Lineage-specific expansions of TET/JBP genes and a new class of DNA transposons shape fungal genomic and epigenetic landscapes.

TET/JBP dioxygenases oxidize methylpyrimidines in nucleic acids and are implicated in generation of epigenetic marks and potential intermediates for DNA demethylation. We show that TET/JBP genes are lineage-specifically expanded in all major clades of basidiomycete fungi, with the majority of copies predicted to encode catalytically active proteins. This pattern differs starkly from the situation in most other organisms that possess just a single or a few copies of the TET/JBP family. In most basidiomycetes, TET/JBP genes are frequently linked to a unique class of transposons, KDZ (Kyakuja, Dileera, and Zisupton) and appear to have dispersed across chromosomes along with them. Several of these elements typically encode additional proteins, including a divergent version of the HMG domain. Analysis of their transposases shows that they contain a previously uncharacterized version of the RNase H fold with multiple distinctive Zn-chelating motifs and a unique insert, which are predicted to play roles in structural stabilization and target sequence recognition, respectively. We reconstruct the complex evolutionary history of TET/JBPs and associated transposons as involving multiple rounds of expansion with concomitant lineage sorting and loss, along with several capture events of TET/JBP genes by different transposon clades. On a few occasions, these TET/JBP genes were also laterally transferred to certain Ascomycota, Glomeromycota, Viridiplantae, and Amoebozoa. One such is an inactive version, calnexin-independence factor 1 (Cif1), from Schizosaccharomyces pombe, which has been implicated in inducing an epigenetically transmitted prion state. We argue that this unique transposon-TET/JBP association is likely to play important roles in speciation during evolution and epigenetic regulation.

Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea.

TET/JBP enzymes oxidize 5-methylpyrimidines in DNA. In mammals, the oxidized methylcytosines (oxi-mCs) function as epigenetic marks and likely intermediates in DNA demethylation. Here we present a method based on diglucosylation of 5-hydroxymethylcytosine (5hmC) to simultaneously map 5hmC, 5-formylcytosine, and 5-carboxylcytosine at near-base-pair resolution. We have used the method to map the distribution of oxi-mC across the genome of Coprinopsis cinerea, a basidiomycete that encodes 47 TET/JBP paralogs in a previously unidentified class of DNA transposons. Like 5-methylcytosine residues from which they are derived, oxi-mC modifications are enriched at centromeres, TET/JBP transposons, and multicopy paralogous genes that are not expressed, but rarely mark genes whose expression changes between two developmental stages. Our study provides evidence for the emergence of an epigenetic regulatory system through recruitment of selfish elements in a eukaryotic lineage, and describes a method to map all three different species of oxi-mCs simultaneously.

5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea.

BACKGROUND: The transition from the vegetative mycelium to the primordium during fruiting body development is the most complex and critical developmental event in the life cycle of many basidiomycete fungi. Understanding the molecular mechanisms underlying this process has long been a goal of research on basidiomycetes. Large scale assessment of the expressed transcriptomes of these developmental stages will facilitate the generation of a more comprehensive picture of the mushroom fruiting process. In this study, we coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development. RESULTS: We evaluated the expression of >3,000 genes in the two respective growth stages and discovered that almost one-third of these genes were preferentially expressed in either stage. This identified a significant turnover of the transcriptome during the course of fruiting body development. Additionally, we annotated more than 79,000 transcription start sites (TSSs) based on the transcriptomes of the mycelium and stage 1 primoridum stages. Patterns of enrichment based on gene annotations from the GO and KEGG databases indicated that various structural and functional protein families were uniquely employed in either stage and that during primordial growth, cellular metabolism is highly up-regulated. Various signaling pathways such as the cAMP-PKA, MAPK and TOR pathways were also identified as up-regulated, consistent with the model that sensing of nutrient levels and the environment are important in this developmental transition. More than 100 up-regulated genes were also found to be unique to mushroom forming basidiomycetes, highlighting the novelty of fruiting body development in the fungal kingdom. CONCLUSIONS: We implicated a wealth of new candidate genes important to early stages of mushroom fruiting development, though their precise molecular functions and biological roles are not yet fully known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea.

Analysis of the Basidiomycete Coprinopsis cinerea reveals conservation of the core meiotic expression program over half a billion years of evolution.

Coprinopsis cinerea (also known as Coprinus cinereus) is a multicellular basidiomycete mushroom particularly suited to the study of meiosis due to its synchronous meiotic development and prolonged prophase. We examined the 15-hour meiotic transcriptional program of C. cinerea, encompassing time points prior to haploid nuclear fusion though tetrad formation, using a 70-mer oligonucleotide microarray. As with other organisms, a large proportion (∼20%) of genes are differentially regulated during this developmental process, with successive waves of transcription apparent in nine transcriptional clusters, including one enriched for meiotic functions. C. cinerea and the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe diverged ∼500-900 million years ago, permitting a comparison of transcriptional programs across a broad evolutionary time scale. Previous studies of S. cerevisiae and S. pombe compared genes that were induced upon entry into meiosis; inclusion of C. cinerea data indicates that meiotic genes are more conserved in their patterns of induction across species than genes not known to be meiotic. In addition, we found that meiotic genes are significantly more conserved in their transcript profiles than genes not known to be meiotic, which indicates a remarkable conservation of the meiotic process across evolutionarily distant organisms. Overall, meiotic function genes are more conserved in both induction and transcript profile than genes not known to be meiotic. However, of 50 meiotic function genes that were co-induced in all three species, 41 transcript profiles were well-correlated in at least two of the three species, but only a single gene (rad50) exhibited coordinated induction and well-correlated transcript profiles in all three species, indicating that co-induction does not necessarily predict correlated expression or vice versa. Differences may reflect differences in meiotic mechanisms or new roles for paralogs. Similarities in induction, transcript profiles, or both, should contribute to gene discovery for orthologs without currently characterized meiotic roles.

Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus).

The mushroom Coprinopsis cinerea is a classic experimental model for multicellular development in fungi because it grows on defined media, completes its life cycle in 2 weeks, produces some 10(8) synchronized meiocytes, and can be manipulated at all stages in development by mutation and transformation. The 37-megabase genome of C. cinerea was sequenced and assembled into 13 chromosomes. Meiotic recombination rates vary greatly along the chromosomes, and retrotransposons are absent in large regions of the genome with low levels of meiotic recombination. Single-copy genes with identifiable orthologs in other basidiomycetes are predominant in low-recombination regions of the chromosome. In contrast, paralogous multicopy genes are found in the highly recombining regions, including a large family of protein kinases (FunK1) unique to multicellular fungi. Analyses of P450 and hydrophobin gene families confirmed that local gene duplications drive the expansions of paralogous copies and the expansions occur in independent lineages of Agaricomycotina fungi. Gene-expression patterns from microarrays were used to dissect the transcriptional program of dikaryon formation (mating). Several members of the FunK1 kinase family are differentially regulated during sexual morphogenesis, and coordinate regulation of adjacent duplications is rare. The genomes of C. cinerea and Laccaria bicolor, a symbiotic basidiomycete, share extensive regions of synteny. The largest syntenic blocks occur in regions with low meiotic recombination rates, no transposable elements, and tight gene spacing, where orthologous single-copy genes are overrepresented. The chromosome assembly of C. cinerea is an essential resource in understanding the evolution of multicellularity in the fungi.

Fluorescent and bimolecular-fluorescent protein tagging of genes at their native loci in Neurospora crassa using specialized double-joint PCR plasmids.

The double-joint polymerase chain reaction (DJ-PCR) is a technique that can be used to construct vectors for targeted genome integration without laborious subcloning steps. Here we report the availability of plasmids that facilitate DJ-PCR-based construction of Neurospora crassa tagging vectors. These plasmids allow the creation of green or red fluorescent protein (GFP or RFP) tagging vectors for protein localization studies, as well as split-yellow fluorescent protein (YFP) tagging vectors for bimolecular fluorescence complementation (BiFC) analyses. We have demonstrated the utility of each plasmid with the tagging of known meiotic silencing proteins. Microscopic analysis of the tagged strains indicates that SMS-2 and QIP form macromolecular complexes in the perinuclear region during meiosis.

Characterization of interactions between and among components of the meiotic silencing by unpaired DNA machinery in Neurospora crassa using bimolecular fluorescence complementation.

Bimolecular fluorescence complementation (BiFC) is based on the complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are united by interactions between proteins covalently linked to them. We have successfully applied BiFC in Neurospora crassa using two genes involved in meiotic silencing by unpaired DNA (MSUD) and observed macromolecular complex formation involving only SAD-1 proteins, only SAD-2 proteins, and mixtures of SAD-1 and SAD-2 proteins.

Meiotic cytogenetics in Coprinus cinereus.

The basidiomycete fungus Coprinus cinereus has naturally synchronous meiosis and is amenable to analysis using an array of well-developed genetic and molecular tools. In this chapter, we explain in detail the two methods most commonly employed for C. cinereus, staining of intact gill segments and chromosome spreads, with an example of the application of each. We describe iron-hematoxylin staining of intact gill segments for the brightfield examination of meiotic progression, and the use of surface spreads and fluorescence in situ hybridization (FISH) to investigate meiotic chromosome pairing. Gill segments can alternatively be stained with DAPI for the determination of meiotic stage, or propidium iodide for the quantitation of nuclear DNA content, and the chromosome fixation and spreading techniques used for FISH are also suitable for immunolocalization studies of chromosomal proteins.

DCL-1 colocalizes with other components of the MSUD machinery and is required for silencing.

In Neurospora, a gene present in an abnormal number of copies is usually a red flag for mischief. One way to deal with these potential intruders is by destroying their transcripts. Widely known as RNA interference (RNAi), this mechanism depends on the "dicing" of a double-stranded RNA intermediate into small-interfering RNA, which in turn guide the degradation of mRNA from the target gene. Quelling is a vegetative silencing system in Neurospora that utilizes such a mechanism. Quelling depends on the redundant activity of two Dicer-like ribonucleases, DCL-1 and DCL-2. Here, we show that Meiotic Silencing by Unpaired DNA (MSUD), a mechanism that silences expression from unpaired DNA during meiosis, requires the dcl-1 (but not the dcl-2) gene for its function. This result suggests that MSUD operates in a similar manner to Quelling and other RNAi systems. DCL-1 colocalizes with SAD-1 (an RdRP), SAD-2, and SMS-2 (an Argonaute) in the perinuclear region.

Introducing student inquiry in large introductory genetics classes.

An appreciation of genetic principles depends upon understanding the individual curiosity that sparked particular investigations, the creativity involved in imagining alternative outcomes and designing experiments to eliminate these outcomes, and the clarity of thought necessary to convince one's scientific peers of the validity of the conclusions. At large research universities, students usually begin their study of genetics in large lecture classes. It is widely assumed that the lecture format, coupled with the pressures to be certain that students become familiar with the principal conclusions of genetics investigations, constrains most if not all departures from the formats textbooks used to explain these conclusions. Here I present several examples of mechanisms to introduce meaningful student inquiry in an introductory genetics course and to evaluate student creative effort. Most of the examples involve altered student preparation prior to class and additional in-class activities, while a few depend upon a smaller recitation section, which accompanies the course from which the examples have been drawn. I conclude that large introductory classes are suitable venues to teach students how to identify scientific claims, determine the evidence that is essential to eliminate alternative conclusions, and convince their peers of the validity of their arguments.

Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea.

The basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.

Novel proteins required for meiotic silencing by unpaired DNA and siRNA generation in Neurospora crassa.

During meiosis in the filamentous fungus Neurospora crassa, unpaired genes are identified and silenced by a process known as meiotic silencing by unpaired DNA (MSUD). Previous work has uncovered six proteins required for MSUD, all of which are also essential for meiotic progression. Additionally, they all localize in the perinuclear region, suggesting that it is a center of MSUD activity. Nevertheless, at least a subset of MSUD proteins must be present inside the nucleus, as unpaired DNA recognition undoubtedly takes place there. In this study, we identified and characterized two new proteins required for MSUD, namely SAD-4 and SAD-5. Both are previously uncharacterized proteins specific to Ascomycetes, with SAD-4 having a range that spans several fungal classes and SAD-5 seemingly restricted to a single order. Both genes appear to be predominantly expressed in the sexual phase, as molecular study combined with analysis of publicly available mRNA-seq datasets failed to detect significant expression of them in the vegetative tissue. SAD-4, like all known MSUD proteins, localizes in the perinuclear region of the meiotic cell. SAD-5, on the other hand, is found in the nucleus (as the first of its kind). Both proteins are unique compared to previously identified MSUD proteins in that neither is required for sexual sporulation. This homozygous-fertile phenotype uncouples MSUD from sexual development and allows us to demonstrate that both SAD-4 and SAD-5 are important for the production of masiRNAs, which are the small RNA molecules associated with meiotic silencing.

SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery.

In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species.

QIP, a protein that converts duplex siRNA into single strands, is required for meiotic silencing by unpaired DNA.

RNA interference (RNAi) depends on the production of small RNA to regulate gene expression in eukaryotes. Two RNAi systems exist to control repetitive selfish elements in Neurospora crassa. Quelling targets transgenes during vegetative growth, whereas meiotic silencing by unpaired DNA (MSUD) silences unpaired genes during meiosis. The two mechanisms require common RNAi proteins, such as RNA-directed RNA polymerases, Dicers, and Argonaute slicers. We have previously demonstrated that, while Quelling depends on the redundant dicer activity of DCL-1 and DCL-2, only DCL-1 is required for MSUD. Here, we show that QDE-2-interacting protein (QIP), an exonuclease that is important for the production of single-stranded siRNA during Quelling, is also required for MSUD. QIP is crucial for sexual development and is shown to colocalize with other MSUD proteins in the perinuclear region.

An expanded family of fungalysin extracellular metallopeptidases of Coprinopsis cinerea.

Proteolytic enzymes, particularly secreted proteases of fungal origin, are among the most important of industrial enzymes, yet the biochemical properties and substrate specificities of these proteins have been difficult to characterize. Genomic sequencing offers a powerful tool to identify potentially novel proteases. The genome of the model basidiomycete Coprinopsis cinereus was found to have an unusually high number of metalloproteases that closely match the M36 peptidase family known as fungalysins. The eight predicted C. cinereus fungalysins divide into two groups upon comparison with fungalysins from other fungi. One member, CcMEP1, is most similar to the single representative fungalysins from the basidiomycetes Phanerochaete chrysosporium, Cryptococcus neoformans, and Ustilago maydis, and to the fungalysin type-protein from Aspergillus fumigatus. The remaining seven C. cinereus predicted fungalysins form a group with similarity to three predicted M36 peptidases of Laccaria bicolor. All eight of the C. cinereus enzymes contain both the signature M36 Pfam domain and the FTP propeptide domain. All contain large propeptides with considerable sequence conservation near a proposed cleavage site. The predicted mature enzymes range in size from 37-46 kDa and have isoelectric points that are mildly acidic to neutral. The proximity of these genes to telomeres and/or to transposable elements may have contributed to the expansion of this gene family in C. cinereus.

VARIATION IN RIBOSOMAL DNA AMONG BIOLOGICAL SPECIES OF ARMILLARIA, A GENUS OF ROOT-INFECTING FUNGI.

Armillaria is a genus of root-infecting fungi composed of several biological species in North America and Europe. To examine relatedness among biological species, ribosomal DNA (rDNA) from one isolate was cloned and rDNAs from 30 isolates were mapped for eight restriction enzymes. The positions of the large (26S) and small (18S) rRNA cistrons were found by Northern hybridizations of total cellular RNA with rDNA subclones and by alignment of maps with conserved restriction sites present in rRNA genes of other fungi. Nine restriction-site and two length polymorphisms were observed. Eight North American (Roman numerals) and five European (species epithets) biological species could be placed in six classes with respect to rDNA maps (rDNA class 1: I and A. ostoyae; class 2: II; class 3: A. borealis; class 4: V, IX, and X; class 5: III, VII, A. lutea, and A. cepistipes; and class 6: VI and A. mellea). Most, but not all, polymorphisms were in intergenic regions.