Kuo, Yu-Lin et al. (2015) International Worm Meeting "Antagonistic function of HPL-2 and SET-2 in olfactory adaptation of the AWC neuron."

Kuo, Yu-Lin, Juang, Bi-Tzen

[International Worm Meeting, 2015]

Sensory neurons are the front-line of an organism to detect environmental changes. For survival purposes, neural circuits must integrate external stimuli and modify output signals to behave appropriately. The process of the modulation is termed neuronal plasticity. To gain insight into the molecular basis for neuronal plasticity, we study the olfactory AWC neuron in C. elegans. The AWC neurons are able to sense attractive volatile chemical compounds such as butanone, which induce animals to move toward to this odor source, termed chemotaxis. Prolonged odor exposure in the absence of food results in the ability to ignore those attractive odors, termed adaptation. In previous work, we found that the putative RNAase III MUT-7 is required for increasing the levels of specific siRNAs to direct the histone associated protein HPL-2 bound to the siRNA's cognate gene in an adult odor-adapted animal. Genetic analysis revealed that HPL-2 and MUT-7 affect adaptation by acting in the same pathway. HPL-2 is heterochromatin protein 1 (HP1) homologs in human and HP1 family protein can interact with methylated lysine 9 of histone H3 (H3K9) to form of heterochromatin and contribute to repression of target gene. The transgenic strain carrying the non-methylated lysine-to-leucine mutation of histone H3K9 in the AWC neuron blocks adaptation. Thus, we asked the importance of histone methylation in behavioral plasticity. Simonet et al. (2007) reported that a histone H3K4 methyltransferase SET-2 antagonizes HPL-2 during the development of the vulva. Likewise, we found that loss of SET-2 suppresses the adaptation defects of both the hpl-2 null and the mut-7 inactivated mutants. The rescue analysis showed that the SET-2 acts in the AWC neuron for modulating odor adaptation. Our work opens up the possibility that the olfactory memory formation is dependent on changes in chromatin modification.

Reinhart BJ et al. (1996) East Coast Worm Meeting "INVESTIGATING THE BASIS OF TWO lin-14 GENETIC ACTIVITIES."

Reinhart BJ, Ruvkun GB

[East Coast Worm Meeting, 1996]

The heterochronic gene lin-14 controls the timing of post-embryonic developmental events, and perturbations in lin-14 gene activity result in the inappropriate timing of stage specific events. Genetic evidence suggests two gene activities for lin-14, lin-14a for proper L1 timing and lin-14b for L2. In addition, the lin-14 gene uses two promoters, A and B, to produce three distinct proteins (A, B1, and B2) by differential splicing of the transcript from the B promoter. We have investigated the possibility that the different proteins are the basis for the lin-14 genetic activities. The lin-14 gene structure and all three transcripts are conserved in C. briggsae. Sequencing loss-of-function mutations has revealed that while null mutations map to a conserved region common to all products, mutations which only effect the lin-14b genetic activity are specific to LIN-14B1 and LIN-14B2. This suggests that LIN-14B1 and LIN-14B2 are necessary for proper L2 timing, and LIN-14A is sufficient for L1 fates. A single lin-14a mutation that has been identified is a point mutation common to all three isoforms. We are currently investigating the rescuing activity of genomic constructs with LIN-14A deleted to determine the necessity of LIN-14A and the sufficiency of LIN-14B. We have examined the possibility that an expression difference from the promoters, rather than biochemical differences among the proteins, is responsible for the lin-14 activities. For example, LIN-14A may be expressed early to allow L1 fates and LIN-14B may be expressed later to allow L2 fates. Two lin-14b mutations are lin-14b nulls, lin-14(n355n534amber) and lin-14(n360), a gamma allele. Neither mutant produces LIN-14B1 or B2 by Western blots with exon-specific antibodies. In both mutants, immunostaining detected LIN-14A protein in all cell types previously determined to express LIN-14. Subsequently, we examined these mutants using lin-4 and lin-14(n355gf) which disrupt lin-4 downregulation of LIN-14 and allow a later expression of LIN-14A. lin-14(n355n534amber) lacks L2 lineages although LIN-14A is expressed at all stages of development. However, the opposite is suggested by lin-4(e912); lin-14(n360), which appears to have wild-type timing. Western analysis and antibody staining suggest this disparity may be due to overall lower LIN-14A expression in lin-14(n355n534) and that LIN-14A can be sufficient for all lin-14 function as seen in lin-4(e912); lin-14(n360). Another possibility is that lin-4(e912) is suppressing the lin-14 phenotype via another gene, such as lin-28. We are currently analyzing lin-4(e912); lin-14(n355n534) to verify that lin-4 is acting directly through lin-14.

Tang LZ et al. (2000) East Coast Worm Meeting "Regulation of the heterochronic gene lin-28 independently of the lin-4 RNA"

Moss EG, Tang LZ, Seggerson K

[East Coast Worm Meeting, 2000]

The heterochronic genes act in a temporal cascade to permit the normal succession and synchrony of developmental events through the larval stages. Two of these genes, lin-14 and lin-28, act early in development to cause L1- and L2-specific events to occur. Both must be repressed to allow subsequent stage-specific events to occur in the L3 and later. lin-4, which encodes a small RNA, is needed for the repression of both of these genes. When lin-4 is absent, both lin-14 and lin-28 expression remain high in late stages, causing a reiteration of early larval fates and retarded development. However, if either lin-14 or lin-28 is removed by mutation in the absence of lin-4, the other is gene is repressed. We have referred to this as the mutual positive feedback loop between lin-14 and lin-28. The existence of this feedback loop indicates that repression of lin-14 and lin-28 can take place independently of the lin-4 RNA. We have characterized the lin-4-independent repression of lin-28. The repression of lin-28 by the lin-4-dependent and lin-4-independent mechanisms appears to occur in the same time frame. LIN-28 decreases after the L1 in wildtype and in a lin-4; lin-14 double mutant, but not in a lin-4 mutant. Repression of lin-28 fails to occur in a lin-14(gf) mutant that is insensitive to lin-4 due to a rearrangement of its 3 UTR this is true even in with a functional lin-4 gene. Therefore, the lin-4 RNA is neither necessary nor sufficient to cause the temporal repression of lin-28. We have determined that the lin-4-independent repression of lin-28 is mediated through the genes 3 UTR through a site that is distinct from the lin-4-complementary element. We have also determined that the lin-28 mRNA is polysome-associated in the repressed state, as is the case for the lin-4-dependent repression, suggesting the two mechanisms of repression are similar, if not identical. It may be that lin-4 exerts its effect by acting simultaneously on lin-14 and lin-28 to enhance the feedback loop repression mechanism. Using a new genetic screen we are seeking mutations in trans-acting factors that are needed for the repression of lin-28.

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

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

[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.

Hong Y et al. (1996) East Coast Worm Meeting "VULVA-SPECIFIC HETEROCHRONIC PHENOTYPE CONFERRED BY A lin-31::lin-14 GENE FUSION"

Ambros VR, Hong Y

[East Coast Worm Meeting, 1996]

lin-31 is required for the normal specification of vulval fates (Miller et al, Genes & Dev, 1993; 7(6):933-47). A lin-31::gfp transgene is expressed in vulva precursor cells (Pn.p) cells beginning in the L2, and is down regulated once the Pn.p cells adopt their fates (Patrick Tan and Stuart Kim, personal comm.). We recently identified a 5.7Kb lin-14 genomic DNA sequence, lin-14R, which rescues lin-14(n179ts) precocious vulval and lateral hypodermal defects when expressed under the control of a col-10 promoter. We constructed a lin-31::lin-14R transcriptional fusion to express lin-14R specifically in Pn.p cells. Transgenic animals carrying the lin-31::lin-14R transgene are egg-laying-defective and exhibit vulval cell lineage defects like that of lin-14(gf) or lin-4(lf) mutants, presumably due to the expression of functional LIN-14 specifically in the vulval cell lineage. lin-31::lin-14Rd, a derivative of lin-31::lin-14R in which 500bp containing Lin-4 RNA binding sequences was removed from lin-14R 3'UTR, causes a more severe retarded vulva phenotype than lin-31::lin-14R. This is presumably a result of continuous expression independent of down-regulation by lin-4. A strain (VT775) containing an integrated lin-31::lin-14Rd transgenic array exhibits nearly 100% vul phenotype. Unlike the vulval defect of other heterochronic mutants, the lin-31::lin-14Rd vulval phenotype is not suppressed by post-dauer development, probably as a result of post-dauer activity of the lin-31 promoter. A gfp fusion derivative of lin-31::lin-14Rd in which GFP is inserted into the carboxy end of LIN-14 results in similar vulva-specific retarded development, indicating that this LIN-14::GFP fusion protein is functional. Preliminary results show that in lin-31::lin-14Rd::gfp lines retarded Pn.p cells express strong GFP up to the late L4. Further study will focus on: 1) analyzing vulva cell lineages in lin-31::lin-14Rd::gfp lines; 2) testing the dependence of the lin-31::lin-14Rd vul phenotype on other genes of the vulval and heterochronic gene pathways; and 3) genetic screening for the revertants from VT775 to identify genes acting downstream of lin-14 in the vulval cell cycle and developmental pathways.

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."

Wightman BC, Ruvkun GB, 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.

Olsen PH et al. (1995) International C. elegans Meeting "The post-transcriptional regulation of lin-14 by lin-4 - polysome analysis."

Olsen PH, Ambros VR

[International C. elegans Meeting, 1995]

lin-14 activity is temporally down-regulated during wild type larval development by the action of a small lin-4 antisense RNA. To investigate the mechanism by which lin-4 is regulating Lin-14 protein levels, the distribution of lin-14 mRNA on size-fractionated polysomes was analyzed. If lin-4 inhibits lin-14 at the level of translational initiation in the late L1 and L2 stages, we would expect to find a decrease in the fraction of large lin-14 RNA-containing polysomes. The principal result is that there is no change in the size distribution of lin-14 polysomes between the L1, when Lin-14 protein is abundant, and the L2, when the steady-state level of Lin-14 protein is essentially undetectable. The second result is that the 22 nucleotide lin-4 RNA is detected in polysome fractions in the approximate molar ratio of 5 to 12 lin-4 RNA's per lin-14 mRNA. To test whether the cosedimentation of lin-4 RNA with lin-14 is specific to the presence of the lin-14 3'UTR, polysomes from C. elegans extracts containing a lin-14 (n355n679) allele that reportedly lacks putative lin-4 binding sites were fractionated. lin-4 RNA was also found to cosediment with this lin-14 (n355n679), though at an apparently lower stoichiometry. Taken together, these results suggest that either Lin-14 protein is made in the L2 but is tagged for rapid degradation, a process signaled by lin-4 RNA or alternatively, the lin-14 polysomes may no longer be translationally competent in the presence of lin-4 RNA. Furthermore, lin-4 RNA may not only be associatedwith lin-14 RNA but also other RNA's or the translational machinery. Experiments are underway to examine these issues.

K Seggerson et al. (1999) International C. elegans Meeting "Translational regulation of the heterochronic gene lin-28 by the lin-4 RNA and a lin-14-dependent positive feedback loop"

EG Moss, JE Johnstone, K Seggerson

[International C. elegans Meeting, 1999]

The heterochronic gene lin-28 is one of several regulators that control the stage-specificity of a variety of postembryonic developmental events throughout the animal. Two other heterochronic genes, lin-4 and lin-14 regulate lin-28 . lin-4 encodes a short RNA that represses lin-28 expression after the L1. Without this repression, retarded development results. However, lin-28 can be repressed after the L1 in the absence of lin-4 if the lin-14 activity is reduced. lin-14 , therefore, is a positive regulator of lin-28 . lin-14 expression is likewise dependent on lin-28 after the L1 (Arasu, et al. 1991). Thus there is a mutual positive regulatory feedback loop between lin-14 and lin-28 , two genes that are both repressed by lin-4 . We've found that, like lin-14 , lin-28 repression by lin-4 occurs by a post-transcriptional mechanism. Further, we've found that the lin-14 dependence of lin-28 expression is also post-transcriptional. By sucrose gradient analysis we've found that the repression via each regulatory circuit does not prevent the lin-28 mRNA from associating with polyribosomes. This is consistent with a mechanism of repression that acts after translation initiation. Also, these observations are consistent with a model proposed by V. Ambros in which a single regulatory mechanism affects lin-28 , and the lin-4 and lin-14 regulatory circuits impinge oppositely on that mechanism. We are conducting experiments to test this model. A lin-4 complementary element (LCE) in the 3' UTR of lin-28 is required for the repression by lin-4 . We mapped the lin-14 -dependence also to the 3' UTR of lin-28 and found that the LCE is not required for lin-14 -dependence. We have constructed lin-28 :GFP transgenes bearing alterations of the 3' UTR and tested these for down-regulation of GFP and lin-28 activity in two genetic backgrounds that reveal each regulatory circuit. The lin-14 -dependence and the lin-4 regulation appear to be mediated by separate cis-acting elements. We would like to know whether these elements could confer repression on a heterologous gene. We are also seeking factors that interact with these elements in order to learn how lin-28 's repression takes place.

Arasu P et al. (1990) Worm Breeder's Gazette "How other heterochronic genes control the lin-14 temporal switch."

Arasu P, Ruvkun GB

[Worm Breeder's Gazette, 1990]

Anti-lin-14 antibodies detect a nuclear protein in specific somatic cells of late embryos and early to mid-L1 animals that quickly disappears during late L1 and later stages. Two major proteins of 76 and 67 kD are detected in immunoblots of protein extracts only from embryos and L1 larvae. These proteins do not correspond to the lin- 14a and lin-14b gene activities (proposed by Ambros & Horvitz, 1987) nor to the lin-14A and lin-14B transcripts (see Burglin et. al., WBG this issue) because both proteins are detected in various lin-14a-b+ and a+b- mutants. We have analyzed the temporal and cellular expression patterns of lin-14 protein accumulation in various heterochronic mutants, i.e. lin-4(e912), lin-28(n719), lin-29(n333), and the double mutants lin-4( e912);lin-28(n719) and lin-14(n536sd);lin-28(n719). lin-4, like lin- 14 semidominant mutants, exhibits a retarded phenotype, displays inappropriately high levels of the 76 and 67 kD lin-14 proteins at all developmental stages, and shows inappropriate lin-14 staining in nuclei at L2, L3, L4 and adult stages. Thus, the lin-4 gene negatively regulates lin-14 protein levels after larval stage 1. Northern blots and RNase protections suggest that this lin-4 regulation of lin-14 is post-transcriptional, as with the lin-14 gain- of-function (gof) mutants; the temporal regulation of both lin-14 transcripts is normal in a lin-4 mutant (Wightman & Ruvkun, unpublished results). In contrast, lin-28 animals have a steeper lin- 14 temporal gradient compared to wild-type; little to no lin-14 protein is visible in the hypodermal and intestinal nuclei of early to mid-L1 animals while the ventral cord neurons, body wall muscle and nerve ring nuclei are comparable in intensity to wild-type. Thus, the lin-28 gene activity positively regulates lin-14 protein levels in specific cell lineages in L1 animals. We do not yet know if this effect is transcriptional or post-transcriptional. These data suggest that at least in the hypodermal and intestinal lineages, lin-14 does not act downstream of lin-28 but instead either up-regulates lin-14 gene expression or controls the stability/activity of the lin-14 protein. Because of the opposing effects of lin-4/lin-14gof and lin- 28 gene activities on lin-14, we examined the temporal regulation of lin-14 expression in double mutants to characterize their epistasis relationship in molecular terms. lin-4;lin-28 and lin-14gof;lin-28 mutants show lin-14 staining in late embryos to mid-L1 stages which fades to undetectable in late L1 and later stage animals. Therefore, while both lin-4/lin-14gof and lin-28 mutations affect lin-14 protein levels, the lin-28 mutation is epistatic to the effects of the lin-4 and lin-14gof mutations on the pattern of lin-14 protein accumulation. This molecular epistasis is consistent with the genetic epistasis of lin-28 to lin-4 and lin-14gof mutations. lin-29 animals show no variation from the wild-type pattern of lin-14 protein confirming that it is indeed downstream of lin-14 in the epistasis pathway. We have also found that in wild-type animals, the down-regulation of lin-14 protein levels is linked to actual developmental time, (not to GMT!). We have investigated this by allowing eggs to hatch and suspending animals at the L1 stage by starvation for up to 125 hours. There is no decline in lin-14 protein levels in these arrested L1s by immunoblot or immunostaining analyses. Northern blots showed that lin- 14 transcript levels are similar in starved L1s compared to newly hatched L1s. However, in the presence of cycloheximide, starved L1s show a steep decline in lin-14 staining at 24 to 48 hours. These data suggest that rather than being stable, lin-14 protein is continually translated in these arrested L1s. If starved L1s are then fed and develop into L2s, the lin-14 protein levels fade as occurs during normal development so that upon feeding, a signal to resume postembryonic development causes a decrease in lin-14 protein levels. This decrease in lin-14 is probably attained by negative post- transcriptional regulation of the lin-14 3'UTR (see Burglin et. al., WBG this issue) The lin-4 gene product is a good candidate for this negative regulatory activity. Our data predicts that the lin-4 gene may become active after this L1 feeding switch. In starved L1s of lin- 28 and lin-14(n179ts25C) mutants, the lin-14 protein staining fades faster than in wild-type suggesting that either a functional lin-14 protein positively autoregulates lin-14 gene expression or protein stability, and that the lin-28 activity is necessary for this effect.

Tan PBO et al. (1998) Cell "MAP kinase signaling specificity mediated by the LIN-1 Ets/LIN-31 WH transcription factor complex during C. elegans vulval induction."

Kim SK, Lackner MR, Tan PBO

[Cell, 1998]

The let-23 receptor/mpk-1 MAP kinase signaling pathway induces the vulva in C. elegans. We show that MPK-1 directly regulates both the LIN-31 winged-helix and the LIN-1 Ets transcription factors to specify the vulval cell fate. lin-31 and lin-1 act genetically downstream of mpk-1, and both proteins can be directly phosphorylated by MAP kinase. LIN-31 binds to LIN-1, and the LIN-1/LIN-31 complex inhibits vulval induction. Phosphorylation of LIN-31 by MPK-1 disrupts the LIN-1/LIN-31 complex, relieving vulval inhibition. Phosphorylated LIN-31 may also act as a transcriptional activator, promoting vulval cell fates. LIN-31 is a vulval-specific effector of MPK-1, while LIN-1 acts as a general effector. The partnership of tissue-specific and general effectors may confer specificity onto commonly used signaling pathways, creating distinct tissue-specific outcomes.