The transcriptional activator complex Hap2/3/4/5 plays a key role in the upshift of genes involved in oxidative metabolism in S. cerevisiae. The gene encoding the Hap4p activator subunit is the only one of which the transcription is regulated in response to available carbon source, and this regulation is the main determinant for the activity of the complex. The present study describes the characterization of the unusually long promoter region of the HAP4 gene as a first step in the unravelling of the mechanisms of transcriptional regulation of HAP4 gene expression. Initially, we generated 5′-HAP4 promoter deletion fused to a minimal CYC1–LacZ reporter and integrated the constructs in the ura site of the genome. However, we were not able to detect effects of 5′-HAP4 promoter deletions in a chromosomal context as there was a problem of insufficient sensitivity. To compensate for this by virtue of increased expression levels, we therefore made use of subfragments of the HAP4 promoter cloned in front of the minimal CYC1–LacZ reporter on episomal vectors, although we are aware that this is unlikely to reflect the complete complexity of expression control in the chromosomal context. This revealed a carbon source-dependent activation by the 265 bp fragment positioned from −741 bp to −1006 bp. The apparently long distance is not unlikely, taking into account that the transcription start site is located more than 250 bp upstream of the ATG (Forsburg and Guarente, 1989). Two other fragments downstream of the 265 bp activating region did not lead to a carbon source-dependent activation of the reporter gene. Based on the presence of a sequence closely resembling a CSRE consensus, a 30 nt region within the 265 bp sequence was selected to test its activating capacity. Indeed, this 30 nt region was able to confer carbon source-dependent regulation in a Cat8p-dependent manner. Deletion of the 30 nt sequence caused a significant decrease in reporter gene activity on both glucose and ethanol/glycerol, although the induction ratio was not affected. This suggests that other sequences within the 265 bp fragment might contribute to carbon-dependent regulation as well. Preliminar results further exploring the 265 bp activating fragment imply that several subfragments of the 265 bp fragment are indeed able to induce transcription in a carbon-dependent manner. Identification of the proteins binding to the 30 nt region has become nearer at hand with the partial purification of protein complexes migrating as high molecular weight structures at 190 and 230 kDa when cross-linked with the 30 nt region. Unfortunately, further purification of the 300 mM KCl elution fraction and attempts to identify possible candidate proteins using mass spectrometry have been unsuccessful until now.
Intriguingly, the carbon source-dependent activation by both the 265 bp region and the 30 nt region within this region, was dependent on the presence of a functional Cat8p, as demonstrated by both a drastically reduced level of reporter gene induction and slightly altered DNA–protein binding in the absence of Cat8p. Cat8p has been reported not only to control expression of genes encoding enzymes of gluconeogenesis (Bojunga et al., 1998; Hedges et al., 1995; Kratzer and Schueller, 1997; Rahner et al., 1999; RandezGil et al., 1997), but also of IDP2, encoding NADP-dependent cytosolic isocitrate dehydrogenase (Bojunga and Entian, 1999) that has no direct role in gluconeogenesis. The role of Cat8p may be a more general one, involving activation of other genes that are strongly derepressed under non-fermentative growth conditions. In this light, it is interesting that HAP4 is also strongly induced when glucose becomes limiting during the diauxic shift (DeRisi et al., 1997), thereby inducing transcription of genes involved in respiration. Idp2p has been suggested to play a defence role in the increase in respiration and concomitant formation of reactive oxygen species (Bojunga and Entian, 1999), in addition to its role in providing reducing equivalents (Minard and McAlister-Henn, 1999). In this respect, transcriptional regulation by Cat8p may thus be an efficient way of coordinating the increase in glyoxylate and gluconeogenic gene transcription with increase in respiratory function and defence mechanisms against reactive oxygen species. Proft et al. (1995) suggest that Cat8p activation might globally affect respiratory metabolism based on the observation that some cat8 mutant alleles showed a loss of cytochrome c oxidase and O2 uptake activity. This is consistent with our findings that activation of HAP4 expression is dependent on Cat8p and recent experiments showing that Hap4p affects mitochondrial biogenesis as a whole (Lascaris et al., 2002, submitted for publication). However, Cat8p is not the sole determinant of carbon source-dependent transcriptional induction of HAP4, as deletion of CAT8 has no detectable effect on the steady-state transcriptional levels of HAP4 under control of its full-length 5′ promoter region. This has been demonstrated by Northern analysis (data not shown), as well as in a genome-wide expression study for targets of Cat8p (Haurie et al., 2001). A possible explanation might be that the role of Cat8p in transcriptional activation of HAP4 could be transient, or redundant to other factors that regulate transcription of HAP4. The fact that protein binding to the 30 nt region could not be competed by the CSRE of ACS1, a target of Cat8p, is an additional indication that the influence of Cat8p is not direct, but mediated via other factors. Based on the results described in this study, it can be concluded that we have just started to lift a corner of the veil that covers a complex regulation of HAP4 gene expression in S. cerevisiae. It might well be possible that the HAP4 promoter harbours additional regulatory features that would fine-tune its expression to regulate the balance between fermentative and oxidative metabolism in yeast.