Expression analysis of Aspergillus nidulans OE::rsmA
ABSTRACT: Investigation of whole genome gene expression level changes in Aspergillus nidulans OE::rsmA compared to wild-type RDIT9.32 (veA). A twelve array study using total RNA recovered from six separate cultures of Aspergillus nidulans wild-type RDIT9.32 (veA) and six separate cultures of Aspergillus nidulans overexpressing rsmA (restorer of secondary metabolism A), using custom-designed, four-plex arrays. The experiment was divided into two runs. In the first run, three biological replicates each of Aspergillus nidulans wild-type RDIT9.32 (veA) and Aspergillus nidulans carrying a plasmid overexpressing rsmA under the control of the gpdA promoter were assayed. In the second run, three biological replicates each of Aspergillus nidulans wild-type RDIT9.32 (veA) and Aspergillus nidulans overexpressing rsmA at the native locus under the control of the gpdA promoter were assayed.
Project description:Investigation of whole genome gene expression level changes in Aspergillus nidulans AN1599 (PbcR) overexpression mutant, compared to the FGSC A4 wild-type strain. Overexpression of the Zn(II)2Cys6 –type transcription factor, AN1599.4 (PbcR, pimaradiene biosynthetic cluster regulator), activates a secondary metabolite gene cluster in Aspergillus nidulans. Activation of the pathway in Aspergillus nidulans lead to a production of ent-pimara-8(14),15-diene. 12x135K array of two separate cultures of FGSC A4 and two separate cultures of oe:AN1599(PbcR) with three separate RNA extractions from each culture. Each 135K array measures expression level of 10,546 genes with 6 probes/transcript. In addition, the array format contains tiling probes for 36 longer transcripts. All probes are in duplicates, giving the total number of 137,562 probes per array.
Project description:Purpose: To explore conservation of gene regulation by the transcription factor clr-2/clrB in Neurospora crassa and Aspergillus nidulans Methods: mRNA from wild type and clr-2/clrB mutants were collected after a culture shift from sucrose/glucose to Avicel (crystaline cellulose) or no carbon media Results: We show that N. crassa and A. nidulans have similair global transcriptional responses to Avicel, with several hundred genes showing specific induction, though the induced genes are more specifically targeted at cellulose for N. crassa and more targeted at hemicellulose and pectin for A. nidulans. clr-2/clrB has a conserved fundamental function in cellulose induction, though the mechanism has diverged. Misexpression of clr-2 is sufficeint for inducer free cellulase secretion in N. crassa, but neither clrB or heterologous clr-2 is sufficient for inducer free cellulase secretion in A. nidulans. Conclusions: Our study demonstrates a conserved and essential role in cellulose utilization for the transcription factor clr-2 in filamentous ascomycetes and demonstrates that manipulation of clr-2 expression can be used to control cellulase expression in some species. Biological triplicates of liquid culture N. crassa and A. nidulans were harvested at 4 hours and 6 hours, respectively, after a switch to media of interest. Global mRNA abundances from liquid cultures of N. crassa and A. nidulans were measured by sequencing on the Illumina Genome Analyzer IIx and HiSeq2000 platforms.
Project description:Light is a major environmental signal regulating many different biological processes. In Aspergillus nidulans light controls asexual and sexual development as well as the production of secondary metabolites. In order to get a global view of genes regulated during asexual development and of genes involved in other light-regulated biological processes, a genome-wide approach was undertaken. Total RNA was isolated from surface-grown, developmentally competent mycelia of the wild-type strain FGSC4 exposed to white light (11 W/m2) for 30 minutes or grown in the dark, labelled, and hybridized to a spotted microarray of A. nidulans.
Project description:The sirA gene encodes a member of sirtuin protein that is NAD(+)-dependent histone deacetylase (HDAC) and ubiquitous in eukaryote. DNA microarray analyses for Aspergillus nidulans FGSCA26 (WT) strain and Gene disruptant of sirA (SirAd) indicated that genes for synthesizing secondary metabolic products such as sterigmatocystin, penicillin G, emericellamide, aspernidine A, xanthone, austinol, and siderophores are down-regulated by SirA. Aspergillus nidulans WT and SirAd strains were cultured in 200 ml of GMM at 30°C for 24, 48, and 72 h, and their total RNA was purified as described above.
Project description:We describe thephosphoproteome of filamentous fungus Aspergillus nidulans. Phosphopeptides were enriched using affinity enrichment using titanium dioxide and separated using a convenient ultralong gradient separations on c18 reverse phase columns. Over 1637 phosphopeptides corresponding to 647 phosphoproteins were identified using using a “high-high” strategy using HCD on the novel Q-exactive platform
Project description:Histone posttranslational modifications (HPTMs) are involved in regulating the synthesis of fungal bioactive compounds. The exact molecular mechanisms of the silencing/activation of secondary metabolism (SM) clusters by these epigenetic events however are not yet fully understood. This work applies a combined approach of quantitative mass spectrometry (LC-MS/MS) and chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) to identify the chromatin landscape in two metabolic states: primary and secondary metabolism. Furthermore, to link the particular chromatin states to the expression of condition specific genes, genome wide transcriptome (RNA-seq) was performed. Strikingly, we found that silent A. nidulans SM clusters are free of repressive H3K9me3 though this heterochromatic mark forms distinguished peaks flanking the many SM clusters. In addition, silent SM clusters do not contain detectable levels of activating histone marks such as H3K4me3, H3K36me3 or H3Ac, which, to some extent, are established upon activation of the clusters. In order to investigate the function of dynamic H3K4 methylation/demethylation in transcription, we characterized the KdmB- Jarid1 family histone demethylase. The in vitro assay using heterologously expressed KdmB showed that it is an active demethylase; moreover, MS/MS as well ChIP-seq approaches revealed that it targets H3K4me3 in vivo mediating transcriptional repression. KdmB positively regulates the expression of 40% of A. nidulans SM genes and this function appears to be independent of its demethylase activity. Our bioinformatics approach revealed two states of H3K4me3 in A. nidulans genome: loci with low levels of this mark are more disposed to differential expression in response to environmental clues, while the genes marked by high H3K4me3 levels are constitutively transcribed in our experimental conditions. Taken together our data reveal important role of H3K4 methylation/demethylation in transcription regulation. Furthermore, this study presents the first genome-wide map of H3K4me3, H3K9me3, H3K36me3 and H3Ac in A. nidulans in different metabolic conditions. Two strains, wild type and kdmB deletion, at two conditions, growth at primary (17h) and secondary (48h), were analysed. Each sample was replicated.
Project description:This SuperSeries is composed of the following subset Series: GSE9275: A tri-species Aspergillus array (nidulans arrays) GSE9276: A tri-species Aspergillus array (niger arrays) GSE9277: A tri-species Aspergillus array (oryzae arrays) Keywords: SuperSeries Refer to individual Series
Project description:Microarray analysis was used to identify the calcium-responsive genes dependent on CrzA in the filamentous fungus Aspergillus nidulans. In order to identify such genes, we conducted the two types of experiment. One was a comparison between wild type with calcium treatment and wild type without calcium treatment. Another was a comparison between wild type with calcium treatment and crzA mutant with calcium treatment. From a comparison between the results of these experiments, we could identify the genes whose expression was induced or repressed in response to calcium in a manner dependent on CrzA. KEY WORD; Aspergillus nidulans, calcium response, crzA Conidia of wild type or crzA mutant strains were incubated at 37C in CD medium for 18h and treated with or without calcium (final concentration; 50 mM) for 15 min. The mycelia were harvested and frozen in liquid nitrogen, ground to a powder, and used for RNA preparation. mRNA was purified and used for hybridization experiments. A total of 2 hybridizations were performed for each microarray experiment described in the summary. The following replicates were carried out: 1. In-slide replicates were carried out for each analyses. 2. Dye swap replicates were carried out for each analyses. The slides were scanned with an Axon GenePix 4000B scanner (Molecular Devices). The resulting TIFF images were imported into GenePix Pro and fluorescent intensity of spots were calculated for each of the Cy3 and Cy5 channels. Global normalization was applied to all analyses. Following normalization, spots whose Cy3 or Cy5 intensity was less than 0 were removed from the data set (the exceptional case was that intensity of the other channel was more than 100). The dye-swap replicates and in-slide replicates were subjected to all analyses. Finally, gene expression ratios (channel 2/channel 1) were calculated for each replicates. Gene expression was considered to be significantly higher or lower whenever the spot intensity changed by at least 2-fold in all four replicates.
Project description:Hypoxia imposes a challenge upon most of the filamentous fungi that require oxygen for proliferation. Here, we used whole genome DNA microarrays to investigate global transcriptional changes in Aspergillus nidulans gene expression after exposure to hypoxia followed by normoxia. Aeration affected the expression of 2,864 genes (27% of the total number of genes in the fungus), of which 50% were either induced or repressed under hypoxic conditions. Up-regulated genes included those for glycolysis, ethanol production, the tricarboxylic acid (TCA) cycle, and for the γ-aminobutyrate (GABA) shunt that bypasses two steps of the TCA cycle. Ethanol and lactate production under hypoxic conditions indicated that glucose was fermented to these compounds via the glycolytic pathway. Since the GABA shunt bypasses the NADH-generating reaction of the TCA cycle catalyzed by oxoglutarate dehydrogenase, hypoxic A. nidulans cells eliminated excess NADH. Hypoxia down-regulated some genes involved in transcription initiation by RNA polymerase II, and lowered the cellular mRNA content. These functions were resumed by reoxygenation, indicating that A. nidulans controls global transcription to adapt to a hypoxic environment. This study is the first to show that hypoxia elicits systematic transcriptional responses in A. nidulans. We transferred A. nidulans cells from normoxic to hypoxic conditions for 6 h, and then back to normoxic conditions to examine the effect of hypoxia on gene expression. Total RNA was prepared for DNA microarray analysis from the cells after 3 and 6 h of exposure to hypoxia, followed by 3 and 6 h of reoxygenation.