Abstract
Differential mRNA display was used to comprehensively screen the murine thymic transcriptome for genes modulated in vivo by dietary zinc. A moderate feeding protocol rendered young adult, outbred mice zinc-deficient and zinc-supplemented without alterations in feeding behavior or growth. However, these levels of deficiency and supplementation altered specific mRNA abundances in a manner detectable by differential display. In total, 240 primer-pair combinations were used to generate >48,000 interpretable cDNA bands derived from thymic total RNA, of which only 265 or 0.55% were identified as zinc-modulated under these moderate dietary conditions. The most strongly zinc-modulated cDNAs identified by display were reamplified and sequenced. No cDNAs encoding zinc-metalloenzymes or zinc-finger transcription factors were identified as zinc-modulated in this global screening. Those zinc-regulated genes independently confirmed by quantitative PCR included: heat shock proteins 40 and 60; heat shock cognate 70; histocompatibility 2, class II antigen A, α; and the T cell cytokine receptor. In addition, a variety of transcription- and translation-related factors (such as ribosomal proteins L3, L5, and L28; nuclear matrix protein 84; matrin cyclophilin; the H3 histone family 3A protein; β2 microglobulin; and a cleavage and polyadenylation factor) were identified as zinc-modulated. These profiling data show that differential expression of genes in the thymus in response to the dietary zinc supply precedes many of the phenotypic effects on thymic function associated with severe zinc restriction or supplementation. Several genes involved in T cell development were identified as regulated by zinc and will be targets to evaluate the effects of zinc on immune function.
Keywords: nutrition‖genomics‖functional genomics‖immunology
Zinc is an essential micronutrient required for vertebrate growth and development and severe zinc deficiency produces pleiotropic effects impacting multiple physiological systems. In particular, the immune system is sensitive to zinc deficiency, which can result in thymic atrophy, lymphopenia, and increased susceptibility to infectious disease (1, 2). In humans, zinc supplementation has been shown to reduce the morbidity of diarrhea (3), respiratory disease (4, 5), nematode infection (6), and sickle cell disease (7). The potential existence of immune dysfunction in individuals with a mild zinc deprivation and possible immune-enhancing benefits of supplemental zinc intake are questions that lack definitive data and warrant future research (8). In addition, convincing explanations for the molecular basis for symptoms associated with deficient or excess consumption of zinc have not been forthcoming. We have addressed those questions here through use of the differential display (DD) technique for profiling of genes expressed in the thymus of zinc-deficient (Zn−) and zinc-supplemented (Zn+) mice.
The rationale was that the differing biochemical roles for zinc should provide a stimulus for a characteristic, altered gene expression pattern when the normal dietary supply is either withdrawn or given in excess. We have successfully applied DD profiling in earlier studies to screen for zinc-modulated genes in the rat small intestine (9), demonstrating that this technology provides the sensitivity to identify physiologically relevant genes. For example, one intestinal cDNA sequence derived from DD identified the up-regulation of the preprouroguanylin gene in zinc deficiency. The active peptide produced from preprouroguanylin is uroguanylin, a natriuretic hormone involved in intestinal fluid secretion, which, when overproduced, could lead to diarrhea. Uroguanylin up-regulation may be related to the zinc-responsive diarrheal disease observed in many parts of the world. The sequence information from DD allowed subsequent cloning of the rat preprouroguanylin gene and further functional studies at the protein level (10–12).
The DD approach for transcription profiling allowed us to conduct a comprehensive survey of the mouse thymus transcriptome. An advantage to DD is its capacity to find novel EST data, both contributing to sequence databases and holding the potential for gene discovery (13). Even with the recent completion of the initial draft of the mouse genome (14) and the tremendous throughput advantages of array analysis, arrays are completely dependent on a priori sequence information, and thus DD best served our criterion for a genome-wide survey, which was not obtainable through available murine arrays.
The profiling presented in this report represents differential mRNA display analyses generated from 240 primer-pair combinations. Approximately 48,000 cDNA bands were separated by denaturing polyacrylamide gels. Given an estimated 15,000 genes actively transcribed in any one cell type, the data, statistically (15), represent a comprehensive screen of the mouse thymic transcriptome during both deprivation and supplementation of dietary zinc.
Materials and Methods
Feeding Studies.
Young-adult (30 ± 3 g) male CD-1 mice (Charles River Breeding Laboratories) were housed and fed as described (16). Treatment groups were provided one of three dietary zinc levels [Zn− (<1 mg/kg), zinc-normal (ZnN; 30 mg/kg), or Zn+ (180 mg/kg)], and animals (n = 5–10 per treatment) were fed the diet for 3 weeks. Blood was collected by cardiac puncture for measurement of the serum zinc concentration. The protocol was approved by the University of Florida Institutional Animal Care and Use Committee. Physiological values are reported as the mean ± SEM and were compared by one-way ANOVA.
RNA Isolation and Differential Display Reactions.
Whole thymus (≈250 mg) was excised and homogenized in TriPure Isolation Reagent (Roche Diagnostics), and total RNA was isolated according to the manufacturer's instructions. RNA concentrations were determined spectrophotometrically, and integrity was verified by agarose electrophoresis and ethidium bromide staining. Equal amounts of RNA were pooled from mice (n = 7) within treatment groups and the pooled samples were DNase treated by using the DNA-free kit (Ambion, Austin, TX).
For these experiments, the entire set of HIEROGLYPH mRNA profile kits (Beckman Coulter) was used for DD reverse transcription and PCR according to the manufacturer's protocols. In total, this includes 12 anchored 3′ primers (AP1–12) and 20 arbitrary 5′ primers (ARP1–20) of defined nucleotide sequence, which together are predicted to comprehensively screen an entire mammalian transcriptome. Specifically, the reaction conditions used pooled, DNase-treated RNA (≈0.1 μg/μl) from each treatment group, reverse transcribed by using 2 μM of a single specific AP and 2 units/μl SuperScript II RNase H− reverse transcriptase (Invitrogen). Subsequent 20-μl PCR amplification reactions were done in triplicate for each sample by using 2 μl of first-strand products for template and 0.05 units/μl AmpliTaq DNA polymerase (Roche Diagnostics) with supplied buffer; PCR reactions had final concentrations of 0.2 μM for each AP and ARP, 20 μM for each of the dNTPs, and 0.125 μCi/μl [α-33P]dATP (DuPont/NEN; 1 Ci = 37 Bq). Cycling parameters were: 95°C for 2 min; 4 cycles of 92°C for 15 sec, 50°C for 30 sec, and 72°C for 2 min; 25 cycles of 92°C for 15 sec, 60°C for 30 sec, and 72°C for 2 min; and 72°C for 7 min.
Denaturing PAGE.
After addition of a denaturing loading dye (95% formamide/0.05% bromophenol blue/0.05% xylene cyanol) and a 2-min, 95°C heat step, PCR products were electrophoresed under two distinct conditions by using a Genomyx LR DNA sequencer (Beckman Coulter). To resolve longer cDNAs, DD reaction products were separated for 16 h through a 340-μm-thick, 4.5% acrylamide gel matrix containing urea (Beckman 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 to Kodak Biomax MR film for display visualization.
Excision, Reamplification, and Identification of DD Products.
Selected bands were circumscribed with a scalpel, rehydrated with <1 μl of deionized H2O, excised, and placed in 100 μl of TE (10 mM Tris/1 mM EDTA) or 1× PCR buffer (10 mM Tris⋅HCl/1.5 mM MgCl2/50 mM KCl) in TE, in light of data from Frost and Guggenheim (17). Reamplification reactions used the universal, full-length M13(-48) (5′-AGCGGATAACAATTTCACACAGGA-3′) and T7 promoter (5′-GCCCTATAGTGAGTCGTATTAC-3′) primers (GIBCO/BRL), which facilitated the direct sequencing of reamplified cDNAs from the M13(-48) primer. Reactions were run with 2 μl of gel band eluate (in a 40 μl final volume), 2× PCR buffer (Roche Molecular Biochemicals), 20 μM each dNTPs, 0.2 μM each M13 and T7 primers, and 0.05 units/μl AmpliTaq polymerase (Roche Diagnostics). Cycling parameters were 95°C for 2 min; 4 cycles of: 92°C for 15 sec, 50°C for 30 sec, 72°C for 2 min; 25 cycles of: 92°C for 15 sec, 60°C for 30 sec, 72°C for 2 min; and 72°C for 7 min.
To assess quality of the reactions, 2 μl of reamplification product was electrophoresed in a 1.5% agarose, 1× TBE gel (0.9 M Tris/0.9 M borate/0.02 M EDTA), stained with SYBR Green I (Molecular Probes), and scanned on a Storm PhosphorImager (Molecular Dynamics). For obtaining sufficiently concentrated PCR product for sequencing, reactions were then repeated under identical conditions in nine 40-μl reactions (using DNA in the original gel band eluate for template). Reaction products were purified and concentrated by using QIAquick PCR purification columns (Qiagen, Valencia, CA) and sent for sequencing at the University of Florida's Sequencing Core. The GenBank databases (18) were queried by using blast (19), run from SEQWEB V.2.02 of the wisconsin package (Accelrys), to evaluate sequence information.
Independent Confirmation by Quantitative PCR Analysis.
Quantitative real-time PCR (Q-PCR) primers and TaqMan probes were designed by using PRIMER EXPRESS V.2.0 (Applied Biosystems). The 18S rRNA assay, used for total RNA normalization, and all one-step RT-PCR reagents were purchased from Applied Biosystems, and all assays were performed on a GeneAmp 5700 Sequence Detection System (Applied Biosystems). Relative quantitation was determined from 4 log10-range standard curves with pooled samples run in triplicate. All assays except 18S rRNA used 900 nM each of the forward and reverse primers and 250 nM of probe. Primers and probe used (forward, reverse, and probe, respectively) were: T cell cytokine receptor [TCCR, GenBank accession no. NM_016671; 5′-GGGAGCCCAGGGATAAAGG-3′, 5′-TGAGCCCAGTCCACCACATAC-3′, and 5′-FAM-CAATGGTTTCCTGGTCCCTTGTTTCCA-BHQ1-3′]; the heat shock protein 40 [Hsp40, accession no. NM_008298; 5′-AATGGAGAAGCGTATGAGGATGA-3′, 5′-ACTGGCCCATTAAGAGGTCTGA-3′, and 5′-FAM-CACCCCAGAGGTGGCGTTCA-BHQ1-3′]; the heat shock protein 60 [Hsp60, accession no. X53584; 5′-TTGCCCTTATCAATGAACTGTGA-3′, 5′-TCAGTCATTTTCTCCAGGTGACTTC-3′, and 5′-FAM-CTCAAGGCAGGTTCCTCACCAATAACTTCAG-BHQ1-3′]; the heat shock cognate protein 70 [Hsc70, accession no. BC006722; 5′-GCTGCCGGGCATTCG-3′, 5′-CCTTAGACATGGTTGCTTGTGTGTAG-3′, and 5′-FAM-TGGTCTCGTCGTCAGCGCAGCT-BHQ1-3′], and the histocompatibility 2, class II antigen A, α [H2-Aα, accession no. BC019721; 5′-GGCCTTGTGGGCATCGT-3′, 5′-TCTGGAGGTGCCACCTGATC-3′, and 5′-FAM-TGGGCACCATCTTCATCATTCAAGGC-BHQ1-3′] (BioSource International, Camarillo, CA). The metallothionein (MT) primers and probe have been described (16).
Results
Animals.
The moderate nature of the 3-week feeding protocol, consistent with earlier studies (16), was underscored by the lack of significant differences in terminal body and thymus weights between dietary treatment groups (data not shown). Serum zinc concentrations in Zn− mice (5.0 ± 0.3 μM) were significantly (P < 0.0001) lower than those of the ZnN (16.1 ± 0.4 μM) and Zn+ (15.1 ± 0.7 μM) mice. Further demonstrating lower zinc status of the Zn− mice was their thymic MT mRNA levels, which were 66% those of the ZnN or Zn+ mice, as measured by Q-PCR.
Differential Display.
All 12 APs, in combination with all 20 ARPs, were used to generate differential mRNA displays of thymic transcripts in Zn−, ZnN, and Zn+ mice. The 240 primer-pair combinations produced ≈48,000 interpretable bands on denaturing polyacrylamide gels. Typically, triplicate reactions from six to seven AP and ARP pair combinations were run on each gel simultaneously and, in total, 86 DD gels were generated to complete the screening of all primer-pair combinations. Presuming an estimated 15,000 actively transcribed genes in any one cell type, and considering the statistical requirements to represent each mRNA transcript by at least one cDNA on a gel from a single primer pair (15), this represents a complete screen of the thymic transcriptome under each of these dietary conditions.
Of the ≈48,000 bands surveyed, 265 bands were observed to be differentially regulated by zinc treatment and were excised for further investigation. Criteria for defining a band as zinc-modulated were pronounced differences between treatment groups, consistency among triplicate reactions, overall band intensity, and a size of >200 nt. After summarizing these criteria, differentially expressed bands were ranked as 1–4 to prioritize the order of reamplification reactions. In this subjective assessment, bands 1 (73) were the most intense, demonstrating extreme differentiation between treatment groups (“on” vs. “off” signals); bands 2 (67) were intense with modest differentiation; bands 3 (52) were less intense, but showed extreme differentiation between treatment groups; and bands 4 (73) were less intense, with modest treatment differentiation. Of the 265 excised differential cDNA bands (Table 1), 90 appeared increased in Zn− mice and another 58 appeared decreased in Zn− mice. Fewer cDNA bands appeared modulated by dietary zinc supplementation, with 36 bands appearing increased and 41 appearing decreased in Zn+ mice. An additional 40 bands appeared to be modulated by both high and low dietary zinc intake, although only one of these demonstrated modulation consistent with a zinc dose–response across all three conditions, i.e., appearing decreased in Zn− and increased in Zn+ mice.
Table 1.
Overall results of differential displays
| Bands | Number | % |
|---|---|---|
| Interpretable bands surveyed | ≈48,000 | |
| Excised differentially displayed bands | 265 | 0.55 (of surveyed) |
| Increased in Zn− | 90 | 34 |
| Decreased in Zn− | 58 | 22 |
| Increased in Zn+ | 36 | 13.5 |
| Decreased in Zn+ | 41 | 15.5 |
| Modulated in both Zn− and Zn+ | 40 | 15 |
To demonstrate the reproducibility of DD, reaction products from AP3 and ARP2 and -3 were generated in two subsequent months and displayed. Profiles were virtually identical in banding patterns (see Figs. 3 and 4, which are published as supporting information on the PNAS web site, www.pnas.org), demonstrating gross reproduction of DD RT-PCR reactions. Furthermore, when sequenced independently, five differential bands (two from the Month 1 Gel and two from the Month 2 Gel, hypothesized to be the same, and a close lower band) were all identified as the same cDNA (Fig. 4). This cDNA coded for mitochondrial NADH dehydrogenase subunit 2 (NADH:ubiquinone oxidoreductase; mt-Nd2), and was found overexpressed in Zn+ mice relative to their Zn− and ZnN counterparts. These cDNA bands are examples of those ranked as 1 by our criteria.
Characterization of Zinc-Modulated Bands.
Optimization generally required for high-efficiency amplification of any one DNA template by PCR was not practical with the number of DD bands excised in these experiments. Our strategy then, beginning with DD bands prioritized as 1, involved an initial assessment of template capacity for reamplification under standard conditions, as well as the number of product bands produced by amplification reaction. Robust reactions that produced a single PCR product were pursued for sequencing. Those cDNAs, which had a 1 priority and that were successfully reamplified, sequenced and identified by blast, are listed in Tables 2 (modulated by Zn− treatment) and 3 (modulated by Zn+ treatment). Single-pass DNA sequencing generally yielded between 100 and 750 bases of high-quality sequence from each cDNA (Tables 2 and 3, third column); however, a few produced a low-quality sequence 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 discontinuous regions. All matches are to mouse sequences unless otherwise 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 sequence 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.
Table 2.
DD transcripts modulated in Zn− mice
| Name | Band* | No. of nt† | Identity, %‡ | GenBank accession no. |
|---|---|---|---|---|
| Increased in Zn− 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 | ||||
| Apoptosis 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 Zn− mice | ||||
| Hsc70, mRNA | 2,4,2 | 718 | 100 | BC006722 |
| Similar to RIKEN cDNA 9530053H05 gene, clone IMAGE:4488629, mRNA | 2,7,1 | 642 | 99 | BC017513 |
| cDNA clone K0707H07-3′, mRNA (dbEST) | 100 | BM244188 | ||
| cDNA clone K0706D12-3′, mRNA (dbEST) | 2,14,1 | 291 | 100 | BM244095 |
| cDNA clone K0700A06-3′, mRNA (dbEST) | 100 | BM243664 | ||
| Retrotransposon L1Md-A101 pORF2, mRNA and L1Md-A2 repetitive element ORF2, mRNA | 3,1,4 | 500 | 98 | AY053456 |
| 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-Aα, mRNA | 4,18,3 | 594 | 97 | NM_010378 |
| 9,6,1 | 729 | 98 | ||
| cDNA clone IMAGE:596239-5′, mRNA (dbEST) | 5,4,3 | 624 | AA138077 | |
| Rearranged immunoglobulin κ light chain | 5,14,1 | 733 | 99 | X67211 |
| 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.
Table 3.
DD transcripts modulated in Zn+ mice
| Name | Band* | No. of nt | Identity, % | GenBank accession no. |
|---|---|---|---|---|
| Increased in Zn+ 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 Zn+ 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 |
| β-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 κ 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 (Cpsf5), mRNA | 7,13,1 | 770 | 99 | NM_026623 |
| 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.
Confirmation of Select DD Clones.
Five DD cDNAs were chosen, based on our interest in their annotated functions (Hsp40, Hsp60, Hsc70, H2Aα, and TCCR), for independent confirmation of regulation using Q-PCR (Fig. 1). In all cases, the direction of zinc modulation observed on the DDs was reproduced. Interestingly, for all three heat shock proteins, depression of expression was seen in both Zn− and Zn+ mice (Fig. 1), although the depression seen in Zn+ mice was not at the magnitude observed in Zn− mice.
Figure 1.
Q-PCR analyses of select DD clones and metallothionein (MT). Assays were performed on triplicate pooled total RNA samples (n = 7 per group) from Zn−, ZnN, and Zn+ mice. Relative quantity calculations used 18S rRNA as the endogenous normalization control. Values are mean of three pooled samples calibrated to ZnN.
Discussion
The data presented here represent a view of differential gene expression in the thymus as the result of alterations in dietary 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 widespread 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 majority of the identified bands excised (≈56%) were influenced by zinc deficiency, whereas ≈29% were influenced by zinc supplementation. 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 altered in a more robust manner and homeostatic mechanisms are unable to sufficiently compensate.
Heat shock proteins act as chaperones for nascent polypeptides, through de novo folding, to yield accurate native conformations [reviewed by Frydman (20)]. From this physiological perspective, it is particularly interesting to note which heat shock proteins (Hsc70, Hsp40, and Hsp60) were found down-regulated in these DD experiments. Briefly, the Hsc70 protein is a 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, interestingly, contains two essential, cysteine-rich zinc-binding domains (20). In contrast to the Hsc70 and Hsp40 systems, which protect nascent polypeptides in the cytoplasm, Hsp60, a member of the large (>800 kDa) barrel-shaped chaperonin family, functions within the mitochondrial matrix (reviewed in ref. 22). The Q-PCR results for Hsp40, Hsp60, and Hsc70 confirmed the DD-observed decreases in Zn− animals and also revealed decreased, albeit less pronounced, message abundance in Zn+ animals. An explanation for down-regulation of the heat shock protein genes is not possible from our experiments. The reduction is probably not a product of a generalized stress response because, in that situation, an increased expression would be anticipated. Thymocytes produce many peptides related to immune function and, therefore, reduction in thymic activity and protein synthesis related to zinc status may reduce the need for these chaperones.
Also confirmed by Q-PCR as decreased in Zn− mice was a subunit of the MHC class II receptor, termed H2-Aα (Fig. 1). H2-Aα was identified twice from DD bands 9,6,1 and 4,18,3; both were markedly decreased in Zn−. Sequence analysis showed that these DD bands are derived from different regions of the same H2-Aα mRNA transcript. The H2-Aα peptide is one of three possible α subunits for the MHC class II molecules in mice, and is encoded within the H-2 gene locus on chromosome 17. Both α and β MHC subunits are required for cell surface expression, and, in the thymus, interactions between MHC receptors and the T cell receptors of developing thymocytes mediate positive and negative selection processes, with MHC class I molecules presenting antigens to CD8+ T cells and MHC class II molecules presenting antigens to the CD4+ T cells. Future experiments may show that reduction in H2-Aα expression contributes to the lymphopenia of zinc deficiency or the pathogen-specific increased susceptibility to infectious disease seen secondary to a zinc deficiency.
The decreased expression of H2-Aα in the thymic stroma of Zn− animals validated our decision to use total RNA extracted from whole thymus, rather than RNA from isolated thymocytes for expression profiling. In this regard, it may be relevant that the β2 microglobulin (β2-M) gene is down-regulated in Zn+ mice. β2-M, of the MHC I molecule, and H2-Aα, of the MHC II molecule, are structurally similar and have marked amino acid sequence homology, but are derived from genes on two different chromosomes (23) and have no significant similarity at the mRNA level. Consequently, the down-regulation of β2-M gene in Zn+ mice and the down-regulation of H2-Aα gene in Zn− mice could both relate to immune dysfunction in these dietary conditions because structural similarities of these proteins suggest some common relationship to zinc that is not currently understood.
Identification of the TCCR as a zinc-modulated transcript is of particular interest because of its tissue-specific expression, which, from among the murine tissues examined, is highest in the thymus and peripheral blood lymphocytes (24, 25). There is relatively little known about this “orphan” receptor of the class I family of cytokine receptors that is defined by a common, conserved, extracellular cytokine binding domain. This family also includes receptors for interleukins and growth factors such as thrombopoietin, erythropoietin, and leptin (25). The data from TCCR knockout mice imply that this receptor is essential for development of Th1 immune responses in vivo (24, 26), which is particularly relevant given research suggesting that an aberrant Th1/Th2 balance occurs in human zinc deficiency (27, 28). Differentiation of naive CD4+ T lymphocytes into armed effector Th1 or Th2 cells occurs in the periphery; consequently, the function of TCCR in the thymus is currently unknown. Research focused on TCCR expression in the thymus may clarify the zinc interaction noted in this study and its relationship to zinc-related cytokine imbalance.
The diagram in Fig. 2 presents our interpretation of how some of the genes found modulated by zinc deficiency in mouse thymus may relate to the immune dysfunction associated with altered zinc status. The results of this study are combined with those from a limited cDNA array profile (16). Zinc deficiency is marked by lymphopenia that results from reduced replenishment of peripheral T lymphocytes with mature, naive T cells exiting the thymus. The up-regulation of the LCK in Zn− animals implies a potential mechanism for this loss of T cells because this tyrosine kinase mediates signal transduction from the CD4 and CD8α coreceptors in a manner that depends on zinc (29, 30). Decreased zinc availability for this interaction limits signaling from the CD4 or CD8α receptors, which might communicate to the nucleus a need to increase transcription of LCK in a compensatory manner. Decreased expression of H2-Aα in Zn− mice is consistent with a feedback loop. MHC class II molecules are expressed by thymic epithelial cells and present self-antigens to the TCR and CD4 coreceptor on developing thymocytes. Thymic epithelial cells may be most sensitive to zinc restriction, and the increase in LCK expression could stem from reduced extracellular signals generated by ligation of the H2-Aα molecules.
Figure 2.
An emerging view of gene transcripts altered in murine thymus in response to 3 weeks of dietary zinc deficiency. Arrows after gene name designate direction of mRNA change in Zn− relative to ZnN mice. Messages changed were: H2-Aα; TCCR; Hsp40; Hsc70; Hsp60; Rpl5 and Rpl28, ribosomal proteins L5 and L28; Api5, apoptosis and inhibitory factor 5; GDP dissociation i3, GDP dissociation inhibitor 3; Nmp84; and Tho2. Transcripts altered by zinc deficiency identified in previous work (16) are italicized, specifically: LCK, lymphocyte-specific tyrosine kinase; MLR, mouse lamina receptor; MCL, myeloid cell leukemia sequence; and RAD23B, DNA repair and recombination protein.
These profiling experiments demonstrate that genes related to T cell function dysregulate in zinc deficiency and could be responsible, in part, for the increased severity of microbial and parasitic infections observed in dietary zinc deficiency, or associated with complications of dialysis and other procedures that deplete zinc reserves. Profiling of genes from the Zn+ mice suggests that zinc excess is more generally associated with effects common to multiple cell types. In any event, the genes identified in this genome profile of murine thymus provide targets of inquiry related to the role of zinc in biology and methods to assess zinc nutrition in animals, including humans.
Supplementary Material
Acknowledgments
We thank Dr. Savita Shankar of the University of Florida's DNA Sequencing Core Laboratory. This work was supported by National Institutes of Health Grant DK 31127, Institute of Food and Agricultural Sciences Funds, and Boston Family Endowment Funds of the University of Florida.
Abbreviations
- AP
anchored primer
- ARP
arbitrary primer
- DD
differential display
- H2-Aα
histocompatibility 2, class II antigen A, α
- Hsc
heat shock cognate protein
- Hsp
heat shock protein
- MT
metallothionein
- Q-PCR
quantitative real-time PCR
- TCCR
T cell cytokine receptor
- Zn−
zinc-deficient
- ZnN
zinc-normal
- Zn+
zinc-supplemented
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