Transcriptional remodeling in response to iron deprivation in Saccharomyces cerevisiae

Abstract

The budding yeast Saccharomyces cerevisiae responds to depletion of iron in the environment by activating Aft1p, the major iron-dependent transcription factor, and by transcribing systems involved in the uptake of iron. Here, we have studied the transcriptional response to iron deprivation and have identified new Aft1p target genes. We find that other metabolic pathways are regulated by iron: biotin uptake and biosynthesis, nitrogen assimilation, and purine biosynthesis. Two enzymes active in these pathways, biotin synthase and glutamate synthase, require an iron-sulfur cluster for activity. Iron deprivation activates transcription of the biotin importer and simultaneously represses transcription of the entire biotin biosynthetic pathway. Multiple genes involved in nitrogen assimilation and amino acid metabolism are induced by iron deprivation, whereas glutamate synthase, a key enzyme in nitrogen assimilation, is repressed. A CGG palindrome within the promoter of glutamate synthase confers iron-regulated expression, suggesting control by a transcription factor of the binuclear zinc cluster family. We provide evidence that yeast subjected to iron deprivation undergo a transcriptional remodeling, resulting in a shift from iron-dependent to parallel, but iron-independent, metabolic pathways.

Figure 1.

Relative mRNA levels of Aft1p target genes by cDNA microarray analysis. Aft1p target genes identified in previous studies and in this study are shown. Aft1p target genes described in this article are marked with an asterisk. Each column represents a single array and each cell represents the ratio of the mRNA levels from the first culture condition to the second culture condition. Transcripts more highly expressed in the first culture are in red, transcripts more highly expressed in the second culture are in green. The scale indicates the magnitude of the expression ratio. Gray cells indicate a missing data point. Genes were grouped according to their functional role in iron metabolism. The complete data set for the microarrays is available at http://microarray-pubs.stanford.edu/iron-_reg/index.shtml. A clustered analysis of all of the genes exhibiting iron- and Aft1p-dependent regulation is presented in Supplementary Figure 1.

Figure 2.

Iron- and Aft1p-dependent transcription of new genes in the Aft1p regulon. (A) Consensus binding sites for Aft1p in the DNA sequences upstream of TIS11, HMX1, COT1, SMF3, and VHT1. Boxed and shaded nucleotides were identical in more than four of the eight sequences. Numbering corresponds to +1 at the putative translation start site. (B) Northern blot analysis of Aft1p regulated genes. Congenic AFT1+, aft1Δ, and AFT1-1^up strains were grown in SD medium (lanes 1–3) or the AFT1+ strain was grown in iron-poor (20 μM), iron-sufficient (100 μM), or iron-enriched (500 μM) medium (lanes 4–6). Total RNA was isolated from cells in the exponential phase of growth and subjected to Northern blot analysis using the indicated probes. The actin signal (ACT1) served as a loading control.

Figure 3.

Failure of biotin synthesis under conditions of iron deprivation. (A) Biotin biosynthetic pathway in yeast. Genes in ovals encode enzymes of biotin biosynthetic pathway. Genes in boxes encode transporters of biotin or biotin precursors. Bio2p contains a 4Fe-4S cluster. (B–E) Effect of iron deprivation on growth of cells synthesizing biotin de novo. The VHT1+ (B and D) and the vht1Δ (C and E) strains were precultured in SD (+Fe, B and C) medium or iron-poor medium (-Fe, D and E) and reinoculated at an A₆₀₀ of 0.1 into the same medium containing the indicated amounts of KAPA or biotin at 100 ng/ml. Aliquots of culture were removed at the indicated intervals and the absorbance at 600 nm was measured. (F) Iron-dependent transcription of BIO2, BIO3, and BIO4. Strains expressing the indicated AFT1 alleles were grown in SD medium and the AFT1+ strain was grown in defined-iron medium supplemented with the indicated concentrations of iron. Total RNA was isolated from exponentially growing cells and Northern blot analysis was performed using the indicated probes.

Figure 4.

Transcriptional activation of genes involved in amino acid and nitrogen source uptake and metabolism under conditions of iron deprivation. (A) Microarray analysis. Data are presented from two arrays in which RNA from wild-type cells grown in iron-poor medium was compared with RNA from cells grown in iron-sufficient medium. A subset of genes with greater than twofold induction in at least one array is shown. The average induction in the two arrays is shown in the column on the right. (B) Northern blot analysis of a subset of the genes identified in A.

Figure 5.

Effect of iron on glutamate biosynthesis. (A) Ammonia assimilation and glutamate synthesis in yeast. Genes encoding glutamate dehydrogenases (GDH1 and GDH3), glutamine synthetase (GLN1), and glutamate synthase (GLT1) and the reactions they catalyze are indicated. Glt1p is a 4Fe-4S cluster protein. (B) Stimulation of glutamate synthase activity by iron supplementation. The parent strain was grown in defined-iron medium containing the indicated amounts of iron, crude cell lysates were prepared, and enzymatic activities were measured as described in MATERIALS AND METHODS. Assays were performed on duplicate samples, each assay was repeated twice, and the data were pooled for analysis. Error bars indicate the average deviation.

Figure 6.

Activation of GLT1 transcription by iron supplementation. (A and B) Strains expressing the indicated AFT1 allele were grown in SD medium (lanes 1–3) or the AFT1+ strain was grown in defined-iron medium containing the indicated amounts of iron (lanes 4–8). Northern blot analysis was performed using probes for GLT1 (A), and GDH3, GDH1, and GLN1 (B). (C) Aft1p-independent activation of GLT1 transcription. A strain expressing the Fet3p/Ftr1p iron transporter under the control of the constitutively active PGK1 promoter was deleted for AFT1. The resulting congenic AFT1+ and aft1Δ strains were grown in defined-iron medium containing the indicated amounts of iron and Northern blot analysis for GLT1 was performed.

Figure 7.

Transcriptional activation of the purine biosynthetic pathway by iron supplementation. (A) Microarray analysis. Data are presented from two arrays in which RNA from wild-type cells grown in iron-enriched medium was compared with RNA from cells grown in iron-sufficient medium. The sets of genes involved in one-carbon metabolism through folate and purine biosynthesis are shown. The average induction in the two arrays is indicated. (B) Northern blot confirmation of iron-dependent activation of purine biosynthetic pathway. Total RNA from wild-type cells grown in the indicated concentrations of iron was isolated, and Northern blot analysis was performed using probes for GCV1, ADE17, and ACT1. (C) Role of adenine and Bas1p on iron-dependent activation of ADE17. Northern blot analysis was performed on cells grown in the indicated concentrations of adenine and iron. BAS+ and bas1Δ strains were grown in 20 mg/l adenine.

Figure 8.

Identification of the iron-dependent activation sequence in the promoter of GLT1. Sequences upstream from the GLT1 open reading frame were fused to lacZ, and the resulting reporter constructs were transformed into the wild-type strain. Black bars indicate putative TATA boxes. Arrows indicate the transcription initiation site. β-Galactosidase activity was measured in lysates from cells grown in 5, 20, 100, and 500 μM iron (plasmids 1–6 and 10) or 10, 100, and 500 μM iron (plasmids 7–9 and 11–14), and the induction of activity from the lowest to highest iron concentration is indicated. Plasmids 1–6, deletions from the 5′ end of the GLT1 promoter fused to the lacZ coding region. Plasmids 7–14, deletions from the 3′ end of the GLT1 promoter fused to the GLT1 TATA boxes of plasmid 5. Plasmids 11–14, shaded boxes refer to the 5′-most, center, and 3′-most third of the region between –550 and –450. Assays were performed in duplicate and each assay was repeated three times. Induction is reported with the average deviation.