellular localization of PML was not altered by shRNA knockdown of MORC3 (supplementary Figure 8B). We also examined the effect of MORC3 repression in p53-expressing Saos-2 cells treated with ADR. In cells transfected with the shRNA control vector, p53 was recruited into PML-NBs 6 h after ADR treatment (14% of PML IV–expressing cells; Figure 5E, top images). However, we could not detect ADR-induced recruitment of p53 into PML-NBs in MORC3-repressed cells (Figure 5E, middle images). In contrast, endogenous CBP localized on PML-NBs in both MORC3-knockdown cells (Figure 5E, bottom images) and cells transfected with control vector (data not shown). To clarify the role of ATPase domain of MORC3 in the localization of Sp100 and p53 to PML-NBs, we transfected HeLa cells with the vector carrying FLAG-MORC3-E35A. Overexpression of the mutant MORC3 protein caused dispersion of Sp100 in 91% of cells (Figure 5F, top images), whereas PML was still localized in NBs (Figure 5F, bottom images).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), little is known about other machinery involved in localizing proteins from the nucleoplasm to subnuclear domains. This is the first reported ATP-dependent molecular mechanism of recruitment of proteins to a subnuclear domain essential for an important biological response.
Induction of Premature Senescence and Recruitment of Nuclear Proteins into PML-NBs by MORC3
Both the p53 and pRB pathways are essential for cells to establish and maintain the senescent state (Serrano et al., 1997; Campisi, 2005). We show that expression of MORC3 induced premature senescence (Figure 1) through activation of p53 (Figure 2). Formation of a ternary complex of p53, CBP, and PML in PML-NBs is essential for this process (Pearson et al., 2000; Bischof et al., 2002). MORC3 mediated the localization of both p53 and CBP into PML-NBs in U-2 OS cells and WI38 cells (Figure 5, A and B) and formed a complex with p53 and PML IV (Supplementary Figure 7). In MORC3-repressed cells treated with ADR, CBP but not p53 localized into PML-NBs (Figure 5E). These results explain why p53 was not efficiently activated in ADR-treated Morc3–/– cells despite its stabilization and suggest that MORC3 mediates p53 recruitment to a CBP and PML complex in the process of premature senescence and p53 activation induced by genotoxic stress. MORC3 was partially localized in PML-NBs by ATRA treatment of NB4 cells (Figure 4D). The partial rather than complete localization of MORC3 could be due to incomplete reorganization of PML-NBs or to the expression of the fusion protein, PML-RAR. Furthermore, overexpression of PML IV also recruited MORC3 (Figure 4F) as well as CBP and p53 into PML-NBs (Pearson et al., 2000). These results suggest that MORC3 recognizes PML to recruit p53 into PML-NBs.
Effect of MORC3 on Posttranslational Modification of p53
Localization of p53 on PML-NBs is thought to mediate its posttranslational modifications, including acetylation of lysine residue 382 (mouse K379) by p300/CBP (Pearson et al., 2000) and phosphorylation of human p53 serine residue 20 (mouse S23) by CHK2 (Louria-Hayon et al., 2003) and serine residue 46 (no equivalent residue in mice) by HIPK2 (D'Orazi et al., 2002; Hofmann et al., 2002). In Morc3–/– cells treated with ADR, p53 acetylation on K373 (mouse K370) and/or K382 but not phosphorylation of S20 was slightly but reproducibly reduced compared with wild type (Figure 3E). However, the magnitude of this reduction does not appear sufficient to explain the lack of p53 activation. Although PML is essential for acetylation of p53 (Fogal et al., 2000; Pearson et al., 2000), MORC3-dependent localization of p53 into PML-NBs is not necessary for its stabilization through phosphorylation and acetylation but is required for its activation in response to genotoxic stress. The relationship between acetylation, phosphorylation, and subnuclear localization of p53 requires further study.
Roles of MORC3 GHL-ATPase Domain
We showed that the ATPase activity-deficient MORC3 mutation, glutamic acid 35 to alanine, caused diffuse nuclear localization of MORC3 and could not increase the activation of p53. We also showed by an ATP-depletion experiment that ATP is necessary for the PML-NBs localization of MORC3. The glutamic acid is conserved in all GHL-ATPase family proteins but not in Histidine kinases of the GHKL-ATPase/kinase superfamily and functions as a base for water activation in ATP hydrolysis (Dutta and Inouye, 2000). GHL-ATPase family proteins exist as homodimers, and hydrolysis of ATP causes a conformational change that regulates intermolecular interaction and association with other molecules. By immunoprecipitation analysis, MORC3 also forms at least a homodimer (Supplementary Figure 7). MORC3 may associate with other molecules in the nucleoplasm or PML-NBs in an ATP-dependent conformation.
Other MORC Family Proteins
It is likely that other MORC family proteins play important roles in shuttling various proteins between nuclear subdomains and the nucleoplasm. In fact, Spo11, which mediates double-strand chromosomes breaks during the first meiotic stage, cannot form foci on chromosomes in Morc1-deficient mice (Romanienko and Camerini-Otero, 2000). Morc1 may thus serve a function analogous to MORC3 in recruiting proteins such as Spo11 onto foci of meiotic chromosomes. The ancient phylogeny of MORC proteins (Inoue et al., 1999) suggests that other members are also involved in intranuclear shuttling mechanisms that remains to be elucidated.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Present address: Department of Pediatrics, University of Michigan Health System, Ann Arbor, MI 48109-0656.
Address correspondence to: Norimitsu Inoue (inoue-no{at}mc.pref.osaka.jp).
Abbreviations used: MORC, microrchidia; PML, promyelocytic leukemia; NBs, nuclear bodies; CBP, CRE-binding protein; MEFs, mouse embryonic fibroblasts; SA--Gal, senescence–associated -galactosidase; bp, base pairs; I-P, internal ribosomal entry site-puromycin resistance gene; dpc, days postcoitum; ADR, adriamycin; ATRA, all-trans-retinoic acid.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), EGFP-MORC3 (MORC3, 
) or EGFP-PML IV (PML IV, 
) were selected with puromycin for 4 d, and the number of the cells was counted at days 3, 6, 9, and 12. Day 0 indicates the first day after the selection. (B) Representative senescence-associated 
