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Objectives Transcriptional regulatory modules are usually modelled via a network, in which nodes correspond to genes and edges correspond to regulatory associations between them. In the model yeast Saccharomyces cerevisiae, the topological properties of such a network are well-described (distribution of degrees, hierarchical levels, organization in network motifs, etc.). To go further on this, our aim was to search for additional information resulting from the new combination of classical representations of transcriptional regulatory networks with more realistic models of the spatial organization of S. cerevisiae genome in the nucleus. Results Taking advantage of independent studies with high-quality datasets, i.e. lists of target genes for specific transcription factors and chromosome positions in a three dimensional space representing the nucleus, particular spatial co-localizations of genes that shared common regulatory mechanisms were searched. All transcriptional modules of S. cerevisiae, as described in the latest release of the YEASTRACT database were analyzed and significant biases toward co-localization for a few sets of target genes were observed. To help other researchers to reproduce such analysis with any list of genes of their interest, an interactive web tool called 3D-Scere (https://3d-scere.ijm.fr/) is provided.

International audience; Aspergillus fumigatus is a major opportunistic fungal pathogen of immunocompromised and immunocompetent hosts. To successfully establish an infection, A. fumigatus needs to use host carbon sources, such as acetate, present in the body fluids and peripheral tissues. However, utilization of acetate as a carbon source by fungi in the context of infection has not been investigated. This work shows that acetate is metabolized via different pathways in A. fumigatus and that acetate utilization is under the regulatory control of a transcription factor (TF), FacB. A. fumigatus acetate utilization is subject to carbon catabolite repression (CCR), although this is only partially dependent on the TF and main regulator of CCR CreA. The available extracellular carbon source, in this case glucose and acetate, significantly affected A. fumigatus virulence traits such as secondary metabolite secretion and cell wall composition, with the latter having consequences for resistance to oxidative stress, antifungal drugs, and human neutrophil-mediated killing. Furthermore, deletion of facB significantly impaired the in vivo virulence of A. fumigatus in both insect and mammalian models of invasive aspergillosis. This is the first report on acetate utilization in A. fumigatus, and this work further highlights the importance of available host-specific carbon sources in shaping fungal virulence traits and subsequent disease outcome, and a potential target for the development of antifungal strategies.IMPORTANCE Aspergillus fumigatus is an opportunistic fungal pathogen in humans. During infection, A. fumigatus is predicted to use host carbon sources, such as acetate, present in body fluids and peripheral tissues, to sustain growth and promote colonization and invasion. This work shows that A. fumigatus metabolizes acetate via different pathways, a process that is dependent on the transcription factor FacB. Furthermore, the type and concentration of the extracellular available carbon source were determined to shape A. fumigatus virulence determinants such as secondary metabolite secretion and cell wall composition. Subsequently, interactions with immune cells are altered in a carbon source-specific manner. FacB is required for A. fumigatus in vivo virulence in both insect and mammalian models of invasive aspergillosis. This is the first report that characterizes acetate utilization in A. fumigatus and highlights the importance of available host-specific carbon sources in shaping virulence traits and potentially subsequent disease outcome.

Several Candida species can undergo a heritable and reversible transition from a ‘white’ state to a mating proficient ‘opaque’ state. This ability relies on highly interconnected transcriptional networks that control cell-type-specific gene expression programs over multiple generations. Candida albicans, the most prominent pathogenic Candida species, provides a well-studied paradigm for the white-opaque transition. In this species, a network of at least eight transcriptional regulators controls the balance between white and opaque states that have distinct morphologies, transcriptional profiles, and physiological properties. Given the reversible nature and the high frequency of white-opaque transitions, it is widely assumed that this switch is governed by epigenetic mechanisms that occur independently of any changes in DNA sequence. However, a direct genomic comparison between white and opaque cells has yet to be performed. Here, we present a whole-genome comparative analysis of C. albicans white and opaque cells. This analysis revealed rare genetic changes between cell states, none of which are linked to white-opaque switching. This result is consistent with epigenetic mechanisms controlling cell state differentiation in C. albicans and provides direct evidence against a role for genetic variation in mediating the switch.

Eukaryotic protein synthesis generally initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but the quantitative importance of this context in different species is unclear. We tested this concept in two pathogenic Cryptococcus yeast species by genome-wide mapping of translation and of mRNA 5′ and 3′ ends. We observed thousands of AUG-initiated upstream open reading frames (uORFs) that are a major contributor to translation repression. uORF use depends on the Kozak sequence context of its start codon, and uORFs with strong contexts promote nonsense-mediated mRNA decay. Transcript leaders in Cryptococcus and other fungi are substantially longer and more AUG-dense than in Saccharomyces. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Analysis of other fungal species shows that such dual-localization is also predicted to be common in the ascomycete mould, Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that quantitatively programs both the expression and the structures of proteins in diverse fungi.

International audience; The development of a tetO/TetR system in the fungus Neurospora crassa is described. The system includes (i) a synthetic gene encoding a TetR variant fused to GFP, and (ii) a standard tetO array integrated homologously, as a proof of principle, near the his-3 gene. The localization of TetR-GFP at the tetO array (observed by fluorescence microscopy) can be disrupted by the application of tetracycline. The full-length array is stable during vegetative growth, but it triggers strong repeat-induced point mutation (RIP) by the RID-dependent as well as the DIM-2-dependent pathways during the sexual phase. Thus, both RIP pathways must be inactivated to allow the faithful inheritance of the unmodified construct. In summary, this study introduces a new molecular tool into Neurospora research, and suggests that the standard tetO array can self-engage in recombination-independent homologous pairing.

Convergent gene pairs can produce transcripts with complementary sequences. We had shown that mRNA duplexes form in vivo in Saccharomyces cerevisiae via interactions of mRNA overlapping 3′-ends and can lead to posttranscriptional regulatory events. Here we show that mRNA duplex formation is restricted to convergent genes separated by short intergenic distance, independently of their 3′-untranslated region (UTR) length. We disclose an enrichment in genes involved in biological processes related to stress among these convergent genes. They are markedly conserved in convergent orientation in budding yeasts, meaning that this mode of posttranscriptional regulation could be shared in these organisms, conferring an additional level for modulating stress response. We thus investigated the mechanistic advantages potentially conferred by 3′-UTR mRNA interactions. Analysis of genome-wide transcriptome data revealed that Pat1 and Lsm1 factors, having 3′-UTR binding preference and participating to the remodeling of messenger ribonucleoprotein particles, bind differently these messenger-interacting mRNAs forming duplexes in comparison to mRNAs that do not interact (solo mRNAs). Functionally, messenger-interacting mRNAs show limited translational repression upon stress. We thus propose that mRNA duplex formation modulates the regulation of mRNA expression by limiting their access to translational repressors. Our results thus show that posttranscriptional regulation is an additional factor that determines the order of coding genes.

Pervasive transcription generates many unstable non-coding transcripts in budding yeast. The transcription of such noncoding RNAs, in particular antisense RNAs (asRNAs), has been shown in a few examples to repress the expression of the associated mRNAs. Yet, such mechanism is not known to commonly contribute to the regulation of a given class of genes. Using a mutant context that stabilised pervasive transcripts, we observed that the least expressed mRNAs during the exponential phase were associated with high levels of asRNAs. These asRNAs also overlapped their corresponding gene promoters with a much higher frequency than average. Interrupting antisense transcription of a subset of genes corresponding to quiescence-enriched mRNAs restored their expression. The underlying mechanism acts in cis and involves several chromatin modifiers. Our results convey that transcription interference represses up to 30% of the 590 least expressed genes, which includes 163 genes with quiescence-enriched mRNAs. We also found that pervasive transcripts constitute a higher fraction of the transcriptome in quiescence relative to the exponential phase, consistent with gene expression itself playing an important role to suppress pervasive transcription. Accordingly, the HIS1 asRNA, normally only present in quiescence, is expressed in exponential phase upon HIS1 mRNA transcription interruption.

International audience; Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA-sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane-associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus.

International audience; Transcription is a source of genetic instability that can notably result from the formation of genotoxic DNA:RNA hybrids, or R-loops, between the nascent mRNA and its template. Here we report an unexpected function for introns in counteracting R-loop accumulation in eukaryotic genomes. Deletion of endogenous introns increases R-loop formation, while insertion of an intron into an intronless gene suppresses R-loop accumulation and its deleterious impact on transcription and recombination in yeast. Recruitment of the spliceosome onto the mRNA, but not splicing per se, is shown to be critical to attenuate R-loop formation and transcription-associated genetic instability. Genome-wide analyses in a number of distant species differing in their intron content, including human, further revealed that intron-containing genes and the intron-richest genomes are best protected against R-loop accumulation and subsequent genetic instability. Our results thereby provide a possible rationale for the conservation of introns throughout the eukaryotic lineage.

The biological impact of alternative splicing is poorly understood in fungi, although recent studies have shown that these microorganisms are usually intron-rich. In this study, we re-annotated the genome of C. neoformans var. neoformans using RNA-Seq data. Comparison with C. neoformans var. grubii revealed that more than 99% of ORF-introns are in the same exact position in the two varieties whereas UTR-introns are much less evolutionary conserved. We also confirmed that alternative splicing is very common in C. neoformans, affecting nearly all expressed genes. We also observed specific regulation of alternative splicing by environmental cues in this yeast. However, alternative splicing does not appear to be an efficient method to diversify the C. neoformans proteome. Instead, our data suggest the existence of an intron retention-dependent mechanism of gene expression regulation that is not dependent on NMD. This regulatory process represents an additional layer of gene expression regulation in fungi and provides a mechanism to tune gene expression levels in response to any environmental modification.

Following phagocytosis, microbes are exposed to an array of antimicrobial weapons that include reactive oxygen species (ROS) and cationic fluxes. This is significant as combinations of oxidative and cationic stresses are much more potent than the corresponding single stresses, triggering the synergistic killing of the fungal pathogen Candida albicans by “stress pathway interference.” Previously we demonstrated that combinatorial oxidative plus cationic stress triggers a dramatic increase in intracellular ROS levels compared to oxidative stress alone. Here we show that activation of Cap1, the major regulator of antioxidant gene expression in C. albicans, is significantly delayed in response to combinatorial stress treatments and to high levels of H2O2. Cap1 is normally oxidized in response to H2O2; this masks the nuclear export sequence, resulting in the rapid nuclear accumulation of Cap1 and the induction of Cap1-dependent genes. Here we demonstrate that following exposure of cells to combinatorial stress or to high levels of H2O2, Cap1 becomes trapped in a partially oxidized form, Cap1OX-1. Notably, Cap1-dependent gene expression is not induced when Cap1 is in this partially oxidized form. However, while Cap1OX-1 readily accumulates in the nucleus and binds to target genes following high-H2O2 stress, the nuclear accumulation of Cap1OX-1 following combinatorial H2O2 and NaCl stress is delayed due to a cationic stress-enhanced interaction with the Crm1 nuclear export factor. These findings define novel mechanisms that delay activation of the Cap1 transcription factor, thus preventing the rapid activation of the stress responses vital for the survival of C. albicans within the host., IMPORTANCE Combinatorial stress-mediated synergistic killing represents a new unchartered area in the field of stress signaling. This phenomenon contrasts starkly with “stress cross-protection,” where exposure to one stress protects against subsequent exposure to a different stress. Previously we demonstrated that the pathogen Candida albicans is acutely sensitive to combinations of cationic and oxidative stresses, because the induction of H2O2-responsive genes is blocked in the presence of cationic stress. We reveal that this is due to novel mechanisms that delay activation of the Cap1 AP-1-like transcription factor, the major regulator of the H2O2-induced regulon. Cap1 becomes trapped in a partially oxidized form following simultaneous exposure to oxidative and cationic stresses. In addition, cationic stress promotes the interaction of Cap1 with the Crm1 nuclear export factor, thus inhibiting its nuclear accumulation. These mechanisms probably explain the potency of neutrophils, which employ multiple stresses to kill fungal pathogens.

Candida albicans can form biofilms that exhibit elevated intrinsic resistance to various antifungal agents, in particular azoles and polyenes. The molecular mechanisms involved in the antifungal resistance of biofilms remain poorly understood. We have used transcript profiling to explore the early transcriptional responses of mature C. albicans biofilms exposed to various antifungal agents. Mature C. albicans biofilms grown under continuous flow were exposed for as long as 2 h to concentrations of fluconazole (FLU), amphotericin B (AMB), and caspofungin (CAS) that, while lethal for planktonic cells, were not lethal for biofilms. Interestingly, FLU-exposed biofilms showed no significant changes in gene expression over the course of the experiment. In AMB-exposed biofilms, 2.7% of the genes showed altered expression, while in CAS-exposed biofilms, 13.0% of the genes had their expression modified. In particular, exposure to CAS resulted in the upregulation of hypha-specific genes known to play a role in biofilm formation, such as ALS3 and HWP1 . There was little overlap between AMB- or CAS-responsive genes in biofilms and those that have been identified as AMB, FLU, or CAS responsive in C. albicans planktonic cultures. These results suggested that the resistance of C. albicans biofilms to azoles or polyenes was due not to the activation of specific mechanisms in response to exposure to these antifungals but rather to the intrinsic properties of the mature biofilms. In this regard, our study led us to observe that AMB physically bound C. albicans biofilms and beta-glucans, which have been proposed to be major constituents of the biofilm extracellular matrix and to prevent azoles from reaching biofilm cells. Thus, enhanced extracellular matrix or beta-glucan synthesis during biofilm growth might prevent antifungals, such as azoles and polyenes, from reaching biofilm cells, thus limiting their toxicity to these cells and the associated transcriptional responses.

International audience; Fungal secondary metabolites are important bioactive compounds but the conditions leading to expression of most of the putative secondary metabolism (SM) genes predicted by fungal genomics are unknown. Here we describe a novel mechanism involved in SM-gene regulation based on the finding that, in Aspergillus nidulans, mutants lacking components involved in heterochromatin formation show de-repression of genes involved in biosynthesis of sterigmatocystin (ST), penicillin and terrequinone A. During the active growth phase, the silent ST gene cluster is marked by histone H3 lysine 9 trimethylation and contains high levels of the heterochromatin protein-1 (HepA). Upon growth arrest and activation of SM, HepA and trimethylated H3K9 levels decrease concomitantly with increasing levels of acetylated histone H3. SM-specific chromatin modifications are restricted to genes located inside the ST cluster, and constitutive heterochromatic marks persist at loci immediately outside the cluster. LaeA, a global activator of SM clusters in fungi, counteracts the establishment of heterochromatic marks. Thus, one level of regulation of the A. nidulans ST cluster employs epigenetic control by H3K9 methylation and HepA binding to establish a repressive chromatin structure and LaeA is involved in reversal of this heterochromatic signature inside the cluster, but not in that of flanking genes.

International audience; Like other multicellular organisms, the model nematode C. elegans responds to infection by inducing the expression of defense genes. Among the genes upregulated in response to a natural fungal pathogen is nlp-29, encoding an antimicrobial peptide. In a screen for mutants that fail to express nlp-29 following fungal infection, we isolated alleles of tpa-1, homologous to the mammalian protein kinase C (PKC) delta. Through epistasis analyses, we demonstrate that C. elegans PKC acts through the p38 MAPK pathway to regulate nlp-29. This involves G protein signaling and specific C-type phospholipases acting upstream of PKCdelta. Unexpectedly and unlike in mammals, tpa-1 does not act via D-type protein kinases, but another C. elegans PKC gene, pkc-3, functions nonredundantly with tpa-1 to control nlp-29 expression. Finally, the tribbles-like kinase nipi-3 acts upstream of PKCdelta in this antifungal immune signaling cascade. These findings greatly expand our understanding of the pathways involved in C. elegans innate immunity.

International audience; LSD1 represses and activates transcription by demethylating histone H3K4me and H3K9me, respectively. Genetic ablation of the S. pombe homologs, splsd1 and splsd2, resulted in slow growth and lethality, respectively, underscoring their physiological importance. spLsd1 and spLsd2 form a stable protein complex, which exhibits demethylase activity toward methylated H3K9 in vitro. Both proteins were associated with the heterochromatin boundary regions and euchromatic gene promoters. Loss of spLsd1 resulted in increased H3K9 methylation accompanied by reduced euchromatic gene transcription and heterochromatin propagation. Removal of the H3K9 methylase Clr4 partially suppressed the slow growth phenotype of splsd1Delta. Conversely, catalytically inactivating point mutations in the splsd1 and splsd2 genes partially mimicked the growth and heterochromatin propagation phenotypes. Taken together, these findings suggest the importance of both enzymatic and nonenzymatic roles of spLsd1 in regulating heterochromatin propagation and euchromatic transcription and also suggest that misregulation of spLsd1/2 is likely to impact the epigenetic state of the cell.

International audience; Biofilms are a source of therapeutic failures because of their intrinsic tolerance to antimicrobials. Candida glabrata is one of the pathogenic yeasts that is responsible for life-threatening disseminated infections and able to form biofilms on medical devices such as vascular and urinary catheters. Recent progresses in the functional genomics of C. glabrata have been applied to the study of biofilm formation, revealing the contribution of an array of genes to this process. In particular, the Yak1 kinase and the Swi/Snf chromatin remodeling complex have been shown to relieve the repression exerted by subtelomeric silencing on the expression of the EPA6 and EPA7 genes, thus allowing the encoded adhesins to exert their key roles in biofilm formation. This provides a framework to evaluate the contribution of other genes that have been genetically linked to biofilm development and, based on the function of their orthologs in Saccharomyces cerevisiae, appear to have roles in adaptation to nutrient deprivation, calcium signaling, cell wall remodeling and adherence. Future studies combining the use of in vitro and animal models of biofilm formation, omics approaches and forward or reverse genetics are needed to expand the current knowledge of C. glabrata biofilm formation and reveal the mechanisms underlying their antifungal tolerance.

International audience; Several nuclear pore-associated factors, including the SUMO-protease Ulp1, have been proposed to prevent the export of intron-containing messenger ribonucleoparticles (mRNPs) in yeast. However, the molecular mechanisms of this nuclear pore-dependent mRNA quality control, including the sumoylated targets of Ulp1, have remained unidentified. Here, we demonstrate that the apparent 'pre-mRNA leakage' phenotype arising upon ULP1 inactivation is shared by sumoylation mutants of the THO complex, an early mRNP biogenesis factor. Importantly, we establish that alteration of THO complex activity differentially impairs the expression of intronless and intron-containing reporter genes, rather than triggering bona fide 'pre-mRNA leakage'. Indeed, we show that the presence of introns within THO target genes attenuates the effect of THO inactivation on their transcription. Epistasis analyses further clarify that different nuclear pore components influence intron-containing gene expression at distinct stages. Ulp1, whose maintenance at nuclear pores depends on the Nup84 complex, impacts on THO-dependent gene expression, whereas the nuclear basket-associated Mlp1/Pml39 proteins prevent pre-mRNA export at a later stage, contributing to mRNA quality control. Our study thus highlights the multiplicity of mechanisms by which nuclear pores contribute to gene expression, and further provides the first evidence that intronic sequences can alleviate early mRNP biogenesis defects.

In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3∆ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope., Author Summary In the genome of eukaryotic cells, domains of active and repressive chromatin alternate along the chromosome arms. Insulator elements are necessary to shield these different environments from each other. In the fission yeast Schizosaccharomyces pombe, the chromatin remodeler Fft3 is required to maintain the repressed subtelomeric chromatin. Here we show that Fft3 maintains nucleosome structure of insulator elements at the subtelomeric borders. We also observe that subtelomeres and insulator elements move away from the nuclear envelope in cells lacking Fft3. The nuclear periphery is known to harbor repressive chromatin in many eukaryotes and has been implied in insulator function. Our results suggest that chromatin remodeling through Fft3 is required to maintain proper chromatin structure and nuclear organization of insulator elements.

International audience; Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.

Cryptic unstable transcripts (CUTs) are widely distributed in the genome of S. cerevisiae. These RNAs generally derive from nonannotated regions of the genome and are degraded rapidly and efficiently by the nuclear exosome via a pathway that involves degradative polyadenylation by a new poly(A) polymerase borne by the TRAMP complex. What is the share of significant information that is encrypted in CUTs and what distinguishes a CUT from other Pol II transcripts are unclear to date. Here we report the dissection of the molecular mechanism that leads to degradation of a model CUT, NEL025c. We show that the Nrd1p-Nab3p-dependent pathway, involved in transcription termination of sno/snRNAs, is required, albeit not sufficient, for efficient degradation of NEL025c RNAs and at least a subset of other CUTs. Our results suggest an important role for the Nrd1p-Nab3p pathway in the control of gene expression throughout the genome.

The biogenesis of cytochrome c oxidase requires coordination between the nucleus and mitochondria because both these compartments provide the structural subunits of this enzyme. In addition, synthesis, membrane insertion and assembly of the mitochondrially encoded subunits are controlled in a concerted way by numerous nuclear-encoded factors, including Oxa1 and Cox18, which play successive roles in Cox2 assembly in Saccharomyces cerevisiae . These two factors share a weak structural similarity and define two sub-branches of the Oxa1/YidC/Alb3 gene family, whose members facilitate the membrane insertion of various hydrophobic proteins into diverse biological membranes. In this study, we have analyzed a second human and a third fission yeast member of the family. We show, by deletion in the fission yeast genome, as well as expression and functional complementation experiments in both yeasts, that these new genes belong to the COX18 rather than to the OXA1 sub-branch. So far, the fission yeast gene cox18Sp+ is the smallest functional member of this gene family. COX18Hs gives rise to various mRNAs with different coding capacities, and we show that cox18Sp+ and COX18Hs are expressed at a low level and appear to be stringently regulated. This transcriptional control contrasts with the constitutive abundance of the OXA1 mRNAs and might reflect major functional differences between these nevertheless structurally related genes.

0006-291X (Print) Journal Article; The transcription of a set of 32 putative xylanase genes from Fusarium graminearum was examined by quantitative PCR after growth on different carbon sources (hop cell wall, xylan, xylose, or carboxymethylcellulose). Growing on plant cell wall medium, this fungus displays a great diversity of expression of xylan-related genes, with 30 being induced. A second level of diversity exists because expression patterns can be very different for loci encoding enzymes with the same activity (the same EC number). The wealth of xylan-degrading enzymes and the differential expression confer on the fungus a great flexibility of reaction to variation in its environment.

Nitric oxide and NO-derived species (RNS) are defence molecules with broad antimicrobial activity. Microorganisms have developed strategies to sense RNS and counteract their damaging effects. We used Saccharomyces cerevisiae, harbouring a deletion of YHB1 that encodes the main NO scavenger enzyme, to study consequences of RNS exposure on whole-genome transcriptional response. The expression of > 700 genes was altered on RNS treatment. No major role for ROS-scavenging enzymes was found, and the respiratory chain, the main site of ROS production, had only minor involvement in the RNS-induced stress. The changes were generally transient and also found after treatment with the respiratory inhibitor myxothiazol. However, 117 genes showed a persistent response that was not observed after myxothiazol treatment. Of these, genes of the glutathione and DNA repair systems, iron homeostasis and transport were found to be upregulated. Severe repression of genes of respiratory chain enzymes was observed. Many of these genes are known to be regulated by the transcription factor Hap1p, suggesting that RNS might interfere with Hap1p activity. We showed also that Msn2/4p and Yap1p, key regulators of the response to general stress and oxidative stress, respectively, played a role in mediating the RNS-induced response.

International audience; yMGV (yeast Microarray Global Viewer) was designed to provide biologists with meaningful information from genome-wide yeast expression data. The database includes most of the available expression data published on yeast microarrays over the last 4 years. It provides customizable tools for the rapid visualization of expression profiles associated with a set of genes from all published experiments. It also allows users to compare the results from different publications so that they can identify genes with common expression profiles. We used yMGV to perform global analyses to find a gene expression profile specific for given biological conditions and to locate functional gene clusters on chromosomes. Other organisms will be added to this database. yMGV is accessible on the web at http://transcriptome.ens.fr/ymgv.

Summary The program of DNA replication, defined by the temporal and spatial pattern of origin activation, is altered during development and in cancers. However, whether changes in origin usage play a role in regulating specific biological processes remains unknown. We investigated the consequences of modifying origin selection on meiosis in fission yeast. Genome-wide changes in the replication program of premeiotic S phase do not affect meiotic progression, indicating that meiosis neither activates nor requires a particular origin pattern. In contrast, local changes in origin efficiencies between different replication programs lead to changes in Rad51 recombination factor binding and recombination frequencies in these domains. We observed similar results for Rad51 when changes in efficiencies were generated by directly targeting expression of the Cdc45 replication factor. We conclude that origin selection is a key determinant for organizing meiotic recombination, providing evidence that genome-wide modifications in replication program can modulate cellular physiology., Graphical Abstract, Highlights • Premeiotic S phase neither activates nor requires a specific origin usage program • Origin selection is a key regulator of the distribution of meiotic recombination • Organization of genome duplication has a functional impact on cellular physiology, Changes in the pattern of DNA replication are observed during development and in various pathologies, but it is unclear whether they contribute to these processes. Wu and Nurse show, by altering replication origin usage in fission yeast, that origin selection is critical for the organization of meiotic recombination.

Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Δ/efg1Δ mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Δ/efg1Δ strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure., Author Summary Pathogenic microbes often evolve complex traits to adapt to their respective hosts, and this evolution is ongoing: for example, microorganisms are developing resistance to antimicrobial compounds in the clinical setting. The ability of the common human pathogenic fungus, Candida albicans, to switch from yeast to hyphal (filamentous) growth is considered a central virulence attribute. For example, hyphal formation allows C. albicans to escape from macrophages following phagocytosis. A well-investigated signaling network integrates different environmental cues to induce and maintain hyphal growth. In fact, deletion of two central transcription factors in this network results in a mutant that is both nonfilamentous and avirulent. We used experimental evolution to study the adaptation capability of this mutant by continuous co-incubation within macrophages. We found that this selection regime led to a relatively rapid re-connection of signaling between environmental cues and the hyphal growth program. Indeed, the evolved mutant regained the ability to filament and its virulence in vivo. This bypass of central transcription factors was based on a single nucleotide exchange in a gene encoding a component of the general transcription regulation machinery. Our results show that even a complex regulatory network, such as the transcriptional network which governs hyphal growth, can be remodeled via microevolution.

International audience; Fission yeast centromeres are transcriptionally silent and form a heterochromatin-like structure essential for normal centromere function; this appears analogous to heterochromatin and position effect variegation in other eukaryotes. Conditional mutations in three genes designated cep (centromere enhancer of position effect) were found to enhance transcriptional silencing within centromeres. Cloning of the cep1(+) and cep2(+) genes by functional complementation revealed that they are identical to the previously described genes pad1(+) and mts2(+), respectively, which both encode subunits of the proteasome 19S cap. Like Mts2 and Mts4, epitope-tagged Cep1/Pad1 localizes to or near the nuclear envelope throughout the cell cycle. The cep mutants display a range of phenotypes depending on the temperature. Silencing within the central domain of centromeres is increased at 36 degrees C. This suggests that the proteasome is involved in regulating silencing and thus centromeric chromatin architecture, possibly by lowering the level of some chromatin-associated protein by ubiquitin-dependent degradation. This is the first report of defective proteasome function affecting heterochromatin-mediated transcriptional silencing. At 36 and 32 degrees C, the cep mutants lose chromosomes at an elevated rate, and at 18 degrees C, the mutants are cryosensitive for growth. Cytological analysis at 18 degrees C revealed a defect in sister chromatid separation while other mitotic events occurred normally, indicating that cep mutations might interfere specifically with the degradation of inhibitor(s) of sister chromatid separation. These observations suggest that 19S subunits confer a level of substrate specificity on the proteasome and raise the possibility of a link between components involved in centromere architecture and sister chromatid cohesion.

International audience; We present here a simple and fast method allowing the isolation of DNA binding sites for transcription factors from families of coregulated genes, with results illustrated in Saccharomyces cerevisiae. Although conceptually simple, the algorithm proved efficient for extracting, from most of the yeast regulatory families analyzed, the upstream regulatory sequences which had been previously found by experimental analysis. Furthermore, putative new regulatory sites are predicted within upstream regions of several regulons. The method is based on the detection of over-represented oligonucleotides. A specificity of this approach is to define the statistical significance of a site based on tables of oligonucleotide frequencies observed in all non-coding sequences from the yeast genome. In contrast with heuristic methods, this oligonucleotide analysis is rigorous and exhaustive. Its range of detection is however limited to relatively simple patterns: short motifs with a highly conserved core. These features seem to be shared by a good number of regulatory sites in yeast. This, and similar methods, should be increasingly required to identify unknown regulatory elements within the numerous new coregulated families resulting from measurements of gene expression levels at the genomic scale. All tools described here are available on the web at the site http://copan.cifn.unam.mx/Computational_Biology/ yeast-tools

International audience; In response to genotoxic stress (GS), Candida albicans can undergo polarized growth and massive genome rearrangements including loss-of-heterozygosity (LOH) events. We evaluated the contribution of the CaRad53p and CaDun1p kinases of the DNA damage checkpoint (DDCP) in these processes. Characterization of C. albicans rad53ΔΔ and dun1ΔΔ mutants revealed that the two kinases were involved in the maintenance of heterozygosity. SNP-RFLP typing and whole-genome sequencing of rad53ΔΔ isolates having undergone a LOH revealed that, according to the chromosome on which LOH had occurred, these were predominantly due to break-induced replication/mitotic cross-over or chromosome loss. Loss of CaRAD53 also resulted in frequent aneuploidies. Deletion of CaDUN1 led to an increase in recombination-dependent LOH but did not trigger aneuploidies. It also increased GS sensitivity but did not impair GS-induced polarized growth contrary to CaRAD53 deletion. Characterization of CaRad53p site-directed mutants demonstrated that its kinase activity and N-terminal phosphorylation sites were crucial for its function in the resistance to GS, maintenance of heterozygosity, cell cycle regulation and polarized growth. Moreover, using phosphomimic mutants, we revealed an uncoupling of the functions of CaRad53p in these different processes, thus providing a novel understanding of how the DDCP may regulate downstream events in response to GS.

International audience; Rho G-proteins are critical for polarized growth, yet little is known about the dynamics of their activation during fungal filamentous growth. We first investigated the roles of Rho1 and Rho2 during Candida albicans filamentous growth. Our results show that Rho1 is required for invasive filamentous growth and that Rho2 is not functionally redundant with Rho1. Using fluorescent reporters, we examined the dynamics of the active form of Rho1 and Cdc42 during initiation and maintenance of hyphal growth. Quantitative analyses indicated that the distribution, but not the level, of these active G-proteins is altered during initial polarization upon germ tube emergence. A comparison of the dynamics of these active G-proteins during budding and hyphal growth indicates that a higher concentration of active Cdc42 was recruited to the germ tube tip than to the bud tip. During hyphal elongation, active Cdc42 remained tightly restricted to the hyphal tip, whereas active Rho1 was broadly associated with the apex and subsequently recruited to the cell division site. Furthermore, our data suggest that phosphoinositide-bis-phosphates are critical to stabilize active Rho1 at the growth site. Together, our results point towards different regulation of Cdc42 and Rho1 activity during initiation and maintenance of filamentous growth.

The choline oxidase ( CHOA ) and betaine aldehyde dehydrogenase ( BADH ) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD + to NADH. Analysis of the AfchoA Δ:: HPH and AfbadA Δ:: HPH single mutants and the AfchoA Δ AfbadA Δ:: HPH double mutant showed that Af ChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. Af ChoAp and Af BadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.

International audience; BACKGROUND:SUN proteins are involved in yeast morphogenesis, but their function is unknown.RESULTS:SUN protein plays a role in the Aspergillus fumigatus morphogenesis. Biochemical properties of recombinant SUN proteins were elucidated.CONCLUSION:Both Candida albicans and Aspergillus fumigatus sun proteins show a β-(1,3)-glucanase activity.SIGNIFICANCE:The mode of action of SUN proteins on β-(1,3)-glucan is unique, new, and original. In yeasts, the family of SUN proteins has been involved in cell wall biogenesis. Here, we report the characterization of SUN proteins in a filamentous fungus, Aspergillus fumigatus. The function of the two A. fumigatus SUN genes was investigated by combining reverse genetics and biochemistry. During conidial swelling and mycelial growth, the expression of AfSUN1 was strongly induced, whereas the expression of AfSUN2 was not detectable. Deletion of AfSUN1 negatively affected hyphal growth and conidiation. A closer examination of the morphological defects revealed swollen hyphae, leaky tips, intrahyphal growth, and double cell wall, suggesting that, like in yeast, AfSun1p is associated with cell wall biogenesis. In contrast to AfSUN1, deletion of AfSUN2 either in the parental strain or in the AfSUN1 single mutant strain did not affect colony and hyphal morphology. Biochemical characterization of the recombinant AfSun1p and Candida albicans Sun41p showed that both proteins had a unique hydrolysis pattern: acting on β-(1,3)-oligomers from dimer up to insoluble β-(1,3)-glucan. Referring to the CAZy database, it is clear that fungal SUN proteins represent a new family of glucan hydrolases (GH132) and play an important morphogenetic role in fungal cell wall biogenesis and septation.

International audience; We investigated the antifungal activity of fused Mannich ketone (FMK) congeners and two of their aminoalcohol derivatives. In particular, FMKs with five-membered saturated rings were shown to have minimum inhibitory concentration (MIC90s) ranging from 0.8 to 6 µg/mL toward C. albicans and the closely related C. parapsilosis and C. krusei while having reduced efficacy toward C. glabrata and almost no efficacy against Aspergillus sp. Transcript profiling of C. albicans cells exposed for 30 or 60 min to 2-(morpholinomethyl)-1-indanone, a representative FMK with a five-membered saturated ring, revealed a transcriptional response typical of oxidative stress and similar to that of a C. albicans Cap1 transcriptional activator. Consistently, C. albicans lacking the CAP1 gene was hypersensitive to this FMK, while C. albicans strains overexpressing CAP1 had decreased sensitivity to 2-(morpholinomethyl)-1-indanone. Quantitative structure-activity relationship studies revealed a correlation of antifungal potency and the energy of the lowest unoccupied molecular orbital of FMKs and unsaturated Mannich ketones thereby implicating redox cycling-mediated oxidative stress as a mechanism of action. This conclusion was further supported by the loss of antifungal activity upon conversion of representative FMKs to aminoalcohols that were unable to participate in redox cycles.

Sfl1p and Sfl2p are two homologous heat shock factor-type transcriptional regulators that antagonistically control morphogenesis in Candida albicans, while being required for full pathogenesis and virulence. To understand how Sfl1p and Sfl2p exert their function, we combined genome-wide location and expression analyses to reveal their transcriptional targets in vivo together with the associated changes of the C. albicans transcriptome. We show that Sfl1p and Sfl2p bind to the promoter of at least 113 common targets through divergent binding motifs and modulate directly the expression of key transcriptional regulators of C. albicans morphogenesis and/or virulence. Surprisingly, we found that Sfl2p additionally binds to the promoter of 75 specific targets, including a high proportion of hyphal-specific genes (HSGs; HWP1, HYR1, ECE1, others), revealing a direct link between Sfl2p and hyphal development. Data mining pointed to a regulatory network in which Sfl1p and Sfl2p act as both transcriptional activators and repressors. Sfl1p directly represses the expression of positive regulators of hyphal growth (BRG1, UME6, TEC1, SFL2), while upregulating both yeast form-associated genes (RME1, RHD1, YWP1) and repressors of morphogenesis (SSN6, NRG1). On the other hand, Sfl2p directly upregulates HSGs and activators of hyphal growth (UME6, TEC1), while downregulating yeast form-associated genes and repressors of morphogenesis (NRG1, RFG1, SFL1). Using genetic interaction analyses, we provide further evidences that Sfl1p and Sfl2p antagonistically control C. albicans morphogenesis through direct modulation of the expression of important regulators of hyphal growth. Bioinformatic analyses suggest that binding of Sfl1p and Sfl2p to their targets occurs with the co-binding of Efg1p and/or Ndt80p. We show, indeed, that Sfl1p and Sfl2p targets are bound by Efg1p and that both Sfl1p and Sfl2p associate in vivo with Efg1p. Taken together, our data suggest that Sfl1p and Sfl2p act as central “switch on/off” proteins to coordinate the regulation of C. albicans morphogenesis., Author Summary Candida albicans can switch from a harmless colonizer of body organs to a life-threatening invasive pathogen. This switch is linked to the ability of C. albicans to undergo a yeast-to-filament shift induced by various cues, including temperature. Sfl1p and Sfl2p are two transcription factors required for C. albicans virulence, but antagonistically regulate morphogenesis: Sfl1p represses it, whereas Sfl2p activates it in response to temperature. We show here that Sfl1p and Sfl2p bind in vivo, via divergent motifs, to the regulatory region of a common set of targets encoding key determinants of morphogenesis and virulence and exert both activating and repressing effects on gene expression. Additionally, Sfl2p binds to specific targets, including genes essential for hyphal development. Bioinformatic analyses suggest that Sfl1p and Sfl2p control C. albicans morphogenesis by cooperating with two important regulators of filamentous growth, Efg1p and Ndt80p, a premise that was confirmed by the observation of concomitant binding of Sfl1p, Sfl2p and Efg1p to the promoter of target genes and the demonstration of direct or indirect physical association of Sfl1p and Sfl2p with Efg1p, in vivo. Our data suggest that Sfl1p and Sfl2p act as central “switch on/off” proteins to coordinate the regulation of C. albicans morphogenesis.

International audience; The het-c locus of the filamentous fungus Podospora anserina controls heterokaryon formation through genetic interaction with alleles of the unlinked loci het-e and het-d. We have isolated four wild-type and two mutant alleles of the het-c locus. A comparison of the predicted proteins encoded by the different wild-type alleles revealed an unusual high level of amino-acid replacements compared to silent polymorphisms but only one amino-acid difference is sufficient to modify the specificity of het-c alleles. Chimeric genes constructed in vitro may exhibit a new specificity different from that of any known wild-type allele.

International audience; The ura4+ gene displays phenotypes consistent with variegated expression when inserted at 11 sites throughout fission yeast centromere 1. An abrupt transition occurs between the zone of centromeric repression and two adjacent expressed sites. Mutations in six genes alleviate repression of the silent-mating type loci and of ura4+ expressed from a site adjacent to the silent locus, mat3-M. Defects at all six loci affect repression of the ura4+ gene adjacent to telomeres and at the three centromeric sites tested. The clr4-S5 and rik1-304 mutations cause the most dramatic derepression at two out of three sites within cen1. All six mutations had only slight or intermediate effects on a third site in the center of cen1 or on telomeric repression. Strains with lesions at the clr4, rik1, and swi6 loci have highly elevated rates of chromosome loss. We propose that the products of these genes are integral in the assembly of a heterochromatin-like structure, with distinct domains, enclosing the entire centromeric region that reduces or excludes access to transcription factors. The formation of this heterochromatic structure may be an absolute requirement for the formation of a fully functional centromere.

Neurospora and higher eukaryotes share a common mechanism for the signal transduction of environmental stimuli. A scenario is given in which the Neurospora WC-1 photoreceptor represents a function orthologous to that of vertebrate nuclear receptors, acting through the association with the HAT NGF-1 via a vertebrate-like LXXLL motif., In Neurospora crassa and other filamentous fungi, light-dependent–specific phenomena are regulated by transcription factors WC-1 and WC-2. In addition to its transcriptional activity, WC-1 is able to directly sense light stimuli through a LOV sensor domain. Its location in the nucleus and heterodimerization with WC-2, together with the presence of a zinc-finger DNA-binding domain and an environmental sensor domain, all resemble the functional evolutionary architecture adopted by vertebrate nuclear receptors (NRs). Here we describe a scenario in which WC-1 represents a functional orthologue of NRs and acts through association with the chromatin-modifying coactivator NGF-1, which encodes a homologue of the yeast Gcn5p acetyltransferase. To support this view, we show a direct association between WC-1 and NGF-1 that depends on a WC-1 region containing a conserved functional LXXLL motif, a signature previously described as being an exclusive feature of NR/coactivator interaction. Our data suggest that a WC-1/NGF-1 complex is preassembled in the dark on light-inducible promoters and that, after exposure to light stimulation, NGF-1–associated HAT activity leads to histone H3 acetylation and transcriptional activation. Finally, we provide evidence for a NGF-1–independent acetylated form of WC-1. Overall our data indicate that Neurospora and higher eukaryotes share a common mechanism for the signal transduction of environmental stimuli.

Transcription-associated recombination (TAR) is crucial for stability among repeated units of rDNA. Several histone deacetylases and a chromatin architectural component control the synthesis of ncRNA and rDNA recombination. The only acetylation state of histone H4 at Lys-16 is sufficient to regulate TAR at rDNA., Transcription-associated recombination is an important process involved in several aspects of cell physiology. In the ribosomal DNA (rDNA) of Saccharomyces cerevisiae, RNA polymerase II transcription–dependent recombination has been demonstrated among the repeated units. In this study, we investigate the mechanisms controlling this process at the chromatin level. On the basis of a small biased screening, we found that mutants of histone deacetylases and chromatin architectural proteins alter both the amount of Pol II–dependent noncoding transcripts and recombination products at rDNA in a coordinated manner. Of interest, chromatin immunoprecipitation analyses in these mutants revealed a corresponding variation of the histone H4 acetylation along the rDNA repeat, particularly at Lys-16. Here we provide evidence that a single, rapid, and reversible posttranslational modification—the acetylation of the H4K16 residue—is involved in the coordination of transcription and recombination at rDNA.

International audience; RNA Pol II transcription termination can occur by at least two alternative pathways. Cleavage and polyadenylation by the CPF/CF complex precedes mRNA transcription termination, while the Nrd1 complex is involved in transcription termination of non-coding RNAs such as sno/snRNAs or cryptic unstable transcripts. Here we show that transcription of RPL9B, one of the two genes coding for the ribosomal protein Rpl9p, terminates by either of these two pathways. The balance between these two pathways is modulated in response to the RPL9 gene copy number, resulting in the autoregulation of RPL9B gene expression. This autoregulation mechanism requires a conserved potential stem-loop structure very close to the polyadenylation sites. We propose a model in which Rpl9p, when in excess, binds this conserved 3'-UTR structure, negatively interfering with cleavage and polyadenylation to the benefit of the Nrd1-dependent termination pathway, which, being coupled to degradation by the nuclear exosome, results in downregulation of RPL9B gene expression.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., In nature, many microorganisms form specialized complex, multicellular, surface-attached communities called biofilms. These communities play critical roles in microbial pathogenesis. The fungal pathogen Candida albicans is associated with catheter-based infections due to its ability to establish biofilms. The transcription factor Bcr1 is a master regulator of C. albicans biofilm development, although the full extent of its regulation remains unknown. Here, we report that Bcr1 is a phosphoprotein that physically interacts with the NDR kinase Cbk1 and undergoes Cbk1-dependent phosphorylation. Mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to alanine markedly impaired Bcr1 function during biofilm formation and virulence in a mouse model of disseminated candidiasis. Cells lacking Cbk1, or any of its upstream activators, also had reduced biofilm development. Notably, mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to glutamate in cbk1Δ cells upregulated the transcription of Bcr1-dependent genes and partially rescued the biofilm defects of a cbk1Δ strain. Therefore, our data uncovered a novel role of the NDR/LATS kinase Cbk1 in the regulation of biofilm development through the control of Bcr1. © 2012 Gutiérrez-Escribano et al., This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2009-11251 to J. C.-B. and BFU2010-15884 to C.R.V.) and the Regional Government of Extremadura (PRI08A017, GRU09001 and GRU10008) to J. C.-B. All Spanish funding was co-sponsored by the European Union FEDER programme. P.G-E. was supported by a pre-doctoral fellowship (FPU program) from the Spanish Government. Work in the C. d’E. laboratory was supported by the European Commission (FINSysB, PITN-GA-2008-214004) and Agence Nationale de la Recherche (KANJI, ANR-08-MIE-033-01). U.Z. was a recipient of post-doctoral fellowship of Institut Carnot «Pasteur Maladies Infectieuses» (Programme Fungi).

Biofilm formation plays an important role in fungal pathogenesis. In this work, we used a genetic screen in order to identify and characterize genes involved in the formation of biofilms by the opportunistic fungal pathogen Candida glabrata. We identified the Cst6p transcription factor as a negative regulator of the EPA6 gene that encodes an adhesin central to C. glabrata biofilm formation. Analysis of single and double mutant strains showed that Cst6p acts in a pathway independent of the Yak1/Sir4 pathway also known to regulate expression of EPA6 and consequently biofilm formation. In contrast, we showed that the chromatin remodelling Swi/Snf complex positively regulates biofilm formation in C. glabrata. RT-qPCR experiments demonstrated that EPA6 expression, and thus biofilm formation, depends on the integrity of the Sir complex. Finally, we showed that Swi/Snf-dependent regulation of biofilm formation is adhesin-specific., This work was supported by a grant from the Institut Pasteur (PTR173).

Aspergillus fumigatus has two chitin synthases ( CSMA and CSMB ) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the Δ csmA strain cell wall was less than half the amount found in the parental strain. In contrast, the Δ csmB mutant strain and, unexpectedly, the Δ csmA /Δ csmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis.

Aspergillus fumigatus is the most common and deadly pulmonary fungal infection worldwide. In the lung, the fungus usually forms a dense colony of filaments embedded in a polymeric extracellular matrix. To identify candidate genes involved in this biofilm (BF) growth, we used RNA-Seq to compare the transcriptomes of BF and liquid plankton (PL) growth. Sequencing and mapping of tens of millions sequence reads against the A. fumigatus transcriptome identified 3,728 differentially regulated genes in the two conditions. Although many of these genes, including the ones coding for transcription factors, stress response, the ribosome, and the translation machinery, likely reflect the different growth demands in the two conditions, our experiment also identified hundreds of candidate genes for the observed differences in morphology and pathobiology between BF and PL. We found an overrepresentation of upregulated genes in transport, secondary metabolism, and cell wall and surface functions. Furthermore, upregulated genes showed significant spatial structure across the A. fumigatus genome; they were more likely to occur in subtelomeric regions and colocalized in 27 genomic neighborhoods, many of which overlapped with known or candidate secondary metabolism gene clusters. We also identified 1,164 genes that were downregulated. This gene set was not spatially structured across the genome and was overrepresented in genes participating in primary metabolic functions, including carbon and amino acid metabolism. These results add valuable insight into the genetics of biofilm formation in A. fumigatus and other filamentous fungi and identify many relevant, in the context of biofilm biology, candidate genes for downstream functional experiments.

International audience; The cell-fate decision leading to gametogenesis is essential for sexual reproduction. In S. cerevisiae, only diploid MATa/α but not haploid MATa or MATα cells undergo gametogenesis, known as sporulation. We find that transcription of two long noncoding RNAs (lncRNAs) mediates mating-type control of sporulation. In MATa or MATα haploids, expression of IME1, the central inducer of gametogenesis, is inhibited in cis by transcription of the lncRNA IRT1, located in the IME1 promoter. IRT1 transcription recruits the Set2 histone methyltransferase and the Set3 histone deacetylase complex to establish repressive chromatin at the IME1 promoter. Inhibiting expression of IRT1 and an antisense transcript that antagonizes the expression of the meiotic regulator IME4 allows cells expressing the haploid mating type to sporulate with kinetics that are indistinguishable from that of MATa/α diploids. Conversely, expression of the two lncRNAs abolishes sporulation in MATa/α diploids. Thus, transcription of two lncRNAs governs mating-type control of gametogenesis in yeast.

International audience; The fungal pathogen Candida albicans forms therapeutically challenging biofilms on biomedical implants. Using a transcript profiling approach genes whose expression is favoured upon biofilm growth compared with planktonic growth have been previously identified. Knock-out mutants for 38 of these genes were constructed, six of which showed a specific defect in biofilm formation. Among these genes, TYE7 that encodes a transcriptional activator of glycolytic genes in planktonic and biofilm growth conditions was identified as being required for the cohesiveness of biofilms. Biofilms formed by the tye7Δ knock-out mutant showed a hyperfilamentous morphology, and growth of this mutant on solid medium under hypoxia was also associated with the production of hyphae. Similar to TYE7 inactivation, inhibition of glycolysis or ATP synthesis using oxalate or an uncoupler, respectively, triggered morphogenesis when a wild-type strain was grown under hypoxia. These treatments also induced the formation of weakly cohesive, hyper-filamentous biofilms by a wild-type strain. Our data indicate that a hypoxic environment is generated within C. albicans biofilms and that continued biofilm development requires a Tye7p-dependent upregulation of glycolytic genes necessary to adapt to hypoxia and prevent uncontrolled hyphal formation. Thus, adaptation to hypoxia is an integral component of biofilm formation in C. albicans.

International audience; The spatial organization of genes and chromosomes plays an important role in the regulation of several DNA processes. However, the principles and forces underlying this nonrandom organization are mostly unknown. Despite its small dimension, and thanks to new imaging and biochemical techniques, studies of the budding yeast nucleus have led to significant insights into chromosome arrangement and dynamics. The dynamic organization of the yeast genome during interphase argues for both the physical properties of the chromatin fiber and specific molecular interactions as drivers of nuclear order.

International audience; Increasing evidence implicates cohesin in the control of gene expression. Here we report the first analysis of cohesin-dependent gene regulation in fission yeast. Global expression profiling of the mis4-367 cohesin loader mutant identified a small number of upregulated and downregulated genes within subtelomeric domains (SD). These 20- to 40-kb regions between chromosome arm euchromatin and telomere-proximal heterochromatin are characterized by a combination of euchromatin (methylated lysine 4 on histone H3/methylated Tysine 9 on histone H3 [H3K4me]) and heterochromatin (H3K9me) marks. We focused our analysis on the chromosome 1 right SD, which contains several upregulated genes and is bordered on the telomere-distal side by a pair of downregulated genes. We find that the expression changes in the SD also occur in a mutant of the cohesin core component Rad21. Remarkably, mutation of Rad21 results in the depletion of Swi6 binding in the SD. In fact, the Rad21 mutation phenocopied Swi6 loss of function: both mutations led to reduced cohesin binding, reduced H3K9me, and similar gene expression changes in the SD. In particular, expression of the gene pair bordering the SD was dependent both on cohesin and on Swi6. Our data indicate that cohesin participates in the setup of a subtelomeric heterochromatin domain and controls the expression of the genes residing in that domain.

Arrays of Sir3 binding sites at telomeres are sufficient to promote long-range trans-telomere interactions., A general feature of the nucleus is the organization of repetitive deoxyribonucleic acid sequences in clusters concentrating silencing factors. In budding yeast, we investigated how telomeres cluster in perinuclear foci associated with the silencing complex Sir2–Sir3–Sir4 and found that Sir3 is limiting for telomere clustering. Sir3 overexpression triggers the grouping of telomeric foci into larger foci that relocalize to the nuclear interior and correlate with more stable silencing in subtelomeric regions. Furthermore, we show that Sir3′s ability to mediate telomere clustering can be separated from its role in silencing. Indeed, nonacetylable Sir3, which is unable to spread into subtelomeric regions, can mediate telomere clustering independently of Sir2–Sir4 as long as it is targeted to telomeres by the Rap1 protein. Thus, arrays of Sir3 binding sites at telomeres appeared as the sole requirement to promote trans-interactions between telomeres. We propose that similar mechanisms involving proteins able to oligomerize account for long-range interactions that impact genomic functions in many organisms.

The Pol I subunits Rpa34 and Rpa49, required for elongation and 35S rRNA production, promote clustering of neighboring Pol I complexes to enhance the rRNA gene transcription cycle., RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I–specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.

Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly being investigated for second-generation biofuel production from lignocellulosic biomass. The induction mechanisms of T. reesei cellulases have been described recently, but the regulation of the genes involved in their transcription has not been studied thoroughly. Here we report the regulation of expression of the two activator genes xyr1 and ace2 , and the corepressor gene ace1 , during the induction of cellulase biosynthesis by the inducer lactose in T. reesei QM 9414, a strain producing low levels of cellulase (low producer). We show that all three genes are induced by lactose. xyr1 was also induced by d -galactose, but this induction was independent of d -galactose metabolism. Moreover, ace1 was carbon catabolite repressed, whereas full induction of xyr1 and ace2 in fact required CRE1. Significant differences in these regulatory patterns were observed in the high-producer strain RUT C30 and the hyperproducer strain T. reesei CL847. These observations suggest that a strongly elevated basal transcription level of xyr1 and reduced upregulation of ace1 by lactose may have been important for generating the hyperproducer strain and that thus, these genes are major control elements of cellulase production.