Hemoglobin and growth signals control HBR1 expression. (A) HBR1-CDC36 region in C. albicans. The SY1 EST used to identify the HBR1 genomic clone overlaps the 3′ end of CaHMX1, a predicted heme oxygenase (5, 49). The arrowhead indicates the mapped transcription start. This orientation and positioning of CDC36 with HBR1 is conserved with the ortholog FAP7 in S. cerevisiae. (B) Hemoglobin increases HBR1 basal expression. C. albicans 44807 cells were grown with and without 500 μg of hemoglobin/ml at 30°C, and RNA was isolated for Northern analysis. The same blot was sequentially hybridized with DNA probes of HBR1 and PGK1. rRNA was used as a loading control (ethidium bromide-stained gel). RNA isolated from strain CAF2-1 gave similar results (data not shown). (C) Hemoglobin and glucose stimulate periodic HBR1 transcriptional activity. CAMP 35 cells were transferred to YNB medium containing 2% glucose with (•) or without (○) 500 μg of hemoglobin/ml and sampled at the indicated times for luciferase activity. This assay was repeated four times with similar results each time. (D) Hemoglobin and glucose signaling to HBR1 are separable. CAMP 35 cells were transferred to YNB medium lacking glucose with (•) or without (○) hemoglobin as indicated in panel C. Luciferase activity (LUX) is reported as light units per 5 × 10⁴ cells.

HBR1 deletion leads to high-frequency white-opaque phase switching. (A) Prototrophic HBR1^+/− strain CAMP 43 growing on phloxine B agar plates (frame 1), showing an opaque colony (center) surrounded by white colonies, from microscopic examination of CAMP 43 (frame 2, opaque; frame 3, white), and Red 3/6 (frame 4, opaque) cells. Bar, 10 μm. (B) Scanning electron micrograph of a CAMP 43 opaque cell, illustrating the surface protrusions or pimples characteristic of opaque cells. Bar, 3 μm. (C) Regulation of phase-specific genes in the HBR1 heterozygote was determined by RT-PCR analysis of RNA isolated from 4-h exponential-phase cultures of strain CAMP 43 grown at 30°C in YNB-glucose. W, white; O, opaque; ACT1, actin. Probes for phase-specific transcription were as follows: white, WH11 (59); opaque, OP4 and SAP1 (38).

Regulation of a1/α2 repression and α-cell gene targets through HBR1. (A) An a1/α2 target is derepressed in an HBR1 heterozygote. Results of qPCR analysis of RNA isolated from 4-h log-phase cells with and without 0.5 mg of hemoglobin/ml are shown. MTL and HBR1 genotypes as well as white (W) and opaque (O) phenotypes are indicated. CAG1 expression from each strain was corrected for the level of the CDC36 internal standard and then normalized to CAF2-1 levels (arbitrarily set at 1). Strain designations (from left to right): CAF2-1, CAMP 43, CAMP 43, CHY477, CAMP 49, and MM278. This figure represents two separate analyses. Levels are reported as ± the SD. (B). YEL007w expression is derepressed in HBR1 heterozygous cells. qPCR analysis was carried out with the same cDNA preparations as described for panel A. Strain designations are as in panel A, except CAMP 43 opaque cells were not tested. Normalized levels are reported as ± the SD. (C and D) Two α-specific genes are down-regulated in CAMP 43 opaque cells. Mating factor α and STE3 mRNA levels were determined using qPCR with the same cDNA preparations as described for panel A. Levels are reported as ± the SD.

Model for the role of HBR1 in MTL gene regulation. (A) Hbr1p expression is stimulated by growth and by signals from the host factor hemoglobin. Normal cellular levels of Hbr1p support MTLα1 and MTLα2 expression and moderately repress MTLα1. Both alleles of HBR1 are necessary to maintain MTLα gene expression and functional levels of the a1/α2 repressor. According to current models (36), this repressor limits expression of CAG1, white-opaque phase switching, and mating. (B) Limiting Hbr1p by removing stimuli controlling its expression or by allelic deletion represses MTLα1 and MTLα2 gene expression. These conditions derepress genes regulated by a1 α2, permit white-opaque phenotypic switching, increase expression of a-cell-specific genes, and decrease α-cell-specific genes. Through this process whereby MTLα1 and MTLα2 expression is down-regulated, the cells acquire a cell characteristics and are capable of mating. Thus, HBR1 regulation of MTL genes is a mechanism that can allow mating without the deletion of MTL genes.

Conservation of the predicted amino acid sequences of HBR1 and FAP7. The alignment of predicted protein sequences of Hbr1p and Fap7p is shown. A consensus sequence was generated using DIALIGN 2.2 (37; http://bibiserv.techfak.uni-bielefeld.de/dialign/) with the following eukaryotic orthologs of Hbr1p: Schizosaccharomyces (CAB52884), Candida (AF466197), Saccharomyces (CAA98740), Caenorhabditis (Q09527), Drosophila (AAF58491), Oryctolagus (AAF09498), Homo sapiens (Q9Y3D8), Mus (BAB29612), Anopheles (EAA06472), and Arabidopsis (BAB10972). All uppercase residues were aligned in the analysis. Consensus residues that were highly conserved are indicated below the aligned yeast sequences. Residues 65 to 68 are the SUMO consensus (43).

Hbr1p is a positive regulator of MTLα expression. (A) HBR1 heterozygosity does not lead to MTL gene deletions. Results of PCR analysis of genomic DNA using primers specific for MTL genes and for the λimm434 region used in construction of parental strain CAI-4 (14) are shown. Lanes 1 and 2, CAMP43, white and opaque, respectively; lane 3, Red 3/6, white; lane 4, CAI-4, white; lane 5, clinical isolate 156, white. (B) MTL α gene expression is not detectable in the HBR1 heterozygote, as shown by an RT-PCR analysis of RNA isolated from 4-h exponential-phase cells cultured in YNB-glucose medium at 30°C. Gene-specific primers listed on the left were used in PCRs of 26 cycles (MTL genes) or 20 cycles (ACT1). Lane 1, strain CAMP 48; lanes 2 and 3, strain CAMP 43; lane 4, strain CAI-4. (C) MTLα genes are induced only during exponential growth. Results of the RT-PCR analysis of RNA isolated from CAF2-1 cells at the indicated times after transfer of stationary-phase cells to YNB-glucose medium are shown. (D) HBR1 overexpression sustains MTLα expression into early stationary phase. Results of RT-PCR analysis of RNA harvested at 4 and 24 h after transfer of stationary-phase cells to low-methionine medium are shown. CAMP 63 contains an HBR1 copy under the control of the MET3 promoter. The MET3 promoter maintained HBR1 RNA at higher levels in the CAMP 63 strain than in strains containing only the native HBR1 promoter (see Fig. 6B). (E) Hemoglobin can sustain MTLα gene expression into stationary phase. Strain CAF2-1 cells were cultivated in the presence or absence of 0.5 mg of hemoglobin/ml, and RNA was harvested at the indicated times. Primer sets for RT-PCR analysis are listed in the figure. MTLα, 26 cycles; rRNA, 15 cycles.

HBR1 regulates a-specific genes and is a negative regulator of MTL a. (A) Two a-specific genes are regulated through HBR1. qPCR analysis was performed using the same cDNA preparations as in Fig. 5. (B) Hbr1p expression represses MTLa1 expression. Strain CAMP 43 contains only one HBR1 allele, and CAMP 63 contains HBR1 expressed at high levels under the control of the MET3 promoter.