Frequent Changes in the Number of Reiterated Ribosomal RNA Genes Throughout the Life Cycle of the Basidiomycete Coprinus Cinereus

We have examined the stability of the tandemly repeated genes that encode the ribosomal RNA in Coprinus cinereus. These genes are contained within two linked HindIII fragments in a 3.0-Mb chromosome. We monitored the size of these fragments in both mitotic and meiotic segregants using the contour-clamped homogeneous electric field (CHEF) method. No length changes were observed in the smaller HindIII fragment (100 kb; 10 repeats) among the DNAs prepared from 46 asexual spore derivatives (oidia) or 128 meiotic segregants (basidiospores from 32 tetrads). However, the larger HindIII fragment (1100 kb; 120 repeats) did exhibit variability. Substantial changes, involving up to 40% of the larger HindIII fragment were recorded in 7 of 46 oidial isolates (including 4 of 22 transformed derivatives). To learn if the changes were confined to the vegetative portion of the life cycle, we examined transmission of HindIII variants through three crosses. In the first two crosses (16 tetrads total), no changes were observed in the large HindIII fragment. However, in the third cross (16 tetrads), each tetrad showed at least one alteration. In half of the tetrads from the third cross, the altered patterns segregated 2:2, suggesting that the changes occurred after mating but prior to premeiotic DNA replication. We conclude that breakage and rejoining reactions within the rDNA are frequent and are not confined to any particular stage of the life cycle. It also appears that certain repeats are sheltered from these events. Finally, marked differences in rDNA stability were observed in the crosses analyzed

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

INAUGURAL ARTICLE by a Recently Elected Academy Member:Lineage-specific expansions of TET/JBP genes and a new class of DNA transposons shape fungal genomic and epigenetic landscapes

5-Methylcytosine in DNA of eukaryotes, such as humans, is an important epigenetic mark. The recently characterized TET/JBP enzymes generate oxidized derivatives of methylcytosine, such as hydroxy-, formyl-, and carboxymethylcytosine in mammals, which serve as further epigenetic marks or intermediates for demethylation. Unlike animals, which contain one to three TET genes, fungi, such as mushrooms and rusts, display lineage-specific expansions with numerous TET/JBP genes, which are often associated with a unique class of transposable elements. We present evidence that expansion and turnover of these elements and associated TET/JBP genes play important roles in genomic organization, epigenetics, and speciation of fungal lineages, especially basidiomycetes (mushrooms, rusts, and smuts). Domesticated versions of these transposons might also participate in genome rearrangements or repair in humans

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

Abstract: 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

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

A prominent epigenetic mechanism for gene regulation is methylation of cytosine bases in DNA. TET enzymes facilitate DNA demethylation by converting 5-methylcytosine (5mC) to oxidized methylcytosines (oxi-mCs). We show that oxi-mCs are generated by conserved TET/JBP enzymes encoded in the genome of the model organism Coprinopsis cinerea and present a method for simultaneous mapping of the three different species of oxi-mCs at near–base-pair resolution. We observe that centromeres and transposable elements exhibit distinctive patterns of 5mC and oxi-mC, and show that gene body 5mC and oxi-mC mark silent paralogous multicopy genes. Our study describes a method to map three species of oxi-mC simultaneously and reveals the colocation of 5mC and oxi-mC at functional elements throughout the C. cinerea genome

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

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