The Histone Acetyltransferase Gcne (GCN5) Plays a Central Role in the Regulation of Aspergillus Asexual Development
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INVESTIGATION The Histone Acetyltransferase GcnE (GCN5) Plays a Central Role in the Regulation of Aspergillus Asexual Development David Cánovas,*,†,1,2 Ana T. Marcos,*,1 Agnieszka Gacek,†,1 María S. Ramos,* Gabriel Gutiérrez,* Yazmid Reyes-Domínguez,†,3 and Joseph Strauss†,‡ *Departmento de Genética, Facultad de Biología, Universidad de Sevilla, 41012, Spain, †Fungal Genetics and Genomics Unit, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna A-3430, Austria, and ‡Department of Health and Environment, Bioresources, Austrian Institute of Technology, Tulln/Donau A-3430, Austria 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 ΔgcnE 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 ΔgcnE strain. Ectopic induction of brlA from a heterologous alcA promoter did not remediate the conidiation defects in the ΔgcnE 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. HROMATIN rearrangements are associated with the lation, and ubiquitination at different positions of the his- Ctranscriptional regulation of gene expression in eukar- tone proteins. In particular, acetylation of lysine 9 or lysine yotes. For example, facultative heterochromatin can be as- 14 in histone H3 has been associated with activation of sociated with the transcriptionally active or silent states of transcription. Acetylation of histones plays two roles in the developmentally regulated loci (Grewal and Jia 2007). This regulation of transcription: it alters the physical properties is achieved in part through histone post translational mod- of the histone–DNA interaction, and it also provides a frame ifications (PTM), which play a very important role in the for the binding of bromodomain proteins that remodel the control of these active or silent chromatin states. Histone chromatin and regulate gene expression (Spedale et al. modifications include acetylation, methylation, phosphory- 2012). These modifications regulate the nucleosome posi- tioning at the gene promoters and the recruitment of the Copyright © 2014 by the Genetics Society of America regulatory proteins. One of these modifiers, the SAGA com- doi: 10.1534/genetics.114.165688 plex, is responsible for the acetylation of several lysine Manuscript received May 1, 2014; accepted for publication June 4, 2014; published Early Online June 6, 2014. residues in the N-terminal region of histones, particularly Supporting information is available online at http://www.genetics.org/lookup/suppl/ histone H3 lysine 9 (H3K9) and histone H3 lysine 14 doi:10.1534/genetics.114.165688/-/DC1. 1These authors contributed equally to this work. (H3K14) (Kuo et al. 1996). The SAGA complex is a multi- 2Corresponding author: Departamento de Genética, Facultad de Biología, Universidad meric protein association with several subunits including de Sevilla, Reina Mercedes 6, 41012 Sevilla, Spain. E-mail: [email protected] 3Present address: Research Centre for Agriculture and Forestry Laimburg, Laimburg Ada2p, Ada3p, Spt3p, and Tra1p (Grant et al. 1997; Spedale 6, Auer/Ora, BZ, 39040, Italy. et al. 2012), where Gcn5p is the subunit with the histone Genetics, Vol. 197, 1175–1189 August 2014 1175 acetyltransferase (HAT) catalytic activity (Grant et al. 1997). Dominguez 2011). For example, it has been demonstrated The SAGA complex is implicated in several functions related that acetylation of histone H3 is required for the synthesis of to transcription, such as transcription initiation and elonga- secondary metabolites in A. nidulans (Reyes-Dominguez tion, histone ubiquitination, and interactions of TATA-binding et al. 2010; Nützmann et al. 2011; Bok et al. 2013; Nützmann proteins. In addition, SAGA has also been implicated in et al. 2013). Reduction of heterochromatin marks leads to messenger RNA (mRNA) export in yeasts and Drosophila higher secondary metabolite production in Aspergillus and (Rodriguez-Navarro et al. 2004; Kurshakova et al. 2007). Fusarium species (Reyes-Dominguez et al. 2010, 2012), and In Saccharomyces cerevisiae, the SAGA complex is involved in it has also been found that it de-represses silent clusters, the transcriptional regulation of 12% of the yeast genome. leading to the production of novel metabolites (Bok et al. Approximately, a third of that 12% of the yeast genome is 2009). In addition, adverse metabolic and morphologic downregulated and two-thirds are upregulated in DGCN5 effects are also observed in histone modifier mutants, for cells (Lee et al. 2000), implying a direct or indirect negative example, deletion of the histone H3K9 methyltransferase role of Gcn5p. Interestingly, a high proportion of genes regu- clrD in Aspergillus fumigatus resulted in reduced radial lated by SAGA are upregulated during the responses to envi- growth and also delayed transcriptional activation of brlA ronmental stresses (such as heat, oxidation, and starvation) and conidiation (Palmer et al. 2008). (Huisinga and Pugh 2004). The SAGA complex is also present Asexual reproduction, also called conidiation, results in in metazoans, where it has diverged and evolved into four the formation of mitotic propagules (conidia), which are the different complexes (two SAGA and two ATAC complexes), infectious particles for pathogenic filamentous fungi. Con- while lower eukaryotes, such as yeasts and other fungi, con- idiation is the most common and proliferative reproductive tain one single SAGA complex. It was hypothesized that this mode in filamentous fungi. For this reason, conidiation has evolution into a diverse set of complexes is involved in cellu- been extensively studied in A. nidulans for several decades lar specialization during development and homeostasis in (for recent reviews see Etxebeste et al. 2010; Park and Yu metazoans (Spedale et al. 2012). The SAGA and ATAC com- 2012; Krijgsheld et al. 2013). Conidiation is controlled by plexes participate in the regulation of genes in response to a central regulatory pathway (Figure 1), encompassing intracellular and extracellular signals: protein kinase C sig- three transcriptional activators: BrlA, AbaA, and WetA (see naling, response to osmotic stress, UV-induced DNA damage, reviews by Adams et al. 1998; Yu et al. 2006). The first arsenite-induced signaling, endoplasmic reticulum stress, and component in this regulatory cascade, BrlA, is essential to nuclear receptor signaling (Spedale et al. 2012). Likewise, drive conidiation (Adams et al. 1988). brlA expression is plants also have multiple HATs. In Arabidopsis,AtGCN5is silent during vegetative growth, and its expression during involved in many developmental processes (Servet et al. conidiation is controlled by a number of genes, including the 2010). fluffy genes. Deletion of any of the fluffy genes gives a typical Although elegant experimental approaches using Neuros- fluffy phenotype with cotton-like colonies, lack of normal pora crassa as a model system have significantly contributed conidia, and reduced levels of brlA expression (Adams to general concepts of DNA methylation, genome defense, et al. 1998; Yu et al. 2006). There are six fluffy genes: fluG and heterochromatin formation (Tamaru and Selker 2001; and flbA–E. fluG encodes a protein similar to bacterial Freitag et al. 2002, 2004; Honda et al. 2010; Rountree and glutamine synthetases (Lee and Adams 1994), and the Selker 2010), studies on transcriptionally related chromatin FluG protein is responsible for the synthesis of the extracel- rearrangements and histone modifications are still scarce in lular factor dehydroaustinol that, in conjunction with the filamentous fungi, a broad group of ecologically, industrially, orsellinic acid derivative diorcinol, induces conidiation and clinically important organisms. In N. crassa, the tran- (Rodriguez-Urra et al. 2012). FluG works upstream of the scriptional activation of the light-inducible gene al-3 flbA-E genes (Yu et al. 2006). Flb genes operate in three requires the acetylation of histone H3K14 by a homolog of parallel routes in A. nidulans to regulate the expression of Gcn5p, NGF-1 (Grimaldi et al. 2006), and in Aspergillus brlA upon induction of conidiation. FlbA is a repressor of the nidulans the SAGA/ADA complex is involved in the acetyla- G-protein signaling, which participates