Genome-Wide Transcriptome and Chromatin Analysis of Aspergillus nidulans Primary and Secondary Metabolism Reveals Crucial Function for a Kdm5-Family Histone Demethylase.
ABSTRACT: 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:A. nidulans kdmA encodes a member of the KDM4 family of jumonji histone demethylase proteins, highly similar to metazoan orthologues both within functional domains and in domain architecture. This family of proteins exhibits demethylase activity toward lysines 9 and 36 of histone H3 and plays a prominent role in gene expression and chromosome structure in many species. Mass spectrometry mapping of A. nidulans histones revealed that around 3% of bulk histone H3 carried trimethylated H3K9 (H3K9me3) but more than 90% of histones carried either H3K36me2 or H3K36me3. KdmA functions as H3K36me3 demethylase and has roles in transcriptional regulation. Genetic manipulation of KdmA levels is tolerated without obvious effect in most conditions, but strong phenotypes are evident under various conditions of stress. Transcriptome analysis revealed that – in submerged early and late cultures – between 25% and 30% of the genome is under KdmA influence, respectively. Transcriptional imbalance in the kdmA deletion mutant may contribute to the lethal phenotype observed upon exposure of mutant cells to low-density visible light on solid medium. While KdmA acts as transcriptional co-repressor of primary metabolism (PM) genes it is required for full expression of several genes involved in biosynthesis of secondary metabolites (SM). Two strains, wild type and kdmA deletion, at two conditions, growth at primary (17h) and secondary (48h), were analyzed. Each sample was replicated.
Project description:Mining of fungal genomes uncovered their great potential for the production of novel secondary metabolites (SMs). However most of them stay silent under standard laboratory cultivation conditions. Co-cultivation of fungi with organism that occur in their natural habitat has shown to be trigger for the activation of such silent SM gene clusters. Recently, we showed that the cultivation of Aspergillus nidulans with the bacterium Streptomyces rapamycinicus leads to the activation of the orsellinic acid gene cluster. Hence we decided to study this interaction further to gain insight into the regulation of SM gene clusters and more specifically to study the chromatin remodelling network actuve upon co-cultivation of the two organisms. This study gives novel insight into the regulation of the orsellinic acid gene cluster and the interaction of the two organisms. To the best of our knowledge this is the first report of mapping the chromatin landscape of microbial interactions, making this study a role model for the analysis of similar systems.
Project description: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:Genome wide transcriptional changes induced by various types of oxidative stresses as well as salt stress were studied in a DatfA mutant and the appropriate control A. nidulans strains. Although a significant number of stereotypically regulated genes was identified (Core Oxidative Stress Response or COSR genes) when the global transcriptional effects of five different oxidative stress conditions were compared the number of co-regulated genes decreased to 13 when NaCl stress was included into the analyses. The appearance of only a few co-regulated genes and the great number of genes regulated merely by one certain type of stress do not support the existence of a S. cerevisiae-type Environmental Stress Response in A. nidulans. Deletion of atfA, a true functional ortholog of fission yeast’s “all-purpose” stress response transcription factor, increased the oxidative stress sensitivity of A. nidulans and affected the transcription of several genes under both unstressed and stressed conditions. The number of genes under AtfA control was quite stress-type dependent; e.g. deletion of atfA altered the transcription of a wide spectrum of genes under menadione sodium bisulfite stress but had only a minor effect on the transcriptome profiles when A. nidulans cultures were exposed to H2O2, tBOOH, NaCl and, especially, to diamide stress. These observations suggest that the function of AtfA in the regulation of various stress responses is much smaller than we thought before or other transcription factors can take over a number of AtfA’s functions when the atfA gene is deleted. It is noteworthy that both oxidative and salt stress induced the transcription of some secondary metabolite gene clusters and the deletion of atfA enhanced the stress responsiveness of further clusters. Surprisingly, certain clusters were down-regulated by the stress conditions tested and the majority of them were not stress-responsive at all. Therefore, stress dependent regulation seems to be a frequent but far not a general feature of the regulation of secondary metabolism in A. nidulans. 14 samples, 7 with the control strain and 7 with an DatfA strain (each series contains samples from untreated as well as menadione, low concentration hidrogen-peroxide, high concentration hidrogen-peroxide, tert-butylhydroperoxide, diamide and NaCl treated cultures)
Project description:We report maps of H3K4me3 and H3ac - activiting expression histone modifications in C6 rat glioma cells. The data was obtained using whole genome high throughput technology. The sequencing was performed on HiSeq Ilumina platform. Examination of H3K4me3 histone modification and H3ac histone modification in C6 rat glioma cell line
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:The main objective was to identifiy genes related to scrA- extragenic suppression of crzA. Thus, through transcriptional profiling, we characterised the transcriptional basis of calcium tolerance in our mutant strain, ΔcrzA/scrA-. To evaluate the effect of calcium on global A. nidulans gene expression, we performed competitive microarray hybridizations using RNA obtained from the wild-type, ΔcrzA and ΔcrzA/scrA- strains before and after short pulses (10 and 30 minutes) of 200 mM calcium chloride. The experiment was further validated by real-time PCR. Two-condition experiment : A. nidulans wild-type, ΔcrzA and ΔcrzA/scrA- strains with 200 mM CaCl2 for 10 and 30 min at 30°C and 150 rpm in 25 mL YAG medium. Firstly, the strains were grown overnight at 30°C and 150 rpm in YAG medium (preculture). Afterwards, the medium was switched by a new media (25 mL) plus calcium and incubated as described above. Control condition refers to the preculture in YAG medium without further incubation.
Project description:Transcriptional profiling of A. nidulans comparing Xylose and Fructose grown on Wild type strain. The main objective was to identifiy genes related to Xylose transport. The experiment was further validated by real-time PCR. Three-condition experiment : A. nidulans strains grown during 16 h on fructose and transfered to xylose for 6, 12 and 24h.
Project description:Transcriptional profiling of A. nidulans comparing the mutant strain alcAypkA grown on inducing or repressing conditions. The main objective was to identifiy genes related to ypkA gene function. The experiment was further validated by real-time PCR and enzymatic assay. Four-condition experiment: A. nidulans strains grown during 16 and 24 h at 37°C in glucose 4% or glycerol 2% threonine 100mM.
Project description:Transcriptional profiling of A. nidulans comparing the mutant strain alcApkcA ΔcnaA and ΔcnaA grown on alcA inducing conditions. The main objective was to identifiy genes related to ΔcnaA phenotype supression by pkcA overexpression. The experiment was further validated by real-time PCR. Two-condition experiment : A. nidulans strains grown during 24 and 48 h at 37oC in glycerol 2% threonine 100mM.