Inactivation of HAP4 Accelerates RTG-Dependent Osmoadaptation in Saccharomyces cerevisiae.

Mitochondrial RTG (an acronym for ReTroGrade) signaling plays a cytoprotective role under various intracellular or environmental stresses. We have previously shown its contribution to osmoadaptation and capacity to sustain mitochondrial respiration in yeast. Here, we studied the interplay between RTG2, the main positive regulator of the RTG pathway, and HAP4, encoding the catalytic subunit of the Hap2-5 complex required for the expression of many mitochondrial proteins that function in the tricarboxylic acid (TCA) cycle and electron transport, upon osmotic stress. Cell growth features, mitochondrial respiratory competence, retrograde signaling activation, and TCA cycle gene expression were comparatively evaluated in wild type and mutant cells in the presence and in the absence of salt stress. We showed that the inactivation of HAP4 improved the kinetics of osmoadaptation by eliciting both the activation of retrograde signaling and the upregulation of three TCA cycle genes: citrate synthase 1 (CIT1), aconitase 1 (ACO1), and isocitrate dehydrogenase 1 (IDH1). Interestingly, their increased expression was mostly dependent on RTG2. Impaired respiratory competence in the HAP4 mutant does not affect its faster adaptive response to stress. These findings indicate that the involvement of the RTG pathway in osmostress is fostered in a cellular context of constitutively reduced respiratory capacity. Moreover, it is evident that the RTG pathway mediates peroxisomes-mitochondria communication by modulating the metabolic function of mitochondria in osmoadaptation.

Interplay between transcriptional regulators and the SAGA chromatin modifying complex fine-tune iron homeostasis.

The human fungal pathogen Candida albicans responds to iron deprivation by a global transcriptome reconfiguration known to be controlled by the transcriptional regulators Hap43 (also known as Cap2), Sef1, and the trimeric Hap2-Hap3-Hap5 complex. However, the relative roles of these regulators are not known. To dissect this system, we focused on the FRP1 and ACO1 genes, which are induced and repressed, respectively, under iron deprivation conditions. Chromatin immunoprecipitation assays showed that the trimeric HAP complex and Sef1 are recruited to both FRP1 and ACO1 promoters. While the HAP complex occupancy at the FRP1 promoter was Sef1-dependent, occupancy of Sef1 was not dependent on the HAP complex. Furthermore, iron deprivation elicited histone H3-Lys9 hyperacetylation and Pol II recruitment mediated by the trimeric HAP complex and Sef1 at the FRP1 promoter. In contrast, at the ACO1 promoter, the HAP trimeric complex and Hap43 promoted histone deacetylation and also limited Pol II recruitment under iron deprivation conditions. Mutational analysis showed that the SAGA subunits Gcn5, Spt7, and Spt20 are required for C. albicans growth in iron-deficient medium and for H3-K9 acetylation and transcription from the FRP1 promoter. Thus, the trimeric HAP complex promotes FRP1 transcription by stimulating H3K9Ac and Pol II recruitment and, along with Hap43, functions as a repressor of ACO1 by maintaining a deacetylated promoter under iron-deficient conditions. Thus, a regulatory network involving iron-responsive transcriptional regulators and the SAGA histone modifying complex functions as a molecular switch to fine-tune tight control of iron homeostasis gene expression in C. albicans.

"Labile" heme critically regulates mitochondrial biogenesis through the transcriptional co-activator Hap4p in Saccharomyces cerevisiae.

Heme (iron protoporphyrin IX) is a well-known prosthetic group for enzymes involved in metabolic pathways such as oxygen transport and electron transfer through the mitochondrial respiratory chain. However, heme has also been shown to be an important regulatory molecule (as "labile" heme) for diverse processes such as translation, kinase activity, and transcription in mammals, yeast, and bacteria. Taking advantage of a yeast strain deficient for heme production that enabled controlled modulation and monitoring of labile heme levels, here we investigated the role of labile heme in the regulation of mitochondrial biogenesis. This process is regulated by the HAP complex in yeast. Using several biochemical assays along with EM and epifluorescence microscopy, to the best of our knowledge, we show for the first time that cellular labile heme is critical for the post-translational regulation of HAP complex activity, most likely through the stability of the transcriptional co-activator Hap4p. Consequently, we found that labile heme regulates mitochondrial biogenesis and cell growth. The findings of our work highlight a new mechanism in the regulation of mitochondrial biogenesis by cellular metabolites.

Mitochondrial cysteinyl-tRNA synthetase is expressed via alternative transcriptional initiation regulated by energy metabolism in yeast cells.

Eukaryotes typically utilize two distinct aminoacyl-tRNA synthetase isoforms, one for cytosolic and one for mitochondrial protein synthesis. However, the genome of budding yeast (Saccharomyces cerevisiae) contains only one cysteinyl-tRNA synthetase gene (YNL247W, also known as CRS1). In this study, we report that CRS1 encodes both cytosolic and mitochondrial isoforms. The 5' complementary DNA end method and GFP reporter gene analyses indicated that yeast CRS1 expression yields two classes of mRNAs through alternative transcription starts: a long mRNA containing a mitochondrial targeting sequence and a short mRNA lacking this targeting sequence. We found that the mitochondrial Crs1 is the product of translation from the first initiation AUG codon on the long mRNA, whereas the cytosolic Crs1 is produced from the second in-frame AUG codon on the short mRNA. Genetic analysis and a ChIP assay revealed that the transcription factor heme activator protein (Hap) complex, which is involved in mitochondrial biogenesis, determines the transcription start sites of the CRS1 gene. We also noted that Hap complex-dependent initiation is regulated according to the needs of mitochondrial energy production. The results of our study indicate energy-dependent initiation of alternative transcription of CRS1 that results in production of two Crs1 isoforms, a finding that suggests Crs1's potential involvement in mitochondrial energy metabolism in yeast.

Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression.

Regulation of mitochondrial biogenesis and respiration is a complex process that involves several signaling pathways and transcription factors as well as communication between the nuclear and mitochondrial genomes. Under aerobic conditions, the budding yeast Saccharomyces cerevisiae metabolizes glucose predominantly by glycolysis and fermentation. We have recently shown that altered chromatin structure in yeast induces respiration by a mechanism that requires transport and metabolism of pyruvate in mitochondria. However, how pyruvate controls the transcriptional responses underlying the metabolic switch from fermentation to respiration is unknown. Here, we report that this pyruvate effect involves heme. We found that heme induces transcription of HAP4, the transcriptional activation subunit of the Hap2/3/4/5p complex, required for growth on nonfermentable carbon sources, in a Hap1p- and Hap2/3/4/5p-dependent manner. Increasing cellular heme levels by inactivating ROX1, which encodes a repressor of many hypoxic genes, or by overexpressing HEM3 or HEM12 induced respiration and elevated ATP levels. Increased heme synthesis, even under conditions of glucose repression, activated Hap1p and the Hap2/3/4/5p complex and induced transcription of HAP4 and genes required for the tricarboxylic acid (TCA) cycle, electron transport chain, and oxidative phosphorylation, leading to a switch from fermentation to respiration. Conversely, inhibiting metabolic flux into the TCA cycle reduced cellular heme levels and HAP4 transcription. Together, our results indicate that the glucose-mediated repression of respiration in budding yeast is at least partly due to the low cellular heme level.

The HAP complex in Fusarium verticillioides is a key regulator of growth, morphogenesis, secondary metabolism, and pathogenesis.

Among eukaryotic organisms, the HAP complex is a conserved, multimeric transcription factor that regulates gene expression by binding to the consensus sequence CCAAT. In filamentous fungi, the HAP complex has been linked to primary and secondary metabolism, but its role in pathogenesis has not been investigated extensively. The overarching goal of this study was to elucidate the role of the HAP complex in Fusariumverticillioides, a ubiquitous and damaging pathogen of maize. To this end, orthologs of core HAP complex genes (FvHAP2, FvHAP3, and FvHAP5) were identified and deleted in F. verticillioides via a reverse genetics approach. Deletion of FvHAP2, FvHAP3, or FvHAP5 resulted in an indistinguishable phenotype among the deletion strains, including reduced radial growth and conidiation, altered colony morphology, and derepression of pigmentation. Additionally, disruption of the HAP complex impaired infection and colonization of maize stalks. Deletion strains were hypersensitive to osmotic and oxidative stress, which suggests the HAP complex of F. verticillioides may mediate responses to environmental stress during pathogenesis. This study directly implicates the HAP complex in primary and secondary metabolism in F. verticillioides and provides one of the first links between the HAP complex and virulence in a plant pathogenic fungus.

Role of the Heme Activator Protein Complex in the Sexual Development of Cryptococcus neoformans.

The CCAAT-binding heme activator protein (HAP) complex, comprising the DNA-binding heterotrimeric complex Hap2/3/5 and transcriptional activation subunit HapX, is a key regulator of iron homeostasis, mitochondrial functions, and pathogenicity in Cryptococcus neoformans, which causes fatal meningoencephalitis. However, its role in the development of human fungal pathogens remains unclear. To elucidate the role of the HAP complex in C. neoformans development, we constructed hap2Δ, hap3Δ, hap5Δ, and hapXΔ mutants and their complemented congenic MATα H99 and MATa YL99a strains. The HAP complex plays a conserved role in iron utilization and stress responses in cells of both mating types. Deletion of any of the HAP complex components markedly enhances filamentation during bisexual mating. However, the Hap2/3/5 complex, but not HapX, is crucial in repressing pheromone production and cell fusion and is thus a critical repressor of sexual differentiation of C. neoformans. Interestingly, deletion of the heterotrimeric complex transcriptionally regulated both positive and negative regulators in the pheromone-responsive Cpk1 mitogen-activated protein kinase (MAPK) pathway. Chromatin immunoprecipitation-quantitative PCR analysis revealed that the HAP complex physically bound to the CCAAT motif of the CRG1 and GPA2 promoter regions. Notably, the HAP complex was differentially localized depending on the mating type in basal conditions; it was enriched in the nuclei of MATα cells but diffused in the cytoplasm of MATa cells. Interestingly, however, a portion of the HAP complex in both mating types relocalized to the cell membrane during mating. In conclusion, the Hap2/3/5 heterotrimeric complex and HapX play major and minor roles, respectively, in repressing the sexual development of C. neoformans in association with the Cpk1 MAPK pathway. IMPORTANCE Cryptococcus neoformans isolates are of two mating types: MATα strains, which are predominant, and MATa strains, isolated from the sub-Saharan African region, where cryptococcosis is most abundant and severe. Here, we demonstrated the function of the CCAAT-binding HAP complex (Hap2/3/5/X) as a transcriptional repressor of Cpk1 pathway-related genes in cells of both mating types. Deletion of any HAP complex component markedly enhanced filamentation without affecting normal sporulation. In particular, deletion of the DNA-binding HAP complex components (Hap2/3/5), but not HapX, markedly enhanced pheromone production and cell fusion efficiency, validating its repressive role in the early stage of mating in C. neoformans. The HAP complex regulates the expression of both negative and positive mating regulators and is thus crucial for the regulation of the Cpk1 MAPK pathway during mating. This study provides insights into the complex signaling networks governing the sexual differentiation of C. neoformans.

The Putative Transcription Factor Gene thaB Regulates Cellulase and Xylanase Production at the Enzymatic and Transcriptional Level in the Fungus Talaromyces cellulolyticus.

Talaromyces cellulolyticus is a promising strain for industrial cellulase production. In this study, the thaB gene, which is a homologue of the hap2/B gene in other filamentous fungi, was isolated and characterized. When grown in the presence of cellulose, culture supernatants of a thaB-disrupted strain (YDTha) exhibited decreased cellulase and xylanase enzymatic activities compared to the control strain. Furthermore, YDTha exhibited lower expression of the genes encoding cellulases and xylanases compared to the control strain. When cellobiose and lactose (soluble carbon sources) were used as carbon sources, the expression of the genes encoding cellulases and xylanases was decreased in both the YDTha and the control strains, though the expression levels in YDTha remained lower than those in the control strain. These results suggested that thaB has a positive role in cellulase and xylanase production in T. cellulolyticus.

The Hap Complex in Yeasts: Structure, Assembly Mode, and Gene Regulation.

The CCAAT box-harboring proteins represent a family of heterotrimeric transcription factors which is highly conserved in eukaryotes. In fungi, one of the particularly important homologs of this family is the Hap complex that separates the DNA-binding domain from the activation domain and imposes essential impacts on regulation of a wide range of cellular functions. So far, a comprehensive summary of this complex has been described in filamentous fungi but not in the yeast. In this review, we summarize a number of studies related to the structure and assembly mode of the Hap complex in a list of representative yeasts. Furthermore, we emphasize recent advances in understanding the regulatory functions of this complex, with a special focus on its role in regulating respiration, production of reactive oxygen species (ROS) and iron homeostasis.

The CCAAT-binding complex (CBC) in Aspergillus species.

BACKGROUND: The CCAAT binding complex (CBC), consisting of a heterotrimeric core structure, is highly conserved in eukaryotes and constitutes an important general transcriptional regulator. Scope of the review. In this review we discuss the scientific history and the current state of knowledge of the multiple gene regulatory functions, protein motifs and structure of the CBC in fungi with a special focus on Aspergillus species. Major conclusions and general significance. Initially identified as a transcriptional activator of respiration in Saccharomyces cerevisiae, in other fungal species the CBC was found to be involved in highly diverse pathways, but a general rationale for its involvement was missing. Subsequently, the CBC was found to sense reactive oxygen species through oxidative modifications of cysteine residues in order to mediate redox regulation. Moreover, via interaction with the iron-sensing bZIP transcription factor HapX, the CBC was shown to mediate adaptation to both iron starvation and iron excess. Due to the control of various pathways in primary and secondary metabolism the CBC is of crucial importance for fungal virulence in both animal and plant hosts as well as antifungal resistance. Consequently, CBC-mediated control affects biological processes that are of high interest in biotechnology, agriculture and infection medicine. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.