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Substitution as a mechanism for genetic robustness: the duplicated deacetylases Hst1p and Sir2p in Saccharomyces cerevisiae.

Hickman MA, Rusche LN - PLoS Genet. (2007)

Bottom Line: Our results imply that after the duplication, SIR2 and HST1 subfunctionalized.Therefore, the ability of Sir2p to substitute for Hst1p probably results from a retained but reduced affinity for the Sum1 complex that is a consequence of subfunctionalization via the duplication, degeneration, and complementation mechanism.These results suggest that the evolutionary path of duplicate gene preservation may be an important indicator for the ability of duplicated genes to contribute to genetic robustness.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America.

ABSTRACT
How duplicate genes provide genetic robustness remains an unresolved question. We have examined the duplicated histone deacetylases Sir2p and Hst1p in Saccharomyces cerevisiae and find that these paralogs with non-overlapping functions can provide genetic robustness against mutations through a substitution mechanism. Hst1p is an NAD(+)-dependent histone deacetylase that acts with Sum1p to repress a subset of midsporulation genes. However, hst1Delta mutants show much weaker derepression of target loci than sum1Delta mutants. We show that this modest derepression of target loci in hst1Delta strains occurs in part because Sir2p substitutes for Hst1p. Sir2p contributes to repression of the midsporulation genes only in the absence of Hst1p and is recruited to target promoters by a physical interaction with the Sum1 complex. Furthermore, when Sir2p associates with the Sum1 complex, the complex continues to repress in a promoter-specific manner and does not spread. Our results imply that after the duplication, SIR2 and HST1 subfunctionalized. The single SIR2/HST1 gene from Kluyveromyces lactis, a closely related species that diverged prior to the duplication, can suppress an hst1Delta mutation in S. cerevisiae as well as interact with Sir4p in S. cerevisiae. In addition, the existence of two distinct protein interaction domains for the Sir and Sum1 complexes was revealed through the analysis of a chimeric Sir2-Hst1 molecule. Therefore, the ability of Sir2p to substitute for Hst1p probably results from a retained but reduced affinity for the Sum1 complex that is a consequence of subfunctionalization via the duplication, degeneration, and complementation mechanism. These results suggest that the evolutionary path of duplicate gene preservation may be an important indicator for the ability of duplicated genes to contribute to genetic robustness.

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Sir2p Contributes to Repression of Sum1p-Repressed Genes in the Absence of Hst1p(A) Expression of the Sum1p-repressed pGAS2-HIS3 reporter was tested in wild-type (LRY1453), hst1Δ (LRY1454), hst1Δ sir2Δ (LRY1422), hst1Δ hst2Δ (LRY1686), hst1Δ hst3Δ (LRY1704), and hst1Δ hst4Δ (LRY1687) strain backgrounds. A ten-fold dilution series of each strain was plated on rich medium (growth) or medium lacking histidine (−histidine) and photographed after 3 d of growth at 30 °C. The most concentrated samples on rich medium and the most dilute samples on selective medium were omitted. Thus, the first spot on complete medium is equivalent to the second spot on medium lacking histidine.(B) Expression of the Sum1p-repressed genes DTR1 and SPS1 was assessed in wild type (W3031-a), hst1Δ (LRY198), hst1Δ sir2Δ (LRY333), hst1-N291A (LRY1306), sir2Δ (LRY1079), and hst1Δ sir3Δ (LRY345) strains. RNA was extracted from logarithmically growing cells and analyzed by quantitative RT-PCR. DTR1 and SPS1 transcript levels were normalized to ACT1 transcript levels and then compared to the wild-type strain to measure gene induction. A value of one (dashed line) corresponds to no induction.
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pgen-0030126-g001: Sir2p Contributes to Repression of Sum1p-Repressed Genes in the Absence of Hst1p(A) Expression of the Sum1p-repressed pGAS2-HIS3 reporter was tested in wild-type (LRY1453), hst1Δ (LRY1454), hst1Δ sir2Δ (LRY1422), hst1Δ hst2Δ (LRY1686), hst1Δ hst3Δ (LRY1704), and hst1Δ hst4Δ (LRY1687) strain backgrounds. A ten-fold dilution series of each strain was plated on rich medium (growth) or medium lacking histidine (−histidine) and photographed after 3 d of growth at 30 °C. The most concentrated samples on rich medium and the most dilute samples on selective medium were omitted. Thus, the first spot on complete medium is equivalent to the second spot on medium lacking histidine.(B) Expression of the Sum1p-repressed genes DTR1 and SPS1 was assessed in wild type (W3031-a), hst1Δ (LRY198), hst1Δ sir2Δ (LRY333), hst1-N291A (LRY1306), sir2Δ (LRY1079), and hst1Δ sir3Δ (LRY345) strains. RNA was extracted from logarithmically growing cells and analyzed by quantitative RT-PCR. DTR1 and SPS1 transcript levels were normalized to ACT1 transcript levels and then compared to the wild-type strain to measure gene induction. A value of one (dashed line) corresponds to no induction.

Mentions: Gene expression data indicate that deletion of HST1 derepresses target genes modestly, compared to the level of derepression observed in a sum1Δ background (unpublished data) [10]. These results suggest that either deacetylation is not critical for gene repression or another deacetylase acts at these promoters in the absence of Hst1p. To identify other deacetylases that may function in the absence of Hst1p, the four other known NAD+-dependent deacetylases, SIR2, HST2, HST3, and HST4 were deleted in combination with HST1. To assay levels of expression in these double deletion backgrounds, a pGAS2-HIS3 reporter was used. The GAS2 promoter is not strongly induced in the absence of Hst1p but is greatly induced in the absence of Sum1p [10]. In addition, the promoter contains a MSE and is reported to bind Sum1p [35]. If another deacetylase contributes to repression at this promoter in the absence of Hst1p, then deletion of both deacetylases should derepress the pGAS2-HIS3 reporter to a greater extent than deletion of Hst1p alone. Increased expression was observed in the hst1Δ sir2Δ double deletion strain compared to the hst1Δ strain (Figure 1A). The other double deletions, hst1Δ hst2Δ, hst1Δ hst3Δ, and hst1Δ hst4Δ, did not display any difference in derepression compared to the single hst1Δ background.


Substitution as a mechanism for genetic robustness: the duplicated deacetylases Hst1p and Sir2p in Saccharomyces cerevisiae.

Hickman MA, Rusche LN - PLoS Genet. (2007)

Sir2p Contributes to Repression of Sum1p-Repressed Genes in the Absence of Hst1p(A) Expression of the Sum1p-repressed pGAS2-HIS3 reporter was tested in wild-type (LRY1453), hst1Δ (LRY1454), hst1Δ sir2Δ (LRY1422), hst1Δ hst2Δ (LRY1686), hst1Δ hst3Δ (LRY1704), and hst1Δ hst4Δ (LRY1687) strain backgrounds. A ten-fold dilution series of each strain was plated on rich medium (growth) or medium lacking histidine (−histidine) and photographed after 3 d of growth at 30 °C. The most concentrated samples on rich medium and the most dilute samples on selective medium were omitted. Thus, the first spot on complete medium is equivalent to the second spot on medium lacking histidine.(B) Expression of the Sum1p-repressed genes DTR1 and SPS1 was assessed in wild type (W3031-a), hst1Δ (LRY198), hst1Δ sir2Δ (LRY333), hst1-N291A (LRY1306), sir2Δ (LRY1079), and hst1Δ sir3Δ (LRY345) strains. RNA was extracted from logarithmically growing cells and analyzed by quantitative RT-PCR. DTR1 and SPS1 transcript levels were normalized to ACT1 transcript levels and then compared to the wild-type strain to measure gene induction. A value of one (dashed line) corresponds to no induction.
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Related In: Results  -  Collection

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pgen-0030126-g001: Sir2p Contributes to Repression of Sum1p-Repressed Genes in the Absence of Hst1p(A) Expression of the Sum1p-repressed pGAS2-HIS3 reporter was tested in wild-type (LRY1453), hst1Δ (LRY1454), hst1Δ sir2Δ (LRY1422), hst1Δ hst2Δ (LRY1686), hst1Δ hst3Δ (LRY1704), and hst1Δ hst4Δ (LRY1687) strain backgrounds. A ten-fold dilution series of each strain was plated on rich medium (growth) or medium lacking histidine (−histidine) and photographed after 3 d of growth at 30 °C. The most concentrated samples on rich medium and the most dilute samples on selective medium were omitted. Thus, the first spot on complete medium is equivalent to the second spot on medium lacking histidine.(B) Expression of the Sum1p-repressed genes DTR1 and SPS1 was assessed in wild type (W3031-a), hst1Δ (LRY198), hst1Δ sir2Δ (LRY333), hst1-N291A (LRY1306), sir2Δ (LRY1079), and hst1Δ sir3Δ (LRY345) strains. RNA was extracted from logarithmically growing cells and analyzed by quantitative RT-PCR. DTR1 and SPS1 transcript levels were normalized to ACT1 transcript levels and then compared to the wild-type strain to measure gene induction. A value of one (dashed line) corresponds to no induction.
Mentions: Gene expression data indicate that deletion of HST1 derepresses target genes modestly, compared to the level of derepression observed in a sum1Δ background (unpublished data) [10]. These results suggest that either deacetylation is not critical for gene repression or another deacetylase acts at these promoters in the absence of Hst1p. To identify other deacetylases that may function in the absence of Hst1p, the four other known NAD+-dependent deacetylases, SIR2, HST2, HST3, and HST4 were deleted in combination with HST1. To assay levels of expression in these double deletion backgrounds, a pGAS2-HIS3 reporter was used. The GAS2 promoter is not strongly induced in the absence of Hst1p but is greatly induced in the absence of Sum1p [10]. In addition, the promoter contains a MSE and is reported to bind Sum1p [35]. If another deacetylase contributes to repression at this promoter in the absence of Hst1p, then deletion of both deacetylases should derepress the pGAS2-HIS3 reporter to a greater extent than deletion of Hst1p alone. Increased expression was observed in the hst1Δ sir2Δ double deletion strain compared to the hst1Δ strain (Figure 1A). The other double deletions, hst1Δ hst2Δ, hst1Δ hst3Δ, and hst1Δ hst4Δ, did not display any difference in derepression compared to the single hst1Δ background.

Bottom Line: Our results imply that after the duplication, SIR2 and HST1 subfunctionalized.Therefore, the ability of Sir2p to substitute for Hst1p probably results from a retained but reduced affinity for the Sum1 complex that is a consequence of subfunctionalization via the duplication, degeneration, and complementation mechanism.These results suggest that the evolutionary path of duplicate gene preservation may be an important indicator for the ability of duplicated genes to contribute to genetic robustness.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America.

ABSTRACT
How duplicate genes provide genetic robustness remains an unresolved question. We have examined the duplicated histone deacetylases Sir2p and Hst1p in Saccharomyces cerevisiae and find that these paralogs with non-overlapping functions can provide genetic robustness against mutations through a substitution mechanism. Hst1p is an NAD(+)-dependent histone deacetylase that acts with Sum1p to repress a subset of midsporulation genes. However, hst1Delta mutants show much weaker derepression of target loci than sum1Delta mutants. We show that this modest derepression of target loci in hst1Delta strains occurs in part because Sir2p substitutes for Hst1p. Sir2p contributes to repression of the midsporulation genes only in the absence of Hst1p and is recruited to target promoters by a physical interaction with the Sum1 complex. Furthermore, when Sir2p associates with the Sum1 complex, the complex continues to repress in a promoter-specific manner and does not spread. Our results imply that after the duplication, SIR2 and HST1 subfunctionalized. The single SIR2/HST1 gene from Kluyveromyces lactis, a closely related species that diverged prior to the duplication, can suppress an hst1Delta mutation in S. cerevisiae as well as interact with Sir4p in S. cerevisiae. In addition, the existence of two distinct protein interaction domains for the Sir and Sum1 complexes was revealed through the analysis of a chimeric Sir2-Hst1 molecule. Therefore, the ability of Sir2p to substitute for Hst1p probably results from a retained but reduced affinity for the Sum1 complex that is a consequence of subfunctionalization via the duplication, degeneration, and complementation mechanism. These results suggest that the evolutionary path of duplicate gene preservation may be an important indicator for the ability of duplicated genes to contribute to genetic robustness.

Show MeSH
Related in: MedlinePlus