Caracterização funcional de fatores de transcrição envolvidos na regulação do metabolismo de glicogênio de Neurospora crassa

Este trabalho descreve a caracterização funcional de alguns fatores de transcrição possivelmente envolvidos na regulação do metabolismo do glicogênio no fungo filamentoso Neurospora crassa. Em uma análise sistemática realizada com 69 linhagens mutantes em genes codificadores para fatores de transcrição, foram identificadas e selecionadas sete linhagens que apresentaram perfis diferentes de acúmulo de glicogênio, em relação à linhagem selvagem, tanto durante o crescimento vegetativo (30°C) como no estresse térmico (45°C). Com o objetivo de verificar o envolvimento dos fatores de transcrição selecionados na expressão dos genes gsn (glicogênio sintase) e gpn (glicogênio fosforilase), experimentos de Northern blot foram realizados e revelaram variações ao nível transcricional em algumas linhagens. A análise por Blast revelou que alguns fatores de transcrição haviam sido previamente estudados em N. crassa (CSP-1, RCO-1, NIT2) ou em outros organismos (PacC, FlbC, Fkh1) e participam na regulação de diferentes processos biológicos. O fator de transcrição PACC e a influência do pH externo sobre a regulação do metabolismo do glicogênio foram investigados mais detalhadamente neste trabalho. Os resultados mostraram que na linhagem selvagem o acúmulo de glicogênio e a expressão do gene gsn foram reprimidos em condições de pH alcalino, enquanto que o gene pacC foi superexpresso nesta condição. A linhagem mutante pacCKO mostrou perder a regulação sobre o acúmulo de glicogênio e expressão do gene gsn, tanto durante o crescimento em pH fisiológico (5,8) como alcalino (7,8). A análise de ligação DNA-proteína mostrou que a proteína PACC de N. crassa recombinante produzida em E. coli foi capaz de se ligar ao motif de DNA para a proteína PACC, presente na região promotora do gene gsn...This work describes the functional characterization of transcription factors likely involved in glycogen metabolism regulation in the filamentous fungus Neurospora crassa. In a systematic analysis performed with 69 strains knocked-out in genes encoding transcription factors, seven strains were identified and selected by presenting profiles of glycogen accumulation different from that existent in the wild-type strain during vegetative growth (30°C) and under heat stress (45°C). In order to verify the involvement of the selected transcription factors in regulation of gsn (codes for glycogen synthase) and gpn (codes for glycogen phosphorylase) genes, Northern blot assays were performed. Differences in gene expression were observed in some strains compared to the wild-type strain. Blast analysis showed that transcription factors have been previously studied either in N. crassa (CSP-1, RCO-1, NIT2) or in other organisms (PacC, FlbC, Fkh1) participating in the regulation of different biological processes. The transcription factor PACC and the influence of the external pH under the regulation of the glycogen metabolism were further investigated. The results showed that in the wild-type strain the glycogen content and the gsn gene expression were repressed under alkaline conditions, while the pacC gene was overexpressed in this condition. The pacCKO strain showed impairments in the glycogen accumulation and gsn gene expression under normal pH (5.8) and alkaline (7.8) conditions. Protein-DNA binding analysis showed that N. crassa PACC recombinant protein produced in E. coli cells was able to bind to the pacC motif present in the gsn promoter. Binding specificity was confirmed by competition assays using an oligonucleotide containing the DNA motif and by binding to a DNA fragment containing the motif mutated by site-directed mutagenesis... (Complete abstract click electronic access below)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Ambient pH Controls Glycogen Levels by Regulating Glycogen Synthase Gene Expression in Neurospora crassa. New Insights into the pH Signaling Pathway

Glycogen is a polysaccharide widely distributed in microorganisms and animal cells and its metabolism is under intricate regulation. Its accumulation in a specific situation results from the balance between glycogen synthase and glycogen phosphorylase activities that control synthesis and degradation, respectively. These enzymes are highly regulated at transcriptional and post-translational levels. The existence of a DNA motif for the Aspergillus nidulans pH responsive transcription factor PacC in the promoter of the gene encoding glycogen synthase (gsn) in Neurospora crassa prompted us to investigate whether this transcription factor regulates glycogen accumulation. Transcription factors such as PacC in A. nidulans and Rim101p in Saccharomyces cerevisiae play a role in the signaling pathway that mediates adaptation to ambient pH by inducing the expression of alkaline genes and repressing acidic genes. We showed here that at pH 7.8 pacC was overexpressed and gsn was down-regulated in wild-type N. crassa coinciding with low glycogen accumulation. In the pacC(KO) strain the glycogen levels and gsn expression at alkaline pH were, respectively, similar to and higher than the wild-type strain at normal pH (5.8). These results characterize gsn as an acidic gene and suggest a regulatory role for PACC in gsn expression. The truncated recombinant protein, containing the DNA-binding domain specifically bound to a gsn DNA fragment containing the PacC motif. DNA-protein complexes were observed with extracts from cells grown at normal and alkaline pH and confirmed by ChIP-PCR analysis. The PACC present in these extracts showed equal molecular mass, indicating that the protein is already processed at normal pH, in contrast to A. nidulans. Together, these results show that the pH signaling pathway controls glycogen accumulation by regulating gsn expression and suggest the existence of a different mechanism for PACC activation in N. crassa.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways

Abstract Background Glycogen and trehalose are storage carbohydrates and their levels in microorganisms vary according to environmental conditions. In Neurospora crassa, alkaline pH stress highly influences glycogen levels, and in Saccharomyces cerevisiae, the response to pH stress also involves the calcineurin signaling pathway mediated by the Crz1 transcription factor. Recently, in yeast, pH stress response genes were identified as targets of Crz1 including genes involved in glycogen and trehalose metabolism. In this work, we present evidence that in N. crassa the glycogen and trehalose metabolism is modulated by alkaline pH and calcium stresses. Results We demonstrated that the pH signaling pathway in N. crassa controls the accumulation of the reserve carbohydrates glycogen and trehalose via the PAC-3 transcription factor, which is the central regulator of the signaling pathway. The protein binds to the promoters of most of the genes encoding enzymes of glycogen and trehalose metabolism and regulates their expression. We also demonstrated that the reserve carbohydrate levels and gene expression are both modulated under calcium stress and that the response to calcium stress may involve the concerted action of PAC-3. Calcium activates growth of the Δpac-3 strain and influences its glycogen and trehalose accumulation. In addition, calcium stress differently regulates glycogen and trehalose metabolism in the mutant strain compared to the wild-type strain. While glycogen levels are decreased in both strains, the trehalose levels are significantly increased in the wild-type strain and not affected by calcium in the mutant strain when compared to mycelium not exposed to calcium. Conclusions We previously reported the role of PAC-3 as a transcription factor involved in glycogen metabolism regulation by controlling the expression of the gsn gene, which encodes an enzyme of glycogen synthesis. In this work, we extended the investigation by studying in greater detail the effects of pH on the metabolism of the reserve carbohydrate glycogen and trehalose. We also demonstrated that calcium stress affects the reserve carbohydrate levels and the response to calcium stress may require PAC-3. Considering that the reserve carbohydrate metabolism may be subjected to different signaling pathways control, our data contribute to the understanding of the N. crassa responses under pH and calcium stresses

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

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 down-regulated 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 identification of some of the transcription factors was coincident with the presence of DNA-binding motifs specific for the transcription factors in the gsn 5'-flanking region, and some of these DNA-binding motifs were demonstrated to be functional by Electrophoretic Mobility Shift Assay (EMSA) experiments. Strains knocked-out in these transcription factors presented impairment in the regulation of gsn expression, suggesting that the transcription factors regulate glycogen accumulation by directly regulating gsn gene expression. Five selected mutant strains showed defects in cell cycle progression, and two transcription factors were light-regulated. The results indicate that there are connections linking different cellular processes, such as metabolism control, biological clock, and cell cycle progression. Molecular & Cellular Proteomics 10: 10.1074/mcp.M111.007963, 1-13, 2011.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

<i>pacC</i> gene knockout and phenotypic analysis of the mutant strain.

<p>(A) Schematic illustration of the <i>pacC</i> gene knockout strategy. (B) Diagnostic PCR for validation of the <i>pacC</i> knockout was performed by using 0090-F1 and 0090-R4 oligonucleotides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044258#pone-0044258-t001" target="_blank">Table 1</a>). (C) Linear growth analysis. Apical extension of basal hyphae was determined in race tubes, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044258#s4" target="_blank">Material and Methods</a>. The results shown are the average of three independent experiments. (D) Melanization. Strains were cultured in 250 mL flasks containing VM medium for 10 days (3 days at 30°C in the dark and 7 days at room temperature in ambient light/dark). Melanization can be visualized as brown pigment formation in the <i>pacC^KO</i> strain. (E) Radial growth analysis. Basal hyphae growth was examined after cultivating the strains on plates containing solid VM medium, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044258#s4" target="_blank">Material and Methods</a>. Growth was expressed as colony diameter.</p

PACC binds specifically to the <i>gsn</i> promoter region in an alkaline pH-independent manner.

<p>(A) Gel shift analysis using crude cellular extracts fractionated on a Heparin-Sepharose column. Crude cellular extracts (CCE) from mycelia submitted or not to alkaline pH stress (pH 7.8) were fractionated by affinity chromatography. Left panel, a protein fraction (35 µg) exhibiting DNA-binding activity was assayed in the presence of specific competitors. Lane 1, <i>pacC</i> probe, no protein added. Lanes 2 and 7, proteins from pH 5.8 and pH 7.8 samples, respectively, in the absence of competitors. Lanes 3 and 8, DNA band shift in the presence of the 146 bp <i>pacC</i> specific competitor. Lanes 4 to 6 and 9 to 11, DNA band shifts in the presence of increasing amounts of the 27 bp DNA oligo <i>pacC</i> as a specific competitor. Right panel, a protein fraction (35 µg) from knocked-out strain crude cellular extract was assayed. Lane 12, <i>pacC</i> probe, no protein added. O, gel origin; SC, specific competitor; FP, free probe. (B) PACC shows the same molecular mass at pH 5.8 and pH 7.8. Crude cellular extracts prepared from wild-type <i>N. crassa</i> submitted or not to alkaline pH stress were analyzed by Western blotting using a polyclonal anti-PACC antibody. The protein α-tubulin (theoretical molecular mass 50 kD) was used as a loading control. (C) Chromatin immunoprecipitation assay using the polyclonal anti-PACC antibody. Genomic DNA samples from wild-type <i>N. crassa</i> submitted or not to pH stress were immunoprecipitated with the anti-PACC antibody and subjected to PCR to amplify a 146 bp DNA fragment of the <i>gsn</i> promoter containing the <i>pacC</i> motif. A plasmid construction containing the entire sequence of the <i>gsn</i> gene, including its 5′- and 3′-flanking regions, was used as a positive control. As a negative control, the immunoprecipitation reactions were performed without the anti-PACC antibody. L, 1 kb DNA ladder.</p

<i>gsn, gpn</i> and <i>pacC</i> gene expression during acid and alkaline pH stress.

<p>Cells from the wild-type and <i>pacC^KO</i> strains were cultivated at pH 5.8 for 24 h and shifted to pH 4.2 and pH 7.8. Samples were collected and used to extract total RNA. Total RNA (15 µg) was separated by electrophoresis in a denaturing formaldehyde gel, transferred to nylon membrane and probed with α-³²P-radiolabeled 678 bp <i>gsn</i> cDNA, or 798 bp <i>gpn</i> cDNA or 639 bp <i>pacC</i> cDNA fragments (gel autoradiographies). The 28 S rRNA was used as a loading control after ethidium bromide staining. The results shown are the average of at least three independent experiments. (A) Analysis of the <i>gsn, gpn</i> and <i>pacC</i> genes in the wild-type strain at different times after pH shifting. After pH stress the remaining cultures were transferred back to physiological conditions (RE, recuperation, pH 5.8) and samples were collected. (B) Analysis of the <i>gsn</i> and <i>gpn</i> genes in the <i>pacC^KO</i> strain compared to the wild-type strain at different times after pH shifting. 0, cell samples before pH shifting (control).</p

Glycogen accumulation and gene expression during combined pH and heat shock stress.

<p>Glycogen and total RNA were extracted from wild-type mycelia cultivated at pH 5.8 and 30°C for 24 h and then shifted to pH 5.8, 4.2 and 7.8 at 45°C for 30 min. After 30 min, the remaining samples were transferred back to the physiological temperature (30°C) at the three pH conditions and incubated for different times (RE, recuperation). (A) Accumulation of glycogen. (B) <i>gsn</i> and <i>pacC</i> gene expression. Total RNA (15 µg) was separated by electrophoresis in a denaturing formaldehyde gel, transferred to nylon membrane and probed with the α-³²P radiolabeled 678 bp <i>gsn</i> cDNA and 639 bp <i>pacC</i> cDNA fragments (gel autoradiographies). The 28 S rRNA was used as a loading control after ethidium bromide staining. The results shown are the average of at least three independent experiments. 0, cell samples before pH shifting (control).</p

Glycogen accumulation during acid and alkaline pH stress.

<p>(A) Glycogen content in the wild-type strain. Glycogen was extracted from mycelia grown under physiological conditions (pH 5.8, control) and under acid (pH 4.2) and alkaline (pH 7.8) stress. After 120 min, the remaining cultures were transferred back to physiological conditions (pH 5.8, RE, recuperation). The results shown are the average of at least three independent experiments. (B) Glycogen content in the <i>pacC^KO</i> strain compared to the wild-type strain. 0, cell samples before the pH shift (control).</p