A Genome-wide Screen for Neurospora crassa Transcription Factors Regulating Glycogen Metabolism

Abstract

Transcription factors play a key role in transcription regulation as they recognize and directly bind to defined sites in promoter regions of target genes, and thus modulate differential expression. The overall process is extremely dynamic, as they have to move through the nucleus and transiently bind to chromatin in order to regulate gene transcription. To identify transcription factors that affect glycogen accumulation in Neurospora crassa, we performed a systematic screen of a deletion strains set generated by the Neurospora Knockout Project and available at the Fungal Genetics Stock Center. In a wild-type strain of N. crassa, glycogen content reaches a maximal level at the end of the exponential growth phase, but upon heat stress the glycogen content rapidly drops. The gene encoding glycogen synthase (gsn) is transcriptionally downregulated when the mycelium is exposed to the same stress condition. We identified 17 deleted strains having glycogen accumulation profiles different from that of the wild-type strain under both normal growth and heat stress conditions. Most of the transcription factors identified were annotated as hypothetical protein, however some of them, such as the PacC, XlnR, and NIT2 proteins, were biochemically well-characterized either in N. crassa or in other fungi. The fungus Neurospora crassa has been widely used as a model organism for the understanding of fundamental aspects of eukaryotic biology. The knowledge of its genome sequence (1) has allowed the identification of proteins required for gene regulation, such as the transcriptional regulatory proteins. An examination of the classes of transcription factors in the N. crassa genome reveals that the organism carries elements shared by simple and complex metazoan models (2). The availability of a set of deletion strains, each carrying a deletion in a specific ORF encoding a transcription factor, allows the screening for genes linked to a particular phenotype. Here we used this mutant strains set to identify transcription factors that either directly or indirectly regulate glycogen metabolism in N. crassa.In many organisms, glycogen is a carbon and energy reserve carbohydrate with an intricate metabolism regulation that senses nutrient availability and other environmental conditions. The amount of glycogen found in a particular situation results from the balance between glycogen synthase and glycogen phosphorylase activities. These enzymes regulate, respectively, the synthesis and degradation of this compound and they are both regulated by phosphorylation. Besides reversible changes in their activities, glycogen levels are also correlated with physiological conditions. In addition, other proteins may also be involved in glycogen accumulation because protein activation resulting from different signaling pathways affects glycogen storage (3,4).In N. crassa, glycogen content reaches a maximal level at the end of the exponential growth phase. However, under stress conditions, such as heat shock, glycogen content drops rapidly (5, 6). The ...

Regulation of Cellulase and Hemicellulase Gene Expression in Fungi

“…pacc gene in N. crassa (ORF NCU00090) is the pacc /RIM101 orthologue, extensively studied in A. nidulans and S. cerevisiae [146]. The transcription factor PacC responds to changes in extracellular pH by activating specific alkaline genes and repressing specific acid genes [147, 148]. …”

“…xlr-1 encodes a member of a TF family containing conserved fungal Zn(2)-Cys(6) binuclear cluster domain with significant amino acid homology to xyr1 in Trichoderma species [148], sharing 57.6% identity with homolog in T. reesei . Recently, Sun et al [143] demonstrated that a deletion of xlr-1 gene abolishes growth of N. crassa in both xylan and xylose containing media, but it slightly affects the growth on Avicel and the production of cellulase activity in the presence of this substrate.…”

“…Another transcription factor identified in N. crassa was NIT2 protein (AreA in A. nidulans ), a member of the GATA factors family, characterized in N. crassa as a positive regulator of genes encoding enzymes for nitrogen source catabolism under nitrogen limiting conditions [149, 150]. Goncalves et al [148] suggested that nit-2 also acts as a repressor of carbon metabolism. They analyzed cis elements present in the gene encoding glycogen synthase ( gsn ) promoter and showed that nit-2 is able to bind these cis elements.…”

The ACEII recombinant Trichoderma reesei QM9414 strains with enhanced xylanase production and its applications in production of xylitol from tree barks

“…The lack of certain regulatory proteins or some key transcription regulator factors causes this difference in target protein expression [51, 52]. Gene expression and regulation studies conducted on filamentous fungi have suggested that, most of the genes encoding for the cellulose and hemicellulose degrading enzymes were majorly regulated during transcriptional stages [53]. …”

Transient Cold Shock Induces Oxidative Stress Events in Antarctic Fungi

“…Trehalose and glycogen accumulation in microbial cells when they are subjected to unfavourable growth conditions could also be used as stress indicators (Parrou et al, 1997;Türkel, 2006;Ocón et al, 2007;Iturriaga et al, 2009;Gonçalves et al, 2011). Both carbohydrates are important storage compounds in fungal vegetative cells and spores (Robinson, 2001;Rúa et al, 2008).…”