Hawkins MG et al. (1993) Worm Breeder's Gazette "A New GATA-Factor Isolated from C. elegans"

McGhee JD, Hawkins MG

[Worm Breeder's Gazette, 1993]

Ges-1 ,a gut-specific esterase first expressed at the 4-8E stage of development is a convenient marker gene to study early intestine development in C. elegans. We are interested in the factors that bind to the control region of the ges-1 gene to bring about its regulation. Footprinting and band-shift experiments using nuclear extracts from embryos (V. Stroeher et al., submitted) have identified a number of protein factors that bind to a region of the ges-1 gene 1.0 -1.3 kB upstream of the ATG. Of particular interest is a region that contains two WGATAR motifs similar to the VPE2 elements identified in the C. elegans vitellogenin genes, which are also expressed in a gut specific manner (MacMorris et al, 1992. Mol. Cell Biol. 12, 1652-1662). Egan et al. (see abstracts of the 1993 International C. elegans meeting) have shown that these GATA motifs are important for the gut-specific expression of ges-1 .We therefore used multimerized GATA-containing double stranded oligonucleotides to screen approximately 300,000 plaques from a C. elegans, mixed stage cDNA expression library ( Okkema et a/., WBG Vol12 ,ii).One clone was identified on the criteria of DNA-protein binding. This clone was then used to isolate a number of apparently close to full length cDNA clones from an independent C. elegans mixed stage cDNA library. The full length cDNA is 1597 nucleotides in length, is SL1 trans-spliced and contains a putative open reading frame of 433 amino acids in length, ie. encoding a protein of approximately 47,000Mr. Sequence analysis has shown the protein to be similar to GATA-factors isolated from a diverse range of species including elt-1 (C. elegans, Spieth et al., 1991. Mol. Cell Biol. 11, 4651-4659), GATA-4 (Mice, Arceci et al., 1993. Mol. Cell Biol. 13, 2235-2246), GATA-3 (Xenopus, Zon etal., 1991. Proc. Natl. Acad. Sci. USA. 88, 10642-10646), GATA-2 (humans, Lee et al., 1991. J. Biol. Chem. 266, 16188-16192) and GATA-1 (chicken, Evans and Felsenfeld, 1989. Cell 58,877-885). The new GATA factor contains two putative zinc-fingers as shown below. The C-terminal zinc-finger shows a similarity of 76%-80% when compared to the C-terminal zinc-fingers of the GATA factors listed above. However, the N-terminal zinc-finger shows a lower degree of similarity when compared to all other known GATA-factors ie. it has the sequence C-V-K-C-N(16)-C-V-N-C instead of the conserved motif C-X-N-C-N(17)-C-N-X-C. Furthermore the inter-finger distance is different from other GATA-factors. Thus we do not know if the protein has two zinc-fingers or one zinc-finger and a pseudo zinc-finger. It has been shown with the other GATA-factors that the C-terminal finger is much more important for DNA binding. For example Ormichinski et al. (1993, Proc. Natl. Acad. Sci. USA. 90, 1676-1680) showed the C-terminal zinc-finger of Chick GATA-1 was necessary for sequence specific DNA-protein interaction. The important question is whether the new gene is involved in regulating ges-1 and we hope to approach this via Tc1 insertion mutants and studying expression patterns. We now have genomic clones but they have yet to be positioned onto the C. elegans physical map.

Hawkins MG et al. (1995) J Biol Chem "elt-2, a second GATA factor from the nematode Caenorhabditis elegans."

McGhee JD, Hawkins MG

[J Biol Chem, 1995]

We have previously shown that a tandem pair of (A/T)GATA(A/G) sequences in the promoter region of the Caenorhabditis elegans gut esterase gene (ges-1) controls the tissue specificity of ges-1 expression in vivo. The ges-1 GATA region was used as a probe to screen a C. elegans cDNA expression library, and a gene for a new C. elegans GATA-factor (named elt-2) was isolated. The longest open reading frame in the elt-2 cDNA codes for a protein of M(r) 47,000 with a single zinc finger domain, similar (approximately 75% amino acid identity) to the C-terminal fingers of all other two-fingered GATA factors isolated to date. A similar degree of relatedness is found with the single-finger DNA binding domains of GATA factors identified in invertebrates. An upstream region in the ELT-2 protein with the sequence C-X2-C-X16-C-X2-C has some of the characteristics of a zinc finger domain but is highly diverged from the zinc finger domains of other GATA factors. The elt-2 gene is expressed as an SL1 trans-spliced message, which can be detected at all stages of development except oocytes; however, elt-2 message levels are 5-10-fold higher in embryos than in other stages. The genomic clone for elt-2 has been characterized and mapped near the center of the C. elegans X chromosome, ELT-2 protein, produced by in vitro transcription-translation, binds to ges-1 GATA-containing oligonucleotides similar to a factor previously identified in C. elegans embryo extracts, both as assayed by electrophoretic migration and by competition with wild type and mutant oligonucleotides. However, there is as yet no direct evidence that elt-2 does or does not control ges-1.

Hawkins MG et al. (1995) International C. elegans Meeting "ELT-2; A GUT-SPECIFIC C. ELEGANS GATA- BINDING PROTEIN."

Chung MA, Hawkins MG, McGhee JD

[International C. elegans Meeting, 1995]

The C. elegans ges-1 gene codes for a gut- specific esterase first expressed at the 4-8E cell stage of development and is a convenient marker to study intestine development.. We are interested in the factors that bind to the control region of the ges-1 gene to bring about its regulation. Promoter mutagenesis experiments has shown two "WGATAR" motifs to be crucial for the correct spatial expression pattern of the ges-1 gene. Expression screening using GATA-binding motifs yielded a positive cDNA clone and sequence analysis has shown the full length cDNA encodes a protein similar to GATA-factors isolated from a diverse range of species. Using the nomenclature of Spieth et al., we have named the new GATA-factor elt-2 (erythroid-like transcription factor 2). The genomic clone for elt-2 has been isolated, sequenced and mapped to a region on chromosome X adjacent to mab- 18. ELT-2 protein has been expressed by in vitro transcription-translation and used to demonstrate sequence specific DNA-binding by gel mobility shift assays. In addition, ELT-2 protein forms sequence specific DNA-protein complexes that essentially comigrate with complexes formed with embryo extracts. elt-2 message can be detected in all stages of development but is present at a 5-10 fold higher level in embryos. We have not been able to detect expression in oocytes. Using Lac-Z reporter constructs we have shown that elt-2 is expressed in an intestine specific manner during development and expression begins when the gut consists of 2 E-cells. To determine the function of elt-2, a Tc1 insertion mutant has been identified but excision experiments have not yet been successful.

Hawkins MG et al. (1993) International C. elegans Meeting "DNA-protein interactions in the control region of the C. elegans ges-1 gene."

Hawkins MG, Schroeder DF, Stoeher VL, McGhee JD

[International C. elegans Meeting, 1993]

JD Kormish et al. (1999) International C. elegans Meeting "A genetic screen for genes involved in gut development and differentiation"

JD McGhee, JD Kormish

[International C. elegans Meeting, 1999]

To investigate genes potentially involved in gut development and differentiation, we are developing a genetic screen for gut obstructed (gob) mutants. The gut specific elt-2 GATA factor appears necessary for correct gut cell development. elt-2 null mutants arrest as L1s with a blocked intestinal lumen and abnormal brush border (1). When elt-2 null mutants are fed on a mixture of fluorescent beads and Escherichia coli , the beads collect as a large "gob" in the posterior pharynx and anterior gut that can be easily detected under the dissecting microscope. We are now using this gut obstructed phenotype to screen for mutants that should potentially identify genes involved in gut development. Preliminary results of this screen are promising, as several candidates have already been isolated. 1) Fukushige T, Hawkins MG and McGhee JD (1998) Dev Biol 198(2):286-302

Xie Z et al. (2020) Biomed Pharmacother "Magnolol alleviates Alzheimer's disease-like pathology in transgenic C. elegans by promoting microglia phagocytosis and the degradation of beta-amyloid through activation of PPAR-."

Xie Z, Zhang Z, Fu Y, Xu J, Zhao J, Yang Q, Wang H, Holscher C, Jiang Y, Zeng H

[Biomed Pharmacother, 2020]

This study aims to investigate whether magnolol (MG), a natural neolignane compound, can prevent AD induced by beta-amyloid (A) and the possible mechanisms involved. MG dose-dependently reduces A deposition, toxicity and memory impairment caused by A in transgenic C. elegans. More importantly, these effects are reversed by GW9662, a selective peroxisome proliferator-activated receptor- (PPAR-) antagonist. MG is more effective in enhancing PPAR- luciferase levels than honokiol (HK). Meanwhile, MG has the potential to bind with the ligand binding domain of PPAR- (PPAR--LBD). As expected, MG inhibited the luciferase activity of NF-B and its target genes of inflammatory cytokines, and this effect was blocked by GW9662. The luciferase activity of Nrf2-ARE expression can be activated by MG and decreased A-induced reactive oxygen species (ROS). The target gene LXR of PPAR- is activated by MG, which upregulates ApoE and promotes microglia phagocytosis and the degradation of A, and these effects were also reversed by GW9662. In summary, MG can attenuate A-induced AD and the underlying mechanism is the reduction of inflammation and promotion of phagocytosis and degradation of A, which is dependent on PPAR-.

Frandsen J et al. (2020) ACS Chem Neurosci "Neural Glyoxalase Pathway Enhancement by Morin Derivatives in an Alzheimer's Disease Model."

Frandsen J, Narayanasamy P, Choi SR

[ACS Chem Neurosci, 2020]

The gyloxalase pathway (GP) is an antioxidant defense system that detoxifies metabolic byproduct methylglyoxal (MG). Through sequential reactions, reduced glutathione (GSH), glyoxalase I (glo-1), and glyoxalase II (glo-2) convert MG into D-lactate. Spontaneous reactions involving MG alter the structure and function of cellular macromolecules through the formation of inflammatory advanced glycation endproducts (AGEs). Accumulation of MG and AGEs in neural cells contributes to oxidative stress (OS), a state of elevated inflammation commonly found in neurodegenerative diseases including Alzheimer's Disease (AD). Morin is a common plant-produced flavonoid polyphenol that exhibits the ability to enhance the GP-mediated detoxification of MG. We hypothesize that structural modifications to morin will improve its inherent GP enhancing ability. Here we synthesized a morin derivative, dibromo-morin (DBM) and formulated a morin encapsulated nanoparticle (MNP) - and examined their efficacy in enhancing neural GP activity. Cultured mouse primary cerebellar neurons and Caenorhabditis elegans were induced to a state of OS with MG, and treated with morin, DBM, and MNP. Results indicated the morin derivatives were more effective compared to the parent compound in neural GP enhancement and preventing MG-mediated OS.

Hashizume O et al. (2024) Dev Biol "Intestinal Mg2+ accumulation induced by cnnm mutations decreases the body size by suppressing TORC2 signaling in Caenorhabditis elegans."

Hashizume O, Funato Y, Kawabe T, Miki H

[Dev Biol, 2024]

Mg²⁺ is a vital ion involved in diverse cellular functions by forming complexes with ATP. Intracellular Mg²⁺ levels are tightly regulated by the coordinated actions of multiple Mg²⁺ transporters, such as the Mg²⁺ efflux transporter, cyclin M (CNNM). Caenorhabditis elegans (C. elegans) worms with mutations in both cnnm-1 and cnnm-3 exhibit excessive Mg²⁺ accumulation in intestinal cells, leading to various phenotypic abnormalities. In this study, we investigated the mechanism underlying the reduction in body size in cnnm-1; cnnm-3 mutant worms. RNA interference (RNAi) of gtl-1, which encodes a Mg²⁺-intake channel in intestinal cells, restored the worm body size, confirming that this phenotype is due to excessive Mg²⁺ accumulation. Moreover, RNAi experiments targeting body size-related genes and analyses of mutant worms revealed that the suppression of the target of rapamycin complex 2 (TORC2) signaling pathway was involved in body size reduction, resulting in downregulated DAF-7 expression in head ASI neurons. As the DAF-7 signaling pathway suppresses dauer formation under stress, cnnm-1; cnnm-3 mutant worms exhibited a greater tendency to form dauer upon induction. Collectively, our results revealed that excessive accumulation of Mg²⁺ repressed the TORC2 signaling pathway in C. elegans worms and suggest the novel role of the DAF-7 signaling pathway in the regulation of their body size.

Vladimir V Bakaev et al. (2003) Worm Breeder's Gazette "An attempt to slow aging in C. elegans. 32. No positive effect of peroxide"

Vladimir V Bakaev, Lyudmila M Bakaeva

[Worm Breeder's Gazette, 2003]

The purpose of this study was to investigate the effect of hydrogen peroxide in water solutions on the nematode life span. In this experiment hydrogen peroxide was used in following dilutions: 100 mg/L, 10 mg/L, 1 mg/L, 0.1 mg/L, 0.01 mg/L and 0.001 mg/L. Three adult animals (3-5 days old) were kept in microtitre wells containing 0.5 ml of liquid medium (with E. coli and without hydrogen peroxide) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (without hydrogen peroxide in medium) every day (one worm in one well) beginning from third day. Then, beginning from 3rd day, these worms were transferred every day in next wells containing medium with hydrogen peroxide in any concentration. This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table. Conclusion: If hydrogen peroxide solution was applied to C. elegans, it was not able to increase their mean longevity significantly in comparison with control.

Fukushige T et al. (1996) West Coast Worm Meeting "A GUT-SPECIFIC GATA TRANSCRIPTION FACTER (ELT-2) IN C. ELEGANS."

Fukushige T, McGhee JD, Hawkins MG, Chung MA

[West Coast Worm Meeting, 1996]

The C. elegans GATA transcription factor elt-2 was cloned by expression screening using GATA binding motifs from the promoter of the gut specific ges-1 gene (1). Previous promoter analysis has shown that these GATA motifs are crucial for the correct gut specific expression of ges-1 (2, 3). Furthermore ELT-2 protein has been demonstrated to bind specifically to these motifs by gel mobility shift assays. Using anti-ELT-2 antibody as well as elt-2::lacZ (or GFP) reporter constructs, we have now shown that elt-2 is indeed expressed in an intestine specific manner during embryonic and post-embryonic development, apparently as early as the 2E cell stage. Production of an elt-2 knockout is proceeding by Tc1 excision in order to test whether elt-2 is necessary for ges-1 expression. However, we have shown that elt-2 is at least sufficient for ges-1 expression by using a heat shock promoter (HSP):: elt-2 construct to produce ectopic ELT-2 expression. Early embryos produced by hsp::elt-2 transgenic worms (with wild type embryos as a control) were incubated at 33oC for 30 min and then incubated at 20oC over-night before assaying for GES-1 activity. Most wild type control embryos hatch without changing phenotype (i.e. GES-1 and gut granules remain localized in the gut). However, embryos in which ELT-2 has been expressed ectopically stop growing at the mid or late gastrulation stage. Most importantly, GES-1 is now expressed throughout the entire embryo, i.e. ectopic ELT-2 can apparently activate ges-1 expression in cells outside of the gut. We have performed a similar experiment to show that elt-2 may also control the C. elegans fkh-1 gene (see John Kalb's abstract). To identify upstream factors that control elt-2, deletion analysis of the elt-2 promoter is proceeding: the current status is that elt-2 expression is controlled by a region between -1.2 kb and -3.5 kb upstream of the elt-2 coding region. Several other aspects of elt-2 regulation are being investigated: (i) whether other GATA transcription factors (eg. elt-1 and elt-3) can replace the function of elt-2, and; (ii) whether elt-2 is auto-regulated. 1. Hawkins and McGhee (1995) J. Biol. Chem. 270, 14666-14671. 2. Egan et al. (1995) Dev. Biol. 170, 397-419. 3. Fukushige et al. (in press) Dev. Biol.