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• W2069503379 abstract "Cdt1 is an essential component for the assembly of a pre-replicative complex. Cdt1 activity is inhibited by geminin, which also participates in neural development and embryonic differentiation in many eukaryotes. Although Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi. Here, we identify the C. elegans geminin, GMN-1. Biochemical analysis reveals that GMN-1 associates with C. elegans CDT-1, the Hox protein NOB-1, and the Six protein CEH-32. GMN-1 inhibits not only the interaction between mouse Cdt1 and Mcm6 but also licensing activity in Xenopus egg extracts. RNA interference-mediated reduction of GMN-1 is associated with enlarged germ nuclei with aberrant nucleolar morphology, severely impaired gametogenesis, and chromosome bridging in intestinal cells. We conclude that the Cdt1-geminin system is conserved throughout metazoans and that geminin has evolved in these taxa to regulate proliferation and differentiation by directly interacting with Cdt1 and homeobox proteins. Cdt1 is an essential component for the assembly of a pre-replicative complex. Cdt1 activity is inhibited by geminin, which also participates in neural development and embryonic differentiation in many eukaryotes. Although Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi. Here, we identify the C. elegans geminin, GMN-1. Biochemical analysis reveals that GMN-1 associates with C. elegans CDT-1, the Hox protein NOB-1, and the Six protein CEH-32. GMN-1 inhibits not only the interaction between mouse Cdt1 and Mcm6 but also licensing activity in Xenopus egg extracts. RNA interference-mediated reduction of GMN-1 is associated with enlarged germ nuclei with aberrant nucleolar morphology, severely impaired gametogenesis, and chromosome bridging in intestinal cells. We conclude that the Cdt1-geminin system is conserved throughout metazoans and that geminin has evolved in these taxa to regulate proliferation and differentiation by directly interacting with Cdt1 and homeobox proteins. Eukaryotic DNA replication is controlled by the stepwise assembly of a prereplicative complex (pre-RC) 1The abbreviations used are: pre-RC, pre-replicative complex; pre-IC, pre-initiation complex; ORC, origin recognition complex; MCM, minichromosome maintenance protein complex; EST, expressed sequence tag; RNAi, RNA interference; dsRNA, double-stranded RNA; GST, glutathione S-transferase. 1The abbreviations used are: pre-RC, pre-replicative complex; pre-IC, pre-initiation complex; ORC, origin recognition complex; MCM, minichromosome maintenance protein complex; EST, expressed sequence tag; RNAi, RNA interference; dsRNA, double-stranded RNA; GST, glutathione S-transferase. and the replication apparatus. The pre-RC includes the origin recognition complex (ORC), the minichromosome maintenance protein complex (MCM), as well as the Cdc6 and Cdt1 proteins. After activation of S phase-promoting kinases, pre-RC converts to the pre-initiation complex (pre-IC). This step includes the loading of Cdc45 and Gins complex, which allows DNA polymerases to assemble with the replication apparatus. Each step in the assembly of pre-RC, pre-IC, and replication apparatus is regulated by checkpoint mechanisms thus maintaining genome integrity and ploidy and avoiding disposition to carcinogensesis (1Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1385) Google Scholar, 2Blow J.J. Hodgson B. Trends Cell Biol. 2002; 12: 72-78Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 3Mendez J. Stillman B. BioEssays. 2003; 25: 1158-1167Crossref PubMed Scopus (169) Google Scholar). Cdt1 is an essential component of licensing reaction conserved in eukaryotes including human, Xenopus, Drosophila, Caenorhabditis elegans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae (4Maiorano D. Moreau J. Mechali M. Nature. 2000; 404: 622-625Crossref PubMed Scopus (292) Google Scholar, 5Nishitani H. Lygerou Z. Nishimoto T. Nurse P. Nature. 2000; 404: 625-628Crossref PubMed Scopus (365) Google Scholar, 6Whittaker A.J. Royzman I. Orr-Weaver T.L. Genes Dev. 2000; 14: 1765-1776PubMed Google Scholar, 7Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (573) Google Scholar, 8Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Devault A. Vallen E.A. Yuan T. Green S. Bensimon A. Schwob E. Curr. Biol. 2002; 12: 689-694Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 10Tanaka S. Diffley J.F. Nat. Cell Biol. 2002; 4: 198-207Crossref PubMed Scopus (216) Google Scholar, 11Zhong W. Feng H. Santiago F.E. Kipreos E.T. Nature. 2003; 423: 885-889Crossref PubMed Scopus (258) Google Scholar). Cdt1 directly associates with MCM2–7 complex to form a pre-RC in late mitosis and early G1 phase. Cdt1-dependent loading of MCM2–7 onto chromatin is the limiting step for preventing reinititation, because overexpression of Cdt1 causes re-replication in Xenopus egg extract (5Nishitani H. Lygerou Z. Nishimoto T. Nurse P. Nature. 2000; 404: 625-628Crossref PubMed Scopus (365) Google Scholar, 12Arias E.E. Walter J.C. Genes Dev. 2005; 19: 114-126Crossref PubMed Scopus (163) Google Scholar, 13Li A. Blow J.J. EMBO J. 2005; 24: 395-404Crossref PubMed Scopus (115) Google Scholar, 14Maiorano D. Krasinska L. Lutzmann M. Mechali M. Curr. Biol. 2005; 15: 146-153Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 15Yoshida K. Takisawa H. Kubota Y. Genes Cells. 2005; 10: 63-73Crossref PubMed Scopus (52) Google Scholar, 16Nishitani H. Lygerou Z. Nishimoto T. J. Biol. Chem. 2004; 279: 30807-30816Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Cdt1 activity is inhibited by geminin. Geminin was first identified in Xenopus egg extract as degradable protein by anaphase-promoting complex and later characterized as the licensing inhibitor through direct interaction with Cdt1 (7Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (573) Google Scholar, 17Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (389) Google Scholar, 18McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar). Furthermore, geminin has been found in various multicellular eukaryotes including human, Xenopus, and Drosophila,to participate in neural development and embryonic differentiation (19Kroll K.L. Salic A.N. Evans L.M. Kirschner M.W. Development (Camb.). 1998; 125: 3247-3258Crossref PubMed Google Scholar, 20Quinn L.M. Herr A. McGarry T.J. Richardson H. Genes Dev. 2001; 15: 2741-2754Crossref PubMed Scopus (145) Google Scholar, 21Del Bene F. Tessmar-Raible K. Wittbrodt J. Nature. 2004; 427: 745-749Crossref PubMed Scopus (211) Google Scholar, 22Luo L. Yang X. Takihara Y. Knoetgen H. Kessel M. Nature. 2004; 427: 749-753Crossref PubMed Scopus (181) Google Scholar). In contrast, geminin homologue has not been found in C. elegans as well as monocellular eukaryotes including S. cerevisiae and S. pombe yet. To better understand the relevance of the geminin-Cdt1 system in higher eukaryotes, we described the domain organization of mouse Cdt1 (8Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The less conserved amino-terminal region is responsible for binding DNA, whereas the central region has geminin binding activity and is conserved among multicellular eukaryotes including C. elegans. The carboxyl-terminal region, also conserved, functions as an Mcm6-binding domain. Because C. elegans CDT-1 has a geminin-binding domain, we undertook to find a C. elegans geminin homologue. Cloning of C. elegans cdt-1, mcm-6, and gmn-1 cDNAs—The amino acid sequence of Drosophila geminin was used to screen the NCBI data base for C. elegans geminin, which yielded a sequence with the accession number Y75B8A.17. To generate full-length C. elegans geminin, two exons were amplified by the PCR with C. elegans genomic DNA as a template and sequence-specific primers (forward primer for exon 1, 5′-CCGAATTCCATATGAGCAGAATCGGCTT-3′; reverse primer for exon 1, 5′-CCCGGTACCGTACTCAAACATCTGCGTCTCCA-3′; forward primer for exon 2, 5′-CCCGGTACCGTGTCCACGCAAACGATCAT-3′; reverse primer for exon 2, 5′-CCGGATCCTACTCGAGTTGCGCGG-3′). The PCR products were digested with EcoRI-KpnI or KpnI-XhoI and inserted into EcoRI- and XhoI-digested pET24b (Novagen) to generate pET-C. elegans gmn-1-His6. cDNAs for C. elegans cdt-1, mcm-6, nob-1, and ceh-32 were amplified by PCR using EST clones yk10c5, yk401h5, and yk678c5 for cdt-1, yk1057e12, and yk1327h09 for mcm-6, yk403d9, and yk467d4 for nob-1, and yk1006f04 and yk340g10 for ceh-32. The following PCR primers were used: cdt-1-forward, 5′-CCGGATCCATATGAAGTTCCCGGGTGACTAGATCC-3′; cdt-1-reverse, 5′-GGCTCGAGATGAAATTTGAGAGATCTTGCTGC-3′; mcm-6-forward, 5′-GGTCTAGATCTATGGACAACATTATCGGCGG-3′; mcm-6-reverse, 5′-CCCGGCCGCTCGAGTTATTCGTCGGCAATAACAT-3′; nob-1-forward, 5′-CCGAATTCCATATGATTTCGGTGATGCAACAAATG-3′; nob-1-reverse, 5′-GGCTCGAGTTAGTTGATCAATCGCTCGATGCAC-3′; ceh-32-forward, 5′-CCGAATTCCATATGTTCACTCCAGAACAGTTCAC-3′; ceh-32-reverse, 5′-GGCTCGAGTTACTCAGATTGAGATGTCGTTGAC-3′. The cdt-1 and mcm-6 PCR products were digested with NdeI-XhoI and inserted into NdeI- and XhoI-digested pET24b-FLAG, which contains the FLAG tag sequence in the 5′-region as described previously (8Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The nob-1 and ceh-32 PCR products were digested with EcoRI-XhoI and inserted into EcoRI- and XhoI-digested pGEX-4T (Amersham Biosciences). All constructs were confirmed by DNA sequencing on an Applied Biosystems 377A automatic DNA sequencer. The nucleotide sequences reported in this paper have been submitted to the GenBank™/DDBJ Data Bank with accession number AB190260 for C. elegans gmn-1. Yeast Two-hybrid Analysis—Yeast two-hybrid analysis was performed as described (8Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). cDNAs for C. elegans cdt-1, mcm-6, gmn-1, and orc-2 (yk1265f03 and yk236f8) were amplified using specific-sequence primers with appropriate restriction sites. PCR products were digested with EcoRI, MunI, SalI, or XhoI and subcloned into compatible sites of pLexA and pB42AD. Expression and Purification of Recombinant Proteins in E. coli— FLAG- and His6-tagged mouse Cdt1, mouse Mcm6, and C. elegans CDT-1, GST-tagged C. elegans NOB-1 and CEH-32, and T7 as well as His6-tagged mouse geminin and C. elegans GMN-1 were overproduced in the E. coli strain BL21(DE3) and purified as described (8Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The recombinant mouse Mcm4/6/7 complex was purified from insect cells as described (8Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The details of glycerol gradient centrifugation have been described previously (23Mizuno T. Ito N. Yokoi M. Kobayashi A. Tamai K. Miyazawa H. Hanaoka F. Mol. Cell. Biol. 1998; 18: 3552-3562Crossref PubMed Scopus (41) Google Scholar). The native molecular weight of gmn-1 was obtained by using the equation of Siegel and Monty (24Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1542) Google Scholar) as described by Saxena et al. (25Saxena S. Yuan P. Dhar S.K. Senga T. Takeda D. Robinson H. Kornbluth S. Swaminathan K. Dutta A. Mol. Cell. 2004; 15: 245-258Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Coimmunoprecipitation Analysis—Fifty ng FLAG-mouse Cdt1, FLAG-C. elegans CDT-1, and FLAG-mouse Mcm6 were mixed in the presence or absence of various amounts of geminin and immunoprecipitated with anti-FLAG M2 antibody-conjugating agarose (Sigma) for 4 h at 4 °C in 150 μl of NET-gel buffer containing 20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.1% Nonidet P-40, and 0.25% gelatin. After washing with NET-gel buffer, precipitates were dissolved in 30 μl of 2× Laemmli sample buffer and subjected to SDS-PAGE and Western blot analysis using anti-Mcm6 (Santa Cruz Biotechnology), anti-FLAG tag (Sigma), and anti-T7 tag (Novagen) antibodies as described (8Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Fifty or 250 ng of GST-tagged NOB-1 and CEH-32 was mixed with GMN-1, and GMN-1 associated with GST-tagged proteins was precipitated with glutathione-Sepharose as described (8Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). A construct with an amino-terminal fragment of mouse Cdt1 (MmCdt1-(1–130) fused to GST was used as a negative control (8Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Licensing Activity Assay Using Xenopus Egg Extracts—Xenopus egg extract and demembranated sperm nuclei were prepared as reported (17Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (389) Google Scholar). Cdt1-depleted extract was prepared starting with an interphase Xenopus egg extract supplemented with 25 mm phosphocreatine, 15 μg ml-1 creatine phosphokinase, and 0.25 mg ml-1 cycloheximide using anti-Xenopus Cdt1 antiserum following a previously described method (17Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (389) Google Scholar). Sperm chromatin competent for the licensing reaction was prepared by incubation of demembranated sperm nuclei (10,000 nuclei μl-1) with the Cdt1-depleted extract for 20 min to allow assembly with ORC and Cdc6, and the resulting chromatin was isolated. The licensing reaction was carried out by a 15-min incubation of a reaction mixture (4 μl) containing competent chromatin, partially purified Mcm2–7 fraction, and GST-fused Xenopus Cdt1 in the absence or presence of 50 ng μl-1 Xenopus geminin or C. elegans GMN-1. After the addition of 10 μl of interphase Xenopus egg extract supplemented with [α-32P]dATP (20 kBq) and an excess of Xenopus geminin (100 ng μl-1), licensing activity was assessed by measuring radioactivity incorporated into newly synthesized DNA during a further 3-h incubation. To measure the association of Mcm4 with chromatin, sperm chromatin was incubated with Mcm2–7 as described above and pelleted from the replication assay, and the amount of Mcm4 bound to the chromatin was determined by Western blot analysis. RNA Interference Assay—Templates for RNAi were prepared by amplification of a genomic fragment of gmn-1/Y75B8A.17 using the exon 1 forward primer and the exon 2 reverse primer, and this fragment was subcloned into the EcoRI/XhoI site of pBluescript. RNAi was performed using double-stranded RNA (dsRNA) derived from the gmn-1 genomic clone. The cloned gmn-1 DNA fragment was amplified with primers containing the T7 RNA polymerase promoter sequence, and dsRNA was synthesized in vitro with T7 RNA polymerase. Delivery of dsRNA into worms was performed by soaking (26Maeda I. Kohara Y. Yamamoto M. Sugimoto A. Curr. Biol. 2001; 11: 171-176Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar) or microinjection (27Kamath R.S. Fraser A.G. Dong Y. Poulin G. Durbin R. Gotta M. Kanapin A. Le Bot N. Moreno S. Sohrmann M. Welchman D.P. Zipperlen P. Ahringer J. Nature. 2003; 421: 231-237Crossref PubMed Scopus (2665) Google Scholar). Initial screens of the C. elegans genome data base using cDNAs for human geminin as queries to search for homologous EST clones was not successful. However, when Drosophila geminin was used as a query, a PSI-BLAST search yielded a single homologous gene, Y75B8A.17. Previous large scale RNAi analyses reported no detectable phenotypes for inactivation of this gene (27Kamath R.S. Fraser A.G. Dong Y. Poulin G. Durbin R. Gotta M. Kanapin A. Le Bot N. Moreno S. Sohrmann M. Welchman D.P. Zipperlen P. Ahringer J. Nature. 2003; 421: 231-237Crossref PubMed Scopus (2665) Google Scholar, 28Gonczy P. Echeverri C. Oegema K. Coulson A. Jones S.J. Copley R.R. Duperon J. Oegema J. Brehm M. Cassin E. Hannak E. Kirkham M. Pichler S. Flohrs K. Goessen A. Leidel S. Alleaume A.M. Martin C. Ozlu N. Bork P. Hyman A.A. Nature. 2000; 408: 331-336Crossref PubMed Scopus (730) Google Scholar). Amino acid alignments showed that the protein has a putative coiled-coil structure. The COILS program predicted that residues 119–150 have a coiled-coil probability >0.9 and form five hepted repeats, and residues 114–171 have a probability >0.5 and form eight hepted repeats. The coiled-coil structure is conserved in human, mouse, Xenopus, and Drosophila geminin (18McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar, 20Quinn L.M. Herr A. McGarry T.J. Richardson H. Genes Dev. 2001; 15: 2741-2754Crossref PubMed Scopus (145) Google Scholar) (Fig. 1A), indicating that the protein encoded by Y75B8A.17 is the C. elegans geminin homologue. We named the gene gmn-1 according to the nomenclature of Caenorhabditis Genetics Center. The open reading frame predicted by EST sequences and by the genome sequence encodes a putative protein of 180 amino acid residues with a calculated molecular mass of 20.2 kDa, which shares 19% identity and 52% similarity with the central coiled-coil region of Drosophila geminin (Fig. 1B). We also identified a single gmn-1 ortholog (CBG23049) in Caenorhabditis briggsae, a related nematode that diverged from C. elegans roughly 100 million years ago (29Stein L.D. Bao Z. Blasiar D. Blumenthal T. Brent M.R. Chen N. Chinwalla A. Clarke L. Clee C. Coghlan A. Coulson A. D'Eustachio P. Fitch D.H. Fulton L.A. Fulton R.E. Griffiths-Jones S. Harris T.W. Hillier L.W. Kamath R. Kuwabara P.E. Mardis E.R. Marra M.A. Miner T.L. Minx P. Mullikin J.C. Plumb R.W. Rogers J. Schein J.E. Sohrmann M. Spieth J. Stajich J.E. Wei C. Willey D. Wilson R.K. Durbin R. Waterston R.H. PloS Biol. 2003; 1: E45Crossref PubMed Scopus (672) Google Scholar). C. elegans gmn-1 and the C. briggsae orthologue share the same exon-intron structure, and their predicted protein products share 56% identity (69% similarity), with higher conservation in the coiled-coil region (73% identity and 84% similarity). To determine whether C. elegans contains a geminin-Cdt1 system similar to that of other multicellular eukaryotes, we further characterized the putative protein, GMN-1. We first carried out a yeast two-hybrid analysis using cDNAs encoding Cdt1 and Mcm6 from both mouse and C. elegans and the geminin candidate GMN-1. GMN-1 was found to associate with C. elegans CDT-1 and mouse Cdt1 and, conversely, mouse geminin associated with C. elegans CDT-1 (Fig. 1C). Similarly, both C. elegans and mouse Mcm6 interacted with both C. elegans and mouse Cdt1. In contrast, C. elegans CDT-1 does not associate with ORC-2, suggesting that the Cdt1-Orc2 interaction is not conserved among metazoans. To confirm the protein-protein interactions suggested by yeast two-hybrid analysis, biochemical characterization was carried out using recombinant proteins. Purified CDT-1, mouse Cdt1, and mouse Mcm6 were mixed with mouse geminin or GMN-1, and bound proteins were precipitated by a FLAG-tag inserted at the amino termini of Cdt1 and Mcm6. Both mouse geminin and GMN-1 were detected in bound fractions (Fig. 2C). In contrast, in the presence of mouse Mcm6, neither protein was co-precipitated. The amount of GMN-1 precipitated in a complex with Cdt1 was less than that of mouse geminin, indicating that the affinity of GMN-1 for CDT-1 is lower than that of mouse geminin. Taken together, we conclude that GMN-1 can bind both mouse and C. elegans Cdt1 proteins and that the Cdt1-geminin interaction is conserved in C. elegans and the mouse. Next, we examined the effect of GMN-1 on the interaction between Mcm6 and Cdt1. In the presence of GMN-1, the amount of mouse Mcm6 co-precipitated with Cdt1 was severely reduced in a dose-dependent manner (Fig. 2D). Thus, like mouse geminin, GMN-1 interferes with the interaction between Cdt1 and Mcm6. During purification procedures, we found that gel filtration of gmn-1 showed it eluting with a molecular mass of about 100 kDa (Fig. 2B). Glycerol gradient centrifugation showed it migrating with an apparent molecular mass of 14 kDa (Fig. 2B). Combining these values in the equation of Siegel and Monty (24Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1542) Google Scholar) with calculation as described by Saxena et al. (25Saxena S. Yuan P. Dhar S.K. Senga T. Takeda D. Robinson H. Kornbluth S. Swaminathan K. Dutta A. Mol. Cell. 2004; 15: 245-258Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) gives a value of 35 kDa for the native molecular mass of gmn-1, suggesting that it exists as a highly elongated, nonglobular dimer, which is consistent with a number of recent reports (25Saxena S. Yuan P. Dhar S.K. Senga T. Takeda D. Robinson H. Kornbluth S. Swaminathan K. Dutta A. Mol. Cell. 2004; 15: 245-258Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 30Ferenbach A. Li A. Brito-Martins M. Blow J.J. Nucleic Acids Res. 2005; 33: 316-324Crossref PubMed Scopus (55) Google Scholar). Since geminin was reported to be a negative regulator for DNA replication, in that it inhibits Cdt1 activity in a licensing assay system using Xenopus egg extracts (7Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (573) Google Scholar, 17Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (389) Google Scholar), we next asked whether GMN-1 also represses Cdt1 activity in this assay. Licensing activity was assayed with increasing amounts of Xenopus Cdt1 in the presence or absence of Xenopus geminin or GMN-1. GMN-1 has a weak but detectable inhibitory effect on licensing activity (Fig. 3A). In addition, the inhibitory effect was overcome by adding more Cdt1, indicating that GMN-1 affects Cdt1 activity. To further understand how GMN-1 inhibits licensing activity, we precipitated chromatin from the licensing system and measured the amount of MCM complex bound to chromatin by Western analysis. GMN-1 reduced the amount of Mcm4 that associates with chromatin (Fig. 3B). Thus, these results strongly suggest that GMN-1 is a functional counterpart of Xenopus geminin. We speculate that its weakly inhibitory effect reflects its lower affinity for mouse and C. elegans Cdt1 proteins. Recently, Zhong et al. (11Zhong W. Feng H. Santiago F.E. Kipreos E.T. Nature. 2003; 423: 885-889Crossref PubMed Scopus (258) Google Scholar) reported that an RNAi-mediated decrease in the expression of C. elegans cul-4, a subunit of the ubiquitin ligase complex, results in CDT-1 accumulation and extensive re-replication. Since geminin is thought to prevent re-replication by controlling Cdt1 function, RNAi experiments to inhibit the expression of gmn-1 were carried out. Although gmn-1 dsRNA delivered by the feeding method did not result in a detectable phenotype, consistent with a previous report (27Kamath R.S. Fraser A.G. Dong Y. Poulin G. Durbin R. Gotta M. Kanapin A. Le Bot N. Moreno S. Sohrmann M. Welchman D.P. Zipperlen P. Ahringer J. Nature. 2003; 421: 231-237Crossref PubMed Scopus (2665) Google Scholar), dsRNA introduced by the soaking method (26Maeda I. Kohara Y. Yamamoto M. Sugimoto A. Curr. Biol. 2001; 11: 171-176Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar) or by microinjection (31Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11430) Google Scholar) caused sterility in ∼20% of animals of the F1 generation (Table I). Further observation of sterile F1 worms by Nomarski microscopy indicated aberrant germ line development. Oogenesis was severely affected, and partially differentiated germ cells accumulated in the proximal arm of the gonad (Fig. 4A). In most of sterile worms, some germ nuclei in the distal region of the gonad (where germ cells undergo mitosis) were enlarged, and their nucleoli were misshapen (Fig. 4B).Table IRNAi phenotype of gmn-1Sterile F1 animalsNumber of P0 animalsSoaking methodControl (H2O)0/252(0%)7gmn-1(RNAi)107/376(28.5%)12MicroinjectionControl (H2O)0/206(0%)6gmn-1(RNAi)112/552(20.3%)9 Open table in a new tab In addition to these germ line defects, mitotic defects were also detected. Intestinal cells of C. elegans become binucleate and polyploid during larval development (32Hedgecock E.M. White J.G. Dev. Biol. 1985; 107: 128-133Crossref PubMed Scopus (141) Google Scholar). In the late L1 stage, most intestinal nuclei divide without accompanying cell division, giving rise to 20 intestinal cells with a total of 30–34 nuclei. In addition, intestinal nuclei go through one round of endoreduplication per molt, so that the ploidy of each nucleus is 32C by the final molt. We found that some RNAi-induced sterile gmn-1 adults (17/99 = 17%) showed one to three pairs of chromosome bridging in a total of 30–34 intestinal nuclei, indicative of a defect in coordination between endoreduplication and the nuclear division cycle (Fig. 4C). Recently, geminin was shown to bind various nuclear proteins including homeodomain proteins of the Hox family, the Six/sine oculis class protein Six3, and polycomb group proteins (21Del Bene F. Tessmar-Raible K. Wittbrodt J. Nature. 2004; 427: 745-749Crossref PubMed Scopus (211) Google Scholar, 22Luo L. Yang X. Takihara Y. Knoetgen H. Kessel M. Nature. 2004; 427: 749-753Crossref PubMed Scopus (181) Google Scholar). To determine whether GMN-1 has an affinity for homeobox transcription factors, physical interactions between GMN-1 and the C. elegans Hox and Six3 proteins were examined. We cloned cDNAs for nob-1 and ceh-32. The nob-1 gene encodes one of three C. elegans posterior group homeodomain transcription factors orthologous to vertebrate Hox9–13 proteins (33Aboobaker A. Blaxter M. Curr. Opin. Genet. Dev. 2003; 13: 593-598Crossref PubMed Scopus (57) Google Scholar). The ceh-32 gene encodes a Six/sine oculis-type homeodomain transcription factors most closely related to Six3/6 subfamily (34Dozier C. Kagoshima H. Niklaus G. Cassata G. Burglin T.R. Dev. Biol. 2001; 236: 289-303Crossref PubMed Scopus (36) Google Scholar). Both homeobox proteins were expressed as GST fusion proteins and purified. GMN-1 is co-precipitated with NOB-1 as well as with CEH-32 in a dose-dependent manner (Fig. 2E). Although the physiological relationship of GMN-1, NOB-1, and CEH-32 is not yet clear, we conclude that GMN-1 can bind homeobox proteins in vitro, at least NOB-1 and CEH-32, implicating it in the control of development or differentiation through direct interactions with homeobox proteins, as has been found in fish and mice. Previous experiments showed that inhibition of Human, Drosophila, and Xenopus geminin confers different phenotypes. Geminin (-/-) Drosophila exhibits a variety of defects including anaphase chromosome bridges, an increase in the proportion of S phase cells in some tissues including the midgut and the ovaries, and a malformed peripheral nervous system (20Quinn L.M. Herr A. McGarry T.J. Richardson H. Genes Dev. 2001; 15: 2741-2754Crossref PubMed Scopus (145) Google Scholar, 35Mihaylov I.S. Kondo T. Jones L. Ryzhikov S. Tanaka J. Zheng J. Higa L.A. Minamino N. Cooley L. Zhang H. Mol. Cell. Biol. 2002; 22: 1868-1880Crossref PubMed Scopus (167) Google Scholar). In contrast, geminin-depleted Xenopus embryos have a unique early embryonic lethal phenotype, in which cells arrest in G2 phase immediately after the midblastula transition (36McGarry T.J. Mol. Biol. Cell. 2002; 13: 3662-3671Crossref PubMed Scopus (73) Google Scholar). In mammalian cells, RNAi of geminin led to overreplication and the formation of giant nuclei (37Melixetian M. Ballabeni A. Masiero L. Gasparini P. Zamponi R. Bartek J. Lukas J. Helin K. J. Cell Biol. 2004; 165: 473-482Crossref PubMed Scopus (213) Google Scholar, 38Zhu W. Chen Y. Dutta A. Mol. Cell. Biol. 2004; 24: 7140-7150Crossref PubMed Scopus (203) Google Scholar). It is unclear why the geminin-depleted phenotype differs between these species. Our findings that inhibition of gmn-1 confers defects in germ line development and in intestinal cells point to a primary role for geminin as a regulator of development and proliferation in metazoans. Interestingly, RNAi-mediated inhibition of the cul-4 gene to allow accumulation of CDT-1 caused blast cells, but not germ cells, to enlarge due to DNA re-replication (11Zhong W. Feng H. Santiago F.E. Kipreos E.T. Nature. 2003; 423: 885-889Crossref PubMed Scopus (258) Google Scholar). It is noteworthy that gmn-1 is mainly expressed in the germ line and in oocytes 2Y. Kohara, personal communication. (39Kim S.K. Lund J. Kiraly M. Duke K. Jiang M. Stuart J.M. Eizinger A. Wylie B.N. Davidson G.S. Science. 2001; 293: 2087-2092Crossref PubMed Scopus (543) Google Scholar). Thus, we speculate that the accumulation of CDT-1 in germ line provoked by cul-4 depletion is controlled by the GMN-1-mediated inhibition of re-replication. The structural domain organization of geminin has been studied recently (25Saxena S. Yuan P. Dhar S.K. Senga T. Takeda D. Robinson H. Kornbluth S. Swaminathan K. Dutta A. Mol. Cell. 2004; 15: 245-258Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 40Lee C. Hong B. Choi J.M. Kim Y. Watanabe S. Ishimi Y. Enomoto T. Tada S. Cho Y. Nature. 2004; 430: 913-917Crossref PubMed Scopus (117) Google Scholar). Crystallographic analyses revealed that geminin adopts a dimerized coiled-coil structure. The amino-terminal region of the coiled-coil dimer is essential for interaction with Cdt1, while the carboxyl-terminal region prevents access of the MCM complex to Cdt1 through steric hindrance (40Lee C. Hong B. Choi J.M. Kim Y. Watanabe S. Ishimi Y. Enomoto T. Tada S. Cho Y. Nature. 2004; 430: 913-917Crossref PubMed Scopus (117) Google Scholar). Moreover, charged residues in the coiled-coil region are also essential for interaction with Cdt1 (25Saxena S. Yuan P. Dhar S.K. Senga T. Takeda D. Robinson H. Kornbluth S. Swaminathan K. Dutta A. Mol. Cell. 2004; 15: 245-258Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). An amino acid alignment of GMN-1 with other geminin homologues suggests that amino-terminal residues and charged residues in the coiled-coil regions crucial for interaction with Cdt1 are well conserved (Fig. 1A, open and closed circles). Moreover, GMN-1 also contains coiled-coil domains in its carboxyl-terminal region, potentially enabling it to disturb interactions between Cdt1 and Mcm6. Therefore, GMN-1 shares structural properties with its mammalian counterparts. Indeed, estimation of native molecular mass of GMN-1 by combining the results of gel filtration and glycerol gradient sedimentation suggests a value of 35 kDa for the native molecular mass of GMN-1, indicative of existence as a highly elongated dimer. However, the affinity of GMN-1 for CDT-1 is rather weak compared with Xenopus or mouse geminin, so its inhibitory effect may be limited. The interaction between GMN-1 and CDT-1 might require another component or may involve post-transcriptional modification, which would be a unique feature of the C. elegans system. In conclusion, we propose that the affinity of Cdt1 for geminin has increased during evolution, with the result that geminin has become a key regulator that prevents re-replication, in addition to its original roles in cell cycle progression, cell growth, and differentiation. We thank the members of the Cellular Physiology Laboratory of RIKEN for helpful discussions, C. Eichinger for careful reading of the manuscript, Y. Ichikawa and R. Nakazawa for DNA sequencing, and Dr. Y. Kohara at the National Institute of Genetics for providing C. elegans EST clones." @default.
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