Adaptive expression responses in the Pol-gamma null strain of S. pombe depleted of mitochondrial genome

Abstract

Background: DNA polymerase gamma(Pol-gamma) has been shown to be essential for maintenance of the mitochondrial genome (mtDNA) in the petite-positive budding yeast Saccharomyces cerevisiae. Budding yeast cells lacking mitochondria exhibit a slow-growing or petite-colony phenotype. Petite strains fail to grow on non-fermentable carbon sources. However, it is not clear whether the Pol-gamma is required for mtDNA maintenance in the petite-negative fission yeast Schizosaccharomyces pombe.

Results: We show that disruption of the nuclear gene pog1+ that encodes Pol-gamma is sufficient to deplete mtDNA in S. pombe. Cells bearing pog1Delta allele require substantial growth periods to form petite colonies. Mitotracker assays indicate that pog1Delta cells are defective in mitochondrial function and EM analyses suggest that pog1Delta cells lack normal mitochondrial structures. Depletion of mtDNA in pog1Delta cells is evident from quantitative real-time PCR assays. Genome-wide expression profiles of pog1Delta and other mtDNA-less cells reveal that many genes involved in response to stimulus, energy derivation by oxidation of organic compounds, cellular carbohydrate metabolism, and energy reserve metabolism are induced. Conversely, many genes encoding proteins involved in amino acid metabolism and oxidative phosphorylation are repressed.

Conclusion: By showing that Pol-gamma is essential for mtDNA maintenance and disruption of pog1+ alters the genome-wide expression profiles, we demonstrated that cells lacking mtDNA exhibit adaptive nuclear gene expression responses in the petite-negative S. pombe.

Figure 1

Disruption of the gene encoding Pol-γ. (A) The phylogenic tree of Pol-γ. (B) Tetrad dissection analysis of spores generated from pog1⁺/pog1Δ diploid cells after incubation at 30°C for ~5 days. The asterisk indicates that an uppermost wild type spore in the 9^th tetrad was implanted into the agar and thus grows slower than other wild type spores. (C) The magnified view of a tetrad in (B). Genotypes of individual spores are indicated. (D) pog1Δ spores form petite colonies after a lengthy incubation term (~15 days) at 30°C.

Figure 2

Growth phenotypes of pog1Δ cells. (A) Abnormal cellular morphologies of pog1Δ cells. Circled number 1 and 2 indicate dumb-bell cell shapes; number 2 and 3 indicate cells with excessive division septum deposition; number 3 indicates anucleate cells. (B) Subcellular localization of Pog1-γ. Cells bearing a sole copy of pog1⁺-GFP were examined for GFP localization using fluorescence microscopy. (C) Live cells stained with mitochondria-specific compound MitoTracker. (D) Approximately 5 μl of 10-fold series diluted cells were spotted onto standard EMM medium (glucose) and glycerol medium (glycerol) and incubated at 30°C for 5–7 days. (E) EM images of wild type and pog1Δ cells. Nc and m stand for nucleus and mitochondrion, respectively.

Figure 3

Comparisons of the copy number of mtDNA and nuclear DNA in various S. pombe strains. The left panel is the plot of fluorescence versus PCR cycle number. The arrows indicate the median of Ct-values for either mtDNA (mt; in green) or nuclear DNA (nc; in purple). The right panel is the box-plot representing the distribution Ct-values by primer pairs either specific to mtDNA (in green) or nuclear DNA (in purple): the minimal (left-end bar), the first quantile (left side of the box), median (central think bar), the third quantile (right side of the box), and the maximal (right-end bar). The copy number of nuclear DNA in various strains is set to 1 as reference for the copy number of mtDNA. The assays were carried out using total DNA samples extracted from wild type (A), pog1Δ (B), ptp1-1 (C), and ptp2-1 (D) cells as indicated.

Figure 4

Characteristics of differentially expressed genes in mtDNA-less strains. (A) and (B) Venn diagrams of the up-regulated genes (3-fold or greater) and down-regulated genes (1/3-fold or less), respectively, in pog1Δ, ptp1-1, and ptp2-1 cells compared to the wild type cells. (C) Enriched gene-ontology categories in the list of the intersection of the up-regulated genes in the mtDNA-less strains. A gene-ontology category is considered if three genes or more in the tested gene list are involved in the category. (D) Enriched gene-ontology categories in the list of the union of the down-regulated genes in the mtDNA-less strains. (E) GO Graph. The relationship of some enriched GO categories is shown. Categories in red and green indicate those enriched with up- and down-regulated genes, respectively.

Figure 5

Expression profiles of pog1Δ, ptp1-1, and ptp2-1 cells. (A) Expression profiles of genes encoding TCA cycle enzymes. The phasogram of gene expression profiles (mtDNA-less cells versus wild type cells) is shown in which rows indicate genes and columns indicate individual repeats (for S.c., columns represent different cell densities). S. pombe gene names are listed in the left and S. cerevisiae (S.c.) are listed in the right. Induced expression levels are shown in red and repressed are in green (S.c. data are based on the study by Epstein et al.). No changes are in black and no data are in grey. The color key is shown at the bottom. (B) TCA cycle. Genes induced are indicated in red while genes repressed are in green. (C-E) Expression profiles of genes encoding proteins involved in cytochrome c reductase, oxidase, and ATP synthase complexes, respectively. The profiles are displayed as in (A).