A MADS box protein consensus binding site is necessary and sufficient for activation of the opaque-phase-specific gene OP4 of Candida albicans - PubMed (original) (raw)

A MADS box protein consensus binding site is necessary and sufficient for activation of the opaque-phase-specific gene OP4 of Candida albicans

S R Lockhart et al. J Bacteriol. 1998 Dec.

Abstract

The majority of strains of Candida albicans can switch frequently and reversibly between two or more general phenotypes, a process now considered a putative virulence factor in this species. Candida albicans WO-1 switches frequently and reversibly between a white and an opaque phase, and this phenotypic transition is accompanied by the differential expression of white-phase-specific and opaque-phase-specific genes. In the opaque phase, cells differentially express the gene OP4, which encodes a putative protein 402 amino acids in length that contains a highly hydrophobic amino-terminal sequence and a carboxy-terminal sequence with a pI of 10.73. A series of deletion constructs fused to the Renilla reniformis luciferase was used to functionally characterize the OP4 promoter in order to investigate how this gene is differentially expressed in the white-opaque transition. An extremely strong 17-bp transcription activation sequence was identified between -422 and -404 bp. This sequence contained a MADS box consensus binding site, most closely related to the Mcm1 binding site of Saccharomyces cerevisiae. A number of point mutations generated in the MADS box consensus binding site as well as a complete deletion of the consensus site further demonstrated that it was essential for the activation of OP4 transcription in the opaque phase. Gel mobility shift assays with the 17-bp activation sequence identified three specific complexes which formed with both white- and opaque-phase cell extracts. Competition with a putative MADS box consensus binding site from the promoter of the coordinately regulated opaque-phase-specific gene PEP1 (SAP1) and the human MADS box consensus binding site for serum response factor demonstrated that one of the three complexes formed was specific to the OP4 sequence.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

Oligonucleotides used for generating deletion constructs and for gel mobility shift assays.

FIG. 2

FIG. 2

The white-opaque transition of the adenine auxotroph Red 3/6 of C. albicans WO-1 and differential expression of OP4 in the opaque phase. (A) Cells of strain Red 3/6 switch spontaneously and reversibly at 25°C at frequencies of approximately 10−3 between a white- and an opaque-colony-forming phenotype. This reversible transition is accompanied by a distinct change in cellular morphology. White-phase cells are round while opaque-phase cells are twice as large, on average, and elongated and contain a large vacuole. (B) Opaque-phase cells of strain Red 3/6 differentially express the gene OP4. Northern blots of white- and opaque-phase cells were probed with OP4 and the constitutively expressed gene ADE2. Even though Red 3/6 is an adenine auxotroph, the mutant ade2 gene is constitutively expressed. Bar, 2 μm.

FIG. 3

FIG. 3

The 854-bp sequence immediately upstream of the translational start site of opaque-phase-specific gene OP4. PRE, consensus sequences of binding sites for Ste12 of S. cerevisiae (14, 16, 23); C-box, a C-rich sequence also identified in the upstream region of the coordinately activated, opaque-phase-specific gene PEP1 (SAP1) (30); MADS box binding site, MADS box consensus binding site homologous with the Mcm1 binding site of S. cerevisiae (2); TATA, TATA box consensus sequence (10); tsp, transcription start point.

FIG. 4

FIG. 4

Homology between the MADS box consensus binding site in the OP4 promoter and MADS box consensus binding site of S. cerevisiae, humans, and plants. Shown are Mcm1 binding site of S. cerevisiae (1), Rlm1 binding site of S. cerevisiae (13), AGAMOUS binding site of Arabidopsis (17), SRF binding site of humans (35), and Mef2a binding site of humans (33). Grey boxes denote conserved nucleotides.

FIG. 5

FIG. 5

Functional characterization of the OP4 promoter through a set of deletion derivatives. (A) Model of the OP4 promoter. Sequences homologous to known regulatory sequences in the promoters of other genes as well as the C box also found in the promoter of the coordinately regulated PEP1 (SAP1) gene are noted. The sequence of the promoter is presented in Fig. 3. ORF, open reading frame. (B) Activity of Rluc under the regulation of the deletion derivatives of the OP4 promoter in the opaque and white phases of switching. s.d., standard deviation; Op., opaque; Wh., white; Diff., difference.

FIG. 6

FIG. 6

Functional characterization of the OP4 MADS box consensus sequence through substitution of consensus sequence with polylinker (OΔMADS) and replacement of the promoter downstream of the consensus sequence with the downstream region of the WH11 promoter (OWhyb). (A) Models of the two constructs OΔMADS and OWhyb. (B) Sequence of the substitution in OΔMADS. The OP4 activation sequence is highlighted. (C) Activity of Rluc under the regulation constructs OΔMADS and OWhyb. Note that CROP31 contain the complete OP4 promoter and that CRW3 is a promoterless construct. Op., opaque; Wh., white; s.d., standard deviation; Diff., difference.

FIG. 7

FIG. 7

Gel mobility shift assays with an oligonucleotide containing the MADS box consensus binding site flanked by 16 upstream and 8 downstream nucleotides of the OP4 promoter and either white- (A) or opaque-phase (B) cell extract. Lanes 1, no protein extract; lanes 2, bovine serum albumin in place of protein extract; lanes 3, white- or opaque-phase protein extract; lanes 4, 5, and 6, increasing concentrations of unlabeled oligonucleotide containing the putative MADS box consensus binding site of OP4; lanes 7, 8, and 9, increasing concentrations of unlabeled oligonucleotides containing the putative MADS box consensus binding site of PEP1 (SAP1); lanes 10, 11, and 12, increasing concentrations of unlabeled oligonucleotide containing the SRF consensus binding site. Double-stranded oligonucleotides used in the gel mobility shift experiments include OP4 (M4 and M4A in Fig. 1), the 38 bp of the OP4 promoter containing the MADS box consensus binding site; PEP1 (SAP1) (M5 and M5A in Fig. 1), the 38 bp of the OP4 promoter in which the OP4 MADS box consensus binding site is replaced with the putative MADS box consensus binding site in the promoter of the gene PEP1 (SAP1) (30), which is coordinately regulated with OP4 (31); and SRF (M1 and M1A), the 38 bp of the OP4 promoter in which the OP4 MADS box consensus binding site is replaced with the SRF binding site of humans (35). Lower exposures discriminated the two bands CIV and CIII in the lanes containing no competitor, OP4 competitor, and 100× SRF competitor (data not shown).

References

    1. Acton T, Zhong H, Vershon A. DNA-binding specificity of Mcm1: operator mutations that alter DNA-bending and transcriptional activities by a MADS box protein. Mol Cell Biol. 1997;17:1881–1889. - PMC - PubMed
    1. Ammerer G. Identification, purification, and cloning of a polypeptide (PRTF/GRM) that binds to mating-specific promoter elements in yeast. Genes Dev. 1990;4:299–312. - PubMed
    1. Anderson J, Soll D. Unique phenotype of opaque cells in the white-opaque transition of Candida albicans. J Bacteriol. 1987;169:5579–5588. - PMC - PubMed
    1. Anderson J, Cundiff L, Schnars B, Gao M, Mackenzie I, Soll D. Hypha formation in the white-opaque transition of Candida albicans. Infect Immun. 1989;57:458–467. - PMC - PubMed
    1. Anderson J, Mihalik R, Soll D. Ultrastructure and antigenicity of the unique cell wall pimple of the Candida opaque phenotype. J Bacteriol. 1990;172:224–235. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources