Dietary zinc modulates expression in murine thymus: Results from a comprehensive differential display screening

J. Bernadette Moore*, Raymond K. Blanchard, and Robert J. Cousins†

Nutritional Genomics Laboratory, Food Science and Human Nutrition Department and Center for Nutritional Sciences, University of Florida, Gainesville, FL 32611-0370

Contributed by Robert J. Cousins, February 3, 2003 Differential mRNA display was used to comprehensively screen the profiling in earlier studies to screen for zinc-modulated in murine thymic transcriptome for genes modulated in vivo by the rat small intestine (9), demonstrating that this technology dietary zinc. A moderate feeding protocol rendered young adult, provides the sensitivity to identify physiologically relevant genes. outbred mice zinc-deficient and zinc-supplemented without alter- For example, one intestinal cDNA sequence derived from DD ations in feeding behavior or growth. However, these levels of identified the up-regulation of the preprouroguanylin gene in deficiency and supplementation altered specific mRNA abundances zinc deficiency. The active peptide produced from pre- in a manner detectable by differential display. In total, 240 primer- prouroguanylin is uroguanylin, a natriuretic hormone involved in pair combinations were used to generate >48,000 interpretable intestinal fluid secretion, which, when overproduced, could lead cDNA bands derived from thymic total RNA, of which only 265 or to diarrhea. Uroguanylin up-regulation may be related to the 0.55% were identified as zinc-modulated under these moderate zinc-responsive diarrheal disease observed in many parts of the dietary conditions. The most strongly zinc-modulated cDNAs iden- world. The sequence information from DD allowed subsequent tified by display were reamplified and sequenced. No cDNAs cloning of the rat preprouroguanylin gene and further functional encoding zinc-metalloenzymes or zinc-finger transcription factors studies at the level (10–12). were identified as zinc-modulated in this global screening. Those zinc-regulated genes independently confirmed by quantitative PCR The DD approach for transcription profiling allowed us to included: heat shock 40 and 60; heat shock cognate 70; conduct a comprehensive survey of the mouse thymus transcrip- histocompatibility 2, class II antigen A, ␣; and the T cell cytokine tome. An advantage to DD is its capacity to find novel EST data, receptor. In addition, a variety of transcription- and translation- both contributing to sequence databases and holding the poten- related factors (such as ribosomal proteins L3, L5, and L28; nuclear tial for gene discovery (13). Even with the recent completion of matrix protein 84; matrin cyclophilin; the H3 histone family 3A the initial draft of the mouse genome (14) and the tremendous protein; ␤2 microglobulin; and a cleavage and polyadenylation throughput advantages of array analysis, arrays are completely factor) were identified as zinc-modulated. These profiling data dependent on a priori sequence information, and thus DD best show that differential expression of genes in the thymus in served our criterion for a genome-wide survey, which was not response to the dietary zinc supply precedes many of the pheno- obtainable through available murine arrays. typic effects on thymic function associated with severe zinc re- The profiling presented in this report represents differential striction or supplementation. Several genes involved in T cell mRNA display analyses generated from 240 primer-pair com- development were identified as regulated by zinc and will be binations. Approximately 48,000 cDNA bands were separated by targets to evaluate the effects of zinc on immune function. denaturing polyacrylamide gels. Given an estimated 15,000 genes actively transcribed in any one cell type, the data, statis- nutrition ͉ genomics ͉ functional genomics ͉ immunology tically (15), represent a comprehensive screen of the mouse thymic transcriptome during both deprivation and supplemen- inc is an essential micronutrient required for vertebrate tation of dietary zinc. Zgrowth and development and severe zinc deficiency produces pleiotropic effects impacting multiple physiological systems. In Materials and Methods particular, the immune system is sensitive to zinc deficiency, Feeding Studies. Young-adult (30 Ϯ 3 g) male CD-1 mice (Charles which can result in thymic atrophy, lymphopenia, and increased River Breeding Laboratories) were housed and fed as described susceptibility to infectious disease (1, 2). In humans, zinc sup- (16). Treatment groups were provided one of three dietary zinc plementation has been shown to reduce the morbidity of diar- levels [ZnϪ (Ͻ1mg͞kg), zinc-normal (ZnN; 30 mg͞kg), or Znϩ rhea (3), respiratory disease (4, 5), nematode infection (6), and (180 mg͞kg)], and animals (n ϭ 5–10 per treatment) were fed the sickle cell disease (7). The potential existence of immune diet for 3 weeks. Blood was collected by cardiac puncture for dysfunction in individuals with a mild zinc deprivation and measurement of the serum zinc concentration. The protocol was possible immune-enhancing benefits of supplemental zinc intake approved by the University of Florida Institutional Animal Care are questions that lack definitive data and warrant future and Use Committee. Physiological values are reported as the Ϯ research (8). In addition, convincing explanations for the mo- mean SEM and were compared by one-way ANOVA. SCIENCES lecular basis for symptoms associated with deficient or excess

consumption of zinc have not been forthcoming. We have APPLIED BIOLOGICAL addressed those questions here through use of the differential Abbreviations: AP, anchored primer; ARP, arbitrary primer; DD, differential display; H2-A␣, display (DD) technique for profiling of genes expressed in the histocompatibility 2, class II antigen A, ␣; Hsc, heat shock cognate protein; Hsp, heat shock Ϫ ϩ protein; MT, metallothionein; Q-PCR, quantitative real-time PCR; TCCR, T cell cytokine thymus of zinc-deficient (Zn ) and zinc-supplemented (Zn ) receptor; ZnϪ, zinc-deficient; ZnN, zinc-normal; Znϩ, zinc-supplemented. mice. *Present address: Food and Nutrition Board, Institute of Medicine, 500 5th Street NW, The rationale was that the differing biochemical roles for zinc Washington, DC 20001. should provide a stimulus for a characteristic, altered gene †To whom correspondence should be addressed at: Food Science and Human Nutrition expression pattern when the normal dietary supply is either Department, University of Florida, 201 FSHN Building, P.O. Box 110370, Gainesville, withdrawn or given in excess. We have successfully applied DD FL 32611-0370. E-mail: cousins@ufl.edu.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0330670100 PNAS ͉ April 1, 2003 ͉ vol. 100 ͉ no. 7 ͉ 3883–3888 Downloaded by guest on September 29, 2021 RNA Isolation and Differential Display Reactions. Whole thymus ing at the University of Florida’s Sequencing Core. The Gen- (Ϸ250 mg) was excised and homogenized in TriPure Isolation Bank databases (18) were queried by using BLAST (19), run from Reagent (Roche Diagnostics), and total RNA was isolated SEQWEB V.2.02 of the WISCONSIN PACKAGE (Accelrys), to evaluate according to the manufacturer’s instructions. RNA concentra- sequence information. tions were determined spectrophotometrically, and integrity was verified by agarose electrophoresis and ethidium bromide stain- Independent Confirmation by Quantitative PCR Analysis. Quantita- ing. Equal amounts of RNA were pooled from mice (n ϭ 7) tive real-time PCR (Q-PCR) primers and TaqMan probes were within treatment groups and the pooled samples were DNase designed by using PRIMER EXPRESS V.2.0 (Applied Biosystems). treated by using the DNA-free kit (Ambion, Austin, TX). The 18S rRNA assay, used for total RNA normalization, and all For these experiments, the entire set of HIEROGLYPH one-step RT-PCR reagents were purchased from Applied Bio- mRNA profile kits (Beckman Coulter) was used for DD reverse systems, and all assays were performed on a GeneAmp 5700 transcription and PCR according to the manufacturer’s proto- Sequence Detection System (Applied Biosystems). Relative Ј cols. In total, this includes 12 anchored 3 primers (AP1–12) and quantitation was determined from 4 log10-range standard curves 20 arbitrary 5Ј primers (ARP1–20) of defined nucleotide se- with pooled samples run in triplicate. All assays except 18S quence, which together are predicted to comprehensively screen rRNA used 900 nM each of the forward and reverse primers and an entire mammalian transcriptome. Specifically, the reaction 250 nM of probe. Primers and probe used (forward, reverse, and conditions used pooled, DNase-treated RNA (Ϸ0.1 ␮g͞␮l) from probe, respectively) were: T cell cytokine receptor [TCCR, each treatment group, reverse transcribed by using 2 ␮Mofa GenBank accession no. NM࿝016671; 5Ј-GGGAGCCCAGG- single specific AP and 2 units͞␮l SuperScript II RNase HϪ GATAAAGG-3Ј,5Ј-TGAGCCCAGTCCACCACATAC-3Ј, reverse transcriptase (Invitrogen). Subsequent 20-␮l PCR am- and 5Ј-FAM-CAATGGTTTCCTGGTCCCTTGTTTCCA- plification reactions were done in triplicate for each sample by BHQ1-3Ј]; the heat shock protein 40 [Hsp40, accession no. using 2 ␮l of first-strand products for template and 0.05 units͞␮l NM࿝008298; 5Ј-AATGGAGAAGCGTATGAGGATGA-3Ј, AmpliTaq DNA polymerase (Roche Diagnostics) with supplied 5Ј-ACTGGCCCATTAAGAGGTCTGA-3Ј, and 5Ј-FAM- buffer; PCR reactions had final concentrations of 0.2 ␮M for CACCCCAGAGGTGGCGTTCA-BHQ1-3Ј]; the heat shock each AP and ARP, 20 ␮M for each of the dNTPs, and 0.125 protein 60 [Hsp60, accession no. X53584; 5Ј-TTGCCCTT- ␮Ci͞␮l[␣-33P]dATP (DuPont͞NEN; 1 Ci ϭ 37 Bq). Cycling ATCAATGAACTGTGA-3Ј,5Ј-TCAGTCATTTTCTCCAG- parameters were: 95°C for 2 min; 4 cycles of 92°C for 15 sec, 50°C GTGACTTC-3Ј, and 5Ј-FAM-CTCAAGGCAGGTTCCT- for 30 sec, and 72°C for 2 min; 25 cycles of 92°C for 15 sec, 60°C CACCAATAACTTCAG-BHQ1-3Ј]; the heat shock cognate for 30 sec, and 72°C for 2 min; and 72°C for 7 min. protein 70 [Hsc70, accession no. BC006722; 5Ј-GCTGC- CGGGCATTCG-3Ј,5Ј-CCTTAGACATGGTTGCTTGTGT- Denaturing PAGE. After addition of a denaturing loading dye (95% GTAG-3Ј, and 5Ј-FAM-TGGTCTCGTCGTCAGCGCAGCT- formamide͞0.05% bromophenol blue͞0.05% xylene cyanol) and BHQ1-3Ј], and the histocompatibility 2, class II antigen A, ␣ a 2-min, 95°C heat step, PCR products were electrophoresed [H2-A␣, accession no. BC019721; 5Ј-GGCCTTGTGGG- under two distinct conditions by using a Genomyx LR DNA CATCGT-3Ј,5Ј-TCTGGAGGTGCCACCTGATC-3Ј, and 5Ј- sequencer (Beckman Coulter). To resolve longer cDNAs, DD FAM-TGGGCACCATCTTCATCATTCAAGGC-BHQ1-3Ј] reaction products were separated for 16 h through a 340-␮m- (BioSource International, Camarillo, CA). The metallothionein thick, 4.5% acrylamide gel matrix containing urea (Beckman (MT) primers and probe have been described (16). Coulter). For resolution of shorter cDNAs, a 6% gel matrix was used for 2.5 h. After rinsing and drying, the DD gel was exposed Results to Kodak Biomax MR film for display visualization. Animals. The moderate nature of the 3-week feeding protocol, consistent with earlier studies (16), was underscored by the lack of Excision, Reamplification, and Identification of DD Products. Selected significant differences in terminal body and thymus weights be- bands were circumscribed with a scalpel, rehydrated with Ͻ1 ␮l tween dietary treatment groups (data not shown). Serum zinc ␮ Ϫ Ϯ ␮ Ͻ of deionized H2O, excised, and placed in 100 lofTE(10mM concentrations in Zn mice (5.0 0.3 M) were significantly (P Tris͞1 mM EDTA) or 1ϫ PCR buffer (10 mM Tris⅐HCl͞1.5 mM 0.0001) lower than those of the ZnN (16.1 Ϯ 0.4 ␮M) and Znϩ ͞ Ϯ ␮ MgCl2 50 mM KCl) in TE, in light of data from Frost and (15.1 0.7 M) mice. Further demonstrating lower zinc status of Guggenheim (17). Reamplification reactions used the universal, the ZnϪ mice was their thymic MT mRNA levels, which were 66% full-length M13(-48) (5Ј-AGCGGATAACAATTTCACA- those of the ZnN or Znϩ mice, as measured by Q-PCR. CAGGA-3Ј) and T7 promoter (5Ј-GCCCTATAGTGAGTCG- TATTAC-3Ј) primers (GIBCO͞BRL), which facilitated the Differential Display. All 12 APs, in combination with all 20 ARPs, direct sequencing of reamplified cDNAs from the M13(-48) were used to generate differential mRNA displays of thymic primer. Reactions were run with 2 ␮l of gel band eluate (in a 40 transcripts in ZnϪ, ZnN, and Znϩ mice. The 240 primer-pair ␮l final volume), 2ϫ PCR buffer (Roche Molecular Biochemi- combinations produced Ϸ48,000 interpretable bands on dena- cals), 20 ␮M each dNTPs, 0.2 ␮M each M13 and T7 primers, and turing polyacrylamide gels. Typically, triplicate reactions from 0.05 units͞␮l AmpliTaq polymerase (Roche Diagnostics). Cy- six to seven AP and ARP pair combinations were run on each cling parameters were 95°C for 2 min; 4 cycles of: 92°C for 15 sec, gel simultaneously and, in total, 86 DD gels were generated to 50°C for 30 sec, 72°C for 2 min; 25 cycles of: 92°C for 15 sec, 60°C complete the screening of all primer-pair combinations. Pre- for 30 sec, 72°C for 2 min; and 72°C for 7 min. suming an estimated 15,000 actively transcribed genes in any one To assess quality of the reactions, 2 ␮l of reamplification cell type, and considering the statistical requirements to repre- product was electrophoresed in a 1.5% agarose, 1ϫ TBE gel (0.9 sent each mRNA transcript by at least one cDNA on a gel from M Tris͞0.9 M borate͞0.02 M EDTA), stained with SYBR Green a single primer pair (15), this represents a complete screen of the I (Molecular Probes), and scanned on a Storm PhosphorImager thymic transcriptome under each of these dietary conditions. (Molecular Dynamics). For obtaining sufficiently concentrated Of the Ϸ48,000 bands surveyed, 265 bands were observed to PCR product for sequencing, reactions were then repeated be differentially regulated by zinc treatment and were excised for under identical conditions in nine 40-␮l reactions (using DNA in further investigation. Criteria for defining a band as zinc- the original gel band eluate for template). Reaction products modulated were pronounced differences between treatment were purified and concentrated by using QIAquick PCR puri- groups, consistency among triplicate reactions, overall band fication columns (Qiagen, Valencia, CA) and sent for sequenc- intensity, and a size of Ͼ200 nt. After summarizing these criteria,

3884 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0330670100 Moore et al. Downloaded by guest on September 29, 2021 Table 1. Overall results of differential displays appearing decreased in Znϩ mice. An additional 40 bands Bands Number % appeared to be modulated by both high and low dietary zinc intake, although only one of these demonstrated modulation Interpretable bands surveyed Ϸ48,000 consistent with a zinc dose–response across all three conditions, Excised differentially displayed bands 265 0.55 (of surveyed) i.e., appearing decreased in ZnϪ and increased in Znϩ mice. Ϫ Increased in Zn 90 34 To demonstrate the reproducibility of DD, reaction products Ϫ Decreased in Zn 58 22 from AP3 and ARP2 and -3 were generated in two subsequent ϩ Increased in Zn 36 13.5 months and displayed. Profiles were virtually identical in banding Decreased in Znϩ 41 15.5 patterns (see Figs. 3 and 4, which are published as supporting Modulated in both ZnϪ and Znϩ 40 15 information on the PNAS web site, www.pnas.org), demonstrat- ing gross reproduction of DD RT-PCR reactions. Furthermore, when sequenced independently, five differential bands (two differentially expressed bands were ranked as 1–4 to prioritize from the Month 1 Gel and two from the Month 2 Gel, hypoth- the order of reamplification reactions. In this subjective assess- esized to be the same, and a close lower band) were all identified ment, bands 1 (73) were the most intense, demonstrating ex- as the same cDNA (Fig. 4). This cDNA coded for mitochondrial treme differentiation between treatment groups (‘‘on’’ vs. ‘‘off’’ NADH dehydrogenase subunit 2 (NADH:ubiquinone oxi- signals); bands 2 (67) were intense with modest differentiation; doreductase; mt-Nd2), and was found overexpressed in Znϩ bands 3 (52) were less intense, but showed extreme differenti- mice relative to their ZnϪ and ZnN counterparts. These cDNA ation between treatment groups; and bands 4 (73) were less bands are examples of those ranked as 1 by our criteria. intense, with modest treatment differentiation. Of the 265 excised differential cDNA bands (Table 1), 90 appeared in- Characterization of Zinc-Modulated Bands. Optimization generally creased in ZnϪ mice and another 58 appeared decreased in ZnϪ required for high-efficiency amplification of any one DNA mice. Fewer cDNA bands appeared modulated by dietary zinc template by PCR was not practical with the number of DD bands supplementation, with 36 bands appearing increased and 41 excised in these experiments. Our strategy then, beginning with

Table 2. DD transcripts modulated in Zn؊ mice Name Band* No. of nt† Identity, %‡ GenBank accession no.

Increased in ZnϪ mice T cell cytokine receptor (TCCR), mRNA 2,17,1 373 99 NM࿝016671 Similar to Tho2 (LOC243171), mRNA 3,1,1 247 97 XM࿝144450 Clone RP24–252G15, complete sequence§ 3,8,1G 391 99 AC122451 Hypothetical protein MGC28284, mRNA 4,19,1 326 97 NM࿝153552 Similar to nuclear matrix protein 84, mRNA 91 BC024951 Clone IMAGE:1514385, mRNA 5,10,2 690 96 BC031349 GDP dissociation inhibitor 3 5,18,6 755 100 BC024971 AK079690 inhibitory protein 5 (Api5), mRNA 7,11,1 469 99 XM࿝123850 96 NM࿝007466 Rat Smhs2 protein, mRNA 8,6,3 502 99 NM࿝134396 Ribosomal protein L5 (Rpl5), mRNA 8,13,2 758 93 XM࿝132197 Clone MGC:46985 IMAGE:5004588, mRNA 9,4,1 269 93 BC037639 Archain 1 (Arcn1), mRNA 11,18,2 720 98 NM࿝145985 Decreased in ZnϪ mice Hsc70, mRNA 2,4,2 718 100 BC006722 Similar to RIKEN cDNA 9530053H05 gene, clone 2,7,1 642 99 BC017513 IMAGE:4488629, mRNA cDNA clone K0707H07-3Ј, mRNA (dbEST) 100 BM244188 cDNA clone K0706D12-3Ј, mRNA (dbEST) 2,14,1 291 100 BM244095 cDNA clone K0700A06-3Ј, mRNA (dbEST) 100 BM243664 Retrotransposon L1Md-A101 pORF2, mRNA and L1Md-A2 3,1,4 500 98 AY053456 repetitive element ORF2, mRNA 98 M13002 DnaJ (Hsp40) homolog, subfamily A, member 1 (DnaJa1), mRNA 3,2,4 477 99 NM࿝008298 Ribosomal protein L28 3,8,2C 320 99 NM࿝009081 H2-A␣, mRNA 4,18,3 594 97 NM࿝010378 9,6,1 729 98 cDNA clone IMAGE:596239-5Ј, mRNA (dbEST) 5,4,3 624 AA138077 SCIENCES Rearranged immunoglobulin ␬ light chain 5,14,1 733 99 X67211 APPLIED BIOLOGICAL cDNA clone K0285F05 (dbEST) 6,15,2 401 94 BM229829 Axonemal dynein heavy chain 8, short form (Dnahc8) 7,6,2G 316 99 AF356521 Hsp60 kDa 10,7,2D 392 99 XM࿝109908 Riken clone A630014C11, 3-day neonate thymus cDNA 11,2,1 344 96 AK041481

*Designation of clone: AP, ARP, band cut, and, if a letter is present, subclone. †Number of nucleotides returned from a single sequencing run of reamplification PCR. ‡Percentage identities of longest homology match returned from BLAST. §Italics indicate cDNAs demonstrating differential expression in alternate zinc treatment.

Moore et al. PNAS ͉ April 1, 2003 ͉ vol. 100 ͉ no. 7 ͉ 3885 Downloaded by guest on September 29, 2021 Table 3. DD transcripts modulated in Zn؉ mice Name Band* No. of nt Identity, % GenBank accession no.

Increased in Znϩ mice RIKEN cDNA 2700023B17 gene, mRNA 2,7,3 468 96 NM࿝025948 Mitochondrial NADH dh subunit 2 3,3,1 677 99 NC࿝001569 Clone RP24–252G15† 3,8,1G 391 99 AC122451 Clone IMAGE:1514385, mRNA 5,10,2 690 96 BC031349 GDP dissociation inhibitor 3 5,18,6 755 100 AK079690 Rat Smhs2 protein, mRNA 8,6,3 502 99 NM࿝134396 Putative repetitive element 9,8,1A 150 94 Unknown Long interspersed L1 repeat 10,8,1B 329 94 X03725 Archain 1 (Arcn1), mRNA 11,18,2 720 98 NM࿝145985 Decreased in Znϩ mice 16S ribosomal RNA (mitochondrial) 1,6,1 234 98 V00665 Similar to matrin cyclophilin (matrin-cyp) 2,8,1 96 90 XM࿝130275 Hypothetical gene supported by BC010584 mRNA XM࿝129835 and and similar to putative protein kinase (LOC193982) 3,7,1 585 99 XM࿝110350 Ribosomal protein L3 (Rpl3), mRNA 3,7,2 157 100 NM࿝013762 Ribosomal protein L28 (Rpl28), mRNA 3,8,2C 320 99 NM࿝009081 ␤-2 microglobulin (B2m), mRNA 4,2,3 746 97 NM࿝009735 Phospholipase C, gamma 1 (Plcg1), mRNA 5,4,5 545 100 XM࿝130636 Rearranged immunoglobulin ␬ light chain 5,14,1 733 99 X67211 Axonemal dynein heavy chain 8 short form (Dnahc8), mRNA 7,6,2G 316 99 AF356521 Cleavage and polyadenylation factor 5, 25-kDa subunit 7,13,1 770 99 NM࿝026623 (Cpsf5), mRNA H3 histone, family 3A (H3f3a), mRNA 7,20,1 787 98 XM࿝147791 Ki-67 cell proliferation antigen 11,9,3 276 96 X82786

*Designation of clone: AP, ARP, band cut, and, if a letter is present, subclone. †Italics designate cDNAs demonstrating differential expression in alternate zinc treatment.

DD bands prioritized as 1, involved an initial assessment of tion of regulation using Q-PCR (Fig. 1). In all cases, the direction template capacity for reamplification under standard conditions, of zinc modulation observed on the DDs was reproduced. as well as the number of product bands produced by amplifica- Interestingly, for all three heat shock proteins, depression of tion reaction. Robust reactions that produced a single PCR expression was seen in both ZnϪ and Znϩ mice (Fig. 1), ϩ product were pursued for sequencing. Those cDNAs, which had although the depression seen in Zn mice was not at the Ϫ a 1 priority and that were successfully reamplified, sequenced magnitude observed in Zn mice. and identified by BLAST, are listed in Tables 2 (modulated by Discussion ZnϪ treatment) and 3 (modulated by Znϩ treatment). Single- pass DNA sequencing generally yielded between 100 and 750 The data presented here represent a view of differential gene bases of high-quality sequence from each cDNA (Tables 2 and expression in the thymus as the result of alterations in dietary 3, third column); however, a few produced a low-quality se- quence and were dropped from further analysis. Sequences were compared by BLAST against the GenBank and dbEST databases with the DD bands’ identity established as the highest-scoring, annotated cDNA or EST sequence, or multiple sequences if there was more than one perfect match. The homology of the longest contiguous match reported by BLAST is given in Tables 2 and 3 and does not necessarily reflect the homology to the entire DD sequence or the level of homology over multiple discontin- uous regions. All matches are to mouse sequences unless oth- erwise noted, and the high degree of matching sequence between the DD sequences and mouse cDNAs and ESTs supports the specificity to which DD PCR can identify individual regulated transcripts. Sequences from two DD bands appears to encode repetitive elements because they produced many genomic se- quence matches that do not correlate with any gene families. The first (Table 2, band 3,1,4) appears to be a retrotransposon related sequence (GenBank accession no. AY053456) that is residual in some mRNA untranslated regions, whereas the second (band 9,8,1A) is currently uncharacterized in the literature. Fig. 1. Q-PCR analyses of select DD clones and metallothionein (MT). Assays were performed on triplicate pooled total RNA samples (n ϭ 7 per group) from Confirmation of Select DD Clones. Five DD cDNAs were chosen, ZnϪ, ZnN, and Znϩ mice. Relative quantity calculations used 18S rRNA as the based on our interest in their annotated functions (Hsp40, endogenous normalization control. Values are mean of three pooled samples Hsp60, Hsc70, H2A␣, and TCCR), for independent confirma- calibrated to ZnN.

3886 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0330670100 Moore et al. Downloaded by guest on September 29, 2021 supply of zinc. Our objective was to identify gene transcripts modulated early in either a moderate dietary zinc restriction or dietary zinc supplementation to elucidate the pathways through which zinc exerts its influence. Zinc-regulated thymic genes are of interest because of the well described immunodeficiency that accompanies zinc deficiency in rodents (1) and humans (2). Our decision to additionally examine the thymic gene expression response to zinc supplementation was prompted by the wide- spread use of zinc supplements and zinc-fortified foods marketed for immune enhancing properties, and the paucity of data regarding the molecular outcomes of such supplementation (8). The cDNAs identified in this DD profile as altered by dietary zinc status represent a very small subset (0.55%) of the total thymic transcripts generated and illustrate the subtle effects of the modest dietary treatments used in these studies. The ma- jority of the identified bands excised (Ϸ56%) were influenced by zinc deficiency, whereas Ϸ29% were influenced by zinc supple- mentation. Therefore, we view these differences as pointing to the specificity of these responses, because larger numbers of genes would be expected to change when physiologic systems are Fig. 2. An emerging view of gene transcripts altered in murine thymus in altered in a more robust manner and homeostatic mechanisms response to 3 weeks of dietary zinc deficiency. Arrows after gene name are unable to sufficiently compensate. designate direction of mRNA change in ZnϪ relative to ZnN mice. Messages Heat shock proteins act as chaperones for nascent polypep- changed were: H2-A␣; TCCR; Hsp40; Hsc70; Hsp60; Rpl5 and Rpl28, ribosomal tides, through de novo folding, to yield accurate native confor- proteins L5 and L28; Api5, apoptosis and inhibitory factor 5; GDP dissociation i3, GDP dissociation inhibitor 3; Nmp84; and Tho2. Transcripts altered by zinc mations [reviewed by Frydman (20)]. From this physiological deficiency identified in previous work (16) are italicized, specifically: LCK, perspective, it is particularly interesting to note which heat shock lymphocyte-specific tyrosine kinase; MLR, mouse lamina receptor; MCL, my- proteins (Hsc70, Hsp40, and Hsp60) were found down-regulated eloid cell leukemia sequence; and RAD23B, DNA repair and recombination in these DD experiments. Briefly, the Hsc70 protein is a protein. constitutively expressed, rather than heat-inducible, member of the Hsp70 family, and is one of the most abundant soluble proteins in the mammalian cell (21). Hsp40 is a chaperone that, for expression profiling. In this regard, it may be relevant that the ␤ ␤ ϩ interestingly, contains two essential, cysteine-rich zinc-binding 2 microglobulin ( 2-M) gene is down-regulated in Zn mice. ␤ ␣ domains (20). In contrast to the Hsc70 and Hsp40 systems, which 2-M, of the MHC I molecule, and H2-A , of the MHC II protect nascent polypeptides in the cytoplasm, Hsp60, a member molecule, are structurally similar and have marked amino acid of the large (Ͼ800 kDa) barrel-shaped chaperonin family, , but are derived from genes on two different functions within the mitochondrial matrix (reviewed in ref. 22). (23) and have no significant similarity at the ␤ The Q-PCR results for Hsp40, Hsp60, and Hsc70 confirmed the mRNA level. Consequently, the down-regulation of 2-M gene DD-observed decreases in ZnϪ animals and also revealed in Znϩ mice and the down-regulation of H2-A␣ gene in ZnϪ decreased, albeit less pronounced, message abundance in Znϩ mice could both relate to immune dysfunction in these dietary animals. An explanation for down-regulation of the heat shock conditions because structural similarities of these proteins sug- protein genes is not possible from our experiments. The reduc- gest some common relationship to zinc that is not currently tion is probably not a product of a generalized stress response understood. because, in that situation, an increased expression would be Identification of the TCCR as a zinc-modulated transcript is anticipated. Thymocytes produce many peptides related to im- of particular interest because of its tissue-specific expression, mune function and, therefore, reduction in thymic activity and which, from among the murine tissues examined, is highest in the protein synthesis related to zinc status may reduce the need for thymus and peripheral blood lymphocytes (24, 25). There is these chaperones. relatively little known about this ‘‘orphan’’ receptor of the class Also confirmed by Q-PCR as decreased in ZnϪ mice was a I family of cytokine receptors that is defined by a common, subunit of the MHC class II receptor, termed H2-A␣ (Fig. 1). conserved, extracellular cytokine binding domain. This family H2-A␣ was identified twice from DD bands 9,6,1 and 4,18,3; also includes receptors for interleukins and growth factors such both were markedly decreased in ZnϪ. Sequence analysis as thrombopoietin, erythropoietin, and leptin (25). The data showed that these DD bands are derived from different regions from TCCR knockout mice imply that this receptor is essential of the same H2-A␣ mRNA transcript. The H2-A␣ peptide is one for development of Th1 immune responses in vivo (24, 26), which of three possible ␣ subunits for the MHC class II molecules in is particularly relevant given research suggesting that an aberrant mice, and is encoded within the H-2 gene locus on Th1͞Th2 balance occurs in human zinc deficiency (27, 28). 17. Both ␣ and ␤ MHC subunits are required for cell surface Differentiation of naive CD4ϩ T lymphocytes into armed effec- expression, and, in the thymus, interactions between MHC tor Th1 or Th2 cells occurs in the periphery; consequently, the

receptors and the T cell receptors of developing thymocytes function of TCCR in the thymus is currently unknown. Research SCIENCES mediate positive and negative selection processes, with MHC focused on TCCR expression in the thymus may clarify the zinc

class I molecules presenting antigens to CD8ϩ T cells and MHC interaction noted in this study and its relationship to zinc-related APPLIED BIOLOGICAL class II molecules presenting antigens to the CD4ϩ T cells. cytokine imbalance. Future experiments may show that reduction in H2-A␣ expres- The diagram in Fig. 2 presents our interpretation of how some of sion contributes to the lymphopenia of zinc deficiency or the the genes found modulated by zinc deficiency in mouse thymus may pathogen-specific increased susceptibility to infectious disease relate to the immune dysfunction associated with altered zinc status. seen secondary to a zinc deficiency. The results of this study are combined with those from a limited The decreased expression of H2-A␣ in the thymic stroma of cDNA array profile (16). Zinc deficiency is marked by lymphopenia ZnϪ animals validated our decision to use total RNA extracted that results from reduced replenishment of peripheral T lympho- from whole thymus, rather than RNA from isolated thymocytes cytes with mature, naive T cells exiting the thymus. The up-

Moore et al. PNAS ͉ April 1, 2003 ͉ vol. 100 ͉ no. 7 ͉ 3887 Downloaded by guest on September 29, 2021 regulation of the LCK in ZnϪ animals implies a potential mech- responsible, in part, for the increased severity of microbial and anism for this loss of T cells because this tyrosine kinase mediates parasitic infections observed in dietary zinc deficiency, or asso- signal transduction from the CD4 and CD8␣ coreceptors in a ciated with complications of dialysis and other procedures that manner that depends on zinc (29, 30). Decreased zinc availability ϩ ␣ deplete zinc reserves. Profiling of genes from the Zn mice for this interaction limits signaling from the CD4 or CD8 recep- suggests that zinc excess is more generally associated with effects tors, which might communicate to the nucleus a need to increase common to multiple cell types. In any event, the genes identified transcription of LCK in a compensatory manner. Decreased ex- in this genome profile of murine thymus provide targets of pression of H2-A␣ in ZnϪ mice is consistent with a feedback loop. MHC class II molecules are expressed by thymic epithelial cells and inquiry related to the role of zinc in biology and methods to present self-antigens to the TCR and CD4 coreceptor on develop- assess zinc nutrition in animals, including humans. ing thymocytes. Thymic epithelial cells may be most sensitive to zinc restriction, and the increase in LCK expression could stem from We thank Dr. Savita Shankar of the University of Florida’s DNA reduced extracellular signals generated by ligation of the H2-A␣ Sequencing Core Laboratory. This work was supported by National molecules. Institutes of Health Grant DK 31127, Institute of Food and Agricultural These profiling experiments demonstrate that genes related to Sciences Funds, and Boston Family Endowment Funds of the University T cell function dysregulate in zinc deficiency and could be of Florida.

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