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Nucleic Acids Res. Apr 1, 2000; 28(7): 1499–1505.
PMCID: PMC102793

SURVEY AND SUMMARY: Saccharomyces cerevisiae basic helix–loop–helix proteins regulate diverse biological processes

Abstract

Basic helix–loop–helix (bHLH) proteins are among the most well studied and functionally important regulatory proteins in all eukaryotes. The HLH domain dictates dimerization to create homo- and heterodimers. Dimerization juxtaposes the basic regions of the two monomers to create a DNA interaction surface that recognizes the consensus sequence called the E-box, 5′-CANNTG-3′. Several 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 juxtaposition of two regions (extensions of helix 1) that are rich in basic residues (Table (Table11).

Table 1.
Sequence alignment of the basic regions of select bHLH proteins

The two basic regions create a DNA-binding interface which interacts with the consensus sequence 5′-CANNTG-3′ (Table (Table2)2) (2,3). Since all bHLH proteins bind this core sequence, certain conserved amino acids in the basic region allow for recognition of the core consensus site. Specifically, a highly conserved glutamic acid (E9 in Table Table1)1) contacts the ‘CA’ nucleotides of the consensus binding site. Other residues in the basic region of each bHLH protein dictate specificity to direct different bHLH proteins to different target sites. This is best evidenced by a mutant form of MyoD that contains a change in the basic region (L14 to R14) (Table (Table1)1) which allows it to recognize a c-Myc binding site (5′-CACGTG-3′) instead of a MyoD binding site (5′-CAGCTG-3′) (4). The altered amino acid contacts the fourth nucleotide of the core consensus binding site.

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

Another important feature of bHLH proteins is their ability to form multiple dimer combinations via the HLH domains. Max can dimerize with itself, c-Myc, Mad and Mxi1 (1). However, there is specificity in terms of the dimers that each bHLH protein can form since c-Myc does not appear to form dimers with MyoD. In the case of Max, the choice for dimerization is dependent on partner availability. The amount of Max is constant and abundant relative to c-Myc or Mad. The c-Myc protein is transiently expressed in growth factor-stimulated cells and the Mad protein is expressed during differentiation. Thus, c-Myc/Max dimers are found in growth-stimulated cells and Mad/Max dimers are observed during differentiation.

The ability of bHLH proteins to form multiple dimer combinations is an efficient mechanism for regulation of gene expression since different dimers are likely to target different sets of genes. Therefore, it will be important to determine how bHLH proteins coordinate gene expression in response to extracellular factors. This requires understanding the regulation of bHLH function and identification of the bHLH target genes. It is also important to identify all of the binding partners for each bHLH protein. While it has been laborious to address these questions in higher eukaryotic systems, yeast now provides a 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.

THE PHO4 SYSTEM

The PHO4 (YFR034C) gene was the first yeast gene shown to encode a bHLH protein (5). Sequence comparison to a large number of mammalian and Drosophila bHLH proteins revealed similarity in the bHLH region. The product of the PHO4 gene binds to a sequence that contains the core bHLH binding site, 5′-CACGTG-3′, which is found in the promoters of several genes involved in phosphate utilization (Table (Table2).2). The ability of Pho4p to bind this sequence was initially demonstrated by mobility shift assays and footprinting experiments using the promoter of the PHO5 gene. This promoter contains two Pho4p binding sites (UASp1 and UASp2) which flank a Pho2p binding site (6). The binding of Pho4p to these sites is required for the ~1000-fold induction of PHO5 gene expression in response to phosphate starvation. Binding of Pho4p to UASp1 and to a lesser degree UASp2 is enhanced cooperatively by binding of Pho2p (7). Pho2p binding is believed to disrupt intra-molecular interactions within an internal repression domain of Pho4p, thereby increasing access to the transcriptional activation domain (7,8).

Curiously, the core binding sites for Pho4p and Cbf1p (another bHLH protein) are identical (Table (Table2).2). Although both proteins bind the same sequence there is no evidence for overlapping 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 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 Table Table1)1) (9). When this Glu is changed to the Cbf1p counterpart (D3 in Table Table1),1), the mutant Pho4p is now able to recognize the Cbf1p optimal binding site (core sequence flanked by the T).

As is the case with c-Myc and MyoD, a highly conserved Glu (E9 in Table Table1)1) in the basic region is important for Pho4p binding to the core sequence. Analysis of the protein crystal structure demonstrated that this conserved Glu binds to the nucleotides ‘CA’ of the consensus binding site (10). Changing this Glu residue to an Asp, Asn or Leu residue completely inhibits binding to the core sequence (Table (Table3).3). Interestingly, changing it to Gln does not affect the ability of Pho4p to bind the core sequence. This is curious given that this Glu residue is conserved in virtually every known bHLH protein (for example, see Table Table11).

Table 3.
Mutations of a conserved Glu (E9) in the basic domains of bHLH proteins

The Pho4p regulatory cascade is also one of the best understood regulatory systems in yeast. The ability of Pho4p to function as a transcriptional activator depends on its phosphorylation state. When cells are grown in the presence of high concentrations of inorganic phosphate, Pho4p is hyper-phosphorylated (11). Pho4p is phosphorylated by a complex, which is encoded by the PHO80 and PHO85 genes. This kinase complex bears remarkable similarity to cyclin/cyclin-dependent kinase (CDK) complexes (11,12). Immunoprecipitation studies show that Pho80p (cyclin) and Pho85p (CDK) interact with Pho4p as a complex (11). The hyper-phosphorylated Pho4p interacts with the export protein Msn5p, which shuttles Pho4p into the cytoplasm (13). Once in the cytoplasm, Pho4p is unable to activate transcription of PHO5. As inorganic phosphate becomes limiting, Pho4p becomes de-phosphorylated. The de-phosphorylated Pho4p is transported back into the nucleus by the import protein Pse1p. There are six serine–proline dipeptides within Pho4p designated SP1–6. Mutational analysis reveals that the serine–proline dipeptides SP2 and SP3 are specifically required for export into the cytoplasm while SP4 is required for import into the nucleus (14).

The phosphorylation of Pho4p in the nucleus under inducing conditions is prevented by the product of the PHO81 gene. The role of this gene was initially suggested by its similarity to a mammalian CDK inhibitor (15). The similarity is restricted to a region that contains ankyrin repeats. Co-immunoprecipitation experiments show that Pho81p interacts with the Pho80p:Pho85p complex and inhibits its activity as a kinase. It is also known that the region containing the ankyrin repeats is sufficient for its inhibitory function (15,16). Consistent with these results is the observation that PHO81 gene expression is also regulated in response to phosphate. Thus, PHO81 gene expression is induced when cells are grown in limiting phosphate (inducing conditions) which reduces the phosphorylation of Pho4p (active state). In support of this, experiments where PHO81 expression is driven by the GAL1 promoter show that induction of PHO5 gene expression requires both induction of PHO81 expression and phosphate starvation (16). Now that the components of this regulatory cascade are in hand, it should be possible to define the signaling mechanism.

THE INO2/INO4 SYSTEM

The INO2 (SCS2, DIE1, YDR123C) and INO4 (YOL108C) genes are required for derepression of phospholipid bio-synthetic gene expression in response to inositol deprivation. Strains containing mutations in either of these two genes are unable to derepress transcription of the INO1 and CHO1 (17,18) phospholipid biosynthetic genes when cells are grown in the absence of inositol. The inability to derepress expression of the INO1 gene results in the characteristic inositol auxotrophy which is the hallmark of ino2 and ino4 mutant strains (17,18). The INO2 and INO4 genes were cloned by complementation of this inositol auxotrophy (19,20). DNA sequence analyses of the cloned genes revealed a high degree of similarity to the bHLH region of the Myc family of proteins (Table (Table1)1) (19,20).

While the products of the INO2 and INO4 genes have been shown to form a heterodimer in vivo (21) and in vitro (22), studies using the yeast two hybrid system suggest that neither protein is capable of homodimerizing (23). A functional analysis of the two proteins reveals that Ino2p contains a transcriptional activation domain (N-terminal) whereas Ino4p does not contain this type of domain (23). Collectively, these observations suggest that Ino4p is required for dimerization with Ino2p, which functions to activate transcription. This organization is reminiscent of the mammalian Myc and Max proteins where Myc has the transcriptional activation function but must dimerize with Max to bind target sequences (1). Another similarity between these two systems is the observation that, like Myc, transcription of the INO2 gene is regulated and that the amount of INO2 expression is limiting relative to INO4 expression (24). It is presumed that the excess of Max over Myc permits it to dimerize with other proteins (Mad and Mxi1) in the absence of Myc (1). If the similarities persist between these two systems, it would suggest that Ino4p also forms multiple dimer combinations with other yeast bHLH proteins.

The mechanism for derepression of the phospholipid bio-synthetic genes in response to inositol deprivation has not been established. However, it is clear that regulation of transcription of the INO2 regulatory gene must play an important role in this response. It has been shown that expression of a cat reporter gene driven by the INO2 promoter is regulated in response to inositol in a pattern that is identical to that of Ino2p target genes such as INO1 and CHO1 (24). That is, INO2-cat expression is maximal when cells are grown in the absence of inositol and repressed when grown in the presence of inositol. As is the case with the Ino2p target genes, expression of the INO2-cat gene is regulated by the INO2 and INO4 genes. However, the regulation of INO2 expression cannot be solely accountable for the response to inositol since expression of the INO1 and CHO1 genes is sensitive to inositol even when INO2 expression is driven by the GAL1 promoter (inositol-insensitive) (25). This inositol-responsive repression is likely to be mediated by the product of the OPI1 gene (17,18). The mechanism of action of the OPI1 gene is still under investigation. However, since OPI1 also represses transcription of the INO2 gene (24), derepression of the target genes necessitates prior derepression of INO2 gene expression. The current model for regulation of phospholipid biosynthetic gene expression has two components. The first 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 negative regulatory gene represses transcription of the INO2 gene and the Ino2p target genes in the presence of inositol.

The product of the SIN3 gene is another factor involved in the regulation of the phospholipid biosynthetic gene expression. This gene functions as a negative regulator of the phospholipid biosynthetic genes (26). Although its mechanism of action is not known, it is clear that Sin3p exerts its effects through the UASINO element suggesting that Sin3p might interact with the Ino2p/Ino4p bHLH complex (27). It is intriguing that the human Sin3p functions as a repressor by interacting with Mad and Mxi1 when they are complexed with Max (28,29).

It has become increasingly obvious that the function of the INO2 and INO4 gene products extends beyond the scope of phospholipid biosynthesis. Several genes, whose expression is regulated by either INO2, or in response to inositol, have been identified (17,18) (Table (Table2).2). The majority of these genes function in some aspect of membrane biogenesis, however, a small number of genes (e.g. ADK1, PHO5, PYK1, PMA1 and MFA1G) do not appear to have any role in this process. The pleiotropic phenotype of ino2 mutant strains also suggests a more general role for Ino2p. These strains have defects in nuclear segregation, bud formation and sporulation, display an aberrant oversized morphology and over-express the PIS1 gene (30,31).

An examination of the promoters for each of the genes whose expression is inositol-responsive, or INO2-dependent, identified a binding site for the Ino2p:Ino4p heterodimer (consensus: 5′-CATGTGAAAT-3′) (Table (Table2).2). This element (called UASINO/ICRE) is necessary and sufficient to bind the Ino2p:Ino4p heterodimer and for inositol-specific regulation (32,33). Predictably, the first six nucleotides of the consensus Ino2p:Ino4p binding site are the core element required for the binding of bHLH proteins. However, an A residue can substitute for the C residue at the first position of the UASINO sequence (Table (Table2)2) (32,33). It is also known that the two nucleotides 5′ to the UASINO element and the nucleotides at positions 7 and 8 play a role in optimizing its function (32).

The potential for Ino4p to form dimers with multiple proteins (discussed above) raises a question concerning the DNA binding specificity of different Ino4p complexes. One answer to this question may be that different complexes recognize variations of the UASINO element. Consistent with this hypothesis, an INO4-dependent/INO2-independent promoter element in the CTR1/HMN1 promoter has the sequence, 5′-CATTTG-3′ (Table (Table2)2) (34). This same sequence has been shown to function as a weak inositol-unresponsive UAS element when fused to a lacZ reporter gene (32).

Another question that needs to be addressed is: what are the other binding partners for Ino4p? Mutant alleles of several of the known yeast bHLH genes are not inositol auxotrophs suggesting that these proteins do not play a direct role in phospholipid biosynthesis [B.P.Ashburner and J.M.Lopes, unpublished data; (35)]. However, both the CTR1/HMN1 and INO1 genes have been shown to contain an INO2-independent, INO4-dependent, UAS element (34,36) suggesting that Ino4p must have an alternate partner. Moreover, another study suggests that Ino4p interacts with a myristoylation-sensitive transcription factor (MSTF) to regulate expression of the INO2, INO4 and FAS1 genes (Table (Table2)2) (37). This study showed that expression of the INO2 and FAS1 genes is elevated in a nmt1 mutant strain (temperature-sensitive defect in N-myristoylation) while INO4 expression is decreased. However, at elevated temperatures expression of the FAS1 gene becomes INO2-independent in a nmt1 mutant strain (37).

THE CBF1 SYSTEM

The CBF1 (CPF1, CEP1, YJR060W) gene encodes a member of the bHLH family which has been designated as Cbf1p, Cpf1p and Cp1p. These proteins were initially identified by their ability to specifically bind a region present in centromeres called CDEI (5′-RTCACRTG-3′) (38,39). The multiple names for this gene and its protein product are partly due to the fact that two proteins of different sizes (39 and 64 kDa) were found to bind the CDEI element suggesting that they might be different proteins. However, the cloning of the CBF1 gene confirmed that these proteins were encoded by the same gene and that the observed difference in sizes of the proteins were likely due to proteolytic degradation of the 64 kDa protein to the 39 kDa form (39). The CBF1 gene encodes a 351 amino acid protein with a predicted molecular weight of 37 kDa (38,39). However, when transcribed and translated in vitro this gene yields a product that migrates as a 60 kDa protein on an SDS–PAGE gel. The altered mobility is presumably due to its unusual amino acid composition, which includes regions that are highly negatively and positively charged (39).

The identification of the CBF1 gene facilitated genetic analyses, which show that Cbf1p is required for chromosomal segregation. Strains bearing cbf1Δ alleles display several phenotypes including slow growth, increased chromosomal loss, sensitivity to microtubule-disrupting drugs (e.g. thiabendazole and benomyl) and methionine auxotrophy (36,39). The methionine-dependent growth was unique because these same strains do not have a growth requirement for tryptophane, adenine, histidine, leucine or uracil (36,39). Since Cbf1p was originally identified as a centromere-binding protein, the methionine auxotrophy was surprising and suggested that Cbf1p has a role in transcriptional regulation of the MET genes. Thus, Cbf1p is a unique member of the bHLH family because it has two distinct functions, chromosome segregation and transcriptional control.

A clear aspect of Cbf1p function is that the centromere function is mechanistically different from the transcription function. This is supported by the existence of mutants that are defective in either centromere function or are methionine auxotrophs (40,41). Moreover, it has been observed that mutant alleles of the SPT21, SIN3, CCR4 and RPD3 genes suppress the methionine auxotrophy of a cbf1 mutant strain but spt21 and sin3 mutant alleles do not suppress the chromosome loss phenotype (42). Thus, the role of Cbf1p in centromere function and transcription must be mechanistically distinct and may involve different Cbf1p domains.

Several lines of investigation suggest that the bHLH domain and an adjacent C-terminal region encoding a leucine zipper domain are required for Cbf1p function. Mutational studies reveal that the bHLH region and the leucine zipper region are both required for Cbf1p dimerization and function (41,43,44). Another observation that ascribes significance to the bHLH–leucine zipper (zip) region is that the CBF1 gene from Kluyveromyces lactis will complement the methionine auxotrophy of a Saccharomyces cerevisiae cbf1Δ allele (45). This is significant because the only appreciable homology between the CBF1 genes of the two organisms resides in the bHLH–zip region (86% identity). A third line of evidence shows that Cbf1p 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 sequence requirements of the CDEI element have been established by examining the effect of point mutants using a chromosomal loss assay (46). These experiments establish that the optimal sequence requirements for the CDEI element are 5′-CACGTG-3′ (Table (Table2).2). As expected this sequence includes the consensus bHLH binding site. The CDEI sequence requirements have not been investigated with respect to either the binding affinity of Cbf1p or the methionine auxotrophy, although several genes have been identified which have CDEI elements in their promoters (4749) (Table (Table2).2). The consensus derived from aligning the CDEI elements found in promoter sequences is indistinguishable from the CDEI sequence requirements obtained by assaying for centromere function.

The specific function of Cbf1p in expression of the methionine biosynthetic genes remains unresolved. Initial reports, using northern blot and primer extension analyses failed to observe any defects in expression of MET25, GAL2 and TRP1 genes in cells containing a cbf1 null mutant allele even though the 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, MET16 and MET25 gene expression (49). These experiments demonstrated that CBF1 is not absolutely required for MET3 and MET25 expression but rather required for complete and efficient derepression. However, the former studies examined MET25 expression immediately after methionine was removed from the media and then again at a very late stage of growth (40). Thus, they were unable to detect the lag in derepression seen in the latter studies (50) and therefore concluded that Cbf1p had no effect on MET25 expression. However unlike the MET25 gene, CBF1 does seem to be absolutely required for MET10, MET14 and MET16 expression (50). Consistent with these studies, another report demonstrated that CBF1 is required for expression of a lacZ reporter gene under the control of the MET16 CDE1 element (48).

Although Cbf1p is a positive regulator of the methionine biosynthetic genes, it does not appear to function as a classic transcriptional activator since a lexA–Cbf1p fusion fails to induce transcription of a reporter gene (50). Instead Cbf1p appears to influence chromatin structure at both the centromeres and the promoters of CDEI-containing genes (e.g. MET25, MET16, TRP1, GAL2) (39,47,51). Cbf1p creates a nucleosome-free region surrounding the CDEI elements (47).

The fact that a cbf1Δ mutant does not completely eliminate MET3, MET10 and MET25 gene expression suggests that other factors may be required. This is borne out by the fact that each of the CDEI-containing promoters is dependent on other transcriptional regulatory proteins such as GCN4 (48) and MET4 (50) for their expression. In the case of the MET16 gene, Cbf1p is nearly 6-fold more effective when the CDEI site is located upstream of the Gcn4p binding site (natural location) relative to when it is placed downstream of the Gcn4p binding site (48). Therefore, the function of Cbf1p may be to recruit Gcn4p to the MET16 promoter.

The idea that Cbf1p recruits other transcriptional activators to the promoters of the MET genes is further supported by studies involving the leucine zipper proteins Met4p and Met28p. Mobility shift assays demonstrated that Met4p and Met28p along with Cbf1p bind to the UASMET16 (51). However, neither Met4p nor Met28p can bind the UASMET16 without Cbf1p. The association and dissociation rates of Cbf1p in the presence and absence of Met28p, suggest that Met28p is required for maintaining Cbf1p on the DNA. These experiments suggest that Cbf1p dimers recruit Met28p and the transcriptional activator protein, Met4p to the CDEI element. Once the Met28p:Met4p is bound to DNA, Met4p activates transcription, while Met28p helps maintain the stability of the complex (51,52).

In spite of all the work that has been done, the results of two previous studies need to be addressed. One study using mutations in the basic region shows that methionine-independent growth does not require DNA binding but that centromere function does require DNA binding (Table (Table3)3) (40). However, another study shows that several mutations in the bHLH–zip region which abolish DNA binding concomitantly results in methionine auxotrophy (Table (Table3)3) (41). It is obvious that more research is needed to understand these results and the complete functional role of Cbf1p.

THE RTG1/RTG3 SYSTEM

The RTG1 (YOL067C) and RTG3 (YBL103C) genes were identified as regulators of CIT2 gene expression (53,54). Expression of the nuclear-encoded CIT2 gene is subject to regulation in response to the functional state of the mitochondria, a process that has been termed retrograde regulation (53). In ρo petite strains (lacking mitochondrial DNA) CIT2 expression is induced 6–30-fold relative to isogenic respiratory competent strains (ρ+). This observation was employed to isolate mutants that fail to induce CIT2 expression in ρo strains. Mutants were isolated by screening for colonies that failed to express a CIT2–lacZ reporter gene in a ρo mutant background. To clone RTG1 and RTG3, the respective mutant strains were transformed with a YCp50-based yeast genomic library. Transformants were screened for their ability to restore expression of the CIT2–lacZ reporter gene. Subsequent sequence analysis revealed a high degree of similarity to several bHLH proteins.

Western blot analysis demonstrated that Rtg1p levels are not increased in a ρo strain (55). However, RTG3 mRNA steady-state levels are derepressed in a ρo strain (54). This regulation is reminiscent of INO2/INO4, suggesting that the constitutive expression of RTG1 may allow it to form multiple dimer combinations.

The cis-acting sequence (UASr; R-box) required for the induction of CIT2 expression is defined by the sequence 5′ -GGTCAC-3′, which does not conform to the classic E-box sequence that binds other bHLH proteins (Table (Table2).2). The unusual DNA binding sequence requirements are not entirely surprising given that the basic region for Rtg1p deviates from the consensus basic regions of other yeast bHLH proteins (Table (Table1).1). The CIT2 promoter contains two inverted copies of the R-box separated by 28 bp. While both R-boxes can form an RTG1/RTG3-dependent complex, the two sites act synergistically in vivo (54).

Activation by a Gal4p–Rtg3p fusion protein can occur in the absence of RTG1 and RTG2, suggesting that Rtg3p is responsible for activation (56). However, a Gal4p–Rtg1p fusion protein cannot activate transcription of the lacZ reporter gene in the absence of RTG3. This evidence suggests that Rtg1p is responsible for recruiting the activation domain of Rtg3p to the UASr element, allowing for transcriptional activation of the CIT2 gene.

Recent experiments have discovered that the tricarboxylic acid cycle genes CIT1, ACO1, IDH1 and IDH2 also require RTG1 and RTG3 for full expression in ρo strains (Table (Table2)2) (57). However unlike CIT2, these genes are also expressed in ρ+ strains and require the HAP2, HAP3 and HAP4 genes for expression.

THE SGC1 SYSTEM

The SGC1 (TYE7, YOR344C) gene is required for expression of glycolytic genes in yeast (Table (Table2)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 required for growth on media containing glucose as a carbon source. A genetic selection for mutants that simultaneously restored both growth on glucose and expression of an ENO1–lacZ reporter gene to a gcr1 mutant strain yielded two dominant SGC1 alleles. The dominant mutant and wild-type alleles of the SGC1 gene were cloned and sequence inspection identified this gene as a member of the bHLH family (35). Northern blot analysis shows that the SGC1-1 mutant allele, in a gcr1 backround, restores expression of several glycolytic genes although the most pronounced effect is on the ENO1, PGK1 and PYK1 genes (35). The SGC1 gene does encode a positive regulator of glycolytic gene expression since an sgc1 mutant strain is defective in expression of the ENO1 gene and its gene product (enolase) (35). It is also clear that Sgc1p and Gcr1p function to stimulate expression of the enolase genes (ENO1 and ENO2) through parallel pathways since a gcr1, sgc1 double mutant strain is more defective in enolase gene expression than either of the single mutant strains. What is not known is whether Sgc1p binds a consensus bHLH binding site. The sequence similarity of the basic region to other bHLH proteins suggests that this is likely to be the case (Table (Table1).1). Interestingly, one of the dominant mutant alleles contained a Glu→Gln change in the basic region (Table (Table3).3). As discussed above, this Glu residue is highly conserved among the yeast bHLH proteins but a change to Gln did not eliminate the DNA binding function of Pho4p (9).

POTENTIAL S.cerevisiae bHLH PROTEINS

The completion of the yeast genome sequencing effort has revealed new putative bHLH proteins. Two yeast genes of known function encode potential bHLH proteins. HMS1 encodes a probable transcription factor with similarities to the Myc family. Over-expression of HMS1 results in filamentous growth and it is therefore believed to be a regulator of pseudohyphal differentiation (58). PHD1 encodes a transcription factor involved in regulating filamentous growth. The overexpression of PHD1 causes invasive filamentous growth in rich media (59). Finally, three ORFs encode potential bHLH proteins: YGR290W, YLR002C and YPL165C. However, it is likely that as our ability to predict protein structure and function improves, the number of bHLH proteins in yeast will increase.

FUTURE DIRECTIONS

As we have seen, bHLH proteins play an integral role in the regulation of a number of important pathways in yeast. However, there are important questions that remain to be answered. First, there is currently little knowledge about the signaling mechanisms that regulate these yeast bHLH systems. There is also currently no published evidence for bHLH proteins forming multiple heterodimer combinations. However, the constitutive expression of both INO4 and RTG1 suggests that multiple heterodimer formation is possible. The discovery of multiple bHLH heterodimers will likely elucidate novel regulatory pathways in yeast. Therefore, this molecular and genetically tractable organism will continue to lend great insight into the function and regulation of bHLH proteins.

ACKNOWLEDGEMENTS

The authors thank Kyle Gardenour and Mohan Rao Kaadige for comments on the manuscript. J.M.L. is supported by a grant from the American Cancer Society (RPG-97-002-01).

REFERENCES

1. Amati B. and Land,H. (1994) Curr. Opin. Genes Dev., 4, 102–108. [PubMed]
2. Ma P.C.M., Rould,M.A., Weintraub,H. and Pabo,C.O. (1994) Cell, 77, 451–459. [PubMed]
3. Ferré-D’Amaré A.R., Prendergast,G.C., Ziff,E.B. and Burley,S.K. (1993) Nature, 363, 38–44. [PubMed]
4. Blackwell T.K., Huang,J., Ma,A., Kretzner,L., Alt,F.W., Eisenman,R.N. and Weintraub,H. (1993) Mol. Cell. Biol., 13, 5216–5224. [PMC free article] [PubMed]
5. Berben G., Legrain,M., Gilliquet,V. and Hilger,F. (1990) Yeast, 6, 451–454. [PubMed]
6. Vogel K., Hörz,W. and Hinnen,A. (1989) Mol. Cell. Biol., 9, 2050–2057. [PMC free article] [PubMed]
7. Barbaric S., Münsterkötter,M., Goding,C. and Hörz,W. (1998) Mol. Cell. Biol., 18, 2629–2639. [PMC free article] [PubMed]
8. Shao D., Creasy,C.L. and Bergman,L.W. (1996) Mol. Gen. Genet., 251, 358–364. [PubMed]
9. Fisher F. and Goding,C.R. (1992) EMBO J., 11, 4103–4109. [PMC free article] [PubMed]
10. Shimizu T., Toumoto,A., Ihara,K., Shimizu,M., Kyohoku,Y., Ogawa,N., Oshima,Y. and Haloshima,T. (1997) EMBO J., 16, 4689–4697. [PMC free article] [PubMed]
11. Kaffman A., Herskowitz,I., Tjian,R. and O’Shea,E.K. (1994) Science, 263, 1153–1156. [PubMed]
12. Hirst K., Fisher,F., McAndrew,P.C. and Goding,C.R. (1994) EMBO J., 13, 5410–5420. [PMC free article] [PubMed]
13. Kaffman A., Rank,N.M., O’Neill,E.M., Huang,L.S. and O’Shea,E.K. (1998) Nature, 396, 482–486. [PubMed]
14. Komeili A. and O’Shea,E.K. (1999) Science, 284, 977–980. [PubMed]
15. Schneider K.R., Smith,R.L. and O’Shea,E.K. (1994) Science, 266, 122–126. [PubMed]
16. Ogawa N., Noguchi,K.-I., Sawai,H., Yamashita,Y., Yompakdee,C. and Oshima,Y. (1995) Mol. Cell. Biol., 15, 997–1004. [PMC free article] [PubMed]
17. Greenberg M.L. and Lopes,J.M. (1996) Microbiol. Rev., 60, 1–20. [PMC free article] [PubMed]
18. Paltauf F., Kohlwein,S.D. and Henry,S.A. (1992) In Jones,E.W., Pringle,J.R. and Broach,J.R. (eds), The Molecular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 415–499.
19. Hoshizaki D.K., Hill,J.E. and Henry,S.A. (1990) J. Biol. Chem., 265, 4736–4745. [PubMed]
20. Nikoloff D.M., McGraw,P. and Henry,S.A. (1992) Nucleic Acids Res., 20, 3253. [PMC free article] [PubMed]
21. Nikoloff D.M. and Henry,S.A. (1994) J. Biol. Chem., 269, 7402–7411. [PubMed]
22. Ambroziak J. and Henry,S.A. (1994) J. Biol. Chem., 269, 15344–15349. [PubMed]
23. Schwank S., Ebbert,R., Rautenstrauß,K., Schweizer,E. and Schüller,H.-J. (1995) Nucleic Acids Res., 23, 230–237. [PMC free article] [PubMed]
24. Ashburner B.P. and Lopes,J.M. (1995) Mol. Cell. Biol., 15, 1709–1715. [PMC free article] [PubMed]
25. Ashburner B.P. and Lopes,J.M. (1995) Proc. Natl Acad. Sci. USA, 92, 9722–9726. [PMC free article] [PubMed]
26. Hudak K.A., Lopes,J.M. and Henry,S.A. (1994) Genetics, 136, 475–483. [PMC free article] [PubMed]
27. Slekar K.H. and Henry,S.A. (1995) Nucleic Acids Res., 23, 1964–1969. [PMC free article] [PubMed]
28. Ayer D.E., Lawrence,Q.A. and Eisenman,R.N. (1995) Cell, 80, 767–776. [PubMed]
29. Schreiber-Agus N., Chin,L., Chen,K., Torres,R., Rao,G., Guida,P., Skoultchi,A.I. and DePinko,R.A. (1995) Cell, 80, 777–786. [PubMed]
30. Anderson M.S. and Lopes,J.M. (1996) J. Biol. Chem., 271, 26596–26601. [PubMed]
31. Hammond C.I., Romano,P., Roe,S. and Tontonoz,P. (1993) Cell. Mol. Biol. Res., 39, 561–577. [PubMed]
32. Bacchawat N., Ouyang,Q. and Henry,S.A. (1995) J. Biol. Chem., 270, 25087–25095. [PubMed]
33. Schüller H.-J., Richter,K., Hoffmann,B., Ebbert,R. and Schweizer,E. (1995) FEBS Lett., 370, 149–152. [PubMed]
34. Li Z. and Brendel,M. (1993) Mol. Gen. Genet., 241, 680–684. [PubMed]
35. Nishi K., Park,C.S., Pepper,A.E., Eichinger,G., Innis,M.A. and Holland,M.J. (1995) Mol. Cell. Biol., 15, 2646–2653. [PMC free article] [PubMed]
36. Swift S. and McGraw,P. (1995) Nucleic Acids Res., 26, 2865–2872.
37. Cok S.J., Martin,C.G. and Gordon,J.I. (1995) Nucleic Acids Res., 23, 1426–1433. [PMC free article] [PubMed]
38. Cai M. and Davis,R.W. (1990) Cell, 61, 437–446. [PubMed]
39. Mellor J., Jiang,W., Funk,M., Rathjen,J., Barnes,C.A., Hinz,T., Hegemann,J.H. and Philippsen,P. (1990) EMBO J., 9, 4017–4026. [PMC free article] [PubMed]
40. Mellor J., Rathjen,J., Jiang,W. and Dowell,S.J. (1991) Nucleic Acids Res., 19, 2961–2969. [PMC free article] [PubMed]
41. Foreman P.K. and Davis,R.W. (1993) Genetics, 135, 287–296. [PMC free article] [PubMed]
42. McKenzie E.A., Kent,N.A., Dowell,S.J., Moreno,F., Bird,L.E. and Mellor,J. (1993) Mol. Gen. Genet., 240, 374–386. [PubMed]
43. Dowell S.J., Tsang,J.S.H. and Mellor,J. (1992) Nucleic Acids Res., 20, 4229–4236. [PMC free article] [PubMed]
44. Masison D.C., O’Connell,K.F. and Baker,R.E. (1993) Nucleic Acids Res., 21, 4133–4141. [PMC free article] [PubMed]
45. Mulder W., Winkler,A.A., Scholten,I.H.J.M., Zonneveld,B.J.M., De Winde,J.H., Steensma,H.Y. and Grivell,L.A. (1994) Curr. Genet., 26, 198–207. [PubMed]
46. Niedenthal R., Stoll,R. and Hegemann,J.H. (1991) Mol. Cell. Biol., 11, 3545–3553. [PMC free article] [PubMed]
47. Kent N.A., Tsang,J.S.H., Crowther,D.J. and Mellor,J. (1994) Mol. Cell. Biol., 14, 5229–5241. [PMC free article] [PubMed]
48. O’Connell K.F., Surdin-Kerjan,Y. and Baker,R.E. (1995) Mol. Cell. Biol., 15, 1879–1888. [PMC free article] [PubMed]
49. Kuras L. and Thomas,D. (1995) FEBS Lett., 367, 15–18. [PubMed]
50. Thomas D., Jacquemin,I. and Surdin-Kerjan,Y. (1992) Mol. Cell. Biol., 12, 1719–1727. [PMC free article] [PubMed]
51. Kuras L., Barbey,R. and Thomas,D. (1997) EMBO J., 16, 2441–2451. [PMC free article] [PubMed]
52. Kuras L. and Thomas,D. (1995) Mol. Cell. Biol., 15, 208–216. [PMC free article] [PubMed]
53. Liao X. and Butow,R.A. (1993) Cell, 72, 61–71. [PubMed]
54. Jia Y., Rothermel,B., Thornton,J. and Butow,R.A. (1997) Mol. Cell. Biol., 17, 1110–1117. [PMC free article] [PubMed]
55. Rothermel B.A., Shyjan,A.W., Etheredge,J.L. and Butow,R.A. (1995) J. Biol. Chem., 270, 29476–29482. [PubMed]
56. Rothermel B.A., Thornton,J.L. and Butow,R.A. (1997) J. Biol. Chem., 272, 19801–19807. [PubMed]
57. Liu Z. and Butow,R.A. (1999) Mol. Cell. Biol., 19, 6720–6728. [PMC free article] [PubMed]
58. Lorenz M.C. and Heitman,J. (1998) Genetics, 150, 1443–1457. [PMC free article] [PubMed]
59. Gimeno C.J. and Fink,G.R. (1994) Mol. Cell. Biol., 14, 2100–2112. [PMC free article] [PubMed]
60. Bajwa W., Meyhack,B., Rudolph,H., Schweingruber,A.-M. and Hinnen,A. (1984) Nucleic Acids Res., 12, 7721–7739. [PMC free article] [PubMed]
61. Bun-ya M., Nishimura,M., Harashima,S. and Oshima,Y. (1997) Mol. Cell. Biol., 11, 3229–3238. [PMC free article] [PubMed]
62. Plankert U., Purwin,C. and Holzer,H. (1991) Eur. J. Biochem., 196, 191–197. [PubMed]
63. Venter U. and Hörz,W. (1989) Nucleic Acids Res., 17, 1353–1369. [PMC free article] [PubMed]
64. Yoshida K., Ogawa,N. and Oshima,Y. (1989) Mol. Gen. Genet., 217, 40–46. [PubMed]

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