Kristina M. Smith

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

Transcription Factors in Light and Circadian Clock Signaling Networks Revealed by Genomewide Mapping of Direct Targets for Neurospora White Collar Complex

ABSTRACT Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.

The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β‐glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1‐modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1‐dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high‐throughput sequencing (‘ChIP‐seq’) showed that lae1 expression was not obviously correlated with H3K4 di‐ or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified.

The Fusarium graminearum Histone H3 K27 Methyltransferase KMT6 Regulates Development and Expression of Secondary Metabolite Gene Clusters

The cereal pathogen Fusarium graminearum produces secondary metabolites toxic to humans and animals, yet coordinated transcriptional regulation of gene clusters remains largely a mystery. By chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing. H3K27me3 was found predominantly in regions that lack synteny with other Fusarium species, generally subtelomeric regions. Di- or trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found in mutually exclusive regions of the genome. To find functions for H3K27me3, we deleted the gene for the putative H3K27 methyltransferase, KMT6, a homolog of Drosophila Enhancer of zeste, E(z). The kmt6 mutant lacks H3K27me3, as shown by western blot and ChIP-seq, displays growth defects, is sterile, and constitutively expresses genes for mycotoxins, pigments and other secondary metabolites. Transcriptome analyses showed that 75% of 4,449 silent genes are enriched for H3K27me3. A subset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in kmt6. A largely overlapping set of genes showed increased expression in kmt6. Almost 95% of the remaining 2,720 annotated silent genes showed no enrichment for either H3K27me3 or H3K4me2/3 in kmt6. In these cases mere absence of H3K27me3 was insufficient for expression, which suggests that additional changes are required to activate genes. Taken together, we show that absence of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity factors. Results from this study provide the framework for novel targeted strategies to control the “cryptic genome”, specifically secondary metabolite expression.

Genes Related to Ion-Transport and Energy Production Are Upregulated in Response to CO2-Driven pH Decrease in Corals: New Insights from Transcriptome Analysis

Since the preindustrial era, the average surface ocean pH has declined by 0.1 pH units and is predicted to decline by an additional 0.3 units by the year 2100. Although subtle, this decreasing pH has profound effects on the seawater saturation state of carbonate minerals and is thus predicted to impact on calcifying organisms. Among these are the scleractinian corals, which are the main builders of tropical coral reefs. Several recent studies have evaluated the physiological impact of low pH, particularly in relation to coral growth and calcification. However, very few studies have focused on the impact of low pH at the global molecular level. In this context we investigated global transcriptomic modifications in a scleractinian coral (Pocillopora damicornis) exposed to pH 7.4 compared to pH 8.1during a 3-week period. The RNAseq approach shows that 16% of our transcriptome was affected by the treatment with 6% of upregulations and 10% of downregulations. A more detailed analysis suggests that the downregulations are less coordinated than the upregulations and allowed the identification of several biological functions of interest. In order to better understand the links between these functions and the pH, transcript abundance of 48 candidate genes was quantified by q-RT-PCR (corals exposed at pH 7.2 and 7.8 for 3 weeks). The combined results of these two approaches suggest that pH≥7.4 induces an upregulation of genes coding for proteins involved in calcium and carbonate transport, conversion of CO2 into HCO3 − and organic matrix that may sustain calcification. Concomitantly, genes coding for heterotrophic and autotrophic related proteins are upregulated. This can reflect that low pH may increase the coral energy requirements, leading to an increase of energetic metabolism with the mobilization of energy reserves. In addition, the uncoordinated downregulations measured can reflect a general trade-off mechanism that may enable energy reallocation.

Functional Analyses of Trichoderma reesei LAE1 Reveal Conserved and Contrasting Roles of This Regulator

The putative methyltransferase LaeA is a global regulator that affects the expression of multiple secondary metabolite gene clusters in several fungi, and it can modify heterochromatin structure in Aspergillus nidulans. We have recently shown that the LaeA ortholog of Trichoderma reesei (LAE1), a fungus that is an industrial producer of cellulase and hemicellulase enzymes, regulates the expression of cellulases and polysaccharide hydrolases. To learn more about the function of LAE1 in T. reesei, we assessed the effect of deletion and overexpression of lae1 on genome-wide gene expression. We found that in addition to positively regulating 7 of 17 polyketide or nonribosomal peptide synthases, genes encoding ankyrin-proteins, iron uptake, heterokaryon incompatibility proteins, PTH11-receptors, and oxidases/monoxygenases are major gene categories also regulated by LAE1. chromatin immunoprecipitation sequencing with antibodies against histone modifications known to be associated with transcriptionally active (H3K4me2 and -me3) or silent (H3K9me3) chromatin detected 4089 genes bearing one or more of these methylation marks, of which 75 exhibited a correlation between either H3K4me2 or H3K4me3 and regulation by LAE1. Transformation of a laeA-null mutant of A. nidulans with the T. reesei lae1 gene did not rescue sterigmatocystin formation and further impaired sexual development. LAE1 did not interact with A. nidulans VeA in yeast two-hybrid assays, whereas it interacted with the T. reesei VeA ortholog, VEL1. LAE1 was shown to be required for the expression of vel1, whereas the orthologs of velB and VosA are unaffected by lae1 deletion. Our data show that the biological roles of A. nidulans LaeA and T. reesei LAE1 are much less conserved than hitherto thought. In T. reesei, LAE1 appears predominantly to regulate genes increasing relative fitness in its environment.

Heterochromatin Is Required for Normal Distribution of Neurospora crassa CenH3

ABSTRACT Centromeres serve as platforms for the assembly of kinetochores and are essential for nuclear division. Here we identified Neurospora crassa centromeric DNA by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) of DNA associated with tagged versions of the centromere foundation proteins CenH3 (CENP-A) and CEN-C (CENP-C) and the kinetochore protein CEN-T (CENP-T). On each chromosome we found an ∼150- to 300-kbp region of enrichment for all three proteins. These regions correspond to intervals predicted to be centromeric DNA by genetic mapping and DNA sequence analyses. By ChIP-seq we found extensive colocalization of CenH3, CEN-C, CEN-T, and histone H3K9 trimethylation (H3K9me3). In contrast, H3K4me2, which has been found at the cores of plant, fission yeast, Drosophila, and mammalian centromeres, was not enriched in Neurospora centromeric DNA. DNA methylation was most pronounced at the periphery of centromeric DNA. Mutation of dim-5, which encodes an H3K9 methyltransferase responsible for nearly all H3K9me3, resulted in altered distribution of CenH3-green fluorescent protein (GFP). Similarly, CenH3-GFP distribution was altered in the absence of HP1, the chromodomain protein that binds to H3K9me3. We conclude that eukaryotes with regional centromeres make use of different strategies for maintenance of CenH3 at centromeres, and we suggest a model in which centromere proteins nucleate at the core but require DIM-5 and HP1 for spreading.

Analysis of clock-regulated genes in Neurospora reveals widespread posttranscriptional control of metabolic potential

Significance Circadian clocks regulate gene expression levels to allow an organism to anticipate environmental conditions. These clocks reside in all the major branches of life and confer a competitive advantage to the organisms that maintain them. The clock in the fungus Neurospora crassa is an excellent model for basic understanding of core circadian architecture as well as for filamentous fungi. Here, we identify genes whose expression is clock regulated; indeed, as much as 40% of the transcriptome may be clock regulated, broadly directing daytime catabolism and nighttime growth. Both transcriptional control and posttranscriptional regulation play major roles in control of cycling transcripts such that DNA binding of transcription factors alone appears insufficient to set the phase of circadian transcription. Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation–based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter–luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.

Bulk Segregant Analysis Followed by High-Throughput Sequencing Reveals the Neurospora Cell Cycle Gene, ndc-1, To Be Allelic with the Gene for Ornithine Decarboxylase, spe-1

ABSTRACT With the advent of high-throughput DNA sequencing, it is now straightforward and inexpensive to generate high-density small nucleotide polymorphism (SNP) maps. Here we combined high-throughput sequencing with bulk segregant analysis to expedite mutation mapping. The general map location of a mutation can be identified by a single backcross to a strain enriched in SNPs compared to a standard wild-type strain. Bulk segregant analysis simultaneously increases the likelihood of determining the precise nature of the mutation. We present here a high-density SNP map between Neurospora crassa Mauriceville-1-c (FGSC2225) and OR74A (FGSC2489), the strains most typically used by Neurospora researchers to carry out mapping crosses. We further have demonstrated the utility of the Mauriceville sequence and our approach by mapping the mutation responsible for the only existing temperature-sensitive (ts) cell cycle mutation in Neurospora, nuclear division cycle-1 (ndc-1). The single T-to-C point mutation maps to the gene encoding ornithine decarboxylase (ODC), spe-1 (NCU01271), and changes a Phe to a Ser residue within a highly conserved motif next to the catalytic site of the enzyme. By growth on spermidine and complementation with a wild-type spe-1 gene, we showed that the defect in spe-1 is responsible for the ts ndc-1 mutation. Based on our results, we propose changing ndc-1 to spe-1ndc, which reflects that this mutation results in an ODC with a specific nuclear division defect.

The fungus Neurospora crassa displays telomeric silencing mediated by multiple sirtuins and by methylation of histone H3 lysine 9

BackgroundSilencing of genes inserted near telomeres provides a model to investigate the function of heterochromatin. We initiated a study of telomeric silencing in Neurospora crassa, a fungus that sports DNA methylation, unlike most other organisms in which telomeric silencing has been characterized.ResultsThe selectable marker, hph, was inserted at the subtelomere of Linkage Group VR in an nst-1 (neurospora sir two-1) mutant and was silenced when nst-1 function was restored. We show that NST-1 is an H4-specific histone deacetylase. A second marker, bar, tested at two other subtelomeres, was similarly sensitive to nst-1 function. Mutation of three additional SIR2 homologues, nst-2, nst-3 and nst-5, partially relieved silencing. Two genes showed stronger effects: dim-5, which encodes a histone H3 K9 methyltransferase and hpo, which encodes heterochromatin protein-1. Subtelomeres showed variable, but generally low, levels of DNA methylation. Elimination of DNA methylation caused partial derepression of one telomeric marker. Characterization of histone modifications at subtelomeric regions revealed H3 trimethyl-K9, H3 trimethyl-K27, and H4 trimethyl-K20 enrichment. These modifications were slightly reduced when telomeric silencing was compromised. In contrast, acetylation of histones H3 and H4 increased.ConclusionWe demonstrate the presence of telomeric silencing in Neurospora and show a dependence on histone deacetylases and methylation of histone H3 lysine 9. Our studies also reveal silencing functions for DIM-5 and HP1 that appear independent of their role in de novo DNA methylation.