Role of SAGA complex subunits in gene regulation of Candida albicans

The SAGA (Spt-Ada-Gcn5-acetyltransferase) is an evolutionary conserved multidomain co-activator complex involved in gene regulation through its histone acetyltransferase (HAT) and deubiquitinase (DUB) functions. It is well studied in Saccharomyces cerevisiae, and recent reports from humans and Drosophila expand its importance from gene transcription regulation to transcription elongation, protein stability and telomere maintenance. In Candida albicans, little is known about the components of the SAGA complex and their influence in morphogenesis and stress response. In this work, we analysed individual components of the SAGA complex, their role in morphogenesis and responses to different signalling cues. We initially analysed conditionally repressed strains of SAGA complex subunits involved in the HAT function of the complex: Tra1, Ngg1, Spt7, Spt8, Taf5, Taf6, Taf9, and Taf10. It appears that the Tra1 might be essential for the viability of C. albicans, as we failed to obtain homozygous deletions although it showed detectible growth in the conditionally repressed strain. Also, we observed that TBP- associated factors are essential in C. albicans, possibly due to their role in the transcription initiation factor TFIID instead of SAGA. We also detected that the Spt8 repressed mutant was extensively invasive in YPD at 300C while a repressed Ngg1 was considerably less invasive compared to its wild type. Also, we have seen that the mutations affecting TBP-binding ability confer susceptibility to drugs, temperature, osmotic, oxidative and DNA damage stress. Further, it seems that the modules of SAGA complex might have antagonistic roles in expression regulation but this needs more in-depth study.

Structure of the human SAGA coactivator complex

The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP6) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer.

The Genetic Profiles of TIF1 and TIF2 Duplicate Genes

<p>As one of the key steps in protein synthesis, translation initiation is subjected to multi-level regulation which is achieved via diverse mechanisms. The cell adjusts protein synthesis accordingly to its status and environment. The degree of contribution of the processes involved in the regulation of translation initiation is still poorly understood. The first part of this study focuses on identifying mechanisms of regulation in a translationally deficient yeast system, impaired by the loss of one or the other of the TIF1/2 duplicate genes, which together code for the eukaryotic initiation factor 4A (eIF4A). A major finding of this research is related to the functional competences associated with the two duplicate members of the gene pair. Although the genetic profile associated with TIF1 highlights a connection with transcriptional process, the majority of transcription-translation inter-talk is allocated with TIF2, along with a dense network of genetic interactions surrounding the SAGA complex. TIF2 is also the only paralog devoted to interactions with a substantial group of functionally related genes involved in early meiotic gene expression. Protein degradation in the global control of protein synthesis represents a fundamental process and accounts for diverse points of control, in particular through ubiquitination/deubiquitination. This research concludes that functional turnover of proteins and the translation/transcription inter-talk emerges as the most significant contributors to the sophistically regulated translational regulation, The second part of this study aims to determine the extent of similarity and divergence between the TIF1 and TIF2 paralogs. Growth of their individual deletion strains was challenged under different chemical and environmental conditions with the intent to explore the relative contributions of each duplicate in response to an extend range of perturbations. The pair of duplicates appeared convincingly robust in coping with these adversities under disparate cellular contexts, thus suggesting a highly conserved and backed-up genetic network. One of the primary treatments made use of lithium, a condition which was hoped to help, along with furthering our understanding of the TIF1 and TIF2 networks, in formulating an explanation on how augmented translation initiation overcomes lithium toxicity. Although this approach did not return results that could be used to address this unresolved topic, evaluation of genetic inhibition and suppression was highly instructive regarding the mechanisms of response triggered upon lithium/galactose stress. Regulation and synchronization of basic cellular processes were affected: emphasis brought on aspects of cell communication highlighted mechanisms articulated by kinase enzymes and the importance of repression of cell cycle progression in control of protein synthesis. Data from the screen also indicated the stress that combined lithium/galactose treatment places on central metabolic pathways, for instance those between the Leloir, gluconeogenesis, and trehalose pathways.</p>

The Genetic Profiles of TIF1 and TIF2 Duplicate Genes

<p>As one of the key steps in protein synthesis, translation initiation is subjected to multi-level regulation which is achieved via diverse mechanisms. The cell adjusts protein synthesis accordingly to its status and environment. The degree of contribution of the processes involved in the regulation of translation initiation is still poorly understood. The first part of this study focuses on identifying mechanisms of regulation in a translationally deficient yeast system, impaired by the loss of one or the other of the TIF1/2 duplicate genes, which together code for the eukaryotic initiation factor 4A (eIF4A). A major finding of this research is related to the functional competences associated with the two duplicate members of the gene pair. Although the genetic profile associated with TIF1 highlights a connection with transcriptional process, the majority of transcription-translation inter-talk is allocated with TIF2, along with a dense network of genetic interactions surrounding the SAGA complex. TIF2 is also the only paralog devoted to interactions with a substantial group of functionally related genes involved in early meiotic gene expression. Protein degradation in the global control of protein synthesis represents a fundamental process and accounts for diverse points of control, in particular through ubiquitination/deubiquitination. This research concludes that functional turnover of proteins and the translation/transcription inter-talk emerges as the most significant contributors to the sophistically regulated translational regulation, The second part of this study aims to determine the extent of similarity and divergence between the TIF1 and TIF2 paralogs. Growth of their individual deletion strains was challenged under different chemical and environmental conditions with the intent to explore the relative contributions of each duplicate in response to an extend range of perturbations. The pair of duplicates appeared convincingly robust in coping with these adversities under disparate cellular contexts, thus suggesting a highly conserved and backed-up genetic network. One of the primary treatments made use of lithium, a condition which was hoped to help, along with furthering our understanding of the TIF1 and TIF2 networks, in formulating an explanation on how augmented translation initiation overcomes lithium toxicity. Although this approach did not return results that could be used to address this unresolved topic, evaluation of genetic inhibition and suppression was highly instructive regarding the mechanisms of response triggered upon lithium/galactose stress. Regulation and synchronization of basic cellular processes were affected: emphasis brought on aspects of cell communication highlighted mechanisms articulated by kinase enzymes and the importance of repression of cell cycle progression in control of protein synthesis. Data from the screen also indicated the stress that combined lithium/galactose treatment places on central metabolic pathways, for instance those between the Leloir, gluconeogenesis, and trehalose pathways.</p>

A unique GCN5 histone acetyltransferase complex controls erythrocyte invasion and virulence in the malaria parasite Plasmodium falciparum

The histone acetyltransferase GCN5-associated SAGA complex is evolutionarily conserved from yeast to human and functions as a general transcription co-activator in global gene regulation. In this study, we identified a divergent GCN5 complex in Plasmodium falciparum, which contains two plant homeodomain (PHD) proteins (PfPHD1 and PfPHD2) and a plant apetela2 (AP2)-domain transcription factor (PfAP2-LT). To dissect the functions of the PfGCN5 complex, we generated parasite lines with either the bromodomain in PfGCN5 or the PHD domain in PfPHD1 deleted. The two deletion mutants closely phenocopied each other, exhibiting significantly reduced merozoite invasion of erythrocytes and elevated sexual conversion. These domain deletions caused dramatic decreases not only in histone H3K9 acetylation but also in H3K4 trimethylation, indicating synergistic crosstalk between the two euchromatin marks. Domain deletion in either PfGCN5 or PfPHD1 profoundly disturbed the global transcription pattern, causing altered expression of more than 60% of the genes. At the schizont stage, these domain deletions were linked to specific down-regulation of merozoite genes involved in erythrocyte invasion, many of which contain the AP2-LT binding motif and are also regulated by AP2-I and BDP1, suggesting targeted recruitment of the PfGCN5 complex to the invasion genes by these specific factors. Conversely, at the ring stage, PfGCN5 or PfPHD1 domain deletions disrupted the mutually exclusive expression pattern of the entire var gene family, which encodes the virulent factor PfEMP1. Correlation analysis between the chromatin state and alteration of gene expression demonstrated that up- and down-regulated genes in these mutants are highly correlated with the silent and active chromatin states in the wild-type parasite, respectively. Collectively, the PfGCN5 complex represents a novel HAT complex with a unique subunit composition including an AP2 transcription factor, which signifies a new paradigm for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.

Connection of core and tail Mediator modules restrains transcription from TFIID-dependent promoters

The Mediator coactivator complex is divided into four modules: head, middle, tail, and kinase. Deletion of the architectural subunit Med16 separates core Mediator (cMed), comprising the head, middle, and scaffold (Med14), from the tail. However, the direct global effects of tail/cMed disconnection are unclear. We find that rapid depletion of Med16 downregulates genes that require the SAGA complex for full expression, consistent with their reported tail dependence, but also moderately overactivates TFIID-dependent genes in a manner partly dependent on the separated tail, which remains associated with upstream activating sequences. Suppression of TBP dynamics via removal of the Mot1 ATPase partially restores normal transcriptional activity to Med16-depleted cells, suggesting that cMed/tail separation results in an imbalance in the levels of PIC formation at SAGA-requiring and TFIID-dependent genes. We propose that the preferential regulation of SAGA-requiring genes by tailed Mediator helps maintain a proper balance of transcription between these genes and those more dependent on TFIID.

SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells

Coactivator complexes regulate chromatin accessibility and transcription. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved coactivator complex. The core module scaffolds the entire SAGA complex and adopts a histone octamer-like structure, which consists of six histone fold domain (HFD)-containing proteins forming three histone fold (HF) pairs, to which the double HFD-containing SUPT3H adds an HF pair. Spt3, the yeast ortholog of SUPT3H, interacts genetically and biochemically with the TATA binding protein (TBP) and contributes to global RNA polymerase II (Pol II) transcription. Here we demonstrate that i) SAGA purified from human U2OS or mouse embryonic stem cells (mESC) can assemble without SUPT3H; ii) SUPT3H is not essential for mESC survival, iii) SUPT3H is required for mESC growth and self-renewal, and iv) the loss of SUPT3H from mammalian cells affects the transcription of only a specific subset of genes. Accordingly, in the absence of SUPT3H no major change in TBP accumulation at gene promoters was observed. Thus, SUPT3H is not required for the assembly of SAGA, TBP recruitment, or overall Pol II transcription, but plays a role in mESC growth and self-renewal. Our data further suggest that yeast and mammalian SAGA complexes contribute to transcription regulation by distinct mechanisms.

Functional characterization of the developmental genes asm2, asm3, and spt3 required for fruiting body formation in the filamentous ascomycete Sordaria macrospora

The formation of fruiting bodies is one of the most complex developmental processes in filamentous ascomycetes. It requires the development of sexual structures that give rise to meiosporangia (asci) and meiotic spores (ascospores) as well as surrounding structures for protection and dispersal of the spores. Previous studies have shown that these developmental processes are accompanied by significant changes of the transcriptome, and comparative transcriptomics of different fungi as well as the analysis of transcriptome changes in developmental mutants have aided in the identification of differentially regulated genes that are themselves involved in regulating fruiting body development. In previous analyses, we used transcriptomics to identify the genes asm2 and spt3, which result in developmental phenotypes when deleted in Sordaria macrospora. In this study, we identified another gene, asm3, required for fruiting body formation, and performed transcriptomics analyses of Δasm2, Δasm3, and Δspt3. Deletion of spt3, which encodes a subunit of the SAGA complex, results in a block at an early stage of development and drastic changes in the transcriptome. Deletion mutants of asm2 and asm3 are able to form fruiting bodies, but have defects in ascospore maturation. Transcriptomics analysis of fruiting bodies revealed a large overlap in differentially regulated genes in Δasm2 and Δasm3 compared to the wild type. Analysis of nuclear distribution during ascus development showed that both mutants undergo meiosis and postmeiotic divisions, suggesting that the transcriptomic and morphological changes might be related to defects in the morphogenesis of structural features of the developing asci and ascospores.

NuA4 and SAGA acetyltransferase complexes cooperate for repair of DNA breaks by homologous recombination

Chromatin modifying complexes play important yet not fully defined roles in DNA repair processes. The essential NuA4 histone acetyltransferase (HAT) complex is recruited to double-strand break (DSB) sites and spreads along with DNA end resection. As predicted, NuA4 acetylates surrounding nucleosomes upon DSB induction and defects in its activity correlate with altered DNA end resection and Rad51 recombinase recruitment. Importantly, we show that NuA4 is also recruited to the donor sequence during recombination along with increased H4 acetylation, indicating a direct role during strand invasion/D-loop formation after resection. We found that NuA4 cooperates locally with another HAT, the SAGA complex, during DSB repair as their combined action is essential for DNA end resection to occur. This cooperation of NuA4 and SAGA is required for recruitment of ATP-dependent chromatin remodelers, targeted acetylation of repair factors and homologous recombination. Our work reveals a multifaceted and conserved cooperation mechanism between acetyltransferase complexes to allow repair of DNA breaks by homologous recombination.