The Fumagillin Gene Cluster, an Example of Hundreds of Genes under veA Control in Aspergillus fumigatus

Aspergillus fumigatus is the causative agent of invasive aspergillosis, leading to infection-related mortality in immunocompromised patients. We previously showed that the conserved and unique-to-fungi veA gene affects different cell processes such as morphological development, gliotoxin biosynthesis and protease activity, suggesting a global regulatory effect on the genome of this medically relevant fungus. In this study, RNA sequencing analysis revealed that veA controls the expression of hundreds of genes in A. fumigatus, including those comprising more than a dozen known secondary metabolite gene clusters. Chemical analysis confirmed that veA controls the synthesis of other secondary metabolites in this organism in addition to gliotoxin. Among the secondary metabolite gene clusters regulated by veA is the elusive but recently identified gene cluster responsible for the biosynthesis of fumagillin, a meroterpenoid known for its anti-angiogenic activity by binding to human methionine aminopeptidase 2. The fumagillin gene cluster contains a veA-dependent regulatory gene, fumR (Afu8g00420), encoding a putative C6 type transcription factor. Deletion of fumR results in silencing of the gene cluster and elimination of fumagillin biosynthesis. We found expression of fumR to also be dependent on laeA, a gene encoding another component of the fungal velvet complex. The results in this study argue that veA is a global regulator of secondary metabolism in A. fumigatus, and that veA may be a conduit via which chemical development is coupled to morphological development and other cellular processes.

veA orthologs have been identified and characterized in other fungi [31,32] including other Aspergillus species, such as A. flavus [33][34][35], A. parasiticus [36] and the model filamentous fungus A. nidulans [37]. These previous studies provided abundant evidence of the role of veA as a regulator of both fungal morphological development and secondary metabolism. In 2003 our group described for the first time the role of veA as a global regulator of secondary metabolism in A. nidulans, including production of the mycotoxin sterigmatocystin [37]. veA also regulates the biosynthesis of other mycotoxins, including aflatoxin, cyclopiazonic acid and aflatrem in Aspergillus flavus [33], the synthesis of trichothecenes in F. graminearum [38], and the production of fumonisins and fusarins in Fusarium spp, specifically F. verticillioides and F. fujikuroi [32,39,40]. However, veA also controls the synthesis of other secondary metabolites known for their beneficial medical applications, for example, the beta-lactam antibiotic penicillin in A. nidulans and P. chrysogenum [37,41] as well as cephalosporin C in Acremonium chrysogenum [42].
In-depth studies of A. nidulans veA and its gene product also revealed mechanistic details of its mode of action. For instance, it is known that the VeA protein is transported to the nucleus by the KapA α-importin, and that this transport is promoted in the absence of light [43,44]. In the nucleus, VeA interacts with light-sensing proteins that also affect secondary metabolism and fungal differentiation, such as the red phytochrome-like protein FphA, which interacts with the blueresponsive proteins LreA-LreB [31,45]. In the nucleus VeA also interacts with VelB and LaeA [46,47]. VelB is another protein in the velvet family [47], and LaeA is a chromatin modifying protein that, like VeA, is required for the synthesis of numerous secondary metabolites [48,49]. In addition, a LaeA-like putative methyltransferase was also described to interact with VeA [50].
A microarray-based transcriptome study showed that A. fumigatus laeA affects the expression of 13 secondary metabolite gene clusters [51]; however, at that time the extent of veA regulation of the activation of secondary metabolite gene clusters was mostly unknown. With the goal of elucidating the full extent of veA-regulation of the A. fumigatus genome, particularly with respect to genes involved in secondary metabolism, we performed RNA sequencing analyses [52] and chemical characterization of A. fumigatus cultures, obtaining results consistent with a global regulatory pattern. This study also contributes to uncovering the regulation of novel secondary metabolite gene clusters in A. fumigatus. For example, an important discovery in our study is that veA and laeA, both of which encode velvet complex components, regulate the recently discovered gene cluster responsible for the synthesis of fumagillin [53]. Fumagillin has been intensely studied due to its potential in the treatment of amebiasis [54], microsporidiosis [55] and most recently, for its anti-angiogenic activity as inhibitor of the human type 2 methionine aminopeptidase (MetAP2) [56,57].

Strains and culture conditions
Aspegillus fumigatus strains used in this study are listed in Table S1. Fungal strains were grown on Czapek Dox media (Difco), unless otherwise indicated, and supplements for the corresponding auxotrophies as needed [58]. Solid medium was prepared by adding 15 g/liter agar. Strains were stored as 30% glycerol stocks at -80°C.

RNA extraction
Total RNA was extracted as previously described [13]. Briefly, conidia from the wild type, deletion veA (∆veA), complementation and over-expression veA (OEveA) strains were inoculated in Czapek-Dox (approximately 10 7 spores/mL) and grown as liquid stationary cultures at 37°C in the dark. Mycelia were collected 48 h and 72 h after inoculation and RNA was extracted using TRIzol (Invitrogen) following the manufacturer's instructions. RNA samples were further purified using QIAgen RNeasy mini kit as previously described [59].
Library preparation and RNA sequencing. RNA-Seq libraries were constructed and sequenced at the Vanderbilt Genome Sciences Resource using the Illumina Tru-seq RNA sample prep kit as previously described [61,62]. In brief, total RNA quality was assessed via Bioanalyzer (Agilent). Upon passing quality control, poly-A RNA was purified from total RNA and the second strand cDNA was synthesized from mRNA. cDNA ends were then blunt repaired and given an adenylated 3' end. Next, barcoded adapters were ligated to the adenylated ends and the libraries were PCR enriched, quantified, pooled and sequenced an on Illumina HiSeq 2000 sequencer.
Read alignment and quantification of gene expression. Illumina TruSeq adapters were trimmed from the 3' end of reads using the scythe software package (available from Buffalo, V. at https://github.com/ucdavis-bioinformatics/ scythe), and low-quality bases were trimmed using the sickle software package (available from Joshi, N. at https:// github.com/ucdavis-bioinformatics/sickle). Reads were aligned to the transcriptome using the bowtie read alignment software for single-end reads, with the maximum mismatches per read set at 2 and a seed length of 28 [63]. Read count per gene was calculated using SAMtools idxstats software [64]. For each sample, gene expression was quantified using the reads per Kilobase of exon per million mapped reads (RPKM) metric [65]. Differential expression was calculated between ∆veA and wild type, between OEveA and wild type, and between the complementation and wild-type strains. Two cutoffs were used to determine differentially regulated genes [61,62]. The first cutoff compared the fold difference between genes by calculating the relative RPKM (rRPKM = RPKM sample1 / RPKM sample2 ) for each gene. The second cutoff compared the proportion of reads mapping to a gene in different samples using Fisher's exact test with Bonferroni's correction for multiple comparisons. A gene was considered differentially regulated if the log 2 rRPKM value was equal to or greater than 2 and the Bonferroni-corrected Fisher's exact p-value was less than 0.05.
Gene ontology categorization. The gene ontology (GO) categorizations of differentially regulated genes were compared against the set of non-differentially regulated genes to identify GO categories that were specifically enriched in differentially upregulated or downregulated gene sets in the three strain comparisons. GO categorizations for each gene were obtained from the AspGD's GOSlim mapper for A. fumigatus Af293 [60]. AspGD's GOSlim mapper contains higher-order GO terms for the process, component, and function sections of GO. All comparisons were performed using Fisher's exact test with Bonferroni's correction for multiple comparisons.
Sliding window analysis. We used a sliding window analysis [61] to determine clusters of genes that were upregulated or downregulated in the A. fumigatus genome in ∆veA versus wild type and OEveA versus wild type comparisons. Briefly, each gene was encoded as upregulated, downregulated or not significantly differentially regulated according to our specified differential regulation cutoffs. Then, we calculated the cumulative binomial probability of observing every window of 24 genes along each chromosome. To account for multiple comparisons, we used an empirically derived false discovery rate (FDR) by randomly permuting the expression data 1,000 times and running the sliding window analysis on each permuted dataset. Our FDR cutoff was set conservatively at 0.01. After all clusters below this FDR cutoff were found, genomically overlapping windows were collapsed into a larger cluster. Due to the window size, regions at the beginning or end of these master clusters may contain stretches of non-differentially regulated genes. We have reported all clusters from the first to last differentially regulated gene found for each cluster and the start and end genes of all significant clusters.

Quantitative RT-PCR analysis
One microgram of total RNA was treated with RQI Dnase to remove possible DNA contamination. Then cDNAs were obtained by reverse transcription using Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega). Quantitative Real Time PCR was performed with an Agilent MX3000p thermocycler using SYBR green Jump Start Taq (Sigma). The primers used for gene expression analysis are listed on Table S2.

Metabolome analysis
Extractions of secondary metabolites.
Secondary metabolites were extracted as previously described [13]. Briefly, liquid Czapek-dox stationary cultures of the wild type, ∆veA, complementation and OEveA strains were grown as described. Supernatants were collected by filtration through sterile Miracloth™ (Calbiochem, USA) from 72 h and 120 h cultures. Fifteen mL of the culture filtrate was extracted with same amount of chloroform. Extracts were allowed to dry and were resuspended in 500µL of methanol; 10 µL aliquots were used for LC-MS analysis.
LC-MS. All solvents and other chemicals used were of analytical grade. All LC-MS analyses were performed on a Shimadzu 2010 EV LC-MS (Phenomenex® Luna, 5μ, 2.0 × 100 mm, C18 column) using positive and negative mode electrospray ionization with a linear gradient of 5-95% MeCN-H 2 O (0.1% formic acid) in 30 minutes followed by 95% MeCN for 15 minutes with a flow rate of 0.1 mL/min. The value of area under curve was observed by EIC (extracted ion chromatogram).

Generation of the fumR (Afu8g00420) deletion strain
Fusion Polymerase Chain Reaction (Fusion PCR) was used to create the deletion cassette of fumR as previously described [66]. First, Aspergillus parasiticus pyrG was PCR amplified from A. parasiticus genomic DNA using primers AparapyrGF-Linker and AparapyrGR-Linker (Table S2). The Aspergillus parasiticus pyrG fragment was ligated into pJET (Fermentas) yielding plasmid pSD38.1 Then, 1.5kb of 5' UTR and 3' UTR of fumR was amplified from A. fumigatus genomic DNA using primer pairs 420P1 & 420P2 and 420P3 & 420P4, respectively (Table S2). Aspergillus parasiticus pyrG was then amplified from pSD38.1 using primers 420P5 and 420P6. Three fragments were fused using primers 420P7 and 420P8 (Table  S2). Protoplast mediated fungal transformation was done as previously described [66] using CEA17ku80 (gift from Robert Cramer) as the host strain. Aspergillus parasiticus pyrG was utilized as selectable marker, resulting in a complete gene replacement of fumR in CEA17ku80. Transformants were first screened using PCR (data not shown) and Southern blot analysis. Other DNA manipulations were done as previously described [67].

Generation of the laeA deletion strain
The laeA deletion DNA cassette was also generated by fusion PCR. Briefly, a 1.5 kb 5' UTR fragment was first amplified from A. fumigatus genomic DNA with primers laeA_p1 and laeA_p2 (Table S2). A 1.3 kb 3' UTR fragment was also amplified from genomic DNA with primers laeA_p3 and laeA_p4 (Table S2). Aspergillus parasiticus pyrG was amplified from pSD38.1 using primers laeA_p5 and lae_p6. The three fragments were fused using primers laeA_p7 and laeA_p8 (Table S2) as previously described [66]. The laeA deletion cassette was transformed into CEA17ku80. Transformants were first screened using PCR (data not shown) and Southern blot analysis.

Hundreds of genes are differentially regulated by veA
Of the 9,784 genes in the A. fumigatus genome [60,68], 453 were upregulated and 1,137 were downregulated in the ∆veA strain when compared with the wild-type strain ( Figure 1; Table  S3). A similar pattern was observed in the OEveA versus wild type comparison, where 335 genes were upregulated and 908 genes were downregulated in the OEveA strain. In sharp contrast, comparison of the complementation strain and wild type showed that the two strains present very similar expression patterns.

Differentially regulated genes are dramatically enriched for secondary metabolism-related processes
Different GO process, component, and function categories are significantly enriched for both upregulated and downregulated genes in the two comparisons (Table 1). Importantly, enrichment analysis using GOSlim categories showed that differentially regulated genes in the ∆veA versus wild type and OEveA versus wild type comparisons have significant functional overlap (Table 1); specifically, 13 of the 19 GO categories that are enriched for either upregulated (3 categories) or downregulated (16 categories) genes in the ∆veA versus wild type comparison are also enriched and in the same direction in the OEveA versus wild type comparison. For example, both secondary metabolic process (GO:0019748) and toxin metabolic process (GO:0009404) GO function categories are enriched in downregulated genes from both comparisons.

Differentially regulated genes are non-randomly distributed across the A. fumigatus genome
We used a sliding window analysis [2] to determine clusters of genes that were upregulated or downregulated for the ∆veA versus wild type and OEveA versus wild type comparisons. In total, 31 downregulated gene clusters and 6 upregulated gene clusters were identified ( Figure 2). Ten downregulated clusters were found independently in both the ∆veA versus wild type and OEveA versus wild type comparisons, suggesting some similarity in phenotype between the αveA and OEveA strains. Twelve of the 31 downregulated clusters and 2 of the 6 upregulated clusters overlap with known or predicted secondary metabolic gene clusters [1,9]. For example, the gene clusters encoding for the secondary metabolites fumagillin, fumitremorgin G, and fumigaclavine C are all differentially regulated in at least one of the two strain comparisons ( Figure 3). All 11 genes in the fumigaclavine C biosynthetic gene cluster are downregulated in both the ∆veA versus wild type and OEveA versus wild type comparisons. In both the ∆veA versus wild type and OEveA versus wild type comparisons, 13 of the 15 genes in the fumagillin gene cluster are downregulated. All genes involved in fumitremorgin G are upregulated on the OEveA versus wild type comparison, but none of these genes are differentially regulated in the αveA versus wild type comparison.

veA regulation profile of secondary metabolite gene clusters in A. fumigatus partially differs from the laeA profile
Previous studies of the model fungus Aspergillus nidulans demonstrated that the veA gene product, VeA, interacts with other proteins in cell nuclei [45,47], among them LaeA. Additionally, Park et al. [69] used tandem affinity techniques [47] to show that the A. fumigatus LaeA co-purifies with A. nidulans VeA, suggesting that A. fumigatus VeA might also interact with A. fumigatus LaeA. LaeA is a putative methyl transferase that affects chromatin conformation [48]. This VeAinteracting protein has also been described as affecting expression of secondary metabolite gene clusters in A. fumigatus [51]. Our results indicate that although the regulation patterns of the two proteins overlap, they are not identical (Table 2). Specifically, five out of the nine clusters that Perrin et al. report as being under full laeA regulation are found to be in windows of differentially regulated genes in both the ∆veA versus wild type and the OEveA versus wild type comparisons. Of the remaining four clusters, two are not found to be differentially regulated and two have different expression patterns in the two comparisons. The Afu6g12040-2080 laeAregulated cluster was found to be part of a window of downregulated genes in the ∆veA versus wild type comparison but not in the OEveA versus wild type comparison. However, all five genes in this cluster are also downregulated in the OEveA versus wild-type comparison, and its lack of detection by the sliding window analysis is due to the gene cluster's small size and the test's stringency. Finally, of the four clusters Perrin et al. describe as being partially regulated by laeA, two are differentially regulated in both veA comparisons, one cluster is differentially expressed in the OEveA versus wild type but not in the ∆veA versus wild type comparison, and one cluster is not differentially expressed in either comparison.  Aspergillus fumigatus has the potential to produce 226 bioactive secondary metabolites [70], and the genes responsible for their synthesis are commonly associated in the form of gene clusters. Recently, Inglis et al. described 39 secondary metabolite gene clusters in the A. fumigatus genome [71], some of which are experimentally characterized and some of which are computationally predicted. The production of a number of secondary metabolites has been shown to be under the control of veA orthologs in different fungal species [32,33,[37][38][39][40][41][42]. In A. fumigatus we recently reported that the expression of gliotoxin genes and gliotoxin production are regulated by veA [13]. Additionally, in our current study, our RNA-seq data indicates that the expression of many secondary metabolite gene clusters is also veAdependent, strongly suggesting that veA affects the synthesis of other natural products in A. fumigatus. For this reason, we also used LC-MS to analyze the production of other compounds in wild type, ∆veA, complementation strain and OEveA cultures. Our data revealed that production of four additional secondary metabolites was also dependent on veA under the experimental conditions assayed, specifically fumagillin, fumitromorgin G, fumigaclavine C and glionitrin A. The production of these four compounds was notably decreased in the ∆veA strain. Relative amounts of fumagillin, fumitremorgin G, fumigaclavine C and glionitrinA in the ∆veA strain were 18%, 23%, 22% and 18% compared to the wild type levels, respectively ( Figure 4). The production of these compounds was also reduced in the OEveA with only 0.3%, 0.1%, 0.7% and 0.8% as compared to wild-type levels, respectively ( Figure 4).

veA controls the fumagillin gene cluster and fumagillin production by regulating the expression of fumR (Afu8g00420)
Due to the medical applications of fumagillin and fumagillinrelated compounds for their potential use in the treatment of amebiasis, microsporidiosis, and for their anti-angiogenic properties, we further characterized the genetic regulation of veA on the fumagillin gene cluster. RNA-seq analysis revealed that veA controls the fumagillin gene cluster, including fumR (Afu8g00420) (Figures 2 and 3), a gene encoding a putative C6 type transcription factor that our previous bioinformatics analysis identified within the fumagillin gene cluster, located in chromosome 8 [53]. We further validated these results by qRT-PCR analysis ( Figure S1). The expression levels of fumR were 12% in ∆veA and 5% in OEveA with respect to the levels in the wild type strain. Expression of Afu8g00370, which encodes a polyketide synthase (PKS) in the fumagillin cluster [53], was also evaluated ( Figure S1). We recently showed that expression of Afu8g00370 is necessary for the production of fumagillin, confirming the predicted role of Afu8g00370 as an indispensable PKS in fumagillin biosynthesis. Our data indicated that expression levels in ∆veA and OEveA strains were only 3% and 0.1%, respectively, compared to wild-type levels.

fumR is necessary for the expression of other genes in the fumagillin gene cluster
To gain insight into the function of the transcription factor encoded by fumR, this gene was deleted by gene replacement techniques using the A. parasiticus pyrG marker as described in the materials and methods section. The ∆fumR strain was confirmed by PCR (data not shown) and Southern blot analysis ( Figure S2). Deletion of fumR did not change growth rate or development in A. fumigatus (data not shown).
Our experiments showed that expression of the PKS gene, Afu8g00370, is regulated by fumR ( Figure 5). The expression of Afu8g00370 in the ΔfumR strain and control strain was analyzed by qRT-PCR at 48 h and 72 h post inoculation. At both time points examined, there was only negligible expression of Afu8g00370 in the ∆fumR mutant as compared to the wild-type levels. Furthermore, our analysis revealed that the expression of other genes in the fumagillin cluster is also under the control of fumR ( Figure 5). We examined the expression of Afu8g00520, the terpene cyclase involved in fumagillin biosynthesis [53] and Afu8g00380, an essential acyltransferase for fumagillin production [53]. In addition to the characterized genes in the fumagillin cluster, we analyzed the expression of other predicted genes in the cluster, including Afu8g00510, Afu8g00500, Afu8g00480, Afu8g470, Afu8g440 and Afu8g00430. Our results indicate that all genes were minimally expressed in the ∆fumR mutant compared to the wild type at both time points tested ( Figure 5). Only Afu8g00470 showed slightly higher expression than the other genes in the cluster in ∆fumR (20% and 36% at 48 h and 72 h post inoculation, respectively) as compared to wild-type levels.

fumR is required for fumagillin biosynthesis
Extracts from wild type and ∆fumR 72 h and 120 h cultures were subjected to LC-MS analysis as described in the material and methods section. The chemical analysis showed a peak corresponding to fumagillin in the wild type, with a retention time of 29.1 minutes ( Figure 6). However, this peak was completely absent in the ∆fumR strain, indicating that A. fumigatus is unable to produce fumagillin in the absence of fumR. The amount of fumagillin production in the wild type increased over time (Figure 6).

laeA regulates expression of fumR and the PKS gene Afu8g00370
The identity of the cluster involved in fumagillin biosynthesis was not elucidated at the time of the microarray study

Figure 3. Expression patterns of the fumagillin (A), fumitremorgin G (B), and fumigaclavine C (C) gene clusters in the ΔveA vs WT and OEveA vs WT comparisons.
Genes labeled with numbers correspond to locus tags without the chromosomal prefix or trailing zeros (i.e., the gene labeled 380 in the fumagillin cluster corresponds to Afu8g00380). The sole exception is fumR (Afu8g00420), the name given to the regulatory gene for the fumagillin cluster characterized in this study. Genes are color-coded by differential expression; blue indicates downregulation, red indicates upregulation, and white indicates no differential regulation. previously carried out with a ∆laeA mutant [51]. In a recent study we described the A. fumigatus fumagillin gene cluster [53], and in the present study we have demonstrated that veA regulates fumR, and that expression of fumR is necessary for the activation of other genes in the cluster. We also examined whether the expression of fumR was also dependent on laeA, analyzing the expression of this gene in a ∆laeA mutant and corresponding control strain. The ∆laeA strain was constructed as described in material and methods, and the strain was verified by Southern blot analysis ( Figure S3). qRT-PCR results indicated a near complete loss of fumR expression in ∆laeA under conditions that allowed its expression in the control strain  (3% and 8% at 48 h and 72 h as compared to wild-type levels) (Figure 7). Expression of Afu8g00370 was also downregulated in ΔlaeA (9% and 7% at 48 h and 72 h as compared to wildtype levels).

Discussion
Invasive aspergillosis is a disease caused by the ubiquitous opportunistic invasive mold Aspergillus fumigatus. In immunocompromised patients, the intraepithelial immune system of the lung is unable to properly eliminate the inhaled conidia, which then germinate [1][2][3][4][5][6]. Despite the prevalence of aspergillosis infection and the improvements in diagnosis, novel effective strategies to reduce Aspergillus infections are still needed. Deciphering the genetic mechanism controlling A. fumigatus cellular processes might provide the basis for the development of new strategies to prevent or treat aspergillosis.
Our study clearly established that veA is a global regulator of the A. fumigatus genome, affecting the expression of hundreds of genes, many of which are involved in secondary metabolism related processes, in non-random genomic locations. Secondary metabolites, also known as natural products, are  part of the fungal chemical arsenal important for habitat adaptation. Some of them are considered virulent factors playing an important role in the pathogen-host interaction. The most studied of these secondary metabolites is gliotoxin, known for its immunosuppressive properties [16,18,[72][73][74], for inhibiting phagocytosis in macrophage, and for induction of apoptosis [29,30]. We recently reported that expression of gliotoxin genes and concomitant gliotoxin production is dependent on veA in A. fumigatus [13]. In this study we demonstrate that veA controls the expression of 14 secondary metabolite gene clusters whose metabolite products are known and an additional 23 putative secondary metabolite gene clusters. Although veA exercised negative regulation on some gene clusters, overall veA acted as a positive regulator. The veA-dependent gene clusters regulatory pattern was not identical to that described for laeA [51], which encodes a VeAinteracting protein in the velvet complex [31,47,69], presenting some differences in their regulatory output. Among the gene clusters regulated by veA are those involved in the synthesis of fumitremorgin G, fumigaclavine C and fumagillin. Production of most of these compounds correlates with the veA regulatory pattern observed for the respective gene clusters, with the exception of fumitremorgin, suggesting that in this case other veA-dependent factors might be needed for production of this compound at wild-type levels. Fumitremorgin G, fumigaclavine C, and fumagillin, together with production of glionitrin A, currently an orphan compound without an associated gene cluster, have been described to be relevant in the A. fumigatus infection process and in other pathologies. Fumitremorgins are associated with dysfunction of the nervous system causing tremors, seizures and abnormal behavior in animals [75,76]. The alkaloid fumigaclavine also causes nervous system damage as well as alteration of the reproductive system [77]. Fumagillin has been associated with invasive aspergillosis due to its effect in slowing ciliary beat frequency [17] and inhibition of endothelial proliferation [17]. Some of these compounds, however, are bioactive molecules with potential or current applications, particularly anti-tumoral compounds such as glionitrin A [78], and the well-known fumagillin and related compounds, with applications against amebiasis [54], microsporidiosis [55], and with known anti-angiogenic activity as inhibitors of the human type 2 methionine aminopeptidase (MetAP2) [56,57]. Our study shows that the expression of most of the genes in the fumagillin gene cluster was negatively affected by either deletion or over-expression of veA. This corresponded with a decrease in fumagillin production in these two strains compared to the wild type.
Further insight into the mechanism regulating the secondary metabolite gene cluster in A. fumigatus may contribute to decreasing the detrimental effects of this fungus as well as increasing the production of valuable secondary metabolites, such as fumagillin. Our study indicated that the fumagillin gene cluster is regulated by a C6 transcription factor gene, now denominated fumR, located within the boundaries of this cluster. Other C6 type transcription factor genes have been found within other secondary metabolite genes clusters, such as the well-known gliZ in the A. fumigatus gliotoxin gene cluster [18] and aflR in the A. nidulans sterigmatocystin cluster [79,80], which have been demonstrated to regulate such clusters. fumR positively regulated the expression of the recently characterized PKS gene, Afu8g00370, the terpene cyclase gene, Afu8g00520, and the acyltransferase Afu8g00380 [53]. Furthermore, fumR also regulates all the other predicted genes in this cluster. Deletion of fumR resulted in complete absence of fumagillin production.
Our study showed that fumR is under the control of veA. Both deletion and over-expression of veA downregulated fumR transcription, suggesting that veA influences the activation of the fumagillin gene cluster through regulation of fumR. Overexpression of veA also had a marked negative effect on the expression of many A. fumigatus secondary metabolite gene clusters. We previously described a similar effect in the veA regulation of the gliotoxin gene cluster [13], showing a decrease of gliotoxin production in both deletion and overexpression strains with respect to wild-type levels. Our RNA sequencing data provide strong evidence supporting this Figure 7. Expression of fumR and PKS370 is regulated by laeA. Total RNA was extracted from WT and ∆laeA strains, grown as stationary liquid Czapek-Dox cultures for 48 h and 72 h. Relative expression of fumR and PKS gene Afu8g00370 was calculated using 2 -ΔΔCt method as described by Schmittgen and Livak [89]. Primers used for the expression analysis are listed in Table S2. The bar represents the mean of three replicates and error bars represent standard error. Expression of 18S was used as internal reference. Values were normalized to expression levels of wild-type levels considered as 1. pattern. The same pattern has also been observed in other Aspergillus species; for example, the production of penicillin has been demonstrated to be negatively affected by either deletion or over-expression of veA in A. nidulans [37,81]. Since VeA is part of a protein complex or complexes [31,45,47,50,69], we hypothesized that a balanced stoichiometry between VeA and other possible partners might be necessary for proper function, including the activation of secondary metabolite gene clusters. It is possible that other VeA-interacting proteins might also modulate the expression of the fumagillin gene cluster. In this current study we also examined whether laeA affects the expression of genes in this cluster. Our results revealed that this is indeed the case; the absence of laeA greatly decreases fumR and Afu8g00370 expression. This indicates that both VeA and LaeA, components of the fungal velvet protein complex, are indispensable for normal expression of the fumagillin gene cluster and fumagillin production in the opportunistic pathogen A. fumigatus.

Conclusion
In this study we have demonstrated that veA is a global genetic regulator in the opportunistic human pathogen A. fumigatus, controlling the expression of hundreds of genes. Among the genes governed by veA are numerous secondary metabolite gene clusters, some of them responsible for the synthesis of natural products considered to be virulent factors during A. fumigatus infection. Interestingly, we also showed that some of these veA-dependent gene clusters are associated with the production of important medical drugs, such as fumagillin, known for its anti-angiogenic properties among other relevant medical applications. All the genes in the fumagillin gene cluster are under the control of the endogenous regulator fumR, which is regulated by veA and laeA, both encoding interacting components in the velvet complex. The findings presented here provide further insight into the regulatory dynamics of the A. fumigatus genome, contributing to settinga basis for novel strategies to decrease the negative effects of A. fumigatus while increasing its potential to produce beneficial compounds. Figure S1.

Supporting Information
qRT-PCR validation of RNA sequencing analysis of the expression of fumR (A) and Afu8g00370 (B) in the wild type, ∆veA, complementation and OEveA. Total RNA was extracted using TRIzol from 72h old stationary cultures grown in Czapek-Dox medium. The relative expression was calculated using 2 -ΔΔCt method as described by Schmittgen and Livak [89]. Primers used for expression analysis are listed in Table S2. The bar represents the mean of three replicates and error bars represent standard error. Expression of 18S was used as internal reference gene. Values were normalized to the expression levels of WT which was considered as 1. (TIF) Figure S2. Targeted fumR deletion. (A) Diagram showing SalI sites (S) in the wild-type fumR locus, and the same locus after gene replacement of fumR by the A. parasiticus pyrG gene used as selection marker for fungal transformation. The fragment used as probe templates for Southern blot analyses is also shown. (B) Southern blot analysis. The ∆fumR deletion construct was transformed in CEA17ku80 (Table S1). Additional transformants also presented the correct band pattern (data not shown). (TIF)  (Table S1). Additional transformants also presented the correct band pattern (data not shown). (TIF)