Aft1p and Aft2p Mediate Iron-responsive Gene Expression in Yeast through Related Promoter Elements*,

Julian C. Rutherford, Shulamit Jaron and Dennis R. Winge {ddagger}

From the Departments of Medicine and Biochemistry, University of Utah Health Sciences Center, Salt Lake City, Utah 84132

Received for publication, January 5, 2003 , and in revised form, May 2, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcription factors Aft1p and Aft2p from Saccharomyces cerevisiae regulate the expression of genes that are involved in iron homeostasis. In vitro studies have shown that both transcription factors bind to an iron-responsive element (FeRE) that is present in the upstream region of genes in the iron regulon. We have used DNA microarrays to distinguish the genes that are activated by Aft1p and Aft2p and to establish for the first time that each factor gives rise to a unique transcriptional profile due to the differential expression of individual iron-regulated genes. We also show that both Aft1p and Aft2p mediate the in vivo expression of FET3 and FIT3 through a consensus FeRE. In addition, both proteins regulate MRS4 via a variant FeRE with Aft2p being the stronger activator from this particular element. Like other paralogous pairs of transcription factors within S. cerevisiae, Aft1p and Aft2p are able to interact with the same promoter elements while maintaining specificity of gene activation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Saccharomyces cerevisiae contains paralogous gene pairs that code for transcription factors that can have both overlapping and distinct functions. These transcription factors bind to the same promoter elements but generate distinct transcriptional profiles. Included in this group are Ace2p/Swi5p, Pdr1p/Pdr3p, and Yap1/Yap2p. Ace2p and Swi5p regulate the expression of cell cycle-specific genes, are 83% identical in their zinc finger DNA-binding domains and recognize the same DNA binding site in vitro (1, 2). However, Ace2p and Swi5p can regulate separate genes, where discrimination between promoter elements is achieved through the interaction of these factors with other DNA-binding proteins (2). Alternatively, in cases where both transcription factors can induce the expression of the same gene, one is often the more potent activator (3). There are genes that require both Ace2p and Swi5p for maximal expression and others that are antagonistically regulated by these factors (4). Pdr1p and Pdr3p share 36% identity and regulate the expression of genes that are involved in pleiotropic drug resistance. Both these factors are able to bind to the same DNA element (PDRE) in vivo as either hetero- or homodimers (5). Differential expression of Pdr1p/Pdr3p target genes may therefore involve different combinations of Pdr1p/Pdr3p at different PDREs (5). Yap1p and Yap2p are 88% identical in their DNA binding regions and bind to the same consensus site (6). Analysis of global gene expression using microarrays has shown that although Yap1p and Yap2p are both involved in the response to cellular stresses, they regulate different regulons (7). The mechanism(s) of Yap1p/Yap2p gene selectivity are not understood but may include variations in the base pairs flanking the consensus Yap binding site (7).

The iron regulon of S. cerevisiae is well characterized and includes genes that are involved in the uptake, compartmentalization and use of iron. These include genes that encode siderophore transporters (ARN1–4), iron reductases (FRE1–6), iron permeases (FTR1, FET4), and multicopper oxidases that are involved in the coordinated oxidation and transport of iron across membranes (FET3, FET5) (812). In addition, three related genes (FIT1-FIT3) encode proteins that localize within the yeast cell wall and whose function is partially related to siderophore uptake (13).

Genes within the iron regulon are induced under low iron conditions and are regulated by the transcription factors, Aft1p and Aft2p (14, 15, 18). Aft1p and Aft2p are 39% identical within their N-terminal regions, which contain the DNA binding domain of each protein (15). The activation domain of Aft1p is within its C-terminal 413–572 amino acid residues which, when fused to the Gal4p DNA binding domain, activates transcription in an iron-independent manner (16). In addition, the N-terminal region of Aft1p contains a nuclear export signal that mediates differential cellular localization of Aft1p in response to iron (16). Aft1p is localized to the cytosol under iron-replete conditions, but is nuclear and active in iron-deficient conditions. Mutant alleles of AFT1 and AFT2 (AFT1-1up and AFT2-1up respectively) have been identified that result in the iron-independent activation of the iron regulon (14, 15). These gain of function mutations result from a single cysteine to phenylalanine substitution in identical regions of Aft1p and Aft2p.

A strain that lacks a functional Aft1p (aft1{Delta}) grows poorly in iron-limiting conditions and exhibits reduced iron uptake and cell surface iron-reductase activity (14, 17). The vector-borne AFT2-1up allele is able to partially restore iron uptake in the aft1{Delta} strain (15). The aft2{Delta} strain has no growth phenotype on iron-limiting medium (15). However, the aft1{Delta} aft2{Delta} strain is more sensitive to iron-limiting conditions than the single aft1{Delta} strain (15, 18). The aft1{Delta} strain also fails to grow with glycerol as a respiratory carbon source, and this phenotype is partially restored by complementation with low copy plasmids containing the AFT2 or AFT2-1up alleles (15). Analysis of global gene expression has shown that the AFT2-1up allele activates the expression of a subset of the iron regulon (15). However, our previous microarray analysis did not compare transcript profiles for both AFT2-1up and AFT1-1up cells. Additionally, the AFT1-1up and the AFT2-1up alleles differentially regulate the expression of two iron-regulated genes (MRS4 and FIT2) (15).

Aft1p mediates transcriptional regulation through an iron-responsive element (FeRE)1 that has the consensus sequence PyPuCACCCPu (14). The FeRE was identified by the in vivo analysis of the FET3 promoter region using lacZ fusion constructs and DNA footprinting (14). Aft1p binds to the FET3 FeRE in vivo in an iron-dependent manner. Sequences similar to the FET3 FeRE were identified in the promoter regions of five other known iron-regulated genes. In vitro assays confirmed the binding of Aft1p to these particular sequences, from which the consensus FeRE sequence was derived (14). SMF3, that encodes a metal transporter, is also iron-regulated in an Aft1p/Aft2p-dependent manner through a consensus FeRE (19). N-terminal truncates of Aft1p and Aft2p bind to the FET3 FeRE in vitro, consistent with both factors regulating the iron regulon through the consensus FeRE (15). Expression of AFT2 from a high copy plasmid, but not the chromosomal copy of AFT2, activates the expression of a lacZ reporter under the control of the FET3 FeRE in an iron-dependent manner (18). However, the high expression of one of a pair of transcription factors can result in the aberrant activation of genes that are specifically regulated by the other factor (2). It is therefore not clear to what extent Aft1p and Aft2p interact with the same responsive element in vivo.

The evidence is therefore consistent with Aft1p and Aft2p having overlapping but non-redundant roles in the transcriptional regulation of the iron regulon in S. cerevisiae. We are interested in understanding the selective advantage of this organism having two iron-responsive transcription factors. In this study, microarray experiments have been used to further define the activities of the gain of function mutants and demonstrate that they give rise to different global transcriptional profiles. We have also analyzed the ability of Aft1p and Aft2p to activate transcription through the same consensus FeRE. In vivo lacZ reporter constructs show clearly that Aft1p and Aft2p activate gene expression through the consensus FeREs in the FET3 and FIT3 promoter regions. Both Aft1p and Aft2p also induce the activation of MRS4 through a variant FeRE. An extended FeRE has been identified that is overrepresented in the genes that are most highly induced by the AFT1-1up and AFT2-1up alleles.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and Culture Conditions—Haploid aft1{Delta}aft2{Delta} strains were isolated following the mating of the strains, BY4741aft2{Delta} (MATa his3{Delta}1 leu2{Delta}0 met15{Delta}0 ura3{Delta}0 aft2::kanMX4) and BY4742aft1{Delta} (MAT{alpha} his3{Delta}1 leu2{Delta}0 lys2{Delta}0 ura3{Delta}0 aft1::kanMX4), which were purchased from Research Genetics. A strain containing a copy of the AFT2-1up allele integrated at the HIS3 locus was generated by transforming a haploid aft1{Delta}aft2{Delta} strain (MAT{alpha} his3{Delta}1 leu2{Delta}0 ura3{Delta}0 aft1::kanMX4 aft2::kanMX4) with plasmid pAFT2-1upINT that had been linearized using MscI and selecting for growth on agar plates lacking histidine. The allele status of the aft1{Delta}aft2{Delta} strains and the integration of the AFT2-1up allele at the HIS3 locus were verified using PCR and DNA sequencing. Cells were grown with 2% glucose as a carbon source, in 1% yeast extract, 2% peptone medium (YPD), complete-synthetic medium (CMD) or, when appropriate, complete-synthetic medium lacking uracil and/or histidine (CMD-Ura, CMD-His, CMD-Ura/His).

Vectors and Fusion Genes—The YCp plasmids, pAFT1-1up and pAFT2-1up are HIS3 derivatives of pAFT1-1up and pAFT2-1up (15) from which the AFT1 (XhoI/SacI) and AFT2 (BamHI/XbaI) sequences have been subcloned into the YCp plasmid, pRS413. Consequently, the gain of function mutant alleles of AFT1 and AFT2 within pAFT1-1up and pAFT2-1up are under the control of their own promoters (15). The integrative vector pAFT2-1upINT was generated by subcloning the AFT2 sequences (XhoI/XbaI) from pAFT2-1up into the YIp plasmid, pRS403. The lacZ reporter constructs, pFC-W and pFC-LM2 were a gift from Andrew Dancis (14). These contain, respectively, a functional and non-functional copy of the FET3 FeRE in a minimal promoter. The plasmid pFET3-lacZ (0.5 kb of the 5'-region of FET3, inclusive of the start codon, fused to the lacZ gene in YEp354, Ref. 20) was a gift from Jerry Kaplan (21). To generate a version of pFET3-lacZ in which the same FeRE that is present in pFC-W was mutated to a non-functional FeRE (CACCC to CAGGG), pFET3-lacZ was used as template for QuikChange mutagenesis (Stratagene) using the primer 5'-GGCAAGGCCCATCTTCAAAAGTGCAGGGATTTGCAGGTGCTCTTATTCTCGCC-3' and its complement to generate pFET3-lacZ (mut). The 0.5-kb fragment containing the FET3 sequences from pFET3-lacZ and pFET3-lacZ (mut) were excised and ligated into the PstI/SmaI sites of Yep354 to generate pFET3-lacZ and pFET3-lacZ (mut) respectively. To generate pFIT3-lacZ (1 kb of 5'-region of FIT3, inclusive of the start codon, fused to the lacZ gene in YEp354) yeast-genomic DNA from strain BY4741 was isolated and used as template for PCR using the primers 5'-CGGGATCCTCCATAAACATTTCCTTTGTC-3' and 5'-CCAAGCTTTCATTTTAGGGATTATTGTTATTAG-3'. The resulting 1-kb fragment was ligated into the BamHI/HindIII sites of Yep354 to generate pSJDW36. Plasmid pSJDW36 was used as template for QuikChange mutagenesis using the primer 5'-CATCAAAAAATATGGGATAGCGCCCTGCGCAACAAACACCCTGCAAAAAAAAATCTAGGACATAGG-3' and its complement to mutate two potential FeREs to non-functional FeREs (CACCC to CAGGG) and thereby generate pSJDW36 (mut). The FIT3 sequences from both pSJDW36 and pSJDW36 (mut) were excised and ligated to the BamHI/HindIII sites of Yep354 to generate pFIT3-lacZ and pFIT3-lacZ (mut), respectively. To generate plasmid pMRS4a-lacZ (1 kb of 5'-region of MRS4, inclusive of the start codon, fused to the lacZ gene in YEp354) yeast-genomic DNA from strain BY4741 was used as template for PCR using the primers 5'-CGCACAGGATCCTCGAAGATAGCGTAGCGTTC-3' and 5'-GGGAGCAAGCTTCATAATATTAACTGATATTTCGGTTG-3' (primer Mrs4a). The resulting 1-kb fragment was ligated into the BamHI/HindIII sites of pHOLLY to generate pJRDW25. Plasmid pJRDW25 was used as template for PCR using primer Mrs4a with, individually, primers 5'-CGCACAGGATCCCATATTTGGAATTCAGC-3', 5'-CGCACAGGATCCCCCACAGGAATCGCTAC-3', 5'-CGCACAGGATCCCGTGTCTCTTTTCGGTA-3', 5'-CGCACAGGATCCGAATGAGAGCATGGCGA-3', and 5'-CGCACAGGATCCCTTTTGCCTACCATTGG-3' to generate sequential truncations of the MRS4 promoter region. The resulting fragments, and the 1-kb BamHI/HindIII fragment from pJRDW25, were ligated into the BamHI/HindIII sites of Yep354 to generate plasmids pMRS4lacZ(a-f). Plasmid pMRS4-lacZd was used as template for QuikChange mutagenesis using the primer 5'-CGAAGACTGAAAGGCAAGAACAGGGTGCTATCTTTTGCCTACCATTG-3' and its complement and primer 5'-GTCTCTTTTCGGTATTTTGGCAGGGTTTCTTGAATGAGAGCATGGC -3' and its complement to generate, respectively, plasmids, pMRS4-lacZd (mut1) and pMRS4-lacZd (mut2) that contain CACCC to CAGGG mutations in individual FeRE-like sequences. To generate a reporter construct that contains the FeRE of MRS4 within the {Delta}UAS CYC1 promoter fused to the lacZ gene, 200 pmol of each of the partial overlapping oligos 5'-tcgaAAGAAAGGGTGCCAAAA-3' and 5'-ctagTTTTGGCACCCTTTCTT-3' were mixed in SSC (final 0.32x), boiled for 15 min, and then incubated overnight at 55 °C. The annealed products were then ligated into the XhoI/XbaI sites of pNB404 (22) to generate pMRS4-FeRE. All PCR-generated sequences were confirmed by DNA sequencing. All yeast transformations were performed using the lithium acetate procedure. In the case of transformations using aft1{Delta}aft2{Delta} strains, cells were pregrown in YPD under nitrogen and agar plates supplemented with FeCl2 (100 µM).

mRNA Quantification by S1 Nuclease Analysis—Strain aft1{Delta}aft2{Delta} (MAT {alpha} his3{Delta}1 leu2{Delta}0 ura3{Delta}0 aft1::kanMX4 aft2::kanMX4) was used for transformation of all lacZ reporter constructs with separately pAFT1-1up, pAFT2-1up or pRS413. Cells were harvested at mid-log phase, and total RNA was extracted using the hot acidic phenol method (31). DNA oligonucleotides, with sequences complementary to lacZ, FIT3 and CMD1 mRNA (calmodulin as an internal loading control), were end-labeled with 32P using T4 polynucleotide kinase (New England Biolabs). S1 analysis was carried out as previously described (1). Briefly, the 32P-labeled oligonucleotides were hybridized with 12 µg of total RNA in HEPES buffer (38 mM HEPES, pH 7.0, 0.3 M NaCl, 1 mM EDTA, 0.1% Triton X-100) at 55 °C overnight. The reactions were then treated with 35 units of S1 nuclease (Promega), and the resulting DNA:RNA double-stranded duplexes were ethanol-precipitated and heat-denatured in formamide buffer. The samples were then separated using an 8% polyacrylamide/8.3 M urea gel.

Microarray Analysis—Strain aft1{Delta}aft2{Delta} (MATa his3{Delta}1 leu2{Delta}0 lys2{Delta}0 met15{Delta}0 ura3{Delta}0 aft1::kanMX4 aft2::kanMX4) was transformed with separately, pAFT1-1up, pAFT2-1up and their corresponding parental vectors as controls (pRS316, pRS416). Cells were grown in 300 ml of CMD-Ura medium and harvested at an OD600 nm of 0.4. Total RNA was isolated by the hot acidic phenol method and mRNA was isolated from total RNA using the PolyATtract mRNA Isolation System IV kit (Promega) following the manufacturer's instructions. mRNA (1.2 µg) was used to generate cDNA probes by reverse transcription (Superscript II, GIBCO) with incorporation of Cy3-dCTP or Cy5-dCTP (Amersham Biosciences). The arrayed slides were produced as follows. The PCR amplification products of all the S. cerevisiae open reading frames (ORFs) were purchased from Research Genetics. Each PCR product was reamplified and purified on glass fiber filter plates (Millipore). The reamplified products representing ~6000 ORFs (4% not represented) were diluted to 50% Me2SO and spotted in duplicate on 3-aminopropyl-methyldiethoxy silane-coated slides using a Generation III Microarray Spotter (Amersham Biosciences). The cDNA-labeled probes were then hybridized onto an arrayed slide and fluorescence was captured with a GEN III dual-laser confocal scanner (Molecular Dynamics). Fluorescent intensities were quantified using Arrayvision 6.0 (Imaging Research). The ratios of signal intensities (Cy5/Cy3) were normalized by the median output signal intensity for all genes, and the normalized data for three independent experiments were averaged. The data were filtered to remove those measurements that were not higher than the slides' background fluorescence and assembled into EXCEL spreadsheets. Analysis of the potential regulatory sequences within the most highly induced genes was carried out using the program GeneSpring 3.2 (Silicon Genetics). The EXCEL spreadsheets containing the normalized, filtered, and averaged data are available as Supplemental Material.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Microarray Analysis of Aft1p and Aft2p Regulons—Previous studies have shown that Aft1p and Aft2p differentially activate the expression of a subset of the iron-regulated genes in S. cerevisiae (15). We were interested to learn the effect of Aft1p and Aft2p on global gene expression in cells of the same genetic background. DNA microarray analysis was initiated to compare the expression profile resulting from the AFT1-1up allele and, separately, the AFT2-1up allele. The aft1{Delta}aft2{Delta} strain was used into which pAFT1-1up, pAFT2-1up and control vectors had been separately transformed. The AFT1-1up/AFT2-1up alleles were expressed from their own promoters in low copy plasmids, so as to minimize the overexpression of the gain of function alleles. The transcriptional profiles of the pAFT1-1up- and the pAFT2-1up-containing strains were separately compared with that of control strains. Each experiment was carried out independently on three occasions. The average induction ratio for each gene was calculated, and the genes that were most highly induced in cells containing the AFT1-1up and AFT2-1up alleles were identified (Tables I and II, Fig. 1). These data will include genes that are the direct targets of Aft1p and/or Aft2p or genes that are activated as the result of the indirect effects of Aft1p/Aft2p. A complete data set can be found in the Supplemental Material.


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TABLE I
Genes induced by AFT1-1up

Microarray analysis was carried out comparing transcript levels of control aft1{Delta}aft2{Delta} cells and cells containing pAFT1-1up. The 25 most highly induced genes are shown. The mean and S.D. of the Cy5/Cy3 ratios of three independent experiments are listed. Also shown are the equivalent data (mean, S.D.) from the experiments involving pAFT2-1up. Listed is the number of consensus FeRE sites (PyPuCACCC) within 1 kb of the start codon of each gene. Notes: (1) AFT1 is not present in the control strain. (2) YDR271C overlaps with CCC2 - a known iron regulated gene. (3) A transcript is not detected (ND) when the signal is not greater than the background signal.

 

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TABLE II
Genes induced by AFT2-1up

Microarray analysis was carried out comparing transcript levels of control aft1{Delta}aft2{Delta} cells and cells containing pAFT2-1up. The 25 most highly induced genes are shown. The mean and the S.D. of the Cy5/Cy3 ratios of three independent experiments are listed. Also shown are the equivalent data (mean, S.D.) from the experiments involving pAFT1-1up. Listed is the number of consensus FeRE sites (PyPuCACCC) within 1 kb of the start codon of each gene. Notes: (1) AFT2 is not present in the control strain (2) YOR225w overlaps with ISU2- a known iron regulated gene (3). A transcript is not detected (ND) when the signal is not greater than the background signal.

 


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FIG. 1.
Summary of the genes induced by Aft1p and Aft2p. The overlap represents those genes that are induced by both Aft1p and Aft2p. Those genes that are known to encode proteins that are involved in iron homeostasis are shaded. AFT1 and AFT2 are excluded.

 

Many of the genes identified as being induced as the result of the AFT1-1up/AFT2-1up alleles are known to code for proteins that are involved in iron metabolism. Some of these genes are induced to a similar extent as the result of either Aft1p or Aft2p (e.g. FRE6, ATX1, COT1). However, the more highly induced genes tend to be activated to a greater extent when Aft1p is present (e.g. FIT3, FIT2, FTR1). There are examples of genes that contain a consensus FeRE but which are only induced as the result of one of the Aft1p/Aft2p factors. These genes include the Aft1p-specific FRE2 and ARN2 and the Aft2p-specific BNA2 and ECM4. BNA2 codes for a protein with similarity to indoleamine 2,3-dioxygenases and Ecm4p is involved in cell wall biosynthesis, although its precise function is not known. Included in the group of genes that are preferentially induced as the result of Aft1p is YOR387C. Interestingly, YOR387C is located within a region of chromosome XV that contains a group of genes (FIT2, FIT3, FRE3) that are highly induced in cells containing either AFT1-1up or AFT2-1up. YOR387C codes for a 206-amino acid protein that shares 93% identity with the mitochondrial Vel1p from S. cerevisiae (23). Common to both experiments are OYE3 and YHL035C, which have not previously been identified as targets of the Aft1p/Aft2p factors. OYE3 codes for a NADPH dehydrogenase and does not contain a consensus FeRE within its 5'-upstream region and consequently may be induced as an indirect result of the Aft1p/Aft2p factors. YHL035C has one consensus FeRE within its 5'-upstream region and codes for a member of the ATP-binding cassette (ABC) family of transporters, however its localization and function are unknown.

Integration of AFT2-1up Allele—To confirm that the use of plasmid-borne constitutive alleles for the microarray experiments was an appropriate strategy, the AFT2-1up locus, under the control of its own promoter and terminator, was integrated into the HIS3 locus of the aft1{Delta}aft2{Delta} strain. S1 nuclease protection assays were used to analyze the expression of FIT3 in the aft1{Delta}aft2{Delta} strain containing a control plasmid or pAFT2-1up, the aft1{Delta}aft2{Delta} strain with the integrated AFT2-1up allele and the aft1{Delta} strain. The expression of FIT3 that resulted from the plasmid-borne AFT2-1up allele was only slightly greater than that of the integrated AFT2-1up allele (Fig. 2). Consequently, it is unlikely that the microarray data include genes that have been artificially induced through overexpression of the AFT1-1up and AFT2-1up alleles. Interestingly, the AFT2-1up allele still responds to iron to some extent when it is present in the chromosome, but that response is lost in the strain containing the plasmid-borne AFT2-1up allele.



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FIG. 2.
Comparison of activation of the expression of FIT3 by the chromosomal and plasmid-borne AFT2-1up allele. S1 nuclease protection assays were used to quantify mRNA level of FIT3. RNA was isolated from the aft1{Delta}aft2{Delta} strain transformed with a control plasmid or pAFT2-1up and the aft1{Delta}aft2{Delta} strain into which the AFT2-1up allele had been integrated into the HIS3 locus (aft1{Delta}AFT2-1up). Each strain was grown in CMD (-His) medium with or without added iron (100 µM) to mid-log phase. RNA was also isolated from the aft1{Delta} strain that had been grown in CMD with and without the iron chelator BPS (100 µM). The upper band for each sample is the FIT3 gene, and the lower band is the calmodulin-loading control (CMD1).

 

In Vivo Analysis of FET3 FeRE—Truncated Aft1p and Aft2p that contain the N-terminal basic regions of each protein, are able to bind to the FET3 FeRE in vitro (15). As both the AFT1-1up and AFT2-1up alleles activate the expression of many of the same genes, we initially used two reporter constructs to learn if both factors act through the same FeRE in vivo. The first reporter construct contains 500 bp of the FET3 5'-upstream region, which has 3 FeRE-like sequences, fused to the lacZ reporter gene (pFET3-lacZ)(Fig. 3B). A mutated fusion gene was generated in which the core CACCC sequence of the FeRE closest to the lacZ start codon was changed to the sequence, CAGGG. This mutation is known to create a non-functional FeRE (14). The second reporter construct consists of the same FET3 single FeRE within a minimal CYC1 promoter/lacZ reporter construct that lacks an upstream activating sequence (pFC-W). The corresponding control vector contains a non-functional CAGGG sequence within the FeRE (pFC-LM2) (14). Plasmids pFET-lacZ and pFC-W, and their corresponding control vectors, were transformed separately into the aft1{Delta}aft2{Delta} strain containing either pAFT1-1up or pAFT2-1up. S1 nuclease protection assays were used to analyze the expression of the lacZ reporter genes. Both the AFT1-1up and AFT2-1up alleles induced the expression of lacZ in pFET-lacZ and pFC-W, but not the control reporter genes containing a non-functional FeRE (Fig. 3C). In both cases, the AFT2-1up induced expression of the reporter genes was weaker than the activation by the AFT1-1up allele. The AFT2-1up allele is a strong activator of the chromosomal FET3 (15), so the minimal effects of this allele with these particular reporter constructs may be due to the lack of upstream sequences of the FET3 promoter region that are absent in pFET3-lacZ and pFC-W.



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FIG. 3.
Aft1p and Aft2p regulate the expression of FET3 through the same FeRE. A, the nucleotide sequences of 3 FeRE-like elements within 500 bp of the start codon of FET3. Nucleotides that deviate from the consensus FeRE sequence identified in Ref. 14 are underlined. B, plasmid pFC-W contains the –251 FeRE that has been inserted into the CYC1 promoter and fused to the lacZ gene. Plasmid pFET3-lacZ contains 500 bp of the upstream region of FET3 fused to the lacZ gene. Ovals represent FeRE-like sequences (filled in) or mutated FeREs (hatched). C, S1 nuclease protection assays were used to quantify mRNA levels of lacZ. RNA was isolated from the aft1{Delta}aft2{Delta} strain that had been separately transformed with pRS413 (control), pAFT1-1up and pAFT2-1up together with the lacZ reporter constructs pFC or pFET3-lacZ and their respective control plasmids [pFC-W and pFET3-lacZ (mut)]. Each strain was grown in CMD (-His/Ura) to midlog phase. The upper band for each sample is the lacZ gene. and the lower band is the calmodulin-loading control (CMD1).

 

In Vivo Analysis of FIT3 FeRE—The most highly induced gene by the AFT1-1up and AFT2-1up alleles is FIT3 (Tables I and II). To analyze the extent that both Aft1p and Aft2p activate the expression of FIT3 through the same FeRE, we generated a reporter construct, containing 1 kb of the FIT3 5'-region fused to lacZ (pFIT3-lacZ). It was anticipated that the larger 5'-upstream region would be representative of the AFT2-1up induced expression of the chromosomal copy of FIT3. There are 4 potential FeRE sites within the 1-kb 5'-region of FIT3 that each deviate from the consensus FeRE by one nucleotide (Fig. 4A). To see if loss of an FeRE would have the same effect for both Aft1p- and Aft2p-dependent activation, we generated a FIT3-lacZ fusion gene that contained mutations in two of these potential FeRE sites. The core CACCC FeRE sequence was changed to the non-functional CAGGG (Fig. 4B). Plasmid pFIT3-lacZ and its control were transformed separately into the aft1{Delta}aft2{Delta} strain that contained either pAFT1-1up or pAFT2-1up. Disruption of the two FeRE sites attenuates both the AFT1-1up and AFT2-1up induced expression of the FIT3-lacZ fusion (Fig. 4C). Both of these FeRE sites deviate from the consensus FeRE in their most 3'-nucleotide, consistent with this nucleotide being unimportant for recognition by Aft1p and Aft2p. In agreement with the microarray data, the AFT1-1up allele is a stronger activator than the AFT2-1up allele of the expression of FIT3. Together, the analysis of the expression of the FET3-lacZ and FIT3-lacZ fusion genes is consistent with the AFT1-1up and AFT2-1up alleles inducing expression through the same core FeRE.



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FIG. 4.
Aft1p and Aft2p regulate the expression of FIT3 through the same FeRE. A, the nucleotide sequences of 4 FeRE-like elements within 1 kb of the start codon of FIT3. Nucleotides that deviate from the consensus FeRE sequence identified in Ref. 14 are underlined. B, plasmid pFET3-lacZ contains 1 kb of the upstream region of FIT3 fused to the lacZ gene. Ovals represent FeRE-like sequences (filled in) or mutated FeREs (hatched). C, S1 nuclease protection assays were used to quantify mRNA levels of lacZ. RNA was isolated from the aft1{Delta}aft2{Delta} strain that had been separately transformed with pRS413 (control), pAFT1-1up and pAFT2-1up together with the lacZ reporter constructs pFIT3-lacZ and its control pFIT3-lacZ (mut). Each strain was grown in CMD (-His/Ura) to mid-log phase. The upper band for each sample is the lacZ gene, and the lower band is the calmodulin-loading control (CMD1).

 

In Vivo Analysis of MRS4 FeRE—We have previously shown that MRS4 is preferentially induced by the AFT2-1up allele (15). Interestingly, the 1-kb 5'-upstream region of MRS4 contains 5 FeRE-like sequences that each deviate from the consensus FeRE by 2 nucleotides (Fig. 5A). To determine whether the AFT1-1up and AFT2-1up alleles activate the expression of MRS4 through these non-consensus FeRE sites, a series of MRS4-lacZ fusions were generated which contain truncations of the MRS4 upstream region (Fig. 5B). These reporter constructs were transformed separately into the aft1{Delta}aft2{Delta} strain containing either pAFT1-1up or pAFT2-1up. The presence of at least two putative FeRE-like sites resulted in AFT2-1up-dependent lacZ expression, whereas the fusion with only one putative FeRE-like site did not (Fig. 5C). Similar results were observed in cells that contained pAFT1-1up (Fig. 5C). Consistent with the microarray data, the AFT2-1up allele is a stronger activator than AFT1-1up of the expression of the MRS4-lacZ fusion (Fig. 5C). To confirm that Aft1p and Aft2p induce expression through one of these two FeRE-like sequences, each sequence within the MRS4-lacZ fusion containing the two proximal sites (–193 and –238 bp upstream of the ATG) was mutated to a non-functional FeRE site. The MRS4-lacZ fusion with the mutation in the –193 site retained full expression, whereas the mutation of the –238 site resulted in the loss of Aft1p- and Aft2p-dependent expression (Fig. 6). Therefore, Aft1p and Aft2p activate the expression of MRS4 through a variant FeRE.



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FIG. 5.
Aft1p and Aft2p regulate the expression of MRS4 through a variant FeRE. A, the nucleotide sequences of 5 FeRE-like elements within 1 kb of the start codon of MRS4. Nucleotides that deviate from the consensus FeRE sequence identified in Ref. 14 are underlined. B, plasmids pMRS4-lacZ (a–f) contain sequential deletions of 1 kb of the upstream region of MRS4 fused to the lacZ gene. Ovals represent FeRE-like sequences. C, S1 nuclease protection assays were used to quantify mRNA levels of lacZ. RNA was isolated from the aft1{Delta}aft2{Delta} strain that had been separately transformed with pRS413 (control), pAFT1-1up and pAFT2-1up together with the lacZ reporter constructs pMRS4-lacZ (a–f). The level of lacZ expresion was qualitatively similar in the strains containing pMRS4-lacZe/f in the presence of pRS413 (control), pAFT1-1up and pAFT2-1up. Each strain was grown in CMD (-His/Ura) to mid-log phase. The upper band for each sample is the lacZ gene, and the lower band is the calmodulin-loading control (CMD1).

 


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FIG. 6.
Aft1p and Aft2p regulate the expression of MRS4 through a variant FeRE. A, plasmid pMRS4-lacZd contains 259 bp of the upstream region of MRS4 fused to the lacZ gene. Ovals represent FeRE-like sequences (filled in) or mutated FeREs (hatched). B, S1 nuclease protection assays were used to quantify mRNA levels of lacZ. RNA was isolated from the aft1{Delta}aft2{Delta} strain that had been separately transformed with pRS413 (control), pAFT1-1up, and pAFT2-1up together with the lacZ reporter construct pMRS4-lacZ d (that contains two FeRE-like sequences) and control plasmids, in which each FeRE-like sequence had been individually mutated to a non-functional FeRE. Each strain was grown in CMD (-His/Ura) to mid-log phase. The upper band for each sample is the lacZ gene, and the lower band is the calmodulin-loading control (CMD1).

 

The preferential expression from the MRS4 FeRE by Aft2p could be due to Aft2p having a higher affinity for that particular site and/or may be dependent on other sequences in the MRS4 promoter region. To analyze the ability of Aft1p and Aft2p to activate transcription via this FeRE in a different promoter context, the sequence TTTTGGCACCCTTTCTT was cloned into the multiple cloning site (MCS) of pNB404, that is within a {Delta}UAS CYC1 promoter that is fused to the lacZ gene. This reporter construct was transformed separately into the aft1{Delta}aft2{Delta} strain containing either pAFT1-1up or pAFT2-1up. The highest expression of the reporter gene was detected in the strain containing the AFT2-1up allele (Fig. 7). A similar reporter construct in which the same sequence was cloned into the MCS of pNB404 in the opposite orientation gave the same pattern of expression (data not shown). The preferential activation of transcription by Aft2p from the MRS4 FeRE is therefore not dependent on other sequences within the MRS4 promoter region.



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FIG. 7.
The MRS4 FeRE is preferentially recognized by Aft2p. RNA was isolated from the aft1{Delta}aft2{Delta} strain that had been separately transformed with the control pRS413 (control), pAFT1-1up (1up) and pAFT2-1up (2up) together with the lacZ reporter construct pMRS4-FeRE (that contains the active UAS identified in Fig. 6 cloned into the {Delta}UAS CYC1 promoter that is fused to lacZ). Each strain was grown in CMD (-His/Ura) to mid-log phase. S1 nuclease protection assays were used to quantify mRNA levels of lacZ. The upper band for each sample is the lacZ gene, and the lower band is the calmodulin loading control (CMD1).

 

Identification of an Extended FeRE—The program GeneSpring was used to identify the extent to which the known FeRE appears within the 5'-upstream region of the genes identified by the microarray analysis. Initially, a search was carried out to identify any potential regulatory sequences within 1 kb of the start codon the genes within Tables I and II (probability cutoff of 0.05). The only sequence that was identified was TGCACCC. This sequence is consistent with the known FeRE except that it lacks the most 3'-nucleotide, which is consistent with our in vivo analysis of the regulation of FIT3. We then identified those genes within Tables I and II that contain the sequence PyPuCACCC (the consensus FeRE lacking the 3'-nucleotide) in either orientation within 1 kb of their start codon. Of the 40 genes that were analyzed, 29 contain at least one copy of this FeRE (10 are common to both lists, Fig. 1). This corresponds to this FeRE being present in 73% of the genes analyzed. A similar analysis of the whole genome identified 17% of all open reading frames (ORFs) having a least one FeRE within the same 5'-upstream region. During this analysis we noted that many of the identified FeRE sequences were preceded by AT-rich regions. We therefore searched for those genes that contain at least one copy of the sequence A/TA/TA/TPyPuCACCC in either orientation within their 5'-upstream region. Of the 40 genes in Tables I and II, 15 genes contain this extended FeRE (38% of genes analyzed) compared with 240 ORFs within the entire genome (4% of the genes analyzed). This represents a significant enrichment of genes that contain an extended FeRE in that group of genes that are induced in cells containing either of the AFT1-1up or AFT2-1up alleles.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
S. cerevisiae contains two paralogous iron-responsive transcription factors, Aft1p and Aft2p (15). In the present study, the AFT1-1up and AFT2-1up alleles were used to compare the global gene expression pattern that is mediated by each factor. We show for the first time that the AFT1-1up and AFT2-1up alleles result in distinct global transcriptional profiles when they are expressed separately within the same strain. The genes that are induced by Aft1p and Aft2p fall into a number of different categories. There are clearly some genes that encode for components of iron metabolism that are induced to an equivalent extent by both Aft1p and Aft2p. However, a group of the most highly activated iron-regulated genes are more highly induced by Aft1p. For example, expression of FET3 is stimulated 25-fold by the AFT1-1up allele compared with 4-fold by the AFT2-1up allele. There are genes that contain a consensus FeRE that are induced by either Aft1p or Aft2p but not both. In the case of the Aft1p-specific genes FRE2 and ARN2, the known iron-related function of these genes is consistent with Aft1p directly regulating these genes. In the case of the Aft2p-specific genes BNA2 and ECM4, it is not known if the FeRE sequences within their promoter regions are active and, consequently, if Aft2p directly regulates these genes. There are also genes that do not contain a consensus FeRE, or an FeRE-like sequence, within their upstream region, that are induced in cells that contain the gain of function alleles. The induction of these genes is assumed to be as the result of the indirect effect of Aft1p and Aft2p. The differential activation of YAP2 by Aft1p and Aft2p is interesting. Yap2p is itself a transcription factor and its own differential expression by Aft1p and Aft2p may be responsible, in part, for the different Aft1p- and Aft2p-dependent global gene expression. The unique transcriptional profiles therefore result from the varying direct gene activation by Aft1p and Aft2p and the resulting indirect effects of the expression of genes that are not in the iron regulon.

In general our microarray data are consistent with other published microarray data that have identified genes that are regulated in response to iron. These include individual genes that are activated by the AFT1-1up allele, genes within the iron regulon that are activated indirectly in response to cobalt stress and genes that are activated in a strain that lacks a functional Yfh1p, a protein that is involved in mitochondrial iron-sulfur cluster formation (10, 12, 13, 14, 25, 26). There are, however, differences between these data sets. For example, the direct analysis of the gain of function mutants found a number of genes that are activated by the Aft1p/Aft2p factors but which are not induced in a yfh1{Delta} strain. These genes include BIO5, OYE3, YHL035c, ECM4, BNA2, and MRS4. In this particular case, these differences in global gene expression may arise from the pleiotropic effects of mitochondrial dysfunction in a yfh1{Delta} strain.

Analysis of the promoter regions of the genes that are most highly induced by the Aft1p/Aft2p factors led to the identification of an extended FeRE. This extended FeRE includes the previously identified consensus FeRE with an additional 5' AT-rich region. It is known that extended regions of homopolymeric (dA:dT) can influence local chromatin structure and thereby influence transcription factor function (24). Further work is required to understand the significance, if any, of this extended FeRE.

Using low copy vectors, we have shown for the first time that Aft1p and Aft2p mediate gene activation through the same FeREs in vivo. Both Aft1p and Aft2p activate the expression of a lacZ reporter gene that is under the control of a minimal promoter that contains the consensus FeRE from FET3. In a similar experiment using another reporter construct, mutation of two FeRE-like sequences within the FIT3 promoter region, results in the reduction of the AFT1-1up or AFT2-1up-dependent activation of a FIT3-lacZ reporter gene. It was noted that both these FeRE sequences deviate from the consensus FeRE at the most 3'-nucleotide. These data are consistent with the observation that a strain containing an AFT1 allele (aft1-1{Delta}) encoding only the N-terminal 440 residues of Aft1p, and thereby lacking the C-terminal transactivation domain, is more sensitive to low iron conditions than the aft1{Delta} strain (17). Such a phenotype might arise if the truncated Aft1p was able to compete successfully with Aft2p for binding to particular FeREs, and thereby prevent Aft2p from activating the iron regulon.

In addition to transcriptional control mediated via a consensus FeRE, we have also shown that both Aft1p and Aft2p regulate MRS4 through a variant FeRE. Mrs4p has been implicated as an important mitochondrial iron transporter (27). MRS4 contains five FeRE-like sequences within its upstream region that contain the core CACCC sequence. The –238 MRS4 site is clearly an active FeRE, as mutation of the CACCC core in this site prevents the activation of a MRS4-lacZ reporter gene by Aft1p and Aft2p. In addition, when this FeRE is placed within a heterologous promoter it mediates transcriptional activation in an Aft2p-specific manner. This is consistent with the preferential activation of MRS4 by Aft2p being due to the variant FeRE itself and not other sequences within the MRS4 promoter region. This site (GGCACCC) differs from the consensus FeRE site as it contains two purines upstream of the CACCC core sequence. It is known that a DNA duplex containing a GGCACCC FeRE non-consensus sequence competes weakly with TGCACCC in an in vitro assay (14). The AFT2-1up allele, as compared with the AFT1-1up allele, is the more potent activator of MRS4. The existence of other functional FeRE sequences that deviate from the consensus FeRE and the possibility of preferential binding to different FeRE sequences by Aft1p and Aft2p are topics for future studies. It is, however, clear that in future Aft1p- and Aft2p-dependent regulation must not be dismissed based on the absence of a consensus FeRE within the promoter region of a particular gene.

The data from this study are consistent with both Aft1p and Aft2p regulating gene expression through similar promoter elements and each being responsible for different transcriptional profiles. To address the question of FeRE-dependent regulation by Aft1p and Aft2p it was necessary to undertake the work using the aft1{Delta}aft2{Delta} strain. This enabled each transcription factor to be studied separately without the concern of competition for the same promoter elements. The challenge now is to understand how Aft1p and Aft2p interact with FeREs when both are present in vivo. The paralogous factors Yap1p/Yap2p and Pdr1p/Pdr3p may bind as heterodimers to their respective palindromic promoter elements. The FeREs are not palindromes, and no work to date has addressed the oligomerization state of Aft1p or Aft2p. If Aft1p and Aft2p can form heterodimers, then generation of different transcriptional profiles would suggest that different global gene expression could be mediated by varying concentrations of Aft1p and Aft2p hetero- and homodimers. If Aft1p and Aft2p do not dimerize then they must bind to the FeREs independently of each other. The question therefore arises, to what extent do the Aft1p/Aft2p factors compete for the same sites and does each factor have the same or different affinities for particular FeREs? In addition, how does the localization of each factor influence the occupancy of particular FeREs? Aft1p moves from the cytosol to the nucleus under low iron conditions. If Aft2p is localized differently, it may be sensing a different cellular pool of iron that may influence the occupancy of the FeREs by Aft1p/Aft2p. We are in the process of analyzing the cellular localization of Aft2p in response to iron.

There are genes that are specifically regulated by one of the Aft1p/Aft2p factors. It is unclear why Aft1p, and not Aft2p, activate the iron responsive genes, FRE2 and ARN2. It is possible that other proteins are involved in the promoter selectivity of Aft1p and Aft2p at some promoters. Alternatively, specific gene activation may relate to the mechanism of transcriptional activation that is employed by Aft1p and Aft2p. There is little sequence conservation in the C-terminal activation domains of each factor. It would be interesting to learn if both Aft1p and Aft2p can bind to the same upstream region of genes that contain multiple FeRE sequences. Conceptually similar to Aft1p/Aft2p hetero- and homodimers binding to the same FeRE, the binding of both Aft1p and Aft2p factors to multiple FeREs in the same promoter region could result in a graded response to iron levels depending on the relative levels of each factor. Transcriptional autoregulation by AFT1 has been demonstrated (AFT2 was not analyzed) (28), and we have confirmed microarray data (29) using S1 analysis, that AFT2, but not AFT1, is induced in response to treatment with the alkylating agent methyl methanesulfonate (data not shown). This is consistent with other microarray data that have shown that paralogous genes that have similar functions can be differentially regulated in response to different environmental conditions (30). Therefore, there may be a selective advantage of an Aft1p- or an Aft2p-dependent transcriptional profile under a particular set of cellular conditions. Further work analyzing promoter occupancy by both Aft1p and Aft2p under different cellular conditions in vivo and detailed footprinting of Aft1p and Aft2p binding to different FeREs in vitro will further our understanding of iron-dependent transcriptional regulation in S. cerevisiae.


    FOOTNOTES
 
* This research was supported by Grant CA 61286 from NCI, National Institutes of Health (to D. R. W.) and Microarray Supplemental Award ES03817 from the National Institutes of Environmental Health Sciences (to D. R. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains EXCEL spreadsheets containing the normalized, filtered, and averaged data. Back

{ddagger} To whom correspondence should be addressed. Tel.: 801-585-5103; Fax: 801-585-5469; E-mail: dennis.winge{at}hsc.utah.edu.

1 The abbreviations used are: FeRE, iron-responsive element; ORF, open reading frame; CMD, complete-synthetic medium; UAS, upstream activation sequence. Back


    ACKNOWLEDGMENTS
 
We thank the excellent technical assistance of the University of Utah Microarray Core Facility led by Dr. Brian Dalley and supported by the Huntsmann Cancer Foundation. We acknowledge National Institutes of Health Grant 5P30-CA 42014 to the Biotechnology Core Facility for DNA synthesis at the University of Utah.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dohrmann, P. R., Butler, G., Tamai, K., Dorland, S., Greene, J. R., Thiele, D. J., and Stillman, D. J. (1992) Genes Dev. 6, 93–104[Abstract]
  2. Dohrmann, P. R., Voth, W. P., and Stillman, D. J. (1996) Mol. Cell. Biol. 16, 1746–1758[Abstract]
  3. Toone, W. M., Johnson, A. L., Banks, G. R., Toyn, J. H., Stuart, D., Wittenberg, C., and Johnston, L. H. (1995) EMBO J. 14, 5824–5832[Abstract]
  4. Doolin, M. T., Johnson, A. L., Johnston, L. H., and Butler, G. (2001) Mol. Microbiol. 40, 422–432[CrossRef][Medline] [Order article via Infotrieve]
  5. Mamnun, Y. M., Pandjaitan, R., Mahe, Y., Delahodde, A., and Kuchler, K. (2002) Mol. Microbiol. 46, 1429–1440[CrossRef][Medline] [Order article via Infotrieve]
  6. Fernandes, L., Rodrigues-Pousada, C., and Struhl, K. (1997) Mol. Biol. Cell 17, 6982–6993
  7. Cohen, B. A., Pilpel, Y., Mitra, R. D., and Church, G. M. (2002) Mol. Biol. Cell 13, 1608–1614[Abstract/Free Full Text]
  8. Martins, L., Jensen, L. T., Simon, J. R., Keller, G., and Winge, D. R. (1997) J. Biol. Chem. 273, 23716–23721[Abstract/Free Full Text]
  9. Dancis, A. (1998) J. Pediatr. 12, S24-S29
  10. Yun, C.-W., Ferea, T., Rashford, J., Ardon, O., Brown, P. O., Botstein, D., Kaplan, J., and Philpott, C. C. (2000) J. Biol. Chem. 275, 10709–10715[Abstract/Free Full Text]
  11. Yun, C.-W., Tiedeman, J. S., Moore, R. E., and Philpott, C. C. (2000) J. Biol. Chem. 275, 16354–16359[Abstract/Free Full Text]
  12. Foury, F., and Talibi, D. (2001) J. Biol. Chem. 276, 7762–7768[Abstract/Free Full Text]
  13. Protchenko, O., Ferea, T., Rashford, J., Tiedeman, J., Brown, P. O., Botstein, D., and Philpott, C. C. (2001) J. Biol. Chem. 276, 49244–49250[Abstract/Free Full Text]
  14. Yamaguchi-iwai, Y., Stearman, R., Dancis, A., and Klausner, D. (1996) EMBO J. 15, 3377–3384[Abstract]
  15. Rutherford, J. C., Jaron S., Ray, E., Brown, P. O., and Winge, D. R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 14322–14327[Abstract/Free Full Text]
  16. Yamaguchi-Iwai, Y., Ueta, R., Fukunaka, A., and Sasaki, R. (2002) J. Biol. Chem. 277, 18914–18918[Abstract/Free Full Text]
  17. Casas, C., Aldea, M., Espinet, C., Gallego, C., Gil, R., and Herero, E. (1997) Yeast 13, 621–637[CrossRef][Medline] [Order article via Infotrieve]
  18. Blaiseau, P.-L., Lesuisse, E., and Camadro, J.-M. (2001) J. Biol. Chem. 276, 34221–34226[Abstract/Free Full Text]
  19. Portnoy, M. E., Jensen, L. T., and Culotta, V. C. (2002) Biochem. J. 362, 119–124[CrossRef][Medline] [Order article via Infotrieve]
  20. Myers, A. M., Tzagoloff, A., Kinney, D. M., and Lusty, C. J. (1986) Gene 45, 299–310[CrossRef][Medline] [Order article via Infotrieve]
  21. Li, L., and Kaplan, J. (2001) J. Biol. Chem. 276, 5036–5043[Abstract/Free Full Text]
  22. Bachhawat, N., Ouyang, Q., and Henry, S. A. (1995) J. Biol. Chem. 270, 25087–25095[Abstract/Free Full Text]
  23. Kumar, A., Agarwal, S., Heyman, J. A., Matson, S., Heidtman, M., Piccirillo, S., Umansky, L., Drawid, A., Jansen, R., Liu, Y., Cheung, K. H., Miller, P., Gerstein, M., Roeder, G. S., and Snyder, M. (2002) Genes Dev. 16, 707–719[Abstract/Free Full Text]
  24. Lascaris, R. F., Groot, E., Hoen, P. B., Mager, W. H., and Planta, R. J. (2000) Nucleic Acids Res. 28, 1390–1396[Abstract/Free Full Text]
  25. Chen, O. S., Hemenway, S., and Kaplan, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 12321–12326[Abstract/Free Full Text]
  26. Stadler, J. A., and Schweyen R. J. (2002) J. Biol. Chem. 277, 39649–39654[Abstract/Free Full Text]
  27. Foury, F., and Roganti, T. (2002) J. Biol. Chem. 277, 24475–24483[Abstract/Free Full Text]
  28. Lee, T. I., Rinaldi, N. J., Robert, F., Odom, D. T., Bar-Joseph, Z., Gerber, G. K., Hannett, N. M., Harbison, C. T., Thompson, C. M., Simon, I., Zeitlinger, J., Jennings, E. G., Murray, H. L., Gordon, D. B., Ren, B., Wyrick, J. J., Tagne, J. B., Volkert, T. L., Fraenkel, E., Gifford, D. K., and Young, R. A. (2002) Science 298, 799–804[Abstract/Free Full Text]
  29. Jelinsky, S. A., Estep, P., Church, G. M., and Samson, L. D. (2000) Mol. Cell. Biol. 20, 8157–8167[Abstract/Free Full Text]
  30. Gasch, A. P., Spellman, P. T., Kao, C. M., Carmel-Harel, O., Eisen, M. B., Storz, G., Botstein, D., and Brown, P. O. (2000) Mol. Biol. Cell 11, 4241–4257[Abstract/Free Full Text]
  31. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1997) Current Protocols in Molecular Biology, Vol. 2, p. 13.0.1, John Wiley & Sons, Inc., New York