Lin K-C et al. (1997) Science "daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans."

Rodan A, Dorman JB, Lin K-C, Kenyon CJ

[Science, 1997]

The wild-type Caenorhabditis elegans nematode ages rapidly, undergoing development, senescence, and death in less than 3 weeks. In contrast, mutants with reduced activity of the gene daf-2, a homolog of the insulin and insulin-like growth factor receptors, age more slowly than normal and live more than twice as long. These mutants are active and fully fertile and have normal metabolic rates. The life-span extension caused by daf-2 mutations requires the activity of the gene daf-16. daf-16 appears to play a unique role in life-span regulation and encodes a member of the hepatocyte nuclear factor 3 (HNF-3)/forkhead family of transcriptional regulators. In humans, insulin down-regulates the expression of certain genes by antagonizing the activity of HNF-3, raising the possibility that aspects of this regulatory system have been conserved.AD - Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0554, USA.FAU - Lin, KAU - Lin KFAU - Dorman, J BAU - Dorman JBFAU - Rodan, AAU - Rodan AFAU - Kenyon, CAU - Kenyon CLA - engSI - GENBANK/AF032112ID - AG11816/AG/NIAPT - Journal ArticleCY - UNITED STATESTA - ScienceJID - 0404511RN - 0 (DAF-16 transcription factor)RN - 0 (DAF-2 protein)RN - 0 (DNA, Complementary)RN - 0 (Nuclear Proteins)RN - 0 (Somatomedins)RN - 0 (Transcription Factors)RN - 0 (forkhead protein)RN - 11061-68-0 (Insulin)RN - EC 2.7.11.- (Receptor, Insulin)SB - IM

K Lin et al. (1999) International C. elegans Meeting "isolation OF ADDITIONAL DAF-2 SUPPRESSORS"

C Kenyon, K Lin

[International C. elegans Meeting, 1999]

The wild-type C.elegans ages rapidly, undergoing development, senescence, and death in less then 3 weeks. In contrast, mutants with reduced activity of daf-2 , a homolog of the insulin and insulin-like growth factor (IGF-1) receptors (Kimura et al., 1997), age more slowly and live more than twice as long (Kenyon et al., 1993). Wild-type DAF-2 activity also promotes growth to adulthood and prevents dauer formation, since severe loss of daf-2 function causes the animals to become dauers in the presence of food. Both the lifespan extension and dauer-constitutive phenotypes caused by daf-2 mutations are dependent on the activity of daf-16 , which encodes an HNF-3/forkhead family member (Ogg et al., 1997; Lin et al., 1997). Previous screens for suppressors of the dauer-constitutive phenotype of daf-2 in our lab have resulted in isolation of just additional alleles of daf-16 (Lin et al., 1997). In order to overcome this problem and isolate additional daf-2 suppressors, we created daf-2(e1370) animals with extra copies of the wild-type daf-16 gene, by injecting a rescuing genomic daf-16 construct and integrating the arrays into the genome. The integrated array fully rescued dauer formation in the daf-16(null);daf-2(e1370) background. We screened a total of 100,000 genomes for suppressors of the dauer constitutive phenotype of daf-2(e1370) , and we're now screening for suppressors of the lifespan extension phenotype. We will report our progress in characterizing the suppressors we isolated.

Lee RC et al. (1993) Cell "The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14."

Lee RC, Feinbaum RL, Ambros VR

[Cell, 1993]

lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans. lin-4 acts by negatively regulating the level of LIN-14 protein, creating a temporal decrease in LIN-14 protein starting in the first larval stage (L1). We have cloned the C. elegans lin-4 locus by chromosomal walking and transformation rescue. We used the C. elegans clone to isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally rescue the lin-4 null allele of C. elegans. Comparison of the lin-4 genomic sequence from these four species and site-directed mutagenesis of potential open reading frames indicated that lin-4 does not encode a protein. Two small lin-4 transcripts of approximately 22 and 61 nt were identified in C. elegans and found to contain sequences complementary to a repeated sequence element in the 3' untranslated region (UTR) of lin-14 mRNA, suggesting that lin-4 regulates lin-14 translation via an antisense RNA-RNA

Ha I et al. (1997) International C. elegans Meeting "STRUCTURE ANALYSIS OF THE C. ELEGANS TEMPORAL GRADIENT GENERATING LIN-14/LIN-4 RNA DUPLEX."

Ruvkun GB, Ha I

[International C. elegans Meeting, 1997]

The C. elegans heterochronic gene lin-14 generates a temporal gradient of the LIN-14 proteins that control stage-specific patterns of cell lineage during development. We propose that lin-4 RNA base-pairs with the lin-14 3' UTR to cause downregulation of translation of the lin-14 mRNA. Mutations in the proposed RNA duplex region of the lin-l4 mRNA that disrupt in vitro RNA duplex formation also disrupt temporal gradient formation in vivo. These data demonstrate that lin-4/lin-14 RNA duplex formation is required for the generation of the LIN-14 temporal gradient. Many loops and bulged structures in RNA have been shown to be important for interacting with proteins. Based on the lin-4/lin-14 RNA duplex model, four of the seven lin-4/lin-14 RNA duplexes are predicted to bulge a lin-4 C residue, and three sites are predicted to form non-bulged RNA duplexes. Reporter genes bearing multimerized bulged C lin-4 binding sites in the 3' UTR downregulate in N2 but not in a lin-4(null). However, similar transgenes bearing multimerized nonbulged lin-4 binding sites show almost no downregulation in N2 or lin-4(null). Interestingly, lin-4 RNA binds strongly in vitro to the non-bulged lin-4 binding sites, but not to the bulged C lin-4 binding sites. These results suggest that a transacting factor(s) may recognize the bulged C of the lin-14/lin-4 duplex in vivo and act to stabilize the lin-14/lin-4 duplex. We are currently in the process of identifying such factors genetically and biochemically.

Ha I et al. (1996) Genes Dev "A bulged lin-4/lin-14 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation."

Ruvkun GB, Wightman BC, Ha I

[Genes Dev, 1996]

The Caenorhabditis elegans heterochronic gene lin-14 generates a temporal gradient of the LIN-14 proteins to control stage-specific patterns of cell lineage during development. Down-regulation of LIN-14 is mediated by the lin-14 3' untranslated region (UTR), which bears seven sites that are complementary to the regulatory lin-4 RNA. Here we report molecular and genetic evidence that RNA duplexes between the lin-4 and lin-14 RNAs form in vivo and are necessary for LIN-14 temporal gradient generation. lin-4 RNA binds in vitro to a lin-14 mRNA bearing the seven lin-4 complementary sites but not to a lin-14 mRNA bearing point mutations in these sites. In vivo, the lin-4 complementary regions are necessary for lin-14 3' UTR-mediated temporal gradient formation. Based on lin-14 3' UTR sequence comparisons between C. elegans and C. briggsae, four of the seven lin-4/lin-14 RNA duplexes are predicted to bulge a lin-4 C residue, and three sites are predicted to form nonbulged RNA duplexes. Reporter genes bearing multimerized bulged C lin-4 binding sites show almost wild-type temporal gradient formation, whereas those bearing multimerized nonbulged lin-4 binding sites do not form a temporal gradient. Paradoxically, lin-4 RNA binds in vitro to nonbulged lin-14 RNA more avidly than to the bulged lin-14 RNA. This suggests that a specific secondary structure of lin-4/lin-14 RNA duplex that may be recognized by an accessory protein, rather than an RNA duplex per se, is required in vivo for the generation of the LIN-14 temporal gradient.

Chamberlin HM et al. (2000) Development "The bromodomain protein LIN-49 and trithorax-related protein LIN-59 affect development and gene expression in Caenorhabditis elegans."

Thomas JH, Chamberlin HM

[Development, 2000]

We have molecularly characterized the lin-49 and lin-59 genes in C. elegans, and found their products are related to Drosophila trithorax group (trx-G) proteins and other proteins implicated in chromatin remodelling. LIN-49 is structurally most similar to the human bromodomain protein BR140, and LIN-59 is most similar to the Drosophila trx-G protein ASH1. In C. elegans, lin-49 and lin-59 are required for the normal development of the mating structures of the adult male tail, for the normal morphology and function of hindgut (rectum) cells in both males and hermaphrodites and for the maintenance of structural integrity in the hindgut and egg-laying system in adults. Expression of the Hox genes egl-5 and mab-5 is reduced in lin-49 and lin-59 mutants, suggesting lin-49 and lin-59 regulate HOM-C gene expression in C. elegans as the trx-G genes do in Drosophila. lin-49 and lin-59 transgenes are expressed widely throughout C. elegans animals. Thus, in contrast to the C. elegans Polycomb group (Pc-G)-related genes mes-2 and mes-6 that function primarily in the germline, we propose lin-49 and lin-59 function in somatic development similar to the Drosophila trx-G genes.

Wightman BC et al. (1993) Cell "Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans."

Wightman BC, Ha I, Ruvkun GB

[Cell, 1993]

During C. elegans development, the temporal pattern of many cell lineages is specified by graded activity of the heterochronic gene Lin-14. Here we demonstrate that a temporal gradient in Lin-14 protein is generated posttranscriptionally by multiple elements in the lin-14 3'UTR that are regulated by the heterochronic gene Lin-4. The lin-14 3'UTR is both necessary and sufficient to confer lin-4-mediated posttranscriptional temporal regulation. The function of the lin-14 3'UTR is conserved between C. elegans and C. briggsae. Among the conserved sequences are seven elements that are each complementary to the lin-4 RNAs. A reporter gene bearing three of these elements shows partial temporal gradient activity. These data suggest a molecular mechanism for Lin-14p temporal gradient formation: the lin-4 RNAs base pair to sites in the lin-14 3'UTR to form multiple RNA duplexes that down-regulate lin-14 translation.

Liu J et al. (1999) Genetics "Structural requirements for the tissue-specific and tissue-general functions of the Caenorhabditis elegans epidermal growth factor LIN-3."

Hill RJ, Tzou P, Sternberg PW, Liu J

[Genetics, 1999]

Caenorhabditis elegans lin-3 encodes a homolog of the epidermal growth factor (EGF) family of growth factors. LIN-3 is the inductive signal for hermaphrodite vulval differentiation, and it is required for animal viability, hermaphrodite fertility, and the specification of anterior cell fates in the male B cell lineage. We describe the cloning of a lin-3 homolog from C. briggsae, sequence comparison of C. elegans lin-3 with C. briggsae lin-3, and the determination of molecular lesions in alleles of C. elegans lin-3, including three new alleles. We also analyzed the severity of phenotypes caused by the new and existing alleles of lin-3. Correlation of mutant phenotypes and their molecular lesions, as well as sequence comparison between two species, reveal that the EGF motif and the N-terminal portion of the cytoplasmic domain are important for the functions of LIN-3 in all tissues, while the C-terminal portion of the cytoplasmic domain is involved in the tissue-specific functions of lin-3. We discuss how the structure of lin-3 contributes to its functions in multiple developmental processes.

Kiyokazu Morita et al. (2007) International Worm Meeting "Suppressor screening of the heterochronic gene lin-66."

Kiyokazu Morita, Min Han

[International Worm Meeting, 2007]

Heterochronic genes regulate developmental timing in C. elegans . In a recent publication, we described our analysis of the lin-66 gene that was identified by isolating two suppressor mutations 1. We showed that lin-66 mutations cause retarded heterochronic phenotypes in seam cell and vulval cell lineages and die at the late L4 stage. Further analysis also indicates that lin-66 regulates the L2 to L3 transition of seam cell development by regulating the expression of lin-28. Our results also suggest that lin-66 regulation of lin-28 expression is parallel to three other mechanisms that regulate lin-28 expression, including LIN-14-mediated up-regulation, miRNA-mediated repression, and DAF-12-involved translational promotion. Although our data suggest that the regulation is mediated by the 3 UTR of lin-28, the mechanism of this regulation is currently not understood. Our further biochemical analysis of lin-66 interacting proteins has not been fruitful. To obtain more insight into the molecular mechanism of lin-66 function, we carried out screens for suppressors of the lin-66(lf) mutation. Because lin-31(lf) can suppress the lethality of lin-66(lf), we used the lin-31; lin-66 double mutant as the starting strain. We screened more than 30, 000 haploid genomes and isolated 7 mutations that suppress the retarded heterochronic phenotype in hypodermal alae formation of lin-66(lf). We are currently mapping the suppressors. We will present the SNP mapping data at the meeting. (1). Morita K and Han M, (2006) Multiple mechanisms are involved in regulating the expression of the developmental timing regulator lin-28 in C. elegans. EMBO J, 25 5794-5804.

P Olsen et al. (1999) International C. elegans Meeting "Developmental regulation and mechanism of action of the lin-4 translational repressor RNA."

V Ambros, P Olsen, E Moss, R Feinbaum, F Liu, HS Silverman

[International C. elegans Meeting, 1999]

lin-4 encodes a small RNA that is complementary to sequences in the 3' untranslated regions of lin-14 and lin-28 mRNAs and that acts to down-regulate LIN-14 and LIN-28 during the L1 and L2. This down-regulation is essential for the proper timing of events in C. elegans larval development. The temporal profile of lin-4 RNA accumulation determines the timing of LIN-14 and LIN-28 protein down-regulation, and thereby controls the timing of postembryonic developmental events. To explore the role of lin-4 upstream sequences in the timing of lin-4 expression, we tested lin-4 deletion constructs for their expression in transgenic worms. These experiments have identified elements essential for lin-4 transcription, and also some apparent negative regulatory elements. A one-hybrid screen for C. elegans proteins that bind to essential lin-4 upstream sequences in yeast cells has identified a zinc-finger DNA binding protein that specifically recognizes lin-4 DNA. We are testing for potential developmental roles for this protein in lin-4 regulation. We have investigated the mechanism of lin-4 RNA action by examining the fate of lin-14 mRNA in vivo during the time that lin-4 RNA is expressed. Our results suggest that the rate of synthesis, the state of polyadenylation, the abundance in the cytoplasmic fraction, and the polysomal sedimentation profile of lin-14 mRNA do not change in response to the accumulation of lin-4 RNA. We conclude that lin-4 RNA represses LIN-14 protein production by acting after initiation of lin-14 mRNA translation. Surprisingly, lin-4 RNA seems to be required for translational repression of LIN-14 and LIN-28 only in the presence of lin-14 and lin-28 activities, suggesting that lin-4 RNA represses translation by modulating a mutual translational feedback circuit between lin-14 and lin-28 . The potential roles of heterochronic genes other than lin-4 in this feedback pathway are being explored.