FungiExpresZ: an intuitive package for fungal gene expression data analysis, visualization and discovery.

Bioinformatics analysis and visualization of high-throughput gene expression data require extensive computer programming skills, posing a bottleneck for many wet-lab scientists. In this work, we present an intuitive user-friendly platform for gene expression data analysis and visualization called FungiExpresZ. FungiExpresZ aims to help wet-lab scientists with little to no knowledge of computer programming to become self-reliant in bioinformatics analysis and generating publication-ready figures. The platform contains many commonly used data analysis tools and an extensive collection of pre-processed public ribonucleic acid sequencing (RNA-seq) datasets of many fungal species, including important human, plant and insect pathogens. Users may analyse their data alone or in combination with public RNA-seq data for an integrated analysis. The FungiExpresZ platform helps wet-lab scientists to overcome their limitations in genomics data analysis and can be applied to analyse data of any organism. FungiExpresZ is available as an online web-based tool (https://cparsania.shinyapps.io/FungiExpresZ/) and an offline R-Shiny package (https://github.com/cparsania/FungiExpresZ).

Aquila: a spatial omics database and analysis platform.

Spatial omics is a rapidly evolving approach for exploring tissue microenvironment and cellular networks by integrating spatial knowledge with transcript or protein expression information. However, there is a lack of databases for users to access and analyze spatial omics data. To address this limitation, we developed Aquila, a comprehensive platform for managing and analyzing spatial omics data. Aquila contains 107 datasets from 30 diseases, including 6500+ regions of interest, and 15.7 million cells. The database covers studies from spatial transcriptome and proteome analyses, 2D and 3D experiments, and different technologies. Aquila provides visualization of spatial omics data in multiple formats such as spatial cell distribution, spatial expression and co-localization of markers. Aquila also lets users perform many basic and advanced spatial analyses on any dataset. In addition, users can submit their own spatial omics data for visualization and analysis in a safe and secure environment. Finally, Aquila can be installed as an individual app on a desktop and offers the RESTful API service for power users to access the database. Overall, Aquila provides a detailed insight into transcript and protein expression in tissues from a spatial perspective. Aquila is available at https://aquila.cheunglab.org.

Chromatin profiling reveals heterogeneity in clinical isolates of the human pathogen Aspergillus fumigatus.

Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains.

Identification of Anticancer Peptides from the Genome of Candida albicans: in Silico Screening, in Vitro and in Vivo Validations.

Anticancer peptides (ACPs) are promising future therapeutics, but their experimental discovery remains time-consuming and costly. To accelerate the discovery process, we propose a computational screening workflow to identify, filter, and prioritize peptide sequences based on predicted class probability, antitumor activity, and toxicity. The workflow was applied to identify novel ACPs with potent activity against colorectal cancer from the genome sequences of Candida albicans. As a result, four candidates were identified and validated in the HCT116 colon cancer cell line. Among them, PCa1 and PCa2 emerged as the most potent, displaying IC50 values of 3.75 and 56.06 µM, respectively, and demonstrating a 4-fold selectivity for cancer cells over normal cells. In the colon xenograft nude mice model, the administration of both peptides resulted in substantial inhibition of tumor growth without causing significant adverse effects. In conclusion, this work not only contributes a proven computational workflow for ACP discovery but also introduces two peptides, PCa1 and PCa2, as promising candidates poised for further development as targeted therapies for colon cancer. The method as a web service is available at https://app.cbbio.online/acpep/home and the source code at https://github.com/cartercheong/AcPEP_classification.git.

Mediator Undergoes a Compositional Change during Transcriptional Activation.

Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter.

The KdmB-EcoA-RpdA-SntB chromatin complex binds regulatory genes and coordinates fungal development with mycotoxin synthesis.

Chromatin complexes control a vast number of epigenetic developmental processes. Filamentous fungi present an important clade of microbes with poor understanding of underlying epigenetic mechanisms. Here, we describe a chromatin binding complex in the fungus Aspergillus nidulans composing of a H3K4 histone demethylase KdmB, a cohesin acetyltransferase (EcoA), a histone deacetylase (RpdA) and a histone reader/E3 ligase protein (SntB). In vitro and in vivo evidence demonstrate that this KERS complex is assembled from the EcoA-KdmB and SntB-RpdA heterodimers. KdmB and SntB play opposing roles in regulating the cellular levels and stability of EcoA, as KdmB prevents SntB-mediated degradation of EcoA. The KERS complex is recruited to transcription initiation start sites at active core promoters exerting promoter-specific transcriptional effects. Interestingly, deletion of any one of the KERS subunits results in a common negative effect on morphogenesis and production of secondary metabolites, molecules important for niche securement in filamentous fungi. Consequently, the entire mycotoxin sterigmatocystin gene cluster is downregulated and asexual development is reduced in the four KERS mutants. The elucidation of the recruitment of epigenetic regulators to chromatin via the KERS complex provides the first mechanistic, chromatin-based understanding of how development is connected with small molecule synthesis in fungi.

Publisher Correction: Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.

The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.

TFIIH phosphorylation of the Pol II CTD stimulates mediator dissociation from the preinitiation complex and promoter escape.

The transition between transcriptional initiation and elongation by RNA polymerase (Pol) II is associated with phosphorylation of its C-terminal tail (CTD). Depletion of Kin28, the TFIIH subunit that phosphorylates the CTD, does not affect elongation but causes Pol II occupancy profiles to shift upstream in a FACT-independent manner indicative of a defect in promoter escape. Stronger defects in promoter escape are linked to stronger effects on preinitiation complex formation and transcription, suggesting that impairment in promoter escape results in premature dissociation of general factors and Pol II near the promoter. Kin28 has a stronger effect on genes whose transcription is dependent on SAGA as opposed to TFIID. Strikingly, Kin28 depletion causes a dramatic increase in Mediator at the core promoter. These observations suggest that TFIIH phosphorylation of the CTD causes Mediator dissociation, thereby permitting rapid promoter escape of Pol II from the preinitiation complex.

Tuning Hsf1 levels drives distinct fungal morphogenetic programs with depletion impairing Hsp90 function and overexpression expanding the target space.

The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen Candida albicans is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of C. albicans HSF1 induces morphogenesis in the absence of external cues. HSF1 depletion compromises Hsp90 function, thereby driving filamentation. HSF1 overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.

A genome-wide analysis of carbon catabolite repression in Schizosaccharomyces pombe.

BACKGROUND: Optimal glucose metabolism is central to the growth and development of cells. In microbial eukaryotes, carbon catabolite repression (CCR) mediates the preferential utilization of glucose, primarily by repressing alternate carbon source utilization. In fission yeast, CCR is mediated by transcriptional repressors Scr1 and the Tup/Ssn6 complex, with the Rst2 transcription factor important for activation of gluconeogenesis and sexual differentiation genes upon derepression. Through genetic and genome-wide methods, this study aimed to comprehensively characterize CCR in fission yeast by identifying the genes and biological processes that are regulated by Scr1, Tup/Ssn6 and Rst2, the core CCR machinery. RESULTS: The transcriptional response of fission yeast to glucose-sufficient or glucose-deficient growth conditions in wild type and CCR mutant cells was determined by RNA-seq and ChIP-seq. Scr1 was found to regulate genes involved in carbon metabolism, hexose uptake, gluconeogenesis and the TCA cycle. Surprisingly, a role for Scr1 in the suppression of sexual differentiation was also identified, as homothallic scr1 deletion mutants showed ectopic meiosis in carbon and nitrogen rich conditions. ChIP-seq characterised the targets of Tup/Ssn6 and Rst2 identifying regulatory roles within and independent of CCR. Finally, a subset of genes bound by all three factors was identified, implying that regulation of certain loci may be modulated in a competitive fashion between the Scr1, Tup/Ssn6 repressors and the Rst2 activator. CONCLUSIONS: By identifying the genes directly and indirectly regulated by Scr1, Tup/Ssn6 and Rst2, this study comprehensively defined the gene regulatory networks of CCR in fission yeast and revealed the transcriptional complexities governing this system.

Universal plasmids to facilitate gene deletion and gene tagging in filamentous fungi.

Gene manipulation is an important routine technique and its efficiency is often a rate-limiting step in research. To facilitate gene manipulation in filamentous fungi, we adapted the S. cerevisiae Gene Deletion and Gene Tagging plasmid collections to include additional selectable markers that make the useful resources applicable to other fungi. Three markers for auxotrophic selection in Aspergillus and related species (the riboB, pyroA and pyrG genes of Aspergillus fumigatus) and a dominant selectable marker for glufosinate resistance (the Bar gene from Streptomyces hygroscopicus) were introduced to the collections. A total of fifty-six plasmids were constructed for all combinations between the four new markers and thirteen epitope tags (viz., 3xHA, 13xMYC, 3xFLAG, FLAG, MYC, T7, HIS, Strep, S, HSV, VSV-G, V5 and GFP). The selectable marker and epitope tag cassettes are positioned between two universal sequences in the plasmids, and therefore, can be amplified by PCR using the same pair of primers. With these plasmids, we have also established a simple and efficient procedure for making gene deletion and gene tagging transformation DNA constructs. The procedure, along with the universal flanking sequences, allows quick and easy interchange of selectable marker and epitope cassettes in transformation DNA constructs for different selection and/or tagging. To demonstrate utility and efficiency of the system, we simultaneously performed C-terminal tagging of HapB - a subunit of the highly conserved Aspergillus nidulans CCAAT binding complex that plays important transcriptional regulatory roles - using ten different epitopes in order to identify those neutral to HapB function in vivo. It is expected that the expanded plasmid collections coupled with the simple construction strategy would facilitate gene manipulation in many fungal species.

PBrowse: a web-based platform for real-time collaborative exploration of genomic data.

Genome browsers are widely used for individually exploring various types of genomic data. A handful of genome browsers offer limited tools for collaboration among multiple users. Here, we describe PBrowse, an integrated real-time collaborative genome browser that enables multiple users to simultaneously view and access genomic data, thereby harnessing the wisdom of the crowd. PBrowse is based on the Dalliance genome browser and has a re-designed user and data management system with novel collaborative functionalities, including real-time collaborative view, track comment and an integrated group chat feature. Through the Distributed Annotation Server protocol, PBrowse can easily access a wide range of publicly available genomic data, such as the ENCODE data sets. We argue that PBrowse represents a paradigm shift from using a genome browser as a static data visualization tool to a platform that enables real-time human-human interaction and knowledge exchange in a collaborative setting. PBrowse is available at http://pbrowse.victorchang.edu.au, and its source code is available via an open source BSD 3 license at http://github.com/VCCRI/PBrowse.

The Cyc8-Tup1 complex inhibits transcription primarily by masking the activation domain of the recruiting protein.

The yeast Tup1-Cyc8 corepressor complex is recruited to promoters by DNA-binding repressors, but the mechanisms by which it inhibits expression of genes involved in various stress pathways are poorly understood. Conditional and rapid depletion of Tup1 from the nucleus leads to concurrent nucleosome depletion and histone acetylation, recruitment of coactivators (Swi/Snf, SAGA, and Mediator), and increased transcriptional activity. Conversely, coactivator dissociation occurs rapidly upon rerepression by Cyc8-Tup1, although coactivator association and transcription can be blocked even in the absence of nucleosomes. The coactivators are recruited to the sites where Tup1 was located prior to depletion, indicating that the repressor proteins that recruit Tup1 function as activators in its absence. Last, Cyc8-Tup1 can interact with activation domains in vivo. Thus, Cyc8-Tup1 regulates transcription primarily by masking and inhibiting the transcriptional activation domains of the recruiting proteins, not by acting as a corepressor. We suggest that the corepressor function of Cyc8-Tup1 makes only a modest contribution to expression of target genes, specifically to keep expression levels below the nonactivated state.

Dual DNA binding and coactivator functions of Aspergillus nidulans†TamA, a Zn(II)2Cys6 transcription factor.

Transcription factors containing DNA binding domains generally regulate transcription by direct interaction with DNA. For most transcription factors, including the fungal Zn(II)2Cys6 zinc binuclear cluster transcription factors, the DNA binding motif is essential for function. However, Aspergillus nidulans TamA and the related Saccharomyces cerevisiae Dal81p protein contain Zn(II)2Cys6 motifs shown to be dispensable for function. TamA acts at several promoters as a coactivator of the global nitrogen GATA transcription factor AreA. We now show that TamA is the major transcriptional activator of gdhA, encoding the key nitrogen metabolism enzyme NADP-glutamate dehydrogenase. Moreover, activation of gdhA by TamA occurs primarily by a mechanism requiring the TamA DNA binding motif. We show that the TamA DNA binding motif is required for DNA binding of FLAG-epitope-tagged TamA to the gdhA promoter. We identify a conserved promoter element required for TamA activation, and show that TamA and AreA are reciprocally required for full binding at the gdhA promoter under conditions where AreA is inactive at most promoters but active at gdhA. Therefore TamA has dual functions as a DNA-binding transcription factor and a non-DNA-binding coactivator. Dual DNA-binding and coactivator functions provide an additional level of combinatorial control to mediate gene-specific expression.

Multiple nuclear localization signals mediate nuclear localization of the GATA transcription factor AreA.

The Aspergillus nidulans GATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in the Saccharomyces cerevisiae functional orthologs Gln3p and Gat1p, and one putative noncanonical RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed areA mutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs individually or collectively did not affect utilization of nitrogen sources or AreA-dependent gene expression and did not prevent AreA nuclear localization. Mutation of the bipartite NLS conferred the inability to utilize alternative nitrogen sources and abolished AreA-dependent gene expression likely due to effects on DNA binding but did not prevent AreA nuclear localization. Mutation of all six NLSs simultaneously prevented AreA nuclear accumulation. The bipartite NLS alone strongly directed GFP to the nucleus, whereas the classical NLSs collaborated to direct GFP to the nucleus. Therefore, AreA contains multiple conserved NLSs, which show redundancy and together function to mediate nuclear import. The noncanonical bipartite NLS is conserved in GATA factors from Aspergillus, yeast, and mammals, indicating an ancient origin.

Extensive divergence of yeast stress responses through transitions between induced and constitutive activation.

Closely related species show a high degree of differences in gene expression, but the functional significance of these differences remains unclear. Similarly, stress responses in yeast typically involve differential expression of numerous genes, and it is unclear how many of these are functionally significant. To address these issues, we compared the expression programs of four yeast species under different growth conditions, and found that the response of these species to stress has diverged extensively. On an individual gene basis, most transcriptional responses are not conserved in any pair of species, and there are very limited common responses among all four species. We present evidence that many evolutionary changes in stress responses are compensated either (i) by the response of related genes or (ii) by changes in the basal expression levels of the genes whose responses have diverged. Thus, stress-related genes are often induced upon stress in some species but maintain high levels even in the absence of stress at other species, indicating a transition between induced and constitutive activation. In addition, ~15% of the stress responses are specific to only one of the four species, with no evidence for compensating effects or stress-related annotations, and these may reflect fortuitous regulation that is unimportant for the stress response (i.e., biological noise). Frequent compensatory changes and biological noise may explain how diverged expression responses support similar physiological responses.

Pan-drug resistance and hypervirulence in a human fungal pathogen are enabled by mutagenesis induced by mammalian body temperature.

The continuing emergence of invasive fungal pathogens poses an increasing threat to public health. Here, through the China Hospital Invasive Fungal Surveillance Net programme, we identified two independent cases of human infection with a previously undescribed invasive fungal pathogen, Rhodosporidiobolus fluvialis, from a genus in which many species are highly resistant to fluconazole and caspofungin. We demonstrate that R. fluvialis can undergo yeast-to-pseudohyphal transition and that pseudohyphal growth enhances its virulence, revealed by the development of a mouse model. Furthermore, we show that mouse infection or mammalian body temperature induces its mutagenesis, allowing the emergence of hypervirulent mutants favouring pseudohyphal growth. Temperature-induced mutagenesis can also elicit the development of pan-resistance to three of the most commonly used first-line antifungals (fluconazole, caspofungin and amphotericin B) in different Rhodosporidiobolus species. Furthermore, polymyxin B was found to exhibit potent activity against the pan-resistant Rhodosporidiobolus mutants. Collectively, by identifying and characterizing a fungal pathogen in the drug-resistant genus Rhodosporidiobolus, we provide evidence that temperature-dependent mutagenesis can enable the development of pan-drug resistance and hypervirulence in fungi, and support the idea that global warming can promote the evolution of new fungal pathogens.

Reprogramming of carbon metabolism by the transcriptional activators AcuK and AcuM in Aspergillus nidulans.

The ability of fungi to use carbon sources metabolized via the TCA cycle requires gluconeogenesis. In Aspergillus nidulans the AcuK and AcuM transcription factors regulate the expression of the gluconeogenic genes acuF, encoding phosphoenolpyruvate carboxykinase, and acuG, encoding fructose-1,6-bisphosphatase. Expressed proteins containing the AcuK/AcuM N-terminal DNA-binding domains bind together in vitro to motifs containing repeats of CGG separated by seven bases (CCGN7CCG) and the functionality of these sequences was verified in vivo by acuF-lacZ reporter studies. Chromatin immunoprecipitation analysis showed inter-dependent DNA binding of the proteins to the promoters of gluconeogenic genes in vivo independent of the carbon source. Deletion of the mdhC gene encoding a cytoplasmic/peroxisomal malate dehydrogenase showed that this activity is not essential for gluconeogenesis and indicated that induction of AcuK/AcuM regulated genes might result from malate accumulation. Deletion of the gene for the alternative oxidase did not affect growth on gluconeogenic carbon sources; however, expression was absolutely dependent on AcuK and AcuM. Orthologues of AcuK and AcuM, are present in a wide range of fungal taxa and the CCGN7CCG motif is present in the 5' of many genes involved in gluconeogenesis indicating a fundamental role for these transcription factors in reprogramming fungal carbon metabolism.

Reconstitution of human RNA interference in budding yeast.

Although RNA-mediated interference (RNAi) is a widely conserved process among eukaryotes, including many fungi, it is absent from the budding yeast Saccharomyces cerevisiae. Three human proteins, Ago2, Dicer and TRBP, are sufficient for reconstituting the RISC complex in vitro. To examine whether the introduction of human RNAi genes can reconstitute RNAi in S. cerevisiae, genes encoding these three human proteins were introduced into S. cerevisiae. We observed both siRNA and siRNA- and RISC-dependent silencing of the target gene GFP. Thus, human Ago2, Dicer and TRBP can functionally reconstitute human RNAi in S. cerevisiae, in vivo, enabling the study and use of the human RNAi pathway in a facile genetic model organism.