RCM-1 | Wnt c59

Regulation of glycogen metabolism by the CRE-1, RCO-1 and RCM-1 proteins in Neurospora crassa. The role of CRE-1 as the central transcriptional regulator

Fernanda Barbosa Cupertino 1, Stela Virgilio 1, Fernanda Zanolli Freitas, Thiago de Souza Candido, Maria Célia Bertolini ⇑
Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, UNESP, 14800-060 Araraquara, SP, Brazil

Abstract

The transcription factor CreA/Mig1/CRE-1 is a repressor protein that regulates the use of alternative car- bon sources via a mechanism known as Carbon Catabolite Repression (CCR). In Saccharomyces cerevisiae, Mig1 recruits the complex Ssn6-Tup1, the Neurospora crassa RCM-1 and RCO-1 orthologous proteins, respectively, to bind to promoters of glucose-repressible genes. We have been studying the regulation of glycogen metabolism in N. crassa and the identiﬁcation of the RCO-1 corepressor as a regulator led us to investigate the regulatory role of CRE-1 in this process. Glycogen content is misregulated in the rco-1KO, rcm-1RIP and cre-1KO strains, and the glycogen synthase phosphorylation is decreased in all strains, showing that CRE-1, RCO-1 and RCM-1 proteins are involved in glycogen accumulation and in the regulation of GSN activity by phosphorylation. We also conﬁrmed the regulatory role of CRE-1 in CCR and its nuclear localization under repressing condition in N. crassa. The expression of all glycogenic genes is misregulated in the cre-1KO strain, suggesting that CRE-1 also controls glycogen metabolism by regulating gene expression. The existence of a high number of the Aspergillus nidulans CreA motif (50 -SYGGRG-30 ) in the glycogenic gene promoters led us to analyze the binding of CRE-1 to some DNA motifs both in vitro by DNA gel shift and in vivo by ChIP-qPCR analysis. CRE-1 bound in vivo to all motifs analyzed demonstrating that it down-regulates glycogen metabolism by controlling gene expression and GSN phosphorylation.

1. Introduction

Microorganisms can grow in a variety of environmental condi- tions due to their wide range of adaptative responses that ensure survival and their use of energy-saving mechanisms. Nutrient responses can inﬂuence different regulatory mechanisms, includ- ing those related to the use of carbon sources. In general, ﬁlamen- tous fungi use glucose as their preferred carbon source while the alternative sugar metabolizing enzymes are repressed in a mecha- nism known as Carbon Catabolite Repression (CCR) (Ruijter and Visser, 1997; Vinuselvi et al., 2012). In recent years, numerous studies have demonstrated the importance of CCR mechanism, especially in the secretory control of hydrolytic enzymes by industrial microorganisms such as Trichoderma reesei (Hypocrea jecorina) and Aspergillus species (reviewed in Aro et al., 2005). The production of cellulolytic and xylanolytic enzymes is regulated by glucose through the action of the transcription factor CreA (Aspergillus nidulans), CRE-1 (Neurospora crassa) or CRE1 (T. reesei). This transcription factor is a repressor protein that regulates the transcription of genes related to the use of alternative carbon sources when glucose is present (de Vries et al., 1999; Mach-Aigner et al., 2008; Sun and Glass, 2011).

The action of the C2H2-zinc ﬁnger protein CreA/CRE-1/Mig1 is well conserved among different fungi. CreA/CRE-1 binds to the 50 -SYGGRG-30 motif (Sun and Glass, 2011; Kulmburg et al., 1993) that displays strong identity to the motif recognized by Mig1 (50 -SYGGGG-30 ) (Lundin et al., 1994). However, the regulation of Cre-mediated repression is complex and apparently varies among fungi. Transcriptional and post-transcriptional events regulate A. nidulans CreA function (Strauss et al., 1999) and phosphorylation regulates H. jecorina CRE1 activity (Cziferszky et al., 2002). Although protein kinases that phosphorylate this transcription factor are unknown, the involvement of the AMPK/Snf1 kinase in the regulation by phosphorylation has been demonstrated (Vautard-Mey and Fèvre, 2000; Ostling and Ronne, 1998). This kinase phosphorylates in vitro the yeast Mig1, however the own H. jecorina CRE1 was not phosphorylated by the same kinase (Cziferszky et al., 2003). The cellular compartmentalization of CreA/CRE-1/Mig1 also differs among fungi. Mig1 location was initially reported to be glucose-dependent, being nuclear in condi- tions of CCR and translocating to the cytoplasm under glucose- limiting conditions (De Vit et al., 1997). The A. nidulans GFP-tagged CreA location was demonstrated to be nuclear in the presence of high glucose (Vautard-Mey et al., 1999; Roy et al., 2008; Brown et al., 2013), however the cytoplasmic localization was strongly inﬂuenced by the nature of the derepressing carbon source (Brown et al., 2013). Similar results have been described for the Fusarium oxysporum GFP-Cre1 fusion protein, which showed nuclear localization during growth on ethanol, a derepressing con- dition (Jonkers and Rep, 2009). More recently, the T. reesei CRE1 was described to recycle between nucleus and cytoplasm depend- ing on the carbon source (Lichius et al., 2014).

This transcription factor plays a direct role controlling the expression of a large number of genes encoding cell wall degrading enzymes. In T. reesei and Aspergillus species, CRE1/CreA, respec- tively, regulates the gene expression of cellullases, hemicellullases and xylanases (Ilmén et al., 1997; Orejas et al., 1999; reviewed in Ruijter and Visser, 1997), while in N. crassa, deletion of the cre-1 gene led to an increase in the production of hydrolytic enzymes involved in cellulose degradation (Sun and Glass, 2011). We have been investigating the regulatory mechanisms involved in N. crassa glycogen metabolism and in a screening of a mutant strains set in transcription factors we identiﬁed the corepressor RCO-1 protein (Gonçalves et al., 2011). RCO-1 was ﬁrst identiﬁed in N. crassa by Yamashiro et al. (1996) as a protein that mediates the repression of conidiation and it is orthologous to the Saccharomyces cerevisiae Tup1, a protein component of the Ssn6-Tup1 complex (Keleher et al., 1992). This complex mediates the repression of genes related to different cellular processes, depending on the DNA-binding pro- tein that recruits it to DNA. In yeast, such complex regulates glu- cose-repressible genes in a Mig1-dependent way, a process involving chromatin remodeling and nucleosome compaction (Treitel and Carlson, 1995; reviewed in Smith and Johnson, 2000). In N. crassa, RCM-1 is the S. cerevisiae Ssn6 orthologous protein and, together with RCO-1, may have a role in regulating glucose- repressible genes. Lee and Ebbole (1998) demonstrated the regulation of the N. crassa con-10 gene by RCO-1 in a medium without glucose. More recently, the RCO-1-RCM-1 complex was described to have a role in photoadaptation (Olmedo et al., 2010) and was identiﬁed as a partner of the transcription factor CSP1, a clock-controlled repressor (Sancar et al., 2011). The iden- tiﬁcation of RCO-1 as likely regulating the glycogen metabolism in N. crassa and the high number of CreA motifs (50 -SYGGRG-30 ) in the promoter region of genes codifying for glycogen metabo- lism enzymes prompted us to start investigating the regulatory role of CRE-1, RCO-1 and RCM-1 in the regulation of glycogen metabolism in N. crassa.

In this report, we demonstrate that CRE-1, RCO-1 and RCM-1 proteins regulate glycogen metabolism by a process in which CRE-1 may play a central role since the gene expression of all glycogen enzymes was misregulated in the cre-1KO mutant strain. Gel mobility assays showed that the recombinant GST::CRE-1 recognized and bound speciﬁcally to the gsn and gpn promoters in vitro and ChIP-qPCR analysis conﬁrmed CRE-1 binding to all glycogenic gene promoters. In addition, CRE-1::GFP bound in vivo to its own promoter but was not able to bind to a DNA fragment lacking a CRE-1 motif.

2. Materials and methods

2.1. Neurospora crassa strains and growth conditions

The N. crassa FGSC#9718 (mat a, mus-51::bar), cre-1KO (FGSC#10372), rco-1KO (FGSC#11371) and rcm-1RIP (FGSC#10215) strains were purchased from the Fungal Genetics Stock Center (FGSC) (McCluskey, 2003). The his-3::Pn-cre-1-gfp strain was a gift from N. L. Glass, University of California, Berkeley, CA, USA (Sun and Glass, 2011). All strains were maintained on solid Vogel’s mini- mal (VM) medium, pH 5.8 (Vogel, 1956) containing 2% sucrose. Conidia from 10-day culture were collected, suspended in sterile water and counted. For vegetative growth, 107 conidia/mL or hyphae homogenates (for rco-1KO and rcm-1RIP strains) were ﬁrst germinated in 60 mL of VM medium +2% sucrose at 30 °C, 250 rpm, for 24 h. After this period, cultures were harvested and the mycelia were frozen in liquid nitrogen and stored at 80 °C. For growth in different carbon sources, 109 conidia/mL were ﬁrst germinated in 1 L of VM medium +2% fructose (non-repressing car- bon source) (Ziv et al., 2008) at 30 °C, 250 rpm, for 24 h. After this period, cultures were harvested and the mycelia were divided in four samples: one was frozen in liquid nitrogen and stored at 80 °C for further processing (control sample) and the remaining were transferred into 400 mL of fresh VM medium containing 2% of glucose, or xylose or glycerol. Samples (125 mL) were harvested after incubation for 2, 4, and 8 h and processed as before. To ana- lyze the effect of 2-deoxy-D-glucose (2-DG) in catabolic repression, conidia (107/mL) from wild-type and cre-1KO strains were inocu- lated into 20 mL of VM medium containing 1% sucrose, or glucose or xylose with or without 1 mM 2-DG and incubated at 30 °C, 250 rpm, for 24 h. The mycelia were harvested, ﬁltered and dried at 98 °C for 16 h. The biomass weight was expressed as a percent-age relative to samples grown without 2-DG.

2.2. Glycogen and protein quantiﬁcation

Mycelia pads were ground to a ﬁne powder in a pre-chilled mor- tar in liquid nitrogen and extracted in lysis buffer (50 mM Tris–HCl, pH 7.6, 100 mM NaF, 1 mM EDTA, 1 mM PMSF, 0.1 mM TCLK, 1 mMbenzamidine, and 1 lg/mL each of pepstatin and aprotinin). Cellular extracts were clariﬁed by centrifugation at 10,000g, for 10 min at 4 °C, and the supernatants were used for glycogen and protein quan- tiﬁcations. Glycogen content was measured according to Freitas et al. (2010). Brieﬂy, 100 lL of the crude extract was precipitated with 20% TCA (ﬁnal concentration) and centrifuged (5000g, 10 min, 4 °C). The glycogen in the supernatant was precipitated with 500 lL of 95% cold ethanol, collected by centrifugation, washed twice with 66% ethanol, dried and digested with a-amylase (10 mg/mL) and amyloglucosidase (30 mg/mL). Free glucose was measured using a glucose oxidase kit and the glycogen content was normalized to total protein. Total protein was quantiﬁed by the Hartree (1972) method, using BSA as standard.

2.3. Glycogen synthase activity

The activity of glycogen synthase was determined by [14C]-glu- cose incorporation, as described by Thomas et al. (1968). Brieﬂy, mycelia pads were ground to a ﬁne powder in nitrogen liquid in a pre-chilled mortar and 200 mg of each sample was extracted in 1 mL of lysis buffer (50 mM Tris HCl, pH 7.5, 100 mM NaF, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine, 1 mM b-mercaptoethanol and 1 lg/mL each of aprotinin, pepstatin and TLCK). Cellular extracts were clariﬁed (7000g, 10 min, 4 °C) and total proteins were quantiﬁed (Hartree, 1972) using BSA as standard. To assay glycogen synthase activity, a volume of 15 lL, containing approxi- mately 30 lg of total protein, was added to 30 lL of reaction buffer [50 mM Tris–HCl, pH 7.8, 20 mM EDTA, 25 mM NaF, 0.67% glycogen, 3 mM UDP-[14C]-glucose (1.8 mCi/mmol), with or without 7.2 mM glucose-6-phosphate (G6P)] and incubated at 30 °C during 15 min. After incubation, 75 lL of each reaction were withdrawn, placed on Whatmann 3 MM ﬁlter paper and washed with cold ethanol (70%) under stirring for 15 min. Two additional washes in ethanol (70%) were done, the ﬁrst for 60 min and the second for 15 min. After washing, the ﬁlters containing the reaction pro- duct were dried and radioactivity was quantiﬁed in a LS 6500 Scintillation Counter (Beckman CoulterTM). One unit of glycogen synthase activity was deﬁned as the amount of enzyme that trans- ferred 1 lmol of glucose to glycogen per minute.

2.4. RNA isolation and gene expression analysis

Total RNA was prepared from mycelia samples according to Sokolovsky et al. (1990) method. RNA (10 lg) from each sample was fractionated on a 2.2 M formaldehyde 1.2% (w/v) agarose gel, stained with ethidium bromide and visualized under UV light to assess the rRNAs integrity. Gene expression analysis was per- formed by quantitative PCR (qPCR). RNA samples (10 lg) were treated with RQ1 RNAse-free DNAse (Promega) and subjected to cDNA synthesis by using the SuperScript III First Strand Synthesis kit (Invitrogen) and oligo (dT) primers, according to the manufac- turers’ instructions. qPCR was performed on the StepOnePlus ™Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and speciﬁc primers for glycogen synthase (gsn), glycogen phosphorylase (gpn), glycogenin (gnn), 1,4-a-glucan branching enzyme (gbn), glycogen debranching enzyme (gdn) and b-tubulin (tub-2) mRNA amplicons (Table 1). Five biological replicates were run and the data were analyzed using the StepOne™ Software v2.1 (Applied Biosystems) in the relative quantiﬁcation standard curve method. The ﬂuorescent dye ROX™ was used as a passive reference to nor- malize the SYBR green reporter dye ﬂuorescent signal. All PCR products had melting curves indicating the presence of a single amplicon. The tubulin b chain (tub-2 gene, NCU04054) was used as the endogenous control in all experiments.

2.5. Cellular localization

For microscopy experiments, conidia from the cre-1KO comple- mented strain (his-3::Pn-cre-1-gfp) were inoculated onto a cover- slip and incubated in liquid VM containing 1% sucrose for 16 h at 30 °C. After this time, the cells were transferred to the following media: VM without carbon source, VM plus 1% sucrose and VM plus 1% xylose and incubated for 1 h at 30 °C. Before transferring, the cells were washed in the same transfer media to remove traces of sucrose. After incubation, mycelia on the coverslips were ﬁxed (3.7% formaldehyde, 50 mM NaH2PO4, pH 7.0, 0.2% (v/v) tween 80), washed with phosphate buffered saline (PBS) and stained with 0.5 lg/mL DAPI for 5 min. The mycelia were washed again in PBS and examined in an AXIO Imager.A2 Zeiss microscope. Images were captured with the AxioCam MRm camera and processed using the AxioVision software. Further processing was done using Adobe Photoshop 7.0.

2.6. cre-1 cDNA cloning and production and puriﬁcation of the recombinant protein

The N. crassa cre-1 gene (ORF NCU08807) encodes a 430 amino acid protein with a theoretical molecular mass of 47 kD. The entire cre-1 cDNA fragment (1293 bp) was ampliﬁed from the pYADE5 cDNA plasmid library (Brunelli and Pall, 1993) with the oligonu- cleotides 8807-F and 8807-R (Table 1) and subcloned into the pMOSBlue cloning vector (GE Healthcare) leading to the pMOS- 8807 plasmid. A 1.3 kb BamHI-EcoRI fragment was removed from pMOS-8807 and inserted into the pGEX-4T1 vector (GE Healthcare) resulting in the pGEX-8807 plasmid. For expression of the non- fused GST and the GST::CRE-1 recombinant protein, Escherichia coli Rosetta (DE3) pLysS cells harboring the pGEX-4T1 or pGEX-8807 plasmid constructions were used, respectively. Cells were grown at 37 °C in 1 L of LB medium to an OD600 of 0.7 and induced with IPTG (ﬁnal concentration 0.4 mM) for 4 h at 37 °C and 200 rpm. For puriﬁcation, cells were suspended in buffer A (10 mM NaH2PO4, 1.8 mM KH2PO4, pH 7.3, 140 mM NaCl, 2.7 mM KCl, 1 mM DTT, 1 mM PMSF and 10 mM benzamidine) containing 0.5% Triton X-100 and 0.5% Tween 20 and lysed by sonication (5 cycles of 30 s sonication and 30 s on ice). After centrifugation, the supernatant was subjected to afﬁnity chromatography on a GSTrap HP column (GE Healthcare) using the ÄKTA Prime puriﬁcation system. The recombinant protein was eluted in buffer B (50 mM Tris–HCl, pH 8, 0, 10 mM glutathione) and dialyzed twice against 1 L of dialysis buffer (10 mM Tris–HCl, pH 7.9, 100 mM KCl, 10% v/v glycerol, 1 mM EDTA and 0.5 mM DTT). The puriﬁed pro- tein was analyzed by 10% SDS–PAGE (Laemmli, 1970) followed by Coomassie Brilliant blue staining and quantiﬁed by the Hartree (1972) method using BSA as standard.

2.7. Electrophoretic mobility shift assay (EMSA)

DNA–protein binding reactions were carried out in 30–80 lL of 1 binding buffer (25 mM HEPES-KOH, pH 7.9, 20 mM KCl, 10% v/v glycerol, 1 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, 12.5 mM benza- midine, and 5 lg/mL each of antipain and pepstatin A) containing 2 lg of poly(dI-dC).(dI-dC) as non-speciﬁc competitor and 3–10 lg of either GST or GST::CRE-1 recombinant protein. A radiolabeled DNA probe ( 104 cpm) was added and the reactions were incu- bated at room temperature for 20 min. Free probe was separated from DNA–protein complexes by electrophoresis on a native 5% polyacrylamide gel in 0.5 TBE buffer at 300 V, 10 mA and 10 °C. After electrophoresis, the gel was dried and autoradiographed. For competition assays, an excess of speciﬁc DNA competitor was added to the binding reactions 10 min prior to incubation with the radiolabeled probe.

2.8. DNA probe and competitors for EMSA

A high number of CreA motif (50 -SYGGRG-30 ) was identiﬁed in the 50 -ﬂanking region of the gsn, gpn, gnn, gbn and gdn genes by
in silico analysis. To obtain the gsn and gpn probes, DNA fragments containing the CreA motif were ampliﬁed from genomic DNA using the oligonucleotides described in Table 1, in the presence of [a-32P]-dATP (3000 Ci/mmol). The probes were puriﬁed on a 2% low-melting point agarose gel. The unlabeled probes were used as speciﬁc DNA competitors. A 27 bp DNA oligonucleotide was also used as a competitor after annealing the complementary oligonu- cleotides oligoCRE-1-F and oligoCRE-1-R (Table 1). The speciﬁc competitors were quantiﬁed by measuring the absorbance at 260 nm. For competition assays, the speciﬁc competitors and the dsDNA CRE-1 oligonucleotide were added to the binding reactions in 10–30-fold and 3–10-fold molar excess, respectively.

2.9. ChIP-qPCR analysis

Chromatin immunoprecipitation assays were performed as described by Tamaru et al. (2003) with modiﬁcations. Brieﬂy, coni- dia from the his-3::Pn-cre-1-gfp strain were grown in 125 mL of liquid VM medium containing 2% sucrose at 30 °C, 250 rpm, for 24 h and the chromatin was ﬁxed by adding formaldehyde to 1% ﬁnal concentration, followed by incubation for 30 min at 30 °C and 250 rpm. Formaldehyde was quenched using 125 mM glycine, at 30 °C, 250 rpm, for 10 min. Sonicated chromatin prepared from each sample was pre-cleared with protein A Mag Sepharose (GE Healthcare) pre-blocked with 0.5% BSA in PBS and then immuno- precipitated with anti-GFP antibody (Sigma) and protein A Mag Sepharose. As a negative control, a mock reaction without antibody was run (no Ab). The DNA concentration was quantiﬁed and 25 ng of each reaction: input DNA, no Ab and IPs (immunoprecipitated DNAs with anti-GFP) were analyzed by absolute quantiﬁcation by quantitative PCR (qPCR). qPCR was performed on the StepOnePlus ™Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems) and speciﬁc oligonucleotides for gsn, gpn, gnn, gbn, and gdn pro- moter regions (Table 1). A cre-1 promoter region containing the CreA motif was used as positive control of binding and an ubiquitin region, lacking the motif were used as negative control of binding. All PCR products had melting curves indicating the presence of a single amplicon.

3. Results

3.1. CRE-1, RCO-1, and RCM-1 proteins regulate glycogen accumulation, glycogen synthase activity and expression of genes encoding glycogenic enzymes

We previously screened a set of N. crassa knockout strains in genes encoding transcription factors and identiﬁed the RCO-1 as a protein that regulates glycogen metabolism (Gonçalves et al., 2011). This protein is the S. cerevisiae Tup1 orthologue. In yeast, Tup1 requires Ssn6, the called Ssn6-Tup1 complex, which, together with the Mig1 transcription factor, acts as a repressor of glucose- repressible genes (Smith and Johnson, 2000). Since the N. crassa CRE-1, RCO-1 and RCM-1 proteins correspond to the S. cerevisiae Mig1, Tup1 and Ssn6 orthologous proteins, respectively, we inves- tigated the glycogen metabolism in the corresponding knockout strains. The rcm-1RIP is a mutant strain, in which the gene was par- tially inactivated by RIP (Repeat Induced Point Mutation); the knockout strain is not viable (Olmedo et al., 2010). Glycogen accumulation and gsn gene expression were previously analyzed in a wild-type strain of N. crassa during vegetative growth (30 °C), and the results showed higher levels after 24 h of growth (de Paula et al., 2002). Here, we analyzed the glycogen content, the glycogen synthase phosphorylation (the rate-limiting enzyme in glycogen synthesis) and the expression of genes encoding the glycogenic enzymes glycogenin (gnn), glycogen synthase (gsn), branching enzyme (gbn), glycogen phosphorylase (gpn), and debranching enzyme (gdn) in the cre-1KO, rco-1KO and rcm-1RIP strains after 24 h of growth.

All mutant strains presented signiﬁcant differences in glycogen accumulation compared to the wild-type strain (Fig. 1A). However, whereas the cre-1KO strain showed increased glycogen levels (two times), the rcm-1RIP strain showed a reduction of three times in glycogen content. The rco-1KO strain showed only a slight increase phosphorylation and, then, less active enzyme. All mutant strains displayed higher ratio when compared to the wild-type strain showing that the enzyme is less phosphorylated, and then more active in the mutant strains (Fig. 1B). The relative gene expression was also analyzed for all genes encoding glycogenic enzymes (Fig. 1C). The cre-1KO strain showed misregulation in the expres- sion of all glycogenic genes. The CRE-1 protein appeared to act as a repressor of glycogen synthesis since gsn, gbn and gnn expression (genes encoding enzymes for the glycogen synthesis) was higher in expression (encodes a degradation enzyme) was lower in the same strain, likely contributing to the high glycogen accumulated by the mutant strain (Fig. 1A). The gnn and gpn expression was dramatically increased in the rco-1KO strain, in agreement with previous results on gpn expression in this mutant strain (Bertolini et al., 2012). Finally, the gbn, gpn and gdn expression was not substantially changed in the rcm-1RIP strain and the gene expression results did not explain the levels of the glycogen accumulated by this strain. It is likely that the lower gsn gene expression contributed to the reduced glycogen levels in this mutant strain.

Fig. 1. Glycogen accumulation, glycogen synthase activity and expression of genes encoding glycogen metabolism enzymes under physiological growth. (A) Glycogen accumulation in wild-type and mutant strains. (B) Glycogen synthase activity (—G6P/+G6P ratio) in the same strains. (C) Gene expression analysis of genes gsn (NCU006687), gpn (NCU07027), gnn (NCU06698), gbn (NCU05429) and gdn (NCU00743) in the same strains. The expression of the tubulin tub-2 gene (NCU04054) was used as the endogenous control for all genes. Mycelia were grown at 30 °C for 24 h in VM medium containing 2% sucrose. Results represent the average of 3–5 independent experiments. The asterisks indicate signiﬁcant differences compared to the wild-type strain (T-test), ⁄p < 0.01 and ⁄⁄p < 0.05.

Based on these results, we concluded that the transcription fac- tor CRE-1 could act as a repressor of the glycogen metabolism in N. crassa, likely by regulating the expression of genes encoding glyco- genic enzymes.

3.2. CRE-1 mediates repression of glycogen metabolism under carbon repressing and non-repressing conditions

Since the transcription factor CreA/CRE-1/Mig1 mediates the CCR mechanism in most fungi and CRE-1 regulates glycogen meta- bolism in N. crassa we decided to investigate whether the regulation was dependent on the growth conditions. First, we con- ﬁrmed that CRE-1 protein mediates CCR by growing the wild-type and cre-1KO strains in VM medium containing either glucose or sucrose (for repressing condition) and in VM containing xylose (for non-repressing condition), in the absence and presence of 2-DG (Fig. 2A). This compound is a non-metabolizable glucose ana- log that can be phosphorylated but cannot be further isomerized. The wild-type strain showed reduced growth in xylose medium containing 2-DG while the growth of the mutant strain was unaf- fected by the presence of 2-DG. These results conﬁrmed that, like in other fungi, N. crassa CRE-1 plays an important regulatory role in CCR.
To analyze whether the repressing and non-repressing carbon sources inﬂuenced the regulation of glycogen metabolism by CRE-1 protein, conidia were ﬁrst grown for 24 h in fructose (dere- pressor carbon source) medium and then transferred to glucose (repressor carbon source), xylose or glycerol (non-repressing car- bon sources). The rco-1KO and rcm-1RIP mutant strains were included in this assay in order to assess whether RCO-1 and RCM-1 proteins were required, together with CRE-1, in the reg- ulation. The Fig. 2B shows that the cre-1KO strain accumulated the highest levels of glycogen regardless of the carbon source, indi- cating that CRE-1 negatively regulates glycogen metabolism under repressing and non-repressing growth conditions. However, the glycogen accumulation in the cre-1KO was more pronounced in the presence of fructose and glucose, the preferable carbon source for glycogen accumulation. The glycogen accumulated by the rco-1KO and rcm-1RIP mutant strains followed the same pattern presented in Fig. 1A, with some variations. In general, the rco-1KO strain showed slightly higher levels while the rcm-1RIP strain showed slightly lower levels than those presented by the wild-type strain (p < 0.01). These results suggest that both RCO-1 and RCM-1 proteins may not have a role in the glycogen metabolism repres- sion mediated by CRE-1. Although the cre-1KO strain accumulated high levels of glycogen in the presence of glucose (repressing con- dition), the glycogen levels accumulated in the presence of glycerol (non-repressing condition) were very similar to those of the other mutant strains, mainly to the rco-1KO strain. Finally, comparison of the different growth conditions, it was possible to observe that alternative carbon sources such as those used in this work (xylose or glycerol) are not good substrates for glycogen synthesis.

Fig. 2. Glycogen accumulation under different carbon sources. (A) Effect of 2-deoxy-D-glucose (2-DG) on the growth of wild-type and cre-1KO mutant strains. Mycelia were grown for 24 h at 30 °C in VM medium containing 1% sucrose (left panel), glucose (middle panel) or xylose (right panel) in the absence or presence of 1 mM 2-DG. Mycelial mats were harvested, dried at 98 °C for 16 h and weighed. (B) Glycogen content in wild-type and mutant strains. Mycelia were grown at 30 °C for 24 h in VM medium containing 2% fructose (C, control) and then transferred to VM medium containing 2% glucose, xylose or glycerol. Mycelia were collected 2, 4 and 8 h after transferring. Results represent the average of at least three independent experiments. The asterisks indicate signiﬁcant difference (T-test, p < 0.01) in the presence and absence of 2-DG (A), and differences between the mutants and wild-type strains in the same growth condition (B).

We analyzed the cellular localization of CRE-1 under repressing and derepressing conditions in a CRE-1::GFP complemented strain in which the protein expression is under control of the native pro- moter. For this, conidia were germinated in VM containing sucrose for 16 h and then transferred to medium containing sucrose (repressing condition) and media containing either xylose (non-re- pressing condition) or lacking any carbon source. In cells grown in sucrose and transferred to sucrose, the CRE-1::GFP localization was predominantly nuclear (Fig. 3A and B), as expected for a repressor carbon source such as sucrose. However, after 1 h of transfer to medium containing xylose, CRE-1::GFP was detected in nuclei and the cytoplasm (Fig. 3C). In VM lacking any carbon source (C-free) the protein was localized both in the nuclei and cytoplasm after 1 h of transfer (Fig. 3D) and it was predominantly outside the nuclei after later time of transferring (4 h, Fig. 3E). The results demonstrated that CRE-1 protein is present in both the nucleus and the cytoplasm in derepressed and starved conditions, showing that the CRE-1 partial absence of nucleus may induce derepression of glucose-repressible genes. Although cycling was observed, com- plete absence of CRE-1 from the nucleus seems not be essential for depression in N. crassa. Our results were similar to those described for A. nidulans by Brown et al. (2013) but differed from those reported by Sun and Glass (2011) in N. crassa, although the later experiments were performed in a different way. The authors observed a nuclear CRE-1 localization in agarose medium lacking any carbon source.

3.3. Recombinant GST::CRE-1 binds to gsn and gpn promoters in vitro

The CreA DNA-binding motif was ﬁrst identiﬁed in the A. nidu- lans gene promoters, including the alcR and alcA for ethanol utilization (Kulmburg et al., 1993) as being the consensus sequence 50 -SYGGRG-30 . In N. crassa genome, this sequence is very common in the genome, however, Sun and Glass (2011) described that genes having adjacent motifs in their promoter regions are more likely to be the direct targets of CRE-1. A search for this motif in the pro- moters of the glycogenic genes (gnn, gsn, gbn, gpn, and gdn) revealed many motifs in all 50 -ﬂanking regions, either adjacent or not (Fig. 4). Some of these motifs (shaded boxes), containing either two or three CRE-1 adjacent motifs in different position of the pro- moter regions, were analyzed by DNA shift using the recombinant GST::CRE-1 protein produced in E. coli. First, we assayed the binding reaction in the labeled gsn 234 bp-probe (gsn 2) containing two adjacent motifs (Fig. 5A) located 1578 and 1601 bp from the ATG start codon. Two DNA–protein complexes of different elec- trophoretic mobility were observed using 3 lg of the recombinant protein (Fig. 5A, lane 3), which were lightly reduced in the pres- ence of unlabeled speciﬁc competitor added prior to the probe (Fig. 5A, lane 4). However, the complexes were strongly reduced by adding the 27 bp oligonucleotide cre-1 (Fig. 5A, lanes 5–8), which is a short dsDNA oligonucleotide containing a single 50 - SYGGRG-30 motif (see Fig. 5B, upper panel). The complexes were not observed when the GST protein was added (Fig. 5A, lane 9) demonstrating that the binding complexes were speciﬁc for CRE-1. An interesting result was obtained when only one motif of the same probe was analyzed (gsn 2, 179 bp). A unique DNA–protein complex was visualized (Fig. 5A, lane 12), which was totally removed in the presence of the CRE-1 motif-containing oligonu- cleotide cre-1 (Fig. 5A, lanes 15 and 16). A similar result was observed when the gsn 3 DNA probe, which contains three adjacent motifs (gsn 3, 166 bp) located 228, 293, and 327 bp from the ATG start codon, was used (Fig. 5B). Three DNA–protein complexes exhibiting different molecular masses were visualized (Fig. 5B, lanes 2 and 3), with the lower mass complex showing weak inten- sity and speciﬁcity. The speciﬁcity was probed using the same oligonucleotide cre-1 (Fig. 5B, lane 7) and the GST protein (Fig. 5B, lane 8). The results in Fig. 5A and B showed that N. crassa CRE-1 recognized the same DNA motif as in A. nidulans but did not require adjacent motifs for binding as previously described (Cubero and Scazzocchio, 1994). From these results, we suggest that each DNA–protein complex may correspond to only one DNA motif.

Fig. 3. Subcellular localization of CRE-1::GFP under repressing and derepressing conditions. The his-3::Pn-cre-1-sfgfp strain (6) was grown on VM medium containing 1% sucrose for 16 h (A) and then transferred to medium containing sucrose (repressing condition) (B), or xylose (derepressing condition) (C) or a medium lacking carbon source (D and E). Images were taken after 1 and 4 h of incubation. Fluorescence was evaluated using a Zeiss Microscope at a magniﬁcation of 100×. Results shown represent one of at least two independent experiments.

Fig. 4. Schematic representations of CreA motifs in the 50 -ﬂanking regions of the glycogenic genes. The black dots indicate the position of the A. nidulans CreA motifs (50 - SYGGRG-30 ) identiﬁed in the 50 -ﬂanking region of the gsn (NCU006687), gpn (NCU07027), gnn (NCU06698), gbn (NCU05429), gdn (NCU00743) and cre-1 (NCU08807) genes. The shaded boxes indicate regions that were analyzed by electrophoretic mobility shift assays (EMSA) and the white dashed boxes the regions analyzed by ChIP-qPCR. gsn was the only gene whose transcription initiation site (T) was experimentally determined (Freitas and Bertolini, 2004).

Fig. 5. Binding of recombinant GST::CRE-1 to the gsn promoter. (A) Upper panel, schematic representation of the gsn 2 probes (179 and 234 bp) with the CreA motifs analyzed. Lower panels, gel shift analysis using different concentrations of GST::CRE-1 and two probes in the absence and presence of speciﬁc competitors (SC and oligo cre- 1). Lanes 1 and 10, gsn 2 probe, no protein added. Lane 9, the protein GST was used as a negative control. (B) Upper panel, schematic representation of the gsn 3 probe and the oligo cre-1 with the CreA motifs. Lower panel, gel shift analysis using different concentrations of GST::CRE-1 in the absence and presence of speciﬁc competitors (SC and oligo cre-1). Lane 1, gsn 3 probe, no protein added. Lane 8, the protein GST was used as a negative control. O, gel origin; SC, speciﬁc competitor; FP, free probe. Results shown represent one of at least two independent experiments.

We also investigated whether GST::CRE-1 was able to recognize and bind in vitro to some CreA motifs identiﬁed in the gpn pro- moter (see Fig. 4, shaded boxes). Initially, a 190 bp-probe (gpn 1) containing three motifs located 1984, 2074 and 2090 bp from the start codon was assayed and three DNA–protein complexes were visualized (Fig. 6A, lane 2). The highest and lowest molecular mass complexes were strongly decreased when the unlabeled probe was added as speciﬁc competitor prior to binding (Fig. 6A, lane 3) and the lowest molecular mass complex was completely abolished in the presence of 20-fold molar excess of the DNA oligonucleotide cre-1 (Fig. 6A, lane 6). Interestingly, the inter- mediate complex was not removed by adding these competitors, which suggested that it was a high afﬁnity DNA–protein complex. Fig. 6B shows the binding reaction of the GST::CRE-1 protein to the gpn 3 probe (138 bp) containing two motifs, localized at 262 and 290 bp from the start codon; three complexes were also visual- ized (Fig. 6B, lane 2). Similar to the results described in Fig. 5A and B, formation of the complexes was either decreased or abolished in the presence of the speciﬁc competitors and the GST protein was unable to bind to the probes (Fig. 6A and B, lanes 7). It is noteworthy that the oligonucleotide cre-1, used as a speciﬁc competitor in all DNA-binding reactions, corresponds to a motif present in the gsn promoter (gsn 3), suggesting that the regions surrounding the DNA motif do not strongly inﬂuence the protein binding. In addition, it should be noted that all CRE-1 DNA motifs (50 -SYGGRG-30 ) analyzed in this work corresponded to different nucleotide sequences. An interesting result was the presence of multiple DNA–protein complexes exhibiting different molecular masses for probes having more than one motif. We speculate that they may represent complexes having distinct conformational structures.

Fig. 6. Binding of recombinant GST::CRE-1 to the gpn promoter. (A) Upper panel, schematic representation of the gpn 1 probe with the CreA motifs analyzed. Lower panel, gel shift analysis using 5.0 lg of GST::CRE-1 in the absence and presence of speciﬁc competitors (SC and oligo cre-1). Lane 1, gpn 1 probe, no protein added. Lane 7, the protein GST was used as a negative control. (B) Upper panel, schematic representation of the gpn 3 probe with the CreA motif analyzed. Lower panel, gel shift analysis using 5.0 lg of GST::CRE-1 in the absence and presence of speciﬁc competitors (SC and oligo cre-1). Lane 1, gpn 3 probe, no protein added. Lane 7, the protein GST was used as a negative control. O, gel origin; SC, speciﬁc competitor; FP, free probe. Results shown represent one of at least two independent experiments.

Fig. 7. ChIP-qPCR. Genomic DNA from his-3::Pn-cre-1-gfp strain grown at 30 °C for 24 h in VM medium containing 2% sucrose was immunoprecipitated with anti-GFP antibody and subjected to qPCR by absolute quantiﬁcation to detect direct targets of CRE-1. DNA fragments ampliﬁcation was analyzed in gsn (A), gpn (B), gdn (C), gnn (D), gbn (E) and cre-1 (F) promoters in the genomic DNA. A region inside the coding sequence of the ubiquitin gene was used as negative control of binding (F). The input DNA was used as positive controls of the reactions. As the negative control, the immunoprecipitation reactions were done without antibodies (no Ab). The results represent the average of experimental triplicate in two biological replicates. The asterisks indicate signiﬁcant differences (T-test, p < 0.001).

3.4. CRE-1 binds to all glycogenic gene promoters in vivo

Chromatin immunoprecipitation-qPCR assays were done to conﬁrm the binding in vivo of CRE-1 to the DNA motifs in the glyco- genic genes (Fig. 7). Using ChIP-qPCR we analyzed whether the DNA motifs were indeed the binding targets for CRE-1. In these experiments we used the cre-1KO complemented strain (his-3::Pn- cre-1-gfp) and anti-GFP antibody. Chromatin was obtained from mycelia grown in VM medium containing sucrose (repressing car- bon source), a condition that favors glycogen accumulation in the cre-1KO strain (see Fig. 1A). As a negative control, a mock reaction without antibody was run and the input DNA was used as positive control of the experiments. As previously described, many CreA motifs were identiﬁed in the 50 -ﬂanking regions of the glycogenic genes and some of them were bound in vitro by the recombinant CRE-1 (Figs. 5 and 6).

The ChIP-qPCR was analyzed in gsn (Fig. 7A), gpn (Fig. 7B), gdn (Fig. 7C), gnn (Fig. 7D) and gbn (Fig. 7E) promoters. The cre-1 pro- moter and a region inside the coding sequence of the ubiquitin gene, lacking the motif, were used as positive and negative control of binding, respectively. The Fig. 7A shows that CRE-1 was not able to bind to the single motif located at 2034 bp from the ATG start codon in the gsn1 promoter region. However, CRE-1 bound to gsn2 and gsn3 promoter regions, which possess two and three CreA adjacent motifs, respectively. CRE-1 also bound speciﬁcally to all regions in gpn (Fig. 7B), gdn (Fig. 7C), gnn (Fig. 7D), and gbn (Fig. 7E) promoters. Interestingly, some regions possess a single motif (gnn and gdn promoters) showing that the CRE-1 transcrip- tion factor can also recognize and bind in vivo to one single motif. It is important to observe the high copy number of the gsn3 (Fig. 7A, right panel), which may suggest a major regulatory role of CRE-1 in the expression of this gene. Finally, CRE-1 bound to its own promoter, which contains three adjacent CreA motifs (see Fig. 4) but did not bind to the ubiquitin gene fragment, which does not have any CreA motifs (Fig. 7F) (considering p < 0.001). These results showed that CRE-1 speciﬁcally recognizes and binds to all glycogenic gene promoters in vivo, therefore regulating their expression in the presence of sucrose (repressive condition).

4. Discussion

A screening of N. crassa strains deleted in transcription factors allowed us to identify transcriptional regulators that are likely involved in glycogen metabolism control. Some of the proteins identiﬁed are described in the literature as involved in alternative cellular processes what raised insights concerning the importance of the energy balance provided by glycogen metabolism in differ- ent biological processes (Gonçalves et al., 2011). In this work, we investigated the link between glycogen metabolism and carbon repression in N. crassa. Carbon catabolic repression (CCR) is a mechanism present in many microorganisms and related to the glucose-preferred effect on the metabolism of other carbon sources. It is mediated by the transcription factor Mig1/CreA/ CRE-1, highly conserved among fungal species, and in S. cerevisiae, Mig1 recruits the repressor complex Tup1-Ssn6 to promoters of glucose-repressible genes (Treitel and Carlson, 1995). In this study, we used the cre-1KO strain to assess the regulation of glycogen metabolism and included the strains rco-1KO and rcm-1RIP, which are the RCO-1 and RCM-1 mutant strains, the orthologues of S. cerevisiae Tup1 and Ssn6, respectively. We previously identiﬁed RCO-1 as likely involved in the regulation of glycogen metabolism (Gonçalves et al., 2011) and also observed a severe defect in glyco- gen accumulation by the cre-1KO strain, which suggested that this transcription factor represses glycogen metabolism in N. crassa. In this work, we demonstrated that the repressor activity mediated by CRE-1 was observed under repressing and non-repressing car- bon conditions, indicating that it was not dependent on the exter- nal carbon source. The accumulation of glycogen in cre-1KO strain could result from misregulation in the expression of genes encod- ing glycogenic enzymes. All genes, except gpn (encodes glycogen phosphorylase), were up-regulated in cre-1KO, indicating that loss of CRE-1 caused derepression of these genes. On the other hand, gpn was down-regulated indicating that CRE-1 also plays a role in gene activation, in agreement with Sun and Glass (2011) and similar to the ﬁndings described for this transcription factor in A. nidulans (Mogensen et al., 2006) and T. reesei (Portnoy et al., 2011). In the Sun and Glass (2011) work, the genes gsn and gpn were described as putative targets of CRE-1 regulation.

To analyze the regulatory role of CRE-1 on gene expression we examined the 50 -ﬂanking regions of all glycogenic genes and many CreA DNA-binding motifs (50 -SYGGRG-30 ) were identiﬁed. Some motifs were examined for protein binding in vitro and in vivo based on their positions and number in the genomic region analyzed. DNA shift experiments showed that the E. coli recombinant GST-tagged CRE-1 was able to bind to DNA fragments from the gsn and gpn promoters containing CRE-1 motifs producing DNA– protein complexes with different molecular masses and afﬁnities, depending on the DNA probe. All DNA-binding reactions were speciﬁc since binding was reduced or even abolished when the DNA oligonucleotide cre-1 containing the CRE-1 binding site was used as a speciﬁc competitor. These ﬁndings revealed that CreA binding sites in the promoters analyzed were indeed the target for the binding of recombinant CRE-1. In addition, the nucleotide sequences in the neighborhood may not play an important role in DNA binding since only a single DNA oligonucleotide competed in all the DNA probes analyzed. Our results are not consistent with ﬁndings previously reported in the literature in some aspects. First, the number of complexes formed was independent of the motif orientation; in A. nidulans two divergently oriented sequences, separated by one base pair, are necessary for binding (Cubero and Scazzocchio, 1994). Other important difference was related to the number of motifs required for CRE-1 binding; while some reports indicated a requirement for multiple motifs for binding (Cubero and Scazzocchio, 1994; Sun and Glass, 2011), in our work, CRE-1 was able to bind in vitro and in vivo to only one motif and there seemed to be a correlation between the number of motifs and the number of complexes in the in vitro assays. We observed binding in vivo of CRE-1 in the motifs present in all promoters, with the exception of a region in the gsn promoter, independently of whether a single motif or not. We conclude that under the physio- logical growth conditions analyzed here (VM medium containing 2% sucrose and 24 h of growth) the majority of CreA motifs existent in the glycogenic gene promoters may be functional in vivo. Based on these results we suggest that CRE-1 is a repressor of glycogen synthesis, likely repressing the expression of gsn and gbn genes, that encode enzymes in the glycogen synthesis, and that in its absence the repressor activity is released and glycogen accumulates.

Since in S. cerevisiae Mig1 recruits the complex Tup1/Ssn6 to glucose-repressed promoters (Treitel and Carlson, 1995), we investigated the role of the RCO-1 and RCM-1 proteins in the reg- ulation of the glycogen metabolism. We demonstrated that both proteins regulate the glycogen metabolism by inﬂuencing in the glycogen synthase phosphorylation and in the expression of the genes that encode enzymes of the glycogen metabolism. Taking in consideration the results when repressing and non-repressing conditions were used (Fig. 2B), we conclude that RCO-1 and RCM-1 proteins do not play a regulatory role in the regulation of glycogen metabolism under repressing and non-repressing growth conditions; however, CRE-1 may play a repressor central role in this process. RCO-1 was previously described to have a minor role in CreA-mediated carbon repression in A. nidulans (Hicks et al., 2001; García et al., 2008). We have not determined whether these proteins interact to each other and we cannot preclude the possibility of additional proteins being required for binding. The Tup1-Ssn6 complex has been implicated in the repression of a large number of genes in S. cerevisiae, although neither Tup1 nor Ssn6 binds directly to DNA (reviewed in Parnell and Stillman, 2011). Different mechanisms have been proposed to explain the repressor function of the Tup1-Ssn6 complex, including interaction with histone deacetylases, inhibition of transcriptional activators and modiﬁcation of chromatin structures. However, in a recent study, Wong and Struhl (2011) proposed that the Tup1-Ssn6 com- plex regulates transcription by blocking the activation domains of DNA-binding proteins, thereby preventing their interaction with transcriptional activators rather than by acting as a corepressor. In N. crassa, the RCO-1-RCM-1 complex was identiﬁed to tran- siently interact with the clock-controlled transcriptional repressor CSP1 (Sancar et al., 2011), regulating its kinetics of phospho- rylation and thus its degradation. Interestingly, the authors described the gene encoding glycogen phosphorylase as a putative target of regulation by CSP-1 and RCO-1.

Our ﬁndings show that CRE-1 mediates the repression of glycogen metabolism under carbon repressing and non-repressing con- ditions, however repressing carbon sources such as glucose are preferred to non-repressing carbon sources such as xylose and glycerol for glycogen accumulation. Glucose is metabolized to pyruvate by glycolysis and further metabolism depends on the growth conditions whether aerobic/anaerobic, whereas xylose is metabolized via the pentose phosphate pathway. Different organ- isms, including ﬁlamentous fungi, need to regulate the metabolic reprogramming of their glucose/carbon metabolism, although the molecular mechanisms involved are not always well understood. The regulation of galactose metabolism by the S. cerevisiae gene GAL1 provides a good example for understanding such regulation. In glucose-rich medium, GAL1 is repressed by the Mig1-Tup1- Ssn6 complex (Nehlin et al., 1991), while in galactose-containing medium without glucose, GAL1 transcription is activated by Gal4 that recruits the SAGA complex (Bhaumik and Green, 2001). The repressed/activated states are the consequence of a distinct chro- matin architecture and epigenetic status such that multiple tran- scriptional regulatory proteins must be required, depending on the DNA region. Han and Emr (2011) showed that in S. cerevisiae conversion of the GAL1 promoter from a repressed state to an acti- vated state is dependent on a cytoplasmic component, phos- phatidylinositol 3,5-bisphosphate [PI(3,5)P2] that interacts with Cti6 protein to assemble the activator complex Cti6-Tup1-Ssn6. More recently, the same group showed that this mechanism con- trols the reprogramming from glycolysis to gluconeogenesis (Han and Emr, 2013). The genes FBP1 (encoding fructose-1,6-biphos- phatase) and ICL1 (encoding isocitrate lyase) are under control of Mig1 repressor and the Tup1-Ssn6 corepressor complex and require PI(3,5)P2 for transcriptional activation.

Although the ﬁndings described here represent an important progress in assessing the CRE-1 transcription factor regulating a speciﬁc metabolic process in N. crassa, more studies are required to understand if there is a role of the RCO-1/RCM-1 complex in this process. One model proposed for the repressor activity of Tup1 protein in yeast suggests that, together with Ssn6, they recruit his- tone deacetylases for chromatin remodeling at the promoter (Parnell and Stillman, 2011). Since the complex needs to interact with a DNA-binding protein, it is assumed that large protein com- plexes must be recruited to ensure the repression of target genes. We have previously identiﬁed that a histone acetyltransferase pro- tein binds to some regions of the gsn promoter (Freitas et al., 2008), a ﬁnding that raises interesting questions regarding the role of chromatin architecture in the regulation of this gene. The answer to this question may reveal fundamental aspects of gene regulation in N. crassa glycogen metabolism.

Acknowledgments

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq), Brazil, for grants and fellowships. We deeply thank NL Glass (University of California, Berkeley, CA, USA) for providing the cre-1KO comple- mented strain. We also thank PA de Castro (from GH Goldman’s lab, Faculdade de Ciências Farmacêuticas, USP, Ribeirão Preto, SP, Brazil) and I Malavazi (Universidade Federal de São Carlos, São Carlos, SP, Brazil) for their support on the microscopic analysis.

References

Aro, N., Pakula, T., Penttila, M., 2005. Transcriptional regulation of plant cell wall degradation by ﬁlamentous fungi. FEMS Microbiol. Rev. 29, 719–739.
Bertolini, M.C., Freitas, F.Z., de Paula, R.M., Cupertino, F.B., Gonçalves, R.D., 2012. Glycogen metabolism regulation in Neurospora crassa. In: Witzany, G. (Ed.), Biocommunication of Fungi. Springer, Dordrecht, pp. 39–55.
Bhaumik, S.R., Green, M.R., 2001. SAGA is an essential in vivo target of the yeast acidic activator Gal4p. Genes Dev. 15, 1935–1945.
Brown, N.A., de Gouvea, P.F., Krohn, N.G., Savoldi, M., Goldman, G.H., 2013. Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production. Biotechnol. Biofuels 6, 91. http://dx.doi.org/10.1186/1754-6834-6-91.
Brunelli, J.P., Pall, M.L., 1993. A series of yeast/Escherichia coli lambda expression vectors designed for directional cloning of cDNAs and cre/lox-mediated plasmid excision. Yeast 9, 1309–1318.
Cubero, B., Scazzocchio, C., 1994. Two different, adjacent and divergent zinc ﬁnger binding sites are necessary for CREA-mediated carbon catabolite repression in the proline gene cluster of Aspergillus nidulans. EMBO J. 13, 407–415.
Cziferszky, A., Mach, R.L., Kubicek, C.P., 2002. Phosphorylation positively regulates DNA binding of the carbon catabolite repressor CRE-1 of Hypocrea jecorina (Trichoderma reesei). J. Biol. Chem. 277, 14688–14694.
Cziferszky, A., Seiboth, B., Kubicek, C.P., 2003. The Snf1 kinase of the ﬁlamentous fungus Hypocrea jecorina phosphorylates regulation-relevant serine residues in the yeast carbon catabolite repressor Mig1 but not in the ﬁlamentous fungal counterpart CRE-1. Fungal Genet. Biol. 40, 166–175.
de Paula, R., de Pinho, C.A., Terenzi, H.F., Bertolini, M.C., 2002. Molecular and biochemical characterization of Neurospora crassa glycogen synthase encoded by gsn cDNA. Mol. Genet. Genomics 267, 241–253.
De Vit, M.J., Waddle, J.A., Johnston, M., 1997. Regulated nuclear translocation of the Mig1 glucose repressor. Mol. Biol. Cell 8, 1603–1618.
de Vries, R.P., Visser, J., de Graaff, L.H., 1999. CreA modulates the XlnR-induced expression on xylose of Aspergillus niger genes involved in xylan degradation. Res. Microbiol. 150, 281–285.
Freitas, F.Z., Bertolini, M.C., 2004. Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock. Mol. Gen. Genomics 272, 550–561.
Freitas, F.Z., Chapeaurouge, A., Perales, J., Bertolini, M.C., 2008. A systematic approach to identify STRE-binding proteins of the gsn glycogen synthase gene promoter in Neurospora crassa. Proteomics 8, 2052–2061.
Freitas, F.Z., de Paula, R.M., Barbosa, L.C.B., Terenzi, H.F., Bertolini, M.C., 2010. CAMP signaling pathway controls glycogen metabolism in Neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation. Fungal Genet. Biol. 47, 43–52.
García, I., Mathieu, M., Nikolaev, I., Felenbok, B., Scazzocchio, C., 2008. Roles of the Aspergillus nidulans homologues of Tup1 and Ssn6 in chromatin structure and cell viability. FEMS Microbiol. Lett. 289, 146–154.
Gonçalves, R.D., Cupertino, F.B., Freitas, F.Z., Luchessi, A.D., Bertolini, M.C., 2011. A genome-wide screen for Neurospora crassa transcription factors regulating glycogen metabolism. Mol. Cell. Proteomics 10, 11. http://dx.doi.org/10.1074/ mcp.M111.007963.
Han, B.K., Emr, S.D., 2011. Phosphoinositide [PI(3,5)P2] lipid-dependent regulation of the general transcriptional regulator Tup1. Genes Dev. 25, 984–995.
Han, B.K., Emr, S.D., 2013. The phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)- dependent Tup1 conversion (PIPTC) regulates metabolic reprogramming from glycolysis to gluconeogenesis. J. Biol. Chem. 288, 20633–20645.
Hartree, E.F., 1972. Determination of protein: a modiﬁcation of the Lowry method that gives a linear photometric response. Anal. Biochem. 48, 422–427.
Hicks, J., Lockington, R.A., Strauss, J., Dieringer, D., Kubicek, C.P., Kelly, J., Keller, N., 2001. RcoA has pleiotropic effects on Aspergillus nidulans cellular development. Mol. Microbiol. 39, 1482–1493.
Ilmén, M., Saloheimo, A., Onnela, M.L., Penttilä, M.E., 1997. Regulation of cellulase gene expression in the ﬁlamentous fungus Trichoderma reesei. Appl. Environ. Microbiol. 63, 1298–1306.
Jonkers, W., Rep, M., 2009. Mutation of CRE-1 in Fusarium oxysporum reverts the pathogenicity defects of the FRP1 deletion mutant. Mol. Microbiol. 74, 1100– 1113.
Keleher, C.A., Redd, M.J., Schultz, J., Carlson, M., Johnson, A.D., 1992. Ssn6-Tup1 is a general repressor of transcription in yeast. Cell 68, 709–719.
Kulmburg, P., Mathieu, M., Dowzer, C., Kelly, J., Felenbok, B., 1993. Speciﬁc binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon catabolite repression in Aspergillus nidulans. Mol. Microbiol. 7, 847–857.
Laemmli, U.K., 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lee, K., Ebbole, D., 1998. Tissue-speciﬁc repression of starvation and stress responses of the Neurospora crassa con-10 gene is mediated by RCO-1. Fungal Genet. Biol. 23, 269–278.
Lichius, A., Seidl-Seiboth, V., Seiboth, B., Kubicek, C.P., 2014. Nucleo-cytoplasmic shuttling dynamics of the transcriptional regulators XYR1 and CRE1 under conditions of cellulose and xylanase gene expression in Trichoderma reesei. Mol. Microbiol. http://dx.doi.org/10.1111/mmi.12824.
Lundin, M., Nehlin, J.O., Ronne, H., 1994. Importance of a ﬂanking AT-rich region in target site recognition by the GC box-binding zinc ﬁnger protein MIG1. Mol. Cell. Biol. 14, 1979–1985.
Mach-Aigner, A.R., Pucher, M.E., Steiger, M.G., Bauer, G.E., Preis, S.J., Mach, R.L., 2008. Transcriptional regulation of xyr1, encoding the main regulator of the xylanolytic and cellulolytic enzyme system in Hypocrea jecorina. Appl. Environ. Microbiol. 74, 6554–6562.
McCluskey, K., 2003. The fungal genetics stock center: from molds to molecules.
Adv. Appl. Microbiol. 52, 245–262.
Mogensen, J., Nielsen, H.B., Hofmann, G., Nielsen, J., 2006. Transcription analysis using high-density micro-arrays of Aspergillus nidulans wild-type and creA mutant during growth on glucose or ethanol. Fungal Genet. Biol. 43, 593–603. Nehlin, J.O., Carlberg, M., Ronne, H., 1991. Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose response. EMBO J. 10, 3373–
3377.
Olmedo, M., Navarro-Sampedro, L., Ruger-Herreros, C., Kim, S.R., Jeong, B.K., Lee, B.U., Corrochano, L.M., 2010. A role in the regulation of transcription by light for RCO-1 and RCM-1, the Neurospora homologs of the yeast Tup1-Ssn6 repressor. Fungal Genet. Biol. 47, 939–952.
Orejas, M., MacCabe, A.P., González, J.A.P., Kumar, S., Ramón, D., 1999. Carbon catabolite repression of the Aspergillus nidulans xlnA gene. Mol. Microbiol. 31, 177–184.
Ostling, J., Ronne, H., 1998. Negative control of the Mig1p repressor by Snf1p- dependent phosphorylation in the absence of glucose. Eur. J. Biochem. 252, 162–168.
Parnell, E.J., Stillman, D.J., 2011. Shields up: the Tup1-Cyc8 repressor complex blocks coactivator recruitment. Genes Dev. 25, 2429–2435.
Portnoy, T., Margeot, A., Linke, R., Atanasova, L., Fekete, E., Sándor, E., Hartl, L., Karaffa, L., Druzhinina, I.S., Seiboth, B., Le Crom, S., Kubicek, C.P., 2011. The CRE-
1 carbon catabolite repressor of the fungus Trichoderma reesei: a master regulator of carbon assimilation. BMC Genomics 12, 269–280.
Roy, P., Lockington, R.A., Kelly, J.M., 2008. CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localization or degradation of CreA. Fungal Genet. Biol. 45, 657– 670.
Ruijter, G.J.G., Visser, J., 1997. Carbon repression in Aspergilli. FEMS Microbiol. Lett.
151, 103–114.
Sancar, G., Sancar, C., Brügger, B., Ha, N., Sachsenheimer, T., Gin, E., Wdowik, S., Lohmann, I., Wieland, F., Höfer, T., Diernfellner, A., Brunner, M., 2011. A global circadian repressor controls antiphasic expression of metabolic genes in Neurospora. Mol. Cell 44, 687–697.
Smith, R.L., Johnson, A.D., 2000. Turning genes off by Ssn6-Tup1: a conserved system of transcriptional repression in eukaryotes. Trends Biochem. Sci. 25, 325–330.
Sokolovsky, V., Kaldenhoff, R., Ricci, M., Russo, V.E.A., 1990. Fast and reliable mini- prep RNA extraction from Neurospora crassa. Fungal Genet. Newsl. 37, 41–43.
Strauss, J., Horvath, H.K., Adballah, B.M., Kindermann, J., Mach, R.L., Kubicek, C.P., 1999. The function of CreA, the carbon catabolite repressor of Aspergillus nidulans, is regulated at the transcriptional and post-transcriptional level. Mol. Microbiol. 32, 169–178.
Sun, J., Glass, N.L., 2011. Identiﬁcation of the CRE-1 cellulolytic regulon in
Neurospora crassa. PLoS ONE 6, e25654.
Tamaru, H., Zhang, X., McMillen, D., Singh, P.B., Nakayama, J., Grewal, S.I., David Allis, C., Cheng, X., Selker, E.U., 2003. Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa. Nat. Genet. 34, 75–79.
Thomas, J.A., Schlender, K.K., Larner, J., 1968. A rapid ﬁlter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal. Biochem. 25, 486–499.
Treitel, M.A., Carlson, M., 1995. Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein. Proc. Natl. Acad. Sci. U.S.A. 92, 3132–3136.
Vautard-Mey, G., Fèvre, M., 2000. Mutation of a putative AMPK phosphorylation site abolishes the repressor activity but not the nuclear targeting of the fungal glucose regulator CRE-1. Curr. Genet. 37, 328–332.
Vautard-Mey, G., Cotton, P., Fèvre, M., 1999. Expression and compartmentation of the glucose repressor CRE-1 from the phytopathogenic fungus Sclerotinia sclerotiorum. Eur. J. Biochem. 266, 252–259.
Vinuselvi, P., Kim, M.K., Lee, S.K., Ghim, C.-M., 2012. Rewiring carbon catabolism repression for microbial cell factory. BMB Rep. 45, 59–70.
Vogel, H.J., 1956. A convenient growth medium for Neurospora crassa (medium N).
Microbiol. Genet. Bull. 13, 42–43.
Wong, K.H., Struhl, K., 2011. The Cyc8-Tup1 complex inhibits transcription primarily by masking the activation domain of the recruiting protein. Genes Dev. 25, 2525–2539.
Yamashiro, C.T., Ebbole, D.J., Lee, B.U., Brown, R.E., Bourland, C., Madi, L., Yanofsky, C., 1996. Characterization of rco-1 of Neurospora crassa, a pleiotropic gene affecting growth and development that encodes a homolog of Tup1 of Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 6218–6228.
Ziv, C., Gorovits, R., Yarden, O., 2008. Carbon source affects PKA-dependent polarity of Neurospora crassa in a CRE-1-dependent and independent manner. Fungal Genet. Biol. 45, 103–116.