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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (ARN14), iron reductases (FRE16), 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 413572 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) 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
strain
(15). The aft2
strain has no growth phenotype on iron-limiting medium
(15). However, the
aft1
aft2
strain is more sensitive to
iron-limiting conditions than the single aft1
strain
(15,
18). The aft1
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vectors and Fusion GenesThe 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 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
aft2
strains, cells were pregrown in YPD
under nitrogen and agar plates supplemented with FeCl2 (100
µM).
mRNA Quantification by S1 Nuclease AnalysisStrain
aft1aft2
(MAT
his3
1 leu2
0 ura3
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 AnalysisStrain
aft1aft2
(MATa his3
1
leu2
0 lys2
0 met15
0
ura3
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
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 AlleleTo 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 aft1aft2
strain. S1
nuclease protection assays were used to analyze the expression of
FIT3 in the aft1
aft2
strain
containing a control plasmid or pAFT2-1up, the
aft1
aft2
strain with the integrated
AFT2-1up allele and the aft1
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.
|
In Vivo Analysis of FET3 FeRETruncated 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 aft1aft2
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.
|
In Vivo Analysis of FIT3 FeREThe 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 aft1aft2
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.
|
In Vivo Analysis of MRS4 FeREWe 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 aft1aft2
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.
|
|
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 UAS
CYC1 promoter that is fused to the lacZ gene. This reporter
construct was transformed separately into the
aft1
aft2
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.
|
Identification of an Extended FeREThe 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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
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) 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
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 aft1aft2
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 |
---|
The on-line version of this article (available at
http://www.jbc.org)
contains EXCEL spreadsheets containing the normalized, filtered, and averaged
data.
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.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|