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Hyphal development in Candida albicans requires two temporally linked changes in promoter chromatin for initiation and maintenance.

Lu Y, Su C, Wang A, Liu H - PLoS Biol. (2011)

Bottom Line: Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear.Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signaling.Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California, Irvine, California, United States of America.

ABSTRACT
Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungal infections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast and hyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here we show that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signaling. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. The sequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signaling provides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments in pathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification.

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The function of Hda1 in sustained hyphal transcription is mediated through Yng2 deacetylation (A) Yng2p is deacetylated through an Hda1-dependent mechanism in vivo. Cells were grown at 30°C to OD600 0.8, then WCEs were collected, immunoprecipitated with anti-Ac-K, and probed with anti-Myc. (B) K175 is the major acetylated lysine residue of Yng2 in vivo. Substitution of K175 with arginine (K175R) diminishes acetylation of Yng2. (C) Effects of K175 substitutions on Yng2 stability. K175R mutation causes decreased protein abundance of Yng2, while K175Q causes no detectable change. (D, E) Morphology and expression levels of hypha-specific genes in YNG2 (HLY4035), yng2K175R (HLY4036), and yng2K175Q (HLY4037) cells during hyphal development in YPD with 10% serum at 37°C. HWP1, ALS3, and ECE1 mRNA levels were determined by qRT-PCR. The signal obtained from ACT1 mRNA was used as a loading control for normalization. (F) Dynamic dissociation of Myc-tagged Yng2 and yng2K175R but not Yng2K175Q from promoters during hyphal development by ChIP with anti-Myc antibodies. (G) ChIP with anti-H3 and anti-acetylated H4 antibodies show temporal dynamics in relative levels of H3 and H4 acetylation at the promoters of hypha-specific genes during hyphal induction in the YNG2 (HLY4035) and yng2K175Q mutant (HLY4037) strains. Levels of H3 (relative H3 occupancy; bound/input) are normalized to the respective control DNA at the ADE2 coding sequence region (bound/input). The 0-h values in the wild-type strain are set to be 1.00. Acetylation levels normalized with respect to H4 levels (H4 acetylation/H3 occupancy) are calculated by dividing the values for acetylated H4 with H3 occupancy values. Data on ALS3 and ECE1 for (E, F, G) are in Figure S4. All data show the average of three independent qPCR experiments with error bars representing the SEM.
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pbio-1001105-g004: The function of Hda1 in sustained hyphal transcription is mediated through Yng2 deacetylation (A) Yng2p is deacetylated through an Hda1-dependent mechanism in vivo. Cells were grown at 30°C to OD600 0.8, then WCEs were collected, immunoprecipitated with anti-Ac-K, and probed with anti-Myc. (B) K175 is the major acetylated lysine residue of Yng2 in vivo. Substitution of K175 with arginine (K175R) diminishes acetylation of Yng2. (C) Effects of K175 substitutions on Yng2 stability. K175R mutation causes decreased protein abundance of Yng2, while K175Q causes no detectable change. (D, E) Morphology and expression levels of hypha-specific genes in YNG2 (HLY4035), yng2K175R (HLY4036), and yng2K175Q (HLY4037) cells during hyphal development in YPD with 10% serum at 37°C. HWP1, ALS3, and ECE1 mRNA levels were determined by qRT-PCR. The signal obtained from ACT1 mRNA was used as a loading control for normalization. (F) Dynamic dissociation of Myc-tagged Yng2 and yng2K175R but not Yng2K175Q from promoters during hyphal development by ChIP with anti-Myc antibodies. (G) ChIP with anti-H3 and anti-acetylated H4 antibodies show temporal dynamics in relative levels of H3 and H4 acetylation at the promoters of hypha-specific genes during hyphal induction in the YNG2 (HLY4035) and yng2K175Q mutant (HLY4037) strains. Levels of H3 (relative H3 occupancy; bound/input) are normalized to the respective control DNA at the ADE2 coding sequence region (bound/input). The 0-h values in the wild-type strain are set to be 1.00. Acetylation levels normalized with respect to H4 levels (H4 acetylation/H3 occupancy) are calculated by dividing the values for acetylated H4 with H3 occupancy values. Data on ALS3 and ECE1 for (E, F, G) are in Figure S4. All data show the average of three independent qPCR experiments with error bars representing the SEM.

Mentions: How does promoter-associated Hda1 prevent Nrg1 from binding to the promoters of hypha-specific genes during hyphal elongation? One potential mechanism is chromatin remodeling. We have previously shown nucleosome reassembly and a decrease in H4 acetylation at the UAS regions of hypha-specific genes during hyphal elongation [50]. The decrease in H4 acetylation could be a result of H4 deacetylation by promoter-associated Hda1 or eviction of NuA4 from the promoters. S. cerevisiae Hda1 is a histone deacetylase specific for H3 and H2B [55]. Therefore, H4 may not be a substrate of the C. albicans Hda1. Yng2, a subunit of the NuA4 histone acetyltransferase HAT complex essential for HAT activity, is acetylated at lysine 170 by NuA4 and deacetylated by Rpd3 in S. cerevisiae [56]. Deacetylation of Yng2 leads to its degradation and eviction of chromatin-bound Yng2 with Esa1, the catalytic subunit of NuA4. In C. albicans, the NuA4 HAT complex is recruited to the promoters of hypha-specific genes [50]. By immunoprecipitation with anti-acetylated-lysine antibodies, we detected acetylated Yng2 in wild-type cells, but observed a dramatic increase in the level of acetylated Yng2 in the hda1 mutant (Figure 4A). This suggests that deacetylation of Yng2 in vivo depends on Hda1 activity. K175 was identified as a candidate lysine residue for acetylation by sequence alignment with S. cerevisiae Yng2. This was confirmed by the loss of acetylation of Yng2 when we substituted K175 with arginine (K175R), a mutation that blocks acetylation (Figure 4B). The loss of acetylation of Yng2K175R also suggests that K175 is the major acetylated lysine residue of Yng2 in C. albicans. The K175R mutation led to a reduced level of Yng2, whereas substituting K175 with glutamine (K175Q, a mutation mimicking constitutive acetylation) did not cause a detectable change of protein abundance relative to wild type (Figure 4C). Unlike the yng2 deletion mutant, neither yng2K175R nor yng2K175Q has any detectable growth defect (unpublished data). We then examined whether Yng2 acetylation affects hyphal development. Both wild-type and yng2K175R cells developed long hyphae, whereas yng2K175Q cells were defective in sustained hyphal development and transcription (Figures 4D,E and S4A), a phenotype similar to that of the hda1 mutant. Therefore, Yng2 deacetylation at K175 is important for sustained hyphal transcription. Conversely, constitutively acetylated Yng2 blocks hyphal filament extension.


Hyphal development in Candida albicans requires two temporally linked changes in promoter chromatin for initiation and maintenance.

Lu Y, Su C, Wang A, Liu H - PLoS Biol. (2011)

The function of Hda1 in sustained hyphal transcription is mediated through Yng2 deacetylation (A) Yng2p is deacetylated through an Hda1-dependent mechanism in vivo. Cells were grown at 30°C to OD600 0.8, then WCEs were collected, immunoprecipitated with anti-Ac-K, and probed with anti-Myc. (B) K175 is the major acetylated lysine residue of Yng2 in vivo. Substitution of K175 with arginine (K175R) diminishes acetylation of Yng2. (C) Effects of K175 substitutions on Yng2 stability. K175R mutation causes decreased protein abundance of Yng2, while K175Q causes no detectable change. (D, E) Morphology and expression levels of hypha-specific genes in YNG2 (HLY4035), yng2K175R (HLY4036), and yng2K175Q (HLY4037) cells during hyphal development in YPD with 10% serum at 37°C. HWP1, ALS3, and ECE1 mRNA levels were determined by qRT-PCR. The signal obtained from ACT1 mRNA was used as a loading control for normalization. (F) Dynamic dissociation of Myc-tagged Yng2 and yng2K175R but not Yng2K175Q from promoters during hyphal development by ChIP with anti-Myc antibodies. (G) ChIP with anti-H3 and anti-acetylated H4 antibodies show temporal dynamics in relative levels of H3 and H4 acetylation at the promoters of hypha-specific genes during hyphal induction in the YNG2 (HLY4035) and yng2K175Q mutant (HLY4037) strains. Levels of H3 (relative H3 occupancy; bound/input) are normalized to the respective control DNA at the ADE2 coding sequence region (bound/input). The 0-h values in the wild-type strain are set to be 1.00. Acetylation levels normalized with respect to H4 levels (H4 acetylation/H3 occupancy) are calculated by dividing the values for acetylated H4 with H3 occupancy values. Data on ALS3 and ECE1 for (E, F, G) are in Figure S4. All data show the average of three independent qPCR experiments with error bars representing the SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139633&req=5

pbio-1001105-g004: The function of Hda1 in sustained hyphal transcription is mediated through Yng2 deacetylation (A) Yng2p is deacetylated through an Hda1-dependent mechanism in vivo. Cells were grown at 30°C to OD600 0.8, then WCEs were collected, immunoprecipitated with anti-Ac-K, and probed with anti-Myc. (B) K175 is the major acetylated lysine residue of Yng2 in vivo. Substitution of K175 with arginine (K175R) diminishes acetylation of Yng2. (C) Effects of K175 substitutions on Yng2 stability. K175R mutation causes decreased protein abundance of Yng2, while K175Q causes no detectable change. (D, E) Morphology and expression levels of hypha-specific genes in YNG2 (HLY4035), yng2K175R (HLY4036), and yng2K175Q (HLY4037) cells during hyphal development in YPD with 10% serum at 37°C. HWP1, ALS3, and ECE1 mRNA levels were determined by qRT-PCR. The signal obtained from ACT1 mRNA was used as a loading control for normalization. (F) Dynamic dissociation of Myc-tagged Yng2 and yng2K175R but not Yng2K175Q from promoters during hyphal development by ChIP with anti-Myc antibodies. (G) ChIP with anti-H3 and anti-acetylated H4 antibodies show temporal dynamics in relative levels of H3 and H4 acetylation at the promoters of hypha-specific genes during hyphal induction in the YNG2 (HLY4035) and yng2K175Q mutant (HLY4037) strains. Levels of H3 (relative H3 occupancy; bound/input) are normalized to the respective control DNA at the ADE2 coding sequence region (bound/input). The 0-h values in the wild-type strain are set to be 1.00. Acetylation levels normalized with respect to H4 levels (H4 acetylation/H3 occupancy) are calculated by dividing the values for acetylated H4 with H3 occupancy values. Data on ALS3 and ECE1 for (E, F, G) are in Figure S4. All data show the average of three independent qPCR experiments with error bars representing the SEM.
Mentions: How does promoter-associated Hda1 prevent Nrg1 from binding to the promoters of hypha-specific genes during hyphal elongation? One potential mechanism is chromatin remodeling. We have previously shown nucleosome reassembly and a decrease in H4 acetylation at the UAS regions of hypha-specific genes during hyphal elongation [50]. The decrease in H4 acetylation could be a result of H4 deacetylation by promoter-associated Hda1 or eviction of NuA4 from the promoters. S. cerevisiae Hda1 is a histone deacetylase specific for H3 and H2B [55]. Therefore, H4 may not be a substrate of the C. albicans Hda1. Yng2, a subunit of the NuA4 histone acetyltransferase HAT complex essential for HAT activity, is acetylated at lysine 170 by NuA4 and deacetylated by Rpd3 in S. cerevisiae [56]. Deacetylation of Yng2 leads to its degradation and eviction of chromatin-bound Yng2 with Esa1, the catalytic subunit of NuA4. In C. albicans, the NuA4 HAT complex is recruited to the promoters of hypha-specific genes [50]. By immunoprecipitation with anti-acetylated-lysine antibodies, we detected acetylated Yng2 in wild-type cells, but observed a dramatic increase in the level of acetylated Yng2 in the hda1 mutant (Figure 4A). This suggests that deacetylation of Yng2 in vivo depends on Hda1 activity. K175 was identified as a candidate lysine residue for acetylation by sequence alignment with S. cerevisiae Yng2. This was confirmed by the loss of acetylation of Yng2 when we substituted K175 with arginine (K175R), a mutation that blocks acetylation (Figure 4B). The loss of acetylation of Yng2K175R also suggests that K175 is the major acetylated lysine residue of Yng2 in C. albicans. The K175R mutation led to a reduced level of Yng2, whereas substituting K175 with glutamine (K175Q, a mutation mimicking constitutive acetylation) did not cause a detectable change of protein abundance relative to wild type (Figure 4C). Unlike the yng2 deletion mutant, neither yng2K175R nor yng2K175Q has any detectable growth defect (unpublished data). We then examined whether Yng2 acetylation affects hyphal development. Both wild-type and yng2K175R cells developed long hyphae, whereas yng2K175Q cells were defective in sustained hyphal development and transcription (Figures 4D,E and S4A), a phenotype similar to that of the hda1 mutant. Therefore, Yng2 deacetylation at K175 is important for sustained hyphal transcription. Conversely, constitutively acetylated Yng2 blocks hyphal filament extension.

Bottom Line: Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear.Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signaling.Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California, Irvine, California, United States of America.

ABSTRACT
Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungal infections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast and hyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here we show that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signaling. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. The sequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signaling provides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments in pathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification.

Show MeSH
Related in: MedlinePlus