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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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The Sir21−255-Hst1201−503p Chimera Functions like Sir2p(A) The ability of Sir2p, Hst1p, and HA-Sir21−255-Hst1201−503p to silence HML was assessed by a mating assay. An hst1Δ sir2Δ (LRY333) strain was transformed with an empty vector (pRS416), HA-SIR2 (pRO298), HST1-HA (pLR30), and HA-Sir21−255-Hst1201−503 (pLR488), and a ten-fold dilution series of each strain was mated against a MATα tester strain (LRY1022). Prototrophic diploids were selected on minimal plates. A 10-fold dilution series of the tester strain was plated on minimal plates as a negative control.(B) The association of HA-Sir21−255-Hst1201−503p with Sir4p was assessed by co-immunoprecipitation. Sir4p was immunoprecipitated from whole-cell extracts from the strains used in (A), and the precipitated material was examined by immunoblotting with an α-mouse HA antibody to detect HA-Sir2p, Hst1p-HA, or HA-Sir21−255-Hst1201−503p.
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pgen-0030126-g007: The Sir21−255-Hst1201−503p Chimera Functions like Sir2p(A) The ability of Sir2p, Hst1p, and HA-Sir21−255-Hst1201−503p to silence HML was assessed by a mating assay. An hst1Δ sir2Δ (LRY333) strain was transformed with an empty vector (pRS416), HA-SIR2 (pRO298), HST1-HA (pLR30), and HA-Sir21−255-Hst1201−503 (pLR488), and a ten-fold dilution series of each strain was mated against a MATα tester strain (LRY1022). Prototrophic diploids were selected on minimal plates. A 10-fold dilution series of the tester strain was plated on minimal plates as a negative control.(B) The association of HA-Sir21−255-Hst1201−503p with Sir4p was assessed by co-immunoprecipitation. Sir4p was immunoprecipitated from whole-cell extracts from the strains used in (A), and the precipitated material was examined by immunoblotting with an α-mouse HA antibody to detect HA-Sir2p, Hst1p-HA, or HA-Sir21−255-Hst1201−503p.

Mentions: To determine whether HA-Sir21−255-Hst1201−503p has Sir2p-like function, the ability to silence the mating-type loci was examined by mating assays. HA-Sir21−255-Hst1201−503p enabled the cells to mate in the absence of Sir2p to a level comparable to that seen with wild-type Sir2p (Figure 7A). The extent of mating was greater with HA-Sir21−255-Hst1201−503p than in cells expressing only wild-type Hst1p (Figure 7A). To test if the mating ability of HA-Sir21−255-Hst1201−503p resulted from an association with the Sir complex, we co-immunoprecipitated Sir4p with HA-Sir2p, Hst1p-HA, and HA-Sir21−255-Hst1201−503p. Results from these experiments showed an interaction between HA-Sir21−255-Hst1201−503p and Sir4p comparable to that of wild-type Sir2p (Figure 7B). We conclude that there is a critical component in the N terminus of Sir2p that specifies Sir2p to interact with the Sir complex. The ability of the chimeric Sir2–Hst1 protein to suppress both hst1Δ and sir2Δ mutations suggests that different regions of the protein are involved in conferring specificity for the Sir and Sum1 complexes.


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

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

The Sir21−255-Hst1201−503p Chimera Functions like Sir2p(A) The ability of Sir2p, Hst1p, and HA-Sir21−255-Hst1201−503p to silence HML was assessed by a mating assay. An hst1Δ sir2Δ (LRY333) strain was transformed with an empty vector (pRS416), HA-SIR2 (pRO298), HST1-HA (pLR30), and HA-Sir21−255-Hst1201−503 (pLR488), and a ten-fold dilution series of each strain was mated against a MATα tester strain (LRY1022). Prototrophic diploids were selected on minimal plates. A 10-fold dilution series of the tester strain was plated on minimal plates as a negative control.(B) The association of HA-Sir21−255-Hst1201−503p with Sir4p was assessed by co-immunoprecipitation. Sir4p was immunoprecipitated from whole-cell extracts from the strains used in (A), and the precipitated material was examined by immunoblotting with an α-mouse HA antibody to detect HA-Sir2p, Hst1p-HA, or HA-Sir21−255-Hst1201−503p.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1937012&req=5

pgen-0030126-g007: The Sir21−255-Hst1201−503p Chimera Functions like Sir2p(A) The ability of Sir2p, Hst1p, and HA-Sir21−255-Hst1201−503p to silence HML was assessed by a mating assay. An hst1Δ sir2Δ (LRY333) strain was transformed with an empty vector (pRS416), HA-SIR2 (pRO298), HST1-HA (pLR30), and HA-Sir21−255-Hst1201−503 (pLR488), and a ten-fold dilution series of each strain was mated against a MATα tester strain (LRY1022). Prototrophic diploids were selected on minimal plates. A 10-fold dilution series of the tester strain was plated on minimal plates as a negative control.(B) The association of HA-Sir21−255-Hst1201−503p with Sir4p was assessed by co-immunoprecipitation. Sir4p was immunoprecipitated from whole-cell extracts from the strains used in (A), and the precipitated material was examined by immunoblotting with an α-mouse HA antibody to detect HA-Sir2p, Hst1p-HA, or HA-Sir21−255-Hst1201−503p.
Mentions: To determine whether HA-Sir21−255-Hst1201−503p has Sir2p-like function, the ability to silence the mating-type loci was examined by mating assays. HA-Sir21−255-Hst1201−503p enabled the cells to mate in the absence of Sir2p to a level comparable to that seen with wild-type Sir2p (Figure 7A). The extent of mating was greater with HA-Sir21−255-Hst1201−503p than in cells expressing only wild-type Hst1p (Figure 7A). To test if the mating ability of HA-Sir21−255-Hst1201−503p resulted from an association with the Sir complex, we co-immunoprecipitated Sir4p with HA-Sir2p, Hst1p-HA, and HA-Sir21−255-Hst1201−503p. Results from these experiments showed an interaction between HA-Sir21−255-Hst1201−503p and Sir4p comparable to that of wild-type Sir2p (Figure 7B). We conclude that there is a critical component in the N terminus of Sir2p that specifies Sir2p to interact with the Sir complex. The ability of the chimeric Sir2–Hst1 protein to suppress both hst1Δ and sir2Δ mutations suggests that different regions of the protein are involved in conferring specificity for the Sir and Sum1 complexes.

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