The Nuclear Receptor HIZR-1 Uses Zinc as a Ligand to Mediate Homeostasis in Response to High Zinc

<div><p>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 <i>Caenorhabditis elegans</i> 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.</p></div

The HZA enhancer directly bound the HIZR-1 DNA-binding domain.

<p><b>(A)</b> Infrared image of an electrophoretic mobility shift assay (EMSA). Labeled HZA enhancer DNA bound partially purified, full-length, wild-type (WT) HIZR-1 protein (arrowheads) but not mutant (G23E, S30L, or R63C) proteins. NPC, no protein control: EV, empty vector. The S30L lane contains a faint, rapidly migrating band of unknown significance. <b>(B)</b> Equivalent WT and mutant HIZR-1 protein amounts demonstrated by western blotting. <b>(C)</b> A constant amount of partially purified, full-length, wild-type HIZR-1 protein was incubated with variable concentrations of labeled, wild-type HZA DNA. Values are the average amount of labeled HZA DNA that displayed retarded migration +/- S.D. determined by image analysis and expressed in arbitrary units (A.U.). At least two technical replicates were performed for each HZA DNA concentration. Retarded migration indicates the DNA is bound to HIZR-1 protein. Nonlinear regression was used to calculate a dissociation constant of 20.4 +/- 6.8 nM. <b>(D)</b> A constant amount of partially purified, full-length, wild-type HIZR-1 protein was incubated with a constant amount of labeled wild-type HZA enhancer DNA. The interaction between the labeled DNA and HIZR-1 protein was competed with no unlabeled DNA (Lane 1) or increasing concentrations (5, 50, 500, or 5,000 nM) of unlabeled wild-type HZA DNA (Lanes 2–5) and unlabeled mutant HZA DNA (Lanes 6–9). The mutant HZA DNA had the identical flanking sequences but the order of the 15 base pair HZA was randomized. Lanes 3–5 display competition of binding, as evidenced by diminishment of the intensity of the retarded band (arrowhead). By contrast, lanes 7–9 do not display diminishment of the intensity of the retarded band, indicating that the mutant HZA DNA does not compete for binding to the HIZR-1 protein.</p

HIZR-1 regulates high zinc homeostasis in the <i>C</i>. <i>elegans</i> intestine.

<p><b>(A)</b> Genetic model. High levels of zinc promote HIZR-1 activity and transcriptional activation of multiple genes including <i>cdf-2</i>, <i>ttm-1b</i> and <i>hizr-1</i>. Increased levels of <i>cdf-2</i> and <i>ttm-1b</i> mRNA promote increased levels of CDF-2 and TTM-1B protein, which reduce levels of cytoplasmic zinc in a parallel negative feedback circuit. Increased levels of <i>hizr-1</i> mRNA promotes increased levels of HIZR-1 protein, creating a positive feedback circuit that enhances the negative feedback system. <b>(B)</b> Molecular model. Dietary zinc (Z) enters intestinal cells, binds the LBD of HIZR-1 and promotes nuclear accumulation, HZA enhancer binding, and transcriptional activation. The nuclear accumulation of HIZR-1 could result from increased HIZR-1 protein levels due to autoregulation and/or translocation of HIZR-1 from the cytoplasm to the nucleus. Increased levels of CDF-2 and TTM-1B promote zinc detoxification by sequestration in lysosome-related organelles [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000094#pbio.2000094.ref013" target="_blank">13</a>] and excretion into the intestinal lumen [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000094#pbio.2000094.ref014" target="_blank">14</a>], respectively. Increased levels of HIZR-1 promote homeostasis by a positive feedback circuit.</p

<i>hizr-1</i> was necessary and sufficient for high-zinc–activated transcription.

<p><b>(A–F)</b> mRNA was isolated from populations of wild-type (white), <i>am286</i> (red), or <i>am285</i> (green) animals cultured with 0 or 200 μM supplemental zinc. <i>cdf-2</i>, <i>ttm-1b</i>, and <i>mtl-1</i> transcript levels were analyzed by qPCR; mRNA levels are expressed in arbitrary units (A.U.) and were normalized to <i>rps-23</i>, a ribosomal protein gene that is not regulated by high zinc. For each panel, the values were normalized by setting the value for wild-type animals at 0 μM supplemental zinc equal to 1.0. Bars represent the average +/- S.D. (<i>n</i> = 3), (*, <i>p</i> < 0.05). <b>(A–C)</b> In wild-type animals, <i>cdf-2</i>, <i>ttm-1b</i>, and <i>mtl-1</i> transcript levels were increased significantly when cultured with 200 μM supplemental zinc compared to 0 μM supplemental zinc, demonstrating that these are high-zinc–activated transcripts. Compared to wild-type animals, <i>am286</i> mutant animals displayed significantly lower mRNA levels when cultured with 200 μM supplemental zinc, indicating a defect of high-zinc–activated transcription. <i>cdf-2</i>, <i>ttm-1b</i>, and <i>mtl-1</i> mRNA levels displayed a small but statistically significant reduction in <i>am286</i> mutant animals cultured on 0 μM supplemental zinc compared to wild type cultured on 0 μM supplemental zinc or <i>am286</i> mutant animals cultured on 200 μM supplemental zinc. <b>(D–F)</b> Compared to wild-type animals, <i>am285</i> mutant animals displayed significantly higher mRNA levels when cultured with 0μM supplemental zinc, indicating constitutive activation of high-zinc–activated transcription. <b>(G,H)</b> Animals were synchronized as embryos and cultured with the indicated concentrations of supplemental zinc or copper for 3 d. Data points represent average animal length +/- S.D. (<i>n</i> = 20) (*, <i>p</i> < 0.05).</p

The HZA enhancer mediated the transcriptional activity of HIZR-1 in animals.

<p><b>(A)</b> Diagrams of the basal <i>pes-10</i> promoter with and without three HZA enhancer elements (not to scale) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000094#pbio.2000094.ref015" target="_blank">15</a>]. <b>(B,C)</b> Representative images of anterior intestine of <i>hizr-1(am285gf)</i> transgenic animals containing <i>pes-10p</i>::<i>gfp-nls</i> (basal <i>pes-10</i>) or <i>3XHZApes-10p</i>::<i>gfp-nls</i> (basal <i>pes-10</i>+3X HZA); intestines are outlined, arrowheads indicate representative GFP-positive nuclei, and scale bars are approximately 25 μm. <b>(D)</b> <i>hizr-1(+)</i>, <i>hizr-1(am286lf)</i>, and <i>hizr-1(am285gf</i>) transgenic animals expressing <i>3XHZApes-10p</i>::<i>gfp-nls</i> were cultured with 0 or 200 μM supplemental zinc and assayed for GFP expression. n, number of animals assayed, *, <i>p</i> < 0.05 compared to <i>hizr-1(+)</i> animals cultured with 0 μM supplemental zinc (Chi-squared test).</p

Zinc directly bound the HIZR-1 ligand-binding domain.

<p><b>(A)</b> Glutathione S-transferase (GST) alone and the ligand-binding domain of HIZR-1 (residues 101–412) fused to GST were expressed in bacteria and partially purified by affinity chromatography. Increasing concentrations of either GST::HIZR-1(101–412 WT) or GST alone were incubated with a fixed concentration of radioactive zinc-65, and the amount of zinc-65 bound to protein was quantified by filter binding and scintillation counting. Values are the average +/- S.D. in counts per minute (CPM). At least two technical replicates were performed for each unique protein concentration. GST::HIZR-1(101–412 WT) displayed saturable binding, and a nonlinear regression was used to calculate a dissociation constant of 1.7 +/- 0.3 μM. X-ray crystallography studies indicate that GST binds one zinc molecule per protein [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000094#pbio.2000094.ref032" target="_blank">32</a>]; our data are consistent with saturable, low-level zinc binding by GST alone. <b>(B)</b> GST alone and the ligand-binding domain of DAF-12 isoform A (residues 440–753) fused to GST were expressed in bacteria, partially purified by affinity chromatography, and analyzed for zinc binding using the method described above. GST again displayed saturable, low-level zinc binding. GST::DAF-12(440–753 WT) LBD displayed binding similar to GST alone, indicating that the LBD of DAF-12 does not bind an appreciable amount of zinc. <b>(C)</b> Partially purified GST::HIZR-1(101–412 WT) was incubated with radioactive zinc-65 and no additional metal (none) or 500 μM nonradioactive zinc, copper, nickel, or manganese. Bars indicate the amount of zinc-65 bound to protein +/- S.D. (<i>n</i> = 3) quantified by filter binding and scintillation counting. The values were normalized by setting the value of the sample with no additional metal equal to 1.0, defined as maximal binding. Low values indicate the nonradioactive metal competes effectively with radioactive zinc-65. For GST::HIZR-1(101–412 WT), compared to nonradioactive zinc, nickel and manganese displayed significantly lower effectiveness as competitors (*, <i>p</i> < 0.05). The value for copper was not significantly different than the value for zinc (<i>p</i> = 0.13), indicating copper effectively competes with zinc for binding.</p

A forward genetic screen for mutations that affect zinc-activated transcription of the <i>cdf-2</i> promoter.

<p><b>(A)</b> A diagram (not to scale) of the <i>cdf-2p</i>::<i>gfp</i> transcriptional reporter construct containing the <i>cdf-2</i> promoter (black line, 1,371bp upstream of the ATG start codon) fused to the coding region of green fluorescence protein (green box). The <i>cdf-2</i> promoter contains a high zinc activation (HZA) enhancer element (orange box, 194 bp upstream of the ATG start codon) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000094#pbio.2000094.ref015" target="_blank">15</a>]. This construct was integrated into the genome of <i>C</i>. <i>elegans</i> to generate transgenic strain WU1391 that functions as a high zinc reporter strain. <b>(B)</b> WU1391 transgenic animals containing the <i>cdf-2p</i>::<i>gfp</i> transcriptional reporter were cultured with standard Noble agar minimal medium (NAMM) (0 μM Supplemental Zinc) or NAMM supplemented with 200 μM supplemental zinc. GFP fluorescence was observed with a compound microscope, and representative images are shown. Animals were oriented with the long axis horizontally—the intestine is a prominent tubular structure spanning the length of the animal, and it is outlined with a dotted white line. Animals cultured in medium with no supplemental zinc displayed low-level fluorescence that is a combination of autofluorescence due to gut granules and low level expression of the <i>cdf-2</i> promoter. By contrast, intestinal fluorescence is prominent in animals cultured in zinc-supplemented medium. Scale bars are approximately 100 μm. <b>(C)</b> A flow chart of the forward genetic screen. WU1391 transgenic hermaphrodites (P₀) were mutagenized by ethyl methanesulfonate (EMS) and allowed to self-fertilize for two generations. F₂ self progeny that are homozygous for newly induced mutations were analyzed. <u><i>Z</i></u>inc-<u><i>a</i></u>ctivated <u><i>t</i></u>ranscription-<u><i>c</i></u>onstitutive (Zat-c) mutants were isolated by screening for animals that displayed fluorescence when cultured on medium with no supplemental zinc. <u><i>Z</i></u>inc-<u><i>a</i></u>ctivated <u><i>t</i></u>ranscription-<u><i>d</i></u>eficient (Zat-d) mutants were isolated by screening for animals that did not display fluorescence when cultured on medium with 200 μM supplemental zinc. <b>(D)</b> WU1391, the Zat-d mutant strains (<i>am279</i>, <i>am280</i>, <i>am286</i>, <i>am287</i>, and <i>am288</i>) and the Zat-c mutant strain (<i>am285</i>) were cultured on medium with 0 or 200 μM supplemental zinc. Fluorescence intensity was quantified using microscopy and is expressed in arbitrary units (A.U.). Bars represent the average fluorescence intensity +/- standard deviation (S.D.) (<i>n</i> = 12–18 animals). Compared to the unmutagenized WU1391 starting strain, all five Zat-d mutant strains displayed significantly reduced fluorescence intensity when cultured with 200 μM supplemental zinc; the one Zat-c mutant strain displayed significantly increased fluorescence intensity when cultured with 0 μM supplemental zinc (*, <i>p</i> < 0.05). The Zat-c mutant strain cultured on 200 μM supplemental zinc displayed significantly increased levels of GFP fluorescence compared to the Zat-c mutant strain cultured on 0 μM supplemental zinc and WU1391 animals cultured on 200 μM supplemental zinc. Thus, the Zat-c mutant strain retains zinc-activated transcription while displaying higher baseline expression levels compared to wild type. <b>(E)</b> Upper, physical map of a portion of <i>C</i>. <i>elegans</i> linkage group X (LGX) with loci positions in base pairs (k = thousand). Zat-d and Zat-c mutations were positioned between <i>egl-15</i> and <i>sma-5</i>. Lower, <i>hizr-1</i> locus (not to scale). Open boxes are exons, shaded regions are untranslated, orange box is the HZA enhancer, black bars locate the DNA-binding (DBD) and ligand-binding (LBD) domains, and lines locate mutations.</p

Zinc promoted nuclear accumulation of HIZR-1 in animals.

<p><i>hizr-1(am286lf)</i> animals expressing full-length HIZR-1(1–412 WT)::GFP or HIZR-1(1–412 D270N GF)::GFP were cultured with no supplemental metals or supplemental zinc or copper. <b>(A–C)</b> Representative images show midbody region; the intestines are outlined, arrowheads indicate representative GFP-positive nuclei, and scale bars are approximately 10 μm. The correspondence of GFP fluorescence and intestinal cell nuclei was established by alternating between using differential interference contrast microscopy to visualize and identify nuclei based on morphology and location and fluorescence microscopy to visualize GFP. The punctate fluorescence in panel A appears to be autofluorescence derived from the gut granules, lysosome-related organelles that accumulate fluorescent material, and is not likely to result from HIZR-1(1–412 WT)::GFP expression. <b>(D)</b> Values are number of GFP-positive alimentary nuclei per animal +/- S.D. (<i>n</i> = 10–20) (*, <i>p</i> < 0.05).</p