Candida albicans repetitive elements display epigenetic diversity and plasticity

Abstract

Transcriptionally silent heterochromatin is associated with repetitive DNA. It is poorly understood whether and how heterochromatin differs between different organisms and whether its structure can be remodelled in response to environmental signals. Here, we address this question by analysing the chromatin state associated with DNA repeats in the human fungal pathogen Candida albicans. Our analyses indicate that, contrary to model systems, each type of repetitive element is assembled into a distinct chromatin state. Classical Sir2-dependent hypoacetylated and hypomethylated chromatin is associated with the rDNA locus while telomeric regions are assembled into a weak heterochromatin that is only mildly hypoacetylated and hypomethylated. Major Repeat Sequences, a class of tandem repeats, are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Marker gene silencing assays and genome-wide RNA sequencing reveals that C. albicans heterochromatin represses expression of repeat-associated coding and non-coding RNAs. We find that telomeric heterochromatin is dynamic and remodelled upon an environmental change. Weak heterochromatin is associated with telomeres at 30 °C, while robust heterochromatin is assembled over these regions at 39 °C, a temperature mimicking moderate fever in the host. Thus in C. albicans, differential chromatin states controls gene expression and epigenetic plasticity is linked to adaptation.

Figure 1. Transcriptional silencing at the C. albicans rDNA locus.

(A) Left panel: Schematic of rDNA::URA3⁺ reporter strain. Right panel: silencing assay of the rDNA::URA3⁺ reporter strain. Ura⁺ (URA3/URA3) and Ura⁻ (ura3Δ/ura3Δ) strains were included as controls. (B) qRT-PCR analyses to measure URA3⁺ transcript levels of the rDNA::URA3⁺ reporter strain relative to actin transcript levels (ACT1). Error bars in each panel: Standard deviation (SD) of three biological replicates.

Figure 2. Sir2-dependent heterochromatin at the rDNA locus.

(A) Left panel: Schematic of rDNA::URA3⁺ reporter strain. Right panel: Silencing assay of the rDNA::URA3⁺ reporter strain in WT and hst1 Δ/Δ isolates. A URA⁺ (URA3⁺) strain was included as a control. (B) Silencing assay of the rDNA::URA3⁺ reporter strain WT and sir2 Δ/Δ isolates. A URA⁺ (URA3⁺) strain was included as a control. (C) qRT-PCR analyses to measure URA3⁺ transcript levels relative to ACT1 transcript levels in rDNA::URA3⁺ WT and sir2 Δ/Δ isolates. A URA3⁺ strain is included as a control (D) qRT-PCR analyses to measure levels of the rDNA non-coding RNA (Novel_Chr3_093) relative to ACT1 in WT and sir2 Δ/Δ isolates. (E,F) qChIP to detect H3K9Ac, H4K16Ac levels associated with the rDNA locus and ACT1 in WT and sir2 Δ/Δ isolates and (G) H3K4me2 levels associated with the rDNA locus and ACT1 in WT, sir2 Δ/Δ and set1 Δ/Δ isolates. Error bars in each panel: Standard deviation (SD) of three biological replicates.

Figure 3. MRS repeats are assembled into transcriptionally-permissive chromatin.

(A) Top panel: Schematic of MRS::URA3⁺ reporter strain. Bottom panel: Silencing assay of the MRS::URA3⁺ reporter strain in WT and sir2 Δ/Δ isolates. A URA⁺ (URA3⁺) strain was included as a control. (B,C) RNA deep-sequencing of sir2 Δ/Δ and WT isolates. (B) Normalised read counts (FPKM) of MRS associated (MRS-C) and proximal (MRS-L and MRS-R) genes were calculated from RNA-seq data for WT and sir2 Δ/Δ isolates. The heat-map depicts the log2 fold ratio of FPKM data between sir2 Δ/Δ and WT isolates. (C) Boxplot showing log2 fold changes in transcriptional expression for MRS-internal (MRS-C) and adjacent (MRS-L and MRS-R) genes between sir2 Δ/Δ and WT isolates. (D,E) qChIP to detect H3K9Ac, H4K16Ac levels associated with the MRS repeats and ACT1 in WT and sir2 Δ/Δ isolates and (F) H3K4me2 levels associated with the MRS repeats and ACT1 in WT and set1 Δ/Δ isolates. Error bars in each panel: Standard deviation (SD) of three biological replicates.

Figure 4. Heterochromatin at C. albicans telomeric regions.

Left panel: Schematic of Tel5::URA3⁺ reporter strain. Right panels: Silencing assay of the Tel5::URA3⁺ reporter strain in WT and sir2 Δ/Δ. (A) URA⁺ (URA3⁺) strain was included as a control. (B,C) RNA deep-sequencing of sir2 Δ/Δ and WT isolates. (B) Normalised read counts (FPKM) of subtelomeric genes were calculated from RNA-seq data for WT and sir2 Δ/Δ isolates. The heat-map depicts the log2 fold ratio of FPKM data between sir2 Δ/Δ and WT isolates. (C) Boxplot showing log2 fold changes in transcriptional expression of subtelomeric genes between sir2 Δ/Δ and WT isolates (D–F) qChIP to detect H3K9Ac and H4K16ac levels associated with Tel5::URA3⁺ and ACT1 in WT and sir2 Δ/Δ strains. (C) H3K4me2 levels associated with Tel5::URA3⁺ and ACT1 in WT and set1 Δ/Δ isolates. Error bars in each panel: Standard deviation (SD) of three biological replicates.

Figure 5. Telomeric heterochromatin is plastic.

(A) Left panel: Schematic of Tel5::URA3⁺ reporter strain. Right panels: Silencing assay assessing transcriptional silencing of the Tel5::URA3⁺ reporter strain in WT and sir2 Δ/Δ isolates at 30 °C, in the presence of 1 mM H₂O₂, 200 ng/μl fluconazole and at 39 °C. A URA⁺ (URA3⁺) strain was included as a control. (B) qRT-PCR analyses to measure Tel5::URA3⁺ transcript levels relative to ACT1 at 30 °C and 39 °C in WT (left panel) and sir2 Δ/Δ (right panel) strains. (C) qChIP to detect H3K9Ac levels associated with Tel5::URA3⁺ and ACT1 in WT and sir2 Δ/Δ isolats at 30 °C (Left panel) and 39 °C (Right panel). Error bars in each panel: Standard deviation (SD) of three biological replicates.