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

Institute of Chemical Engineering, University of Technology of Vienna, Gumpendorferstrasse 1a, A-1060 Vienna, Austria.
Molecular Microbiology (Impact Factor: 4.42). 05/2012; 84(6):1150-64. DOI: 10.1111/j.1365-2958.2012.08083.x
Source: PubMed


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.

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Available from: Scott Baker, Oct 12, 2015
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    • "In these cases we either employ bead-beating (T. reesei, Karimi-Aghcheh et al., 2013; Seiboth et al., 2012) or grinding under liquid nitrogen followed by native ChIP (L. maculans, Soyer et al., 2014). "
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    ABSTRACT: The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein-DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin, and H3K9me3, which is considered a mark for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen. Copyright © 2015 Elsevier Inc. All rights reserved.
    Fungal Genetics and Biology 04/2015; 79. DOI:10.1016/j.fgb.2015.03.006 · 2.59 Impact Factor
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    • "The deletion of lae1 results in the complete loss of induction in multiple cellulase, b-glucosidases and xylanases, while the overexpression of lae1 results in increased expression. Furthermore, expression of xyr1, encoding the main inducer of lignocellulolytic enzyme-encoding genes, was shown to be LAE1 dependent (Seiboth et al., 2012). Comparative studies of the influence of LaeA on lignocellulolytic enzyme production in Aspergilli and N. crassa have not been undertaken due to the absence of CAZyme gene clusters in the respective genomes. "
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    ABSTRACT: The utilisation of lignocellulosic plant biomass as an abundant, renewable feedstock for green chemistries and biofuel production is inhibited by its recalcitrant nature. In the environment, lignocellulolytic fungi are naturally capable of breaking down plant biomass into utilisable saccharides. Nonetheless, within the industrial context, inefficiencies in the production of lignocellulolytic enzymes impede the implementation of green technologies. One of the primary causes of such inefficiencies is the tight transcriptional control of lignocellulolytic enzymes via carbon catabolite repression. Fungi coordinate metabolism, protein biosynthesis and secretion with cellular energetic status through the detection of intra- and extra-cellular nutritional signals. An enhanced understanding of the signals and signalling pathways involved in regulating the transcription, translation and secretion of lignocellulolytic enzymes is therefore of great biotechnological interest. This comparative review describes how nutrient sensing pathways regulate carbon catabolite repression, metabolism and the utilisation of alternative carbon sources in Saccharomyces cerevisiae and ascomycete fungi.
    Fungal Genetics and Biology 07/2014; 72. DOI:10.1016/j.fgb.2014.06.012 · 2.59 Impact Factor
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    • "In order to allow the ration engineering of new strains of T. reesei with enhance enzyme production levels, a great interest has been to elucidate the molecular mechanisms operating at the transcriptional network that controls the expression of cellulase genes in response to the cognate environmental conditions [6], [10], [11], [12], [13], [14]. These efforts have allowed the identification of many regulatory proteins and signalling pathways that are responsible for the coordination of cellulase expression in this fungus [15], [16], [17], [18], [19], [20]. For some of the enzymes mentioned above, at least three mechanistic steps take place at the promoter regions: chromatin reorganization, de-repression and induction [2], [6]. "
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    ABSTRACT: In this work, we report the in silico identification of the cis-regulatory elements for XYR1 and CRE1 proteins in the filamentous fungus Trichoderma reesei, two regulators that play a central role in the expression of cellulase genes. Using four datasets of condition-dependent genes from RNA-seq and RT-qPCR experiments, we performed unsupervised motif discovery and found two short motifs resembling the proposed binding consensus for XYR1 and CRE1. Using these motifs, we analysed the presence and arrangement of putative cis-regulatory elements recognized by both regulators and found that shortly spaced sites were more associated with XYR1- and CRE1-dependent promoters than single, high-score sites. Furthermore, the approach used here allowed the identification of the previously reported XYR1-binding sites from cel7a and xyn1 promoters, and we also mapped the potential target sequence for this regulator at the cel6a promoter that has been suggested but not identified previously. Additionally, seven other promoters (for cel7b, cel61a, cel61b, cel3c, cel3d, xyn3 and swo genes) presented a putative XYR1-binding site, and strong sites for CRE1 were found at the xyr1 and cel7b promoters. Using the cis-regulatory architectures nearly defined for XYR1 and CRE1, we performed genome-wide identification of potential targets for direct regulation by both proteins and important differences on their functional regulons were elucidated. Finally, we performed binding site mapping on the promoters of differentially expressed genes found in T. reesei mutant strains lacking xyr1 or cre1 and found that indirect regulation plays a key role on their signalling pathways. Taken together, the data provided here sheds new light on the mechanisms for signal integration mediated by XYR1 and CRE1 at cellulase promoters.
    PLoS ONE 06/2014; 9(6):e99366. DOI:10.1371/journal.pone.0099366 · 3.23 Impact Factor
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