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Fungal Genetics and Biology 49 (2012) 626–634 Contents lists available at SciVerse ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi Characterization of PIR1, a GATA family transcription factor involved in iron responses in the white-rot fungus Phanerochaete chrysosporium Paulo Canessa, Felipe Muñoz-Guzmán, Rafael Vicuña, Luis F. Larrondo ⇑ Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile a r t i c l e i n f o Article history: Received 6 March 2012 Accepted 26 May 2012 Available online 15 June 2012 Keywords: Phanerochaete chrysosporium Iron Multicopper oxidases Biomass Transcription factor a b s t r a c t Iron, although toxic in excess, is an essential element for biological systems. Therefore, its homeostasis is of critical importance and tight mechanisms participate in its acquisition by microbial organisms. Lately, the relevance of this metal for biomass conversion by wood-degrading fungi has been gaining increasing attention. Iron plays a critical role as cofactor of key enzymes such as lignin and manganese peroxidases in lignin-degrading white-rot fungi, while Fe(II) also serves a pivotal role in Fenton reactions that are central in cellulose depolymerization by brown-rotters. It has been hypothesized that multicopper oxidases with ferroxidase activity might participate in controlling the bioavailability of iron in the hyphal proximity, fine-tuning Fenton chemistry and balancing lignin versus cellulose degradation. In order to further explore the dynamics of iron regulation in the well known white-rot fungus Phanerochaete chrysosporium, we analyzed the mRNA levels of the multicopper oxidases genes (mcos) in response to iron supplementation, confirming down-regulation of their expression in response to this metal. To gain a better understanding on the transcriptional mechanisms mediating this effect, we searched for a gene encoding a GATA-type transcription factor with homology to URBS1, the major transcriptional regulator of iron homeostasis in Ustilago maydis. Due to the limitation of experimental tools in P. chrysosporium, the alleged Phanerochaete iron regulator (PIR1) was studied by complementation of a Neurospora SRE/ URBS1-deficient strain, where phenotypic and molecular characteristics of this transcriptional regulator could be easily assessed. In addition, using a genome-wide in silico strategy, we searched for putative cisacting iron-responsive elements in P. chrysosporium. Some of the identified genes showed reduced transcript levels after 30 min in the presence of the metal, consistent with an SRE/URBS1-mediated mechanism, and suggesting a broad effect of iron on the regulation of several cellular processes. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction The white-rot fungus Phanerochaete chrysosporium possesses an extensive array of extracellular oxidases and peroxidases. These enzymatic activities comprise a highly complex oxidative system thought to be involved in lignin depolymerization, in which the connection between peroxidases and availability of H2O2 has been substantially supported. Besides these enzymes, the overall extracellular oxidative system of P. chrysosporium includes both cation and phenoxyl radicals, cation ions such as Mn2+ and Fe2+/Fe3+ and two distinct, but potentially related enzyme activities, namely multicopper oxidases (MCOs) and cellobiose dehydrogenase (CDH, see below) (Kersten and Cullen, 2006). Blue copper-containing proteins known as MCOs are a widely distributed family of metalloproteins undertaking a diverse range ⇑ Corresponding author. Address: Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile (Mailing). Fax: +56 2 2225515. E-mail address: [email protected] (L.F. Larrondo). 1087-1845/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.fgb.2012.05.013 of biological functions. They include mammalian ceruloplasmin, plant ascorbate oxidases, laccases, fungal Fet3 ferroxidases (Solomon et al., 1996; Claus, 2003; Baldrian, 2006) and a recently discovered new branch within the MCO family, first described in P. chrysosporium with the characterization of multicopper oxidase one (Pc-MCO1) (Larrondo et al., 2003). This enzyme was later found to be broadly present in fungi (Hoegger et al., 2006; Kües and Rühl, 2011). Nevertheless, its precise physiological role remains obscure. It must be kept in mind that, in contrast to most white-rot fungi, P. chrysosporium does not produce a conventional laccase. In order to gain insight into the biological function of Pc-MCO1, we have analyzed its biochemical and structural properties (Larrondo et al., 2003), as well as its copper-dependent expression levels (Canessa et al., 2008). Extracellular Pc-MCO1 can efficiently oxidize iron and aromatic amines, but not phenolic compounds (Larrondo et al., 2003). On the other hand, and conceptually opposing Pc-MCO1 ferroxidase activity, it has been shown that extracellular CDH is capable of generating hydroxyl radicals by reducing Fe3+ and producing H2O2 (Kremer and Wood, 1992a,b; Henriksson P. Canessa et al. / Fungal Genetics and Biology 49 (2012) 626–634 et al., 1995). Nevertheless, both the precise role of Pc-MCO1 in iron metabolism and the putative functional coupling between these two enzymes remains uncertain. Notably, iron is particularly important for wood-degrading fungi. This metal not only plays a key role in the central metabolism, but also serves as critical cofactor of several of the enzymes that are involved in biomass conversion. Accordingly, iron plays a pivotal function in the heme-containing peroxidases known as manganese peroxidase (MnP) and lignin peroxidase (LiP) (encoded by 5 and 10 genes in P. chrysosporium, respectively; Martinez et al., 2004). In addition, wood-rotting microorganisms such as P. chrysosporium can produce hydroxyl radical through the Fenton reaction (Fe2+ + H2O2 + H+ ? Fe3+ + H2O + OH) and not surprisingly, there is evidence that supports a role for the Fenton chemistry in the degradation of lignocellulose by this fungus (Kremer and Wood, 1992a,b; Backa et al., 1993; Wood, 1994; Henriksson et al., 1995; Tanaka et al., 1999). Fenton-based mechanisms have been suggested to be key in cellulose depolymerization by brown-rot fungi like Postia placenta (Baldrian and Valaskova, 2008). Analysis of the Postia genome has revealed a poor repertoire of cellulase encoding genes, placing iron-based chemistry and iron regulation as crucial elements in cellulose degradation by this fungus (Martinez et al., 2009). Lignocellulose breakdown by white-rot and brown-rot fungi differs markedly. Whereas the former selectively degrade lignin leaving cellulose less affected, the latter extensively degrade cellulose hardly modifying the lignin constituent of the substrate (reviewed by Arantes et al., 2012). It is in this context that we have previously suggested that the ferroxidase activity of the extracellular Pc-MCO1 may fine-tune chemically based cellulose degradation in P. chrysosporium, by controlling the levels of ferrous iron available for the Fenton chemistry (Larrondo et al., 2003, 2007a). Thus, it is expected to find appropriate regulatory mechanisms to balance intracellular iron involved in various physiological processes (Ehrensberger and Bird, 2011) with those required for the generation of reactive oxygen species (ROS) outside the hyphae in a lignocellulose degradation context. Importantly, genes involved in iron homeostasis in different fungi have been proven to be crucial for full-display of pathogenic traits and virulence, as shown for Aspergillus fumigatus (Schrettl and Haas, 2011; Haas, 2012), Cryptococcus neoformans (Jung and Kronstad, 2011), Candida albicans (Almeida et al., 2009) and Ustilago maydis (Eichhorn et al., 2006). In the latter organism, the GATA family transcription factor URBS1 was first described as a master transcriptional regulator mediating repression of gene expression in the presence of iron (Voisard et al., 1993). The ortholog in different organisms include SRE in Neurospora crassa (Harrison and Marzluf, 2002), SREP in Penicillium chrysogenum (Haas et al., 1997), SREA in Aspergillus nidulans (Haas et al., 1999) and SREB in Blastomyces dermatitidis (Gauthier et al., 2010), among others. Although these transcription factors have received increasing attention in pathogenic fungi, little is known regarding their activity and role in fungi that largely depend on iron for their biomass converting activities, as it is the case of wood-rotters (MacDonald et al., 2012). As a first approach to build a conceptual framework to understand the role of ferroxidases and iron in wood degradation, we analyzed the mRNA levels of all multicopper oxidases genes in this fungus, including also Pc-fet3, the gene encoding a fungal canonical ferroxidase involved in iron acquisition (Larrondo et al., 2007a). The results showed a down-regulation of their respective transcript levels in the presence of iron. In addition, a SRE-like GATA-type transcription factor was identified and functionally characterized using a Neurospora strain deficient in its major regulator of iron metabolism. An in silico genome-wide strategy led to the identification of several genes possessing cis-regulatory sequences in their respective promoter regions, some of which also 627 showed decreased transcript levels upon addition of iron to the culture medium. 2. Materials and methods 2.1. Strain and culture conditions P. chrysosporium homokaryotic strain RP-78 was obtained from the Center for Mycology Research, Forest Products Laboratory, Madison, WI, USA P. chrysosporium spores were inoculated on potato-dextrose agar plates and grown for 1 week at 39 °C. Spores were collected by flooding the agar plates with 5 ml sterile water. Thereafter, spores were inoculated in 20 ml carbon-limited stationary liquid cultures as described (Brown et al., 1993, see details in Supplementary Information). Cultures were harvested after 4 days of incubation. When indicated, they were supplemented with FeCl3 to a final concentration of 0.25 mM, for the indicated times. N. crassa wildtype (74A) and Dsre (FGSC#11268; DNCU07728.2, a) were obtained from the Fungal Genetics Stock Center, Kansas City, MO, USA N. crassa spores were inoculated on 50 ml of liquid culture medium (LCM) composed of 1 Vogel’s salts (Vogel, 1956), 0.5% arginine, 50 ng/mL biotin with sucrose at 2% as a carbon source. This medium was supplemented with FeCl3 to a final concentration of 0.25 mM when indicated (see below). Finally, the obtained cultures were processed as mentioned for P. chrysosporium. For the yeast recombinational cloning procedure (see below), Saccharomyces cerevisiae FY839 was employed. 2.2. RNA extraction and Real-time quantitative RT-PCR (RT-qPCR) In the case of P. chrysosporium cultures, the mycelia obtained from five independent flasks (processed as a batch) were separated from the culture fluid by filtration through Miracloth (Calbiochem) and frozen in liquid nitrogen. The frozen mycelium was ground to a powder, and mRNA was obtained from total RNA as described (see details in Canessa et al., 2008). To ensure the absence of genomic DNA (gDNA) contamination, the obtained poly(A) mRNA was further purified using the RQ1 RNase-free DNase (Promega), at 37 °C for 1 h, accordingly with the manufacturer’s directions. To check for potential gDNA contamination, RT-minus reactions were carried out in real time using SYBR Green detection chemistry. No quantification cycle (Cq) values were obtained, at least under the cycle conditions used (see below). Thereafter, mRNA quantification was determined using RiboGreen dye (Invitrogen) according to the manufacture’s instructions. Only those mRNA samples that did not show gDNA contamination were reverse transcribed using the MMLV reverse transcriptase (Invitrogen) for 45 min at 42 °C, according to manufacturer’s instructions. The obtained cDNA was diluted two-fold. The complete procedure and control experiments intended for RT-qPCR have been described (Canessa et al., 2008). For N. crassa total RNA extraction, the procedure previously described (Chen et al., 2009) was employed. Transcript quantification was achieved using the SensiMixPlus SYBR Green kit (25 ll reactions; Quantace) and the Mx3000P detection system (Stratagene) using the MxPro software (version 4.01), as described in each manufacturer’s manual. Primer sequences for MCO encoding genes, as well as their predicted Tm values and amplicon lengths, were previously reported (Canessa et al., 2008) and are described in Supplementary Information. Additional primer pairs were designed using the Primer3plus software (see below; Untergasser et al., 2007). The RT-qPCR was performed as follows: 10 min at 95 °C followed by 40 cycles of 15s at 95 °C, 15 s at 58 °C and 15 s at 72 °C, followed by a melting cycle from 55 °C to 95 °C (0.2 °C/s) to check for amplification specificity. Cq values were acquired during the annealing period of the 628 P. Canessa et al. / Fungal Genetics and Biology 49 (2012) 626–634 RT-qPCR. Standard quantification curves with several serial 10-fold dilutions of RT-qPCR products were employed to calculate the amplification efficiency (E) of each gene according to the equation E = [10(-1/ slope] 1. These values were used to obtain a more accurate ratio between the gene of interest (GOI) and the expression of the reference gene employed for normalization, using the formula previously described (Livak and Schmittgen, 2001). Levels of mRNA from the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene were used for normalization, as previously described (Canessa et al., 2008). Under the culture condition tested, no significant differences (p < 0.05) were observed for this reference gene in cultures supplement with iron. The same result was observed for tubulin and actin mRNAs, thus showing that reference genes were not affected by the addition of the metal (data not shown). Expression values are referred to the culture not supplemented with iron (control). All experiments were performed in three biological and two technical replicates. iron-mediated transcriptional regulation (fgenesh1_pg.C_scaffold_3000073, Protein ID 2079, Fig. 2). The identified gene model was validated by RT-PCR, cloning and sequencing. The primers employed were 50 -GCAATCCTGTCTCGGCTCGAG-30 (forward) and 50 AATCACATTGGCAGCAGCCGCG-30 (reverse). Two specific PCR products were obtained, belonging to transcripts displaying alternative splicing. These were named as PIR1a and PIR1b. The primers employed to discriminate between these two variants (Fig. 3) were 50 -CAGGCGGCTGGTGCTGCTAAT-30 (forward) and 50 -GCAGCATTGAGAGGAGGCA-30 (reverse). Due to the high GC content present in the C-terminal half of the gene model, no PCR product was obtained using a conventional PCR protocol (data not shown). Consequently, a touchdown PCR protocol (Korbie and Mattick, 2008) was used, employing the following PCR program: an initial denaturation step of 95 °C during 5 min, followed by 33 cycles at 95 °C during 1 min, variable annealing temperatures in each cycle during 1 min, and 72 °C during 2.5 min. Hybridization temperatures were lowered 0.3 °C in each PCR cycle, starting from 70 °C. 2.3. Identification of an SRE-like GATA-type transcription factor gene model 2.4. N. crassa transformation and functional complementation By means of BLAST, and using the amino acid sequence of the URBS1 transcription factor from the basidiomycete Ustilago maydis (GenBank AAB05617; Voisard et al., 1993) as bait, we identified a gene model encoding a putative transcription factor involved in In order to functionally characterize the mentioned SRE-like gene model, a N. crassa complementation strategy was employed. For this, the strain X47–6 (mus-52::hyg^r, ras-1bd, A) was sexually crossed with the strain #11268 (NCU07728.2::hyg^r, a) using Fig. 1. Effect of iron on the expression of MCOs encoding genes. (A) P. chrysosporium was grown for 4 days in C-starved medium, after which it was harvested (0, control) or exposed to FeCl3 (250 lM) for the indicated times. After the addition of this salt, cultures were harvested and mRNA was purified and analyzed by RT-qPCR. Values are referred to the culture not treated with iron. Bars represent the mean of three biological replicates (±SD). Asterisks indicate significant differences (p < 0.05) of gene expression levels in comparison with control cultures. (B) Schematic representation of the Pc-fet3/Pc-ftr1 divergent promoter. Arrows indicate the transcriptional orientation of both genes. Rectangular boxes represent SRE-like GATA elements. Overlapped elements are depicted in light-green boxes, while single elements are indicated in gray. (C) Multiple alignment of the identified sequences showed in (B). Their respective position and orientation is also indicated (R: reverse, F: forward). (D) Computed consensus sequence (obtained from http://weblogo.berkeley.edu/logo.cgi), using the alignment (C) as entry. P. Canessa et al. / Fungal Genetics and Biology 49 (2012) 626–634 629 Fig. 2. Identification of the pir1 gene encoding a putative GATA-type transcription factor involved in iron-mediated regulation in P. chrysosporium. One gene model, fgenesh1_pg.C_scaffold_3000073, Protein ID 2079, was identified by means of BLASTp (see methods) to be the closest homolog to URBS1 (A). Exons are shown in dashedboxes, while introns are depicted using black lines. The aforementioned gene model was corrected and validated using RT-PCR. The diagrams depict the presence of the introns as confirmed by sequencing of the RT-PCR products. Two products differing only in the presence (B) or absence (C) of a 66 bp intron, located towards the 30 region, were identified. The figure also shows the presence of an additional exon in the N-terminal region not previously identified in the original gene model, as well as the absence of two exons located closely to the C-terminal half of the gene model. Westergard-agar medium (Westergaard and Mitchell, 1947). A mus-52::hyg^r, NCU07728.2::hyg^r progeny was selected, genotyped and subsequently used for transformation and heterologous complementation employing the cDNA of both pir1a and pir1b. For this purpose, a genetic construction was assembled containing at both the 50 and 30 end 1000 bp of sequence with homology to the Dsre locus, a sequence for the V5 epitope and a bar resistance cassette, thus allowing the integration of pir1a or pir1b in the same genomic region by the replacement of the hph resistance cassette present in the SRE KO strain (NCU07728.2::hyg^r). The genetic constructions for pir1a or pir1b were obtained using yeast recombinational cloning (Oldenburg et al., 1997). A schematic representation of the genetic constructs and recombination procedure is presented in Supplementary Fig. S1. 2.5. Western blot SDS–PAGE and Western blot techniques were conducted essentially as previously reported (Mancilla et al., 2010). Briefly, 20 lg of protein extracts, obtained as described (Garceau et al., 1997), were run on a 10% SDS–PAGE and transferred to a nitrocellulose membrane. After blocking in TS buffer (Tris 50 mM, NaCl 150 mM; pH 7.5) with skim milk (5% w/v final) the membrane was incubated with anti-V5 antibody (Invitrogen) and then Goat Anti-Mouse Fig. 3. Heterologous complementation of a GATA-type transcription factor from P. chrysosporium in N. crassa. (A) After N. crassa transformation with pir1a and pir1b, total RNA was obtained and RT-PCR performed from the Dsre strain (lane 1) or the transformed strains Dsre::pir1aV5-bar and Dsre::pir1bV5-bar (lanes 2 and 3, respectively). Primers were designed in order to discriminate for the presence of the 66 bp intron (depicted in Fig. 2B, see Material and methods). As shown in the Figure, no splicing of the 66 bp intron is observed for the transgene in the Dsre:: pir1bV5-bar transformed strain. (B) Western blot analysis confirming the presence of the transcription factors. Lanes 1 and 2 contain a nuclear protein extract from the Dsre::pir1aV5-bar and Dsre:: pir1bV5-bar strains, respectively. Lane 3 = positive control (FRQV5-protein). No cross-hybridization was observed in a nuclear protein extract preparation obtained from the Dsre strain (data not shown). (C) Phenotypic characterization of the complemented strains, using LCM medium supplemented with 250 lM FeCl3. (D) Molecular characterization of the complemented strains. The levels of the N. crassa fet3 gene were analyzed by RT-qPCR in the mentioned strains. Levels are referred to the wildtype strain not supplemented with iron (n = 2). Asterisks indicate significant differences (p < 0.05) of gene expression levels in comparison with control cultures. 630 P. Canessa et al. / Fungal Genetics and Biology 49 (2012) 626–634 IgG (H + L)-HRP conjugate antibody (BIO-RAD). The antibodies were used in 1:1500 and 1:2000 dilutions, respectively. The blot was developed by chemiluminescence using the SuperSignal West Femto (Thermo Scientific) and CL-XPosure X-ray films (Pierce). 2.6. Genome-wide in silico search of SRE-like GATA elements Manual inspection of the P. chrysosporium fet3-ftr1 divergent promoter (813 bp from each gene translation start site) led to the identification of putative cis-acting regulatory elements that have been described in the iron-mediated regulation of transcript levels in other fungi (An et al., 1997; Pelletier et al., 2003, 2007; Jung et al., 2006). Thus, we performed a genome-wide search of the 50 -KGATAA-30 element using a custom-made PSSM (PositionSpecific Scoring Matrix), calculated based on the alignment of the observed elements present in the aforementioned promoter (see Fig. 1). Thereafter, the entire set of promoter regions for P. chrysosporium were retrieved and scanned using the matrix scan tool of the Regulatory Sequence Analysis Tool (RSAT) software (van Helden, 2003; Turatsinze et al., 2008). Promoter regions were searched in both strands, from 800 to ATG (default limits), preventing overlaps with upstream ORFs. Cis-Regulatory element Enriched Regions (CRERs) were analyzed and ranked based on their calculated p-value. The p-value threshold was set to that obtained for the fet3-ftr1 divergent promoter. Selected genes (Supplementary Table 1) were validated by RT-qPCR as described above. Primer sequences, as well as predicted Tm values and amplicon length are indicated in the mentioned table. 3. Results 3.1. Effect of iron on mco transcript levels P. chrysosporium possesses four clustered mco genes (Larrondo et al., 2004). In order to establish the effect of Fe3+ on their expression, the fungus was grown in C-starved liquid cultures supplemented with iron (see methods) for the indicated times. As a control, we also analyzed the effect of this metal on fet3 mRNA levels, since its expression has been reported to decrease upon iron supplementation (Larrondo et al., 2007a). As shown in Fig. 1A, transcript levels of all multicopper oxidases encoding genes in P. chrysosporium are reduced in response to iron, with those of mco4 and fet3 being the lowest. Expression of mco3 was not analyzed since it is transcriptionally inactive in the RP-78 strain, due to an insertion of a transposable element known as Pcret1 (Larrondo et al., 2007b). Manual examination of the promoter regions of the mco genes led to the identification of putative cis-acting elements related to the SRE/URBS1-like GATA transcription factor involved in ironmediated transcriptional regulation in several fungi (An et al., 1997; Pelletier et al., 2003, 2007; Jung et al., 2006). Of them, only those within the Pc-fet3/Pc-ftr1 divergent promoter (eight elements, within a 813 bp promoter region; Fig. 1B and C) adhere to the 50 -KGATAA-30 consensus sequence (Fig. 1D) previously reported for the URBS1 transcription factor from U. maydis (Voisard et al., 1993; An et al., 1997; Eichhorn et al., 2006) or the SRE transcription factor from N. crassa (Harrison and Marzluf, 2002). 3.2. Identification and characterization of a SRE/URBS1-like GATA-type transcription factor The preceding observation, as well as the regulation of the expression of mco genes by iron, prompted us to search for a transcription factor responding to this metal in P. chrysosporium. By using the URBS1 sequence as a BLASTp query against the Phanero- chaete genome, the gene model fgenesh1_pg.C_scaffold_3000073, Protein ID 2079 was identified (Fig. 2). Manual inspection of the genome database suggested a putatively incomplete N-terminal half of the encoded protein. Consequently, we amplified by RTPCR, cloned and sequenced the full-length ORF, correcting the computer-assisted gene model prediction present in the genome database (see Fig. 2). Interestingly, two different splicing variants, namely pir1a and pir1b, were verified, the latter possessing a non-spliced 66 bp intron located towards the 30 region (see Fig. 2B and C). This intron encodes a sequence of 22 aa whose presence does not seem to disrupt any identifiable protein domain (data not shown). Consequently, both splicing variants were chosen for further functional characterization in N. crassa by complementation of a Dsre strain. Multiple alignment of both predicted protein sequences revealed that the closest homologs corresponded to the putative SRE transcription factors from the basidiomycetes Schizophyllum commune and Coprinopsis cinereus (35.2 and 30.9 % identity for PIR1a, 35.2 and 30.2 % identity for PIR1b, respectively; Supplementary Fig. S2, Tamura et al., 2011). Conservation around the two zinc-fingers GATA DNA binding domains (DBD) (canonical motif C-X(2)-C-X(17)-C-X(2)-C) could be observed between aa 338–362 and 561–585 from the consensus sequence (corresponding to aa 104–128 and 307–331 in both PIR1a and PIR1b, respectively; Supplementary Fig. S3). All sequences showed a Cys-rich region (CRR) of conserved residues (C-X(5)-C-X(8)-C-X(2)-C) between these two DBD. Upstream from this region, PIR1a and PIR1b exhibited a 31 aa insertion (located between residues 155–185 in both proteins; see Supplementary Fig. S4). On the other hand, inspection of the C-terminal half of the analyzed transcription factor sequences failed to show the presence of conserved motifs (data not shown). PIR1a and PIR1b were transformed into a N. crassa Dsre strain. For this purpose, Dmus-52 was included in the genetic background to facilitate homologous recombination at the sre original locus. The transforming DNAs were targeted to replace the hph sequence that substitutes the sre coding sequence in the sre-knockout strain (details in methods, Supplementary Fig. S1). Thus, the P. chrysosporium cDNAs replaced the N. crassa sre coding sequence and were expressed under the control of the endogenous promoter and the 50 and 30 regulatory sequences. As shown in Fig. 3A, both constructs were transcribed and no splicing was observed for the longer version of pir1 (Dsre::pir1bV5-bar) in N. crassa. In addition, the presence of the V5 C-terminal tag allowed the identification of the corresponding encoded proteins by Western blot analysis (Fig. 3B) (Larrondo et al., 2009). Unexpectedly, additional bands were detected. Based on the C-terminal position of the tag and size difference between both cDNAs, most likely these bands arise from internal translation start sites. As shown in Fig. 3C, when N. crassa was grown in the presence of 250 lM Fe3+, a clear phenotypic difference was observed between the wildtype and the Dsre strains. While the former strain exhibited a white-pale mycelium, the mutant strain showed intense-orange coloration. Interestingly, both Dsre::pir1aV5-bar and Dsre::pir1bV5-bar strains showed an intermediate phenotype, with only a slight-orange coloration. While the changes in coloration were quite suggestive, further proof of functional complementation was obtained by examining Neurospora’s canonical target genes of SRE. The mRNA levels of the fet3 gene from N. crassa were analyzed in both the wildtype and the Dsre strains. As observed in Fig. 3D, the iron induced down-regulation of fet3 expression was also dependent on the presence of the SRE protein. Importantly, high basal expression levels were observed in the Dsre background, which were even higher in the presence of iron. Nevertheless, both PIR1a and PIR1b restored the iron-mediated down-regulation of fet3 in N. crassa, in clear contrast to the situation obtained in the Dsre strain. P. Canessa et al. / Fungal Genetics and Biology 49 (2012) 626–634 3.3. Genome-wide in silico identification of putative SRE-like GATA elements The entire collection of predicted promoter regions of P. chrysosporium was analyzed in the search for putative SRE-like GATA elements. The described strategy (see methods) led to the identification of 28 gene models possessing at least one CRER showing a more significant p-value than that obtained for the fet3/ftr1 promoter region. The complete list of identified genes is available in Supplementary Table 1. Among them, eight were located on Scaffold 4 and five in Scaffold 5 (including the fet3/ftr1 genes). In order to validate the prediction, 14 genes were selected for expression analysis in the presence of iron using RT-qPCR. These genes were selected based on the prediction ranking and putative function (Supplementary Table 1). Despite significant efforts, nonspecific RT-qPCR amplicons were obtained for five of these 14 genes (Supplementary Table 1). Among these five genes, a putative membrane-bound ferric reductase protein (gene model 2772) was identified, in addition to a predicted 3-ketosphinganine reductase protein and two cytochromes p450. Interestingly, an RNA binding protein showed lower mRNA levels in cultures supplemented with iron. However a serine/threonine protein kinase located in opposite transcriptional orientation (at 398 bp from the translation start site) did not show significant differences in its mRNA levels. Gene models showing reduced transcript levels in cultures supplemented with iron also included a protein putatively involved in sexual differentiation (gene model ID 131282) and a predicted sugar phosphatase (gene model ID 129484). An overview of these results in presented in Fig. 4. 4. Discussion A previous hypothesis had stated that MCOs from P. chrysosporium might influence the bioavailability of Fe(II), fine-tuning Fenton chemistry and thus balancing lignin versus cellulose degradation through their ferroxidase activity. In agreement with this idea, we found that Fe3+ does influence mcos expression. Iron not only decreases the transcript levels of the canonical fungal ferroxidase fet3 as previously demonstrated (Larrondo et al., 2007a), but also those of the gene encoding for MCO1, an extracellular enzyme that possesses strong Fe2+ oxidation capability with biochemical properties similar to those of Fet3 (Larrondo et al., 2003). The same observation is valid for the genes encoding for MCO2 and MCO4. Particularly intriguing in the case of the latter enzyme, multiple alignments of all MCOs have shown the presence of a Glu residue equivalent to that of the Fet3 from Saccharomyces cerevisiae involved in iron oxidation (Bonaccorsi di Patti et al., 2000), with the sole exception of MCO4 (Larrondo et al., 2003, 2007a). Notably, both fet3 and mco4 mRNA levels showed that fastest down-regulation kinetics upon iron addition to the culture medium. In the case of fet3, the presence of 8 putative cis-acting GATA elements may explain, at least in part, this prompt response. The same observation is valid for the ftr1 gene (Larrondo et al., 2007a). However, the genome-wide strategy described here to identify cis-acting regulatory elements was unable to detect CisRegulatory element Enriched Regions (CRERs) in the promoters of mco1, mco2 and mco4. Manual inspection of their respective promoters (1000 bp from ATG, in comparison with the 800 bp strategy described in Methods) led to the identification of four 50 -GATA-30 elements (irrespective of the surrounding sequences) in the promoters of mco1 and mco2, whereas only three were found in the promoter of mco4. Accordingly, the number of putative elements did not correlate with the effect exerted by iron on mco transcript levels. As expected, all these putative elements do not 631 strictly adhere to the consensus sequence reported here (Fig. 1D), thus explaining the lack of identification by the employed in-silico strategy. Consequently, it is highly likely that the putative iron-related GATA consensus sequence computed from the fet3-ftr1 promoter may only explain a partial collection of iron-regulated genes. Nevertheless, based on the data presented here, we cannot rule out iron-mediated post-transcriptional effects (Vergara and Thiele, 2008) or even SRE-mediated regulation on other transcription factors that may explain an effect on the observed transcript levels (see below). Similar bioinformatic approaches have been performed in other fungi including Aspergillus fumigatus and Blastomyces dermatitidis (Schrettl et al., 2008; Gauthier et al., 2010). In these organisms, the classic GATA transcription factor binding site has been found spread all-across the genome. Interestingly, in A. fumigatus, U. maydis and B. dermatitidis both ‘‘canonical’’ and ‘‘extended’’ motifs have been described, differing form the consensus sequence reported in this work, but sharing the GATA central core (Eichhorn et al., 2006; Schrettl et al., 2008; Gauthier et al., 2010). This central core has been demonstrated to be essential for the DNA binding activity of the SRE transcription factor of N. crassa (Harrison and Marzluf, 2002). In order to further explore the molecular components involved in iron–mediated responses, we searched in P. chrysosporium for a gene encoding for a putative SRE-like transcription identifying two splicing variants. The pir1a and pir1b sre ortholog sharply differ in their intron–exon composition when compared to well-characterized SRE transcription factors. The same conclusion can be reached from the vast majority of the SRE sequences depicted in the Supplementary Fig. S2 (employing the Comparative Fungal Genomics Platform (CFGP); Park et al., 2008). Those from N. crassa, B. dermatitidis, Histoplasma capsulatum and A. nidulans possess two small introns interrupting the zinc finger domains (Gauthier et al., 2010). On the other hand, pir1 possesses four intronic sequences, with introns 1 and 2 being considerably long (ca. 400 bp) for Phanerochaete standards (Martinez et al., 2004). Intron 3 is alternatively spliced giving rise to two main transcripts, a situation reminiscent of the altered splicing previously described for the mco genes (Larrondo et al., 2004). In contrast to most GATA transcriptional regulators, which contain only one zinc finger domain (Haas et al., 1999), SRE family members – including both PIR1a and PIR1b – exhibit two zinc finger binding domains, flanking a Cys-rich region (CRR), possessing four conserved cysteine residues. The latter have been demonstrated to coordinate iron binding in the SRE ortholog of H. capsulatum (Chao et al., 2008), thus affecting its DNA binding affinity. As shown here, among all transcriptional regulators analyzed, only PIR1a and PIR1b showed an N-terminal insertion in front of the CRR with no significant similarity with any known polypeptide (data not shown). This region is also partially present in the hypothetical SRE proteins from Yarrowia lipolytica and Pleurotus ostreatus. As such, the putative function of this sequence, if any, remains to be determined. One intriguing possibility is that this short region may modulate DNA binding not only affecting the distance between the two DBD, but also influencing iron coordination due to its proximity to the CRR. In the case of SRE from N. crassa, it has been demonstrated that the protein has a clear preference for binding DNA in which two GATA elements are positioned within a certain distance (Harrison and Marzluf, 2002). Despite the aforementioned differences, both PIR1a and PIR1b were able to complement the N. crassa Dsre strain, although not reaching full wildtype levels. The potential effect of the N-terminal insertion in front of the CRR in PIR1 and its possible outcome on this phenotype remains to be determined. Nonetheless, as seen in Fig. 3D, there are contrasting differences between the wildtype and Dsre strains: fet3 mRNA levels show a 2.5-fold increment in the absence of iron in the mutant, thus demonstrating an either 632 P. Canessa et al. / Fungal Genetics and Biology 49 (2012) 626–634 Fig. 4. Transcript levels of selected genes possessing the GATA consensus sequence reported for the fet3/ftr1 genes. ID 129484, predicted sugar phosphatase; ID 131282, sexual differentiation process protein; ID 3242, putative RNA-binding protein; ID 3241, serine/threonine protein kinase (in divergent transcriptional orientation with ID3242); ID 804, M-phase inducer phosphatase; ID 6539, basic-leucine zipper (bZIP) transcription factor; ID 127915, an amidase; and ID 138068, a mitochondrial ATPdependent protease. Expression values are referred to the culture not treated with iron. Bars represent the mean of three biological replicates (±SD). Asterisks indicate significant differences (p < 0.05) of gene expression levels in comparison with control cultures. direct or indirect effect of SRE in the fet3 basal mRNA levels. This could reflect, for instance, the participation of a transcriptional activator normally repressed by SRE in Neurospora, that might be only partially regulated in a Dsre strain transformed with PIR1a or PIR1b. SRE orthologs in some fungi have been described as regulated at the mRNA level. For instance, the sre genes from A. nidulans, H. capsulatum and B. dermatitidis are upregulated in the presence of the metal (Chao et al., 2008; Haas et al., 1999; Gauthier et al., 2010), while those from P. chrysosporium (this work, data not shown), N. crassa, U. maydis, Candida albicans and S. pombe are not (Zhou et al., 1998; Voisard et al., 1993; Lan et al., 2004; Pelletier et al., 2002). On the other hand, the presence of alternative splicing in the pir1 gene indicates organism-specific regulatory mechanisms that are at least absent in N. crassa, since the expression of pir1b does not lead to the splicing of the intron present in this variant. Interestingly, this intron is inserted within a Pro-Arg rich region present in most of the analyzed transcription factors including P. Canessa et al. / Fungal Genetics and Biology 49 (2012) 626–634 PIR1b, but partially absent in PIR1a due to the lack of 22 aa (data not shown). As far as we are aware, pir1 is the only sre/urbs1 ortholog described to date showing alternative splicing. In this regard, those from S. pombe, A. nidulans and Penicillium chrysogenum present two transcriptional start sites (Pelletier et al., 2002; Haas et al., 1999, 1997), but none of them seems to undergo alternative splicing. The findings presented here, as well as those from other fungi, reemphasized the importance of iron in fungal biology, highlighting the complex regulatory networks around the metal. Moreover, SRE transcription factors have lately been shown to control more than just iron homeostasis. For instance, for the fungal pathogens C. neoformans and B. dermatitidis, the SRE orthologs are essential for pathogenicity, regulating critical virulence-associated genes. In addition, they are also required for thermal tolerance, capsule production, mating and growth-phase transitions (Jung et al., 2006; Gauthier et al., 2010, Jung and Kronstad, 2011, Hwang et al., 2012). In turn, in S. cerevisiae the iron-responsive transcription factor Aft1 has been implicated in the regulation of ironindependent cellular processes such as chromosome stability (Hamza and Baetz, 2012). In this regard, the non-bias genomewide strategy employed in this work allowed us to identify in P. chrysosporium potential SRE target genes not necessarily associated with iron metabolism. This differs from commonly employed methods that use functional categories enrichments. Our approach also took advantage of a well-defined divergent promoter region controlling the expression of two coregulated iron-related genes. Among the identified genes, reduced expression levels upon iron supplementation were observed for a predicted sugar phosphatase, as well as two potential regulatory proteins including a RNA binding protein and a sexual differentiation protein of the OPT oligopeptide transporter superfamily. These results did not exclude proteins with iron-associated functions. For instance, among the list of SRE potential target genes, we identified two cytochrome P450 and a ferric reductase protein possessing six putative transmembrane domains. Further work is needed in order to confirm whether this enzyme facilitates hydroxyl radical production by reducing Fe3+ thus counteracting MCOs iron oxidation, or if it is mainly involved in iron acquisition. Although the Fenton chemistry is normally associated with Fe2+, copper-mediated hydroxyl radical formation reaction is also feasible. As copper containing proteins, MCOs are regulated at the protein level by copper (Prohaska, 2011). The MCO encoding genes in P. chrysosporium are also regulated at the transcriptional level by both Cu2+ availability and the copper-dependent transcription factor Pc-ACE1 (Canessa et al., 2008). In contrast to the observed effect of iron on mRNA levels, Cu2+ produces an increment in the transcripts levels of mco1 and mco2, but not of mco4 (Canessa et al., 2008). In a hypothetical scenario, if this observation translates into higher protein quantities when highcopper levels become available, the four copper atoms coordinated at each MCO active site will sequester more metal. Thus, hydroxyl radical formation might be counteracted by both reducing the copper levels available for the chemical reaction and by increasing the P. chrysosporium Fe2+ oxidation potential through the enzyme’s enzymatic activity. The availability of numerous resources for gene manipulation in Neurospora, including among others a knockout collection and tagging strategies (Dunlap et al., 2007; Larrondo et al., 2009) potentiates the analysis of diverse biological problems in this organism. Hence, we are currently trying to understand the influence of other transcription factors and environmental stimuli on sre expression and on iron homeostasis. In addition to this approach, in the near future we expect to test PIR1 binding to selected P. chrysosporium promoters (including mcos), as well to assess the effect of iron on global gene expression. 633 In summary, we have identified and characterized a GATA-type transcription factor potentially involved in iron-mediated gene expression in P. chrysosporium. Denominated PIR1 (Phanerochaete iron regulator 1), this transcription factor shows alternative splicing and complements a N. crassa Dsre strain. 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