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The deacetylase Sir2 from the yeast Clavispora lusitaniae lacks the evolutionarily conserved capacity to generate subtelomeric heterochromatin.

Froyd CA, Kapoor S, Dietrich F, Rusche LN - PLoS Genet. (2013)

Bottom Line: However, we found no evidence that ClHst1 associates with subtelomeric regions or impacts gene expression directly.After subsequent species diversification, the SIR2 paralog was apparently lost in the C. lusitaniae lineage.Thus, C. lusitaniae presents an opportunity to discover how subtelomeric chromatin can be reconfigured.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, Duke University, Durham, North Carolina, United States of America.

ABSTRACT
Deacetylases of the Sir2 or sirtuin family are thought to regulate life cycle progression and life span in response to nutrient availability. This family has undergone successive rounds of duplication and diversification, enabling the enzymes to perform a wide variety of biological functions. Two evolutionarily conserved functions of yeast Sir2 proteins are the generation of repressive chromatin in subtelomeric domains and the suppression of unbalanced recombination within the tandem rDNA array. Here, we describe the function of the Sir2 ortholog ClHst1 in the yeast Clavispora lusitaniae, an occasional opportunistic pathogen. ClHst1 was localized to the non-transcribed spacer regions of the rDNA repeats and deacetylated histones at these loci, indicating that, like other Sir2 proteins, ClHst1 modulates chromatin structure at the rDNA repeats. However, we found no evidence that ClHst1 associates with subtelomeric regions or impacts gene expression directly. This surprising observation highlights the plasticity of sirtuin function. Related yeast species, including Candida albicans, possess an additional Sir2 family member. Thus, it is likely that the ancestral Candida SIR2/HST1 gene was duplicated and subfunctionalized, such that HST1 retained the capacity to regulate rDNA whereas SIR2 had other functions, perhaps including the generation of subtelomeric chromatin. After subsequent species diversification, the SIR2 paralog was apparently lost in the C. lusitaniae lineage. Thus, C. lusitaniae presents an opportunity to discover how subtelomeric chromatin can be reconfigured.

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ClHst1 has a limited and indirect impact on gene expression.(A) Expression levels were assessed for genes identified by RNA-seq as induced in Clhst1Δ strains. Expression was assessed by quantitative RT-PCR in ClHST1 (LRY2544, white) and four independently constructed Clhst1Δ strains (LRY2623, light gray; LRY2671; medium gray; LRY2672, dark gray; LRY2673, black). Levels of mRNA for each gene were first normalized to ClPRI2 (CLUG_00368) and then expressed relative to the WT ClHST1 strain. (B) Expression of genes repressed in hst1Δ strains was measured as in part A. For fold repression, the inverse of the fold expression was calculated. (C) The association of ClHst1 with promoters of candidate genes was examined by chromatin IP. Anti-myc antibody was used to immunoprecipitate proteins from ClHST1 (LRY2826) or ClHST1-MYC (LRY2858) yeast.
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pgen-1003935-g007: ClHst1 has a limited and indirect impact on gene expression.(A) Expression levels were assessed for genes identified by RNA-seq as induced in Clhst1Δ strains. Expression was assessed by quantitative RT-PCR in ClHST1 (LRY2544, white) and four independently constructed Clhst1Δ strains (LRY2623, light gray; LRY2671; medium gray; LRY2672, dark gray; LRY2673, black). Levels of mRNA for each gene were first normalized to ClPRI2 (CLUG_00368) and then expressed relative to the WT ClHST1 strain. (B) Expression of genes repressed in hst1Δ strains was measured as in part A. For fold repression, the inverse of the fold expression was calculated. (C) The association of ClHst1 with promoters of candidate genes was examined by chromatin IP. Anti-myc antibody was used to immunoprecipitate proteins from ClHST1 (LRY2826) or ClHST1-MYC (LRY2858) yeast.

Mentions: Outside of roles at the rDNA array and subtelomeric loci, ScHst1 and KlSir2 repress transcription of particular genes through a promoter-specific mechanism. Consequently, deletion of ScHST1 or KlSIR2 results in the induction of scores of genes [[24], [35], [62]; Hasan and Rusche, unpublished]. To investigate the possibility that ClHst1 has a similar function and to determine in an unbiased way which genes are targets of ClHst1, the RNA-seq data described above were used to identify genes whose expression changed 3-fold or more when ClHST1 was deleted. These genes were re-analyzed by quantitative reverse transcriptase PCR (Figures 7A and B). A total of 13 genes changed upon the deletion of ClHST1 and are summarized in Table 1. This number was unexpectedly low, given the number of genes regulated by ScSir2, ScHst1, and KlSir2. In addition, the expression of some of these genes was inconsistent in subsequent RT-PCR analyses (Figure S3), suggesting they are not direct targets of ClHst1. Moreover, although seven of the genes were induced in the absence of ClHst1, as expected for targets of a transcriptional repressor, the remaining six genes were repressed. The functions of the genes were also surprising. ScHst1 and KlSir2 repress genes such as the middle sporulation genes and NAD+ biosynthetic genes [24], [33], [34]. In contrast, genes affected by ClHst1 are related to flocculation or adhesion, cell wall biogenesis, and membrane transport. Some genes are also involved in stress response.


The deacetylase Sir2 from the yeast Clavispora lusitaniae lacks the evolutionarily conserved capacity to generate subtelomeric heterochromatin.

Froyd CA, Kapoor S, Dietrich F, Rusche LN - PLoS Genet. (2013)

ClHst1 has a limited and indirect impact on gene expression.(A) Expression levels were assessed for genes identified by RNA-seq as induced in Clhst1Δ strains. Expression was assessed by quantitative RT-PCR in ClHST1 (LRY2544, white) and four independently constructed Clhst1Δ strains (LRY2623, light gray; LRY2671; medium gray; LRY2672, dark gray; LRY2673, black). Levels of mRNA for each gene were first normalized to ClPRI2 (CLUG_00368) and then expressed relative to the WT ClHST1 strain. (B) Expression of genes repressed in hst1Δ strains was measured as in part A. For fold repression, the inverse of the fold expression was calculated. (C) The association of ClHst1 with promoters of candidate genes was examined by chromatin IP. Anti-myc antibody was used to immunoprecipitate proteins from ClHST1 (LRY2826) or ClHST1-MYC (LRY2858) yeast.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814328&req=5

pgen-1003935-g007: ClHst1 has a limited and indirect impact on gene expression.(A) Expression levels were assessed for genes identified by RNA-seq as induced in Clhst1Δ strains. Expression was assessed by quantitative RT-PCR in ClHST1 (LRY2544, white) and four independently constructed Clhst1Δ strains (LRY2623, light gray; LRY2671; medium gray; LRY2672, dark gray; LRY2673, black). Levels of mRNA for each gene were first normalized to ClPRI2 (CLUG_00368) and then expressed relative to the WT ClHST1 strain. (B) Expression of genes repressed in hst1Δ strains was measured as in part A. For fold repression, the inverse of the fold expression was calculated. (C) The association of ClHst1 with promoters of candidate genes was examined by chromatin IP. Anti-myc antibody was used to immunoprecipitate proteins from ClHST1 (LRY2826) or ClHST1-MYC (LRY2858) yeast.
Mentions: Outside of roles at the rDNA array and subtelomeric loci, ScHst1 and KlSir2 repress transcription of particular genes through a promoter-specific mechanism. Consequently, deletion of ScHST1 or KlSIR2 results in the induction of scores of genes [[24], [35], [62]; Hasan and Rusche, unpublished]. To investigate the possibility that ClHst1 has a similar function and to determine in an unbiased way which genes are targets of ClHst1, the RNA-seq data described above were used to identify genes whose expression changed 3-fold or more when ClHST1 was deleted. These genes were re-analyzed by quantitative reverse transcriptase PCR (Figures 7A and B). A total of 13 genes changed upon the deletion of ClHST1 and are summarized in Table 1. This number was unexpectedly low, given the number of genes regulated by ScSir2, ScHst1, and KlSir2. In addition, the expression of some of these genes was inconsistent in subsequent RT-PCR analyses (Figure S3), suggesting they are not direct targets of ClHst1. Moreover, although seven of the genes were induced in the absence of ClHst1, as expected for targets of a transcriptional repressor, the remaining six genes were repressed. The functions of the genes were also surprising. ScHst1 and KlSir2 repress genes such as the middle sporulation genes and NAD+ biosynthetic genes [24], [33], [34]. In contrast, genes affected by ClHst1 are related to flocculation or adhesion, cell wall biogenesis, and membrane transport. Some genes are also involved in stress response.

Bottom Line: However, we found no evidence that ClHst1 associates with subtelomeric regions or impacts gene expression directly.After subsequent species diversification, the SIR2 paralog was apparently lost in the C. lusitaniae lineage.Thus, C. lusitaniae presents an opportunity to discover how subtelomeric chromatin can be reconfigured.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, Duke University, Durham, North Carolina, United States of America.

ABSTRACT
Deacetylases of the Sir2 or sirtuin family are thought to regulate life cycle progression and life span in response to nutrient availability. This family has undergone successive rounds of duplication and diversification, enabling the enzymes to perform a wide variety of biological functions. Two evolutionarily conserved functions of yeast Sir2 proteins are the generation of repressive chromatin in subtelomeric domains and the suppression of unbalanced recombination within the tandem rDNA array. Here, we describe the function of the Sir2 ortholog ClHst1 in the yeast Clavispora lusitaniae, an occasional opportunistic pathogen. ClHst1 was localized to the non-transcribed spacer regions of the rDNA repeats and deacetylated histones at these loci, indicating that, like other Sir2 proteins, ClHst1 modulates chromatin structure at the rDNA repeats. However, we found no evidence that ClHst1 associates with subtelomeric regions or impacts gene expression directly. This surprising observation highlights the plasticity of sirtuin function. Related yeast species, including Candida albicans, possess an additional Sir2 family member. Thus, it is likely that the ancestral Candida SIR2/HST1 gene was duplicated and subfunctionalized, such that HST1 retained the capacity to regulate rDNA whereas SIR2 had other functions, perhaps including the generation of subtelomeric chromatin. After subsequent species diversification, the SIR2 paralog was apparently lost in the C. lusitaniae lineage. Thus, C. lusitaniae presents an opportunity to discover how subtelomeric chromatin can be reconfigured.

Show MeSH
Related in: MedlinePlus