In Saccharomyces cerevisiae, heme directly mediates the effects of oxygen on transcription through the heme activator protein Hap1. In the absence of heme, Hap1 is bound by at least four cellular proteins, including Hsp90 and Ydj1, forming a higher-order complex, termed HMC, and its activity is repressed. Here we purified the HMC and showed by mass spectrometry that two previously unidentified major components of the HMC are the Ssa-type Hsp70 molecular chaperone and Sro9 proteins. In vivo functional analysis, combined with biochemical analysis, strongly suggests that Ssa proteins are critical for Hap1 repression in the absence of heme. Ssa may repress the activities of both Hap1 DNA-binding and activation domains. The Ssa cochaperones Ydj1 and Sro9 appear to assist Ssa in Hap1 repression, and only Ydj1 residues 1 to 172 containing the J domain are required for Hap1 repression. Our results suggest that Ssa-Ydj1 and Sro9 act together to mediate Hap1 repression in the absence of heme and that molecular chaperones promote heme regulation of Hap1 by a mechanism distinct from the mechanism of steroid signaling.

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Heme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen. In the yeast Saccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain. To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated. Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation. All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme. More importantly, we show that a novel class of repression modules-RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244-is critical for Hap1 repression in the absence of heme. Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC. These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme.

The yeast heme activator protein Hap1 binds to DNA and activates transcription of genes encoding functions required for respiration and for controlling oxidative damage, in response to heme. Hap1 contains a DNA-binding domain with a C6 zinc cluster motif, a coiled-coil dimerization element, typical of the members of the yeast Gal4 family, and an acidic activation domain. The regulation of Hap1 transcription-activating activity is controlled by two classes of Hap1 elements, repression modules (RPM1-3) and heme-responsive motifs (HRM1-7). Previous indirect evidence indicates that Hap1 may repress transcription directly. Here we show, by promoter analysis, by chromatin immunoprecipitation, and by electrophoretic mobility shift assay, that Hap1 binds directly to DNA and represses transcription of its own gene by at least 20-fold. We found that Hap1 repression of the HAP1 gene occurs independently of heme concentrations. While DNA binding is required for transcriptional repression by Hap1, deletion of Hap1 activation domain and hemeregulatory elements has varying effects on repression. Further, we found that repression by Hap1 requires the function of Hsp70 (Ssa), but not Hsp90. These results show that Hap1 binds to its own promoter and represses transcription in a heme-independent but Hsp70-dependent manner.

In the absence of heme, Hap1 is associated with molecular chaperones such as Hsp90 and Ydj1 and forms a higher order complex termed HMC. Heme disrupts this complex and permits Hap1 to bind to DNA with high affinity, thereby activating transcription. Heme regulation of Hap1 activity is analogous to the regulation of steroid receptors by steroids, which involves molecular chaperones. Steroid receptors often exist as monomers when associated with molecular chaperones in the absence of ligand but as dimers when activated by steroids. Furthermore, previous studies indicate that dimerization might be important for heme activation of Hap1. We therefore determined whether Hap1 is a monomer or oligomer in the absence of heme. By coeluting two Hap1 size variants and by comparing DNA binding properties of the HMC and Hap1 dimer, we show that Hap1 is a preexisting dimer in the HMC. Further, increasing overexpression of Hap1 caused progressive increases in Hap1 DNA binding and transcriptional activities. Our data suggest that in the absence of heme, Hap1 exists as a dimer, and the two subunits act cooperatively in DNA binding. Hap1 repression is caused, at least in part, by inhibition of the DNA binding activity of the preexisting dimer.Dimerization is a common mechanism by which the activity of numerous important biological macromolecules can be regulated. These molecules include receptors for growth hormones (1, 2), steroid hormones (3), other cellular signals (4, 5), and numerous transcription factors (6, 7). The transcriptional activators of the yeast Gal4 family also require dimerization for DNA binding and transcriptional activation (8,9). This family includes at least 52 transcription factors that control a wide array of diverse processes ranging from carbon source utilization to oxygen utilization and drug resistance (8, 9). These members all contain a C6 zinc cluster that recognizes a CGG triplet (9 -15). Although the DNA binding properties of the C6 zinc cluster proteins are well characterized, the molecular mechanisms by which these members act to control transcription in response to various signals are largely unclear. Interestingly, recent data suggest that, like steroid hormone receptors, certain members of the yeast Gal4 family such as Hap1 and Pdr1 (16,17) are regulated by Hsp90 and Hsp70 molecular chaperones. In particular, Hap1 is a heme-responsive transcriptional activator, which promotes transcription of genes required for respiration and for controlling oxidative damage in response to oxygen/heme (18 -20). In the absence of heme, Hap1 is bound to cellular proteins including Hsp82 (the yeast homologue of Hsp90) and Ydj1, forming a higher order complex termed HMC 1 (17,21,22). Hap1 DNA binding and transcriptional activities are repressed in this complex. Heme disrupts the HMC and permits Hap1 to bind to DNA as a dimer with high affinity, thereby activating transcription. The formation and disruption of the HMC are the key events in Hap1 repression in the absence of heme and subsequent activation by heme. ...