© 2000 Oxford University Press Nucleic Acids Research, 2000, Vol. 28, No.

7 1499–1505

SURVEY AND SUMMARY

Saccharomyces cerevisiae basic helix–loop–helix
proteins regulate diverse biological processes
Kelly A. Robinson and John M. Lopes1,*
Department of Molecular and Cellular Biochemistry, Loyola University of Chicago, 2160 South First Avenue,
Maywood, IL 60153, USA and 1Department of Biological Sciences, Wayne State University, 5047 Gullen Mall,
Detroit, MI 48202, USA

Received December 16, 1999; Revised and Accepted February 14, 2000

ABSTRACT of each bHLH protein dictate specificity to direct different

bHLH proteins to different target sites. This is best evidenced
Basic helix–loop–helix (bHLH) proteins are among
by a mutant form of MyoD that contains a change in the basic
the most well studied and functionally important
region (L14 to R14) (Table 1) which allows it to recognize a c-Myc
regulatory proteins in all eukaryotes. The HLH binding site (5′-CACGTG-3′) instead of a MyoD binding site
domain dictates dimerization to create homo- and (5′-CAGCTG-3′) (4). The altered amino acid contacts the
heterodimers. Dimerization juxtaposes the basic fourth nucleotide of the core consensus binding site.
regions of the two monomers to create a DNA inter-
action surface that recognizes the consensus Table 1. Sequence alignment of the basic regions of
sequence called the E-box, 5′-CANNTG-3′. Several select bHLH proteins
bHLH proteins have been identified in the yeast
Saccharomyces cerevisiae using traditional genetic
methodologies. These proteins regulate diverse
biological pathways. The completed sequence of the
yeast genome, combined with novel methodologies
allowing whole-genome expression studies, now
offers a unique opportunity to study the function of
these bHLH proteins. It is the purpose of this review
to summarize the current knowledge of bHLH protein
function in yeast.

GENERAL PROPERTIES OF BASIC

HELIX–LOOP–HELIX (bHLH) PROTEINS
In mammals, bHLH proteins have critical roles in development,
cell growth, differentiation and apoptosis (1). The inaugural
members of this family of proteins were c-Myc and MyoD.
Both proteins have been studied extensively with respect to
functional domains and DNA binding specificity. From these
studies it is clear that the role of the HLH domain is to create
dimer combinations by interactions between the amphipathic
helices (2,3). The consequence of dimerization is the juxta-
position of two regions (extensions of helix 1) that are rich in Another important feature of bHLH proteins is their ability
basic residues (Table 1). to form multiple dimer combinations via the HLH domains.
The two basic regions create a DNA-binding interface which Max can dimerize with itself, c-Myc, Mad and Mxi1 (1).
interacts with the consensus sequence 5′-CANNTG-3′ (Table 2) However, there is specificity in terms of the dimers that each
(2,3). Since all bHLH proteins bind this core sequence, certain bHLH protein can form since c-Myc does not appear to form
conserved amino acids in the basic region allow for recognition of dimers with MyoD. In the case of Max, the choice for dimerization
the core consensus site. Specifically, a highly conserved is dependent on partner availability. The amount of Max is
glutamic acid (E9 in Table 1) contacts the ‘CA’ nucleotides of constant and abundant relative to c-Myc or Mad. The c-Myc
the consensus binding site. Other residues in the basic region protein is transiently expressed in growth factor-stimulated

*To whom correspondence should be addressed. Tel: +1 313 993 7816; Fax: +1 313 577 6891; Email: jlopes@sun.science.wayne.edu
1500 Nucleic Acids Research, 2000, Vol. 28, No. 7

cells and the Mad protein is expressed during differentiation. using the promoter of the PHO5 gene. This promoter contains
Thus, c-Myc/Max dimers are found in growth-stimulated cells two Pho4p binding sites (UASp1 and UASp2) which flank a
and Mad/Max dimers are observed during differentiation. Pho2p binding site (6). The binding of Pho4p to these sites is
The ability of bHLH proteins to form multiple dimer combi- required for the ~1000-fold induction of PHO5 gene expression in
nations is an efficient mechanism for regulation of gene response to phosphate starvation. Binding of Pho4p to UASp1
expression since different dimers are likely to target different and to a lesser degree UASp2 is enhanced cooperatively by
sets of genes. Therefore, it will be important to determine how binding of Pho2p (7). Pho2p binding is believed to disrupt
bHLH proteins coordinate gene expression in response to intra-molecular interactions within an internal repression
extracellular factors. This requires understanding the regulation of domain of Pho4p, thereby increasing access to the transcriptional
bHLH function and identification of the bHLH target genes. It activation domain (7,8).
is also important to identify all of the binding partners for each
bHLH protein. While it has been laborious to address these Table 3. Mutations of a conserved Glu (E9) in the basic domains of
questions in higher eukaryotic systems, yeast now provides a bHLH proteins
unique opportunity to quickly examine these issues using
genome-wide approaches. The answers to some of these questions
are already becoming clear from past recent studies of yeast
bHLH proteins. Here, we review the current knowledge of
yeast bHLH proteins and their target genes as a prelude to the
anticipated onslaught of information obtained from genome-
wide strategies.

Table 2. Compilation of S.cerevisiae bHLH proteins and their targets

Curiously, the core binding sites for Pho4p and Cbf1p

(another bHLH protein) are identical (Table 2). Although both
proteins bind the same sequence there is no evidence for over-
lapping functions. Over-expression of the PHO4 gene does not
complement the methionine auxotrophy of a cbf1 mutant strain
and over-expression of the CBF1 gene does not regulate PHO5
gene expression (9). An explanation for this apparent discrepancy
lies in the fact that the specificity for binding is dictated by
sequences that flank the core bHLH binding site. For example,
a T flanking 5′ to the core sequence inhibits the binding of
Pho4p but not Cbf1p (9). It is therefore not surprising to find
that the genes regulated by Pho4p contain UAS elements that
lack the T residue and that Cbf1p target sites typically contain
aThe core bHLH sequence is underlined. the T residue (9). The inability of Pho4p to recognize the core
sequence preceded by the T residue is known to be due to the
presence of a specific Glu residue in the basic region (E3 in
THE PHO4 SYSTEM Table 1) (9). When this Glu is changed to the Cbf1p counterpart
The PHO4 (YFR034C) gene was the first yeast gene shown to (D3 in Table 1), the mutant Pho4p is now able to recognize the
encode a bHLH protein (5). Sequence comparison to a large Cbf1p optimal binding site (core sequence flanked by the T).
number of mammalian and Drosophila bHLH proteins As is the case with c-Myc and MyoD, a highly conserved
revealed similarity in the bHLH region. The product of the Glu (E9 in Table 1) in the basic region is important for Pho4p
PHO4 gene binds to a sequence that contains the core bHLH binding to the core sequence. Analysis of the protein crystal
binding site, 5′-CACGTG-3′, which is found in the promoters structure demonstrated that this conserved Glu binds to the
of several genes involved in phosphate utilization (Table 2). nucleotides ‘CA’ of the consensus binding site (10). Changing
The ability of Pho4p to bind this sequence was initially demon- this Glu residue to an Asp, Asn or Leu residue completely
strated by mobility shift assays and footprinting experiments inhibits binding to the core sequence (Table 3). Interestingly,
 Nucleic Acids Research, 2000, Vol. 28, No. 7 1501

changing it to Gln does not affect the ability of Pho4p to bind While the products of the INO2 and INO4 genes have been
the core sequence. This is curious given that this Glu residue is shown to form a heterodimer in vivo (21) and in vitro (22),
conserved in virtually every known bHLH protein (for example, studies using the yeast two hybrid system suggest that neither
see Table 1). protein is capable of homodimerizing (23). A functional
The Pho4p regulatory cascade is also one of the best under- analysis of the two proteins reveals that Ino2p contains a
stood regulatory systems in yeast. The ability of Pho4p to function transcriptional activation domain (N-terminal) whereas Ino4p
as a transcriptional activator depends on its phosphorylation does not contain this type of domain (23). Collectively, these
state. When cells are grown in the presence of high concentrations observations suggest that Ino4p is required for dimerization
of inorganic phosphate, Pho4p is hyper-phosphorylated (11). with Ino2p, which functions to activate transcription. This
Pho4p is phosphorylated by a complex, which is encoded by organization is reminiscent of the mammalian Myc and Max
the PHO80 and PHO85 genes. This kinase complex bears proteins where Myc has the transcriptional activation function
remarkable similarity to cyclin/cyclin-dependent kinase but must dimerize with Max to bind target sequences (1).
(CDK) complexes (11,12). Immunoprecipitation studies show Another similarity between these two systems is the observation
that Pho80p (cyclin) and Pho85p (CDK) interact with Pho4p as that, like Myc, transcription of the INO2 gene is regulated and
a complex (11). The hyper-phosphorylated Pho4p interacts that the amount of INO2 expression is limiting relative to INO4
with the export protein Msn5p, which shuttles Pho4p into the expression (24). It is presumed that the excess of Max over
cytoplasm (13). Once in the cytoplasm, Pho4p is unable to activate Myc permits it to dimerize with other proteins (Mad and Mxi1)
transcription of PHO5. As inorganic phosphate becomes in the absence of Myc (1). If the similarities persist between
limiting, Pho4p becomes de-phosphorylated. The de-phospho- these two systems, it would suggest that Ino4p also forms
rylated Pho4p is transported back into the nucleus by the multiple dimer combinations with other yeast bHLH proteins.
import protein Pse1p. There are six serine–proline dipeptides The mechanism for derepression of the phospholipid bio-
within Pho4p designated SP1–6. Mutational analysis reveals synthetic genes in response to inositol deprivation has not been
that the serine–proline dipeptides SP2 and SP3 are specifically established. However, it is clear that regulation of transcription
required for export into the cytoplasm while SP4 is required for of the INO2 regulatory gene must play an important role in this
import into the nucleus (14). response. It has been shown that expression of a cat reporter
The phosphorylation of Pho4p in the nucleus under inducing gene driven by the INO2 promoter is regulated in response to
conditions is prevented by the product of the PHO81 gene. The inositol in a pattern that is identical to that of Ino2p target genes
role of this gene was initially suggested by its similarity to a such as INO1 and CHO1 (24). That is, INO2-cat expression is
mammalian CDK inhibitor (15). The similarity is restricted to maximal when cells are grown in the absence of inositol and
a region that contains ankyrin repeats. Co-immunoprecipitation repressed when grown in the presence of inositol. As is the
experiments show that Pho81p interacts with the case with the Ino2p target genes, expression of the INO2-cat
Pho80p:Pho85p complex and inhibits its activity as a kinase. It gene is regulated by the INO2 and INO4 genes. However, the
is also known that the region containing the ankyrin repeats is regulation of INO2 expression cannot be solely accountable for
sufficient for its inhibitory function (15,16). Consistent with the response to inositol since expression of the INO1 and
these results is the observation that PHO81 gene expression is CHO1 genes is sensitive to inositol even when INO2 expression is
also regulated in response to phosphate. Thus, PHO81 gene driven by the GAL1 promoter (inositol-insensitive) (25). This
expression is induced when cells are grown in limiting phos- inositol-responsive repression is likely to be mediated by the
phate (inducing conditions) which reduces the phosphorylation product of the OPI1 gene (17,18). The mechanism of action of
of Pho4p (active state). In support of this, experiments where the OPI1 gene is still under investigation. However, since OPI1
PHO81 expression is driven by the GAL1 promoter show that also represses transcription of the INO2 gene (24), derepression of
induction of PHO5 gene expression requires both induction of the target genes necessitates prior derepression of INO2 gene
PHO81 expression and phosphate starvation (16). Now that the expression. The current model for regulation of phospholipid
components of this regulatory cascade are in hand, it should be biosynthetic gene expression has two components. The first
possible to define the signaling mechanism. component is that derepression of INO2 gene expression is
required for derepression of target gene expression in the
absence of inositol. The second component is that the OPI1
THE INO2/INO4 SYSTEM negative regulatory gene represses transcription of the INO2
The INO2 (SCS2, DIE1, YDR123C) and INO4 (YOL108C) gene and the Ino2p target genes in the presence of inositol.
genes are required for derepression of phospholipid bio- The product of the SIN3 gene is another factor involved in
synthetic gene expression in response to inositol deprivation. the regulation of the phospholipid biosynthetic gene expression.
Strains containing mutations in either of these two genes are This gene functions as a negative regulator of the phospholipid
unable to derepress transcription of the INO1 and CHO1 biosynthetic genes (26). Although its mechanism of action is
(17,18) phospholipid biosynthetic genes when cells are grown not known, it is clear that Sin3p exerts its effects through the
in the absence of inositol. The inability to derepress expression UASINO element suggesting that Sin3p might interact with the
of the INO1 gene results in the characteristic inositol auxo- Ino2p/Ino4p bHLH complex (27). It is intriguing that the
trophy which is the hallmark of ino2 and ino4 mutant strains human Sin3p functions as a repressor by interacting with Mad
(17,18). The INO2 and INO4 genes were cloned by comple- and Mxi1 when they are complexed with Max (28,29).
mentation of this inositol auxotrophy (19,20). DNA sequence It has become increasingly obvious that the function of the
analyses of the cloned genes revealed a high degree of INO2 and INO4 gene products extends beyond the scope of
similarity to the bHLH region of the Myc family of proteins phospholipid biosynthesis. Several genes, whose expression is
(Table 1) (19,20). regulated by either INO2, or in response to inositol, have been
1502 Nucleic Acids Research, 2000, Vol. 28, No. 7

identified (17,18) (Table 2). The majority of these genes function confirmed that these proteins were encoded by the same gene
in some aspect of membrane biogenesis, however, a small and that the observed difference in sizes of the proteins were
number of genes (e.g. ADK1, PHO5, PYK1, PMA1 and likely due to proteolytic degradation of the 64 kDa protein to
MFA1G) do not appear to have any role in this process. The the 39 kDa form (39). The CBF1 gene encodes a 351 amino
pleiotropic phenotype of ino2 mutant strains also suggests a acid protein with a predicted molecular weight of 37 kDa
more general role for Ino2p. These strains have defects in (38,39). However, when transcribed and translated in vitro this
nuclear segregation, bud formation and sporulation, display an gene yields a product that migrates as a 60 kDa protein on an
aberrant oversized morphology and over-express the PIS1 SDS–PAGE gel. The altered mobility is presumably due to its
gene (30,31). unusual amino acid composition, which includes regions that
An examination of the promoters for each of the genes are highly negatively and positively charged (39).
whose expression is inositol-responsive, or INO2-dependent, The identification of the CBF1 gene facilitated genetic analyses,
identified a binding site for the Ino2p:Ino4p heterodimer which show that Cbf1p is required for chromosomal segregation.
(consensus: 5′-CATGTGAAAT-3′) (Table 2). This element Strains bearing cbf1∆ alleles display several phenotypes
(called UASINO/ICRE) is necessary and sufficient to bind the including slow growth, increased chromosomal loss, sensitivity
Ino2p:Ino4p heterodimer and for inositol-specific regulation to microtubule-disrupting drugs (e.g. thiabendazole and
(32,33). Predictably, the first six nucleotides of the consensus benomyl) and methionine auxotrophy (36,39). The methionine-
Ino2p:Ino4p binding site are the core element required for the dependent growth was unique because these same strains do
binding of bHLH proteins. However, an A residue can substitute not have a growth requirement for tryptophane, adenine,
for the C residue at the first position of the UASINO sequence histidine, leucine or uracil (36,39). Since Cbf1p was originally
(Table 2) (32,33). It is also known that the two nucleotides 5′ identified as a centromere-binding protein, the methionine
to the UASINO element and the nucleotides at positions 7 and 8 auxotrophy was surprising and suggested that Cbf1p has a role
play a role in optimizing its function (32). in transcriptional regulation of the MET genes. Thus, Cbf1p is
The potential for Ino4p to form dimers with multiple a unique member of the bHLH family because it has two
proteins (discussed above) raises a question concerning the distinct functions, chromosome segregation and transcriptional
DNA binding specificity of different Ino4p complexes. One control.
answer to this question may be that different complexes recognize A clear aspect of Cbf1p function is that the centromere function
variations of the UASINO element. Consistent with this hypothesis, is mechanistically different from the transcription function.
an INO4-dependent/INO2-independent promoter element in This is supported by the existence of mutants that are defective
the CTR1/HMN1 promoter has the sequence, 5′-CATTTG-3′ in either centromere function or are methionine auxotrophs
(Table 2) (34). This same sequence has been shown to function (40,41). Moreover, it has been observed that mutant alleles of
as a weak inositol-unresponsive UAS element when fused to a the SPT21, SIN3, CCR4 and RPD3 genes suppress the methionine
lacZ reporter gene (32). auxotrophy of a cbf1 mutant strain but spt21 and sin3 mutant
Another question that needs to be addressed is: what are the alleles do not suppress the chromosome loss phenotype (42).
other binding partners for Ino4p? Mutant alleles of several of Thus, the role of Cbf1p in centromere function and transcription
the known yeast bHLH genes are not inositol auxotrophs must be mechanistically distinct and may involve different
suggesting that these proteins do not play a direct role in Cbf1p domains.
phospholipid biosynthesis [B.P.Ashburner and J.M.Lopes, Several lines of investigation suggest that the bHLH domain
unpublished data; (35)]. However, both the CTR1/HMN1 and and an adjacent C-terminal region encoding a leucine zipper
INO1 genes have been shown to contain an INO2-independent, domain are required for Cbf1p function. Mutational studies
INO4-dependent, UAS element (34,36) suggesting that Ino4p reveal that the bHLH region and the leucine zipper region are
must have an alternate partner. Moreover, another study both required for Cbf1p dimerization and function (41,43,44).
suggests that Ino4p interacts with a myristoylation-sensitive Another observation that ascribes significance to the bHLH–
transcription factor (MSTF) to regulate expression of the leucine zipper (zip) region is that the CBF1 gene from
INO2, INO4 and FAS1 genes (Table 2) (37). This study Kluyveromyces lactis will complement the methionine auxotrophy
showed that expression of the INO2 and FAS1 genes is of a Saccharomyces cerevisiae cbf1∆ allele (45). This is significant
elevated in a nmt1 mutant strain (temperature-sensitive defect because the only appreciable homology between the CBF1
in N-myristoylation) while INO4 expression is decreased. genes of the two organisms resides in the bHLH–zip region
However, at elevated temperatures expression of the FAS1 (86% identity). A third line of evidence shows that Cbf1p
gene becomes INO2-independent in a nmt1 mutant strain (37). generated by translation in vitro binds DNA as a dimer and that
the leucine zipper region is required for dimerization and
binding to the CDE1 element (43).
THE CBF1 SYSTEM
The sequence requirements of the CDEI element have been
The CBF1 (CPF1, CEP1, YJR060W) gene encodes a member established by examining the effect of point mutants using a
of the bHLH family which has been designated as Cbf1p, chromosomal loss assay (46). These experiments establish that
Cpf1p and Cp1p. These proteins were initially identified by the optimal sequence requirements for the CDEI element are
their ability to specifically bind a region present in centromeres 5′-CACGTG-3′ (Table 2). As expected this sequence includes
called CDEI (5′-RTCACRTG-3′) (38,39). The multiple names the consensus bHLH binding site. The CDEI sequence require-
for this gene and its protein product are partly due to the fact ments have not been investigated with respect to either the
that two proteins of different sizes (39 and 64 kDa) were found binding affinity of Cbf1p or the methionine auxotrophy,
to bind the CDEI element suggesting that they might be although several genes have been identified which have CDEI
different proteins. However, the cloning of the CBF1 gene elements in their promoters (47–49) (Table 2). The consensus
 Nucleic Acids Research, 2000, Vol. 28, No. 7 1503

derived from aligning the CDEI elements found in promoter mutations in the basic region shows that methionine-
sequences is indistinguishable from the CDEI sequence independent growth does not require DNA binding but that
requirements obtained by assaying for centromere function. centromere function does require DNA binding (Table 3) (40).
The specific function of Cbf1p in expression of the methionine However, another study shows that several mutations in the
biosynthetic genes remains unresolved. Initial reports, using bHLH–zip region which abolish DNA binding concomitantly
northern blot and primer extension analyses failed to observe results in methionine auxotrophy (Table 3) (41). It is obvious
any defects in expression of MET25, GAL2 and TRP1 genes in that more research is needed to understand these results and the
cells containing a cbf1 null mutant allele even though the complete functional role of Cbf1p.
promoters of all three genes contain CDEI elements (39,40).
Other studies show a pronounced effect of a cbf1 null mutant
allele on the derepression rates of MET3, MET10, MET14, THE RTG1/RTG3 SYSTEM
MET16 and MET25 gene expression (49). These experiments The RTG1 (YOL067C) and RTG3 (YBL103C) genes were
demonstrated that CBF1 is not absolutely required for MET3 identified as regulators of CIT2 gene expression (53,54).
and MET25 expression but rather required for complete and Expression of the nuclear-encoded CIT2 gene is subject to
efficient derepression. However, the former studies examined regulation in response to the functional state of the mitochondria,
MET25 expression immediately after methionine was removed
a process that has been termed retrograde regulation (53). In ρo
from the media and then again at a very late stage of growth
petite strains (lacking mitochondrial DNA) CIT2 expression is
(40). Thus, they were unable to detect the lag in derepression
seen in the latter studies (50) and therefore concluded that induced 6–30-fold relative to isogenic respiratory competent
Cbf1p had no effect on MET25 expression. However unlike the strains (ρ+). This observation was employed to isolate mutants
MET25 gene, CBF1 does seem to be absolutely required for that fail to induce CIT2 expression in ρo strains. Mutants were
MET10, MET14 and MET16 expression (50). Consistent with isolated by screening for colonies that failed to express a
these studies, another report demonstrated that CBF1 is CIT2–lacZ reporter gene in a ρo mutant background. To clone
required for expression of a lacZ reporter gene under the RTG1 and RTG3, the respective mutant strains were transformed
control of the MET16 CDE1 element (48). with a YCp50-based yeast genomic library. Transformants were
Although Cbf1p is a positive regulator of the methionine screened for their ability to restore expression of the CIT2–lacZ
biosynthetic genes, it does not appear to function as a classic reporter gene. Subsequent sequence analysis revealed a high
transcriptional activator since a lexA–Cbf1p fusion fails to degree of similarity to several bHLH proteins.
induce transcription of a reporter gene (50). Instead Cbf1p Western blot analysis demonstrated that Rtg1p levels are not
appears to influence chromatin structure at both the centromeres increased in a ρo strain (55). However, RTG3 mRNA steady-
and the promoters of CDEI-containing genes (e.g. MET25, state levels are derepressed in a ρo strain (54). This regulation
MET16, TRP1, GAL2) (39,47,51). Cbf1p creates a nucleosome- is reminiscent of INO2/INO4, suggesting that the constitutive
free region surrounding the CDEI elements (47). expression of RTG1 may allow it to form multiple dimer
The fact that a cbf1∆ mutant does not completely eliminate combinations.
MET3, MET10 and MET25 gene expression suggests that other The cis-acting sequence (UASr; R-box) required for the
factors may be required. This is borne out by the fact that each induction of CIT2 expression is defined by the sequence
of the CDEI-containing promoters is dependent on other 5′ -GGTCAC-3′, which does not conform to the classic E-box
transcriptional regulatory proteins such as GCN4 (48) and sequence that binds other bHLH proteins (Table 2). The
MET4 (50) for their expression. In the case of the MET16 gene, unusual DNA binding sequence requirements are not entirely
Cbf1p is nearly 6-fold more effective when the CDEI site is surprising given that the basic region for Rtg1p deviates from
located upstream of the Gcn4p binding site (natural location) the consensus basic regions of other yeast bHLH proteins
relative to when it is placed downstream of the Gcn4p binding (Table 1). The CIT2 promoter contains two inverted copies of
site (48). Therefore, the function of Cbf1p may be to recruit the R-box separated by 28 bp. While both R-boxes can form an
Gcn4p to the MET16 promoter.
RTG1/RTG3-dependent complex, the two sites act synergistically
The idea that Cbf1p recruits other transcriptional activators
in vivo (54).
to the promoters of the MET genes is further supported by
Activation by a Gal4p–Rtg3p fusion protein can occur in the
studies involving the leucine zipper proteins Met4p and
Met28p. Mobility shift assays demonstrated that Met4p and absence of RTG1 and RTG2, suggesting that Rtg3p is responsible
Met28p along with Cbf1p bind to the UASMET16 (51). However, for activation (56). However, a Gal4p–Rtg1p fusion protein
neither Met4p nor Met28p can bind the UASMET16 without cannot activate transcription of the lacZ reporter gene in the
Cbf1p. The association and dissociation rates of Cbf1p in the absence of RTG3. This evidence suggests that Rtg1p is responsible
presence and absence of Met28p, suggest that Met28p is for recruiting the activation domain of Rtg3p to the UASr
required for maintaining Cbf1p on the DNA. These experiments element, allowing for transcriptional activation of the CIT2
suggest that Cbf1p dimers recruit Met28p and the transcriptional gene.
activator protein, Met4p to the CDEI element. Once the Recent experiments have discovered that the tricarboxylic
Met28p:Met4p is bound to DNA, Met4p activates transcription, acid cycle genes CIT1, ACO1, IDH1 and IDH2 also require
while Met28p helps maintain the stability of the complex RTG1 and RTG3 for full expression in ρo strains (Table 2) (57).
(51,52). However unlike CIT2, these genes are also expressed in ρ+
In spite of all the work that has been done, the results of two strains and require the HAP2, HAP3 and HAP4 genes for
previous studies need to be addressed. One study using expression.
1504 Nucleic Acids Research, 2000, Vol. 28, No. 7

THE SGC1 SYSTEM regulatory pathways in yeast. Therefore, this molecular and
genetically tractable organism will continue to lend great
The SGC1 (TYE7, YOR344C) gene is required for expression
insight into the function and regulation of bHLH proteins.
of glycolytic genes in yeast (Table 2) (35). Expression of the
glycolytic genes (e.g. ENO1 and ENO2) normally requires the
product of the GCR1 positive regulatory gene, which is also ACKNOWLEDGEMENTS
required for growth on media containing glucose as a carbon The authors thank Kyle Gardenour and Mohan Rao Kaadige
source. A genetic selection for mutants that simultaneously for comments on the manuscript. J.M.L. is supported by a grant
restored both growth on glucose and expression of an ENO1–lacZ from the American Cancer Society (RPG-97-002-01).
reporter gene to a gcr1 mutant strain yielded two dominant
SGC1 alleles. The dominant mutant and wild-type alleles of
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