Coordinated Regulation of Intestinal Functions in C. elegans by LIN-35/Rb and SLR-2

Citation: Kirienko NV, McEnerney JDK, Fay DS ( Coordinated Regulation of Intestinal Functions in C. elegans by LIN-35/Rb and SLR-2 Natalia V. Kirienko 0 John D. K. McEnerney 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 LIN-35 is the sole C. elegans representative of the pocket protein family, which includes the mammalian Retinoblastoma protein pRb and its paralogs p107 and p130. In addition to having a well-established and central role in cell cycle regulation, pocket proteins have been increasingly implicated in the control of critical and diverse developmental and cellular processes. To gain a greater understanding of the roles of pocket proteins during development, we have characterized a synthetic genetic interaction between lin-35 and slr-2, which we show encodes a C2H2-type Zn-finger protein. Whereas animals harboring single mutations in lin-35 or slr-2 are viable and fertile, lin-35; slr-2 double mutants arrest uniformly in early larval development without obvious morphological defects. Using a combination of approaches including transcriptome profiling, mosaic analysis, starvation assays, and expression analysis, we demonstrate that both LIN-35 and SLR-2 act in the intestine to regulate the expression of many genes required for normal nutrient utilization. These findings represent a novel role for pRb family members in the maintenance of organ function. Our studies also shed light on the mechanistic basis of genetic redundancy among transcriptional regulators and suggest that synthetic interactions may result from the synergistic misregulation of one or more common targets. - The Retinoblastoma protein, pRb, was among the first recognized tumor suppressor proteins [13], and loss or repression of pRb function is thought to play a causative role in most human cancers [48]. The role of pRb as a tumor suppressor has been largely attributed to its functions in cell cycle regulation, which it carries out in conjunction with its two family members, p107 and p130, collectively known as the pocket proteins [911]. Pocket proteins act primarily as transcriptional repressors and physically associate with diverse array of transcription factors [12]. The most thoroughly characterized of these interactions is with E2F family members, which leads to the repression of E2F-target genes, a group that includes many genes required for entry and progression through S-phase [1316]. Correspondingly, LIN-35, the sole pocket protein ortholog in C. elegans, carries out analogous cell cycle functions during larval stages of development [1721]. In addition, a growing number of studies have demonstrated non2cell cycle roles for pRb family members, which in some cases may prove relevant to the tumor-suppressing activity of pocket proteins [12,19,22,23]. In the case of LIN-35, the majority of these functions are revealed only when LIN-35 activity is compromised in specific mutant backgrounds. This phenomenon can be explained on the basis of genetic or functional redundancy, a widespread feature of eukaryotic genomes, which is attributable to the complex and overlapping nature of many regulatory networks. The first described, and still most thoroughly characterized, genetically redundant function of LIN-35 is its role restricting epidermal cells from inappropriately acquiring vulval cell fates [22,24,25]. More specifically, when lin-35, a member of the class B group of synthetic multivulval (SynMuv) genes, is simultaneously inactivated with individual members of the SynMuv A or C classes [24,26], hyperinduction of vulval cells is observed. In contrast, single mutants in most SynMuv genes, including lin-35, do not display observable defects in vulval development. LIN-35 also redundantly regulates pharyngeal and vulval morphogenesis [2729], asymmetric cell divisions [30], cell fates in the somatic gonad [31], larval growth and development [30,32,33], and the promotion of cell death [34]. Furthermore, lin-35 functions non-redundantly in the control of germline gene repression [35] and germline apoptosis [36] and to modulate sensitivity to RNAi [35,37]. In addition, transcriptome profiling has suggested potential roles for LIN-35 in intestinal and neuronal development, although direct evidence for functions in these tissues has been lacking [21]. Here we describe a novel role for LIN-35 in the intestine of C. elegans. Specifically, we find that LIN35, in conjunction with the Zn-finger protein SLR-2, acts within intestinal cells to regulate the expression of genes required for proper nutrient utilization. lin-35/Rb and slr-2 Are Genetically Redundant A previously described genetic screen was used to identify genes that function redundantly with lin-35 [20]. Briefly, we chemically mutagenized lin-35(n745) mutants that carry an unstable extrachromosomal array (kuEx119), which expresses wild-type lin-35 together with the sur-5::GFP marker. Following F2 clonal Genetic or functional redundancy is a widespread feature of eukaryotic genomes and may be largely attributable to the complex and overlapping nature of many regulatory networks. Despite the prevalence and importance of this phenomenon, the mechanistic bases underlying genetic redundancy have remained elusive, particularly within the context of multicellular developing organisms. To gain a deeper understanding of this phenomenon, we have focused on a synthetic genetic interaction between lin35, a C. elegans member of the pRb/pocket-protein tumor suppressor family and slr-2, a Zn-finger protein. Whereas single mutants in either lin-35 or slr-2 develop normally, lin-35; slr-2 double mutants display a highly penetrant synthetic growth arrest during early larval development. We show that this arrest is specifically due to an inability to utilize standard nutrient sources. Using a wide range of approaches including transcriptome profiling, mosaic analysis, and expression analysis, we demonstrate that both LIN-35 and SLR-2 act within the intestine to regulate the expression of many genes that function in nutrient utilization. We also show that the basis of the synthetic interaction between lin-35 and slr-2 is not merely due to regulon overlap, but likely results from the synergistic misregulation of multiple shared transcriptional targets in the intestine. selection, we identified strains with synthetic interactions by the presence of visible phenotypes in progeny that failed to inherit the array. One allele, ku297, defines a locus that we have designated as slr-2 (for synthetic with lin-35/Rb). slr-2 single mutants are largely indistinguishable from wild type, although we observed weak-tomoderate elongation defects at low frequencies (the Dpy phenotype). In contrast, lin-35; slr-2 double mutants exhibit uniform early-larval arrest (Figure 1A, 1B, Table 1). To verify tha (...truncated)