Sperm activation is a fascinating example of cell differentiation, in which immotile spermatids undergo a rapid and dramatic transition to become mature, motile sperm. Because the sperm nucleus is transcriptionally silent, this transition does not involve transcriptional changes. Although Caenorhabditis elegans is a leading model for studies of sperm activation, the mechanisms by which signaling pathways induce this transformation remain poorly characterized. Here we show that a conserved transmembrane zinc transporter, ZIPT-7.1, regulates the induction of sperm activation in Caenorhabditis nematodes. The zipt-7.1 mutant hermaphrodites cannot self-fertilize, and males reproduce poorly, because mutant spermatids are defective in responding to activating signals. The zipt-7.1 gene is expressed in the germ line and functions in germ cells to promote sperm activation. When expressed in mammalian cells, ZIPT-7.1 mediates zinc transport with high specificity and is predominantly located on internal membranes. Finally, genetic epistasis places zipt-7.1 at the end of the spe-8 sperm activation pathway, and ZIPT-7.1 binds SPE-4, a presenilin that regulates sperm activation. Based on these results, we propose a new model for sperm activation. In spermatids, inactive ZIPT-7.1 is localized to the membranous organelles, which contain higher levels of zinc than the cytoplasm. When sperm activation is triggered, ZIPT-7.1 activity increases, releasing zinc from internal stores. The resulting increase in cytoplasmic zinc promotes the phenotypic changes characteristic of activation. Thus, zinc signaling is a key step in the signal transduction process that mediates sperm activation, and we have identified a zinc transporter that is central to this activation process. Sperm are specialized cells with transcriptionally silent DNA that has been packaged for delivery into the egg. In their final step of development, immature sperm undergo a rapid transition from nonmotile cells to mature, motile sperm capable of fertilization. The signals that trigger this change are not clearly understood. By identifying mutants in the roundworm Caenorhabditis elegans that are defective in sperm activation, we discovered a conserved transmembrane protein, ZIPT-7.1, that transports zinc and promotes sperm activation in both sexes. ZIPT-7.1 is expressed in the germ line and functions there to control sperm activation. When expressed ectopically in mammalian cells, the protein specifically transports zinc across membranes and localizes primarily to membranes within the cell. Previous genetic studies had identified two pathways that mediate sperm activation in C. elegans, and our results suggest that zipt-7.1 acts at the end of one of these two, the spe-8 pathway. We propose that when this pathway triggers sperm activation, it acts through ZIPT-7.1, which mediates the release of zinc from internal stores in the immature sperm. This released zinc functions as a second messenger to promote the differentiation of mature, motile sperm.

The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance. What are the mechanisms used by animals to cope with stressful environments that inflict damage or restrict essential processes such as growth, development, and reproduction? One strategy is changes in physiology that increase stress resistance, and an extreme version of this strategy is diapause, an alternative developmental state that is enduring and stress resistant. In the nematode C. elegans, stress tolerance and entry into a diapause state called dauer larvae are mediated by the conserved insulin/IGF-1 pathway. Specifically, the FOXO transcription factor DAF-16 promotes stress tolerance and dauer larvae development. However, the targets of DAF-16 that mediate these processes remain largely elusive. Using an unbiased forward genetic screen to discover new mediators of stress tolerance, we identified natc-1, a novel target of DAF-16 and the insulin/IGF-1 pathway. natc-1 encodes a conserved subunit of the N-terminal acetyltransferase C (NAT) complex. The NatC complex modifies target proteins by acetylating the N-terminus. We demonstrated that natc-1 mediates diapause entry and stress tolerance. Furthermore, we elucidated regulation of NatC by demonstrating that natc-1 is a direct transcriptional target that is repressed by DAF-16. These findings may be relevant to other animals because both the insulin/IGF-1 signaling pathway and the NAT system are conserved during evolution.

Significance: Zinc is essential for animal life, and zinc levels are carefully regulated by homeostatic mechanisms. In the model organism Caenorhabditis elegans , the HIZR-1 nuclear receptor directly binds zinc and mediates high zinc homeostasis by regulating transcription. Cadmium is structurally similar to zinc but is a prevalent environmental toxin. Here, we explore how animals respond to these similar metals. The results suggest that cadmium hijacks the high zinc homeostasis response by directly binding and activating HIZR-1. This is an example of cadmium functioning as an activating ligand for a zinc-regulated protein in contrast to the prevailing model that cadmium binding causes protein dysfunction. These results elucidate the interplay between these two metals and reveal an unexpected mechanism of cadmium transcriptional control.

Zinc is essential for biological systems, and aberrant zinc metabolism is implicated in a broad range of 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 fluctuating levels of zinc have not been fully defined. Here, we identified 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 evolutionarily conserved enhancer element present in multiple zinc-inducible genes, the high zinc activation (HZA) element. The HZA was consistently adjacent to a GATA element that mediates expression in intestinal cells. Functional studies using transgenic animals demonstrated that this modular system of DNA enhancers mediates tissue-specific transcriptional activation in response to high levels of dietary zinc. We used this information to search the genome and successfully identified novel zinc-inducible genes. 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. These findings define new mechanisms of zinc homeostasis and tissue-specific regulation of transcription.

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. Here we describe a novel Caenorhabditis elegans nuclear receptor, HIZR-1, that is a high zinc sensor in an animal and the master regulator of high zinc homeostasis. The essential micronutrient zinc acts as a HIZR-1 ligand, and activated HIZR-1 increases transcription of genes that promote zinc efflux and storage. The results identify zinc as the first inorganic molecule to function as a physiological ligand for a nuclear receptor and direct environmental sensing as a novel function of nuclear receptors. Zinc is an essential nutrient for all life forms, and maintaining zinc homeostasis is critical for survival. However, little is known about how animals sense changes in zinc availability and make adjustments to maintain homeostasis. In particular, logic dictates there must be a mechanism for zinc sensing, but it has not been defined in animals. We discovered that the nuclear receptor transcription factor HIZR-1 is the master regulator of high zinc homeostasis in the roundworm Caenorhabditis elegans. In response to high dietary zinc, HIZR-1 activates transcription of multiple genes that encode a network of proteins that store and detoxify excess zinc. Furthermore, our results suggest HIZR-1 itself is the high zinc sensor, since it directly binds zinc ions in the ligand-binding domain that regulates transcriptional activation. These findings advance the understanding of zinc homeostasis and nuclear receptor biology. Nuclear receptors were initially characterized as receptors for hormones such as estrogen, indicating complex animals use these transcription factors to monitor their internal environment. However, nuclear receptors are present in simple organisms that lack endocrine signaling, suggesting these transcription factors might have a primordial function in sensing the external environment. Our results identify a new class of nuclear receptor ligands, the inorganic ion zinc, and a new function for nuclear receptors in directly sensing levels of a nutrient. We speculate that nutrient homeostasis mediated by direct binding of nutrients to the ligand-binding domain is a primordial function of nuclear receptors, whereas endocrine signaling in complex animals mediated by direct binding of hormones to the ligand-binding domain is a derived function of nuclear receptors that appeared later in evolution.