Cbf11 and Mga2 function together to activate transcription of lipid metabolism genes and promote mitotic fidelity in fission yeast

Abstract

Within a eukaryotic cell, both lipid homeostasis and faithful cell cycle progression are meticulously orchestrated. The fission yeast Schizosaccharomyces pombe provides a powerful platform to study the intricate regulatory mechanisms governing these fundamental processes. In S. pombe, the Cbf11 and Mga2 proteins are transcriptional activators of non-sterol lipid metabolism genes, with Cbf11 also known as a cell cycle regulator. Despite sharing a common set of target genes, little was known about their functional relationship. This study reveals that Cbf11 and Mga2 function together in the same regulatory pathway, critical for both lipid metabolism and mitotic fidelity. Deletion of either gene results in a similar array of defects, including slow growth, dysregulated lipid homeostasis, impaired cell cycle progression (cut phenotype), abnormal cell morphology, perturbed transcriptomic and proteomic profiles, and compromised response to the stressors camptothecin and thiabendazole. Remarkably, the double deletion mutant does not exhibit a more severe phenotype compared to the single mutants. In addition, ChIP-nexus analysis reveals that both Cbf11 and Mga2 bind to nearly identical positions within the promoter regions of target genes. Interestingly, Mga2 binding appears to be dependent on the presence of Cbf11 and Cbf11 likely acts as a tether to DNA, while Mga2 is needed to activate the target genes. In addition, the study explores the distribution of Cbf11 and Mga2 homologs across fungi. The presence of both Cbf11 and Mga2 homologs in Basidiomycota contrasts with Ascomycota, which mostly lack Cbf11 but retain Mga2. This suggests an evolutionary rewiring of the regulatory circuitry governing lipid metabolism and mitotic fidelity. In conclusion, this study offers compelling support for Cbf11 and Mga2 functioning jointly to regulate lipid metabolism and mitotic fidelity in fission yeast.

Fig 1. Cells lacking Cbf11 and/or Mga2 display similar, non-additive cellular defects.

(A) Cells of the indicated genotypes were stained with DAPI to visualize nuclei. Cyan arrows point to the cells displaying the ‘cut’ phenotype and indicate positions where a nucleus is trapped in a septum and/or cell wall (top row); cells with fragmented and/or diffuse nuclei are marked with yellow asterisks (middle row); filamentous cells with septation defects are highlighted with dotted outlines (bottom row). Representative images from 6 independent experiments are shown. Scale bar represents 10 μm. (B) Quantification of ‘cut’ phenotype occurrence. Mean ± SD values, as well as individual data points for ≥6 independent experiments are shown. Significance was determined by a Welch Two Sample t-test.

Fig 2. The absence of Cbf11 and/or Mga2 results in similar transcriptome and proteome changes.

RT-qPCR (A) and RNA-seq (B) show that lipid metabolism genes are downregulated in Δcbf11 and/or Δmga2 mutants and this effect is not additive in the double deletion mutant. Means values ± SD, as well as individual data points for ≥3 independent experiments are shown in panel (A). Mean values of 3 independent RNA-seq experiments are shown in panel (B). Significance of changes was determined by a one-sided Mann-Whitney U test. Venn diagrams showing that the sets of genes (C) and proteins (D) expressed differentially in Δcbf11 and/or Δmga2 cells significantly overlap (Fisher’s exact test of RNA-seq and MS proteomics results, respectively). Only protein-coding genes and proteins showing at least 1.5-fold or 2-fold and statistically significant change in expression compared to WT are included in panel (C) and (D), respectively. (E) Both genetic and chemical perturbation of lipid metabolism result in deregulation of the non-coding transcriptome. Gene expression was determined by RNA-seq and compared to WT. Three independent experiments were performed. For each gene, the magnitude of change in expression was plotted against its mean expression value across all samples. Statistically significant differentially expressed genes are shown in cyan (other genes) or green (lncRNAs). The two gray vertical lines indicate the interval of 2-fold up- or downregulation.

Fig 3. Cells lacking Cbf11 and/or Mga2 display nutrient-responsive decrease in lipid droplet (LD) content.

(A) Cells of the indicated genotypes were stained with BODIPY 493/503 to visualize neutral lipids. Note the heterogeneity in LD content in Δcbf11 and/or Δmga2 mutants grown in the complex YES medium. The aberrant LD content is largely suppressed when cells are grown in the minimal EMM medium. Representative microscopic images from 3 independent experiments are shown. DIC overlay is shown to mark cell boundaries. Scale bar represents 10 μm. (B, C) Quantification of LD staining intensity and LD numbers per unit of cell volume. Mean ± SD values, as well as individual data points for ≥3 independent experiments are shown. Significance was determined by a Welch Two Sample t-test.

Fig 4. Cells lacking Cbf11 and/or Mga2 are sensitive to thiabendazole (TBZ) and camptothecin (CPT).

Ten-fold serial dilutions of cultures of the indicated genotypes were plated on YES medium containing TBZ or CPT. Representative images from 2 independent experiments are shown.

Fig 5. Mga2 binds to DNA in a Cbf11-dependent manner.

(A) Scheme of three considered DNA binding regimes of Cbf11 and Mga2. See the main text for more detailed description. Image created with BioRender.com. (B) In vivo binding of Cbf11 and Mga2 TAP-tagged proteins to the promoters of selected lipid metabolism genes was analyzed by ChIP-nexus. Mean strand-specific coverage profile of 3 independent experiments for Cbf11 and Mga2 in the indicated genetic backgrounds, and a strand-specific coverage profile for untagged WT cells (negative control) are visualized in the Integrated Genome Viewer (IGV; Broad Institute). Gene orientation is indicated by three arrowheads; the maximum Y-axis value for each track is indicated on the right side of each panel.

Fig 6. Phylogenetic distribution of Cbf11 and Mga2 homologs in Fungi.

In the phylum Basidiomycota, members of the Cbf11 and Mga2 families are found together in almost all species. However, in the phylum Ascomycota, Cbf11 homologs are typically absent, except for the Taphrinomycotina group to which the fission yeasts belong (S. pombe position indicated with a light blue arrow). The taxonomy of some fungal species is uncertain (white part of the tree). Image created with iTOL v6.