A Novel C₂H₂ Transcription Factor that Regulates <i>gliA</i> Expression Interdependently with GliZ in <i>Aspergillus fumigatus</i>

<div><p>Secondary metabolites are produced by numerous organisms and can either be beneficial, benign, or harmful to humans. Genes involved in the synthesis and transport of these secondary metabolites are frequently found in gene clusters, which are often coordinately regulated, being almost exclusively dependent on transcription factors that are located within the clusters themselves. Gliotoxin, which is produced by a variety of <i>Aspergillus</i> species, <i>Trichoderma</i> species, and <i>Penicillium</i> species, exhibits immunosuppressive properties and has therefore been the subject of research for many laboratories. There have been a few proteins shown to regulate the gliotoxin cluster, most notably GliZ, a Zn₂Cys₆ binuclear finger transcription factor that lies within the cluster, and LaeA, a putative methyltransferase that globally regulates secondary metabolism clusters within numerous fungal species. Using a high-copy inducer screen in <i>A. fumigatus</i>, our lab has identified a novel C₂H₂ transcription factor, which plays an important role in regulating the gliotoxin biosynthetic cluster. This transcription factor, named GipA, induces gliotoxin production when present in extra copies. Furthermore, loss of <i>gipA</i> reduces gliotoxin production significantly. Through protein binding microarray and mutagenesis, we have identified a DNA binding site recognized by GipA that is in extremely close proximity to a potential GliZ DNA binding site in the 5′ untranslated region of <i>gliA</i>, which encodes an efflux pump within the gliotoxin cluster. Not surprisingly, GliZ and GipA appear to work in an interdependent fashion to positively control <i>gliA</i> expression.</p></div

High-copy expression of <i>gipA</i> induces gliotoxin production.

<p>All cultures were grown in repressing conditions. Total RNA was isolated and quantified by dot blot analysis in triplicate. Gliotoxin levels were quantified by RP-HPLC using a standard curve and calculated as mg gliotoxin/g dry mycelial mass. All data sets are normalized to AMA.GL. (a) mRNA transcript levels of several gliotoxin cluster genes after 48 hrs of growth relative to AMA.GL. The results of one representative experiment of three independent experiments are shown as mean ± SD. (b) Gliotoxin levels in growth medium relative to AMA.GL. The data presented is an average of three biological replicates, shown as mean ± SD. The asterisk (*) indicates a statistically significant difference (p-value<0.05), compared to AMA.GL, calculated by one-way ANOVA and Tukey comparison test.</p

Characterization of the putative GipA DNA binding site.

<p>(a) Consensus sequence representing the putative DNA binding site for GipA obtained by protein binding microarray analysis. (b) Layout of putative GipA and GliZ binding sites in the <i>gliA</i> promoter region, relative to the <i>gliA</i> start site. Putative GliZ tandem repeats are purple and bolded and the putative GipA DNA binding site is underlined in orange. (c) Fold increase of LacZ levels of each strain in repressing conditions, relative to the AMA empty vector control. The results of one representative experiment of six independent experiments are shown as mean ± SD. (d) β-galactosidase activity of AMA.GL, AMA.BSM1, and AMA.BSM2 in both repressing and non-repressing conditions. The results of one representative experiment of three independent experiments are shown as mean ± SD. The asterisk (*) indicates a statistically significant difference (p-value<0.05) for each data set, compared to the AMA control strain, calculated by one-way ANOVA and Tukey comparison test.</p

Schematic of the three rounds of the high-copy inducer screen.

<p>(a) Round one: transformation of the genomic library into Af293.1-GL. (b) Round two: transformation of single plasmids from the genomic library into Af293.1-GL. (c) Round three: transformation of single genes into Af293.1-GL.</p

Genes commonly involved in secondary metabolism and possibly regulated by GipA.

<p>Genes commonly involved in secondary metabolism and possibly regulated by GipA.</p

GliZ and GipA are dependent on each other for <i>gliA</i> induction.

<p>Cultures were grown in non-repressing conditions and mRNA was quantified by dot blot analysis in triplicate. Data are normalized to AMA.G (pyrG+ background). These graphs are an average of three biological replicates, shown as mean ± SD. (a) mRNA levels of <i>gliP</i> in all backgrounds relative to AMA.G. (b) mRNA levels of <i>gliA</i> in all backgrounds relative to AMA.G. The asterisk (*) indicates a statistically significant difference (p-value<0.05), compared to AMA.G, calculated by one-way ANOVA and Tukey comparison test.</p

Layout of two potential GipA binding sites that are embedded in putative GliZ binding sites in the <i>gliZ</i> promoter.

<p>GliZ putative trinucleotide repeats are purple and bolded, the putative GipA binding sites are underlined in orange, and binding cluster 1 additionally contains an AreA recognition element (underlined in yellow).</p

Characterization of <i>gipA</i> cDNA.

<p>(a) Schematic of cDNA size and composition. Yellow bars signify μORFs and the green bars display the two zinc finger domains. The size indicated for the coding region includes the one intron. (b) Northern hybridization of <i>gipA</i> from total RNA. WT is wild-type (Af1160) and (R) signifies complemented strains. (c) Cladogram of GipA homologues. <i>N. fischeri</i> is <i>Neosartorya fischeri</i>.</p