Cubillas, C. et al. (2019) International Worm Meeting "The nuclear receptor HIZR-1 regulates HLH-30 to promote lysosome remodeling during high zinc homeostasis."

Diwan, A., Kornfeld, K., Murphy, J., Cubillas, C., Schneider, D.

[International Worm Meeting, 2019]

Zinc is an essential element for animals, but excess zinc is toxic. To achieve detoxification, excess zinc is stored in organelles: zincosomes in mammals and lysosome-related organelles called bilobed granules in C. elegans. Worms cultured in high zinc conditions activate a regulatory cascade involving HIZR-1, a nuclear receptor that directly senses zinc, the HZA enhancer that is located in the promoter regions of zinc activated genes and binds HIZR-1, and CDF-2, a transmembrane zinc transporter localized to lysosomes that loads zinc into bilobed granules. To define the transcriptional response to high zinc and HIZR-1, we performed RNA-seq and identified many genes that were regulated by high zinc and required hizr-1. One prominent group of hizr-1 activated target genes were canonical lysosome-genes such as lgg-1, lgg-2, sqst-1, sqst-3, and multple cathepsin-coding genes. Furthermore, HLH-30, the master regulator of the autophagy-lysosomal pathway (ALP) in C. elegans, was a target gene. The hlh-30 promoter has a HZA, indicating direct regulation by HIZR-1. Based on these results, we hypothesized that high zinc triggers a cascade of transcription factors, HIZR-1 and HLH-30, and together they control transcription of multiple lysosomal target genes leading to formation of bilobed granules. Consistent with this model, high dietary zinc promoted nuclear accumulation of HLH-30 in intestinal nuclei, indicating activation. Single mutants of hlh-30 and hizr-1 were hypersensitive to high dietary zinc, and the hizr-1;hlh-30 double mutant displayed additive hypersensitivity, indicating the genes function non-redundantly to promote high zinc tolerance. HLH-30 binds the E-box in DNA. By analyzing the presence of HZA and E-boxes, we defined genes that are predicted to be direclty regulated by one or both of HIZR-1 and HLH-30, defining a regulatory network necessary to achieve zinc homeostasis. These studies define a new mechanism of high zinc homeostasis by directly linking high zinc sensing to lysosome remodeling.

Cubillas, C. et al. (2015) International Worm Meeting "A modular system of DNA enhancer elements mediates tissue-specific activation of transcription by high dietary zinc in C. elegans."

Roh, H., Cubillas, C., Kornfeld, K.

[International Worm Meeting, 2015]

Zinc is essential for biological systems, and abnormal zinc metabolism is implicated in multiple human diseases. To maintain homeostasis in response to fluctuating levels of dietary zinc, animals regulate gene expression; however, mechanisms that mediate the transcriptional response to such zinc variations have not been fully defined.Here we describe the identification of DNA enhancer elements that mediate intestine-specific transcriptional activation in response to high levels of dietary zinc in C. elegans. Using bioinformatics, we characterized an evolutionary conserved enhancer element present in multiple zinc-inducible genes, the high zinc activation (HZA) element. The HZA location was consistently adjacent to a GATA element that mediates gene expression in intestinal cells. Functional studies of zinc regulated genes (GFP fusions and RNA expression analyses) demonstrated that this modular system of DNA enhancers mediates tissue-specific transcriptional activation in response to high levels of dietary zinc. To characterize the mechanism of enhancer function, we demonstrated that the GATA transcription factor ELT-2 and the mediator subunit MDT-15 are necessary for zinc-responsive transcriptional activation.We used this information to search the genome and successfully identified several novel zinc-inducible genes. A subset of these genes is predicted to be involved in retinoic acid metabolism, such as bcmo-2 and cyp-13a7. These genes have a HZA and are responsive to high levels of dietary zinc, suggesting that retinoic acid may play a role in high zinc detoxification. We are currently combining bioinformatics and RNA-seq approaches to identify the complete set of zinc-regulated transcripts in C. elegans under high dietary zinc conditions.

Earley, B.J. et al. (2017) International Worm Meeting "The Nuclear Receptor HIZR-1 Uses Zinc as a Ligand to Mediate Homeostasis in Response to High Zinc."

Kornfeld, K., Warnhoff, K., Cubillas, C., Earley, B.J.

[International Worm Meeting, 2017]

Nuclear receptors were originally defined as endocrine sensors in humans, leading to the identification of the nuclear receptor superfamily. Despite intensive efforts, most nuclear receptors have no known ligand, suggesting new ligand classes remain to be discovered. Furthermore, nuclear receptors are encoded in the genomes of primitive organisms that lack endocrine signaling, suggesting the primordial function may have been environmental sensing. We identified a novel Caenorhabditis elegans nuclear receptor, HIZR-1, and showed that it is a high zinc sensor in an animal and the master regulator of high zinc homeostasis. To analyze the transcriptional response to high zinc, we characterized four genes activated by high dietary zinc: the zinc-binding metallothionein genes mtl-1 and mtl-2, and the cation diffusion facilitator (CDF) zinc transporter genes cdf-2 and ttm-1b. We showed that transcriptional activation of these genes is mediated by the high zinc activation (HZA) element, a DNA enhancer. We performed a forward genetic screen to identify mediators of high zinc activated transcription that resulted in the discovery of the high zinc activated nuclear receptor (HIZR-1). hizr-1 encodes a nuclear receptor transcription factor that has an evolutionarily conserved DNA-binding domain (DBD) and ligand-binding domain (LBD). We demonstrated that HIZR-1 is both necessary and sufficient to activate transcription of endogenous zinc-homeostasis genes in response to high dietary zinc. We used genetic and biochemical approaches to analyze HIZR-1 function. The LBD directly bound zinc, which promoted nuclear accumulation and activation of the protein, indicating the LBD regulates protein activity and zinc is a physiological ligand; the DBD directly bound the HZA enhancer, which mediates transcriptional activation of multiple genes involved in zinc homeostasis. These findings advance the understanding of zinc biology by identifying a sensor for high zinc in animals and elucidate homeostatic systems by defining a positive feedback loop embedded in a negative feedback circuit. To develop a comprehensive understanding of the homeostatic response mediated by hizr-1, we are currently using multiple techniques to identify the full set of hizr-1 target genes. Thus far, we have identified more than 100 genes transcriptionally activated by high zinc conditions, including multiple genes involved in autophagy, lipophagy, insulin production/processing and carotenoid metabolism. The analysis of HIZR-1 expands the understanding of the NR superfamily by (1) identifying transition metals as a new class of physiological ligand that is distinct from previously described classes such as steroids and lipids and (2) identifying direct nutrient sensing as a new function that may represent a primordial role of NRs.

Earley, B.J. et al. (2017) International Worm Meeting "hizr-1 mediates a subset of the cadmium-activated stress response."

Kornfeld, K., Cubillas, C., Lyons, M.D., Earley, B.J.

[International Worm Meeting, 2017]

Exposure to the toxic metal cadmium is a global health problem. There is an urgent need to develop therapeutic strategies that target cadmium injury, but the mechanisms responsible for cadmium toxicity and the organismal response to cadmium remain poorly understood. Cadmium is chemically similar to zinc, since they are in the same column of the periodic table. By analyzing the transcriptional response to high dietary zinc, we discovered the High Zinc Activation (HZA) enhancer that mediates transcriptional activation. Furthermore, we demonstrated that the HZA also responds to cadmium. HIZR-1 is a nuclear receptor transcription factor that directly binds the HZA enhancer, thereby activating multiple zinc homeostasis genes in response to high dietary zinc. Loss-of-function mutants in hizr-1 fail to activate zinc homeostasis genes, and hizr-1 mutants are hypersensitive to high zinc toxicity. We hypothesized that HIZR-1 might also function as a cadmium receptor. Consistent with this model, we demonstrated that cadmium binds the ligand binding domain of HIZR-1 with comparable affinity to that of zinc. Using transgenic animals expressing a HIZR-1::GFP fusion protein, we showed that HIZR-1 accumulates in the intestinal nuclei following exposure to both zinc and cadmium but not a control metal, manganese. To characterize the global transcriptional response to cadmium and the role of hizr-1, we are currently identifying cadmium activated transcripts. We have identified multiple cadmium induced genes that are hizr-1 dependent as well as many that are hizr-1 independent. To determine the function of these genes, we are currently performing genetic studies of cadmium sensitivity in mutant animals. Surprisingly, hizr-1 mutants are not hypersensitive to cadmium toxicity, suggesting that targets of hizr-1 promote high zinc resistance but not cadmium resistance. Thus, cadmium may hijack the high zinc homeostasis response, and this response may not be protective in the face of cadmium toxicity.

Earley, B. et al. (2019) International Worm Meeting "Cadmium is an activating ligand of HIZR-1."

Lyon, M., Cubillas, C., Earley, B., Ahmad, R., Alcantar, A., Warnhoff, K., Kornfeld, K., Phillips, C.

[International Worm Meeting, 2019]

Exposure to the toxic metal cadmium is a global health problem that causes substantial morbidity and mortality in humans. The long-term goal of this research is to understand how organisms detect and respond to cadmium, and how these processes relate to the biology of the structurally similar but physiological metal zinc. The mechanism of cadmium toxicity is not well established, but a prominent theory is that cadmium binds proteins and other bio-molecules, displacing physiologic metals such as zinc, leading to dysfunction. When C. elegans are cultured in excess zinc conditions, zinc binds and activates the nuclear receptor transcription factor HIZR-1. HIZR-1 translocates to the nucleus and directly binds the High Zinc Activation (HZA) DNA enhancer upstream of multiple zinc homeostasis genes. Using transgene GFP promoter fusions, we showed that the HZA element is necessary and sufficient for cadmium-regulated transcription, and that hizr-1 is also necessary for cadmium-regulated transcription. We hypothesized that cadmium directly binds the ligand-binding domain of HIZR-1, leading to transcriptional activation. Consistent with this model, competitive binding assays revealed that HIZR-1 binds cadmium directly. Furthermore, HIZR-1::GFP accumulated in the nucleus upon cadmium exposure. We used multiple approaches to identify ~150 cadmium activated genes; about 30% were dependent on hizr-1 for activation, whereas others were hizr-1 independent. To determine the function of this hizr-1-medited transcriptional response, we analyzed hizr-1(lf) mutants. hizr-1(lf) by itself did not strongly affect cadmium tolerance, but in a sensitized background hizr-1(lf) caused hypersensitivity to cadmium toxicity. These results show that cadmium is an activating ligand of HIZR-1 rather than a destabilizing toxin. The HIZR-1-mediated transcriptional response is only weakly protective against cadmium, suggesting it evolved primarily to perform high zinc homeostasis rather than cadmium detoxification.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[International Worm Meeting, 2015]

Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.

Kiontke, Karin C. et al. (2009) International Worm Meeting "New Caenorhabditis species: phylogeny and evolution."

Fitch, David H. A., Felix, Marie-Anne, Kiontke, Karin C.

[International Worm Meeting, 2009]

Recently, nine new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to nineteen, eleven of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10 and 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Although none of them is the sister species of C. elegans, C. sp. 5 and C. sp. 9 are close relatives of C. briggsae. C. sp. 9 can hybridize with C. briggsae in the laboratory. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. Three of these species, C. sp. 7, C. sp. 9 and C. sp. 11 have been chosen for genome sequencing. Four further new species branch off before C. japonica within a monophyletic clade which also comprises C. sp. 3 and C. drosophilae. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and seven from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. Other characters, like the shape of the stoma and the male tail, introns, susceptibility to RNAi and genome size are being evaluated in the context of the phylogeny. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last few years. There is no indication that we are even close to knowing all species in this genus.