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SIR2 modifies histone H4-K16 acetylation and affects superhelicity in the ARS region of plasmid chromatin in Saccharomyces cerevisiae.

Chiani F, Di Felice F, Camilloni G - Nucleic Acids Res. (2006)

Bottom Line: Here we report that a plasmid introduced into sir2Delta cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells.This effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not depend on the number or positioning of nucleosomes on plasmids.A model proposing interference with the replication machinery is discussed.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, Rome, Italy.

ABSTRACT
The mutation of the SIR2 gene in Saccharomyces cerevisiae has been associated with a series of different phenotypes including loss of transcriptional silencing, genome instability and replicative aging. Thus, the SIR2 gene product is an important constituent of the yeast cell. SIR2 orthologues and paralogues have been discovered in organisms ranging from bacteria to man, underscoring the pivotal role of this protein. Here we report that a plasmid introduced into sir2Delta cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells. This effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not depend on the number or positioning of nucleosomes on plasmids. Our results indicate that this new phenotype is due to the lack of Sir2p histone deacetylase activity in the sir2Delta strain, because only the H4-K16 residue of the histone octamer undergoes an alteration of its acetylation state. A model proposing interference with the replication machinery is discussed.

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(A) Overexpression of the SIR2 gene leads to positively supercoiled topoisomers. WT cells transformed with pAR44 (carrying the coding region of Sir2p under GAL10 promoter) were induced (Sir2++) or not (WT) with galactose. After a 2 h induction, plasmid DNA was extracted, purified and electrophoresed as reported in Figure 1. The sir2Δ sample is included as a reference for negatively supercoiled topoisomers. (B) sir4Δ behaves like a sir2Δ mutant with respect to altering plasmid topology, implicating the SIR2–SIR4 complex. WT and sir4Δ cells were transformed with p415GAL, grown to exponential phase and plasmid DNA purified as in Figure 1. DNA samples were subjected to 2D electrophoresis as in Figure 1. Bands outside the arc trajectory represent open circle molecules. The ind values reported at the bottom of each topoisomer distribution indicate the increased negative supercoil distribution evaluated by densitometric analysis. (C) Sir3p does not affect plasmid topology. Sir2Δ, WT, sir3Δ and sir3Δ over-expressing Sir2p cells were transformed with pRS316, grown to exponential phase and plasmid DNA purified as in Figure 1. Over-expression of Sir2p in the sir3Δ strain, was engineered as for the experiments shown in Figure 1A (sample sir2++). DNA samples were subjected to 2D electrophoresis as in Figure 1. Bands outside the arc trajectory represent open circle molecules. The ind values reported at the bottom of each topoisomer distribution indicate the increased negative supercoil distribution evaluated by densitometric analysis.
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fig2: (A) Overexpression of the SIR2 gene leads to positively supercoiled topoisomers. WT cells transformed with pAR44 (carrying the coding region of Sir2p under GAL10 promoter) were induced (Sir2++) or not (WT) with galactose. After a 2 h induction, plasmid DNA was extracted, purified and electrophoresed as reported in Figure 1. The sir2Δ sample is included as a reference for negatively supercoiled topoisomers. (B) sir4Δ behaves like a sir2Δ mutant with respect to altering plasmid topology, implicating the SIR2–SIR4 complex. WT and sir4Δ cells were transformed with p415GAL, grown to exponential phase and plasmid DNA purified as in Figure 1. DNA samples were subjected to 2D electrophoresis as in Figure 1. Bands outside the arc trajectory represent open circle molecules. The ind values reported at the bottom of each topoisomer distribution indicate the increased negative supercoil distribution evaluated by densitometric analysis. (C) Sir3p does not affect plasmid topology. Sir2Δ, WT, sir3Δ and sir3Δ over-expressing Sir2p cells were transformed with pRS316, grown to exponential phase and plasmid DNA purified as in Figure 1. Over-expression of Sir2p in the sir3Δ strain, was engineered as for the experiments shown in Figure 1A (sample sir2++). DNA samples were subjected to 2D electrophoresis as in Figure 1. Bands outside the arc trajectory represent open circle molecules. The ind values reported at the bottom of each topoisomer distribution indicate the increased negative supercoil distribution evaluated by densitometric analysis.

Mentions: In order to verify the hypothesis that Sir2p directly affects the topology of the plasmids, we studied the topological state of the plasmids previously analyzed in strains with SIR2 placed under heterologous control to allow a different rate of transcription. We transformed WT cells (W303-1A) with the plasmid pAR44 containing the SIR2 gene under the GAL10 promoter (37); these cells were then transformed with pRS316, grown in minimal medium containing 2% glucose to exponential phase, collected, washed, split in two equal parts and shifted for 2 h to minimal medium with 2% galactose or 2% glucose, in order to either induce or repress SIR2 gene transcription from the pAR44 plasmid. It is important to emphasize that the pRS316 plasmid on which the topological analysis is performed does not contain any sequence responsive to the presence of galactose. DNA enriched in circular forms was prepared as described above and run on a 2D agarose gel. DNA samples from WT cells transformed with pAR44 (un-induced and induced with galactose) were loaded on the same gel; DNA from sir2Δ cells was also loaded as a reference. The DNAs were transferred to a nitrocellulose filter and hybridized to a pRS316 probe (detailed in Materials and Methods). Figure 2A shows the comparison of topoisomer distributions among sir2Δ, WT and SIR2++ (over-expression) strains. The average distribution shifts from more negative (sir2Δ) to more positive topological forms (SIR2++), correlating with the increase in the level of Sir2p in the cells. Note the arc shape: shifted toward the left in sir2Δ cells, centered in WT cells not induced with galactose, and shifted toward the right for DNA from cells induced with galactose to overproduce Sir2p. In this experiment, the ind value ranges from 1.3 (relative to WT) to 0.5. Taken together, these results demonstrate that supercoiling of DNA topoisomers depends on the amount of Sir2p present inside the cells, and that increasing Sir2p moves the distribution toward positive topoisomers (Figure 2A, sample SIR2++); the absence of Sir2p produces the opposite result (Figure 2A, sample sir2Δ) inducing more negative supercoiling.


SIR2 modifies histone H4-K16 acetylation and affects superhelicity in the ARS region of plasmid chromatin in Saccharomyces cerevisiae.

Chiani F, Di Felice F, Camilloni G - Nucleic Acids Res. (2006)

(A) Overexpression of the SIR2 gene leads to positively supercoiled topoisomers. WT cells transformed with pAR44 (carrying the coding region of Sir2p under GAL10 promoter) were induced (Sir2++) or not (WT) with galactose. After a 2 h induction, plasmid DNA was extracted, purified and electrophoresed as reported in Figure 1. The sir2Δ sample is included as a reference for negatively supercoiled topoisomers. (B) sir4Δ behaves like a sir2Δ mutant with respect to altering plasmid topology, implicating the SIR2–SIR4 complex. WT and sir4Δ cells were transformed with p415GAL, grown to exponential phase and plasmid DNA purified as in Figure 1. DNA samples were subjected to 2D electrophoresis as in Figure 1. Bands outside the arc trajectory represent open circle molecules. The ind values reported at the bottom of each topoisomer distribution indicate the increased negative supercoil distribution evaluated by densitometric analysis. (C) Sir3p does not affect plasmid topology. Sir2Δ, WT, sir3Δ and sir3Δ over-expressing Sir2p cells were transformed with pRS316, grown to exponential phase and plasmid DNA purified as in Figure 1. Over-expression of Sir2p in the sir3Δ strain, was engineered as for the experiments shown in Figure 1A (sample sir2++). DNA samples were subjected to 2D electrophoresis as in Figure 1. Bands outside the arc trajectory represent open circle molecules. The ind values reported at the bottom of each topoisomer distribution indicate the increased negative supercoil distribution evaluated by densitometric analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636471&req=5

fig2: (A) Overexpression of the SIR2 gene leads to positively supercoiled topoisomers. WT cells transformed with pAR44 (carrying the coding region of Sir2p under GAL10 promoter) were induced (Sir2++) or not (WT) with galactose. After a 2 h induction, plasmid DNA was extracted, purified and electrophoresed as reported in Figure 1. The sir2Δ sample is included as a reference for negatively supercoiled topoisomers. (B) sir4Δ behaves like a sir2Δ mutant with respect to altering plasmid topology, implicating the SIR2–SIR4 complex. WT and sir4Δ cells were transformed with p415GAL, grown to exponential phase and plasmid DNA purified as in Figure 1. DNA samples were subjected to 2D electrophoresis as in Figure 1. Bands outside the arc trajectory represent open circle molecules. The ind values reported at the bottom of each topoisomer distribution indicate the increased negative supercoil distribution evaluated by densitometric analysis. (C) Sir3p does not affect plasmid topology. Sir2Δ, WT, sir3Δ and sir3Δ over-expressing Sir2p cells were transformed with pRS316, grown to exponential phase and plasmid DNA purified as in Figure 1. Over-expression of Sir2p in the sir3Δ strain, was engineered as for the experiments shown in Figure 1A (sample sir2++). DNA samples were subjected to 2D electrophoresis as in Figure 1. Bands outside the arc trajectory represent open circle molecules. The ind values reported at the bottom of each topoisomer distribution indicate the increased negative supercoil distribution evaluated by densitometric analysis.
Mentions: In order to verify the hypothesis that Sir2p directly affects the topology of the plasmids, we studied the topological state of the plasmids previously analyzed in strains with SIR2 placed under heterologous control to allow a different rate of transcription. We transformed WT cells (W303-1A) with the plasmid pAR44 containing the SIR2 gene under the GAL10 promoter (37); these cells were then transformed with pRS316, grown in minimal medium containing 2% glucose to exponential phase, collected, washed, split in two equal parts and shifted for 2 h to minimal medium with 2% galactose or 2% glucose, in order to either induce or repress SIR2 gene transcription from the pAR44 plasmid. It is important to emphasize that the pRS316 plasmid on which the topological analysis is performed does not contain any sequence responsive to the presence of galactose. DNA enriched in circular forms was prepared as described above and run on a 2D agarose gel. DNA samples from WT cells transformed with pAR44 (un-induced and induced with galactose) were loaded on the same gel; DNA from sir2Δ cells was also loaded as a reference. The DNAs were transferred to a nitrocellulose filter and hybridized to a pRS316 probe (detailed in Materials and Methods). Figure 2A shows the comparison of topoisomer distributions among sir2Δ, WT and SIR2++ (over-expression) strains. The average distribution shifts from more negative (sir2Δ) to more positive topological forms (SIR2++), correlating with the increase in the level of Sir2p in the cells. Note the arc shape: shifted toward the left in sir2Δ cells, centered in WT cells not induced with galactose, and shifted toward the right for DNA from cells induced with galactose to overproduce Sir2p. In this experiment, the ind value ranges from 1.3 (relative to WT) to 0.5. Taken together, these results demonstrate that supercoiling of DNA topoisomers depends on the amount of Sir2p present inside the cells, and that increasing Sir2p moves the distribution toward positive topoisomers (Figure 2A, sample SIR2++); the absence of Sir2p produces the opposite result (Figure 2A, sample sir2Δ) inducing more negative supercoiling.

Bottom Line: Here we report that a plasmid introduced into sir2Delta cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells.This effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not depend on the number or positioning of nucleosomes on plasmids.A model proposing interference with the replication machinery is discussed.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, Rome, Italy.

ABSTRACT
The mutation of the SIR2 gene in Saccharomyces cerevisiae has been associated with a series of different phenotypes including loss of transcriptional silencing, genome instability and replicative aging. Thus, the SIR2 gene product is an important constituent of the yeast cell. SIR2 orthologues and paralogues have been discovered in organisms ranging from bacteria to man, underscoring the pivotal role of this protein. Here we report that a plasmid introduced into sir2Delta cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells. This effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not depend on the number or positioning of nucleosomes on plasmids. Our results indicate that this new phenotype is due to the lack of Sir2p histone deacetylase activity in the sir2Delta strain, because only the H4-K16 residue of the histone octamer undergoes an alteration of its acetylation state. A model proposing interference with the replication machinery is discussed.

Show MeSH
Related in: MedlinePlus