LS Kaltenbach et al. (1999) International C. elegans Meeting "tlp-1 encodes a variant TATA-binding protein that is essential for embryogenesis"

M Domeier, SE Mango, LS Kaltenbach

[International C. elegans Meeting, 1999]

One component of the basal transcription machinery is a 700kD complex called TFIID. TFIID is composed of TATA-binding protein (TBP; CeTBP in C. elegans 1 ) and several TBP-associated factors. The prevailing view is that this complex is expressed in all cells, at all stages, and that it plays an important role in promoter recognition and recruitment of additional components of the basal machinery to target genes. We have identified a variant of TBP (called tlp-1 for T BP- l ike p rotein) in a yeast two-hybrid screen designed to identify proteins that interact with conserved C-terminal sequences of the pharynx identity factor PHA-4 2 . TBP variants have also been found in flies, humans and rodents 3 . Given the ubiquitous and fundamental role of TBP during transcription, why do animals need a second TBP-like protein? Our data suggest that tlp-1 has an essential function during embryogenesis. First, we examined tlp-1 expression. We defined the tlp-1 gene structure and constructed a tlp-1::GFP chimera. This gene is expressed widely from early embryogenesis until adulthood. By contrast, CeTBP::GFP expression does not initiate until the 3-fold stage. Second, we used RNA interference to determine the loss-of-function phenotypes of tlp-1 and CeTBP . The phenotype of most tlp-1(RNAi) embryos mimics the effects of blocking RNA polymerase II 4 : expression of an early zygotic GFP reporter construct is extinguished, E and MS blastomeres fail to gastrulate normally, and embryos arrest at about the 100-cell stage with little overt differentiation. CeTBP(RNAi) embryos, on the other hand, progress through embryogenesis and arrest as fully differentiated L1 larvae. In summary, we propose that i) PHA-4 activates transcription by targeting the basal transcription machinery via the PHA-4 C-terminus and ii) variant TBP-like proteins play an essential role during embryogenesis to mediate RNA polymerase II-dependent transcription. 1. Lichsteiner and Tjian, 1993. 2. Thanks to Bob Barstead and Phillip James for yeast two-hybrid reagents. 3. Crowley et al., 1993; Hansen et al., 1997; Ohbayashi et al., 1999. 4. Edgar et al, 1994; Powell-Coffman et al., 1996. Thanks to Spencer Wright for technical assistance.

Kaltenbach LS et al. (2005) Dev Dyn "Contribution of the amino and carboxyl termini for PHA-4/FoxA function in Caenorhabditis elegans."

Updike DL, Mango SE, Kaltenbach LS

[Dev Dyn, 2005]

FoxA transcription factors are central regulators of gut development in all animals that have been studied. Here we examine the sole Caenorhabditis elegans FoxA protein, which is called pha-4. We describe the molecular characterization of five pha-4 mutations and characterize their associated phenotypes. Two nonsense mutations are predicted to truncate PHA-4 after the DNA binding domain and remove the conserved carboxyl terminus. Surprisingly, animals harboring these mutations are viable, provided the mutant mRNAs are stabilized by inactivating the nonsense-mediated decay pathway. Two additional nonsense mutations reveal that the DNA binding domain is critical for activity. A missense mutation predicted to alter the PHA-4 amino terminus leads to a dramatic reduction in pha-4 activity even though the protein is expressed appropriately. We suggest that the PHA-4 amino terminus is essential for PHA-4 function in vivo, possibly as a transactivation domain, and can compensate for loss of the carboxyl terminus. We also provide evidence for autoregulation by PHA-4. Developmental Dynamics, 2005. (c) 2005 Wiley-Liss, Inc.

Kaltenbach LS et al. (1998) West Coast Worm Meeting "TAKING APART PHA-4: FUNCTIONS OF THE AMINO AND CARBOXYL REGIONS IN PHARYNX DEVELOPMENT"

Mango SE, Horner MA, Kaltenbach LS

[West Coast Worm Meeting, 1998]

Specification of pharyngeal and rectal precursors requires pha-4, a member of the winged-helix transcription factor family (1). This family of proteins includes the developmental regulators fork head in Drosophila and HNF-3 in mammals. Whereas the winged-helix DNA binding domain has been characterized extensively, relatively little is known about the roles of other parts of these proteins in transcriptional regulation. To elucidate the functions of regions outside the DNA binding domain, we have begun characterizing five different pha-4 alleles. The PHA-4 protein contains two additional regions outside the DNA binding domain: an amino terminal region that has sequence similarity with FORK HEAD protein and a carboxyl terminal region that has sequence similarity with HNF-3 and Ce-DISTAL-LESS. Our characterization of five pha-4 alleles indicates that the amino terminus is critical for pha-4 function whereas the carboxyl terminus is dispensible for viability. A single missense mutation in the amino terminus results in a leucine to phenylalanine substitution that renders PHA-4 largely inactive. Antibody staining indicates that the protein is expressed normally in mutant embryos. Therefore, it is likely that this mutation marks a region within PHA-4 that is critical for activity. Analysis of phenotypes associated with four nonsense alleles indicates that the PHA-4 DNA binding domain, but not the carboxyl terminus is essential for viability. These alleles are predicted to generate successive truncations from the carboxyl terminus through the DNA binding domain. To analyze the phenotype associated with each of these alleles, we constructed pha-4; smg-3 double mutants. Mutations in smg-3 disrupt the mRNA surveillance system that recognizes and degrades aberrant mRNAs, allowing accumulation of nonsense-bearing mRNAs and their protein products (2). We found that removing any portion of the DNA binding domain blocked the ability of pha-4 to promote pharyngeal development. Surprisingly, embryos carrying PHA-4 proteins that lacked the entire carboxyl terminus (but retained the DNA binding domain) were viable in combination with an allele of smg-3. These worms can be maintained as a homozygous strain although they are not as healthy as wild type worms. We are currently examining mutant worms for the presence of all pharyngeal cells. In summary, we have shown that the PHA-4 DNA binding domain but not the carboxyl terminus is essential for pharynx organogenesis. Furthermore, a point mutation delineates a site in the amino terminus required for PHA-4 activity. Our current goal is to identify protein(s) that interact with the amino and carboxyl termini of PHA-4. We are using two approaches to identify these factors: 1) a genetic screen for pha-4 suppressors and 2) a yeast two-hybrid screen using the PHA-4 amino or carboxyl regions as bait. Thanks to Jim Priess for one of the pha-4 alleles and Bob Barstead for the two-hybrid library. 1. Mango, S.E., Lambie, E.J. & Kimble, J. Development 120, 3019- 3031 (1994); Kalb, J.M., et al. Development 125, 2171-2180 (1997); Horner, M., et al. in press . 2. Hodgkin, J., Papp, A., Pulak, R., Ambros, V. & Anderson, P. Genetics 123, 301-313 (1989); Pulak, R. & Anderson, R.P. Genes Dev. 7, 1885-1897 (1993).

Kaltenbach LS et al. (2000) Mol Cell "The TBP-like factor CeTLF is required to activate RNA polymerase II transcription during C. elegans embryogenesis."

Kaltenbach LS, Horner MA, Mango SE, Rothman JH

[Mol Cell, 2000]

Metazoans possess two TATA-binding protein homologs, the general transcription factor TBP and a related factor called TLF. Four models have been proposed for the role of TLF in RNA polymerase II (Pol II) transcription: (1) TLF and TBP function redundantly, (2) TLF antagonizes TBP, (3) TLF is a tissue-specific TBP, or (4) TLF and TBP have distinct activities. Here we report that CeTLF is required to express a subset of Pol II genes and associates with at least one of these genes in vivo. CeTLF is also necessary to establish bulk transcription during early embryogenesis. Since CeTLF and CeTBP are expressed at comparable levels in the same cells, these findings suggest CeTLF performs a unique function in activating Pol II transcription distinct from that of CeTBP.

Adachi H et al. (2000) J Gerontol A Biol Sci Med Sci "Effects of tocotrienols on life span and protein carbonylation in Caenorhabditis elegans."

Ishii N, Adachi H

[J Gerontol A Biol Sci Med Sci, 2000]

To assess the efficiency of tocotrienols against oxidative damage, we have demonstrated in a model-system nematode, Caenorhahditis elegans, that tocotrienol administration reduced the accumulation of protein carbonyl (a good indicator of oxidative damage during aging) and consequently extended the mean life span (LS), but nut the maximum LS. Conversely, or-tocopherol acetate did not affect these parameters, ris a way to evaluate the protective ability of tocotrienols against oxidative stress, the life spans of animals administrated tocotrienols before or after exposure to ultraviolet B-induced oxidative stress were measured. Ultraviolet B irradiation shortened the mean LS of animals, whereas preadministration of tocotrienols recovered the mean LS to that of unirradiated animals. Interestingly, postadministration also extended the mean LS mure than that of unirradiated animals, and administration through the LS conferred greater protection. Thus, the administration of tocotrienols to animals results in a reduction of oxidative stress risks. These data indicated that tocotrienols merit further investigation as possible agents for antiaging and oxidative stress prevention. In addition, they suggest that C. elegans will continue to provide provocative clues into the mechanisms of aging.

Vance AA et al. (1994) Worm Breeder's Gazette "Monoclonal Antibodies Against Caenorhabditis elegans Extracellular Matrix."

Otsuka AJ, Lee M, Cheung HT, Vance AA

[Worm Breeder's Gazette, 1994]

Monoclonal Antibodies against Caenorhabditis elegans Extracellular Matrix. Myeongwoo Lee, Annernarie A. Vance, Anthony J. Otsuka, and Hou Tak Cheung. Department of Biological Sciences, Illinois State University, Normal, Illinois, 6179W120. Extracellular matrix (ECM) is a complex molecular meshwork of proteins and carbohydrates that is found between cells. ECM gives tissues mechanical strength, elasticity, and shape. The major classes of ECM components are collagens, noncollagenous glycoproteins, and proteoglycans. Available evidence indicates that ECM play an irnportant role in the development of an organism. Until recently, human or murine experimental models were used in most of the structural and functional studies on the ECM components. Because of the complexity and long life span of higher vertebrates, it is difficult to study the impact of ECM components on development. We chose C. elegans as an experimental model to study the role of ECM components in development because of its simple body plan, well-known genetics, and well-established cell lineages. To identify and characterize the components of the C. elegans ECM, a panel of monoclonal antibodies was prepared against a C. elegans ECM extract by 3.4M NaCI extraction and followed by 2M urea treatment. Three monoclonal antibodies, LS 22, LS 25, and LS 28 have been characterized by immunofluorescence and immunoblotting. In immunofluorescence, LS 22 stained throughout the hemlaphrodite gonad, as well as oocytes from the disrupted gonads. In immunoblotting experiments, LS 22 recogr.uzed a series of epitopes at 110, X5, 67, 52, and 45 kD under reducing conditions. However, LS 22 purified a 67.5 kD species by the immuno-affinity chromatography. Furthermore, LS 22 crossreacted with the mouse EHS tumor matrix, a basement membrane analog from Engelbreth-Holm-Swarm sarcoma. In situ immunolocalization using a second antibody, LS 25, showed that it strongly stained body wall muscle structures with a striated pattern resembling A-bands, the egg-laying muscle near vulva, and the lumen of the intestine. In the wild-type embryo, LS 25 stained two-cell wide arrays corresponding to the muscle quadrants of the one- fold embryo. In immunoblotting, LS 25 stained series of protein bands ranging in molecular weight from 135 kD to 35 kD with the most intense bands at 135, 50, and 40 kD under reducing conditions. Since LS 25 recognized body wall muscles in immunofluorescence staining, we screened the C elegans muscle mutants such as unc-54 (el 90) and unc-45(e286) with LS 25. LS 25 stained muscle filaments in these mutants with disorganized filament patterns and localized intestinal lumen structures with spotted patterns. Another monoclonal antibody, designated LS 28, stained hermaphrodite gonad structures in the immunofluorescence studies. The staining extended throughout the gonad with no crossreaction to the intestine and other structures such as muscle and pharynx. Furthermore, LS 28 showed no staining with embryos and fertilized eggs. The antigenic epitope of LS 28 was detected as a single band of 55 kD in immunoblotting with C. elegans ECM extracts. This antibody also crossreacts with the mouse EHS matrix and a purified mouse ECM component, laminin at 205 kD bands. The crossreactivity of the monoclonal antibodies with known C. elegans ECM components has not been tested yet. It will be of interest to determine the nature of LS 22 and LS 28 crossreactivity with both the worm gonad and mouse ECM. We are in the process of screening mutants defective in muscle cell attachment and ECM components with these antibodies.

Volovik Y et al. (2012) Aging Cell "Temporal requirements of heat shock factor-1 for longevity assurance."

Bejerano-Sagie M, Dubnikov T, Joyce D, Volovik Y, Cohen E, Dillin A, Kapernick EA, Maman M

[Aging Cell, 2012]

Reducing the activity of the insulin/IGF-1 signaling pathway (IIS) modifies development, elevates stress resistance, protects from toxic protein aggregation (proteotoxicity), and extends lifespan (LS) of worms, flies, and mice. In the nematode Caenorhabditis elegans, LS extension by IIS reduction is entirely dependent upon the activity of the transcription factors DAF-16 and the heat shock factor-1 (HSF-1). While DAF-16 determines LS exclusively during early adulthood, it is required for proteotoxicity protection also during late adulthood. In contrast, HSF-1 protects from proteotoxicity during larval development. Despite the critical requirement for HSF-1 for LS extension, the temporal requirements for this transcription factor as a LS determinant are unknown. To establish the temporal requirements of HSF-1 for longevity assurance, we conditionally knocked down hsf-1 during larval development and adulthood of C.elegans and found that unlike daf-16, hsf-1 is foremost required for LS determination during early larval development, required for a lesser extent during early adulthood and has small effect on longevity also during late adulthood. Our findings indicate that early developmental events affect LS and suggest that HSF-1 sets during development of the conditions that enable DAF-16 to promote longevity during reproductive adulthood. This study proposes a novel link between HSF-1 and the longevity functions of the IIS.

Arumugam S et al. (2014) Exp Parasitol "Localization of a filarial phosphate permease that is up-regulated in response to depletion of essential Wolbachia endobacteria."

Pfarr KM, Arumugam S, Hoerauf A

[Exp Parasitol, 2014]

Wolbachia of filarial nematodes are essential, obligate endobacteria. When depleted by doxycycline worm embryogenesis, larval development and worm survival are inhibited. The molecular basis governing the endosymbiosis between Wolbachia and their filarial host is still being deciphered. In rodent filarial nematode Litomosoides sigmodontis, a nematode encoded phosphate permease gene (Ls-ppe-1) was up-regulated at the mRNA level in response to Wolbachia depletion and this gene promises to have an important role in Wolbachia-nematode endosymbiosis. To further characterize this gene, the regulation of phosphate permease during Wolbachia depletion was studied at the protein level in L. sigmodontis and in the human filaria Onchocerca volvulus. And the localization of phosphate permease (PPE) and Wolbachia in L. sigmodontis and O. volvulus was investigated in untreated and antibiotic treated worms. Depletion of Wolbachia by tetracycline (Tet) resulted in up-regulation of Ls-ppe-1 in L. sigmodontis. On day 36 of Tet treatment, compared to controls (Con), >98% of Wolbachia were depleted with a 3-fold increase in mRNA levels of Ls-ppe-1. Anti-Ls-PPE serum used in Western blots showed up-regulation of Ls-PPE at the protein level in Tet worms on day 15 and 36 of treatment. Immunohistology revealed the localization of Wolbachia and Ls-PPE in the embryos, microfilariae and hypodermis of L. sigmodontis female worms and up-regulation of Ls-PPE in response to Wolbachia depletion. Expression of O. volvulus phosphate permease (Ov-PPE) studied using anti-Ov-PPE serum, showed up-regulation of Ov-PPE at the protein level in doxycycline treated Wolbachia depleted O. volvulus worms and immunohistology revealed localization of Ov-PPE and Wolbachia and up-regulation of Ov-PPE in the hypodermis and embryos of doxycycline treated worms. Ls-PPE and Ov-PPE are upregulated upon Wolbachia depletion in same tissues and regions where Wolbachia are located in untreated worms, reinforcing a link between Wolbachia and this nematode encoded protein. The function of nematode phosphate permease in the endosymbiosis is unknown but could involve transportation of phosphate to Wolbachia, which encode all the genes necessary for de novo nucleotide biosynthesis. Electron microscopic localization of PPE and Wolbachia and RNAi mediated knock-down of PPE in filarial nematodes will bring further insights to the functions of PPE in the Wolbachia-nematode symbiosis.

Wanyuan Ao et al. (2001) International C. elegans Meeting "What is TLF doing during the C. elegans cell cycle?"

Wanyuan Ao, S E Mango

[International C. elegans Meeting, 2001]

Transcription is finely regulated during the cell cycle. In most organisms, genes are actively transcribed during interphase, but not during mitosis when DNA is condensed, nascent transcripts are released, and transcription factors are inactivated by phosphorylation and discharged from DNA. How then do cells re-establish the appropriate transcriptional repertoire in newly born daughter cells? One attractive idea is that cells ‘mark’ genes to specify those that are actively being transcribed during interphase. The molecular nature of the mark is currently unknown. We are using C. elegans embryos to investigate cell cycle regulation of RNA Polymerase II (Pol II) transcription. Like other organisms, C. elegans probably inactivates transcription at mitosis since i) Pol II in mitotic cells lacks a phosphorylated epitope normally associated with transcriptional elongation (1, 2) and ii) transcription factors are dispersed at this stage of the cell cycle (L. Kaltenbach and S.E.M., unpublished). Since embryonic blastomeres cycle very rapidly (~15-30 minutes), we envision that gene marking and rapid re-establishment of transcription after mitosis are critical for normal embryonic development. One appealing candidate for a marking protein is the TATA-binding protein (TBP) paralog TLF. TLF is required for Pol II transcription during C. elegans embryogenesis, suggesting it performs a unique function distinct from that of TBP (2, 3). TLF is required to activate a subset of Pol II promoters and facilitates the re-establishment of transcription after mitosis (2). In addition, preliminary data indicate that TLF may associate with mitotic chromosomes during the cell cycle (L. Kaltenbach and S.E.M., unpublished). This association contrasts with TBP, which is released during mitosis (L. Kaltenbach and S.E.M., unpublished). These observations suggest that TLF has a critical role during the cell cycle, possibly to mark genes for their timely reactivation during the next interphase. Our current goal is to test this hypothesis using both in viv o and in vitro approaches. Seydoux and Dunn, Development 124, 2191-2201 (1997) Kaltenbach et al., Molecular Cell, 6, 705-713 (2000). Dantonel et al., Molecular Cell 6, 714- (2000)

Heider U et al. (2006) Int J Med Microbiol "Differential display of genes expressed in the filarial nematode Litomosoides sigmodontis reveals a putative phosphate permease up-regulated after depletion of Wolbachia endobacteria."

Blaxter M, Heider U, Hoerauf A, Pfarr KM

[Int J Med Microbiol, 2006]

Mutualist symbiotic Wolbachia endobacteria are found in most filarial nematodes. Wolbachia are essential for embryogenesis and for larval development into adults, and thus represent a new target for anti-filarial drug development. Tetracycline antibiotics deplete Wolbachia in animal model filaria Litomosoides sigmodontis and Brugia pahangi, as well as in the human parasites Brugia malayi, Onchocerca volvulus and Wuchereria bancrofti. Very little is known about the molecular details of the symbiotic interaction between Wolbachia and filarial nematodes. Nematode genes that respond to anti-Wolbachia antibiotic treatment may play important roles in the symbiosis. Differential display PCR was used to detect several candidate genes that are up-regulated after 3, 6, 15, 30 and 36 days of tetracycline treatment. One of these genes, Ls-ppe-1, was similar to a family of phosphate permeases, and had putative orthologues in O. volvulus and B. malayi. Ls-ppe-1 steady-state mRNA levels were elevated by day 3-6 of treatment, and remained elevated through to 70 days post-treatment. In Caenorhabditis elegans, the knockdown of a homologous phosphate permease results in embryonic lethality, with the production of degenerating embryos, a phenotype also seen in filarial nematodes after depletion of Wolbachia with tetracycline. The potential role of Ls-ppe-1 in the nematode-bacterial symbiosis is discussed.