PLoS Genet. 2016 Dec 5;12(12):e1006453. doi: 10.1371/journal.pgen.1006453.

Investigating Conservation of the Cell-Cycle-Regulated Transcriptional Program in the Fungal Pathogen, Cryptococcus neoformans.

Kelliher CM1, Leman AR1, Sierra CS1, Haase SB1,*

1 Department of Biology, Duke University, Durham, North Carolina, 27708, United States of America

* Corresponding author: Steve Haase, Department of Biology, e-mail: shaase@duke.edu

 

Abstract:

The pathogenic yeast Cryptococcus neoformans causes fungal meningitis in immune-compromised patients. Cell proliferation in the budding yeast form is required for Cneoformans to infect human hosts, and virulence factors such as capsule formation and melanin production are affected by cell-cycle perturbation. Thus, understanding cell-cycle regulation is critical for a full understanding of virulence factors for disease. Our group and others have demonstrated that a large fraction of genes in Saccharomyces cerevisiae is expressed periodically during the cell cycle, and that proper regulation of this transcriptional program is important for proper cell division. Despite the evolutionary divergence of the two budding yeasts, we found that a similar percentage of all genes (~20%) is periodically expressed during the cell cycle in both yeasts. However, the temporal ordering of periodic expression has diverged for some orthologous cell-cycle genes, especially those related to bud emergence and bud growth. Genes regulating DNA replication and mitosis exhibited a conserved ordering in both yeasts, suggesting that essential cell-cycle processes are conserved in periodicity and in timing of expression (i.e. duplication before division). In Scerevisiae cells, we have proposed that an interconnected network of periodic transcription factors (TFs) controls the bulk of the cell-cycle transcriptional program. We found that temporal ordering of orthologous network TFs was not always maintained; however, the TF network topology at cell-cycle commitment appears to be conserved in Cneoformans. During the Cneoformans cell cycle, DNA replication genes, mitosis genes, and 40 genes involved in virulence are periodically expressed. Future work toward understanding the gene regulatory network that controls cell-cycle genes is critical for developing novel antifungals to inhibit pathogen proliferation.

PMID: 27918582

 

Supplement:

The cell cycle is essential for growth and division, which can be leveraged in an infectious disease context to develop novel therapies. The cell cycle is well characterized in the genetic model system Saccharomyces cerevisiae. Nearly 20% of genes in the genome are transcribed at a specific time during the S. cerevisiae cell cycle, and this program of genes is regulated by cell-cycle transcription factors (TFs) [1]. Cryptococcus neoformans is an opportunistic human fungal pathogen, and gene expression dynamics during a normal cell cycle were previously uncharacterized. In this study, we synchronized populations of Saccharomyces cerevisiae and Cryptococcus neoformans var. grubii cells for the cell cycle in rich media conditions and compared transcriptome dynamics between the two distantly related yeasts.

We found that ~20% of genes in both yeasts are periodically expressed during the cell cycle (Figure 1). Given these two programs of cell-cycle genes, we asked the following:

1) How many genes are scored as periodically expressed during the cell cycle? We provide a table of all expressed genes in S. cerevisiae and C. neoformans with corresponding periodicity ranking (S1-S2 Tables). For the purposes of our comparative analyses, we used a cutoff to identify the most periodically expressed genes. However, we also demonstrate that some genes below the periodicity cutoff appear to be weakly cell-cycle regulated (S1 Figure). The RNA expression datasets are viewable for both yeasts on FungiDB (http://fungidb.org/fungidb/) for experts in the fungal genetics community to determine if their gene or pathway of interest may be cell-cycle regulated.

2) Are any Cryptococcus neoformans genes that are required for virulence also periodically expressed during the cell cycle? We found 40 periodic genes that were previously identified in genetic screens for C. neoformans virulence defects (S3 Figure and Table). We highlight periodic enzymes involved in chitin synthesis and MAPK signaling in a recent review [2].

3) Many proteins in S. cerevisiae are known to play a role in specific cell-cycle events in G1, S, G2, or M phase. We found that the transcripts encoding DNA replication, spindle assembly, and mitosis genes have similar expression patterns during the cell cycle in both S. cerevisiae and C. neoformans cells, which suggests that the function of these genes is conserved through evolution (Figure 4 D-I, S6 Table). However, we found that genes involved in bud formation and growth are not transcribed with matching cell-cycle dynamics (Figure 4 A-C, S6 Table), which suggests that the process of bud initiation and growth between the two yeasts may involve different gene products or different timing during the cell cycle.

4) Do a conserved set of cell-cycle transcription factors (TFs) control cell-cycle gene expression? Finally, we asked if TFs are conserved in periodic expression and timing between the two yeasts (Table 1, Figures 5-6). We found evidence that TFs functioning at G1/S phase appear to be conserved in S. cerevisiae and C. neoformans by sequence homology (S4 Table), RNA expression peak time (Table 1), transcript dynamics (Figure 6), and a shared promoter motif enriched in putative target genes (S8 Figure). However, downstream of G1/S phase, we did not find convincing evidence for TF sequence homology or transcript dynamics. Instead, we propose that novel TFs may regulate cell-cycle gene expression in S-G2-M phases of C. neoformans cells (Figure 5).

 

 

Figure 1. Summary of key findings on transcription during the yeast cell-cycle. ~20% of genes in S. cerevisiae and C. neoformans are expressed periodically during the cell cycle: 1,246 genes in S. cerevisiae (A) and 1,134 periodic genes in C. neoformans (B). Genes in each heatmap are ordered along the y-axis by peak time of expression in the respective dataset. Each row represents a unique transcript measured by RNA-Sequencing, and each column is a sampled time point in minutes. Periodic genes were used to compare C. neoformans to previous knowledge about the S. cerevisiae cell cycle.

 

References:

  1. Kelliher CM, Leman AR, Sierra CS, Haase SB. Investigating Conservation of the Cell-Cycle-Regulated Transcriptional Program in the Fungal Pathogen, Cryptococcus neoformans. PLoS Genet. 2016; 12(12): e1006453.
  1. Kelliher CM, Haase SB. Connecting virulence pathways to cell-cycle progression in the fungal pathogen Cryptococcus neoformans. Curr Genet. 2017. doi: 10.1007/s00294-017-0688-5.