A Mechanistic Basis for the Coordinated Regulation of Pharyngeal Morphogenesis in Caenorhabditis elegans by LIN-35/Rb and UBC-18–ARI-1

Fay DS (2009) A Mechanistic Basis for the Coordinated Regulation of Pharyngeal Morphogenesis in Caenorhabditis elegans by LIN-35/Rb and UBC-18-ARI-1. PLoS Genet 5(6): e1000510. doi:10.1371/journal.pgen.1000510 A Mechanistic Basis for the Coordinated Regulation of Pharyngeal Morphogenesis in Caenorhabditis elegans by LIN-35/Rb and UBC-18-ARI-1 Kumaran Mani 0 David S. Fay 0 Stuart K. Kim, Stanford University Medical Center, United States of America 0 Department of Molecular Biology, College of Agriculture, University of Wyoming , Laramie, Wyoming , United States of America Genetic redundancy, whereby two genes carry out seemingly overlapping functions, may in large part be attributable to the intricacy and robustness of genetic networks that control many developmental processes. We have previously described a complex set of genetic interactions underlying foregut development in the nematode Caenorhabditis elegans. Specifically, LIN-35/Rb, a tumor suppressor ortholog, in conjunction with UBC-18-ARI-1, a conserved E2/E3 complex, and PHA-1, a novel protein, coordinately regulates an early step of pharyngeal morphogenesis involving cellular re-orientation. Functional redundancy is indicated by the observation that lin-35; ubc-18 double mutants, as well as certain allelic combinations of pha1 with either lin-35 or ubc-18, display defects in pharyngeal development, whereas single mutants do not. Using a combination of genetic and molecular analyses, we show that sup-35, a strong recessive suppressor of pha-1-associated lethality, also reverts the synthetic lethality of lin-35; ubc-18, lin-35; pha-1, and ubc-18 pha-1 double mutants. SUP-35, which contains C2H2-type Zn-finger domains as well as a conserved RMD-like motif, showed a dynamic pattern of subcellular localization during embryogenesis. We find that mutations in sup-35 specifically suppress hypomorphic alleles of pha-1 and that SUP-35, acting genetically upstream of SUP-36 and SUP-37, negatively regulates pha-1 transcription. We further demonstrate that LIN-35, a transcriptional repressor, and UBC-18-ARI-1, a complex involved in ubiquitin-mediated proteolysis, negatively regulate SUP-35 abundance through distinct mechanisms. We also show that HCF-1, a C. elegans homolog of host cell factor 1, functionally antagonizes LIN-35 in the regulation of sup-35. Our cumulative findings piece together the components of a novel regulatory network that includes LIN-35/Rb, which functions to control organ morphogenesis. Our results also shed light on general mechanisms that may underlie developmental genetic redundancies as well as principles that may govern complex disease traits. - Funding: This work was supported by GM06686 from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Genetic redundancy describes the phenomenon in which the combined inactivation of two distinct genes produces a phenotype that is not observed in either single mutant. One of the current challenges facing geneticists and developmental biologists alike is to understand the underlying bases of genetic redundancy at the molecular level. This may in many cases prove to be a difficult undertaking given the complexity of regulatory networks and the many difficulties associated with establishing clear connections between seemingly disparate genes. Nonetheless, redundancy is an issue of great biological importance, as evidenced in C. elegans, where most genes fail to show obvious or highly penetrant phenotypes following inhibition or inactivation [13]. To date, the most intensively studied case of genetic redundancy in C. elegans involves the Synthetic Multivulval (SynMuv) genes (for a review, see [4]. The SynMuv genes can in most cases be divided into two principal non-overlapping groups, termed class A and class B [5]. Inhibition of individual class A or class B genes does not typically alter normal patterns of vulval cell induction in hermaphrodites. In contrast, the combined loss in activity of any class Aclass B gene pair leads to the ectopic induction of vulval tissue (the Muv phenotype). In addition, a class C group of SynMuv genes has recently been identified; mutations in class C genes are synthetic with mutations in both class A and class B SynMuv genes [6]. Extensive work has shed considerable light on the role of SynMuv genes in vulval development. Namely, most class A and B genes act within the hypodermis, a multi-nucleate epidermal tissue that lies adjacent to the developing vulval precursor cells (VPCs), where they redundantly inhibit the expression of the EGF-like ligand, LIN-3 [7]. Secreted LIN-3 induces vulval cell development through activation of a conserved EGFRRasMap kinase pathway in the VPCs [8]. Thus, in the absence of both class A and class B SynMuv activity, abnormally high levels of LIN-3, secreted by the hypodermis, leads to the hyperinduction of vulval cell fates. Based on studies in C. elegans, Drosophila, and mammals, the large majority of proteins encoded by the class B SynMuv gene family function within a conserved set of structurally related transcriptional repressor complexes that include DRM (Dp, Rb and MuvB) and NuRD (nucleosome remodeling and histone deacetylase; One of the more puzzling aspects of genetics is that the inactivation of many genes fails to produce strong deleterious effects on the organisms that carry those genes. In some cases, however, the combined inactivation of two or more such genes can lead to the expression of robust abnormal phenotypes. These types of synthetic genetic interactions are thought to reflect the presence of functional overlap or redundancy between the involved genes. The root mechanisms that underlie synthetic interactions are thought to be complex and are in most cases poorly understood. Our work here focuses on one case study where we have uncovered the molecular basis underlying a complex set of genetic redundancies in C. elegans. More specifically, we have discovered a novel regulatory network that connects eight genes controlling embryonic foregut development in the nematode C. elegans. By solving mechanisms of this nature, our analysis provides a means for understanding more generally the principles that govern genetic redundancies. Our work also provides insight into the bases of complex disease traits, where the combined interactions of multiple genetic factors leads to outcomes that determine health or disease. reviewed by [4,9]. Among the components that are common to these complexes are LIN-35, the sole C. elegans Retinoblastoma protein (pRb) family ortholog, and EFL-1, a member of the E2F family of transcription factors [1012]. Similar to its role in other systems, LIN-35 acts in large part to mediate the transcriptional repression of E2F target genes [13]. Nevertheless, the pre (...truncated)