The histone acetyltransferase GcnE (GCN5) plays a central role in the regulation of Aspergillus asexual development

Abstract

Acetylation of histones is a key regulatory mechanism of gene expression in eukaryotes. GcnE is an acetyltransferase of Aspergillus nidulans involved in the acetylation of histone H3 at lysine 9 and lysine 14. Previous works have demonstrated that deletion of gcnE results in defects in primary and secondary metabolism. Here we unveil the role of GcnE in development and show that a ∆gcnE mutant strain has minor growth defects but is impaired in normal conidiophore development. No signs of conidiation were found after 3 days of incubation, and immature and aberrant conidiophores were found after 1 week of incubation. Centroid linkage clustering and principal component (PC) analysis of transcriptomic data suggest that GcnE occupies a central position in Aspergillus developmental regulation and that it is essential for inducing conidiation genes. GcnE function was found to be required for the acetylation of histone H3K9/K14 at the promoter of the master regulator of conidiation, brlA, as well as at the promoters of the upstream developmental regulators of conidiation flbA, flbB, flbC, and flbD (fluffy genes). However, analysis of the gene expression of brlA and the fluffy genes revealed that the lack of conidiation originated in a complete absence of brlA expression in the ∆gcnE strain. Ectopic induction of brlA from a heterologous alcA promoter did not remediate the conidiation defects in the ∆gcnE strain, suggesting that additional GcnE-mediated mechanisms must operate. Therefore, we conclude that GcnE is the only nonessential histone modifier with a strong role in fungal development found so far.

Keywords: Aspergillus; Gcn5; GcnE; SAGA; asexual development; brlA; conidiation; fluffy genes; histone acetylation.

Figure 1

Simplified model of the genetic regulation of conidiation. Only some of the regulators studied in this work are shown for clarity. FluG is responsible for the synthesis of an extracellular factor that induces the rest of the fluffy genes in the three parallel routes. FlbE (not shown) interacts and activates FlbB. FlbB and FlbD are transcription factors that jointly bind to the promoter of brlA, activating its transcription. FlbC is another transcription factor activating the expression of brlA. FlbA is a regulator of G-protein activity that positively regulates the transcription of brlA. Activation of brlA is necessary and sufficient to induce conidiation. Ovals indicate the promoter regions, and in front of brlA correspond to the two sites analyzed by ChIP.

Figure 2

The ∆gcnE mutant is impaired in conidiation. (A) Wild type (WT) and ∆gcnE strains were grown vegetatively for 18 hr, and then conidiation was induced in complete medium. Progression of the developmental program was followed under the stereo microscope at the indicated time points. Conidiophore heads were evident after 10 hr of induction in the wild-type strain. Yellow conidia were evident 24 hr after induction. No such structures were seen in the ∆gcnE strain even after 72 hr of induction. (B) Comparision of the conidiation phenotype of wild-type and ∆gcnE strain with the phenotypes of the mutants in the central regulatory pathway (∆brlA, ∆abaA, or ∆wetA) after 4 days of growth. The brlA mutant produced the stalk cells and then continued growing rather than developing the conidiophore vesicles, metulae, phialides, and conidia. Mutations in abaA and wetA interfered in later stages of conidiophore development and were capable of producing white structures corresponding to the vesicles, metulae, and phialides. The ∆gcnE strain resembles a ∆brlA phenotype. (C) SEM images of the wild-type and ∆gcnE strains grown for 1 week. A very low density of immature conidiophores can be observed in the ∆gcnE strain, compared to the complete development of the wild-type conidiophores. Bar, 50 µm. (D) Details of SEM images comparing the wild-type conidiophores with the aberrant ∆gcnE conidiophore morphologies (indicated by arrows). Arrows indicate details of aberrant conidiophores. The double-line arrow points to a severe example where sterigmata cells seem to bud off from a hyphal or stalk cell. A higher magnification of this example is shown as a separate image at the top right. Bar, 10 µm, except for the top right image where the bar corresponds to 5 µm.

Figure 3

Differences in growth rate do not explain the conidiation defects in ∆gcnE. (A) Growth of wild-type and ∆gcnE strains was followed on complete and minimal solid media over a period of 5 days. The linear growth rate of the mutant was only slightly lower in comparison with the wild type on both media. The growth rate is shown as the increment in the colony diameter on solid media per day. Error bars show the standard error of at least three independent experiments performed in duplicates. (B) Wild type and ∆gcnE strains were point-inoculated on complete media plates and allowed to grow at 37 °C. Plates were photographed after the colonies reached the same size.

Figure 4

Global expression analysis of wild-type and ∆gcnE strains growing under vegetative or conidiation conditions. Both strains were grown vegetatively for 18 hr, and then conidiation was induced for 10 hr. The global expression of genes under the four conditions (wild-type vegetative, WT-VEG; wild type-conidiation, WT-CON; ∆gcnE vegetative, GCN-VEG; ∆gcnE conidiation, GCN-CON) was compared by using microarray hibridization. A total of 1162 differentially expressed genes were identified by ANOVA. (A) A dendogram was obtained by centroid linkage clustering using euclidean distances of the 1162 differentially regulated genes in the 12 samples (four conditions with three biological replicates each). The ∆gcnE strain grown under conidiation conditions was more similar to vegetative growth than to the conidiating wild type. (B) PC analysis of the genes differentially regulated under at least one of the four different conditions. The x-axis shows the first PC with a variation of 49% due to the growth mode (vegetative vs. conidiation). The y-axis shows the second PC with a variation of 26% due to the genetic background (wild type vs ∆gcnE). The results obtained by clustering (A) and PC (B) analysis are in agreement.

Figure 5

brlA is not expressed and acetylation of histone H3K9/K14 at the brlA promoter is reduced in the ∆gcnE strain. (A) Both wild-type and ∆gcnE strains were grown vegetatively for 18 hr, and then conidiation was induced for 10 or 72 hr. RNA was isolated and gene expression was quantified by RT-qPCR. Data are shown normalized to the tubulin gene (benA) as an internal standard. (B) ChIP was carried out by immunoprecipitation of cross-linked DNA with an antibody recognizing acetylated histone H3K9ac and H3K14ac, followed by qPCR analysis of the promoter regions. brlA showed an increase in the immunoprecipitated DNA in both distal (brlAp1) and proximal (brlAp3) regions of the promoter in the wild type. In the ∆gcnE strain, acetylation levels were grossly reduced and conidiation-specific increases were not observed. Values were normalized to input DNA (before immunoprecipitation) and are shown as the mean with standard errors of the mean of at least three biologically independent experiments.

Figure 6

Expression of the fluffy genes during conidiation is deregulated in the ∆gcnE strain. Both wild-type and ∆gcnE strains were grown vegetatively for 18 hr, and then conidiation was induced for 10 or 72 hr. RNA was isolated and gene expression was quantified by RT-qPCR. Data are shown normalized to the tubulin gene (benA) as an internal standard. Values are the mean and standard error of the mean of at least three independent experiments.

Figure 7

GcnE has additional as-yet-unidentified targets mediating the developmental effects. (A) Wild-type and ∆gcnE strains were pregrown for 24 hr before addition of different concentrations of orcinol (50 µg to 50 mg) on top of the colony. Plates were incubated for 3 additional days and photographed. The highest concentration of orcinol had some slightly negative effects on colony development in both strains. (B) Strains indicated at the left were pregrown for 24 hr in liquid media under repressing conditions (glucose) and then transferred to fresh liquid medium containing inducing threonine or repressing glucose, and incubation was continued for an additional 24 hr. Fungal pellets were photographed under the light microscope. The parental strains harbored either a construct overexpressing brlA from the alcA promoter (OE::brlA) or the gcnE deletion (∆gcnE). Two independent strains of the cross progeny (DKA234, DKA235) were used in this experiment. Black arrows indicate conidiophore-like structures, black arrowheads point to individual conidia produced in liquid cultures, and white dotted arrows point to vegetative hyphal tips. (C) Strains were pregrown as in B for 24 hr under repressing condintions but then transferred to solid medium containing threonine or glucose, and incubation was continued for 1 day. Fungal colonies were photographed under a stereo microscope at the same magnification. The OEbrlA strain (brlA⁺; alcA(p)::brlA) conidiated on glucose plates due to brlA expression from its native promoter. Two independent strains of the progeny were also used in this experiment. (D) Strains pregrown for 24 hr under repressing condintions (as in B) were transferred to solid medium containing threonine or glucose, and incubation was continued. Plates were photographed after 3 days of growth. Growth inhibition could be observed in the strains overexpressing brlA in both the wild-type and ∆gcnE background only under brlA-inducing conditions (threonine).