Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Aug;173(4):1939-50.
doi: 10.1534/genetics.106.055491. Epub 2006 Jun 18.

New alleles of SIR2 define cell-cycle-specific silencing functions

Mirela Matecic  1 , Kristen Martins-Taylor, Merrit Hickman, Jason Tanny, Danesh Moazed, Scott G Holmes
Affiliations

New alleles of SIR2 define cell-cycle-specific silencing functions

Mirela Matecic et al. Genetics. 2006 Aug.

Abstract

The establishment of transcriptional silencing in yeast requires cell-cycle progression, but the nature of this requirement is unknown. Sir2 is a protein deacetylase that is required for gene silencing in yeast. We have used temperature-sensitive alleles of the SIR2 gene to assess Sir2's contribution to silencing as a function of the cell cycle. When examined in vivo, these conditional alleles fall into two classes: one class exhibits a loss of silencing when raised to the nonpermissive temperature regardless of cell-cycle position, while the second class exhibits a mitosis-specific silencing defect. Alleles of the first class have a primary defect in protein deacetylase activity, while the alleles of the second class are specifically defective in Sir2-Sir4 interactions at nonpermissive temperatures. Using a SIR2 temperature-sensitive allele, we show that silencing can be established at the HML locus during progression through the G2/M-G1 interval. These results suggest that yeast heterochromatin undergoes structural transitions as a function of the cell cycle and support the existence of a critical assembly step for silent chromatin in mitosis.

PubMed Disclaimer

Figures

F<sc>igure</sc> 1.—
Figure 1.—
Conditional alleles of SIR2. (A) Levels of α1 and ACT1 mRNA were measured by RT–PCR in strains bearing different alleles of SIR2. Cultures were maintained in log phase for >10 generations at the listed temperatures, and then RNA was collected and analyzed as described in materials and methods. α1 indicates steady-state levels of message transcribed from the HML locus; ACT1 message is shown as a control. RNA was analyzed from a wild-type strain, a strain lacking the SIR2 gene, and strains bearing the indicated alleles of SIR2. (B) Protein extracts made from cells bearing the indicated SIR2 alleles were subjected to a Western blot using an antibody specific for the Sir2 protein, as described in materials and methods. For the cycling cells, parallel cultures were grown to log phase at either 23° or 37°. G1/early S-blocked cells were grown to early log phase at 23° and then blocked with α-factor. Half the culture was maintained at 23°, while the other half was shifted to 37° for 3 hr. G2/M-blocked cells were grown to early log phase at 23° and then blocked with nocodazole. Half the culture was maintained at 23°, while the other half was shifted to 37° for 3 hr. (C) For each of the experiments shown in B, duplicate gels were run and stained with Coomassie to confirm consistent loading of samples from lane to lane.
F<sc>igure</sc> 1.—
Figure 1.—
Conditional alleles of SIR2. (A) Levels of α1 and ACT1 mRNA were measured by RT–PCR in strains bearing different alleles of SIR2. Cultures were maintained in log phase for >10 generations at the listed temperatures, and then RNA was collected and analyzed as described in materials and methods. α1 indicates steady-state levels of message transcribed from the HML locus; ACT1 message is shown as a control. RNA was analyzed from a wild-type strain, a strain lacking the SIR2 gene, and strains bearing the indicated alleles of SIR2. (B) Protein extracts made from cells bearing the indicated SIR2 alleles were subjected to a Western blot using an antibody specific for the Sir2 protein, as described in materials and methods. For the cycling cells, parallel cultures were grown to log phase at either 23° or 37°. G1/early S-blocked cells were grown to early log phase at 23° and then blocked with α-factor. Half the culture was maintained at 23°, while the other half was shifted to 37° for 3 hr. G2/M-blocked cells were grown to early log phase at 23° and then blocked with nocodazole. Half the culture was maintained at 23°, while the other half was shifted to 37° for 3 hr. (C) For each of the experiments shown in B, duplicate gels were run and stained with Coomassie to confirm consistent loading of samples from lane to lane.
F<sc>igure</sc> 1.—
Figure 1.—
Conditional alleles of SIR2. (A) Levels of α1 and ACT1 mRNA were measured by RT–PCR in strains bearing different alleles of SIR2. Cultures were maintained in log phase for >10 generations at the listed temperatures, and then RNA was collected and analyzed as described in materials and methods. α1 indicates steady-state levels of message transcribed from the HML locus; ACT1 message is shown as a control. RNA was analyzed from a wild-type strain, a strain lacking the SIR2 gene, and strains bearing the indicated alleles of SIR2. (B) Protein extracts made from cells bearing the indicated SIR2 alleles were subjected to a Western blot using an antibody specific for the Sir2 protein, as described in materials and methods. For the cycling cells, parallel cultures were grown to log phase at either 23° or 37°. G1/early S-blocked cells were grown to early log phase at 23° and then blocked with α-factor. Half the culture was maintained at 23°, while the other half was shifted to 37° for 3 hr. G2/M-blocked cells were grown to early log phase at 23° and then blocked with nocodazole. Half the culture was maintained at 23°, while the other half was shifted to 37° for 3 hr. (C) For each of the experiments shown in B, duplicate gels were run and stained with Coomassie to confirm consistent loading of samples from lane to lane.
F<sc>igure</sc> 2.—
Figure 2.—
Sir2 function in G1 phase. (A and B) SIR2 alleles with cell-cycle-specific silencing defects. Cells grown to log phase at 23° were arrested with α-factor (10 μg/ml) in G1 phase. When at least 95% of cells exhibited an unbudded morphology, each culture was divided. One half was maintained at 23°, while the other was shifted to 37°. Silencing at HML was assessed by the bud morphology of the culture; unsilenced cells failed to arrest and form buds. The percentage of unbudded cells (α-factor-arrested cells) is plotted vs. time measured from the division of the culture. HML is efficiently silenced in all cultures maintained at 23° (Figure 2A). Silencing is lost in G1 at the nonpermissive temperature (37°) only in strains bearing the sir2-614 allele or the sir3-8 allele (Figure 2B). (C) α1 mRNA levels in G1-arrested strains. Cells grown to log phase at 23° were arrested with α-factor (10 μg/ml) in G1 phase. When at least 95% of cells exhibited an unbudded morphology, hydroxyurea (20 mg/ml) was added and each culture was divided. One-half was maintained at 23°, while the other was shifted to 37°. Following 4 hr of incubation, RNA was collected and RT–PCR was used to assay α1 and ACT1 message levels. RNA was analyzed from a wild-type strain, a strain lacking the SIR2 gene, and strains bearing the indicated alleles of SIR2.
F<sc>igure</sc> 2.—
Figure 2.—
Sir2 function in G1 phase. (A and B) SIR2 alleles with cell-cycle-specific silencing defects. Cells grown to log phase at 23° were arrested with α-factor (10 μg/ml) in G1 phase. When at least 95% of cells exhibited an unbudded morphology, each culture was divided. One half was maintained at 23°, while the other was shifted to 37°. Silencing at HML was assessed by the bud morphology of the culture; unsilenced cells failed to arrest and form buds. The percentage of unbudded cells (α-factor-arrested cells) is plotted vs. time measured from the division of the culture. HML is efficiently silenced in all cultures maintained at 23° (Figure 2A). Silencing is lost in G1 at the nonpermissive temperature (37°) only in strains bearing the sir2-614 allele or the sir3-8 allele (Figure 2B). (C) α1 mRNA levels in G1-arrested strains. Cells grown to log phase at 23° were arrested with α-factor (10 μg/ml) in G1 phase. When at least 95% of cells exhibited an unbudded morphology, hydroxyurea (20 mg/ml) was added and each culture was divided. One-half was maintained at 23°, while the other was shifted to 37°. Following 4 hr of incubation, RNA was collected and RT–PCR was used to assay α1 and ACT1 message levels. RNA was analyzed from a wild-type strain, a strain lacking the SIR2 gene, and strains bearing the indicated alleles of SIR2.
F<sc>igure</sc> 2.—
Figure 2.—
Sir2 function in G1 phase. (A and B) SIR2 alleles with cell-cycle-specific silencing defects. Cells grown to log phase at 23° were arrested with α-factor (10 μg/ml) in G1 phase. When at least 95% of cells exhibited an unbudded morphology, each culture was divided. One half was maintained at 23°, while the other was shifted to 37°. Silencing at HML was assessed by the bud morphology of the culture; unsilenced cells failed to arrest and form buds. The percentage of unbudded cells (α-factor-arrested cells) is plotted vs. time measured from the division of the culture. HML is efficiently silenced in all cultures maintained at 23° (Figure 2A). Silencing is lost in G1 at the nonpermissive temperature (37°) only in strains bearing the sir2-614 allele or the sir3-8 allele (Figure 2B). (C) α1 mRNA levels in G1-arrested strains. Cells grown to log phase at 23° were arrested with α-factor (10 μg/ml) in G1 phase. When at least 95% of cells exhibited an unbudded morphology, hydroxyurea (20 mg/ml) was added and each culture was divided. One-half was maintained at 23°, while the other was shifted to 37°. Following 4 hr of incubation, RNA was collected and RT–PCR was used to assay α1 and ACT1 message levels. RNA was analyzed from a wild-type strain, a strain lacking the SIR2 gene, and strains bearing the indicated alleles of SIR2.
F<sc>igure</sc> 3.—
Figure 3.—
Sir2 function during mitosis. (A) α1 mRNA levels in nocodazole-arrested strains. Log-phase cultures grown at 23° were blocked at the G2/M boundary with nocodazole. Each culture was then divided; half was maintained at 23°, and the other half was shifted to 37°. After maintaining cultures at their respective temperatures for 5 hr, RNA was collected and RT–PCR was used to assay α1 and ACT1 message, as described in materials and methods. (B) α1 mRNA levels in strains progressing from G2/M to early S phase. Log-phase cultures grown at 23° were blocked in G2/M with nocodazole. Each culture was then divided; half was maintained at 23°, and the other half was shifted to 37°. Cultures were retained at the nocodazole block for 4 hr and then released at their respective temperatures into media containing hydroxyurea. Eight hours following release from the nocodazole block, RNA was collected and RT–PCR was used to assay α1 and ACT1 message.
F<sc>igure</sc> 3.—
Figure 3.—
Sir2 function during mitosis. (A) α1 mRNA levels in nocodazole-arrested strains. Log-phase cultures grown at 23° were blocked at the G2/M boundary with nocodazole. Each culture was then divided; half was maintained at 23°, and the other half was shifted to 37°. After maintaining cultures at their respective temperatures for 5 hr, RNA was collected and RT–PCR was used to assay α1 and ACT1 message, as described in materials and methods. (B) α1 mRNA levels in strains progressing from G2/M to early S phase. Log-phase cultures grown at 23° were blocked in G2/M with nocodazole. Each culture was then divided; half was maintained at 23°, and the other half was shifted to 37°. Cultures were retained at the nocodazole block for 4 hr and then released at their respective temperatures into media containing hydroxyurea. Eight hours following release from the nocodazole block, RNA was collected and RT–PCR was used to assay α1 and ACT1 message.
F<sc>igure</sc> 4.—
Figure 4.—
Two-hybrid assays of Sir2–Sir4 interactions. (A) Sir2 fusion proteins maintain function. A mating assay was performed on strains expressing Sir2 proteins fused at their N terminus to the Gal4 activation domain. An equal number of cells from each culture were spotted onto plates spread with a MATα haploid strain. These plates were incubated overnight at 23° or 37° to allow mating, replica plated to media selecting for diploids, and then incubated an additional night at their respective temperature, when the photos shown were taken. “Growth” shows the same haploid cultures spotted onto nonselective plates lacking a mating partner. Equivalent growth controls were done at 23° and 37° with identical results. pOAD is the base vector expressing only the Gal4-activation domain. (B) Sir2–Sir4 interactions. Each row is labeled with the activation domain fusion used; pOAD is the vector control expressing only the Gal4-activation domain. Each column lists the binding domain fusion used; pOBD2 is the vector control expressing only the Gal4-binding domain. Cultures were grown and plated at the temperatures indicated. This experiment was conducted in a strain lacking the endogenous SIR2, SIR3, and SIR4 genes. Equal numbers of cells were applied to media selecting for expression of a HIS3 reporter gene containing Gal4-binding sites in its promoter. Duplicate platings on media selecting only for the plasmids bearing the binding domain and activation domain fusions ensured that platings were equal (not shown). The Sir4–Δ730N binding domain fusion lacks the N-terminal 730 amino acids of Sir4. (C) An experiment was conducted exactly as described in B in a strain lacking the SIR3 and SIR4 genes.
F<sc>igure</sc> 4.—
Figure 4.—
Two-hybrid assays of Sir2–Sir4 interactions. (A) Sir2 fusion proteins maintain function. A mating assay was performed on strains expressing Sir2 proteins fused at their N terminus to the Gal4 activation domain. An equal number of cells from each culture were spotted onto plates spread with a MATα haploid strain. These plates were incubated overnight at 23° or 37° to allow mating, replica plated to media selecting for diploids, and then incubated an additional night at their respective temperature, when the photos shown were taken. “Growth” shows the same haploid cultures spotted onto nonselective plates lacking a mating partner. Equivalent growth controls were done at 23° and 37° with identical results. pOAD is the base vector expressing only the Gal4-activation domain. (B) Sir2–Sir4 interactions. Each row is labeled with the activation domain fusion used; pOAD is the vector control expressing only the Gal4-activation domain. Each column lists the binding domain fusion used; pOBD2 is the vector control expressing only the Gal4-binding domain. Cultures were grown and plated at the temperatures indicated. This experiment was conducted in a strain lacking the endogenous SIR2, SIR3, and SIR4 genes. Equal numbers of cells were applied to media selecting for expression of a HIS3 reporter gene containing Gal4-binding sites in its promoter. Duplicate platings on media selecting only for the plasmids bearing the binding domain and activation domain fusions ensured that platings were equal (not shown). The Sir4–Δ730N binding domain fusion lacks the N-terminal 730 amino acids of Sir4. (C) An experiment was conducted exactly as described in B in a strain lacking the SIR3 and SIR4 genes.
F<sc>igure</sc> 4.—
Figure 4.—
Two-hybrid assays of Sir2–Sir4 interactions. (A) Sir2 fusion proteins maintain function. A mating assay was performed on strains expressing Sir2 proteins fused at their N terminus to the Gal4 activation domain. An equal number of cells from each culture were spotted onto plates spread with a MATα haploid strain. These plates were incubated overnight at 23° or 37° to allow mating, replica plated to media selecting for diploids, and then incubated an additional night at their respective temperature, when the photos shown were taken. “Growth” shows the same haploid cultures spotted onto nonselective plates lacking a mating partner. Equivalent growth controls were done at 23° and 37° with identical results. pOAD is the base vector expressing only the Gal4-activation domain. (B) Sir2–Sir4 interactions. Each row is labeled with the activation domain fusion used; pOAD is the vector control expressing only the Gal4-activation domain. Each column lists the binding domain fusion used; pOBD2 is the vector control expressing only the Gal4-binding domain. Cultures were grown and plated at the temperatures indicated. This experiment was conducted in a strain lacking the endogenous SIR2, SIR3, and SIR4 genes. Equal numbers of cells were applied to media selecting for expression of a HIS3 reporter gene containing Gal4-binding sites in its promoter. Duplicate platings on media selecting only for the plasmids bearing the binding domain and activation domain fusions ensured that platings were equal (not shown). The Sir4–Δ730N binding domain fusion lacks the N-terminal 730 amino acids of Sir4. (C) An experiment was conducted exactly as described in B in a strain lacking the SIR3 and SIR4 genes.
F<sc>igure</sc> 5.—
Figure 5.—
Complementation of SIR2 alleles. Plasmids bearing the SIR2 allele listed at the left were introduced into strains bearing integrated alleles of the SIR2 alleles listed at the top. Mating assays were performed at 23° and 37° as described in the Figure 4 legend.
F<sc>igure</sc> 6.—
Figure 6.—
Establishment of silencing in M phase. A strain bearing the sir2-614 allele was grown at 23° and blocked at G2/M with nocodazole. After >90% of the cells in the culture exhibited a large-bud morphology, the culture was divided and subjected to the indicated temperature shifts. At the release point, cells were applied to solid media containing α-factor. Released from the nocodazole-induced block, large-budded cells continued through the cell cycle and were either sensitive to α-factor, forming shmoos, or not sensitive to α-factor, forming buds (cells that neither budded nor formed shmoos, always <10% of the total, were not counted). The percentage of large-budded cells in which at least one of the cell–cell pair exhibited sensitivity to α-factor by forming a shmoo is indicated. Data shown are the cumulative results of two independent experiments. At least 90 large-budded cells were assayed for each condition. The difference between 40 and 66% is significant (χ2, P < 0.001), given the number of events assayed for each condition.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Afshar, G., and J. P. Murnane, 1999. Characterization of a human gene with sequence homology to Saccharomyces cerevisiae SIR2. Gene 234: 161–168. - PubMed
    1. Aparicio, O. M., and D. E. Gottschling, 1994. Overcoming telomeric silencing: a trans-activator competes to establish gene expression in a cell cycle-dependent way. Genes Dev. 8: 1133–1146. - PubMed
    1. Bedalov, A., T. Gatbonton, W. P. Irvine, D. E. Gottschling and J. A. Simon, 2001. Identification of a small molecule inhibitor of Sir2p. Proc. Natl. Acad. Sci. USA 98: 15113–15118. - PMC - PubMed
    1. Brachmann, C. B., J. M. Sherman, S. E. Devine, E. E. Cameron, L. Pillus et al., 1995. The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability. Genes Dev. 9: 2888–2902. - PubMed
    1. Braunstein, M., A. B. Rose, S. G. Holmes, C. D. Allis and J. R. Broach, 1993. Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev. 7: 592–604. - PubMed

Publication types

MeSH terms

Substances