Molecular Microbiology (2012) 84(4), 748–765 䊏
doi:10.1111/j.1365-2958.2012.08058.x First published online 16 April 2012
The histone chaperone ASF1 is essential for sexual development in the filamentous fungus Sordaria macrospora Stefan Gesing,1 Daniel Schindler,1 Benjamin Fränzel,2 Dirk Wolters2 and Minou Nowrousian1* 1 Lehrstuhl für Allgemeine und Molekulare Botanik and 2 Lehrstuhl für Analytische Chemie, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany.
Summary Ascomycetes develop four major types of fruiting bodies that share a common ancestor, and a set of common core genes most likely controls this process. One way to identify such genes is to search for conserved expression patterns. We analysed microarray data of Fusarium graminearum and Sordaria macrospora, identifying 78 genes with similar expression patterns during fruiting body development. One of these genes was asf1 (anti-silencing function 1), encoding a predicted histone chaperone. asf1 expression is also upregulated during development in the distantly related ascomycete Pyronema confluens. To test whether asf1 plays a role in fungal development, we generated an S. macrospora asf1 deletion mutant. The mutant is sterile and can be complemented to fertility by transformation with the wild-type asf1 and its P. confluens homologue. An ASF1–EGFP fusion protein localizes to the nucleus. By tandem-affinity purification/mass spectrometry as well as yeast twohybrid analysis, we identified histones H3 and H4 as ASF1 interaction partners. Several developmental genes are dependent on asf1 for correct transcriptional expression. Deletion of the histone chaperone genes rtt106 and cac2 did not cause any developmental phenotypes. These data indicate that asf1 of S. macrospora encodes a conserved histone chaperone that is required for fruiting body development.
Introduction The life cycle of multicellular eukaryotes often comprises cellular differentiation processes leading to complex Accepted 27 March, 2012. *For correspondence. E-mail minou.
[email protected]; Tel. (+49) 234 3224588; Fax (+49) 234 3214184.
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three-dimensional structures. The formation of fruiting bodies in filamentous ascomycetes is an example of such a differentiation process. Fruiting bodies are produced during the sexual cycle to ensure protection and dispersal of the sexual spores. They comprise a number of specialized cell types developing from a relatively uniform vegetative mycelium (Bistis et al., 2003). In recent years, a variety of forward and reverse genetics approaches have been used to analyse the molecular basis of this differentiation process, but a unified model for its genetic control has yet to emerge (Pöggeler et al., 2006a). Because co-ordinated gene expression of participating genes accompanies cell differentiation, patterns of differential expression can serve as indicators of genes that are key players during the process of interest. Accordingly, functional genomics techniques have been used to study fruiting body development, aided by the growing number of sequenced ascomycete genomes (Nowrousian, 2007; 2010). The disadvantage of such large-scale expression studies is that they often yield large numbers of differentially expressed genes, not all of which are directly important to the target process. One solution to this problem is to increase the sensitivity for selecting functionally relevant genes by applying additional criteria. An option is to compare expression data of related species and choose for further analysis those genes that show conserved expression patterns, based on the assumption that evolutionary conservation is a powerful criterion for functional significance (Stuart et al., 2003). Such an approach is feasible in the case of fruiting body development because the four morphological types of fruiting bodies formed by ascomycetes are derived from a common ancestor and constitute homologous structures (Lumbsch, 2000; Pöggeler et al., 2006a). Thus, a reasonable hypothesis is that a set of common core genes controls fruiting body development. Based on this principle, in a previous study we compared the expression of several genes during sexual development in Sordaria macrospora and Pyronema confluens (Nowrousian and Kück, 2006). P. confluens belongs to the Pezizales, a basal group of ascomycetes, and forms apothecia as fruiting bodies, whereas S. macrospora is a member of the derived group of Sordariales, which form the more complex perithecia. The results of this smallscale study supported the assumption that there might be
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significant similarity between expression patterns during fungal fruiting body development, even between distantly related ascomycetes. In the present study, we extended these analyses to a larger scale and compared published microarray hybridization data from sexual development in Fusarium graminearum (Qi et al., 2006) and S. macrospora (Nowrousian et al., 2007a). Among the genes that are transcriptionally upregulated during fruiting body development in both fungi is the predicted asf1 (anti-silencing function 1) gene. asf1 encodes a conserved histone chaperone that was first identified via overexpression in Saccharomyces cerevisiae as a protein involved in chromatin-mediated gene inactivation (Le et al., 1997). It was later shown to be involved in the assembly and disassembly of nucleosomes by complex formation with the core histones H3 and H4, and thus to be associated with the co-ordination of chromatin-mediated processes like replication, transcription and DNA repair (Munakata et al., 2000; Mousson et al., 2007; Krebs and Tora, 2009; Avvakumov et al., 2011). In recent years asf1 has been investigated in a number of eukaryotes, but its role in filamentous fungi has not been addressed. Because of its conserved expression pattern during sexual development in two filamentous fungi, we predicted a central role for asf1 in the differentiation of fruiting bodies. Therefore, we chose asf1 for a more detailed functional analysis and addressed the following key questions: (i) Does an orthologue of asf1 exist in the distantly related ascomycete P. confluens, and is it also differentially expressed? (ii) Is asf1 necessary for fruiting body development in the model organism S. macrospora? (iii) Does the predicted asf1 gene in filamentous fungi also encode a histone chaperone?
Results Comparative analysis of gene expression during sexual development in two filamentous fungi In a previous study, we compared the expression of nine genes during fruiting body development in S. macrospora and P. confluens (Nowrousian and Kück, 2006). The results indicated that there might be a significant conservation of gene expression during fungal sexual development. To extend this analysis to a larger scale, we compared microarray hybridization results from published analyses of sexual development in F. graminearum (Qi et al., 2006) and S. macrospora (Nowrousian et al., 2007a). Both species form perithecia as fruiting bodies, and in both studies, fruiting body development was compared with vegetative growth. The comparative analysis identified 473 F. graminearum genes that were differentially expressed during sexual development and for which expression data were available for S. macrospora. Among
Fig. 1. Comparative analysis of gene expression during sexual development in F. graminearum and S. macrospora. Seventy-eight genes with similar expression patterns in both species (i.e. up- or downregulated in fruiting body morphogenesis versus vegetative growth in both fungi) were subjected to FunCat classification (Ruepp et al., 2004). The graph shows the FunCat categories and corresponding category number that the 78 genes represent. For each category, the fold representation compared with the representation among all predicted proteins in the genome is given for up- and downregulated genes from this category. Categories that are significantly overrepresented (P < 0.05) among the up- or downregulated genes are marked with an asterisk. A dashed horizontal line indicates +1 (representation equal to the genomic representation). The regulated genes belonging to each category are given in Table S1.
these genes, 78 displayed similar expression patterns, i.e. either up- or downregulated during fruiting body morphogenesis in both species (Table S1). FunCat classification (Ruepp et al., 2004) of these 78 genes revealed that genes belonging to the FunCat categories of metabolism and energy were significantly overrepresented among the downregulated genes; genes with predicted functions in transcription/RNA processing, protein activity regulation, cell fate (differentiation/apoptosis), and cell wall biogenesis were significantly overrepresented among the upregulated genes (Fig. 1). These expression patterns are consistent with the hypothesis that fruiting body formation in filamentous fungi requires a metabolically ‘competent’ mycelium that supplies the developing fruiting bodies with nutrients (Wessels, 1993; Pöggeler et al., 2006a). Among the genes that were upregulated during sexual development in both F. graminearum and S. macrospora was the predicted asf1 (anti-silencing function 1) gene (Fig. 2). asf1 was first identified as a gene involved in
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Fig. 2. Expression analysis of asf1 orthologues in different fungi. A. asf1 expression in P. confluens (P.c.), S. macrospora (S.m.), N. crassa (N.c.) and F. graminearum (F.g.). In each case the wild type was grown under vegetative and sexual growth conditions. The data are shown as expression ratios for sexual versus vegetative development. Transcript levels were determined by quantitative real-time PCR in the case of P. confluens and N. crassa asf1 (mean of at least three independent biological replicates; P. confluens: 4-day growth in surface culture, in constant light for sexual mycelia and in darkness for vegetative mycelia; N. crassa: 6 days in constant light, in surface culture for sexual mycelia and submerged for vegetative mycelia) and compared with expression data of S. macrospora and F. graminearum asf1 that were obtained by microarray analysis and published previously (Qi et al., 2006; Nowrousian et al., 2007a) (S. macrospora: 4–5 days in constant light, in surface culture for sexual mycelia and submerged for vegetative mycelia; F. graminearum: 96 h after induction for sexual structures, 4 days for vegetative mycelia). B and C. Expression of asf1 during a time-course of sexual development (3–5 days) and during vegetative growth in S. macrospora (B) and P. confluens (C). Transcript levels were determined by quantitative real-time PCR with two independent biological replicates. Growth conditions were as described in (A). As a control, transcript levels of S. macrospora ppg2 encoding a pheromone precursor and P. confluens Pc2.1G07 encoding a putative exoglucanase were determined. Both genes are strongly upregulated during sexual development compared with vegetative growth as was shown previously (Mayrhofer et al., 2006; Nowrousian and Kück, 2006).
asf1 homologues are conserved in distantly related filamentous fungi and differentially expressed during sexual development
derepression of the silent mating-type loci in S. cerevisiae (Le et al., 1997). It was later shown to be a conserved eukaryotic histone chaperone with a role in the regulation of transcription, DNA replication and other chromatinrelated functions (Munakata et al., 2000; Mousson et al., 2007; Krebs and Tora, 2009). asf1 has been investigated in a number of eukaryotes; however, its role in filamentous fungi has not been addressed. Its conserved expression pattern during sexual development in two filamentous fungi suggested a role in this process; therefore, we chose asf1 as a candidate for a more detailed analysis.
Both S. macrospora and F. graminearum are members of the Sordariomycetes and form perithecia as fruiting bodies. To address the question of whether asf1 expression is also conserved in a more distantly related fungus, we investigated the presence and expression of asf1 in P. confluens. This fungus belongs to the Pezizomycetes, a basal group of ascomycetes, and forms apothecia as fruiting bodies. For cloning P. confluens asf1, a 450 bp cDNA fragment was amplified using oligonucleotides derived from the open reading frame (ORF) of Neurospora crassa asf1, followed by BLAST analysis identifying the predicted Aspergillus terreus asf1 (Accession No. XP_001213807) as the closest homologue of the P. confluens sequence (data not shown). Subsequently an inverse PCR experiment was performed to obtain a 3.2 kb region of P. confluens genomic DNA comprising the asf1 gene (Pcasf1). Introns were verified by amplifying and sequencing P. confluens asf1 cDNA fragments covering the full-length asf1 ORF and parts of the 5′ and 3′ UTR (untranslated region) respectively. The P. confluens asf1 ORF comprises 801 bp interrupted by three introns (Accession No. HQ700586). It encodes a predicted protein of 266 amino acids (Fig. S1). The structure of the S. macrospora asf1 orthologue is similar, it encodes a protein of 269 amino acids. In this fungus, the open reading frame of 810 bp is interrupted by two introns; the © 2012 Blackwell Publishing Ltd, Molecular Microbiology, 84, 748–765
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introns and the UTRs of ~ 231 (5′ UTR) and 558 (3′ UTR) bases were determined from RNA-Seq data (Fig. S2). A multiple alignment of the P. confluens ASF1 and the corresponding orthologues of 11 ascomycetes, two basidiomycetes, two zygomycetes, two chytrids, and one animal clearly emphasizes typical characteristics of ASF1 (Mousson et al., 2007), namely a highly conserved N-terminus and a species-specific C-terminus (Fig. S1). The alignment also shows that the previously noted longer C-terminus of the yeast ASF1 (Mousson et al., 2007) is a feature shared between all investigated ascomycetes, but not other fungal groups (Fig. S1). Quantitative real-time PCR was used to determine the transcript levels of P. confluens asf1 under conditions allowing sexual development and conditions that result in vegetative growth. The corresponding expression ratios were compared with expression data for asf1 orthologues in F. graminearum, S. macrospora and N. crassa, indicating a conserved pattern of upregulated asf1 expression during sexual development in all four fungi (Fig. 2A). Furthermore, a time-course analysis of asf1 expression in S. macrospora and P. confluens indicates that asf1 is most strongly expressed after 3 days, and expression declines after this point (Fig. 2B and C). Interestingly, in both fungi, this time point marks the onset of fruiting body development. Taken together, these data indicate that asf1 homologues are highly conserved in filamentous ascomycetes and upregulated at the onset of sexual development. Thus, asf1 was a candidate of choice for further functional analysis. Phenotypic characterization of an S. macrospora asf1 deletion mutant, and complementation analysis reveal a function of asf1 during development of ascomycetes To address the question of whether asf1 plays a role in fungal morphogenesis, an S. macrospora asf1 (SMAC_08608) deletion mutant was generated by homologous recombination with an hph cassette flanked by upstream and downstream sequences of the asf1 ORF (see Experimental procedures). Microscopic and macroscopic investigations of the knockout strain revealed several developmental defects (Fig. 3). Whereas wild-type mycelium develops ascogonia after 2 days, protoperithecia after 4 days, and perithecia with mature ascospores after 7–8 days, the asf1 deletion mutant is sterile (Fig. 3A). While the mutant still develops ascogonia after 2 days, it produces only few protoperithecia that never differentiate into mature perithecia, not even after more than 20 days of incubation under conditions allowing sexual development. Additionally, deletion of asf1 leads to a reduction in vegetative growth of about 50% (Fig. 3B). Retransformation of the deletion strain with the wild-type asf1 gene completely restored the wild-type phenotype, thus verifying that the
developmental phenotype of the deletion strain indeed results from the missing ASF1 function (Fig. 3). Furthermore, we succeeded in complementing the deletion strain’s developmental defects almost completely by transformation with the P. confluens asf1 gene, indicating conservation of the role of asf1 during development among filamentous ascomycetes (Fig. 3). Sexual development of transformants complemented with the P. confluens asf1 was completed after 8 days, and was thus only marginally slower than the wild-type strain. Vegetative growth was also restored to about 80% of that of the wild-type strain. One of the reasons that transformants with the P. confluens asf1 are somewhat slower in recovering the fertility defects than transformants with the S. macrospora asf1 might be that the C-termini of the ASF1 proteins from S. macrospora and P. confluens are rather dissimilar because of the large evolutionary distance between these two species. It has been hypothesized for other organisms that while the highly conserved N-terminus is essential for histone interactions, the C-terminus might be involved in speciesspecific processes of interaction and regulation (English et al., 2005; Mousson et al., 2005; 2007; Avvakumov et al., 2011). However, taken together, knockout and complementation analyses revealed a conserved function of asf1 during development of filamentous ascomycetes. asf1 is necessary for correct expression of developmental genes ASF1 in yeast and animals has been implicated in a variety of processes that require chromatin restructuring, including DNA replication and transcription (Mousson et al., 2007). Therefore, we analysed the expression of known developmental genes or genes upregulated during development to address the question whether the S. macrospora asf1 is involved in development-dependent transcription (Fig. 4). We analysed gene expression in the asf1 deletion mutant and two complemented transformants that overexpress asf1 ~ 10- to 40-fold compared with the wild type (Fig. 4A). In S. cerevisiae, asf1 overexpression leads to activation of the silent mating type loci (Le et al., 1997); however, in S. macrospora, there are no silent copies of the mating type locus, only one active locus (Pöggeler et al., 1997b). Therefore, we analysed if asf1 deletion or overexpression had an effect on Smta-1 and SmtA-2, the two genes from the mating type locus that are essential for sexual development (Pöggeler et al., 2006b; Klix et al., 2010). Quantitative real-time PCR analysis showed that both Smta-1 and SmtA-2 are upregulated in the asf1 knockout strain, and that they are also upregulated in a complemented transformant with a 10-fold asf1 overexpression (Dasf1::NTAP-asf1, Fig. 4A), but upregulation is reduced in a complemented transformant with a higher overexpression of asf1 (Dasf1::egfp-asf1). A similar effect
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Fig. 3. Phenotypic characterization of the S. macrospora Dasf1 mutant and complemented transformants. A. Analysis of sexual development. For the microscopic investigation of ascogonium and protoperithecium development, the S. macrospora wild type (wt), the asf1 deletion mutant (Dasf1), and transformants complemented with the S. macrospora asf1 (Dasf1::Smasf1) or the P. confluens asf1 (Dasf1::Pcasf1) were grown for 2 and 4 days, respectively, on BMM-coated microscope slides according to Engh et al. (2007b). For the investigation of mature perithecia, strains were analysed after 7–8 days of growth on BMM; in the small boxes on the top left of each photograph, enlarged selections of each overview are shown. In cases of perithecium development, ascus rosettes were prepared and microscopic pictures were taken as shown in the small boxes on the bottom right sides. Wild type, strain S91327; Dasf1, strain S90177; Dasf1::Smasf1, strain T48.2SN; Dasf1::Pcasf1, strain T18.4. B. Analysis of vegetative growth. Strains were grown in race tubes on solid BMM according to Nowrousian and Cebula (2005) and the growth front was marked every 24 h over 7 days. Compared with the wild-type strain S91327 (wt), the asf1 deletion mutant S90177 (Dasf1) shows strongly reduced vegetative growth. By transformation of the deletion mutant with S. macrospora asf1 (SmASF1; strain T48.2) and P. confluens asf1 (PcASF1; strain T18.4) wild-type growth rates were restored.
could be observed for the expression of the pheromone precursor genes ppg1 and ppg2 that were shown previously to be required for fruiting body development (Mayrhofer et al., 2006). Thus, the effect of asf1 on mating type and pheromone gene expression seems to be rather repressing. However, this is not the case for all develop-
mental genes, as expression of pro41, a gene encoding an ER membrane protein essential for sexual development (Nowrousian et al., 2007a), is not significantly affected by either deletion or overexpression of asf1 (Fig. 4A). To address the question whether asf1 has an effect on genes for secondary metabolism, similar, e.g. to the global © 2012 Blackwell Publishing Ltd, Molecular Microbiology, 84, 748–765
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Fig. 4. Expression of developmentally regulated genes in the S. macrospora Dasf1 mutant and complemented transformants. A. Quantitative real-time PCR analysis of expression of developmental genes in the asf1 knockout strain and complemented transformants. Transcript levels are given relative to the wild type, the mean and standard deviation (positive values; for better visualization standard deviations for negative expression ratios are shown in the negative direction) of two independent biological replicates are shown. Dashed lines indicate twofold up- or downregulation. For asf1, no expression was detected in the asf1 deletion mutant as expected (indicated by an asterisk). The following strains were grown for 3 days: Dasf1, S90177; Dasf1::NTAP-asf1, S99578; Dasf1::egfp-asf1, S99588. The number 00527 indicates the polyketide synthase gene SMAC_00527. B. Northern blot analysis of the app (abundant perithecial protein) gene. Strains as indicated in A were grown for 3–5 days.
regulator gene laeA in Aspergillus fumigatus (Perrin et al., 2007), we analysed the expression of four genes involved in polyketide biosynthesis. SMAC_00527 (SMU2918) and fbm1 are located within a cluster of 13 putative polyketide biosynthesis genes and encode a putative polyketide synthase and a monooxygenase respectively. All genes from this cluster are strongly upregulated during sexual development, and deletion of fbm1 leads to delayed maturation of fruiting bodies (Nowrousian, 2009). The genes pks and tih encode a polyketide synthase and trihydroxynaphtalen reductase, respectively, that are required for melanin formation, and therefore for the black colouring of fruiting bodies and ascospores (Engh et al., 2007a; Nowrousian et al., 2012). In contrast to polyketide biosynthesis genes in other fungi, these two genes are not part of a cluster, but are located separately within the S. macrospora genome. Interestingly, expression analysis showed that the clustered genes SMAC_00527 and fbm1 are strongly downregulated in the asf1 mutant and expression is restored in complemented transformants, whereas expression of pks
and tih is upregulated in the deletion mutant and the overexpression strains (Fig. 4A). This might indicate that asf1 is necessary for the activation of secondary metabolite gene clusters, and that non-clustered genes are either repressed (e.g. the pheromone genes, pks and tih) or not affected (e.g. pro41) by asf1. However, expression analysis of the app gene showed that asf1 is also required for transcriptional upregulation of genes that are not involved in secondary metabolism and are not clustered (Fig. 4B). In the wild type, app is only expressed under conditions of fruiting body development, and it is downregulated in several mutants that are blocked at the stage of protoperithecium formation (Nowrousian et al., 2007b). Northern blot analysis of app expression during a developmental time-course showed that the app transcript is not detectable in the asf1 mutant, but expression is restored in the complemented transformants (Fig. 4A). Thus, asf1 is required for correct expression of several developmentally regulated genes, including but not limited to clustered genes for secondary metabolism.
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Fig. 5. Fluorescence microscopic analysis of S. macrospora ASF1 localization. The asf1 deletion mutant (strain S90297) was transformed with plasmids pSN193 and pRH2B expressing an asf1–egfp and an hH2B–TdTomato fusion construct respectively (top row). As a control the wild type (S91237) was transformed with plasmids p1783-1nat and pRH2B expressing egfp and an hH2B–TdTomato fusion construct respectively (bottom row). EGFP alone localizes to the cytoplasm, while the merged images show colocalization of ASF1–EGFP and HH2B–TdTomato, confirming that the S. macrospora ASF1 is a nuclear protein. Bar, 20 mm.
Fluorescence microscopy demonstrates nuclear localization of the S. macrospora ASF1 The function of ASF1 as a histone chaperone requires its localization in the nucleus. To test whether the S. macrospora ASF1 indeed localizes to the nucleus, fluorescence microscopy was performed with a deletion strain complemented with plasmid pSN193 expressing an S. macrospora asf1–egfp fusion construct (Fig. 5). pSN193 restored fertility in the transformants, indicating that the ASF1–EGFP fusion protein is functional (data not shown). Additionally, the strain was co-transformed with plasmid pRH2B, encoding histone H2B fused to TdTomato (HH2B–TdTomato), which was used as a nuclear marker. As a negative control, fluorescence microscopy was performed with wild-type transformants expressing only egfp from plasmid p1783-1nat, and HH2B–TdTomato from plasmid pRH2B. The merged images show the expected localization of EGFP to the cytoplasm in the control strain; the ASF1–EGFP fusion protein in the complemented transformants localizes to the nucleus as indicated by colocalization with the red fluorescing HH2B–TdTomato, confirming the nuclear localization of S. macrospora ASF1 (Fig. 5). Tandem-affinity purification and mass spectrometry identify putative S. macrospora ASF1 interaction partners Based on its high degree of homology to characterized ASF1 proteins in other organisms, and its nuclear localization, S. macrospora ASF1 seemed likely also to function as a histone chaperone. To acquire further evidence for such a role, we performed tandem-affinity purification (TAP) in combination with mass spectrometry (MS) by multidimensional protein identification technology (MudPIT) to identify ASF1 interaction partners. The combination of TAP and MudPIT allows a highly specific identification of in vivo interacting proteins, because of the two-step affinity
purification, and the detection of proteins under native conditions, whereby direct and indirect interactions within complete protein complexes can be identified (Rigaut et al., 1999; Wolters et al., 2001). For TAP analysis, plasmid pDS27.9 was constructed encoding an N-terminal fusion of the S. macrospora ASF1 with a codon optimized TAP-tag from Aspergillus nidulans (Busch et al., 2007) under control of a constitutive promoter (Fig. 6A). Transformation of the sterile S. macrospora asf1 mutant with the plasmid restored fertility, indicating that the construct was functional. NTAP–ASF1 expression of several transformants was tested by SDS-PAGE and immunodetection with an antiCBP antibody. A single spore isolate (S99578) from a transformant with strong NTAP–ASF1 signal was chosen for TAP analysis. The crude extract of S99578 contained the NTAP–ASF1 fusion with the expected size of about 50 kDa (Fig. 6B), whereas the final eluate showed a distinct band of about 40 kDa representing the NTAP–ASF1 without protein A, as well as another band at 25 kDa and several weak bands representing the TEV protease and putative interaction partners of ASF1 respectively. TAP analysis was performed three times independently (three biological replicates), and the pellets of the final elutions were analysed by MudPIT. In each case, about five different peptides of ASF1 were recognized, thus clearly identifying ASF1 in the final elution (Table 1). Figure 6C shows the coverage of ASF1 peptides detected by MS and the corresponding regions of the protein. No peptides from the C-terminal region of ASF1 were detected, most likely because of the trypsin digestion performed as a preliminary step of MS. The density of trypsin cleavage sites in the C-terminal part of ASF1 is low, resulting in larger fragments that are not detected efficiently by MS. In addition to ASF1, several other S. macrospora proteins were identified. To differentiate between putative interaction partners and contaminants, we compared our results to results obtained by using two other © 2012 Blackwell Publishing Ltd, Molecular Microbiology, 84, 748–765
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Fig. 6. Tandem-affinity purification with S. macrospora ASF1, and verification of interactions by yeast two-hybrid analysis. A. The S. macrospora ASF1 was fused with an N-terminal TAP-tag. The TAP-tag comprises protein A for the first purification step with IgG beads, a TEV protease cleavage site and a calmodulin-binding peptide (CBP) for the second purification step with calmodulin beads. B. SDS-PAGE (top) and immunodetection (bottom) of NTAP–ASF1 were performed according to Bloemendal et al. (2012). The crude extract (CE) of the NTAP–ASF1-expressing strain S99578 comprises the total of soluble protein before purification. After each purification step, an aliquot of the flow-through (WS1 and WS2) and of the elution (TEV elution and final elution) was separated by SDS-PAGE. Using an anti-CBP antibody, the NTAP–ASF1 with the expected size of 50 kDa was detected in CE and WS1. IgG-bound protein was eluted by TEV cleavage and collected in the TEV elution; however, NTAP–ASF1 was not detectable in this step because of the high dilution factor. For the second purification step, the TEV eluate was incubated with calmodulin beads. The corresponding flow-through (WS2) did not contain any band representing NTAP–ASF1, indicating that bulk of the fusion protein remained bound to the calmodulin beads. Finally, NTAP–ASF1 and its putative interaction partners were eluted and precipitated. The resulting pellet was resuspended and an aliquot was used for SDS-PAGE. This shows a distinct band of about 40 kDa representing the NTAP–ASF1 without protein A (CBP-ASF1, also detected by immunoblotting), as well as another band at 25 kDa and several weak bands representing the TEV protease and putative interaction partners of ASF1 respectively. C. Peptide coverage of ASF1 during mass spectrometry. The regions of the S. macrospora ASF1 (top) and the corresponding peptides (bottom) that were identified are labelled in grey. D. Yeast two-hybrid analyses of interactions of ASF1 and proteins identified by TAP-tag/MudPIT. Yeast strains expressing a fusion construct of ASF1 and the activation domain of the GAL4 transcription factor (based on pGADT7) and fusion constructs of ASF1, histone H3, histone H4, the 14-3-3 protein or RSC8, respectively, with the binding domain of the GAL4 transcription factor (based on pGBDKT7) were grown on SD10 minimal medium (-trp, -leu, selecting for plasmids), SD17 minimal medium (-trp, -leu, -ade, -his), or analysed using the lacZ test (based on SD10-grown colonies). Growths on SD17, and blue colorization of colonies in the lacZ test indicate interaction of the proteins fused to the activation and binding domains.
S. macrospora proteins as baits for TAP analysis that are not localized to the nucleus: SPG20 and PRO22 (I. Teichert and S. Bloemendal, pers. comm.). Proteins identified with one of these proteins as baits and common TAP contaminants (Gingras et al., 2005) were excluded from our results. This exclusion step left 27 proteins that were identified with two or more different peptides in at least one TAP experiment as putative ASF1 interaction partners (Table S2). A functional classification of these proteins by the FunCat service of the MIPS (Ruepp et al., 2004) confirmed the assumption that ASF1 is a nuclear histone chaperone: about 45% were categorized as cell cycle and DNA-processing proteins, and 40% were associated with putative functions in transcription (data not shown). Of
these proteins, more than 50% were predicted to be localized in the nucleus. Verification of ASF1 interaction with histones H3 and H4 by yeast two-hybrid analysis Several putative ASF1 interaction partners are of particular interest because their homologues are associated with chromatin- and/or ASF1-related functions in other organisms; among these are the core histones H2A, H2B, H3 and H4 (Table 1). Histones H3 and H4 were previously shown to directly interact with ASF1 in other organisms (Mousson et al., 2005; English et al., 2006). To address the question of whether this also is the case in
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Table 1. Putative ASF1 interaction partners with chromatin-related functions that were identified by mass spectrometry following tandem-affinity purification (all isolated proteins are given in Table S2). Orthologue in S. macrospora locus tag
# ident.a
Ø Pept.b
PFAMc
N. crassac
S. cerevisiaec
SMAC_08608 (ASF1) SMAC_02364
3
5
ASF1
NCU09436 (putative ASF1)
ASF1
3
6
Histone
HHF1 or HHF2
SMAC_02363 SMAC_08324 SMAC_08323 SMAC_01844
2 2 3 2
1,5 2 2 2
Histone Histone Histone SWIRM
SMAC_04577 SMAC_06775 SMAC_00082
1 1 1
2 2 2
14-3-3 protein CoA ligase ATP-dependent DNA ligase domain
NCU01634 and NCU00212 (histone H4 genes hH4-1 and hH4-2) NCU01635 (histone H3) NCU02437 (histone H2A) NCU02435 (histone H2B) NCU08003 (related to subunit RSC8 of nucleosome remodelling complex RSC) NCU02806 (putative 14-3-3 protein) NCU06785 (putative ATP citrate lyase-subunit 1) NCU06265 (hypothetical protein)
HHT1 HTA1 or HTA2 HTB1 or HTB2 RSC8 – LSC1 DNL4
a. # ident., number of TAP analyses in which the interaction was identified. b. Ø Pept., average number of different peptides identified by mass spectrometry. c. Results of PFAM (protein family) and BLASTP analyses for the identification of protein families or orthologues in N. crassa and S. cerevisiae, respectively, with e-values < 10-5.
S. macrospora, we performed yeast two-hybrid analyses. Fusion constructs were generated of the yeast GAL activation and binding domains, respectively, with cDNAs for ASF1, H3 and H4 (see Experimental procedures). Plasmids were co-transformed into the yeast strain PJ69-4a. Transactivation tests were negative for all constructs. Interaction analyses were carried out on minimal medium and with the lacZ test (Rose and Botstein, 1983; James et al., 1996; Kaufman et al., 1997). As expected, histones H3 and H4 interact with each other (data not shown). ASF1 showed strong interactions with both histones H3 and H4, but not with itself (Fig. 6D). Taken together, these data indicate that ASF1 acts as a functional histone chaperone in filamentous ascomycetes by forming a complex with the core histones H3 and H4. We also tested whether ASF1 interacts with RSC8 and the 14-3-3 protein, both of which were identified by TAP-tag/MS (Table 1). However, using yeast two-hybrid analyses, no direct interaction of ASF1 with these proteins was found (Fig. 6D). Phenotypic characterization of S. macrospora deletion mutants of the histone chaperone genes rtt106 and cac2 Studies in yeast and animals have identified several H3/H4 histone chaperones in addition to ASF1 that are involved in nucleosome assembly and dissociation (Avvakumov et al., 2011). Among these are RTT106, and the protein complex CAF-1 (chromatin assembly factor 1). Current models predict a role for CAF-1 mostly in replication-dependent chromatin assembly (Kaufman et al., 1997; Takami et al., 2007), although recent reports indicate an additional role in transcription (Kim et al., 2009; Heyd et al., 2011). RTT106, similar to ASF1, is implicated both in replication-dependent
and in replication-independent processes (Huang et al., 2005; Kim et al., 2007; Mousson et al., 2007; Takahata et al., 2009; Silva et al., 2012). To address the question whether these histone chaperones are also necessary for fruiting body formation, we generated S. macrospora deletion mutants in the genes rtt106 (SMAC_03589) and cac2 (SMAC_01629). cac2 encodes a subunit of the CAF-1 complex (Table S3) that was shown to be essential for the function of this complex in yeast and vertebrates, and directly binds ASF1 (Kaufman et al., 1997; Krawitz et al., 2002; Mello et al., 2002; Takami et al., 2007). RTT106 is also present in a complex with ASF1 in yeast, this interaction is mediated by the histones H3 and H4 (Lambert et al., 2010). The S. macrospora cac2 and rtt106 genes both are transcriptionally downregulated during vegetative growth when compared with sexual development, and the strongest expression was observed during sexual development after three days (Fig. 7A), similar to what was found for asf1 (Fig. 2). Interestingly though, and in contrast to what was observed for the asf1 mutant, neither Drtt106 nor Dcac2 showed any developmental phenotype; vegetative growth and fruiting body formation are normal in these strains (Fig. 7B). This might indicate that the effect of the asf1 deletion on development is specific, and not a general effect of histone chaperone deletions.
Discussion Comparative functional genomics to identify developmental genes Fruiting bodies are complex, three-dimensional structures that serve as organs of sexual development in filamentous asco- and basidiomycetes. In recent years, genes involved © 2012 Blackwell Publishing Ltd, Molecular Microbiology, 84, 748–765
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Fig. 7. Expression analysis of S. macrospora cac2 and rtt106 and phenotypic characterization of the corresponding deletion mutants. A. Expression of cac2 and rtt106 during a time-course of sexual development (3–5 days) and during vegetative growth. Transcript levels were determined by quantitative real-time PCR with two independent biological replicates. Growth conditions were as described in Fig. 2. B. Analysis of sexual development. Strains were grown for 7 days on BMM, in the small boxes (bottom right) of each photograph, enlarged selections of each overview are shown. Perithecia are visible as black dots on the surface of the cultures. For comparison, the sterile asf1 deletion mutant was included in the analysis. Wild type, strain S91327; Dasf1, strain S90177; Dcac2, strain DS7185; Drtt106, strain DS6980.
in fruiting body development of several model ascomycetes like A. nidulans, Podospora anserina, N. crassa and S. macrospora were identified by both forward and reverse genetic approaches (Pöggeler et al., 2006a; Kück et al., 2009). Forward genetics studies use mutants to identify genes that when disrupted result in a specific phenotype. For example, the complementation of sterile S. macrospora mutants, or the sequencing of mutant
genomes led to the identification of several developmental genes, and allowed the characterization of their specific functions within this differentiation process (Masloff et al., 1999; Nowrousian et al., 1999; 2007a; 2012; Pöggeler and Kück, 2004; Kück, 2005; Engh et al., 2007b; Bloemendal et al., 2010). Reverse genetic approaches typically rely on the identification of candidate genes for further analysis via their expression patterns as determined by high-throughput methods, e.g. microarrays. While these analyses facilitate the simultaneous screening of many genes, they suffer from a high number of false-positives, i.e. genes that are only co-regulated instead of being directly involved in the target process. Therefore, increasing the sensitivity for selection of functionally relevant genes is of interest, particularly by combining functional genomics analysis with additional criteria such as identifying genes with conserved expression patterns in two or more species. This concept is based on the hypothesis that evolutionary conservation of expression is an important criterion for a gene’s functional relevance (Stuart et al., 2003; Snel et al., 2004; Kalinka et al., 2010); therefore, comparative functional genomics is a useful tool for identifying genes that are key to the process of interest. As noted, we have recently carried out a small pilot study to verify that conserved expression patterns exist during fruiting body development in the distantly related ascomycetes S. macrospora and P. confluens (Nowrousian and Kück, 2006). Furthermore, it was shown that a putative polyketide biosynthesis gene with conserved expression pattern in S. macrospora and N. crassa is involved in sexual development (Nowrousian, 2009). Here, we present a larger study by comparing publicly available microarray data from S. macrospora and F. graminearum (Qi et al., 2006; Nowrousian et al., 2007a). Interestingly, genes involved in metabolism and energy-related processes are overrepresented among the genes that are downregulated during sexual development in both species, while genes with relevance for differentiation processes, such as transcription/RNA processing, protein activity regulation, cell fate (differentiation/apoptosis), and cell wall biogenesis are significantly overrepresented among the upregulated genes. These findings support a long-standing hypothesis that the fungal mycelium accumulates energy during vegetative growth and that a metabolically ‘competent’ mycelium subsequently supports the growing fruiting bodies (Moore-Landecker, 1992; MacKenzie et al., 1993; Wessels, 1993; Pöggeler et al., 2006a). One of the genes showing a conserved pattern of upregulated expression in both species was the predicted asf1 (anti-silencing function) gene. asf1 encodes a highly conserved eukaryotic histone chaperone specific for histones H3 and H4. We demonstrated that asf1 is not only upregulated during fruiting body development in S. mac-
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rospora and F. graminearum but also in N. crassa and the distantly related ascomycete P. confluens. The latter is a member of the Pezizales and forms apothecia, whereas the other three species belong to the Sordariomycetes and produce perithecia. Thus, asf1 upregulation is not restricted to fungi that form perithecia, but is a conserved trait that can also be found in an ascomycete that develops a more basal type of fruiting body. Further analyses showed that asf1 is an essential gene for sexual development in S. macrospora. Additionally, an asf1 deletion mutant of S. macrospora can be complemented to fertility with the P. confluens asf1 gene, indicating conservation of asf1 function in filamentous fungi. Thus, our study confirms that evolutionary conserved expression patterns can serve as strong indicators of genes that are functionally relevant in a target process. The S. macrospora asf1 encodes a conserved histone chaperone that is essential for sexual development In general, histone chaperones are involved in the organization of chromatin by mediating the assembly of histones (H2A, H2B, H3 and H4) and DNA into nucleosomes, as well as the disassembly of intact nucleosomes into their components (Das et al., 2010). On the one hand, this compaction of genomic DNA into chromatin ensures the protection of the genetic material; on the other hand, it prevents the rapid access of DNA-interacting factors within the scope of central cellular processes. Thus, the co-ordinated positioning of nucleosomes by histone chaperones has a strong effect on replication (Sogo et al., 1986), transcription (Schwartz and Ahmad, 2005) and DNA repair (Moggs et al., 2000). The histone chaperone ASF1 was first described in the yeast S. cerevisiae (Le et al., 1997; Singer et al., 1998; Tyler et al., 1999) and has since been characterized in a number of eukaryotic species, such as the plant Arabidopsis thaliana, the worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster and the mammalians Homo sapiens and Mus musculus (Grigsby et al., 2009; Pilyugin et al., 2009; Sunavala-Dossabhoy and De Benedetti, 2009; Zhu et al., 2011); however, to the best of our knowledge, this study is the first of ASF1 in filamentous fungi. The S. macrospora asf1 deletion mutant generated in this study exhibits broad developmental defects. The knockout strain is sterile and shows growth retardation. The latter finding is comparable to results from previous studies with deletion of ASF1 in S. cerevisiae, which led to a reduced cell division rate (Le et al., 1997; Singer et al., 1998; Tyler et al., 1999). Interestingly, asf1 knockouts in other eukaryotes like Schizosaccharomyces pombe, D. melanogaster and DT-40 chicken cell cultures are lethal (Moshkin et al., 2002; Umehara et al., 2002; Sanematsu et al., 2006). Mousson et al. (2007) hypothesized that in
S. cerevisiae other histone chaperones, such as CAF-1, partially take over the role of ASF1, and a similar effect might occur in filamentous ascomycetes. However, to the best of our knowledge, the S. macrospora asf1 knockout mutant is the only viable multicellular eukaryote with an asf1 deletion so far, making it an ideal model organism for the study of ASF1 function with respect to cell differentiation and development. We showed that ASF1 is localized in the nucleus and interacts with histones H3 and H4, indicating that it is a functional histone chaperone. Histones H3 and H4 are the main targets of ASF1 in other organisms, where ASF1 directly interacts with the C-termini of both H3 and H4 (Tagami et al., 2004; English et al., 2005; 2006; Natsume et al., 2007). In addition to H3 and H4, we purified the histones H2A and H2B in the TAP experiments. A dimer of H2A and H2B interacts with an H3–H4 tetramer and DNA to build the nucleosome (Das et al., 2010). A direct interaction between ASF1 and H2A and H2B has not been observed; however, the presence of both H2A and H2B in the final protein eluate might result from the potential of the TAP-tag approach to identify indirect interactions within large protein complexes (Rigaut et al., 1999). Additional evidence for a role of the S. macrospora ASF1 in chromatin organization comes from the identification of a putative RSC8 protein as part of a complex containing ASF1. RSC8 is a subunit of the nucleosome remodelling complex RSC from S. cerevisiae. The RSC complex is a member of the SWI/SNF family of chromatin remodelers and essential for viability of yeast during mitotic growth (Cairns et al., 1996; Treich and Carlson, 1997; Lorch et al., 1998; Neely and Workman, 2002). It is generally agreed that histone chaperones interact with ATP-dependent chromatin remodelers during the energetically unfavoured process of nucleosome disassembly that requires the disruption of the histone–DNA interaction (Lorch et al., 2006). Another putative ASF1 interaction partner identified in the TAP analysis is a 14-3-3 protein. Members of the 14-3-3 protein family bind other, typically phosphorylated proteins and can induce conformational changes in their binding partners. They are involved in numerous cellular processes including the DNA-damage response and cell cycle regulation (Yahyaoui and Zannis-Hadjopoulos, 2009), and can interact with histones and histone deacetyltransferases (Healy et al., 2011). We could not show a direct interaction of ASF1 with RSC8 or the 14-3-3 protein in yeast two-hybrid analyses; however, this might be due to the fact that the TAP method allows identification of large protein complexes, not all subunits of which directly interact with each other. Further putative ASF1 interaction partners that were identified by TAP-tag analysis and that might be involved in chromatin-related functions are an ATP-dependent DNA ligase, and an ATP citrate lyase subunit. The yeast homo© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 84, 748–765
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logue of the purified DNA ligase (DNL4) is involved in the repair of double-strand breaks mediated by the NHEJsystem (non-homologous end joining), coupled to meiosis of S. cerevisiae (Schär et al., 1997). ATP-citrate lyases (ACLs) mediate the synthesis of acetyl-coenzyme A, and the ACL subunit identified in the TAP-tag analysis was shown previously to be essential for sexual development in S. macrospora (Nowrousian et al., 1999). Furthermore, in mammalian cells, ACL is involved in regulation of histone acetylation and thus gene expression, as a response to stimulation by growth factors within cellular differentiation and with respect to the bioenergetic status of the cell (Wellen et al., 2009). It is tempting to speculate that ASF1 of filamentous ascomycetes also is involved in the connection of metabolism and differentiation processes. However, ACL1 was also found in TAP-tag studies in our laboratory using other proteins as bait, therefore it might be a frequent contaminant within TAP protein extracts (I. Teichert, pers. comm.). Verification of the putative ASF1-ACL1 interaction by yeast two-hybrid analysis was inconclusive because of transactivation activity of the acl1-containing constructs (data not shown). Additional putative interaction partners of ASF1 that were found in other organisms are the histone chaperones RTT106 and CAF-1 (Mello et al., 2002; Lambert et al., 2010). Similar to asf1, rtt106 and the gene for the CAF-1 subunit cac2 are transcriptionally upregulated during sexual development in S. macrospora; however, the deletion mutants are normal with respect to growth and sexual development. This might indicate that the sterility of the asf1 deletion mutant is not a general effect of histone chaperone mutants, and it points to a specific role of asf1 during fruiting body development. However, another explanation for the lack of developmental phenotypes in Dcac2 and Drtt106 might be that certain histone chaperones might functionally substitute each other, or that these genes are required under conditions outside of the laboratory. Further studies, e.g. including double-deletion mutants or different growth conditions will be needed to clarify the role of histone chaperones in fungal development. Taken together, the results presented here indicate that the S. macrospora ASF1 acts as a functional histone chaperone. Therefore, it might have a central role in chromatin-related processes including transcription (Park and Luger, 2008; Bai and Morozov, 2010; Weake and Workman, 2010). In S. cerevisiae, ASF1 is involved in chromatin-mediated activation and repression of single genes as well as genome-wide transcriptional regulation (Le et al., 1997; Adkins et al., 2004; 2007; Zabaronick and Tyler, 2005; Rufiange et al., 2007; Williams et al., 2008; Minard et al., 2011a,b). We analysed the expression of developmental genes in the S. macrospora asf1 deletion mutant and overexpressing strains, and found that asf1 is required for correct transcriptional activation of some, and
repression of other genes. Among the genes that are upregulated in the asf1 mutant are the mating type genes Smta-1 and SmtA-2, and the pheromone precursor genes ppg1 and ppg2, all of which are required for sexual development (Mayrhofer et al., 2006; Pöggeler et al., 2006b; Klix et al., 2010). Upregulation of ppg1 and ppg2 was also observed in the sterile mutants pro1, pro11 and pro22, all of which only form protoperithecia, similar to the Dasf1 mutant; and it was speculated whether this effect is caused by the lack of a signal to shut down pheromone gene expression after a certain developmental stage has been reached (Nowrousian et al., 2005). We also specifically looked at secondary metabolism genes, because deregulation of secondary metabolite gene clusters in deletion mutants of the global regulator laeA is correlated with broad developmental defects in several filamentous fungi (Kale et al., 2008; Hoff et al., 2010; Sarikaya Bayram et al., 2010; Wiemann et al., 2010). In contrast to many other filamentous fungi, there are only two putative polyketide biosynthesis clusters present in the S. macrospora genome; all other secondary metabolism genes are located at separate positions (nonclustered) within the genome. Of the two clusters, one is developmentally upregulated and contains the fbm1 gene that was shown to be involved in fruiting body maturation, whereas the other cluster was most likely acquired by horizontal gene transfer and is not developmentally regulated (Nowrousian, 2009; Nowrousian et al., 2010). We analysed the expression of two genes from the developmentally regulated cluster, both of which are strongly downregulated in the asf1 deletion mutant. In contrast to this, the two non-clustered polyketide biosynthesis genes pks and tih are upregulated. However, expression analysis of the developmentally regulated gene app, which is strongly downregulated in the asf1 mutant, showed that transcriptional activation by asf1 is not restricted to clustered secondary metabolism genes. Whether the observed effects of asf1 on the transcription of developmental genes are directly caused by changes in chromatin structure mediated by ASF1 or are more indirect effects remains to be elucidated. However, one can speculate that the broad developmental defects of the S. macrospora asf1 deletion mutant at least in part result from ASF1 functioning as a global transcriptional regulator. For future studies, it will be interesting to explore if there are connections between changes in nucleosome architecture that ASF1 and other histone chaperones might mediate, and transcriptional activation by development-specific transcription factors.
Experimental procedures Strains, growth conditions and transformation Escherichia coli strain XL1-blue MRF’ (Jerpseth et al., 1992) served as host strain for cloning and propagation of recombi-
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nant plasmids constructed by standard laboratory techniques (Sambrook and Russel, 2001). Alternatively, S. cerevisiae strain PJ69-4A (James et al., 1996) was used as host for homologous recombination experiments performed according to Colot et al. (2006). All fungal strains used in this study are listed in Table S4. Standard growth conditions and transformation protocols for S. macrospora were as described previously (Masloff et al., 1999; Nowrousian et al., 1999). Growth conditions allowing vegetative or sexual development of S. macrospora for subsequent RNA extraction and Northern blot or quantitative real-time PCR, as well as the inoculation of strains in race tubes for growth-rate determination have been described by Nowrousian and Cebula (2005). Standard growth conditions for P. confluens were according to Nowrousian and Kück (2006). The wild type was incubated in constant light for fruiting body development or in constant darkness or shaken culture for vegetative growth.
Cloning procedures All plasmids used in this study are listed in Table S5 and oligonucleotides are given in Table S6. For complementation of the asf1 deletion strain and localization analysis of ASF1 (see below) vectors pSN79.40 and pSN193 were generated using standard techniques (Sambrook and Russel, 2001). Briefly, the full-length S. macrospora asf1 was amplified from genomic DNA with oligonucleotides SMU9436-Nco1 and SMU9436-Nco1, thus introducing NcoI sites at both ends of the asf1 ORF. The PCR fragment was then cloned into pDrive (Qiagen) and sequenced. Subsequently the NcoI fragment containing asf1 was cloned into vectors pN-EGFP/NcoI and p1783/NcoI allowing the expression of an EGFP–ASF1 or an ASF1–EGFP fusion protein respectively. The P. confluens asf1 gene (Pcasf1) was cloned through a combination of heterologous and inverse PCR. First, a part of the ORF was amplified by heterologous PCR using N. crassaderived oligonucleotides PCU9436-Fw1 and PCU9436-Rv. The resulting fragment was cloned into pDrive and sequenceverified. Based on this sequence, inverse PCR (Ochman et al., 1988; Nowrousian et al., 2007a) allowed the amplification of regions adjacent to the fragment. For this purpose, appropriate restriction enzymes were selected by Southern analysis using a probe obtained by PCR on P. confluens genomic DNA with oligonucleotides PCU9436probe-fw and PCU9436probe-rv. Genomic P. confluens DNA was then digested with SacI and HindIII, and resulting fragments were self-ligated using T4 DNA ligase (Roche). Subsequently, genomic DNA fragments were amplified with HotMasterTaq polymerase (5 PRIME) using oliqonucleotide pairs invPCR9436-2/invPCR9436-3 and invPCR9436-1/ invPCR9436-2 respectively. The PCR fragments were cloned into vector pDrive (Qiagen) and sequenced. In order to annotate the Pcasf1 gene, the corresponding cDNA was amplified using primer pairs PCU9436-fw1/PCU9436-rv, PCU9436-I3fw/PCU9436-I3-fw, PCU9436-I4-fw/PCU9436-I4-fw and PCU9436-5-utr-fw/PCU9436-5-utr-rv. The fragments were cloned into pDrive (Qiagen) and sequence-verified. The resulting sequences were compared with the genomic Pcasf1 sequence, thus verifying the positions of introns. For complementation analysis, plasmid pSG46.2 was constructed expressing an N-terminal fusion of the P. confluens
asf1 and egfp under control of the A. nidulans gpd promoter: Briefly, the full-length gene was amplified using oligonucleotides PCU9436-compl-1 and PCU9436-compl-2. The resulting fragments were cloned into pDrive (Qiagen) and sequenced. Subsequently the NcoI fragment containing the Pcasf1-ORF was ligated with pN-EGFP/NcoI. For TAP experiments in S. macrospora, plasmid pDS21, containing a TAP-tag for N-terminal fusion (NTAP) under control of the constitutive A. nidulans gpd promoter, was generated by homologous recombination in S. cerevisiae as described (Colot et al., 2006). Briefly, the NTAP was amplified by PCR using oligonucleotides pRSnat_5′NTAP and pRSnat_3′NTAP and vector pEHN8_NTAP (Bloemendal et al., 2010) as a template. For the recombination reaction the resulting fragment and AleI-linearized plasmid pRSnatEGFP (V. Klix and S. Pöggeler, unpubl. data) were transformed into S. cerevisiae strain PJ69-4A (James et al., 1996) as described previously (Bloemendal et al., 2010). Homologous recombination in yeast resulted in plasmid pDS21 that was digested with SpeI, and used for a further homologous recombination step together with full-length S. macrospora asf1, obtained by PCR on genomic DNA with oligonucleotides asf1_ntap_fw and asf1_ntap_rv. The resulting plasmid pDS27.9 contains an NTAP-tagged asf1. For yeast two-hybrid analyses, plasmids expressing fusion constructs of S. macrospora cDNAs and the GAL4 activation (AD) or binding domain (BD), respectively, were constructed by standard techniques. Briefly, cDNAs for the S. macrospora asf1, and the genes for histones H3 and H4, RSC8, and the 14-3-3 protein were amplified by PCR, cloned into pDrive and sequence-verified. Full-length cDNAs were then cloned into NdeI/EcoRI-digested vectors pGAD-T7 and pGBK-T7 (Clontech).
Construction of deletion mutants for asf1, rtt106 and cac2 The generation of an S. macrospora asf1 deletion strain by homologous recombination was done as described previously (Nowrousian and Cebula, 2005). Briefly, the asf1 (SMAC_08608) flanking regions 773 bp upstream and 1051 bp downstream of the asf1 open reading frame were amplified using oligonucleotide pairs 9436-ko1/9436-ko2 and 9436-ko3/9436-ko4 respectively. The upstream fragment was then inserted into PstI/BamHI-digested vector pSF27-34 and the downstream region into SalI/XbaI sites of the resulting construct to yield the asf1 knockout vector pKO-Asf1 that carries a Hygromycin B resistance cassette flanked by the asf1 upstream and downstream sequences. Knockout constructs for rtt106 (SMAC_03589) and cac2 (SMAC_01629) were generated by homologous recombination in yeast as described (Colot et al., 2006). The knockout constructs were transformed into an S. macrospora Dku70 strain (Pöggeler and Kück, 2006). Homologous integration events were verified by PCR and Southern blot analysis according to Nowrousian and Cebula (2005). Primary transformants carrying the deletion alleles were crossed against the S. macrospora wild type to generate a knockout strain without the Dku70 background. Single spore isolates S90177, S90279 and S90533 were chosen for further analysis of Dasf1, single © 2012 Blackwell Publishing Ltd, Molecular Microbiology, 84, 748–765
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spores DS6521 and DS6980 for Drtt106, and single spores DS7185, DS7195 and DS7521 for Dcac2.
Preparation of nucleic acids Nucleic acids of S. macrospora, RNA of N. crassa and DNA of P. confluens were isolated as described previously (Yarden and Yanofsky, 1991; Pöggeler et al., 1997a). RNA of P. confluens was prepared according to Nowrousian and Kück (2006) using the RNeasy lipid tissue mini kit (Qiagen) with a modified manufacturer’s protocol.
Quantitative real-time PCR Quantitative real-time PCR was carried out in a DNA Engine Opticon 2 (MJ Research) or a StepOnePlus (Applied Biosystems) with qPCR MasterMix for SybrGreen (Eurogentec or Promega) as described previously by Nowrousian et al. (2005). Oligonucleotide primers are given in Table S6.
Comparative analysis of microarray data Previously published microarray data from F. graminearum (Qi et al., 2006) and S. macrospora (Nowrousian et al., 2007a) were used for comparison. Briefly, differentially expressed genes from F. graminearum (Table S1 from Qi et al., 2006) were used to identify corresponding N. crassa homologues using BLAST searches (Altschul et al., 1990). In those cases for which corresponding probes existed on the N. crassa microarrays (Tian et al., 2007) that were used for the S. macrospora cross-species hybridization, expression in S. macrospora was compared with that in F. graminearum. Gene expression was regarded as similar if a gene was up- or downregulated during sexual development compared with vegetative growth in F. graminearum and regulated in the same direction in S. macrospora (Table S1). Functional classification of genes with similar regulation patterns was done using the ‘Functional Distribution of Gene Lists’ function of the MIPS FunCat catalogue (Ruepp et al., 2004) (http://mips.helmholtzmuenchen.de/proj/funcatDB/search_main_frame.html).
TAP and MS Sordaria macrospora strains were grown in P-Flasks with BMM liquid medium for 3 days at 27°C. Mycelium was harvested, deep frozen in liquid nitrogen and pulverized. The preparation of protein crude extracts was performed in protein extraction buffer [100 mM Tris-HCL pH 7.6, 250 mM NaCl, 10% glycerol, 0.5% NP-40, 2 mM EDTA, 2 mM DTT; Protease Inhibitor Cocktail IV (1:100 Calbiochem), 1 mM PMSF; 1 mM Benzamidin] as described previously (Kaufman et al., 1997). Purification of TAP-tagged proteins from crude extracts was done as described by Busch et al. (2007) and Bayram et al. (2008) with minor modifications as described by Bloemendal et al. (2012). TEV digestion was carried out with 350 U MobiTEV protease (Mobitec) in the presence of 1 mM Protease Inhibitor Cocktail IV (Calbiochem) at 4°C overnight on a spin wheel. The final eluate was precipitated with TCA (trichloroacetic acid). MS analysis of the acetone
washed pellet was performed using the MudPIT technology and Proteome Discoverer software (version 1.2) for interpretation of MS data as described previously (Kaufman et al., 1997). The data were searched against the predicted S. macrospora proteins (Nowrousian et al., 2010). Accepted results were characterized by a high peptide confidence with a score of 10.
Immunodetection Protein crude extracts were separated by 14% SDS-PAGE (Laemmli, 1970) and transferred onto PVDF membranes by Western blotting. Immunodetection of NTAP–ASF1 was performed with an anti-calmodulin-binding peptide antibody (1:2000, Milipore) and an anti-rabbit horseradish peroxidase (HRP)-linked secondary antibody (Cell Signaling Technologies) according to the manufacturer’s protocols.
Yeast two-hybrid interaction analysis For the verification of TAP results by yeast two-hybrid experiments, S. cerevisiae strain PJ69-4A (James et al., 1996) was transformed with AD- and BD-fusion constructs, and the resulting transformants were selected by leucine and tryptophane prototrophy respectively. Tests for transactivation and interaction tests were performed as described previously (Rose and Botstein, 1983; Jacobsen et al., 2002). Briefly, yeast strains expressing fusion constructs were grown on SD17 minimal medium (-trp, -leu, -ade, -his); growth indicates interaction of the proteins fused to the AD and BD domains. For analysis by lacZ test, strains were grown on medium without tryptophane and leucine (selecting for plasmids). Blue colorization of colonies in the lacZ test indicates interaction.
Microscopy All microscopic investigations were performed after growth of S. macrospora strains on BMM- or SWG-coated glass slides for 2–8 days as described by Engh et al. (2007b). For light and fluorescence microscopy an AxioImager microscope (Zeiss, Jena, Germany) was used with a Photometrix Cool SnapHQ camera (Roper Scientific) for capturing images. Images were then processed with MetaMorph (Molecular Devices) and Photoshop CS4 (Adobe).
Sequencing and sequence analysis Sequencing was performed by the sequencing service of the Faculty for Chemistry and Biochemistry, Ruhr-Universität Bochum, or by GATC Biotech AG (Konstanz, Germany). Alignments for the comparison of two or more protein or nucleotide sequences were executed using LALIGN (http://www.ch. embnet.org/software/LALIGN_form.html; Huang et al., 1990) and CLUSTAL W (Thompson et al., 1994) respectively. For the assignment of protein families to given protein sequences, the models from the PFAM database were searched using the HMMER (version 2.3.2) program HMMPFAM (Eddy, 1998; Green et al., 2005). The search for homologous nucleotide
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and protein sequences was performed using the BLAST algorithm (Altschul et al., 1997) searching the public databases provided by the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi), the Broad Institute (http://www.broadinstitute.org/annotation/ genome/neurospora/Blast.html) or the MIPS (http://mips. helmholtz-muenchen.de).
Nucleotide sequence accession numbers Sequence data of P. confluens asf1 gene locus were submitted to GenBank (NCBI) under Accession No. HQ700586.
Acknowledgements We thank Swenja Ellßel, Silke Nimtz and Susanne Schlewinski for excellent technical assistance, and Prof. Dr Ulrich Kück for general support. We also thank Dr Ines Teichert and Dr Sandra Bloemendal for kindly providing their TAP results as a negative control, as well as Prof. Dr Stefanie Pöggeler and Dr Volker Klix for cloning vectors for homologous recombination in yeast. This study was supported by the Deutsche Forschungsgemeinschaft (DFG, projects No. 407/2-1 and No. 407/4-1).
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