lin-35/Rb and ubc-18, an E2 ubiquitin-conjugating enzyme, function redundantly to control pharyngeal morphogenesis in C. elegans

David S. Fay ) 1 Edward Large 1 Min Han 0 Monica Darland 1 0 Howard Hughes Medical Institute and Department of Molecular, Cellular, and Developmental Biology, University of Colorado , Boulder, CO 80309-0347 , USA 1 Department of Molecular Biology, University of Wyoming , PO Box 3944, Laramie, WY 82071-3944 , USA SUMMARY The retinoblastoma gene product has been implicated in the regulation of multiple cellular and developmental processes, including a well-defined role in the control of cell cycle progression. The Caenorhabditis elegans retinoblastoma protein homolog, LIN-35, is also a key regulator of cell cycle entry and, as shown by studies of synthetic multivulval genes, plays an important role in the determination of vulval cell fates. We demonstrate an additional and unexpected function for lin-35 in organ morphogenesis. Using a genetic approach to isolate lin-35 synthetic-lethal mutations, we have identified redundant roles for lin-35 and ubc-18, a gene that encodes an E2 Functional disruption of the retinoblastoma gene product (pRb) has been implicated as a causal event in the genesis of a wide range of human cancers (reviewed by Sherr, 1996; Nevins, 2001). pRb and its structurally related family members, p107 and p130, play key roles in the regulation of several fundamental cellular processes, including cell cycle entry and the induction of apoptosis (reviewed by Kaelin, 1999; Morris and Dyson, 2001). The ability of pRb to regulate these events is linked directly to its activity as a transcriptional repressor. Specifically, pRb binds to E2F family members and inhibits the expression of E2F target genes (reviewed by Dyson, 1998; Harbour and Dean, 2000). These targets include positive-acting cell cycle regulators, such as cyclins E and A (DeGregori et al., 1995; Duronio and OFarrell, 1995; Ohtani et al., 1995; Schulze et al., 1995), genes that are required for DNA synthesis (Dou et al., 1994; DeGregori et al., 1995), and mediators of apoptosis (DeGregori et al., 1997; Hsieh et al., 1997; Tsai et al., 1998). pRb transcriptional repression of E2F targets occurs through a number of distinct mechanisms, many of which involve the recruitment of enzymes that modify chromatin structure. These include histone deacetylase (Brehm et al., 1998; Luo et al., 1998; Magnaghi-Jaulin et al., 1998), members of the nucleosome remodeling complex (Dunaief et al., 1994; Strober et al., 1996; Zhang et al., 2000) and proteins required for histone methylation (Nielsen et al., 2001). ubiquitin-conjugating enzyme closely related to human UBCH7. lin-35 and ubc-18 cooperate to control one or more steps during pharyngeal morphogenesis. Based on genetic and phenotypic analyses, this role for lin-35 in pharyngeal morphogenesis appears to be distinct from its cell cyclerelated functions. lin-35 and ubc-18 may act in concert to regulate the levels of one or more critical targets during C. elegans development. In addition to cell-cycle regulation, in vitro and tissue culture studies have shown that pRb associates with a diverse set of proteins, many of which regulate the expression of genes required for tissue-specific differentiation (reviewed by Morris and Dyson, 2001). For example, pRb enhances the DNA binding and transactivation activities of NF-IL6 (Chen et al., 1996b) and the C/EBP (Chen et al., 1996a) family of transcription factors to promote adipocyte and leukocyte differentiation, respectively. pRb also promotes muscle differentiation by augmenting the activity of MyoD (Gu et al., 1993) and through inhibition of the transcriptional repressor HBP1 (Tevosian, 1997; Shih et al., 1998). Finally, pRb may bind and regulate the activities of a number of additional factors, including the paired homeodomain-containing proteins Pax3, Pax5, Chx10 and Mhox (Wiggan et al., 1998; Eberhard and Busslinger, 1999); several hormone-responsive transcription factors, including the glucocorticoid receptor (Singh et al., 1995); and the osteoblast transcription and differentiation factor, CBFA1 (Thomas et al., 2001). Whether or not the majority of these reported activities represent authentic in vivo functions for Rb remains to be determined. Acting in concert with transcriptional regulatory factors, the ubiquitin-mediated degradation pathway has emerged as the other principal mechanism by which cells control the abundance of individual proteins. The process is carried out by three classes of enzymes (termed E1, E2 and E3) that act sequentially to catalyze the attachment of ubiquitin, a highly conserved ~76 amino acid protein, to the protein substrate targeted for degradation (reviewed by Weissman, 2001). The process is initiated by E1 enzymes (also known as ubiquitinactivating enzymes), which form a thiol-ester bond with the Cterminal glycine of ubiquitin in an ATP-dependent manner. The E2 or UBC (for ubiquitin-conjugating or ubiquitin-carrier) enzyme then accepts ubiquitin from the E1 via a transthiolation reaction involving the C terminus of ubiquitin. Finally, the transfer of ubiquitin from E2 to a lysine on the target protein is catalyzed by the E3 ubiquitin ligase. E3 enzymes can further be subdivided into two separate families containing either a HECT or a RING finger domain. E3s with a HECT domain form thiol-ester intermediates with ubiquitin prior to attachment to the target protein (Huibregtse et al., 1995), whereas E3s with a RING finger mediate the direct transfer of ubiquitin from E2 to the target protein (Joazeiro and Weissman, 2000). In either case, the majority of ubquitylated proteins are subsequently degraded by the 26S proteasome. A recent analysis of the C. elegans genome identified 20 genes encoding predicted E2/UBC enzymes along with three UBC variants (Jones et al., 2002). This compares with 12 UBCs in S. cerevisiae, 25 in Drosophila and 26 that have thus far been identified in the human proteome. Thus, complexity in the ubiquitylation process begins at the level of UBCs and is further amplified by the large number of potential E3 genes found in the genomes of most higher eukaryotes; the C. elegans genome encodes for >150 RING-finger or HECT domain proteins. Interestingly, RNA-mediated interference (RNAi) experiments of the 23 C. elegans UBC genes revealed functions for only four of them (Jones et al., 2002). RNAi of these genes [let-70 (ubc-2), ubc-9, ubc-12, and ubc-14], all of which are conserved in yeast, results in developmental arrest at various stages. Thus, a large proportion of UBCs in C. elegans may be functionally redundant, either with each other or with other cellular factors that act to regulate protein levels. Using a genetic screen to identify mutations causing synthetic phenotypes with lin-35/Rb in C. elegans, we have previously reported the identification of mutations in fzr-1, a regulatory subunit of the APC proteasome (Fay et al., 2002). lin-35; fzr-1 double mutants display a hyperproliferation phenotype that affects virtually all cell types examined. We now desc (...truncated)