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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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Plasmid chromatin organization does not differ between WT cells and the sir2Δ mutant. WT and sir2Δ cells transformed with pRS316 were grown to exponential phase and subjected to MNase digestion after permeabilization with nystatin. Afterwards, total DNA was extracted, purified and subjected to agarose gel electrophoresis. DNA fragments were transferred onto a nitrocellulose filter and hybridized to AMP (A) or LINK (B) probes in order to reveal nucleosome spacing in two different regions of pRS316. Triangles indicate increasing (or decreasing) amounts of MNase used in the assay. The drawing at the bottom of the figure (C) describes interpretation of the data. Thick-lined ellipses indicate nucleosomes with a clear spacing profile; dotted-line ellipses indicate nucleosomes with less defined spacing profiles.
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fig3: Plasmid chromatin organization does not differ between WT cells and the sir2Δ mutant. WT and sir2Δ cells transformed with pRS316 were grown to exponential phase and subjected to MNase digestion after permeabilization with nystatin. Afterwards, total DNA was extracted, purified and subjected to agarose gel electrophoresis. DNA fragments were transferred onto a nitrocellulose filter and hybridized to AMP (A) or LINK (B) probes in order to reveal nucleosome spacing in two different regions of pRS316. Triangles indicate increasing (or decreasing) amounts of MNase used in the assay. The drawing at the bottom of the figure (C) describes interpretation of the data. Thick-lined ellipses indicate nucleosomes with a clear spacing profile; dotted-line ellipses indicate nucleosomes with less defined spacing profiles.

Mentions: Altering the number of nucleosomes, as well as their distribution or conformation, could influence DNA topology. For this reason, we investigated whether nucleosome spacing on a plasmid was altered in the sir2Δ mutant. WT or sir2Δ cells transformed with pRS316, grown to exponential phase, were processed for spheroplast formation and permeabilized with nystatin as reported previously (41). The permeabilized spheroplasts were subjected to micrococcal nuclease (MNase) digestion as reported (32). DNA was then purified and loaded on an agarose gel. After electrophoresis, samples were transferred onto a nitrocellulose filter and hybridized to different probes in order to detect the presence of nucleosomes in two major regions of the plasmid. As shown in Figure 3A, when the filter was hybridized to the AMP probe, described previously (for details see Materials and Methods), organization into regularly spaced nucleosomes (closed ellipses in the schematic drawing shown in Figure 3C) involving at least four particles was evident. Since the spacing analysis is not oriented, we can conclude that this organization occurs both at the left and at the right sides of the probe. In addition, no differences are detected between the WT and the sir2Δ profiles. When the AMP probe was stripped from the filter followed by re-hybridization to the LINK probe (annealing from 1878 to 2001 of the pRS316 sequence) we observed a slightly different profile (Figure 3B). In this case, only 2–3 nucleosomal particles with defined spacing are visible, most likely reflecting a less organized chromatin structure (dotted ellipses in the schematic drawing reported in Figure 3C). However, in this region also we observe no significant differences between the WT and sir2Δ profiles. Considering that with this analysis we are able to scan ∼2000 nt for each probe and that pRS316 is ∼4 kb long, we conclude that there is no dramatic difference in the nucleosome arrangement between WT and sir2Δ strains.


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)

Plasmid chromatin organization does not differ between WT cells and the sir2Δ mutant. WT and sir2Δ cells transformed with pRS316 were grown to exponential phase and subjected to MNase digestion after permeabilization with nystatin. Afterwards, total DNA was extracted, purified and subjected to agarose gel electrophoresis. DNA fragments were transferred onto a nitrocellulose filter and hybridized to AMP (A) or LINK (B) probes in order to reveal nucleosome spacing in two different regions of pRS316. Triangles indicate increasing (or decreasing) amounts of MNase used in the assay. The drawing at the bottom of the figure (C) describes interpretation of the data. Thick-lined ellipses indicate nucleosomes with a clear spacing profile; dotted-line ellipses indicate nucleosomes with less defined spacing profiles.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Plasmid chromatin organization does not differ between WT cells and the sir2Δ mutant. WT and sir2Δ cells transformed with pRS316 were grown to exponential phase and subjected to MNase digestion after permeabilization with nystatin. Afterwards, total DNA was extracted, purified and subjected to agarose gel electrophoresis. DNA fragments were transferred onto a nitrocellulose filter and hybridized to AMP (A) or LINK (B) probes in order to reveal nucleosome spacing in two different regions of pRS316. Triangles indicate increasing (or decreasing) amounts of MNase used in the assay. The drawing at the bottom of the figure (C) describes interpretation of the data. Thick-lined ellipses indicate nucleosomes with a clear spacing profile; dotted-line ellipses indicate nucleosomes with less defined spacing profiles.
Mentions: Altering the number of nucleosomes, as well as their distribution or conformation, could influence DNA topology. For this reason, we investigated whether nucleosome spacing on a plasmid was altered in the sir2Δ mutant. WT or sir2Δ cells transformed with pRS316, grown to exponential phase, were processed for spheroplast formation and permeabilized with nystatin as reported previously (41). The permeabilized spheroplasts were subjected to micrococcal nuclease (MNase) digestion as reported (32). DNA was then purified and loaded on an agarose gel. After electrophoresis, samples were transferred onto a nitrocellulose filter and hybridized to different probes in order to detect the presence of nucleosomes in two major regions of the plasmid. As shown in Figure 3A, when the filter was hybridized to the AMP probe, described previously (for details see Materials and Methods), organization into regularly spaced nucleosomes (closed ellipses in the schematic drawing shown in Figure 3C) involving at least four particles was evident. Since the spacing analysis is not oriented, we can conclude that this organization occurs both at the left and at the right sides of the probe. In addition, no differences are detected between the WT and the sir2Δ profiles. When the AMP probe was stripped from the filter followed by re-hybridization to the LINK probe (annealing from 1878 to 2001 of the pRS316 sequence) we observed a slightly different profile (Figure 3B). In this case, only 2–3 nucleosomal particles with defined spacing are visible, most likely reflecting a less organized chromatin structure (dotted ellipses in the schematic drawing reported in Figure 3C). However, in this region also we observe no significant differences between the WT and sir2Δ profiles. Considering that with this analysis we are able to scan ∼2000 nt for each probe and that pRS316 is ∼4 kb long, we conclude that there is no dramatic difference in the nucleosome arrangement between WT and sir2Δ strains.

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