The Yeast Heme-responsive Transcriptional Activator Hap1 Is a Preexisting Dimer in the Absence of Heme

Abstract

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. ...

Nuclear translocation of haeme oxygenase-1 is associated to prostate cancer

“…In response to haeme, these transcription factors bind to activation sequences of numerous genes encoding functions required for respiration and for controlling oxidative damage (Hon et al, 1999). As with other heat-shock proteins (Segui-Simarro et al, 2003), the transport of HO-1 could involve either interaction of the enzyme nuclear localisation signal with the nuclear pore complex or with other cytoplasmic components that would deliver the protein (Li Volti et al, 2004).…”

Inducer-Dependent Nuclear Localization of a Zn(II)2Cys6Transcriptional Activator, AmyR, inAspergillus nidulans

“…Most Zn(II) 2 Cys 6 factors localize constitutively to the nucleus, and consequently transcriptional activation by these factors is regulated in the nucleus at the level of DNA binding 16,17) or by intermolecular or intra-molecular masking of the activation domain. 18,19) AmyR is one of the rare examples of regulated nuclear localization.…”

“…These include Gal4p, Pdr1p, Pdr3p, Put3p, Leu3p, Lys14p, and Hap1p of S. cerevisiae, and PrnA and AlcR of A. nidulans. [8][9][10][11][12][13][14][15] Transcriptional activation by these factors in response to inducing stimuli is controlled by mechanisms acting within the nucleus to modulate DNA binding activity 16,17) or to regulate the accessibility of the transcriptional activation domain. 18,19) A. nidulans AflR and NirA are rare examples of Zn(II) 2 Cys 6 transcription factors that show regulated nuclear localization.…”

Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases

“…Studies have revealed that Hap1 contains several important functional elements and that heme regulation of Hap1 involves the regulation of DNA-binding and transcriptionactivating activities of Hap1 (Figure 2A) [14,[28][29][30][31]. The C6 zinc cluster motif, which is located near the N-terminus, and the dimerization domain mediate Hap1 DNA binding, while the seven heme-responsive motifs (HRMs) and three repression modules (RPMs) are responsible for coupling heme regulation with Hap1 activation (Figure 2, Table 1) [14,29,[31][32][33][34].…”

“…The repression of Hap1 in the absence of heme is mediated by the three RPMs, which promote the formation of a higher order Hap1 complex, containing the molecular chaperones and co-chaperones Hsp90, Hsp70, Sro9 and Ydj1 [28,31,36]. This Hap1 multichaperone complex acts to control transcription in response to heme [14,29,30,34]. Hap1 has seven HRMs that can bind heme and mediate heme activation of Hap1 [29].…”