Identification of an active RNAi pathway in Candida albicans.

RNA interference (RNAi) is a fundamental regulatory pathway with a wide range of functions, including regulation of gene expression and maintenance of genome stability. Although RNAi is widespread in the fungal kingdom, well-known species, such as the model yeast Saccharomyces cerevisiae, have lost the RNAi pathway. Until now evidence has been lacking for a fully functional RNAi pathway in Candida albicans, a human fungal pathogen considered critically important by the World Health Organization. Here, we demonstrated that the widely used C. albicans reference strain (SC5314) contains an inactivating missense mutation in the gene encoding for the central RNAi component Argonaute. In contrast, most other C. albicans isolates contain a canonical Argonaute protein predicted to be functional and RNAi-active. Indeed, using high-throughput small and long RNA sequencing combined with seamless CRISPR/Cas9-based gene editing, we demonstrate that an active C. albicans RNAi machinery represses expression of subtelomeric gene families. Thus, an intact and functional RNAi pathway exists in C. albicans, highlighting the importance of using multiple reference strains when studying this dangerous pathogen.

Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, ß- and γ- TLO gene function.

The Candida albicans genome contains between ten and fifteen distinct TLO genes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo in C. albicans we deleted all fourteen TLO genes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in the tloΔ mutant strain compared to the parent, while RNA-seq analysis showed that the tloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, the tloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, ß- and γ-TLO clades resulted in the complementation of the mutant phenotypes, but to different degrees. TLOα1 could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLO genes examined (i.e., TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression of TLOß2 in the tloΔ mutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloß2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegrated TLO genes in a tloΔ/med3Δ double mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range of C. albicans cellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans.

Stress combined with loss of the Candida albicans SUMO protease Ulp2 triggers selection of aneuploidy via a two-step process.

A delicate balance between genome stability and instability ensures genome integrity while generating genetic diversity, a critical step for evolution. Indeed, while excessive genome instability is harmful, moderated genome instability can drive adaptation to novel environments by maximising genetic variation. Candida albicans, a human fungal pathogen that colonises different parts of the human body, adapts rapidly and frequently to different hostile host microenvironments. In this organism, the ability to generate large-scale genomic variation is a key adaptative mechanism triggering dangerous infections even in the presence of antifungal drugs. Understanding how fitter novel karyotypes are selected is key to determining how C. albicans and other microbial pathogens establish infections. Here, we identified the SUMO protease Ulp2 as a regulator of C. albicans genome integrity through genetic screening. Deletion of ULP2 leads to increased genome instability, enhanced genome variation and reduced fitness in the absence of additional stress. The combined stress caused by the lack of ULP2 and antifungal drug treatment leads to the selection of adaptive segmental aneuploidies that partially rescue the fitness defects of ulp2Δ/Δ cells. Short and long-read genomic sequencing demonstrates that these novel genotypes are selected via a two-step process leading to the formation of novel chromosomal fragments with breakpoints at microhomology regions and DNA repeats.

On and Off: Epigenetic Regulation of C. albicans Morphological Switches.

The human fungal pathogen Candida albicans is a dimorphic opportunistic pathogen that colonises most of the human population without creating any harm. However, this fungus can also cause life-threatening infections in immunocompromised individuals. The ability to successfully colonise different host niches is critical for establishing infections and pathogenesis. C. albicans can live and divide in various morphological forms critical for its survival in the host. Indeed, C. albicans can grow as both yeast and hyphae and can form biofilms containing hyphae. The transcriptional regulatory network governing the switching between these different forms is complex but well understood. In contrast, non-DNA based epigenetic modulation is emerging as a crucial but still poorly studied regulatory mechanism of morphological transition. This review explores our current understanding of chromatin-mediated epigenetic regulation of the yeast to hyphae switch and biofilm formation. We highlight how modification of chromatin structure and non-coding RNAs contribute to these morphological transitions.

The Genome of the CTG(Ser1) Yeast Scheffersomyces stipitis Is Plastic.

Microorganisms need to adapt to environmental changes, and genome plasticity can lead to rapid adaptation to hostile environments by increasing genetic diversity. Here, we investigate genome plasticity in the CTG(Ser1) yeast Scheffersomyces stipitis, an organism with an enormous potential for second-generation biofuel production. We demonstrate that S. stipitis has an intrinsically plastic genome and that different S. stipitis isolates have genomes with distinct chromosome organizations. Real-time evolution experiments show that S. stipitis genome plasticity is common and rapid since extensive genomic changes with fitness benefits are detected following in vitro evolution experiments. Hybrid MinION Nanopore and Illumina genome sequencing identify retrotransposons as major drivers of genome diversity. Indeed, the number and position of retrotransposons are different in different S. stipitis isolates, and retrotransposon-rich regions of the genome are sites of chromosome rearrangements. Our findings provide important insights into the adaptation strategies of the CTG(Ser1) yeast clade and have critical implications in the development of second-generation biofuels. These data highlight that genome plasticity is an essential factor for developing sustainable S. stipitis platforms for second-generation biofuels production. IMPORTANCE Genomes contain genes encoding the information needed to build the organism and allow it to grow and develop. Genomes are described as stable structures where genes have specific positions within a chromosome. Changes in gene dosage and position are viewed as harmful. However, it is becoming increasingly clear that genome plasticity can benefit microbial organisms that need to adapt rapidly to environmental changes. Mechanisms of genome plasticity are still poorly understood. This study focuses on Scheffersomyces stipitis, a yeast that holds great potential for second-generation biofuel production generated from forestry and agriculture waste. We demonstrate that S. stipitis chromosomes are easily reshuffled and that chromosome reshuffling is linked to adaptation to hostile environments. Genome sequencing demonstrates that mobile genetic elements, called transposons, mediate S. stipitis genome reshuffling. These data highlight that understanding genome plasticity is important for developing sustainable S. stipitis platforms for second-generation biofuels production.

Chromatin-Mediated Regulation of Genome Plasticity in Human Fungal Pathogens.

Human fungal pathogens, such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, are a public health problem, causing millions of infections and killing almost half a million people annually. The ability of these pathogens to colonise almost every organ in the human body and cause life-threating infections relies on their capacity to adapt and thrive in diverse hostile host-niche environments. Stress-induced genome instability is a key adaptive strategy used by human fungal pathogens as it increases genetic diversity, thereby allowing selection of genotype(s) better adapted to a new environment. Heterochromatin represses gene expression and deleterious recombination and could play a key role in modulating genome stability in response to environmental changes. However, very little is known about heterochromatin structure and function in human fungal pathogens. In this review, I use our knowledge of heterochromatin structure and function in fungal model systems as a road map to review the role of heterochromatin in regulating genome plasticity in the most common human fungal pathogens: Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans.

Chromatin Profiling of the Repetitive and Nonrepetitive Genomes of the Human Fungal Pathogen Candida albicans.

Eukaryotic genomes are packaged into chromatin structures that play pivotal roles in regulating all DNA-associated processes. Histone posttranslational modifications modulate chromatin structure and function, leading to rapid regulation of gene expression and genome stability, key steps in environmental adaptation. Candida albicans, a prevalent fungal pathogen in humans, can rapidly adapt and thrive in diverse host niches. The contribution of chromatin to C. albicans biology is largely unexplored. Here, we generated the first comprehensive chromatin profile of histone modifications (histone H3 trimethylated on lysine 4 [H3K4me3], histone H3 acetylated on lysine 9 [H3K9Ac], acetylated lysine 16 on histone H4 [H4K16Ac], and γH2A) across the C. albicans genome and investigated its relationship to gene expression by harnessing genome-wide sequencing approaches. We demonstrated that gene-rich nonrepetitive regions are packaged into canonical euchromatin in association with histone modifications that mirror their transcriptional activity. In contrast, repetitive regions are assembled into distinct chromatin states; subtelomeric regions and the ribosomal DNA (rDNA) locus are assembled into heterochromatin, while major repeat sequences and transposons are packaged in chromatin that bears features of euchromatin and heterochromatin. Genome-wide mapping of γH2A, a marker of genome instability, identified potential recombination-prone genomic loci. Finally, we present the first quantitative chromatin profiling in C. albicans to delineate the role of the chromatin modifiers Sir2 and Set1 in controlling chromatin structure and gene expression. This report presents the first genome-wide chromatin profiling of histone modifications associated with the C. albicans genome. These epigenomic maps provide an invaluable resource to understand the contribution of chromatin to C. albicans biology and identify aspects of C. albicans chromatin organization that differ from that of other yeasts.IMPORTANCE The fungus Candida albicans is an opportunistic pathogen that normally lives on the human body without causing any harm. However, C. albicans is also a dangerous pathogen responsible for millions of infections annually. C. albicans is such a successful pathogen because it can adapt to and thrive in different environments. Chemical modifications of chromatin, the structure that packages DNA into cells, can allow environmental adaptation by regulating gene expression and genome organization. Surprisingly, the contribution of chromatin modification to C. albicans biology is still largely unknown. For the first time, we analyzed C. albicans chromatin modifications on a genome-wide basis. We demonstrate that specific chromatin states are associated with distinct regions of the C. albicans genome and identify the roles of the chromatin modifiers Sir2 and Set1 in shaping C. albicans chromatin and gene expression.

Sir2 regulates stability of repetitive domains differentially in the human fungal pathogen Candida albicans.

DNA repeats, found at the ribosomal DNA locus, telomeres and subtelomeric regions, are unstable sites of eukaryotic genomes. A fine balance between genetic variability and genomic stability tunes plasticity of these chromosomal regions. This tuning mechanism is particularly important for organisms such as microbial pathogens that utilise genome plasticity as a strategy for adaptation. For the first time, we analyse mechanisms promoting genome stability at the rDNA locus and subtelomeric regions in the most common human fungal pathogen: Candida albicans In this organism, the histone deacetylase Sir2, the master regulator of heterochromatin, has acquired novel functions in regulating genome stability. Contrary to any other systems analysed, C. albicans Sir2 is largely dispensable for repressing recombination at the rDNA locus. We demonstrate that recombination at subtelomeric regions is controlled by a novel DNA element, the TLO Recombination Element, TRE, and by Sir2. While the TRE element promotes high levels of recombination, Sir2 represses this recombination rate. Finally, we demonstrate that, in C. albicans, mechanisms regulating genome stability are plastic as different environmental stress conditions lead to general genome instability and mask the Sir2-mediated recombination control at subtelomeres. Our data highlight how mechanisms regulating genome stability are rewired in C. albicans.

The Chromatin of Candida albicans Pericentromeres Bears Features of Both Euchromatin and Heterochromatin.

Centromeres, sites of kinetochore assembly, are important for chromosome stability and integrity. Most eukaryotes have regional centromeres epigenetically specified by the presence of the histone H3 variant CENP-A. CENP-A chromatin is often surrounded by pericentromeric regions packaged into transcriptionally silent heterochromatin. Candida albicans, the most common human fungal pathogen, possesses small regional centromeres assembled into CENP-A chromatin. The chromatin state of C. albicans pericentromeric regions is unknown. Here, for the first time, we address this question. We find that C. albicans pericentromeres are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Pericentromeric chromatin is associated with nucleosomes that are highly acetylated, as found in euchromatic regions of the genome; and hypomethylated on H3K4, as found in heterochromatin. This intermediate chromatin state is inhibitory to transcription and partially represses expression of proximal genes and inserted marker genes. Our analysis identifies a new chromatin state associated with pericentromeric regions.

Candida albicans repetitive elements display epigenetic diversity and plasticity.

Transcriptionally silent heterochromatin is associated with repetitive DNA. It is poorly understood whether and how heterochromatin differs between different organisms and whether its structure can be remodelled in response to environmental signals. Here, we address this question by analysing the chromatin state associated with DNA repeats in the human fungal pathogen Candida albicans. Our analyses indicate that, contrary to model systems, each type of repetitive element is assembled into a distinct chromatin state. Classical Sir2-dependent hypoacetylated and hypomethylated chromatin is associated with the rDNA locus while telomeric regions are assembled into a weak heterochromatin that is only mildly hypoacetylated and hypomethylated. Major Repeat Sequences, a class of tandem repeats, are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Marker gene silencing assays and genome-wide RNA sequencing reveals that C. albicans heterochromatin represses expression of repeat-associated coding and non-coding RNAs. We find that telomeric heterochromatin is dynamic and remodelled upon an environmental change. Weak heterochromatin is associated with telomeres at 30 °C, while robust heterochromatin is assembled over these regions at 39 °C, a temperature mimicking moderate fever in the host. Thus in C. albicans, differential chromatin states controls gene expression and epigenetic plasticity is linked to adaptation.

The RFTS domain of Raf2 is required for Cul4 interaction and heterochromatin integrity in fission yeast.

Centromeric heterochromatin assembly in fission yeast is critical for faithful chromosome segregation at mitosis. Its assembly requires a concerted pathway of events whereby the RNA interference (RNAi) pathway guides H3K9 methylation to target sequences. H3K9 methylation, a hallmark of heterochromatin structure, is mediated by the single histone methyltransferase Clr4 (equivalent to metazoan Suv3-9), a component of the CLRC complex. Loss of or defects in CLRC components disrupts heterochromatin formation due to loss of H3K9 methylation, thus an intact, fully functional CLRC complex is required for heterochromatin integrity. Despite its importance, little is known about the contribution of the CLRC component Raf2 to H3K9 methylation and heterochromatin assembly. We demonstrate that Raf2 is concentrated at centromeres and contrary to other analyses, we find that loss of Raf2 does not affect CENP-ACnp1 localisation or recruitment to centromeres. Our sequence alignments show that Raf2 contains a Replication Foci Targeting Sequence (RFTS) domain homologous to the RFTS domain of the human DNA methyltransferase DNMT1. We show that the Raf2 RFTS domain is required for centromeric heterochromatin formation as its mutation disrupts H3K9 methylation but not the processing of centromeric transcripts into small interfering RNAs (siRNAs) by the RNAi pathway. Analysis of biochemical interactions demonstrates that the RFTS domain mediates an interaction between Raf2 and the CLRC component Cul4. We conclude that the RFTS domain of Raf2 is a protein interaction module that plays an important role in heterochromatin formation at centromeres.

Distinct roles for Sir2 and RNAi in centromeric heterochromatin nucleation, spreading and maintenance.

Epigenetically regulated heterochromatin domains govern essential cellular activities. A key feature of heterochromatin domains is the presence of hypoacetylated nucleosomes, which are methylated on lysine 9 of histone H3 (H3K9me). Here, we investigate the requirements for establishment, spreading and maintenance of heterochromatin using fission yeast centromeres as a paradigm. We show that establishment of heterochromatin on centromeric repeats is initiated at modular 'nucleation sites' by RNA interference (RNAi), ensuring the mitotic stability of centromere-bearing minichromosomes. We demonstrate that the histone deacetylases Sir2 and Clr3 and the chromodomain protein Swi6(HP1) are required for H3K9me spreading from nucleation sites, thus allowing formation of extended heterochromatin domains. We discovered that RNAi and Sir2 along with Swi6(HP1) operate in two independent pathways to maintain heterochromatin. Finally, we demonstrate that tethering of Sir2 is pivotal to the maintenance of heterochromatin at an ectopic locus in the absence of RNAi. These analyses reveal that Sir2, together with RNAi, are sufficient to ensure heterochromatin integrity and provide evidence for sequential establishment, spreading and maintenance steps in the assembly of centromeric heterochromatin.

Raf1 Is a DCAF for the Rik1 DDB1-like protein and has separable roles in siRNA generation and chromatin modification.

Non-coding transcription can trigger histone post-translational modifications forming specialized chromatin. In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC, composed of Clr4, Raf1, Raf2, Cul4, and Rik1. CLRC mediates H3K9 methylation and siRNA production; it also displays E3-ubiquitin ligase activity in vitro. DCAFs act as substrate receptors for E3 ligases and may couple ubiquitination with histone methylation. Here, structural alignment and mutation of signature WDxR motifs in Raf1 indicate that it is a DCAF for CLRC. We demonstrate that Raf1 promotes H3K9 methylation and siRNA amplification via two distinct, separable functions. The association of the DCAF Raf1 with Cul4-Rik1 is critical for H3K9 methylation, but dispensable for processing of centromeric transcripts into siRNAs. Thus the association of a DCAF, Raf1, with its adaptor, Rik1, is required for histone methylation and to allow RNAi to signal to chromatin.

Building centromeres: home sweet home or a nomadic existence?

Centromere assembly and propagation is governed by genetic and epigenetic mechanisms. A centromere-specific histone H3 variant, CENP-A is strongly favored as the epigenetic mark that specifies centromere identity. Despite the critical importance of centromere function, centromeric sequences are not conserved. This has prompted exploration of other genomic and chromatin features to gain an understanding of where CENP-A is deposited. In this review we highlight recent papers that advance our understanding of how the cell builds a centromere. We focus on what influences the choice of site for CENP-A deposition and therefore the site of centromere formation. We then briefly discuss how centromeres are propagated once the site of centromere assembly is chosen.

Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast.

RNA interference (RNAi) is widespread in eukaryotes and regulates gene expression transcriptionally or post-transcriptionally. In fission yeast, RNAi is tightly coupled to template transcription and chromatin modifications that establish heterochromatin in cis. Exogenous double-stranded RNA (dsRNA) triggers seem to induce heterochromatin formation in trans only when certain silencing proteins are overexpressed. Here, we show that green fluorescent protein (GFP) hairpin dsRNA allows production of high levels of Argonaute-associated small interfering RNAs (siRNAs), which can induce heterochromatin formation at a remote locus. This silencing does not require any manipulation apart from hairpin expression. In cells expressing a ura4(+)-GFP fusion gene, production of GFP siRNAs causes the appearance of ura4 siRNAs from the target gene. Production of these secondary siRNAs depends on RNA-dependent RNA polymerase Rdp1 (RDRP(Rdp1)) function and other RNAi pathway components. This demonstrates that transitivity occurs in fission yeast and implies that RDRP(Rdp1) can synthesize RNA from targeted RNA templates in vivo, generating siRNAs not homologous to the hairpin.

Molecular biology: silencing unlimited.

Heterochromatin domains are essential for normal chromosome functions. The Eri1 ribonuclease is a negative regulator of the RNA interference machinery; recent studies have shown that, in fission yeast lacking Eri1, heterochromatin formation is more promiscuous.

Nuclear pore components are involved in the transcriptional regulation of dosage compensation in Drosophila.

Dosage compensation in Drosophila is dependent on MSL proteins and involves hypertranscription of the male X chromosome, which ensures equal X-linked gene expression in both sexes. Here, we report the purification of enzymatically active MSL complexes from Drosophila embryos, Schneider cells, and human HeLa cells. We find a stable association of the histone H4 lysine 16-specific acetyltransferase MOF with the RNA/protein containing MSL complex as well as with an evolutionary conserved complex. We show that the MSL complex interacts with several components of the nuclear pore, in particular Mtor/TPR and Nup153. Strikingly, knockdown of Mtor or Nup153 results in loss of the typical MSL X-chromosomal staining and dosage compensation in Drosophila male cells but not in female cells. These results reveal an unexpected physical and functional connection between nuclear pore components and chromatin regulation through MSL proteins, highlighting the role of nucleoporins in gene regulation in higher eukaryotes.

X-chromosome targeting and dosage compensation are mediated by distinct domains in MSL-3.

In Drosophila, dosage compensation of X-linked genes is achieved by transcriptional upregulation of the male X chromosome. Genetic and biochemical studies have demonstrated that male-specific lethal (MSL) proteins together with roX RNAs regulate this process. Here, we show that MSL-3 is essential for cell viability and that three domains in the protein have distinct roles in dosage compensation. The chromo-barrel domain (CBD) is not necessary for MSL targeting to the male X chromosome but is important for male viability and equalization of X-linked gene transcription. The polar region cooperates with the CBD in MSL-3 function, whereas the MRG domain is responsible for targeting the protein to the X chromosome. Our results demonstrate that MSL-3 localization to the male X chromosome and transcriptional upregulation of X-linked genes are two separable functions of the MSL-3 protein.

A general precursor ion-like scanning mode on quadrupole-TOF instruments compatible with chromatographic separation.

MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode--implemented on a quadrupole-TOF instrument--that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain.

Structure of the chromo barrel domain from the MOF acetyltransferase.

We report here the structure of the putative chromo domain from MOF, a member of the MYST family of histone acetyltransferases that acetylates histone H4 at Lys-16 and is part of the dosage compensation complex in Drosophila. We found that the structure of this domain is a beta-barrel that is distinct from the alpha + beta fold of the canonical chromo domain. Despite the differences, there are similarities that support an evolutionary relationship between the two domains, and we propose the name "chromo barrel." The chromo barrel domains may be divided into two groups, MSL3-like and MOF-like, on the basis of whether a group of conserved aromatic residues is present or not. The structure suggests that, although the MOF-like domains may have a role in RNA binding, the MSL3-like domains could instead bind methylated residues. The MOF chromo barrel shares a common fold with other chromatin-associated modules, including the MBT-like repeat, Tudor, and PWWP domains. This structural similarity suggests a probable evolutionary pathway from these other modules to the canonical chromo domains (or vice versa) with the chromo barrel domain representing an intermediate structure.