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The Candida albicans Histone Acetyltransferase Hat1 Regulates Stress Resistance and Virulence via Distinct Chromatin Assembly Pathways.

Tscherner M, Zwolanek F, Je S, Sedlazeck FJ, Petryshyn A, Frohner IE, Mavrianos J, Chauhan N, von Haeseler A, Kuchler K - PLoS Pathog. (2015)

Bottom Line: Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment.Remarkably, the oxidative stress phenotype of hat1Δ/Δ cells is a species-specific trait only found in C. albicans and members of the CTG clade.The reduced azole susceptibility appears to be conserved in a wider range of fungi.

View Article: PubMed Central - PubMed

Affiliation: Department for Medical Biochemistry, Medical University of Vienna, Max F. Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria.

ABSTRACT
Human fungal pathogens like Candida albicans respond to host immune surveillance by rapidly adapting their transcriptional programs. Chromatin assembly factors are involved in the regulation of stress genes by modulating the histone density at these loci. Here, we report a novel role for the chromatin assembly-associated histone acetyltransferase complex NuB4 in regulating oxidative stress resistance, antifungal drug tolerance and virulence in C. albicans. Strikingly, depletion of the NuB4 catalytic subunit, the histone acetyltransferase Hat1, markedly increases resistance to oxidative stress and tolerance to azole antifungals. Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment. Furthermore, hat1Δ/Δ cells, despite showing growth defects in vitro, display reduced susceptibility to reactive oxygen-mediated killing by innate immune cells. Thus, clearance from infected mice is delayed although cells lacking Hat1 are severely compromised in killing the host. Interestingly, increased oxidative stress resistance and azole tolerance are phenocopied by the loss of histone chaperone complexes CAF-1 and HIR, respectively, suggesting a central role for NuB4 in the delivery of histones destined for chromatin assembly via distinct pathways. Remarkably, the oxidative stress phenotype of hat1Δ/Δ cells is a species-specific trait only found in C. albicans and members of the CTG clade. The reduced azole susceptibility appears to be conserved in a wider range of fungi. Thus, our work demonstrates how highly conserved chromatin assembly pathways can acquire new functions in pathogenic fungi during coevolution with the host.

No MeSH data available.


Lack of Hat1 accelerates induction of oxidative stress genes.(A) Catalase induction rate is strongly increased in hat1Δ/Δ cells. CAT1 expression levels were measured by RT-qPCR after induction with 1.6 mM H2O2 at the indicated time points. Transcript levels were normalized to the expression level of the reference gene (RG) PAT1. Data are shown as mean + SD from 3 independent experiments. (B) Histone density at the CAT1 locus is reduced in cells lacking Hat1. Histone H3 occupancy was determined by ChIP at the CAT1 promoter region (a) and the CDS (b). (C) Loss of Hat1 leads to increased RNAPII recruitment at the CAT1 locus. RNAPII levels were determined by ChIP at the CAT1 CDS. (D) Induction rate of glutathione-utilizing enzymes is increased in hat1Δ/Δ cells. GPX1 (a) and GST1 (b) expression levels were determined by RT-qPCR at the indicated time points. Experiment was performed as described in (A). (E) Lack of Hat1 leads to increased RNAPII recruitment at the GPX1 and GST1 loci. RNAPII levels were determined by ChIP at the GPX1 (a) and GST1 (b) genes. (F+G) Loss of Cac2 increases the induction rate of both GPX1 and GST1 following H2O2 treatment. Experimental conditions were used as described in (A).
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ppat.1005218.g006: Lack of Hat1 accelerates induction of oxidative stress genes.(A) Catalase induction rate is strongly increased in hat1Δ/Δ cells. CAT1 expression levels were measured by RT-qPCR after induction with 1.6 mM H2O2 at the indicated time points. Transcript levels were normalized to the expression level of the reference gene (RG) PAT1. Data are shown as mean + SD from 3 independent experiments. (B) Histone density at the CAT1 locus is reduced in cells lacking Hat1. Histone H3 occupancy was determined by ChIP at the CAT1 promoter region (a) and the CDS (b). (C) Loss of Hat1 leads to increased RNAPII recruitment at the CAT1 locus. RNAPII levels were determined by ChIP at the CAT1 CDS. (D) Induction rate of glutathione-utilizing enzymes is increased in hat1Δ/Δ cells. GPX1 (a) and GST1 (b) expression levels were determined by RT-qPCR at the indicated time points. Experiment was performed as described in (A). (E) Lack of Hat1 leads to increased RNAPII recruitment at the GPX1 and GST1 loci. RNAPII levels were determined by ChIP at the GPX1 (a) and GST1 (b) genes. (F+G) Loss of Cac2 increases the induction rate of both GPX1 and GST1 following H2O2 treatment. Experimental conditions were used as described in (A).

Mentions: Therefore, we investigated CAT1 induction upon treatment with H2O2. Interestingly, lack of Hat1 as well as Cac2 caused increased CAT1 expression, indicating that these two proteins negatively regulate the induction kinetics of CAT1 (S2E Fig). However, induction levels in the rtt109Δ/Δ control strain were similar to the wild-type (S2E Fig). Since lack of Hat1 affected induction levels of CAT1, we investigated the induction kinetics of this gene in detail. Thus, we determined transcript levels upon treatment with H2O2 over time. Interestingly, lack of Hat1 resulted in a significantly faster induction of the CAT1 gene when compared to the wild-type (Fig 6A). Since Hat1 is involved in the deposition of histones into chromatin, we determined the effect of HAT1 deletion on the histone density at the CAT1 locus by chromatin immunoprecipitation (ChIP) using antibodies against histone H3. Without treatment, the hat1Δ/Δ mutant showed a reduced histone density at the CAT1 promoter (Fig 6Ba). There was no difference in the occupancy in the CAT1 coding sequence (CDS) between the mutant and the wild-type (Fig 6Bb). Interestingly, however, treatment with H2O2 decreased histone density at the promoter as well as in the CDS significantly faster in hat1Δ/Δ cells when compared to the wild-type (Fig 6B). This explains the increased induction rate in the mutant, since nucleosomes represent a physical barrier for RNA polymerase II (RNAPII) and reduced nucleosome density can facilitate transcription [6,35,57]. Thus, lower histone density in the hat1Δ/Δ mutant could promote higher RNAPII processivity leading to increased mRNA production. Alternatively, elevated mRNA levels could also be due to increased RNAPII recruitment, which could be facilitated by the reduction in nucleosome density at the promoter. To clarify whether elevated mRNA levels are the consequence of increased RNAPII recruitment, we determined RNAPII levels at the CAT1 gene using ChIP. Interestingly, we detected increased recruitment of RNAPII to the CAT1 CDS, thus explaining the elevated mRNA levels (Fig 6C).


The Candida albicans Histone Acetyltransferase Hat1 Regulates Stress Resistance and Virulence via Distinct Chromatin Assembly Pathways.

Tscherner M, Zwolanek F, Je S, Sedlazeck FJ, Petryshyn A, Frohner IE, Mavrianos J, Chauhan N, von Haeseler A, Kuchler K - PLoS Pathog. (2015)

Lack of Hat1 accelerates induction of oxidative stress genes.(A) Catalase induction rate is strongly increased in hat1Δ/Δ cells. CAT1 expression levels were measured by RT-qPCR after induction with 1.6 mM H2O2 at the indicated time points. Transcript levels were normalized to the expression level of the reference gene (RG) PAT1. Data are shown as mean + SD from 3 independent experiments. (B) Histone density at the CAT1 locus is reduced in cells lacking Hat1. Histone H3 occupancy was determined by ChIP at the CAT1 promoter region (a) and the CDS (b). (C) Loss of Hat1 leads to increased RNAPII recruitment at the CAT1 locus. RNAPII levels were determined by ChIP at the CAT1 CDS. (D) Induction rate of glutathione-utilizing enzymes is increased in hat1Δ/Δ cells. GPX1 (a) and GST1 (b) expression levels were determined by RT-qPCR at the indicated time points. Experiment was performed as described in (A). (E) Lack of Hat1 leads to increased RNAPII recruitment at the GPX1 and GST1 loci. RNAPII levels were determined by ChIP at the GPX1 (a) and GST1 (b) genes. (F+G) Loss of Cac2 increases the induction rate of both GPX1 and GST1 following H2O2 treatment. Experimental conditions were used as described in (A).
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ppat.1005218.g006: Lack of Hat1 accelerates induction of oxidative stress genes.(A) Catalase induction rate is strongly increased in hat1Δ/Δ cells. CAT1 expression levels were measured by RT-qPCR after induction with 1.6 mM H2O2 at the indicated time points. Transcript levels were normalized to the expression level of the reference gene (RG) PAT1. Data are shown as mean + SD from 3 independent experiments. (B) Histone density at the CAT1 locus is reduced in cells lacking Hat1. Histone H3 occupancy was determined by ChIP at the CAT1 promoter region (a) and the CDS (b). (C) Loss of Hat1 leads to increased RNAPII recruitment at the CAT1 locus. RNAPII levels were determined by ChIP at the CAT1 CDS. (D) Induction rate of glutathione-utilizing enzymes is increased in hat1Δ/Δ cells. GPX1 (a) and GST1 (b) expression levels were determined by RT-qPCR at the indicated time points. Experiment was performed as described in (A). (E) Lack of Hat1 leads to increased RNAPII recruitment at the GPX1 and GST1 loci. RNAPII levels were determined by ChIP at the GPX1 (a) and GST1 (b) genes. (F+G) Loss of Cac2 increases the induction rate of both GPX1 and GST1 following H2O2 treatment. Experimental conditions were used as described in (A).
Mentions: Therefore, we investigated CAT1 induction upon treatment with H2O2. Interestingly, lack of Hat1 as well as Cac2 caused increased CAT1 expression, indicating that these two proteins negatively regulate the induction kinetics of CAT1 (S2E Fig). However, induction levels in the rtt109Δ/Δ control strain were similar to the wild-type (S2E Fig). Since lack of Hat1 affected induction levels of CAT1, we investigated the induction kinetics of this gene in detail. Thus, we determined transcript levels upon treatment with H2O2 over time. Interestingly, lack of Hat1 resulted in a significantly faster induction of the CAT1 gene when compared to the wild-type (Fig 6A). Since Hat1 is involved in the deposition of histones into chromatin, we determined the effect of HAT1 deletion on the histone density at the CAT1 locus by chromatin immunoprecipitation (ChIP) using antibodies against histone H3. Without treatment, the hat1Δ/Δ mutant showed a reduced histone density at the CAT1 promoter (Fig 6Ba). There was no difference in the occupancy in the CAT1 coding sequence (CDS) between the mutant and the wild-type (Fig 6Bb). Interestingly, however, treatment with H2O2 decreased histone density at the promoter as well as in the CDS significantly faster in hat1Δ/Δ cells when compared to the wild-type (Fig 6B). This explains the increased induction rate in the mutant, since nucleosomes represent a physical barrier for RNA polymerase II (RNAPII) and reduced nucleosome density can facilitate transcription [6,35,57]. Thus, lower histone density in the hat1Δ/Δ mutant could promote higher RNAPII processivity leading to increased mRNA production. Alternatively, elevated mRNA levels could also be due to increased RNAPII recruitment, which could be facilitated by the reduction in nucleosome density at the promoter. To clarify whether elevated mRNA levels are the consequence of increased RNAPII recruitment, we determined RNAPII levels at the CAT1 gene using ChIP. Interestingly, we detected increased recruitment of RNAPII to the CAT1 CDS, thus explaining the elevated mRNA levels (Fig 6C).

Bottom Line: Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment.Remarkably, the oxidative stress phenotype of hat1Δ/Δ cells is a species-specific trait only found in C. albicans and members of the CTG clade.The reduced azole susceptibility appears to be conserved in a wider range of fungi.

View Article: PubMed Central - PubMed

Affiliation: Department for Medical Biochemistry, Medical University of Vienna, Max F. Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria.

ABSTRACT
Human fungal pathogens like Candida albicans respond to host immune surveillance by rapidly adapting their transcriptional programs. Chromatin assembly factors are involved in the regulation of stress genes by modulating the histone density at these loci. Here, we report a novel role for the chromatin assembly-associated histone acetyltransferase complex NuB4 in regulating oxidative stress resistance, antifungal drug tolerance and virulence in C. albicans. Strikingly, depletion of the NuB4 catalytic subunit, the histone acetyltransferase Hat1, markedly increases resistance to oxidative stress and tolerance to azole antifungals. Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment. Furthermore, hat1Δ/Δ cells, despite showing growth defects in vitro, display reduced susceptibility to reactive oxygen-mediated killing by innate immune cells. Thus, clearance from infected mice is delayed although cells lacking Hat1 are severely compromised in killing the host. Interestingly, increased oxidative stress resistance and azole tolerance are phenocopied by the loss of histone chaperone complexes CAF-1 and HIR, respectively, suggesting a central role for NuB4 in the delivery of histones destined for chromatin assembly via distinct pathways. Remarkably, the oxidative stress phenotype of hat1Δ/Δ cells is a species-specific trait only found in C. albicans and members of the CTG clade. The reduced azole susceptibility appears to be conserved in a wider range of fungi. Thus, our work demonstrates how highly conserved chromatin assembly pathways can acquire new functions in pathogenic fungi during coevolution with the host.

No MeSH data available.