Telomere position effect is regulated by heterochromatin-associated proteins and NkuA in Aspergillus nidulans

Gene-silencing mechanisms are being shown to be associated with an increasing number of fungal developmental processes. Telomere position effect (TPE) is a eukaryotic phenomenon resulting in gene repression in areas immediately adjacent to telomere caps. Here, TPE is shown to regulate expression of transgenes on the left arm of chromosome III and the right arm of chromosome VI in Aspergillus nidulans. Phenotypes found to be associated with transgene repression included reduction in radial growth and the absence of sexual spores; however, these pleiotropic phenotypes were remedied when cultures were grown on media with appropriate supplementation. Simple radial growth and ascosporogenesis assays provided insights into the mechanism of TPE, including a means to determine its extent. These experiments revealed that the KU70 homologue (NkuA) and the heterochromatin-associated proteins HepA, ClrD and HdaA were partially required for transgene silencing. This study indicates that TPE extends at least 30 kb on chromosome III, suggesting that this phenomenon may be important for gene regulation in subtelomeric regions of A. nidulans

Secondary Metabolism and Development Is Mediated by LlmF Control of VeA Subcellular Localization in <em>Aspergillus nidulans</em>

<div><p>Secondary metabolism and development are linked in <em>Aspergillus</em> through the conserved regulatory velvet complex composed of VeA, VelB, and LaeA. The founding member of the velvet complex, VeA, shuttles between the cytoplasm and nucleus in response to alterations in light. Here we describe a new interaction partner of VeA identified through a reverse genetics screen looking for LaeA-like methyltransferases in <em>Aspergillus nidulans</em>. One of the putative LaeA-like methyltransferases identified, LlmF, is a negative regulator of sterigmatocystin production and sexual development. LlmF interacts directly with VeA and the repressive function of LlmF is mediated by influencing the localization of VeA, as over-expression of <em>llmF</em> decreases the nuclear to cytoplasmic ratio of VeA while deletion of <em>llmF</em> results in an increased nuclear accumulation of VeA. We show that the methyltransferase domain of LlmF is required for function; however, LlmF does not directly methylate VeA <em>in vitro</em>. This study identifies a new interaction partner for VeA and highlights the importance of cellular compartmentalization of VeA for regulation of development and secondary metabolism.</p> </div

LlmF does not methylate the velvet complex <i>in vitro</i>.

<p>(A) An <i>in vitro</i> methylation assay was conducted by incubation of the recombinant proteins with ³H-SAM for 1 hour at 30°C. The reactions were stopped by addition of SDS sample buffer and electrophoresed on Bis-Tris SDS-PAGE gels. The gels were treated with fluorography enhancing solution (En³Hance, Perkin Elmer), dried, and exposed to a tritium phosphor screen for 2 weeks. GST-RmtA and human recombinant histone H4 (NEB) served as a positive control. Methylation of histone H4 by GST-RmtA was visualized via ³H autoradiography. (B) Using the same <i>in vitro</i> methylation assay, members of the velvet complex (VeA, VelB, and KapA) were tested as methylation substrates for LlmF. Under these conditions, LlmF was unable to methylate any of the proteins tested.</p

A model illustrates that LlmF controls VeA subcellular localization through methylation of an unknown substrate.

<p>Cytoplasmic VeA-VelB dimer is recognized by the importin α (KapA) and subsequently imported through the nuclear pore complex. After nuclear import, KapA dissociates where the VeA-VelB dimer functions to activate sexual development, VelB forms a dimer with VosA to repress asexual development, and the LaeA-VeA-VelB heterotrimeric complex forms that activates secondary metabolism. The data presented here depicts a role for a LlmF-VeA transient complex repressing the nuclear import of VeA primarily through the putative methylation activity of LlmF. The red light sensing phytochrome, FphA, also controls the subcellular localization of VeA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003193#pgen.1003193-Purschwitz1" target="_blank">[20]</a>, however it is unknown if FphA and LlmF share a pathway or independently regulate VeA subcellular localization.</p

LlmF does not interact with VelB or KapA and only full-length LlmF interacts with VeA.

<p>(A) A directed yeast-two-hybrid analysis indicates that LlmF is not able to interact with VelB, full length KapA, or KapA⁵⁰, which is missing the first 79 amino acids containing the importin β interaction domain. A positive interaction is indicated by a blue change in color via the <i>lacZ</i> reporter when grown on media containing X-gal. (B) Five truncation mutants of LlmF were constructed and tested against full length VeA in the yeast-two-hybrid assay. (C) Similarly, five different VeA truncations were created based on the locations of the predicted domain structure; the velvet domain is located from amino acid position 29–235 and the PEST domain is located from 458–573. The VeA truncations were tested in the yeast-two-hybrid assay against full length LaeA, VelB, and LlmF. (D) VeA is capable of interacting with LlmF^SAM, which contains a mutation in motif I of the SAM binding site of LlmF.</p

LaeA-like methyltransferases expression over different development stages.

<p>Expression profiling of all of the LaeA-like methyltransferases via northern blot indicates that some are expressed at very low levels (<i>llmD, llmG</i>, and <i>llmJ</i>), some are expressed constantly through development (<i>llmA, llmB</i>, and <i>llmI</i>), and <i>llmC</i> is expressed only during late asexual development. Controls for developmental time points were as follows: <i>brlA</i> was used as a control for asexual induction, <i>mutA</i> for sexual development, and actin (<i>actA</i>) as a loading control. Expression of <i>llmF</i> increases during both late vegetative and asexual development, however it does not increase during sexual development.</p

Recombinant LlmF binds the methyl donor molecule <i>S</i>-adenosyl methionine.

<p>(A) Multiple sequence alignment of LlmF with previously characterized LaeA proteins from <i>A. nidulans</i>, <i>A. flavus</i>, <i>A. fumigatus</i>, <i>Cochliobolus heterostrophus</i>, and <i>Fusarium fujikuroi</i> identifies that LlmF harbors a conserved <i>S</i>-adenosyl methionine (SAM) binding domain <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003193#pgen.1003193-Kagan1" target="_blank">[26]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003193#pgen.1003193-Katz1" target="_blank">[61]</a>. The SAM binding domain can be further broken down into four motifs that correspond to β-strands in the binding pocket: motif I, post-motif I, motif II, and motif III. LlmF and LaeA proteins contain these conserved motifs. Asterisks indicate the conserved glycine residues in motif I that were previously mutated in LaeA to render the protein inactive <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003193#pgen.1003193-Bok3" target="_blank">[17]</a>. (B) An ultraviolet (UV) crosslinking assay was used to test the SAM binding site prediction and show that both LaeA and LlmF are capable of binding SAM. ³H-SAM was incubated with recombinantly purified GST, GST-LaeA, GST-LlmF and LlmF for 30 minutes at room temperature. To crosslink ³H-SAM into the binding site of the enzyme, some samples were incubated on ice while being exposed to UV light for 30 minutes and then all samples were subsequently electrophoresed on a 10% Tricine SDS-PAGE gel, transferred to nitrocellulose membrane, and then exposed to a tritium phosphor screen. Active site crosslinking of SAM was confirmed by incubation with the active site competitive inhibitor, <i>S</i>-adenosyl-homocysteine (SAH) at a concentration of 1 µM.</p

Reverse genetics identified LaeA-like methyltransferases in <i>A. nidulans</i>.

<p>Putative methyltransferases were identified from the genome annotation of <i>A. nidulans</i> at the Broad Institute, based on the presence of a predictive <i>S</i>-adenosyl methionine (SAM) binding domain. A ClustalW multiple sequence alignment was preformed on 88 amino acid sequences that corresponded to 50 identified from the <i>A. nidulans</i> genome, 37 previously characterized from <i>Saccharomyces cerevisiae</i> and <i>Schizosaccharomyces pombe</i>, and one from <i>A. flavus</i> (StcP). Bootstrapping analysis was done using the neighbor joining method with 1000 trials and a seed of 111 (MegAlign, DNASTAR), the numbers at each node indicate bootstrap values, and dashed lines represent collapsed nodes. This analysis identified nine uncharacterized loci that are homologous to LaeA. These nine loci have been named LaeA-like methyltransferases. AN5091 (LlmE) has previously been published and therefore is not included in this manuscript <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003193#pgen.1003193-Palmer1" target="_blank">[24]</a>.</p

Expression of the velvet complex members is not increased in ΔllmF strains.

<p>Mycelia was grown in liquid shaking culture for 20 hours in minimal media and then subsequently transferred to solid minimal media plates. Asexual development was induced by incubation in constant light for 24 hours while sexual development was induced by incubation in constant darkness for 48 hours. Total RNA was extracted from these conditions and expression was analyzed with a northern blot. Actin (<i>actA</i>) was included as a loading control. Numbers represent relative expression levels that have been normalized to actin from each condition and then normalized to wild type expression for each growth condition (asexual or sexual). Quantification was done using ImageJ software. Strains are as follows: WT = RJMP144.6, Δ<i>laeA</i> = RJMP153.7, Δ<i>llmF</i> = RJMP144.9, and OE <i>llmF</i> = RJMP143.5.</p

The methyltransferase domain of LlmF is required for negative regulation of sexual development and sterigmatocystin.

<p>(A) Cultures grown for five days under sexual developmental induction were imaged under a dissecting microscope. Wild type and Δ<i>llmF</i> strains produce abundant cleistothecia and few conidia, while OE <i>llmF</i> strains produce abundant conidia and few cleistothecia under these conditions. Additionally, the OE <i>llmF^SAM</i> mutant displays a Δ<i>llmF</i> phenotype indicating the requirement of the SAM binding domain. (B) Quantification of spores produced under sexual developmental conditions (materials and methods) supports the macroscopic images, as the Δ<i>llmF</i> strain produces an increased ratio of ascospores to conidia (sexual to asexual) and the OE <i>llmF</i> strain produces a decrease in this ratio. These data demonstrate that the OE <i>llmF^SAM</i> mutant has a sporulation ratio similar to that of the Δ<i>llmF</i> strain. Lowercase letters refer to statistical significance that measured with a student T-test of significance using Prism (Graphpad). (C) Analysis of sterigmatocystin was done in light and dark conditions as described in the materials and methods. Relative quantification of sterigmatocystin was achieved using ImageJ software and the bar graph indicates the average of two replicates. Notably, OE <i>llmF^SAM</i> does not repress the production of sterigmatocystin that is displayed in the OE <i>llmF</i> strain. (D) Strains were confirmed by northern analysis of <i>llmF</i> and <i>actA</i> transcripts from RNA extracted from sexual developmental induction.</p