A Differential Genome-Wide Transcriptome Analysis: Impact of Cellular Copper on Complex Biological Processes like Aging and Development

Abstract

The regulation of cellular copper homeostasis is crucial in biology. Impairments lead to severe dysfunctions and are known to affect aging and development. Previously, a loss-of-function mutation in the gene encoding the copper-sensing and copper-regulated transcription factor GRISEA of the filamentous fungus Podospora anserina was reported to lead to cellular copper depletion and a pleiotropic phenotype with hypopigmentation of the mycelium and the ascospores, affected fertility and increased lifespan by approximately 60% when compared to the wild type. This phenotype is linked to a switch from a copper-dependent standard to an alternative respiration leading to both a reduced generation of reactive oxygen species (ROS) and of adenosine triphosphate (ATP). We performed a genome-wide comparative transcriptome analysis of a wild-type strain and the copper-depleted grisea mutant. We unambiguously assigned 9,700 sequences of the transcriptome in both strains to the more than 10,600 predicted and annotated open reading frames of the P. anserina genome indicating 90% coverage of the transcriptome. 4,752 of the transcripts differed significantly in abundance with 1,156 transcripts differing at least 3-fold. Selected genes were investigated by qRT-PCR analyses. Apart from this general characterization we analyzed the data with special emphasis on molecular pathways related to the grisea mutation taking advantage of the available complete genomic sequence of P. anserina. This analysis verified but also corrected conclusions from earlier data obtained by single gene analysis, identified new candidates of factors as part of the cellular copper homeostasis system including target genes of transcription factor GRISEA, and provides a rich reference source of quantitative data for further in detail investigations. Overall, the present study demonstrates the importance of systems biology approaches also in cases were mutations in single genes are analyzed to explain the underlying mechanisms controlling complex biological processes like aging and development.

A Genome-Wide Longitudinal Transcriptome Analysis of the Aging Model Podospora anserine

“…556 genes of the grisea transcriptome were identified to be down-regulated with a factor of at least 3 and a p-value of ≤0.01 for differential expression [22]. The 1,202 genes, identified as being down-regulated during aging in this study, have 89 genes in common.…”

“…Previously we used SuperSAGE successfully to characterize the transcriptome of a specific long-lived mutant of P. anserina and compared it to the transcriptome of the wild type. Validation by qRT-PCR demonstrated the reliability of this method [22]. The data of the current longitudinal study, in which RNA was isolated from the same fungal individuals after a defined period of growth and subjected to a genome-wide SuperSAGE analyses, identified autophagy as a quality control pathway up-regulated late in the life of P. anserina at a time when transcripts, encoding components of other pathways (e.g., proteasome), are down-regulated.…”

“…Sequence tag identification and annotation, and basal statistics were performed by GenXPro (Frankfurt) as described in [22]. The raw data have been deposited in the European Bioinformatics Institute’s ArrayExpress public data repository (http://www.ebi.ac.uk/arrayexpress/) [26] and are available under accession number E-MTAB-2016.…”

Effect of paraquat-induced oxidative stress on gene expression and aging of the filamentous ascomycete Podospora anserina

“…Since transcription of PaCtr3 , coding for a high affinity copper transporter, is dependent on the availability and activity of GRISEA, both in senescent wild-type strains as well as in the grisea mutant, copper-uptake is only possible via low affinity import. As a consequence, a switch from a copper-dependent standard to an iron-dependent alternative respiration, a strongly reduced generation of the mitochondrial superoxide anion and pronounced changes in gene expression are observed in the mutant 22 23 41 .…”

“…This function may link genetic, environmental and stochastic processes involved in aging and lifespan control and can help to explain unexpected and counter-intuitive experimental data. After having investigated genome-wide transcriptome profiles of P. anserina wild-type cultures of different age and of a long-lived mutant in which mitochondrial ROS generation is reduced compared to the wild type 22 23 , we now set out to analyze the impact of increased oxidative stress on global gene expression. Using paraquat (PQ) as a generator of the superoxide anion at the mitochondrial respiratory chain 24 25 , the site at which this ROS is generated also during normal aging, we experimentally induced strong cellular oxidative stress in wild-type cultures of different age and found that this treatment has profound effects on gene expression, growth and lifespan.…”