Figure 1.
The transcriptional signature of capsule induction.
Shown is a heat map of gene expression (blue, low expression; yellow, high expression) for the 880 genes whose expression, as assessed by microarray analysis, trends with capsule size. Cell growth conditions (see Methods) for each column are indicated below the heat map (see Results for abbreviations) and average capsule radius is plotted above (gray bars). The correlation of gene expression and capsule size is plotted at the right.
Figure 2.
Correlation of gene expression and capsule size for selected genes.
Data is shown for three genes that demonstrate correlation between gene expression and capsule size.
Figure 3.
Cells lacking ADA2 display reduced capsule size under inducing conditions.
Panel A, negative staining with India ink of KN99α cells (WT), the indicated deletion strains, and the complemented ada2Δ mutant (ADA2). All images are at the same magnification. Scale bar, 5 µm. Panel B, histogram of capsule size for the ada2Δ mutant (red) and WT (black) populations. Capsule radius is represented in microns.
Figure 4.
Cryptococcal Ada2 is localized to the nucleus.
Wild type cells (WT) and cells modified to express HA epitope-tagged Ada2 from the native locus (ADA2-HA) were labeled with an antibody against HA (αHA, red), and counter-stained with DAPI (blue) to show the location of chromatin. All images were acquired at the same settings and are shown at the same magnification. Scale bar, 1 µm.
Figure 5.
Histone acetylation is markedly reduced in the absence of Ada2.
Shown are immunofluorescence micrographs of wild type (WT), ada2Δ, and complemented ada2Δ (ADA2) cells grown in capsule inducing conditions for 90 min and then probed with antibody to H3K9 (αH3-K9). All images were acquired at the same settings and are shown at the same magnification; scale bar, 1 µm.
Figure 6.
Ada2 is required for growth under certain stress conditions.
Ten-fold serial dilutions of the indicated strains were grown in the conditions shown (see Methods for details). Top panel, growth on rich medium (YPD) at the temperatures indicated above the images; middle panels (four rows of images), growth on YPD with the indicated stressor at the temperatures shown at the right; bottom panel (two rows of images), growth on minimal medium (YNB) or YNB with the indicated stressor at the temperatures shown at the right.
Figure 7.
Ada2 is required for normal hyphal development.
Wild type (no strain designation), ada2Δ, and complemented ada2Δ (ADA2) strains of opposite mating type were mixed and grown under conditions that induce mating (see Methods). Patches were imaged after 13 days.
Figure 8.
Ada2 is required for growth and virulence in mice.
Panel A, C57Bl/6 mice were intranasally inoculated with 1.25 × 104 cells of the indicated strains, and total colony forming units (CFU) were isolated from the lungs after one hour (black bars) or one week (gray bars). The mean±maximum and minimum is shown. Panel B, survival curve of A/Jcr mice that were similarly inoculated with 105 cells of wild type (black), ada2Δ (red), or complemented ada2Δ (gray). Like those infected with ada2Δ, all mice that were infected in the same study with cap59 survived the entire period (not shown).
Table 1.
Genes downstream of Ada2 implicated in processes related to mating or virulence.
Figure 9.
Ada2-dependent acetylation of H3K9 is enriched near gene transcription start sites.
ChIP-Seq was performed on wild type (WT) and the ada2Δ mutant to identify genes located in the proximity of acetylated H3K9. Panel A, a histogram of peaks that occur within 500 bp of the transcription start site of all identified genes [45]. Panel B, an example of ChIP-Seq data aligned to a gene model of GAT204, which was identified as Ada2-dependent by both ChIP-Seq and RNA-Seq. The y-axis represents normalized coverage (reads per million mapped) for samples defined in the text. Coverage is shown for 2 standard deviations above the mean input sample coverage and above. Note that the input DNA profiles are similar for WT and mutant cells, while specific H3K9 associated sequences show TSS-associated peaks only in the WT.
Figure 10.
A model of Ada2 within the broader network of capsule, mating, and antiphagocytic responses (see text for details).
Links from Cir1 and Nrg1 are supported by RNA-Seq data presented here. Links from Hog1 and Pbs2 are supported by published microarray data [47]; links from Gat201 are supported by published data from microarrays and ChIP-chip [24]; and links from Ada2 are supported by RNA-Seq and ChIP-chip data presented here. Red ovals, transcription factors; blue rounded rectangles, signaling proteins; green rounded rectangles, other proteins; green lines, stimulation of transcription; red lines, inhibition of transcription; solid black arrow, phosphorylation; P, phosphate; dashed arrow, catalysis by Pbs2.