Unknown,Transcriptomics,Genomics,Proteomics

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A genetic and functional investigation of the Zn2Cys6 transcription factors RSE2 and RSE3 in Podospora anserina

ABSTRACT: Abstract In Podospora anserina, the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase (aox) when the mitochondrial electron transport chain is impaired. In parallel they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase (pck) and fructose-1,6-biphosphatase (fbp). Orthologues of these transcription factors are present in a wide range of filamentous fungi and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organisation of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 in 5 positions in rse3. Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-ter activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3. It showed that in addition of aox, fbp and pck, RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn2Cys6 transcription factor, a flavohemoglobin and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids and M-NM-1-ketoglutarate pools. With GPL10116: 7 conditions each with four biological replicates: wt, rse2Y326D, rse3G642V, rse2Y326D-rse3G642V, M-NM-^Trse2, M-NM-^Trse3 and M-NM-^Trse2-M-NM-^Trse3; common reference is a pool of four conditions M48h, M96h, C48h and C96h ; Conditions are labelled in Cy3 and the common reference in Cy5

ORGANISM(S): Podospora anserina

SUBMITTER: Annie Sainsard-Chanet

PROVIDER: E-GEOD-51360 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To gain insight into the targets of the PaMpk1 and PaMpk2 MAPK, we here determine the transcription profile of the mutants inactivated for PaMpk1, PaMpk2 and PaNox1 during vegetative growth at stages where they are required for proper development, in comparison with wild type. Microarrays representing 10556 coding sequences (CDS) predicted by the P. anserina genome project were used to estimate the transcription profiles (transcriptomes) during vegetative growth of three-day-old homokaryotic cultures of M-bM-^HM-^FPaMpk1, M-bM-^HM-^FPaMpk2 and the IDC343 PaNox1 mutants in comparison to wild type. Three zones corresponding to three developmental stages can be defined in such wild-type culture. From the growing edge the colony, the first five milimeters (zone 1) are constituted of hyphae lacking pigment and growing on the substrate. In this zone, very few hyphae are aerial and appressorium-like structures are not differentiated. In the second intermediate zone (zone 2), profuse aerial hyphae and appressorium-like structures are differentiated. This zone is about 7 mm wide and corresponds to the area where most perithecia are differentiated during sexual development of heterokaryotic mycelia. The zone that is most distal from the edge lack aerial hyphae (zone 3) accumulates a larger amount of pigment and differentiates few perithecia during sexual reproduction. It is about 1 cm wide. These three zones are easily visualized by the naked eye for sample recovery. The M-bM-^HM-^FPaMpk1, M-bM-^HM-^FPaMpk2 and the IDC343 PaNox1 mutants lack a clear zonation, since the three genes are involved in the signalling of stationary phase . Materials from these mutants were nonetheless recovered as three samples corresponding to the same zones, based mostly on the distance from the growing edge but also from subtle morphological differences. With GPL10116 : reference design, 4 strains (WT, M-bM-^HM-^FPaMpk1, M-bM-^HM-^FPaMpk2 and IDC343 PaNox1 with 3 growth conditions (zone1,2 and 3) each with four biological replicates, the common reference is a pool of four conditions M48h, M96h, C48h and C96h ; Conditions are labelled in Cy3 and the common reference in Cy5

Project description:Abstract BACKGROUND: Mating-type loci in yeasts and ascomycotan filamentous fungi (Pezizomycotina) encode master transcriptional factors that play a critical role in sexual development. Genome-wide analyses of mating-type-specification circuits and mating-type target genes are available in Saccharomyces cerevisiae and Schizosaccharomyces pombe; however, no such analyses have been performed in heterothallic (self-incompatible) Pezizomycotina. The heterothallic fungus Podospora anserina serves as a model for understanding the basic features of mating-type control. Its mat+ and mat- mating types are determined by dissimilar allelic sequences. The mat- sequence contains three genes, designated FMR1, SMR1 and SMR2, while the mat+ sequence contains one gene, FPR1. FMR1 and FPR1 are the major regulators of fertilization, and this study presents a genome-wide view of their target genes and analyzes their target gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: The transcriptomic profiles of the mat+ and mat- strains revealed 157 differentially transcribed genes, and transcriptomic analysis of fmr1(-) and fpr1(-) mutant strains was used to determine the regulatory actions exerted by FMR1 and FPR1 on these differentially transcribed genes. All possible combinations of transcription repression and/or activation by FMR1 and/or FPR1 were observed. Furthermore, 10 additional mating-type target genes were identified that were up- or down-regulated to the same level in mat+ and mat- strains. Of the 167 genes identified, 32 genes were selected for deletion, which resulted in the identification of two genes essential for the sexual cycle. Interspecies comparisons of mating-type target genes revealed significant numbers of orthologous pairs, although transcriptional profiles were not conserved between species. CONCLUSIONS/SIGNIFICANCE: This study represents the first comprehensive genome-wide analysis of mating-type direct and indirect target genes in a heterothallic filamentous fungus. Mating-type transcription factors have many more target genes than are found in yeasts and exert a much greater diversity of regulatory actions on target genes, most of which are not directly related to mating. With GPL10116 : 4 conditions each with four biological replicates :mat+, mat-, fpr1-, fmr1-; common reference is a pool of four conditions M48h, M96h, C48h and C96h ; Conditions are labelled in Cy3 and the common reference in Cy5

Project description:A genome wide microarray, covering the 10546 presently known and predicted CDS, has been constructed with the Agilent in-situ synthesized 60-mers technology. 1) A library of 10 oligonucleotides per CDS has been design by the manufacturer. 2) A computational step has been performed to determine 3' position, intron position and cross-hybridization rate of all probes. These data have been compiled to attribute a score to each probe and thus, to select a first set of 4 oligonucleotides per CDS. 3) Microarray v.2 (GPL10115), manufactured with this design, have been used for a selection procedure based on the use of transcriptomic experimental data generated with diverse culture conditions. To maximize the number of expressed CDS, experimental data were generated with a large range of growth conditions covering crucial physiological changes from key steps of vegetative growth and sexual development.For each condition, four biological replicates were done to increase the statistical power of the analysis. An oligonucleotide scoring has been settled to select the best oligonucleotide per CDS. The final array design, Microarray v.3 (GPL10116), contained 10,539 probes for nuclear CDS. The array was enriched with 17 mitochondrial CDS probes that did not undergo the experimental screening. Each probe was replicated four times on each 44K array to improve the statistical significance of results. Before using in industrious transcriptomic experiment, ultimate qualification steps have been carried out. First, DNA extracted from the/ S/ and /s /strains was hybridized with the final design Microarray v.III. The apha_ORF and /het/ genes were identified as polymorphic between /s/ and /S/ strains (|FC|>2; a/ =/0.001) confirming previous genetic and molecular analyses (Turcq B etal.,1990*,* /Current genetics, /Sellem et al., 1990, Mol. Gen. Genet.). In second qualification step, a self-to-self hybridization experiment, replicated three times, has been /per/formed with a commonreference: 89.3% of the probes displayed a signal-to-standard-deviation ratio >3. The final microarray design is referenced as AMADID 018343. Microarray slides are available to the research community from Agilent. We developed oligonucleotide microarrays for the filamentous fungus Podospora anserina as part of the ANR program project grant ANR-05-BLAN-0385 (project SexDevMycol), coordinated by R. Debuchy. This project was aimed to analyze the sexual development of /P. anserina/ with a variety of approaches, including transcriptomic profiling. Different versions of microarrays were tested on mycelium and sexual development time courses, to evaluate their perfomance in comparing transcriptomic profiles of vegetative vs sexual stages, and in analyzing transcriptomic profiles evolution during sexual development. Complete analyses were performed with the microarray final version (v.III) and several manuscripts dealing with these topics are in preparation. The s (small s) and S (big S) strains of Podospora anserina are different geographical isolates of the same species. Both strains can cross and have identical ITS (accession #: AY278557 and GU391422) confirming that they belong to the same species. The S strain has been completely sequenced (Espagne et al., Genome Biology, 2008, 9, R77 ) and its genome is currently at the finishing step (P. Silar and P. Wincker, unpublished results). The genome of the s strain is not sequenced, and only a few genes are known. The major known difference is at the level of the s locus controling vegetative incompatibility (Turcq et al., Current genetics 1990, 17:297-303). PART1 (with GPL10115): reference design, 5 conditions each with four biological replicates : M24h, M48h, M96h, C24h, C48h; common reference is a pool of four conditions M48h, M96h, C48h and C96h ; Conditions are labelled in Cy3 and the common reference in Cy5 PART2 (with GPL10116): DNA hybridization with a direct design, wt s versus wt S, dye swap (four technical replicates); RNA Homotypic hybridization with the common reference sample (three technical replicates)

Project description:DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase (PEPCk), led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2 and IolR. Determination of the PEPCk activity of the deletion mutants M-NM-^TramA, M-NM-^TgntR1M-NM-^TgntR2, and M-NM-^TiolR indicated that RamA represses pck during growth on glucose about twofold, whereas GntR1, GntR2, and IolR activate pck expression about twofold, irrespective whether glucose or acetate served as carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in divergent orientation to a iol gene cluster, encoding proteins involved in myo-inositol uptake and degradation. Comparative DNA microarray analysis of the M-NM-^TiolR mutant and the parental wild-type revealed strongly elevated (>100-fold) mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT and iolR itself, which presumably is subject to negative autoregulation. A consensus DNA-binding motif was identified (5M-bM-^@M-^Y-KGWCHTRACA-3M-bM-^@M-^Y) which corresponds well to those of other GntR-type regulators of the HutC family. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism. To identify genes that are potentially regulated by IolR, the transcriptome profile of the iolR deletion strain was compared to that of the WT using DNA microarray analysis. The strains were grown in CGXII minimal medium with 4% (wt/vol) glucose as sole carbon source and RNA was isolated from cells harvested in the early exponential growth phase (OD600 of about 5). The transcriptome comparison was performed in triplicate starting from independent cultures. The second replicate included a dye-swap.

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A genetic and functional investigation of the Zn2Cys6 transcription factors RSE2 and RSE3 in Podospora anserina

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