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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) The histone deacetylase activity of Sir2p alters acetylation of the H4-K16 residue in the plasmid ARS. WT and sir2Δ cells transformed with p415GAL were subjected to the standard treatment for ChIP. Coamplification products of four different DNA regions were reported: ARSH4 refers to a region encompassing the ARSH4 and adjacent plasmid DNA of p415GAL; Link refers to a region encompassing the polylinker of p415GAL; ACT1 refers to the actin gene in its chromosomal location and Tel IV refers to a subtelomeric (telomer IV, L-arm). INPUT (1–3): coamplification of three doubled amounts of samples from whole cell extract (without immunoprecipitation). Samples IP, lanes 4–6: whole cell extract immunoprecipited with anti K9-H3 antibodies and coamplified in three doubled amounts; lanes 7–9 samples as in 4–6 immunoprecipited with anti H3-K14; lanes 10–12, samples as in 4–6 immunoprecipited with anti H4-K16; WT and sir2Δ indicate samples treated for ChIP analysis from WT and sir2Δ cells. (B) Shows a graphic representation comparing the ratio of the sir2Δ/WT ratio normalized to relative INPUTS for ARSH4, Polylinker, ACT1 and TEL-IV.
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fig4: (A) The histone deacetylase activity of Sir2p alters acetylation of the H4-K16 residue in the plasmid ARS. WT and sir2Δ cells transformed with p415GAL were subjected to the standard treatment for ChIP. Coamplification products of four different DNA regions were reported: ARSH4 refers to a region encompassing the ARSH4 and adjacent plasmid DNA of p415GAL; Link refers to a region encompassing the polylinker of p415GAL; ACT1 refers to the actin gene in its chromosomal location and Tel IV refers to a subtelomeric (telomer IV, L-arm). INPUT (1–3): coamplification of three doubled amounts of samples from whole cell extract (without immunoprecipitation). Samples IP, lanes 4–6: whole cell extract immunoprecipited with anti K9-H3 antibodies and coamplified in three doubled amounts; lanes 7–9 samples as in 4–6 immunoprecipited with anti H3-K14; lanes 10–12, samples as in 4–6 immunoprecipited with anti H4-K16; WT and sir2Δ indicate samples treated for ChIP analysis from WT and sir2Δ cells. (B) Shows a graphic representation comparing the ratio of the sir2Δ/WT ratio normalized to relative INPUTS for ARSH4, Polylinker, ACT1 and TEL-IV.

Mentions: Most of the phenotypes shown by sir2Δ mutants arise from the lack of histone deacetylase function (8). This enzymatic activity yields hyperacetylated chromatin (54). In order to determine whether the topological alterations we observe are also due to hyperacetylation of plasmid chromatin, we set up ChIP experiments to measure the acetylation level of the histone residues modified by Sir2p on the plasmids studied; we considered two regions in the plasmid analyzed (ARSH4 and polylinker) and also two chromosomal regions as reference (ACT1 and TEL IV). WT and sir2Δ cells were grown in minimal medium to exponential phase. After cross-linking with formaldehyde, whole cell extracts were prepared and the DNA–protein samples were immunoprecipitated with specific antibodies [anti-Acetyl-K9(H3), anti-Acetyl-K14(H3) and anti-Acetyl-K16(H4)]; the cross-linking was reversed and the DNA purified. A multiplex PCR was set up, coamplifying with four pairs of primers corresponding to the following sites: (i) the ARSH4 region of the p415GAL plasmid (ARSH4), (ii) the linker region of the p415GAL plasmid (LINK), (iii) the promoter region of ACT1 gene (ACT1) and (iv) the Tel IV subtelomeric region (TEL IV). As shown in Figure 4A, no enrichment was observed for the polylinker and ACT1 regions in the two strains (negative controls) compared to the respective input values (lanes 1–3, INPUT); in the telomeric region (Tel IV), where Sir2p is known to act (51), the acetylated form of all the histone residues studied is strongly enriched (lanes 4–12 in sir2Δ). As far as the ARSH4 region is concerned, we observed that in the sir2Δ strain, the only hyperacetylated residue relative to WT was H4-K16 (lanes 10–12, in sir2Δ). A quantitative representation of the data are shown in Figure 4B, where the ratios of sir2/WT values for each residue studied (normalized to their INPUT) is reported. Taken together, these data demonstrate that in the sir2Δ mutant, where topological shifts have been observed (Figures 1 and 2), plasmid chromatin is hyperacetylated at H4-K16, suggesting that SIR2-dependent deacetylation of plasmid chromatin may play a role in the alteration of superhelicity.


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) The histone deacetylase activity of Sir2p alters acetylation of the H4-K16 residue in the plasmid ARS. WT and sir2Δ cells transformed with p415GAL were subjected to the standard treatment for ChIP. Coamplification products of four different DNA regions were reported: ARSH4 refers to a region encompassing the ARSH4 and adjacent plasmid DNA of p415GAL; Link refers to a region encompassing the polylinker of p415GAL; ACT1 refers to the actin gene in its chromosomal location and Tel IV refers to a subtelomeric (telomer IV, L-arm). INPUT (1–3): coamplification of three doubled amounts of samples from whole cell extract (without immunoprecipitation). Samples IP, lanes 4–6: whole cell extract immunoprecipited with anti K9-H3 antibodies and coamplified in three doubled amounts; lanes 7–9 samples as in 4–6 immunoprecipited with anti H3-K14; lanes 10–12, samples as in 4–6 immunoprecipited with anti H4-K16; WT and sir2Δ indicate samples treated for ChIP analysis from WT and sir2Δ cells. (B) Shows a graphic representation comparing the ratio of the sir2Δ/WT ratio normalized to relative INPUTS for ARSH4, Polylinker, ACT1 and TEL-IV.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: (A) The histone deacetylase activity of Sir2p alters acetylation of the H4-K16 residue in the plasmid ARS. WT and sir2Δ cells transformed with p415GAL were subjected to the standard treatment for ChIP. Coamplification products of four different DNA regions were reported: ARSH4 refers to a region encompassing the ARSH4 and adjacent plasmid DNA of p415GAL; Link refers to a region encompassing the polylinker of p415GAL; ACT1 refers to the actin gene in its chromosomal location and Tel IV refers to a subtelomeric (telomer IV, L-arm). INPUT (1–3): coamplification of three doubled amounts of samples from whole cell extract (without immunoprecipitation). Samples IP, lanes 4–6: whole cell extract immunoprecipited with anti K9-H3 antibodies and coamplified in three doubled amounts; lanes 7–9 samples as in 4–6 immunoprecipited with anti H3-K14; lanes 10–12, samples as in 4–6 immunoprecipited with anti H4-K16; WT and sir2Δ indicate samples treated for ChIP analysis from WT and sir2Δ cells. (B) Shows a graphic representation comparing the ratio of the sir2Δ/WT ratio normalized to relative INPUTS for ARSH4, Polylinker, ACT1 and TEL-IV.
Mentions: Most of the phenotypes shown by sir2Δ mutants arise from the lack of histone deacetylase function (8). This enzymatic activity yields hyperacetylated chromatin (54). In order to determine whether the topological alterations we observe are also due to hyperacetylation of plasmid chromatin, we set up ChIP experiments to measure the acetylation level of the histone residues modified by Sir2p on the plasmids studied; we considered two regions in the plasmid analyzed (ARSH4 and polylinker) and also two chromosomal regions as reference (ACT1 and TEL IV). WT and sir2Δ cells were grown in minimal medium to exponential phase. After cross-linking with formaldehyde, whole cell extracts were prepared and the DNA–protein samples were immunoprecipitated with specific antibodies [anti-Acetyl-K9(H3), anti-Acetyl-K14(H3) and anti-Acetyl-K16(H4)]; the cross-linking was reversed and the DNA purified. A multiplex PCR was set up, coamplifying with four pairs of primers corresponding to the following sites: (i) the ARSH4 region of the p415GAL plasmid (ARSH4), (ii) the linker region of the p415GAL plasmid (LINK), (iii) the promoter region of ACT1 gene (ACT1) and (iv) the Tel IV subtelomeric region (TEL IV). As shown in Figure 4A, no enrichment was observed for the polylinker and ACT1 regions in the two strains (negative controls) compared to the respective input values (lanes 1–3, INPUT); in the telomeric region (Tel IV), where Sir2p is known to act (51), the acetylated form of all the histone residues studied is strongly enriched (lanes 4–12 in sir2Δ). As far as the ARSH4 region is concerned, we observed that in the sir2Δ strain, the only hyperacetylated residue relative to WT was H4-K16 (lanes 10–12, in sir2Δ). A quantitative representation of the data are shown in Figure 4B, where the ratios of sir2/WT values for each residue studied (normalized to their INPUT) is reported. Taken together, these data demonstrate that in the sir2Δ mutant, where topological shifts have been observed (Figures 1 and 2), plasmid chromatin is hyperacetylated at H4-K16, suggesting that SIR2-dependent deacetylation of plasmid chromatin may play a role in the alteration of superhelicity.

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