The heme activator protein Hap1 represses transcription by a heme-independent mechanism in Saccharomyces cerevisiae

Abstract

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 heme-regulatory 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.

Figure 1.—

(A) The repression of the HAP1 promoter reporter by Hap1 mutants. The domain structures of the Hap1 mutants with point mutation or deletion are shown. Wild-type Hap1 is shown to contain the C6 zinc cluster motif (Zn), the dimerization element (DE), three repression modules (RPM1–3), seven heme-responsive motifs (HRM1–6 and HRM7), and the activation domain (ACT). Mutant Hap1-18 contains a mutation of Ser-to-Arg at amino acid position 63. The deleted regions in mutants Δheme, H7-d1, H7-d2, and smHap1 are shown by angled lines. HB, HG, and HK contain residues 1–444, 1–746, and 1–1308, respectively. The expression plasmids for Hap1 mutants were transformed into Δhap1 cells bearing the full HAP1 promoter-lacZ reporter or the UAS1/CYC1-lacZ reporter, which contains the UAS1 sequence to which Hap1 binds, but not the UAS2 sequence to which the Hap2/3/4/5 complex binds (Guarente et al. 1984; Turcotte and Guarente 1992). Cells were grown in selective medium, and β-galactosidase activities were detected. The data shown are averages from at least three independent transformants. (B) Western blot showing the levels of wild-type and mutant Hap1 proteins. Extracts prepared from cells expressing wild-type Hap1 (lane 1), Hap1-18 (lane 2), Δheme (lane 3), H7-d1 (lane 4), H7-d2 (lane 5), smHap1 (lane 6), HB (lane 7), and HK (lane 8) were subjected to electrophoresis in a 7.5% SDS-polyacrylamide gel and transferred to a polyvinyl difluoride membrane. The levels of Hap1 proteins were detected by probing Western blots with antibodies against Hap1.

Figure 2.—

Deletion analysis of the HAP1 promoter. Shown in the top half are the sequence structures of the various constructed HAP1 promoter reporter genes. Yeast wild-type and Δhap1 cells were transformed with the indicated reporter plasmid. Cells were grown in selective medium, and β-galactosidase activities were detected. The plotted data are averages from at least three independent transformants.

Figure 3.—

Hap1 binding to the HAP1 promoter region encompassing nucleotide sequence −50 to −300. Extracts prepared from Δhap1 cells with the HAP1 gene deleted (lane 1) or from cells expressing high levels of Hap1 (lanes 2–7), or the purified GST-Hap1 protein (100 nm, lanes 9 and 10) were incubated with radiolabeled DNA encompassing nucleotide sequence −50 to −300 of the HAP1 promoter, in the absence (lanes 1 and 2) or presence of preimmune serum (lane 3), Hap1 antibodies (lane 4), unlabeled DNA containing high-affinity Hap1-binding site 7/1 (lanes 5 and 10), unlabeled DNA encompassing nucleotide sequence −1 to −461 of the HAP1 promoter (lane 6), or unlabeled DNA encompassing nucleotide sequence −1 to −380 of the HAP1 promoter (lane 7). The free DNA probe is shown in lane 8. The reaction mixtures were analyzed on a 4% nondenaturing polyacrylamide gel. The positions of the Hap1-DNA complex (Hap1 and GST-Hap1) and the bands representing free probe (Free) are marked. The purified GST-Hap1 fusion protein contains the GST residues 1–222 (26 kD) and the Hap1 residues 1–171 (Zhang and Guarente 1994c) and is smaller than full-length Hap1 (residues 1–1483) expressed in yeast cells. Thus, the GST-Hap1-DNA complex migrated faster than the Hap1-DNA complex in the gel.

Figure 4.—

ChIP-chip analysis indicates that the promoter DNA of the HAP1 gene is preferentially associated with Hap1 in vivo. ChIP-chip analysis was performed as described in materials and methods. Shown is the genomic sequence map around the HAP1 locus. The boxes represent the sequences that are preferentially detected by coimmunoprecipitated DNAs with Hap1 antibodies. Three sequences, iYLR255C encompassing the promoter region of the HAP1 gene, YLR256W encompassing the coding sequence of the HAP1 gene, and iYLRWdelta14, which is distal to the HAP1 gene, are found to be preferentially enriched by coimmunoprecipitation.

Figure 5.—

The effect of Ssa (A) and Hsp90 (B) on the repression of the HAP1 promoter by Hap1. Yeast cells with SSA2, SSA3, and SSA4 genes deleted (a2a3a4); cells expressing a low level of Hsp90 (iLEP); and their wild-type counterparts were transformed with the full-length HAP1 promoter-lacZ reporter plasmid. Cells were grown in selective medium, and β-galactosidase activities were detected. The plotted data are averages from at least three independent transformants. For controls, the activities of the Hap2/3/4/5-driven UAS2UP1-lacZ (Forsburg and Guarente 1989), HIS4-lacZ (Hinnebusch 1984), and UAS1/CYC1-lacZ (Guarente et al. 1984; Turcotte and Guarente 1992) reporters in a2a3a4 cells were also measured and shown. The activities of the Hap2/3/4/5-driven UAS2UP1-lacZ (Forsburg and Guarente 1989) and HIS4-lacZ (Hinnebusch 1984) in the iLEP strain have been shown previously (Zhang et al. 1998). Note that the strains used in A and B are derived from different laboratories and have different genotypes (see materials and methods). This difference likely accounts for the fourfold difference in the HAP1-lacZ reporter activity in the wild-type control strains in A (JN55) and B (W303).