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Fungal Genomics section, Laboratory of Microbiology, Wageningen University, Dreijenlaan 2, NL-6703 HA Wageningen, The Netherlands
Correspondence
Leo H. de Graaff
leo.degraaff{at}wur.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Gielkens et al., 1999; Hasper et al., 2000, 2002). XlnR belongs to the zinc binuclear cluster family of transcription factors, found exclusively in fungi. Activation mechanisms of several zinc binuclear transcription factors have been described. An example of a well-characterized transcription regulator is GAL4 from Saccharomyces cerevisiae. GAL4 binds to the promoters of gal genes, but is prevented from activating transcription by the inhibitory protein GAL80. Induction by D-galactose requires the action of the signal transducer protein GAL3. The GAL3–GAL80 interaction occurs in the cytoplasm and activates GAL4 in the nucleus (Blank et al., 1997; Peng & Hopper, 2002). Another mechanism of regulation exists for LEU3 from S. cerevisiae, the regulator of the leucine biosynthetic pathway. LEU3 undergoes intramolecular changes mediated by 
-isopropylmalate (-IPM), leading to unmasking of the activation domain and subsequent activation of transcription (Wang et al., 1997).
Only two regions of similarity between XlnR and other members of the GAL4 family have been found: the (Zn2Cys6) DNA-binding domain, and the amino acid motif Arg-Arg-Arg-Leu-Trp-Trp, which is a fungal-specific transcription-factor domain of unknown function (Suárez et al., 1995). In silico analysis of the XlnR amino acid sequence predicted the presence of a putative coiled-coil domain (Lupas et al., 1991), directly C-terminal to the DNA-binding domain, and a second coiled-coil region at the C-terminal end of the protein. In a non-xylanase-producing mutant, a single amino acid mutation was found in the latter coiled-coil region in XlnR (van Peij et al., 1998a). Two other loss-of-function mutations have been found in the C-terminal region of XlnR, which might indicate that this region contains the activation domain. In addition to these loss-of-function mutants, another mutant, constitutive in xylanase activity, has been isolated (A. A. Hasper & L. H. de Graaff, unpublished results). Transcription studies have shown that this mutation also affects the response of XlnR to D-glucose, leading to the suggestion that the C-terminal region of the protein, in which this constitutive mutation has been found, is involved in modulation of XlnR activity. To gain an insight into the mechanism by which XlnR regulates transcription of the target genes, several deletion mutants have been constructed, based on the position of putative functional domains in XlnR. These mutants, as well as XlnR mutants harbouring single amino acid mutations, have been analysed for xylanase activity. Finally, the cellular localization of XlnR has been determined using a fluorescent GFP-tag.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The media used were based on minimal medium (MM) (Pontecorvo et al., 1953) and were, when necessary, supplemented with 1·5 mM leucine, 8 µM nicotinamide and/or 5 mM uridine. In transformation experiments, the recipient strain was A. niger NW199 (fwnA6, leuA5, goxC17, pyrA6, 
xlnR : : pIM240). For transformation, A. niger was inoculated at 106 spores ml–1 and grown in shake flasks (250 r.p.m.) at 30 °C, using a starting pH of 6. Transformation was carried out as described by Kusters-van Someren et al. (1991). After transformation, for some of the xlnR transformants, a single-copy integration of the construct, containing the mutated gene at the pyrA locus, was selected by Southern blot analysis. Mutations in the xlnR gene were introduced via site-directed mutagenesis either of plasmid pIM4444, which harbours a 3·8 kb XbaI fragment containing the pyrA gene, or of pIM4474, which harbours a gfp : : xlnR fusion construct (A. A. Hasper & L. H. de Graaff, unpublished results) (Table 2). For analysis of endoxylanase activity, transformants were plated onto MM plates containing 25 mM D-xylose or 25 mm D-glucose, and 0·1 % (v/w) azurin-dyed and cross-linked (AZCL) xylan (Megazyme). Spore solutions of the selected transformants were diluted in Saline/Tween (ST) to concentrations of 10 000, 1000, 100 and 10 spores µl–1. Of each dilution, 5 µl was transferred to the AZCL-xylan plates. After incubation at 30 °C for 2 days, blue haloes were formed by the release of soluble dyed xylan oligomers from the insoluble substrate.
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. Sequencing reactions were performed with the Thermo-Sequenase fluorescence-labelled primer cycle sequencing kit (Amersham Pharmacia Biotech) and xlnR-specific Cy5-labelled oligonucleotides. Sequencing reactions were analysed on an ALFexpress sequencer (Amersham Pharmacia Biotech). Fungal genomic DNA was isolated as previously described by de Graaff et al. (1988), and Southern blot analysis was performed according to standard methods (Sambrook et al., 1989), using the VacuGene XL vacuum blotting system (Amersham Pharmacia Biotech).
Northern blot analysis.
The eight strains used for Northern blot analysis were pre-cultured in MM supplemented with 100 mM D-fructose, and each was transferred to two shake flasks, containing either MM with 25 mM D-xylose or mm with 25 mm D-glucose. Total RNA was isolated from powdered mycelia using TRIzol reagent (Invitrogen), according to the manufacturer's recommendations. For Northern blot analysis, 10 µg total RNA was glyoxylated and separated on a 1·6 % (w/v) agarose gel. After capillary blotting onto a Hybond-N+ membrane (Amersham), the RNA concentration was checked by staining the filters with 0·2 % (w/v) methylene blue solution. The filters were pre-hybridized at 68 °C in a solution containing 0·9 M NaCl, 90 mM trisodium citrate, 5x Denhardt's solution (Sambrook et al., 1989), 10 mM EDTA, 0·5 % (w/v) SDS and 100 µg single-stranded herring sperm DNA per ml, followed by hybridization for 18 h in the same solution at 68 °C. The 0·9 kb EcoRI–XhoI fragment (van Peij et al., 1998b) of the xlnB gene was labelled with the Megaprime DNA labelling system (Amersham Biosciences) and used as probe. The blots were washed in a solution containing 30 mM NaCl, 3 mM trisodium citrate and 0·1 % (w/v) SDS.
Fluorescence microscopy.
Samples of A. niger mycelia used for fluorescence microscopy were prepared by inoculating 200 µl MM, containing 10 mM D-xylose, 10 mM D-glucose and appropriate supplements, with approximately 250 spores of A. niger transformants expressing GFP–XlnR fusion proteins. Inoculated samples were grown in chambered cover glasses (Nalgene Nunc International) for 24 to 30 h at 30 °C. Samples of mycelia were assayed for green fluorescence using a Zeiss Axiovert 100M microscope with the appropriate Zeiss filter combination (excitation filter 470/40 dichroic 510 and LP 520). Nuclei in the mycelia were visualized using 1 : 20 dilution DAPI-stain in Vectashield mounting medium (Vector Laboratories).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-helix structure inactivates XlnR isolated several XlnR loss-of-function mutants to clone the xlnR gene via complementation. One of the mutants harbours an amino acid substitution, Leu650Pro, in the putative C-terminal coiled-coil region (Fig. 1a). Since the introduction of a proline is known to disturb the helical structure of a coiled-coil domain, this mutation indicates that the coiled-coil structure is important for proper functioning of XlnR. To study this further, amino acid Leu650 was substituted by isoleucine in plasmid pIM4445, which harbours xlnR and a 3·8 kb fragment containing pyrA (A. A. Hasper & L. H. de Graaff, unpublished results). The construct was used to transform NW199, an xlnR-disruption mutant, and a single-copy integration was selected by Southern analysis. The xylanase activity of this mutant was examined by AZCL-plate screening (Fig. 2). In contrast to the Leu650Pro mutant, in the Leu650Ile mutant (Fig. 2b) the amino acid substitution did not have a big effect on xylanase activity. Northern blot analysis showed that transcription of xlnB slightly increased in the Leu650Ile mutant in relation to the wild-type (Fig. 3), when D-xylose was used as carbon source. A database search for conserved amino acid motifs showed that, in the putative coiled-coil region, a potential tyrosine phosphorylation site (Patschinsky et al., 1982), KEFEARY, is found from amino acid position 658 to 664. A stop mutation within this potential motif, Tyr664stop, resulted in an XlnR loss-of-function mutant (Figs 1b, 2d and 3). To examine whether phosphorylation of Tyr664 plays a role in XlnR activity, the tyrosine was replaced by phenylalanine (Fig. 1a). This amino acid substitution does not allow phosphorylation at this position, but maintains the secondary structure of the coiled-coil and the aromatic character of the residue at this position. In the Tyr664Phe mutant, xylanase activity on both D-xylose (Fig. 2c) and D-glucose (Fig. 2k) was slightly decreased in relation to the wild-type (Fig. 2j), but did not result in a loss of xylanase activity. Northern blot analysis of xlnB gene expression showed a decrease in expression in the Tyr664Phe mutant, relative to the wild-type.
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), deletion mutants (Fig. 1b) were made by introducing stop mutations in a gfp–xlnR fusion driven by the pkiA promoter to obtain higher expression levels. Three stop mutations were introduced: Tyr83stop, located between the DNA-binding site and the putative basic cluster nuclear localization signal (NLS), Asn635stop upstream and Leu668stop downstream of the predicted C-terminal coiled-coil region. In order to create inducing conditions, the mutants were grown on D-xylose, and the cellular localization of XlnR was determined. Confocal fluorescence microscopy demonstrated that the XlnR deletion mutant Tyr83stop remained in the cytoplasm (Fig. 4b). In agreement with this, no xylanase activity was observed in this deletion mutant (Fig. 2e). In the wild-type, clear blue haloes resulting from xylan degradation were visible (Fig. 2a). Fig. 4(a) shows the nuclear localization of the wild-type Gfp–XlnR fusion protein, and Fig. 4(f) shows the non-fluorescent wild-type as a control. After deletion of the C-terminal portion, including the putative C-terminal coiled-coil domain (Asp635stop), XlnR was also located in the cytoplasm, although a low signal was found in the nucleus (Fig. 4c). The dark non-fluorescent circles in the two mutants, Tyr83stop and Asn635stop, represent vacuoles. AZCL-xylan screening showed low xylanase activity (Fig. 2f), which is in agreement with the low amount of XlnR present in the nucleus. Deletion of the C-terminal portion downstream of the putative coiled-coil domain (Leu668stop) resulted in nuclear localization of XlnR (Fig. 4d), as in the wild-type (Fig. 4a). The xylanase activity in this mutant (Leu668stop) was strongly increased (Fig. 2g) in comparison to the wild-type. It appears that the first 667 amino acids are sufficient to give a fully functional XlnR, and that the C-terminal coiled-coil region of XlnR is involved in nuclear import of the protein.
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), which is in agreement with the finding that XlnR is not transported to the nucleus. However, since no fluorescence was observed, determination of cellular localization was not possible. Although there is only one copy of the mutated gfp : : xlnR fusion gene present in the chromosome, transcription is driven by the constitutive pkiA promoter, which should result in increased levels of protein. Furthermore, no fluorescence was observed in two other mutants harbouring a mutation in the coiled-coil region, Leu650Pro and Tyr664stop. The first mutant contains multiple copies of the gfp : : xlnR gene, and the second mutant includes a single-copy integration of the gene, driven by the pkiA promoter, at the pyrA locus. All these mutations resulted in non-xylanase-producing mutants.
The classical monopartite basic cluster nuclear localization signal in the N-terminal region of XlnR is not functional
Most proteins described thus far are directed to the nucleus by a single classical type of nuclear localization signal (NLS) (Christophe et al., 2000). In the N-terminal region of XlnR, four basic clusters can be found: 1 and 2 upstream, and 3 and 4 downstream of the Zn2Cys6 DNA-binding domain (Fig. 5a). Neither cluster 1 and 2 nor cluster 3 and 4 conform to the bipartite NLS consensus sequence, (K/R)2–X10–12–(K/R)3, of which the NLS of nucleoplasmin is the prototype (Nigg, 1997) (Fig. 5c). In Aspergillus nidulans, the transport of PrnA into the nucleus requires a tripartite nuclear localization sequence (Pokorska et al., 2000). The three basic clusters that form the NLS in PrnA (1, 2 and 3) (Fig. 5b) are separated by only five and eight amino acids. Since clusters 2 and 3 at the N-terminus of XlnR are separated by as many as 37 amino acids, it is not likely that the N-terminal region of XlnR harbours a tripartite NLS of the type found in PrnA. Based on the motifs of karyopherin that recognize NLSs, Pokorska et al. (2000) suggested that there might be a relationship between the number of basic motifs comprising an NLS and the permitted distance between them. Cluster 3, formed by residues 87 to 90, is the only basic stretch that matches the consensus sequence of a monopartite NLS, (K/R)4–6, the prototype for which is the NLS of the SV40 large T antigen (Nigg, 1997).
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). An example of a protein that harbours two regions involved in nuclear import is the homeoprotein thyroid transcription factor (TTF-1). TTF-1 harbours both a classical monopartite NLS and a motif involved in nuclear import that is unrelated to known NLSs (Christophe-Hobertus et al., 1999). To investigate the functionality of the monopartite NLS in XlnR, the two lysines in this cluster were deleted and one arginine was replaced by a glycine, via site-directed mutagenesis in the pkiA : gfp : xlnR construct (88–90, Fig. 1b). The mutated gene was introduced in the xlnR strain NW199, and cellular localization was determined using fluorescence microscopy. Fig. 4(e) shows that partial deletion and mutation of the putative monopartite NLS in the N-terminal region of XlnR did not affect nuclear localization of the protein. Although the nuclear localization of XlnR in this mutant was not affected, no xylanase activity was found (Fig. 2i).
The C-terminal portion downstream of the second coiled-coil domain of XlnR is involved in transcription regulation
Two xylanase non-producing mutants isolated by van Peij et al. (1998a) were shown to have single amino acid substitutions in the C-terminal region of XlnR. This led to the suggestion that this part of the protein is involved in regulation of XlnR activity. In one of the mutants, the tyrosine at position 864 was substituted by aspartate. Since phosphorylation is a general mechanism for responding to activating or inactivating signals, for example as in Pho4 from S. cerevisiae (Komeili & O'shea, 1999), the tyrosine at position 864 was replaced by phenylalanine. The phenylalanine will not disturb the secondary structure of XlnR but cannot be phosphorylated. Analysis of AZCL-plate xylanase activity in the Tyr864Phe mutant showed strongly decreased xylanase activity on D-xylose, while the level of the XlnB transcript was not significantly altered (Figs 3, 6a and 6c). This was in contrast to the xylanase activity of the Tyr864Phe mutant on D-glucose, which was comparable to the wild-type (Fig. 6a, c). The xylanase activity of the wild-type grown on D-glucose-containing plates can be explained by the local consumption of D-glucose, resulting in de-repression and induction of xylanolytic genes by AZCL-xylan. In the xlnR strain, xylanase activity was found surrounding low-spore inocula grown on D-xylose, but this was not the case for higher-spore inocula. Determination of xylanase activity via AZCL-plate screening is a semi-quantitative method, however, and therefore cannot explain the halo formation observed with the low-spore inoculates of the xlnR strain.
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). This has also recently been shown by other studies (A. A. Hasper & L. H. de Graaff, unpublished results). Northern blot analysis showed an increase of the xlnB transcript in this mutant, relative to the wild-type. Also, a high xylanase activity was found under repressing conditions in a mutant harbouring a stop mutation at position Leu668 (Fig. 6e). The higher xylanase activity of the Leu668stop mutant (detected in AZCL plates), compared to the Val756Phe mutant, was probably due to the higher copy number of the xlnR gene in this mutant. These two mutants show that the C-terminal region of XlnR, downstream of the coiled-coil region, might be involved in modulation of XlnR activity in the presence of D-glucose. The two loss-of-function mutations within the last 60 amino acids of XlnR also suggest that this region is relevant to the function of XlnR. To examine this further, two C-terminal deletion mutants of XlnR were constructed (Fig. 1b), based on the position of the two loss-of-function mutations, Leu823Ser and Tyr864Asp (Fig. 1a). In one mutant, amino acids 802–836, encompassing Leu823, were deleted. In the second mutant, a C-terminal fragment of 78 amino acids was removed by introducing a stop codon at position Gly797. Analysis of the xylanase activity on AZCL plates with D-xylose showed that deletion of the 78 C-terminal amino acids resulted in increased xylanase activity, compared to the wild-type (Fig. 6f). In contrast, deletion of amino acids 802–836 resulted in complete loss of xylanase activity on D-xylose (Fig. 6g), as in the xlnR mutant NW199 (Fig. 6b). These different results could be explained by a regulation mechanism through inter- or intramolecular interactions within the C-terminal region. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) had already indicated the functional importance of this region. The additional mutation and deletion studies described in this paper show that maintaining the structure of this domain is essential to the proper functioning of XlnR (Figs 2b, 2c, 2d, 2h and 3). Coiled-coil regions have been found in other transcriptional activators. They are often located near the N-terminus and involved in dimerization, such as in the NirA or PrnA transcription factors of A. nidulans (Strauss et al., 1998, Cazelle et al., 1998). In some transcription factors, such as PrnA, a second putative coiled-coil region is predicted in the C-terminal part of the protein. However, no function has been described for this region in XlnR. Cellular localization of an XlnR mutant, in which the C-terminal region, including the coiled-coil domain, was deleted, showed that this domain is involved in nuclear import of the protein. This suggests the presence of a nuclear-targeting signal, although no classical NLS motif could be found in this region. Several sequence motifs have been reported to mediate the nuclear import of proteins besides the classical basic type of NLS, which is the most common. An example is the M9 sequence from the hnRNP A1 and A2 proteins (Pollard et al., 1996). This sequence is recognized by transportin (Trn), a close homologue of importin-
, which alone, or in a heterodimeric complex with importin-, is responsible for the translocation of proteins harbouring classical NLSs through the nuclear pore (Truant & Cullen, 1999; Palmeri & Malim, 1999). A consensus Trn-interaction motif has been determined to be (YFW)XXJXSXZG(PK)(MLV)(KR) (Bogerd et al., 1999). This motif could not be found in the XlnR putative coiled-coil region.
Another example of a protein with an alternative NLS is the FAS-associated factor qFAF, a nuclear protein of unknown function. Its nuclear-targeting signal resides in a region that includes an -helix (Fröhlich et al., 1998), which also appears to be the case for XlnR. Previously, a putative basic cluster NLS was identified in the N-terminal region of XlnR, but cellular localization of an XlnR mutant, in which this potential NLS was mutated, showed that it is not involved in nuclear localization (Fig. 4e). The region in which this basic cluster is located is a potential coiled-coil domain. The low amount of the XlnR mutant Asn635stop present in the nucleus may be due to another unidentified functional NLS that participates in nuclear localization, within the first 635 amino acids of the protein. Alternatively, the region involved in nuclear import might interact with part of the protein downstream of the putative coiled-coil domain. Thus, the precise regions or domains involved in nuclear localization remain to be determined. Since the XlnR-binding site has been found to bind to a non-palindromic consensus in the promoter, it has been suggested that XlnR binds as a monomer (van Peij, 1998a). Therefore, the N-terminal coiled-coil domain is not likely to be involved in dimerization, even though it is closely linked to the DNA-binding domain. Nevertheless, the XlnR mutant in which the putative NLS had been mutated was not active. Apparently, this putative helical region is also important to the function of XlnR.
In order to measure xylanase activity in the different xlnR mutants, AZCL-plate screening was performed, since this is a straightforward and rapid method to determine the xylanase activity of each sample, although it is not optimal for quantification. Northern blot analysis, on the other hand, is a more accurate method for the quantification of gene expression, but shows the expression of one xylanase only (xlnB), whereas the AZCL-plate method shows the activity of all xylanases. This may explain the discrepancy between the results obtained for the Tyr864Phe mutant by the Northern blot and AZCL-plate methods.
The AZCL-plate screening and Northern blot results of several mutants (Figs 2 and 3) suggest that the putative tyrosine phosphorylation site from amino acid positions 658 to 664 is not involved in activation or inactivation of XlnR protein.
Several members of the Zn2Cys6 transcription factor family have been shown to harbour a C-terminal regulatory domain. In GAL4, the activation domain resides between amino acids 768 and 881, the last 113 residues of the protein (Keegan et al., 1986; Ma & Ptashne, 1987). In LEU3 (886 amino acids), the activation domain is located between residues 861 and 886, and in PUT3 (979 amino acids), the activator of the proline utilization pathway, the C-terminus between residues 890 and 979 has been shown to include domains for activation and regulation (des Etages et al., 2001). In the current study, regulatory mutations were found within the last 120 amino acids of the XlnR C-terminus. Deletion of the C-terminal region downstream of the predicted coiled-coil region leaves a fully active protein, as seen in the XlnR mutant Tyr668stop. This indicates that this region probably responds to repressing signals rather than inducing signals. This is in agreement with the theory that the mutation Val756Phe, which results in xylanase expression under repressing conditions, disturbs a D-glucose inhibitory domain (A. A. Hasper & L. H. de Graaff, unpublished results). Characterization of two deletion mutants, one of which lacked an internal fragment of 34 amino acids, residues 802–836, and in the other of which the last 78 residues were deleted, led to a hypothetical model in which the C-terminal region is involved in regulating the activity of XlnR. In this model, a proposed D-glucose inhibition domain in the C-terminal region downstream of Leu668 responds to repressing signals via intra- or intermolecular interactions, which turn XlnR into an inactive state. This response mechanism is abolished by mutation Val756Phe. The strongly increased xylanase activity in the Leu668stop mutant might be the result of the presence of multiple copies of the mutated XlnR gene integrated in the chromosome. Alternatively, this effect might be explained by the involvement of the C-terminal region in the regulation of XlnR. It seems that a disturbed or deleted coiled-coil domain in the full-length protein affects the stability of XlnR.
Our results have led to the new model of XlnR regulation and transport signals presented in Fig. 7(a).
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ACKNOWLEDGEMENTS |
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Received 11 June 2003;
revised 13 January 2004;
accepted 22 January 2004.
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  A. R. Stricker, K. Grosstessner-Hain, E. Wurleitner, and R. L. Mach Xyr1 (Xylanase Regulator 1) Regulates both the Hydrolytic Enzyme System and D-Xylose Metabolism in Hypocrea jecorina Eukaryot. Cell, December 1, 2006; 5(12): 2128 - 2137. [Abstract] [Full Text] [PDF]
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 S. MacPherson, M. Larochelle, and B. Turcotte A Fungal Family of Transcriptional Regulators: the Zinc Cluster Proteins Microbiol. Mol. Biol. Rev., September 1, 2006; 70(3): 583 - 604. [Abstract] [Full Text] [PDF]
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