The regulation of EFG1 in white–opaque switching in Candida albicans involves overlapping promoters

Authors


Summary

EFG1 , which encodes a trans -acting factor, is expressed as a more abundant 3.2 kb transcript in the white phase and as a less abundant 2.2 kb transcript in the opaque phase of the white–opaque transition in Candida albicans . To understand how alternative phase-specific mRNAs are transcribed from the same gene locus, the 2320 bp upstream region of the gene was functionally characterized by analysing the ­activity of deletion derivatives in a luciferase-based reporter system. The white phase-specific promoter contained three discrete sequences involved in white phase-specific activation, between −2022 and −1809 bp (AR1), between −1809 and −1727 bp (AR2) and between −922 and −840 bp (AR3). A higher resolution deletion and mutation analysis of AR2 revealed two regions between −1809 and −1787 bp and between −1764 and −1728 bp that are responsible for AR2 activation. Targeting of promoter constructs to the ectopic ADE2 genomic site and the 3′ end of the EFG1 genomic site revealed a positional requirement for white phase-regulated activation specific for the AR2 region of the promoter. Gel mobility shift assays using AR2 revealed a white phase-specific activation complex. No discrete activation sequences were identified in the overlapping promoter of the opaque phase-specific EFG1 transcript. The strength of opaque phase activation was directly proportional to the length of the promoter. Northern analysis excluded the possibility of an opaque phase-specific repressor. These results demonstrate overlapping promoters for white and opaque phase-specific expression of the gene for the transcription factor Efg1, with distinctly different mechanisms of phase-specific activation.

Introduction

Candida albicans is capable of switching spontaneously and reversibly between a white phase and an opaque phase that are distinguishable by colony morphology, cellular morphology and gene expression ( Slutsky et al., 1987 ; Soll, 1992 ; 2002a,b ). Only strains homozygous for the mating type locus ( MATa/MATa or MATα/MATα) are capable of white–opaque switching ( Lockhart et al., 2002a ; Miller and Johnson, 2002 ), and only strains expressing the opaque phase phenotype mate with high efficiency ( Lockhart et al., 2002b ; Miller and Johnson, 2002 ). White–opaque switching involves the differential expression of a variety of white phase- and opaque phase-specific genes ( Morrow et al., 1992 ; 1993 ; ­Srikantha and Soll, 1993; Balan et al., 1997 ; Sanglard et al., 1999 ; Soll, 2002a,b ). Transcription of EFG1 , which encodes a transcription factor ( Lo et al., 1997 ; Stoldt et al., 1997 ), however, is regulated in a unique fashion in the white–opaque transition ( Srikantha et al., 2000 ). It is expressed in both phases, but the levels of expression and the size of the transcripts differ. EFG1 encodes a transcription factor with homologues in Saccharomyces cerevisiae ( Gimeno and Fink, 1994 ), Aspergillus nidulans ( Miller et al., 1992 ), Neurospora crassa ( Aramayo et al., 1996 ), Penicillium marneffei ( Borneman et al., 2002 ) and Candida glabrata (S. A. Lachke, T. Srikantha and D. R. Soll, unpublished observation). Overexpression of EFG1 in strain WO-1 of C. albicans stimulates opaque phase cells to switch to the white phase, and reduced expression results in an elongate cellular morphology similar to that of the opaque phase phenotype, but lacking opaque phase-specific pimples ( Sonneborn et al., 1999 ). Deletion of EFG1 results in cells that at first appeared to be blocked in the opaque phase phenotype ( Srikantha et al., 2000 ). However, a shift from 25°C to 42°C caused mass con­version of these cells to the white phase phenotype, including deactivation of opaque phase-specific genes and activation of white phase-specific genes ( Srikantha et al., 2000 ). White phase cells of the EFG1 null mutant, however, still formed elongate cells that were shaped like opaque phase cells, but lacked opaque phase pimples ( Srikantha et al., 2000 ). EFG1 is therefore not involved in the actual switch event. Rather, it functions downstream of the switch event in the regulation of a subset of white phase-specific genes involved in the generation of the round white cell shape.

Although it was originally reported that EFG1 was differentially expressed only in the white phase (Sonneborn et al., 1999), it was subsequently demonstrated that EFG1 is in fact transcribed as a more abundant 3.2 kb mRNA in the white phase and as a far less abundant 2.2 kb mRNA in the opaque phase, and that each mRNA was transcribed from a different start site (Srikantha et al., 2000). To begin to understand how C. albicans differentially transcribes alternative phase-specific mRNAs from the same gene locus, we functionally characterized the 2320 bp upstream regulatory region of EFG1 by analysing the activity of deletion derivatives in a luciferase-based reporter system, and identified a major white phase-­specific activation sequence. This sequence, AR2, was then used to identify a white phase-specific activation complex in gel mobility shift experiments. The results of these studies demonstrate overlapping promoters with distinct mechanisms of activation for white and opaque phase-specific expression of the gene for the transcription factor Efg1.

Results

Cloning the EFG1 promoter

A genomic library of C. albicans strain WO-1 was screened with a 380 bp region of the EFG1 open reading frame (ORF), and lambda clone λEF39.1 containing the 6 kb upstream sequence of EFG1 was selected for further analysis. The 2.4 kb nucleotide sequence upstream of the ATG translation start site was sequenced (Fig. 1), and the sequence was compared with both the S. cerevisiae database and the global eukaryotic promoter database in order to identify sequences homologous to known cis-acting elements. The EFG1 upstream region contained a number of consensus sequences of cis-acting elements that play regulatory roles in other organisms (Fig. 1). These elements included three TATA box protein-binding motifs (Cavallini et al., 1988) at −1221, −551 and −253 bp, four putative pheromone response elements (PRE) (Dolon       et al.,       1989)       at       −1801,       −1521,       −1155       and −1039 bp, six putative E-box elements (Leng et al., 2001) at −2008, −1951, −1657, −1547, −836 and −503 bp, one putative TEA (TEF-1, Tec1 and AbaA motif) DNA consensus sequence (TCS) (Baur et al., 1997; Madhani and Fink, 1997) at −1792 bp, one putative RBF1 site (Ishii et al., 1997) at −1731 bp and one MATα2 binding site (Keleher et al., 1988) at −194 bp (Fig. 1). One of the TATA boxes was 45 bp upstream of the transcription start site for the white phase-specific transcript, and the other was 22 bp upstream of the transcription start site for opaque phase-specific transcription (Fig. 1).

Figure 1.

The 2320 bp sequence upstream of the translational start site of EFG1 . Landmark sites are indicated by boxes and arrows. WhTSP, OpTSP, transcription start sites for the white and opaque phase-specific transcripts ­respectively.

Positional effect on phase-specific expression

In the initial analysis, the 1369 bp of the EFG1 promoter region was cloned upstream of the Renilla reniformis luciferase gene RLUC in the reporter plasmid pCRW3 (Srikantha et al., 2000). When targeted to the ADE2 locus by vector linearization at the ADE2 gene, RLUC activity was higher in opaque phase cells than in white phase cells, which was the reverse relationship revealed by Northern analyses of EFG1 transcript levels (Srikantha et al., 2000). However, when targeted to the EFG1 locus, which placed RLUC directly downstream of the entire 5′ upstream region of EFG1, RLUC activity was higher in the white phase than in the opaque phase, the expected relationship (Srikantha et al., 2000). These observations indicated either that phase-specific regulation of the promoter required residence at the native EFG1 chromosome location, or that it required promoter sequences upstream of −1369 bp.

To resolve this issue, the vector pCRW3E was constructed, which contained a multiple cloning site upstream of RLUC, a 1208 bp sequence of the EFG1 3′ locus that encompassed the last 633 bp of the EFG1 ORF and 575 bp of the region immediately downstream of the EFG1 ORF, and the ADE2 gene (Fig. 2A). The 2320 bp region upstream from the EFG1 translation start site was cloned into pCRW3E upstream of RLUC. This vector could, through selective linearization, be targeted to the ADE2 locus (Fig. 2B) or the 3′ end of the EFG1 locus (Fig. 2C). When targeted to the ADE2 locus, RLUC activity was 12-fold higher in the opaque phase than in the white phase but, when targeted to the 3′ end of the EFG1 locus, RLUC activity was sixfold higher in the white phase than in the opaque phase (Table 1), a proportion consistent with the developmentally regulated expression pattern (Srikantha et al., 2000). These results indicate first that the 2320 bp region upstream of EFG1 contains the promoter elements necessary for white phase-regulated expression and, secondly, that phase-regulated expression can only be achieved at the resident EFG1 locus, revealing a positional effect for phase-specific expression. It should be noted that, although the level of opaque phase expression was only slightly higher at the EFG1 locus than at the ADE2 locus (2.9-fold), white phase expression was dramatically higher (200-fold) at the EFG1 locus (Table 1). This result further indicates that the major positional effect is on white phase-specific promoter activity, not opaque phase-specific promoter activity.

Figure 2.

A physical map of the ADE2 -based integration vector pCRW3E along with its predicted patterns of site-specific integration at the ADE2 and EFG1 loci.
A. Restriction map of pCRW3E.
B. The configuration of the ADE2 locus and the integrated pCRW3E plasmid linearized at a unique XbaI site in the ADE2 gene.
C. The configuration of the EFG1 locus in C. albicans and the integrated pCRW3E plasmid linearized at a unique DraIII site in the EFG1 3′ sequence in the plasmid.

Table 1. .  Functional analysis of the EFG1 promoter at the ectopic ADE2 locus and the resident EFG1 locus.
Target locusPlasmidNo. of coloniesSwitch phenotype RLUC activity( RLUC /30 s µg −1 ) Fold diff with pCRW3ERatio ofWh/Op
  1. The 2320 bp sequence upstream of EFG1 was cloned upstream of RLUC in the vector pCRW3E.

ADE2 pCRW3E containing promoter2White8.5 × 103 ± 6.5 × 102  240.08
2Opaque1.0 × 105 ± 1.0 × 104 291 
EFG1 pCRW3E containing promoter3White1.7 × 106 ± 2.1 × 10548606.0
2Opaque2.9 × 105 ± 8.6 × 104 829 
ADE2 pCRW3E lacking promoter2White3.5 × 102 ± 1.2 × 102
2Opaque3.4 × 102 ± 1.6 × 102 

Functional characterization of the EFG1 promoter

To characterize the EFG1 promoter functionally through the analysis of deletion derivatives, we constructed a URA3-based vector, as this vector could then be used to test promoter function in a variety of mutant backgrounds, which have been created in C. albicans primarily using ura3-based strategies. The vector pA43.1RE contains URA3, the 1208 bp 3′ region of the EFG1 locus that encompassed the last 633 bp of the EFG1 ORF and 575 bp of the region immediately downstream of the EFG1 ORF, and a multiple cloning site immediately upstream of the RLUC reporter gene (Fig. 3A). Deletion derivatives of the 2320 bp upstream region of EFG1 were inserted into the multiple cloning site of pA43.1RE. The resulting vectors were linearized at the EFG1 3′ sequence and used to transform the ura3 WO-1 derivative TS3.3 (Fig. 3B) (Srikantha et al., 2000). For each deletion derivative, 10 independent transformants were chosen for Southern analysis to verify site-specific integration at the EFG1 locus. When digested with BglII and hybridized with the radiolabelled 3′ end of EFG1, the transformant blots contained 11 and 5 kb bands, which represented the expected fragments containing the endogenous EFG1 gene of TS3.3 (data not shown). In addition, all transformant blots contained a 0.5 kb band that represented the vector-derived ‘E’ fragment of the plasmid (Fig. 3B). All transformant blots also contained one variable sized band ‘R’, which contained the deletion derivatives of the EFG1 promoter (Fig. 3B).

Figure 3.

A physical map of the URA3 -based integration vector pA43.1RE along with its predicted pattern of site-specific integration.
A. Restriction map of pA43.1RE.
B. The configuration of the EFG1 locus in C. albicans and the integrated pA43.1RE plasmid linearized at a unique DraIII site in the EFG1 3′ end sequence in the plasmid. The positions of BglII fragments ‘E’ and ‘R’ are noted.

The 11 and 5 kb bands containing sequences from the endogenous EFG1 locus of strain TS3.3 were approximately equal in intensity in all individual transformants and in untransformed TS3.3. However, the vector-specific bands (E and R) of some transformant clones containing the same promoter construct differed in intensity, indicating differences in the number of vector inserts. As BglII sites were involved in the generation of fragments with variable intensity in the Southern blots, and there were no variations in the size of fragments between independent clones of the same construct, we concluded that differences in intensity represented differences in the number of tandem repeats of the vector inserted at the EFG1 locus. This conclusion was supported by the observation that RLUC activity correlated with the varying intensity of inserts (data not shown). We therefore selected for analysis multiple transformants of each construct with E and R bands that exhibited intensities reflecting single-copy insertions.

In Fig. 4A, a model is presented of the EFG1 promoter in which landmark sequences are identified. In Fig. 4B, RLUC activities are presented for the series of deletion derivatives in the white and opaque phase and, in Fig. 5, RLUC activity is plotted for both white and opaque phases as a function of promoter construct length. RLUC activity in the Ef1 transformants harbouring the entire 2320 bp upstream region of EFG1 was considered to be ‘maximum’ (100%) expression in both white and opaque phases. RLUC activity in the white phase of Ef1 was sixfold higher than in the opaque phase (Fig. 4B). The effects of deletions on white phase-specific RLUC expression will be considered first. Deletion of the first 298 bp of the promoter region, between −2320 to −2022 bp (Ef2 and Ef3), resulted in a reduction in RLUC activity of ≈ 10% (Figs 4B and 5). Deletion of the next 213 bp of the promoter region (Ef4, Ef4.1, Ef5, Ef5.1), generating a total deletion of 511 bp (−2320 to −1809 bp), resulted in a reduction in RLUC activity of 65% (Figs 4B and 5). The remaining 35% of RLUC activity, however, was still 1400 times higher than the level of the promoterless construct pA43.1RE and approximately three times the activity of the same construct in the opaque phase (Figs 4B and 5). Deletion of the next 82 bp (Ef6), generating a total deletion of 593 bp (−2320 to −1727 bp), resulted in a dramatic decrease in RLUC activity to 1% of Ef1 activity (Figs 4B and 5). Deletions between −1727 and −922 bp (Ef6.1 to Ef12.4), generating a total deletion of 1398 bp (−2320 to −922 bp), did not significantly affect this already low level of activity (Figs 4B and 5). However, a deletion between −922 and −840 bp (EC9), generating a total deletion of 1480 bp (−2320 to −840 bp), resulted in a decrease in RLUC activity from 1% to 0.2% Ef1 activity (Figs 4B and 5). Deletion of the next 675 bp (EPB9 to EPB2), generating a total deletion of −2320 to −165 bp, had no further effect on activity, which remained at ≈ 0.2% of the Ef1 level. This analysis therefore revealed three activation regions, ‘AR1’ between −2022 and −1809 bp, ‘AR2’ between −1809 and 1727 bp, and ‘AR3’ between −922 and −840 bp (Figs 3 and 4). AR1 contained two putative E-box elements, AR2 contained one putative PRE element, one putative TCS element and one putative RBF1 element, and AR3 contained no identifiable regulatory consensus sequences (Fig. 4A). It should be noted that in no case did a deletion result in an increase in RLUC activity in the white phase, which would have been indicative of a repression sequence. However, this result would also have been obtained if repression and activation sites overlapped. Therefore, these results do not exclude repression sequences in the opaque phase-specific EFG1 promoter.

Figure 4.

Functional characterization of the EFG1 promoter through the analysis of deletion derivatives.
A. Model of the EFG1 promoter. Sequences homologous to known cis-acting regulatory elements in the promoters of other genes are noted.
B. Activity of RLUC under the regulation of the described deletion derivatives of the EFG1 promoter in the white phase and the opaque phase. E, E box element; P, PRE element; T, TCS element; T, TATA box consensus sequence; M, Matα2p binding site, R, Rbf1p binding site; W-TSP, O-TSP, white phase and opaque phase transcription start points respectively; s.d., standard deviation; Op., opaque; Wh., white.

Figure 5.

Logarithmic plot of RLUC activity as a function of the length of the deletion of the EFG1 promoter for the deletion derivatives described in Fig. 4 . AR1, AR2, AR3, activation regions 1, 2 and 3 respectively; all other abbreviations are explained in the legend to Fig. 4 . Open circles represent white phase RLUC activity, whereas closed circles represent opaque phase RLUC activity.

The effects of promoter deletions on opaque phase-specific RLUC expression were different from those on white  phase  expression.  Progressive  deletions  between −2022 and −165 bp resulted in a gradual decrease in RLUC activity (Figs 4 and 5). Deletion of the first 25% of the upstream region, spanning −2320 and −1727 bp (Ef6), resulted in a 22% reduction in RLUC activity, deletion of approximately the first 50% of the upstream region, spanning −2320 and −1115 bp (EPB12), resulted in a 57% reduction in RLUC activity, and deletion of the first 75% of the upstream region, between −2320 and −712 bp (EPB9), resulted in a 71% reduction in RLUC activity (Figs 5B and 6). Therefore, no discrete activation sites were identifiable in the opaque phase promoter. Rather, a linear relationship was revealed between promoter length and RLUC activity.

Figure 6.

High-resolution functional characterization of AR2.
A. The activity of RLUC under the regulation of the described deletion derivatives of the EFG1 promoter in the white and opaque phases. Deletion derivatives extend to −1 bp of the EFG1 promoter in all derivatives. PRE site (light grey) and TCS site (hatched).
B. The activity of RLUC under the regulation of AR2 containing mutations in critical residues. Point mutations are in bold print.

High-resolution analysis of the activation region AR2

The AR2 region spanning −1809 and −1727 bp of the EFG1 promoter therefore contained the major white phase-specific activation sequence. Deletion of this 82 bp region reduced RLUC activity from 5.7 × 105 to 2.1 × 104, a 27-fold reduction (Figs 4B and 5). The same deletion construct, however, had a negligible effect on opaque phase-specific RLUC activity (Figs 4B and 5). This white phase-specific activation region harboured a PRE element between −1802 and −1796 bp and a TCS element between −1799 and −1788 bp. We therefore performed a more detailed deletion analysis as well as a point mutation analysis of this region. Deletion of 20 bp between −1809 and −1786 bp (B3), generating a total deletion of 531 bp (−2320 to −1789 bp) and eliminating both PRE and TCS elements, resulted in a loss of approximately half the white phase RLUC activity of the Ef6 construct, which contained the entire AR2 region (Fig. 6A). Mutations in critical residues of either the PRE site or the TCS site also resulted in an ≈  50% reduction in white phase RLUC activity (Fig. 6B), the same reduction obtained by deleting both elements (Fig. 6A), indicating that the region harbouring the PRE and TCS sites contributed to the activation observed in this region. Deletion of an additional 7 bp, between −1786 and −1779 bp (B2), resulted in no further loss of activity (Fig. 6A). Further deletion of 15 bp between −1779 and −1764 (Ef5.2) gave no further reduction in activity (Fig. 6A). An internal deletion derivative between −1785 and −1758 (ID1), which left the rest of the EFG1 promoter intact, had no effect on promoter activity (Fig. 6A), which was consistent with the results of sequential deletions. Further deletion of the AR2 region between −1764 and −1727 bp (Ef6) resulted in an 18-fold reduction in activity, resolving a second activation site in the AR2 region.   This   region   harboured   a   putative   RBF1   site   at −1731 bp.

Northern analysis of the RLUC transcripts in white and opaque phase cells

Measurements of RLUC activity do not distinguish between white and opaque phase-specific transcripts. Therefore, if a deletion that reduced white phase-specific transcription also increased opaque phase-specific transcription in the white phase, measurements of RLUC activity would not distinguish it. We therefore performed Northern analysis of the RLUC transcripts in white and opaque phase for select deletion derivatives through AR1 and AR2. For the full-length promoter (Ef1), the RLUC transcript in the white phase was 2.3 kb and abundant; in the opaque phase, the RLUC transcript was 1.3 kb and far less abundant (Fig. 7), reflecting the strengths of the alternative phase-specific EFG1 promoters and the difference in transcript size of endogenous EFG1 gene expression in the two phases. The B3 deletion derivative, between −2320 and −1786 bp, reduced the level of the 2.3 kb RLUC transcript in the white phase by ≈  50% (Fig. 7), approximately the same reduction observed in RLUC activity (Fig. 6). The Ef6 deletion derivative, between −2320 and −1727 bp, reduced the 2.3 kb RLUC transcript in the white phase to a negligible level (Fig. 7), approximately the same reduction observed in RLUC activity (Fig. 4). However, the level of the 1.3 kb RLUC transcript in opaque phase cells remained relatively unchanged through all the deletions (Fig. 7), which was again consistent with RLUC activity measurements (Fig. 4).     Deletions     through     Ef6,     between     −2320     and −1727 bp, did not upregulate either the 2.3 kb white phase-specific transcript in the opaque phase or the 1.3 kb opaque phase-specific transcript in the white phase.

Figure 7.

Northern blot analysis of the RLUC transcript in select ­deletion derivatives in the white and opaque phases. Total RNA was probed with the entire RLUC ORF. The white phase RLUC transcript was 2.3 kb, and the opaque phase RLUC transcript was 1.3 kb. The RLUC probe cross-hybridized to the 18S rRNA, generating a band between the two RLUC bands, which should be disregarded. The Northern blot was rehybridized to the entire EFG1 ORF. Ethidium bromide-stained rRNA is provided for the assessment of loading.

A white phase-specific complex interacts with the AR2 region

To test whether the upstream region harbouring AR2 binds to white phase-specific factors, electrophoretic gel mobility shift assays were performed with an end-labelled 130 bp fragment, spanning −1819 to −1689 bp of the EFG1 promoter, and protein extracts from white and opaque phase cells. This DNA fragment contained AR2 (1809–1727) plus a 38 bp downstream region. When increasing concentrations of cell-free protein extract from white phase cells were used in the assay, increasing levels of one white phase-specific complex formed (Fig. 8A). Very low levels of a similar complex formed with opaque phase cell extract (Fig. 8A). These low levels could be explained in part by contamination of opaque phase cell populations with white phase cells resulting from spontaneous switching (Anderson and Soll, 1987; Slutsky et al., 1987). No similar complexes were detected in control reactions in which either no protein extract was added (buffer) or bovine serum albumin (BSA) was added (Fig. 8B). When radiolabelled white phase-specific complex formation was challenged with 100-fold and 200-fold excesses of the unlabelled 130 bp fragment, the levels of labelled complex were reduced but, when challenged with a 200-fold excess of a 76 bp truncated fragment downstream of −1688 bp, which is immediately downstream of AR2, there was no competition (Fig. 8B), indicating that the white phase-specific complex formed only with a sequence in AR2. Furthermore, there was no competition with the ORF of the white phase-specific gene WH11 (data not shown), further supporting the specificity of binding between the 130 bp fragment from AR2 and white cell protein.

Figure 8.

Gel mobility shift and competition assays with the 130 bp DNA fragment containing the white phase-specific activation sequence AR2 of the EFG1 promoter and phase-specific protein extracts.
A. Gel mobility shift with a fixed concentration of end-labelled AR2 and increasing amounts of protein extracts from white or opaque phase cells. Numbers above the lanes indicate µg of white or opaque phase cell protein in the binding reaction mixture.
B. Competition experiment. The end-labelled AR2 fragment was challenged with specific competitor DNA (unlabelled homologous fragment) (SC) or non-specific competitor DNA (NSC). A fixed concentration of the end-labelled fragment plus 13.2 µg of either white or opaque phase cell protein extract was incubated in binding buffer with the indicated molar ratios of SC or NSC.
C. Assay of the end-labelled AR2 fragment with cell protein extracts from either C. albicans strain SC5314 or its cph1 null mutant derivative, JKC19. Numbers above the lanes indicate µg of cell protein in the binding reaction mixture. Buffer, binding buffer with no extract; BSA, 5 µg of BSA alone.

As Ste12p, the S. cerevisiae homologue of C. albicans Cph1p, binds to a PRE site in S. cerevisiae (Dolon et al., 1989), the possibility was entertained that the putative PRE site located at −1801 bp, in an activation region of AR2, may play a role in EFG1 activation. We therefore tested whether a white phase-specific complex formed between      the      130 bp      fragment      spanning      −1819      and −1689 bp of the EFG1 promoter and protein extract from C. albicans strain JKC19, a cph mutant of C. albicans (Liu et al., 1994). Protein extracts of the cph null mutant, the parental wild-type strain SC5314 and white phase cells of strain WO-1 all formed protein complexes of the same intensity, which migrated similarly in agarose gels (Fig. 7C). Therefore, even though a PRE site located in AR2 apparently contributes to EFG1 activation, Cph1p is not essential for binding of the white phase-specific complex to AR2 DNA.

Discussion

Although EFG1 does not appear to play a role in the actual switch event regulating the transition from the white to the opaque or from the opaque to the white phenotype in C. albicans, it does play a role in the expression of the white phase cell phenotype (Srikantha et al., 2000). Although EFG1 null mutants are capable of switching reversibly between the white and opaque phenotypes, in the white phase, mutant cells are unusually elongate rather than round, suggesting that EFG1 is involved in generating the round shape of white phase cells. Consistent with this observation, Sonneborn et al. (1999) reported that EFG1 was differentially transcribed in the white phase. However, subsequent Northern blot and 5′ random amplification of cDNA ends (RACE) analyses by Srikantha et al. (2000) revealed that, although EFG1 was transcribed as a 3.2 kb transcript in the white phase, it was also transcribed as a less abundant 2.2 kb transcript in the opaque phase. Srikantha et al. (2000) also presented evidence that, although the cis-acting sequences necessary for the transcription of the 3.2 kb EFG1 mRNA were distal to the 1.2 kb leader sequence immediately upstream of the EFG1 translation start site, cis-acting sequences regulating the 2.2 kb transcript were in the 1.2 kb leader sequence. These results suggested that the upstream region of EFG1 contained overlapping promoters for the expression of the white phase-specific and opaque phase-specific transcripts. Overlapping promoters have also been demonstrated for the α and β mRNAs of the EFG1 homologue StuA in Aspergillus nidulans (Wu and Miller, 1997).

The white phase-specific promoter

We have identified through a functional analysis of deletion derivatives three discrete cis-acting activation regions in the white phase-specific EFG1 promoter that we have designated AR1, AR2 and AR3. AR1 defines a region spanning −2022 and −1810 bp, in which progressive deletions result in the gradual loss of RLUC activity. Deletion of this entire region results in a 65% loss in reporter activity. This region contains two E box binding sites that cannot alone account for progressive loss of RLUC activity through the entire region. AR2 defines an 82 bp region spanning −1809 and −1727 bp. Deletion of the entire AR2 region results in a 27-fold decrease in activity, resulting in only 1% of the activity of the full-length promoter. High-resolution sequential deletions and an internal deletion of AR2 further delineated two activation subregions of AR2, one between −1809 and −1785 bp and the other between −1764 and −1727 bp. AR2 may therefore be considered the major activation region of the white phase-specific promoter. It contains an overlapping PRE and TCS element in the 5′ subregion, and an RBF1 element in the 3′ subregion. These elements have been shown to play regulatory roles in cell type-specific expression in C. albicans, and in cellular differentiation in A. nidulans, S. cerevisiae and C. albicans (Adrianopoulos and Timberlake, 1994; Liu et al., 1994; Gavrias et al., 1996; Ishii et al., 1997; ­Schweizer et al., 2000).

AR3    defines    a    region    of    82 bp,    between    −922    and −840 bp, the deletion of which reduces the level of white phase expression an additional fivefold. No known cis-acting sequences were identified in this region. Finally, a deletion spanning −165 and −74 bp reduced activity further to that of the promoterless construct.

The opaque phase-specific promoter

In contrast to the white phase-specific promoter, we obtained no evidence of discrete activation sequences that control EFG1 expression in the opaque phase. RLUC activity decreased progressively with progressive deletions between −2022 and −165 bp of the EFG1 promoter. The loss of reporter activity was roughly proportional to the extent of each deletion. Interestingly, deletion of AR2, which reduced RLUC activity to 1% of full-length promoter activity in the white phase, had no similar precipitous effect on opaque phase expression. In fact, the construct with the AR2 deletion exhibited 10 times higher RLUC activity in the opaque phase than in the white phase. As in the case of the white phase promoter, a deletion between −165 and −74 completely abolished promoter activity, indicating that a phase-independent element required for basal transcription resides in this region. It should be emphasized that opaque phase-specific EFG1 promoter activity did not involve discrete activation sequences, but rather appeared to involve additive effects of the entire 2000 bp upstream region. This conclusion is based on the analysis of 24 progressive deletion ­derivatives.

A white phase positional effect

An earlier study by Srikantha et al. (2000) demonstrated that inserting the reporter plasmid containing 1369 bp of the EFG1 promoter in the ectopic ADE2 locus resulted in a reversal of the white/opaque expression ratio of RLUC. Although opaque phase expression was similar at the ADE2 and EFG1 loci, white phase expression was reduced dramatically at the ADE2 locus, and therefore accounted for the reversal. Because, in this earlier study (Srikantha et al., 2000), insertion of the reporter construct, containing the 1369 bp upstream region, into the EFG1 locus restored the entire EFG1 promoter upstream of the reporter gene, it did not discriminate between a requirement for sequences upstream of −1369 bp of the EFG1 promoter and a positional effect at the EFG1 locus. Here, we inserted the expression vector containing the 2320 bp region upstream of EFG1 at the 3′ end of the EFG1 locus. Our results clearly demonstrate a positional effect. Correct phase-regulated expression was obtained at the 3′ end of the EFG1 locus and not at the ADE2 locus. There are several examples of similar positional effects on promoter function in other systems (Allen et al., 1990; al-Shawi et al., 1990; Sabath et al., 1993; Clark et al., 1994; ­Robertson et al., 1995; Cranston et al., 2001; Ramirez et al., 2001).

Deletion through AR2 of the EFG1 promoter resulted in changes in activity similar to that obtained when the intact promoter was inserted at the ADE2 locus. Although RLUC activity in the opaque phase was relatively unaffected, RLUC activity in the white phase was dramatically reduced, leading to a change in the phase-specific expression ratio. These results suggest that the upstream region of the EFG1 promoter, which contains AR1 and AR2, requires residence at the EFG1 locus for proper activation. However, the possibility of enhancers upstream of −2320 bp has not been excluded, as all analysed deletion constructs were inserted at the 3′ end of EFG1 and, hence, were downstream of the entire native 5′ upstream region of the resident EFG1 gene.

A white phase-specific activation complex at AR2

We have demonstrated by gel retardation experiments that AR2 forms a phase-specific complex with white phase cell extract of strain WO-1. This complex formed, but at dramatically reduced levels, using opaque phase extract. We could not discriminate between the possibility that the low levels observed resulted from contamination of low levels of white phase cells in opaque phase cell populations, which occurs through switching (Anderson and Soll, 1987; Slutsky et al., 1987), and the possibility that opaque phase cells do form low levels of the complex.

Previous analysis of the promoter of the white phase-specific gene WH11 by gel retardation experiments also revealed white phase-specific complexes (Srikantha et al., 2000). Although the distal activation sequence of the WH11 promoter formed one white phase-specific complex, the proximal activation sequence formed two white phase-specific complexes. The WH11 promoter formed no opaque phase-specific complexes. A comparison between the WH11 and EFG1 white phase activation regions revealed that each contains an Rbf1p binding site. In addition, the Rbf1p site is in the proximal activation sequence (PAS) of the WH11 promoter and in the proximal component of the AR2 region of the EFG1 promoter. The role of RBF1 in the co-ordinate regulation of white phase-specific genes is now under investigation.

In summary, we demonstrated previously that EFG1 is expressed as different-sized transcripts in the white and opaque phases of switching (Srikantha et al., 2000). Here, we have demonstrated that phase-specific transcription of EFG1 is regulated by overlapping promoters with distinctly different activation mechanisms. It should be noted that EFG1 is not the only phase-regulated gene expressed as alternative molecular weight transcripts in the white–opaque transition. The deacetylase HOS3 is transcribed as a 2.5 kb transcript in the white phase and as a less abundant, lower molecular weight 2.3 kb transcript in the opaque phase (Srikantha et al., 2001). The HOS3 promoter is now being functionally characterized to assess whether overlapping white–opaque promoters represent a general regulatory strategy in the switching process.

Experimental procedures

Maintenance of stock cultures

Candida albicans wild-type strain WO-1 ( Slutsky et al., 1987 ), strain Red3/6, an ade2 auxotroph ( Srikantha et al., 1996 ), and strain TS3.3, a ura3 auxotroph ( Srikantha et al., 2000 ), were maintained at 25°C on agar containing modified Lee's medium ( Bedell and Soll, 1979 ) or modified Lee's medium supplemented with either 0.6 mM adenine sulphate or 0.01 mM uridine in cases of auxotrophy. JKC19, a cph1 / cph1 null mutant ( Liu et al., 1994 ), which was a generous gift from Dr H. Liu of the University of California, Irvine, was maintained at 25°C on agar containing yeast extract, peptone and dextrose. Transformants that acquired either adenine or uridine prototrophy were subsequently maintained in the absence of adenine sulphate or uridine respectively.

Isolation of the EFG1 promoter

A 380 bp region of the EFG1 ORF was used as a probe to screen a λEMBL3A genomic library of C. albicans strain WO-1 (Srikantha and Soll, 1993). Out of ≈ 50 000 plaques screened, 50 putative lambda clones were identified, and two clones, λ14.1 and λ39.1, which contained ≈ 10 and 12 kb of insert DNA, respectively, were selected by Southern analysis. A gene-walking strategy was used to isolate the 5′ flanking region of EFG1 from λ14.1 and λ39.1, using a combination of the primer EC1 with either the lambda left-arm-specific primer ELA or the lambda right-arm-specific primer ERA (Table 2). A 2.4 kb sequence upstream of the ATG start site was obtained from λ39.1 and used in these studies.

Table 2. .  Oligonucleotides used for generating deletion constructs and for gel mobility shift assays.
NameOligonucleotideEstimated product size (–bp) with EC8
EfN15′-TCGAACAGGATGCAACTACA-3′−2320
Ef25′-ACAATCATGATCTCTATGTG-3′−2118
Ef35′-GTATGCGTTAGTCTCAATTG-3′−2022
Ef45′-GCAGTTGTTATTGACAACTT-3′−1952
Ef4.15′-CTTTCACTACAACCTAATTA-3′−1892
Ef55′-GCATTACTCATTGTGCATCT-3′−1834
Ef5.15′-CTATTTGCTATTTCATTTTC-3′−1809
Ef5.25′-CGCCAACAATAGAAACTAAT-3′−1764
Ef65′-GCATATATGCATATACCAAT-3′−1730
Ef6.15′-TAGTTACAAAAGCGGTAGTG-3′−1685
Ef75′-GCATTCAGTCATACATATAC-3′−1624
Ef85′-CATTGCTGTTGGAATCTCTT-3′−1518
Ef95′-ATCTTGTAATCTTGTTATAC-3′−1414
EPB125′-AGACTTGGTTGGTATTAATA-3′−1115
EPB12.15′-AAATAGAATAGGATTTTATT-3′−1073
EPB12.25′-GTTTAAAGTCTTTCCGAGTA-3′−1028
EPB12.45′-TTGCTCAAAAGTCTGGAAAG-3′−922
EC95′-CAACCAATTGCCCTACCCAT-3′−840
EPB95′-GCTACCAAACTGACACAGGT-3′−712
EPB55′-CATTCACACTCAATACACTTC-3′−337
EPB25′-AGCCTTTTTTGCCTCCCACA-3′−165
EPB15′-CAACCAACCAACCCTTAACCC-3′−74
ID1 Rev5′-TCAACAAAGTGAAAATGAAATAGCA-3′ 
EF5GMSA5′-CATCTTTTTTCTATTTGCTAT-3′ 
LR6p5′-TTGGTATTGGGCAGTATTGCTA-3′ 
EC85′-TAATATGGGTTATATTCTTGG-3′ 

Construction of plasmids

A 1208 bp fragment containing the last 633 bp of the EFG1 ORF plus 575 bp immediately downstream from the EFG1 ORF was inserted into the ADE2-containing vector, pCRW3 (Srikantha et al., 1996), to obtain pCRW3E (Fig. 2A). The URA3-based vector pA43.1 (T. Srikantha and D. R. Soll, unpublished) containing the PCK1 promoter was used as a backbone to construct pA43.1R (Fig. 3A). The PCK1 promoter was removed from pA43.1 by SmaI–ApaI digestion. An RLUC ORF, generated by polymerase chain reaction (PCR) using pCRW3 as a template, was substituted in its place. The same EFG1 3′ end region used in pCRW3E (Fig. 2A) was inserted upstream of the RLUC ORF to generate pA43.1RE (Fig. 3A).

Construction of promoter derivatives

For experiments designed to explore positional effects on promoter function, the 2320 bp region immediately upstream of the EFG1 ORF was inserted in the correct direction at the multiple cloning site immediately upstream of the Renilla reniformis luciferase gene (RLUC) in pCRW3E (Fig. 3). To characterize the EFG1 promoter functionally, the same 2320 bp region as well as deletion and mutation derivatives of this sequence were inserted in the correct direction at the multiple cloning site immediately upstream of RLUC in pA43.1RE (Fig. 1). Deletion and mutation derivatives were generated by PCRs using the oligonucleotide primers described in Table 2 and λ39.1 as template. The PCR products were directionally inserted into pA43.1RE as PstI–SmaI fragments. To generate point mutations in the AR2 region of the EFG1 promoter, oligonucleotides carrying two or more specific point mutations were used in PCRs with λ39.1 as template. All plasmid constructs containing deletion and mutation derivatives were verified by sequencing.

For integrative transformation, 25 µg of plasmid was linearized within either the EFG1 3′ end or the ADE2 gene (Fig. 3). In the case of pCRW3E, XbaI was used to linearize the plasmid within ADE2 (Fig. 2B), whereas DraIII was used to linearize it within the EFG1 3′ end (Fig. 2C). Transformations were performed by the lithium acetate method as described previously (Schiestl and Gietz, 1989). Each transformant was analysed by Southern blot analysis to confirm single-copy insertions before being used in subsequent analyses.

To create the internal deletion within the EFG1 promoter, we cloned the entire EFG1 promoter (−2320 bp) in the T-Easy vector (Promega) and PCR amplified the resulting plasmid construct using the outwardly moving oligomers ID1 Rev and EF5.2. The resulting PCR product was religated and sequenced to confirm the 15 bp deletion between −1779 and −1764. This internal deletion construct in T-Easy was used as a template for PCR amplification using EfN1 and EC8 oligomers. The resulting PCR product was used as an insert for cloning in pA43.1RE.

Southern and Northern blot analysis

Southern and Northern blot analyses were performed according to methods described in detail previously (Srikantha et al., 1998; 2000). Prehybridization and hybridization procedures were performed according to the methods of Church and Gilbert (1984).

In vitro luciferase assays

Luciferase activity was assayed according to the methods of Srikantha et al. (1996). In brief, white and opaque phase cells were grown to late log phase in modified Lee's liquid medium and washed once with sterile distilled water and once with luciferase buffer [0.5 M NaCl, 0.1 M K2HPO4, pH 6.7, 1 mM EDTA, 0.6 mM sodium azide, 1 mM phenylmethylsulphonyl fluoride (PMSF), 0.02% BSA] (Srikantha et al., 1996). Cells were lysed as described previously (Srikantha et al., 1996). Luciferase buffer (100 µl) containing 0.5 µM coelenterazine (Molecular Probes) was mixed with 2 µl of undiluted extract. Light emission was measured at 480 nm in the integration mode for 30 s in a Monolight 2010 luminometer (Analytical Luminescence Laboratory). Relative light units (RLUs) were defined as light emitted per 30 s per µg of protein. Protein concentrations were estimated using a modified Lowry assay (Sigma Chemical).

Gel mobility shift assays

The methods used have been described in detail elsewhere (Srikantha et al., 1995). In brief, C. albicans cells were grown to late log phase in modified Lee's liquid medium at 25°C and lysed in a bead beater as described previously (Srikantha et al., 1995). The extracts were then cleared by centrifugation for 2 h at 150 000 g, and the supernatants were dialysed against a solution containing 20 mM Hepes, pH 7.9, 5 mM EDTA, 20% glycerol and 1 mM PMSF). Cell extracts contained between 10 and 12 mg of protein ml−1 as determined by the modified Lowry assay. A PCR-amplified DNA fragment between the −1819 and −1689 region of the EFG1 promoter was end-labelled with [γ32-P]-dATP and used as a DNA probe. Basic binding reactions were performed at 25°C in a total volume of 15 µl of a solution containing 25 mM Hepes (pH 7.9), 50 mM NaCl, 1 mM EDTA, 0.05% Nonidet P-40, 1 mM dithiothreitol, 5% glycerol, 5 µg of poly-(dI–dC) and various amounts of protein extract. The mixture was preincubated for 10 min, the radiolabelled DNA probe (100–300 pg) was then added, and the mixture was incubated for 20 min at 25°C. After incubation, a solution of binding buffer containing xylene–cyanol and bromophenol blue was added, and the reaction mixture was loaded immediately onto a 6% non-denaturing gel (39:1 acrylamide to bisacrylamide) in 0.5× TBE (44.5 mM Tris-HCl, pH 8.1, 44.5 mM H3BO3, 1 mM EDTA) that had been prerun for 1 h at 110 V. Gel electrophoresis was performed at a constant 100 V at 4°C until the marker had migrated to the bottom of the gel. Gels were then transferred to Whatman 3MM paper and autoradiographed.

Acknowledgements

The authors are indebted to Dr Shawn Lockhart for technical advice. This research was supported by NIH grant AI2392.

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