Evelina Y. Basenko

EuPathDB: the eukaryotic pathogen genomics database resource

The Eukaryotic Pathogen Genomics Database Resource (EuPathDB, http://eupathdb.org) is a collection of databases covering 170+ eukaryotic pathogens (protists & fungi), along with relevant free-living and non-pathogenic species, and select pathogen hosts. To facilitate the discovery of meaningful biological relationships, the databases couple preconfigured searches with visualization and analysis tools for comprehensive data mining via intuitive graphical interfaces and APIs. All data are analyzed with the same workflows, including creation of gene orthology profiles, so data are easily compared across data sets, data types and organisms. EuPathDB is updated with numerous new analysis tools, features, data sets and data types. New tools include GO, metabolic pathway and word enrichment analyses plus an online workspace for analysis of personal, non-public, large-scale data. Expanded data content is mostly genomic and functional genomic data while new data types include protein microarray, metabolic pathways, compounds, quantitative proteomics, copy number variation, and polysomal transcriptomics. New features include consistent categorization of searches, data sets and genome browser tracks; redesigned gene pages; effective integration of alternative transcripts; and a EuPathDB Galaxy instance for private analyses of a users data. Forthcoming upgrades include user workspaces for private integration of data with existing EuPathDB data and improved integration and presentation of host–pathogen interactions.

Effects of S6-Strain Mycoplasma gallisepticum Inoculation at 10, 22, or 45 Weeks of Age on the Digestive and Reproductive Organ Characteristics of Commercial Egg-Laying Hens

Experimental inoculation of commercial laying hens with the S6-strain of Mycoplasma gallisepticum (S6MG) at 20 wk of age, while being maintained under ideal conditions, has previously been shown to affect the lengths and weights of various portions of the reproductive tract. Two trials were conducted in the current study to compare the effects of S6MG inoculation prior to lay at 10 wk of age, during onset of lay at 22 wk of age, and during lay at 45 wk of age on the digestive and reproductive organs of commercial layers similarly housed and maintained under ideal conditions. In each trial, liver weight, liver moisture and lipid concentration, incidence of fatty liver hemorrhagic syndrome, ovary weight, ovarian mature follicle numbers, weights and lengths of the oviduct and oviductal regions, and weights and lengths of the small intestine and small intestinal regions were examined at 60 wk of hen age. At 60 wk, liver lipid concentration was depressed, and isthmus weight, as a percentage of total oviduct weight, was increased in birds that had been inoculated with S6MG at 45 wk. Alterations in liver lipid content and weight of the isthmal portion of the oviduct may occur in response to S6MG inoculation during the later stages of production in layers housed under ideal conditions.

Genome-wide redistribution of H3K27me3 is linked to genotoxic stress and defective growth

Significance Regulators of chromatin structure play critical roles in DNA-based processes. Lysine (K) Methyltransferase 1 (KMT1) homologs perform methylation of H3 lysine-9 and are best known for their essential role in heterochromatin formation and transcriptional silencing. Heterochromatin formation is also important for maintenance of genome stability, although the mechanisms are not well understood. We report that altered activity of Polycomb repressive complex-2 (PRC2), a histone lysine-27 methyltransferase complex, is responsible for genotoxic stress, poor growth, and defective development in KMT1-deficient mutants of Neurospora crassa. Mammalian KMT1 and PRC2 are required for development and are frequently mutated in cancer. This work provides information about the cellular consequences of KMT1 and PRC2 deficiency and provides insights into the regulatory and functional relationships of these conserved enzymes. H3K9 methylation directs heterochromatin formation by recruiting multiple heterochromatin protein 1 (HP1)-containing complexes that deacetylate histones and methylate cytosine bases in DNA. In Neurospora crassa, a single H3K9 methyltransferase complex, called the DIM-5,-7,-9, CUL4, DDB1 Complex (DCDC), is required for normal growth and development. DCDC-deficient mutants are hypersensitive to the genotoxic agent methyl methanesulfonate (MMS), but the molecular basis of genotoxic stress is unclear. We found that both the MMS sensitivity and growth phenotypes of DCDC-deficient strains are suppressed by mutation of embryonic ectoderm development or Su-(var)3-9; E(z); Trithorax (set)-7, encoding components of the H3K27 methyltransferase Polycomb repressive complex-2 (PRC2). Trimethylated histone H3K27 (H3K27me3) undergoes genome-wide redistribution to constitutive heterochromatin in DCDC- or HP1-deficient mutants, and introduction of an H3K27 missense mutation is sufficient to rescue phenotypes of DCDC-deficient strains. Accumulation of H3K27me3 in heterochromatin does not compensate for silencing; rather, strains deficient for both DCDC and PRC2 exhibit synthetic sensitivity to the topoisomerase I inhibitor Camptothecin and accumulate γH2A at heterochromatin. Together, these data suggest that PRC2 modulates the response to genotoxic stress.

Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination

Some human cancers maintain their telomeres using the alternative lengthening of telomeres (ALT) mechanism; a process thought to involve recombination. Different types of recombinational telomere elongation pathways have been identified in yeasts. In senescing yeast telomerase deletion (ter1-Δ) mutants with very short telomeres, it has been hypothesized that copying a tiny telomeric circle (t-circle) by a rolling circle mechanism is the key event in telomere elongation. In other cases more closely resembling ALT cells, such as the stn1-M1 mutant of Kluyveromyces lactis, the telomeres appear to be continuously unstable and routinely reach very large sizes. By employing two-dimensional gel electrophoresis and electron microscopy, we show that stn1-M1 cells contain abundant double stranded t-circles ranging from ∼100 to 30 000 bp in size. We also observed small single-stranded t-circles, specifically composed of the G-rich telomeric strand and tailed circles resembling rolling circle replication intermediates. The t-circles most likely arose from recombination events that also resulted in telomere truncations. The findings strengthen the possibility that t-circles contribute to telomere maintenance in stn1-M1 and ALT cells.

Recombination Can either Help Maintain Very Short Telomeres or Generate Longer Telomeres in Yeast Cells with Weak Telomerase Activity

ABSTRACT Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for RAD52 for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles.

FungiDB: An Integrated Bioinformatic Resource for Fungi and Oomycetes.

FungiDB (fungidb.org) is a free online resource for data mining and functional genomics analysis for fungal and oomycete species. FungiDB is part of the Eukaryotic Pathogen Genomics Database Resource (EuPathDB, eupathdb.org) platform that integrates genomic, transcriptomic, proteomic, and phenotypic datasets, and other types of data for pathogenic and nonpathogenic, free-living and parasitic organisms. FungiDB is one of the largest EuPathDB databases containing nearly 100 genomes obtained from GenBank, Aspergillus Genome Database (AspGD), The Broad Institute, Joint Genome Institute (JGI), Ensembl, and other sources. FungiDB offers a user-friendly web interface with embedded bioinformatics tools that support custom in silico experiments that leverage FungiDB-integrated data. In addition, a Galaxy-based workspace enables users to generate custom pipelines for large-scale data analysis (e.g., RNA-Seq, variant calling, etc.). This review provides an introduction to the FungiDB resources and focuses on available features, tools, and queries and how they can be used to mine data across a diverse range of integrated FungiDB datasets and records.

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb)

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species.

The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa

LSH/DDM1 enzymes are required for DNA methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. The filamentous fungus Neurospora crassa is a tractable system that encodes a single LSH/DDM1 homolog (NCU06306). We report that the Neurospora LSH/DDM1 enzyme is encoded by mutagen sensitive-30 (mus-30), a locus identified in a genetic screen over 25 years ago. We show that MUS-30-deficient cells have normal DNA methylation, but are hypersensitive to DNA damaging agents. MUS-30 is a nuclear protein, consistent with its predicted role as a chromatin remodeling enzyme, and levels of MUS-30 are increased following DNA damage. MUS-30 co-purifies with Neurospora WDR76, a homolog of yeast Changed Mutation Rate-1 and mammalian WD40 repeat domain 76. Deletion of wdr76 rescued DNA damage-hypersensitivity of Δmus-30 strains, demonstrating that the MUS-30-WDR76 interaction is functionally important. DNA damage-sensitivity of Δmus-30 is partially suppressed by deletion of methyl adenine glycosylase-1, a component of the base excision repair machinery (BER); however, the rate of BER is not affected in Δmus-30 strains. We found that MUS-30-deficient cells are not defective for DSB repair, and we observed a negative genetic interaction between Δmus-30 and Δmei-3, the Neurospora RAD51 homolog required for homologous recombination. Together, our findings suggest that MUS-30, an LSH/DDM1 homolog, is required to prevent DNA damage arising from toxic base excision repair intermediates. Overall, our study provides important new information about the functions of the LSH/DDM1 family of enzymes.

Histone H1 Limits DNA Methylation in Neurospora crassa

Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in the wild-type and the ΔhH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the ΔhH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa.