A histone deacetylase adjusts transcription kinetics at coding sequences during Candida albicans morphogenesis - PubMed (original) (raw)

A histone deacetylase adjusts transcription kinetics at coding sequences during Candida albicans morphogenesis

Denes Hnisz et al. PLoS Genet. 2012.

Abstract

Despite their classical role as transcriptional repressors, several histone deacetylases, including the baker's yeast Set3/Hos2 complex (Set3C), facilitate gene expression. In the dimorphic human pathogen Candida albicans, the homologue of the Set3C inhibits the yeast-to-filament transition, but the precise molecular details of this function have remained elusive. Here, we use a combination of ChIP-Seq and RNA-Seq to show that the Set3C acts as a transcriptional co-factor of metabolic and morphogenesis-related genes in C. albicans. Binding of the Set3C correlates with gene expression during fungal morphogenesis; yet, surprisingly, deletion of SET3 leaves the steady-state expression level of most genes unchanged, both during exponential yeast-phase growth and during the yeast-filament transition. Fine temporal resolution of transcription in cells undergoing this transition revealed that the Set3C modulates transient expression changes of key morphogenesis-related genes. These include a transcription factor cluster comprising of NRG1, EFG1, BRG1, and TEC1, which form a regulatory circuit controlling hyphal differentiation. Set3C appears to restrict the factors by modulating their transcription kinetics, and the hyperfilamentous phenotype of SET3-deficient cells can be reverted by mutating the circuit factors. These results indicate that the chromatin status at coding regions represents a dynamic platform influencing transcription kinetics. Moreover, we suggest that transcription at the coding sequence can be transiently decoupled from potentially conflicting promoter information in dynamic environments.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. The C. albicans Set3C is a coding sequence histone deacetylase.

(A) Architecture of the S. cerevisiae Set3C. The subunits among which physical interaction was confirmed in C. albicans are colored green (see Figure S1B). (B) Physical interaction of Set3 and Hos2. Set3-3HA was immunoprecipitated from whole cell extracts and the interaction was probed by immunoblot detection of a Hos2-9myc allele. (C) Read density profiles of one replicate of a Set3-9myc and an untagged control ChIP-Seq experiment. Genes were divided into binding targets (“targets”) and non-targets (see Materials and Methods). Transcription start site (TSS) denotes the start codon and Transcription termination site (TTS) denotes the stop codon. The read density values between the TSS and TTS were calculated to a percentage scale, and 500 bases upstream of the TSS and downstream of the TTS were included. On the bottom panel only genes with a coding region longer than 1 kilobase were included. (D) Definition of CaSet3C refined target gene set. Each dot corresponds to one ORF. Binding targets of RNAPII transcribed genes are defined as having an at least 2-fold enrichment on one axis and an at least 1.5-fold on the other axis (blue box). Target tRNA loci are defined as having an at least 1.5-fold enrichment on both axes. The complete dataset is found in Tables S4 and S5. “_r_” denotes a Pearson's correlation coefficient. (E) The Set3C functions as histone deacetylase in vivo. Top panel: validation of Set3 and Hos2 binding using the indicated probes around the PFK1 and tR(CCG)1 loci by qPCR. Values are normalized to a fragment of the ADE2 locus. Bottom panel: ChIP experiments were performed with antibodies against acetylated histone H4 and the C-terminus of histone H3. The qPCR values at the probe positions were normalized to a fragment of the telomere of Chromosome 7. The ratio of the signal of the acetylated H4 ChIP and H3 ChIP is shown on the y-axis. Data are shown as mean+SD of three independent experiments. Statistical significance was determined by two-tailed t-test relative to the control values. *P<0.05, **P<0.01, ***P<0.001.

Figure 2

Figure 2. The Set3C decorates highly transcribed genes.

(A) Correlation of Set binding and RNA expression for the target gene set (defined on Figure 1D). Each dot corresponds to one gene. The distribution of expression values of the genes belonging to each functional category is shown on the right panel. “_r_” denotes a Pearson's correlation coefficient. TF stands for the transcription factor cluster (see text). Statistical significance was determined by the Mann-Whitney U-test relative to the “all genes” set. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. (B) Transcript profile of _set3_Δ/Δ cells by RNA-Seq. The fold change in RNA expression between _set3_Δ/Δ and wild type cells at each gene is plotted against the expression level of the gene in wild type cells. The direct binding targets and their functional groups are highlighted. The distribution of fold changes of the genes belonging to each functional category is shown on the right panel. Statistical significance was determined by the Mann-Whitney U-test relative to the “all genes” set. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

Figure 3

Figure 3. Set3C recruitment predicts induction and depletion predicts repression.

(A) Microscopic images of cells undergoing yeast-to-hypha differentiation. The cells at each time point do not correspond to the cells at the other time points. Scale bar corresponds to 5 µm. (B) Transcript landscape of hyphal cells 30 minutes after induction. The fold change in RNA expression between hyphal and yeast cultures at each gene is plotted against the expression level of the gene in wild type yeast cells measured by RNA-Seq. Each dot represents one gene. Set3C binding targets were defined by Set3C ChIP-Seq experiments (see Materials and Methods). The Set3C target genes are divided into yeast-specific, hypha-specific and constitutively bound subgroups. The distribution of RNA fold changes of the genes belonging to each category is shown on the right panel. Statistical significance was determined by the Mann-Whitney U-test relative to the “all targets” set. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. (C) Correlation of RNA fold change and differential ChIP enrichment signals. Each dot corresponds to one gene, and only the genes defined as Set3C binding targets in at least one phase are shown. “_r_” denotes a Pearson's correlation coefficient. (D) qPCR verification of the correlation on (C). Histone H4 has two loci in C. albicans (HHF1 and HHF22), and the primers used in the qPCR bind alleles of both. Data are shown as mean+SD of three independent experiments. Statistical significance was determined by two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001. (E) Comparison of the gene induction profiles of wild type and _set3_Δ/Δ cells undergoing hyphal differentiation. Fold change between the hyphal and yeast phases for the two genotypes are plotted on the two axes. Each dot corresponds to one gene. The categories of Set3C binding targets are defined as on (B). “_r_” denotes a Pearson's correlation coefficient, and “m” denotes the slope of the linear regression.

Figure 4

Figure 4. The Set3C is a co-factor of glycolysis and morphogenesis regulators.

Statistical analysis of the overlaps of the target genes of selected transcription factors with the target gene set of Set3C in the individual phases. The TF target sets are shown in blue, the Set3C target sets in red, and the TFs are grouped according to the functions they are implicated in. The target sets of the TFs were imported from the following reports: Efg1, Ndt80, Rob1, Brg1, Tec1 and Bcr1 from , Zap1 from , Gal4 and Tye7 from , Cbf1, Fhl1, Ifh1, Tbf1 and Rap1 from . The area of the circles and overlaps are proportional to the number of genes they consist of. For each overlap the P-value of hypergeometric testing is shown. Transcription factors that are themselves binding targets of Set3C in at least one morphological phase are placed in blue boxes.

Figure 5

Figure 5. The Set3C modulates morphogenesis through a TF cluster.

(A) qRT-PCR quantification of the indicated transcripts in wild type and _set3_Δ/Δ yeast cells induced to differentiate into hyphae 10, 20, 30 and 60 minutes after induction. The transcript levels are normalized against RIP1 expression and the level of the respective gene in yeast cells (0 min). The right panel shows the quantitative difference between the values in the two genotypes at each individual time point. Average values of four independent experiments are shown. (B) Transcript levels of BRG1, TEC1, NRG1 and EFG1 in differentiating wild type and _set3_Δ/Δ yeast cells as on (A) quantified by qRT-PCR. IHD1 is hyphal-induced non-Set3C-target control gene. In the bottom right panel the percentage of cells germinating at the starting cultures and 60 minutes post induction is shown as a control. Data are shown as mean+SD of four independent experiments. Statistical significance was determined by two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001. (C) Removal of one BRG1 allele reverts hyperfilamentation of _set3_Δ/Δ cells under intermediate inducing conditions. Shown are photographs of single colonies. YPD at 30°C supports yeast-phase growth, YPD+FCS at 37°C supports hyphal growth, YPD at 37°C represents “intermediate” conditions. Scale bar corresponds to 2 mm.

Figure 6

Figure 6. Four phase-specific Set3C-target TFs form a core circuit.

(A) qRT-PCR analysis of Nrg1, Tec1, Efg1 and Brg1 binding at the EFG1, BRG1, TEC1 and NRG1 promoters. ChIP experiments were performed in yeast phase for Nrg1 and hyphal phase for Tec1, Efg1 and Brg1. Data are shown as mean+SD of three independent experiments. Statistical significance was determined by two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001. (B) Simplified model of the transcription circuit overlayed with a chromatin pathway to regulate C. albicans morphogenesis. The big circle represents the transcript profiles of the four regulators with the indicated scaling. The arrows representing regulatory information are derived from the ChIP experiments at (A). The arrows of Efg1 in the yeast phase are taken from the genome-wide binding data in . The arrows representing autoregulation of each four regulators are omitted for simplicity. Note that in _set3_Δ/Δ cells, the architecture of the TF-circuit remains intact, yet the temporary differences in the regulator mRNA levels are represented by the differences in the grayscale.

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