Inaugural Article: Modulation of intestinal gene expression by dietary zinc status: Effectiveness of cDNA arrays for expression profiling of a single nutrient deficiency

Nutrients play a significant role in health promotion through participation in many diverse physiological mechanisms. With growing awareness of entire genomes and the resources available to investigate this information, there are increased opportunities to examine the impact of nutrition on gene expression. Regulation of gene expression by minerals, vitamins, and specific macronutrients is well established but, in most cases, this influence has been clearly documented for only a very small number of genes (1). Although nutritional science is beginning to embrace the tools of genomics, including cDNA arrays, few attempts at large-scale or global evaluation of nutritional gene regulation have been reported. Consequently, it remains to be established whether the currently identified nutritionally regulated genes are unique, or whether others are similarly modulated. Alternatively, there may be populations or families of genes similarly affected by changes in nutritional status. An additional challenge confronting global gene expression profiling in integrated physiological systems is the detection of relatively subtle, albeit metabolically significant, levels of modulation expected compared with the more drastic alterations found in the pathological changes associated with cancer or infectious disease or resulting from acute stress. The molecular function of zinc affects a wide variety of physiological systems, including those necessary for proper immune function and growth (2, 3). However, the specific pathways that become dysfunctional when dietary zinc supply is inadequate or excessive, because of dietary supplements, have not been identified. Furthermore, specific indices or markers for zinc status assessment of individual human patients or populations are lacking (3, 4). Metallothionein (MT) genes are very sensitive to zinc status and have been well characterized, providing a good model for highly responsive zinc-regulated gene expression (reviewed in ref. 5). Zinc exerts its effects directly on transcription of MT through metal-responsive gene promoter elements and their associated metal-sensing transcription factors (MTFs), such as MTF-1 (reviewed in ref. 6). Interestingly, null mutation of the MTF-1 gene is lethal at day 14 of gestation (7), whereas MT gene deletion is not lethal (8, 9), suggesting that direct zinc regulation of gene transcription via MTF-1 extends beyond MT regulation to other essential physiological systems. In addition to direct effects on gene transcription modulated by zinc, there are likely to be indirect, or secondary, effects on gene expression produced by the alteration of zinc-dependent functions that influence other gene regulation pathways (10). Our laboratory has successfully identified dietary zinc-regulated genes from the small intestine by using subtractive library hybridization as well as mRNA differential display (11, 12). Unlike cDNA arrays, these methods do not rely on existing genome database information and therefore can detect previously unknown genes that are differentially expressed and, in the case of differential display, with high sensitivity for low-abundance mRNAs (10, 12). Differential display has allowed us to identify intestinal uroguanylin precursor as an up-regulated gene in zinc deficiency (13). Subsequently, we confirmed these mRNA screening data and extended them with measurements of uroguanylin peptides by both Western analysis and immunohistochemistry (14, 15). However, differential display requires extensive experimental manipulation; consequently, limited numbers of genes can be screened in a short time span. In this report, we have tested the feasibility of using cDNA arrays to compare global changes in expression of genes of known function that occur in the early stages of a single micronutrient deficiency, specifically zinc deficiency, to complement our ongoing analysis of zinc-regulated mammalian genes.