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C. Elegans G1 Cyclin/CDK 4851 Primers MP 164 (5′-CCAAAACAATGCCGCATATTCCGAC-3′) and A

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C. Elegans G1 Cyclin/CDK 4851 Primers MP 164 (5′-CCAAAACAATGCCGCATATTCCGAC-3′) and A Development 126, 4849-4860 (1999) 4849 Printed in Great Britain © The Company of Biologists Limited 1999 DEV5301 Regulation of postembryonic G1 cell cycle progression in Caenorhabditis elegans by a cyclin D/CDK-like complex Morgan Park and Michael W. Krause* Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room B1-04, Bethesda, Maryland 20892-0510, USA *Author for correspondence (e-mail: [email protected]) Accepted 13 August; published on WWW 6 October 1999 SUMMARY In many organisms, initiation and progression through the transgenic animals is sufficient to activate a S-phase G1 phase of the cell cycle requires the activity of G1-specific reporter gene. We observe no embryonic defects associated cyclins (cyclin D and cyclin E) and their associated cyclin- with depletion of either of these two gene products, dependent kinases (CDK2, CDK4, CDK6). We show here suggesting that their essential functions are restricted to that the Caenorhabditis elegans genes cyd-1 and cdk-4, postembryonic development. We propose that the cyd-1 encoding proteins similar to cyclin D and its cognate cyclin- and cdk-4 gene products are an integral part of the dependent kinases, respectively, are necessary for proper developmental control of larval cell proliferation through division of postembryonic blast cells. Animals deficient the regulation of G1 progression. for cyd-1 and/or cdk-4 activity have behavioral and developmental defects that result from the inability of the postembryonic blast cells to escape G1 cell cycle arrest. Key words: Caenorhabditis elegans, Cell cycle, Cyclin D, CDK, cyd- Moreover, ectopic expression of cyd-1 and cdk-4 in 1, cdk-4 INTRODUCTION CDK2, respectively, are responsible for initiating progression from G1 to S phase by phosphorylating cellular substrates. D- The formation of a complex multicellular organism requires type cyclins act as growth factor sensors and regulators of G1 the control of cell proliferation within the context of initiation and progression (Matsushime et al., 1992; Baldin et environmental and developmental signaling. The G1 phase of al., 1993; Sherr, 1993, 1994). Mitogens induce the synthesis of the cell cycle is a key regulatory point that controls cell cycle cyclin D and the assembly of cyclin D with its catalytic CDK decisions and previous studies in several different systems have partners to form an active complex that will promote passage defined the basic machinery involved in the initiation of cell through the restriction point (Matsushime et al., 1991, 1992, cycle entry and progression through the cell cycle (Sherr, 1993, 1994). The formation of the G1 cyclin/CDK complex is the 1994; Hunter and Pines, 1994). During G1, cells are responsive rate-limiting step in early G1 progression (Ohtsubo and to extracellular cues that control proliferation, differentiation, Roberts, 1993; Quelle et al., 1993; Resnitzky et al., 1994). quiescence, senescence and apoptosis (Sherr, 1994). The Once formed, the activity of this complex is also regulated by commitment to cell division is linked to the restriction point CDK inhibitory proteins (p21/27, INK4; Sherr and Roberts, (START) in late G1 (Pardee, 1989). In mammalian cells, 1995), by phosphorylation state (through the action of passage through this regulatory point requires the presence of CAK, CDC25, WEE1; Lew and Kornbluth, 1996), and by mitogenic signaling, but once past this point, the cells no longer degradation via ubiquitin-mediated pathways (King et al., require mitogenic signaling and they are irreversibly 1996; Hoyt, 1997). committed to enter S phase and to complete the cell cycle Although the molecular mechanisms of cell cycle control are (Pardee, 1989; Sherr, 1994, 1996). well understood and evolutionarily conserved, there currently Progression through the cell cycle is controlled by the is a poor understanding of how regulators of the cell cycle are activity of cyclin-dependent kinases (CDKs) in association integrated into the development of a multicellular organism to with a regulatory cyclin subunit (Hunt, 1991; Nigg, 1995). ensure the correct number of cells are generated at the correct Previous studies have shown that a distinct set of cyclin/ CDK place and time. Recently, organisms such as mice, Xenopus and complexes act during the G1 phase to drive cells through Drosophila have been utilized to study cell cycle regulation in START (Dulic et al., 1992; Koff et al., 1992; Xiong et al., the context of animal developmental programs. Caenorhabditis 1992). D-type and E-type cyclins, in association with their elegans is also emerging as an attractive system to address cognate cyclin-dependent kinases, CDK4 (or CDK6) and questions of developmental cell cycle control. The entire 4850 M. Park and M. W. Krause lineage and timing of cell divisions in C. elegans is known, however, we have been unable to confirm SL1 or SL2 trans-splicing both embryonically and postembryonically, providing single- to this site. The coding sequences corresponding to the cyd-1 and cdk- cell resolution to studies of cell division (Sulston and Horvitz, 4 genes have been deposited in Genbank (AF053067 and AF083878, 1977; Sulston et al., 1983). For example, there are 55 respectively). embryonically born blast cell nuclei that can divide during Gene expression constructs larval development in response to developmental cues. All of Genomic sequence corresponding to the putative promoter regions of these divisions can be monitored by direct observation and with cyd-1 and cdk-4 were cloned separately into a GFP expression vector reporter genes or antibodies specifically marking a subset of (pPD 95.67; A. Fire, G. Seydoux, J. Ahnn, and S. Q. Xu, personal these blast cell lineages. This comparatively simple and defined communication). Regions containing non-coding and coding pattern of cell divisions, coupled with the molecular, genetic sequences were amplified from genomic wild-type N2 DNA by and genomic resources, makes C. elegans an ideal system to PCR. cyd-1 was amplified using primers MP135 (5′-GAAGGCTC- study cell cycle regulation. GCATGTCGAGTTG-3′) and MP136 (5′-CGCGGATCCATAGTA- Several genes affecting the initiation and progression of the GGAACGATGCGCAC-3′). cdk-4 was amplified using primers cell cycle during postembryonic development have been MP147 (5′-CGCGGATCCCGAAGGATCTCCATTTTCTAC-3′) and ′ ′ identified in C. elegans. The heterochronic genes define a MP148 (5 -CGCGGATCCCAATTCATACATCCATTCTGTGG-3 ). developmental pathway regulating the timing of many Each reporter gene construct (cyd-1::GFP and cdk-4::GFP) contains 3.3 kb of non-coding sequence upstream of the ATG start and the first postembryonic cell cycles (Ambros and Horvitz, 1984; Euling (cdk-4) or first two (cyd-1) coding exon sequences fused in frame to and Ambros, 1996; Ambros, 1997). Precocious and retarded the green fluorescent protein (GFP). To rescue the cdk-4(gv3) heterochronic mutants accelerate or delay, respectively, the mutation, the cdk-4::GFP construct was modified by inserting the timing of postembryonic divisions. In addition, two negative cdk-4 cDNA, containing a FLAG tag, between the putative cdk-4 regulators of G1 cell cycle progression have been identified. promoter and GFP-encoding sequences. Transgenic animals The cul-1 gene, encoding one of several cullins in C. elegans, containing these reporter or rescuing constructs were generated by is required for cell cycle exit (Kipreos et al., 1996). The cullins germline transformation using a dominant roller marker (pRF4) as a are part of the ubiquitin-mediated degradation pathway and co-transformation marker (Mello and Fire, 1995). For cdk-4(gv3) rescue, the construct was introduced into the szTI balanced strain presumably act to degrade G1 cyclins like the related yeast (S. cerevisiae) gene product CDC53 (Mathias et al., 1996, KM48 (see below) and viable Rollers cloned to select for rescued homozygous cdk-4(gv3) lines. Jackson, 1996). The cki-1 gene product that is related to the CIP/KIP family of cyclin-dependent kinase inhibitors (Hong et Immunoprecipitations al., 1998) encodes a second negative regulator of G1 A HA-tagged CYD-1 expression construct was generated by PCR using progression in C. elegans. Reduction or loss of function for the cyd-1 EST yk118d3 as template and primers MP155 (5′-CGC- either cul-1 or cki-1 activity results in hyperproliferation of the TCTAGAATGCACTTTGAGTCGTCGTCGGC-3′) and MP156 (5′- postembryonic blast cells. GCGTCTAGAAGCGTAATCTGGAACATCGTATGGGTATAAAGT- In this study, we show that two C. elegans proteins similar CTTGAAGATCTTC-3′). The HA-tag is encoded in primer MP156 and to the G1-specific cyclin, cyclin D, and its cognate cyclin- is added to the carboxy-terminus of CYD-1. A FLAG-tagged CDK-4/6 construct was generated by PCR using cdk-4 EST yk492e2 as template dependent kinases, CDK4 and CDK6, are responsible for ′ initiating postembryonic cell division. By analogy to other and primers MP161 (5 -CGCGGATCCACCATGTGCGAGAATCT- TTATGGAGAGGAGTAC-3′) and MP158 (5′-GCGTCTAGACTTGT- systems, these two factors may function in a complex and we CATCGTCGTCCTTGTAGTCCTTGTTGAAGTTGATTTGC-3′). The show by co-immunoprecipitation that they can interact when FLAG tag is encoded in primer MP158 and is added to the carboxy- co-expressed in insect cells. Depletion of these factors in a cdk- terminus of CDK-4/6. These tagged constructs were cloned into the 4 deletion mutant or by RNA-mediated interference (RNAi) for Fastbac expression vector in the BAC-to-BAC system (Gibco-BRL). cyd-1 or cdk-4, leads to phenotypic defects that are restricted Sf9 cells were infected with Baculovirus extracts and cells harvested to postembryonic development. Our results suggest that cyd-1 48 hours postinfection. Immunoprecipitation used anti-HA antibody and cdk-4 are critical in C. elegans for the developmental (Santa Cruz Biotechnology) and anti-FLAG (Sigma) antibodies. control of G1 cell cycle progression in postembryonic blast Immunoprecipitates were separated by SDS-PAGE using a 4-20% cells. gradient gel (Novex) and analyzed by Western blot using the ABC detection system (Vector labs).
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