Coordinate genomic association of transcription factors controlled by an imported quorum sensing peptide in Cryptococcus neoformans

Abstract

Qsp1 is a secreted quorum sensing peptide required for virulence of the fungal meningitis pathogen Cryptococcus neoformans. Qsp1 functions to control cell wall integrity in vegetatively growing cells and also functions in mating. Rather than acting on a cell surface receptor, Qsp1 is imported to act intracellularly via the predicted oligopeptide transporter Opt1. Here, we identify a transcription factor network as a target of Qsp1. Using whole-genome chromatin immunoprecipitation, we find Qsp1 controls the genomic associations of three transcription factors to genes whose outputs are regulated by Qsp1. One of these transcription factors, Cqs2, is also required for the action of Qsp1 during mating, indicating that it might be a shared proximal target of Qsp1. Consistent with this hypothesis, deletion of CQS2 impacts the binding of other network transcription factors specifically to Qsp1-regulated genes. These genetic and genomic studies illuminate mechanisms by which an imported peptide acts to modulate eukaryotic gene expression.

Fig 1. LIV3, NRG1, and CQS2 act downstream of QSP1.

A) Colony morphology of knockout strains streaked on YPAD agar at 25, 30, or 37°C for 6, 5 or 4 days, respectively. B) Anti-Qsp1 immunoblot of supernatant harvested from overnight cultures in YPAD. C) A Qsp1-secreting donor strain is patched on the left, and colonies from a wrinkled acceptor strain streaked to the right are tested for its ability to be complemented by Qsp1 from the donor strain. D) Schematic for how strains were tested for their ability to survive different concentrations of the cell wall stressor SDS. E) Single mutants for each of the genes shown were tested for their ability to survive increasing concentrations of the cell wall stressor SDS. Water dilutions are shown to the right. Plates were allowed to grow up at room temperature for 4 days. F) Each genotype shown was tested for their ability to survive increasing concentrations of the cell wall stressor SDS. 1 uM synthetic Qsp1 peptide was added to the indicated cultures (+) from the time of inoculation, or not (-). Water dilutions of each culture are shown to the right as a measure of cell input. Plates were allowed to grow up at room temperature for 4 days.

Fig 2. Cqs2, Nrg1, and Liv3 are part of a transcription factor network that shares targets with Qsp1.

A) Schematic of how cultures were grown and harvested for RNA-seq. B) Number of significantly differentially expressed genes in qsp1Δ vs. wild type as determined by DE-seq2 analysis. C) Comparisons of significantly differentially expressed genes from DEseq2 analysis of each mutant compared to wild type (WT) and their P-values shown above the arrows. Solid lines and bold text indicate that the overlap is significant (p<0.05), dotted lines and blue text indicate a non-significant P-value (p≥0.05).

Fig 3. Qsp1 affects Cqs2, Liv3, and Nrg1 binding to a common set of promoters.

A) Expression of Cqs2, Liv3, and Nrg1 protein levels in wild type or qsp1Δ mutants at OD1 in minimal media. Two biological replicates are shown for each condition. B) Schematic for how ChIP-seq cultures were grown and harvested. C) ChIP-Seq data was visualized using the Integrative Genomics Viewer software. Binding across part of chromosome 1 is shown. D) Network diagram of promoters bound by Nrg1, Liv3, and Cqs2 in wild type cells in YNB at OD1. E) Overlaps between promoter sets significantly enriched for Liv3, Cqs2, or Nrg1 binding and their significance. F) Overlaps between promoter sets differentially bound (>1.5-fold) by Liv3, Cqs2, or Nrg1 in the qsp1Δ mutant compared to wild type. G) Breakdown of promoters that are bound more or less by a given transcription factor in qsp1Δ compared to wild type, filtered by promoters significantly bound in either genotype. Significant overlaps between groups are noted with the P-value. H) Overlaps between promoter sets that are >1.5-fold less bound by Liv3, Cqs2, or Nrg1 in a qsp1Δ mutant compared to wild type, and their significance. Only promoters that are called as bound in either genotype by k-means analysis are shown.

Fig 4. Qsp1 regulation of binding of Cqs2, Nrg1, and Liv3 to promoters correlates with a change in expression.

A) Overlap between promoters bound by each transcription factor in wild type and genes that are differentially expressed in the corresponding transcription factor mutant, at OD1 in minimal media. B) Overlap between promoters differentially bound by each transcription factor and genes that are differentially expressed in each qsp1Δ mutant relative to wild type, at OD1 in minimal media. C) Heatmap of genes that are differentially bound by all three TFs Cqs2, Liv3, or Nrg1, and their respective log2-fold expression difference in qsp1Δ compared to wild type. Non-significant differences are colored in white, significant decreases in mutant are shown in blue, and significant increases in qsp1Δ over wild type are shown in yellow. D) Degree of overlap between sets of genes that are differentially bound by a transcription factor and genes that are differentially expressed in a qsp1Δ mutant relative to wild type, and those that are differentially bound by another transcription factor and differentially expressed in qsp1Δ mutants. E) Overlap between gene sets that are differentially bound by all three transcription factors (Liv3, Cqs2, and Nrg1) in the qsp1Δ mutant compared to wild type and genes that are significantly changed, decreased, or increased in the qsp1Δ mutant over wild type.

Fig 5. Cqs2 and Nrg1 impact binding of other transcription factors in the network to promoters, and this impact on binding is similar to the impact of Qsp1 on binding.

A) Protein levels of FLAG-tagged Cqs2, Liv3, and Nrg1 in each transcription factor deletion strain background. Average of two biological replicates is shown. B) ChIP-Seq data was visualized using the Integrative Genomics Viewer software. Binding across part of chromosome 1 is shown. C) Breakdown of promoters that are bound more or less by a given transcription factor in qsp1Δ compared to wild type, filtered by promoters significantly bound in either genotype. D) Overlaps between promoter sets that are less bound by each transcription factor in the qsp1Δ mutant and promoter sets that are less bound by each transcription factor in the nrg1Δ or cqs2Δ mutants.

Fig 6. Model for how Qsp1 triggers changes in gene expression in Cryptococcus neoformans.

Following import into the cytoplasm, Qsp1 alters the binding of Nrg1 and Liv3 by modulating the ability of Cqs2 to bind promoters, thereby causing changes to gene expression. Dotted lines indicate functional rather than physical interactions.