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Sem1p and Ubp6p orchestrate telomeric silencing by modulating histone H2B ubiquitination and H3 acetylation.

Qin S, Wang Q, Ray A, Wani G, Zhao Q, Bhaumik SR, Wani AA - Nucleic Acids Res. (2009)

Bottom Line: Here, we show that proteasome-associated Sem1p and its interacting partner, Ubp6p (a deubiquitinating enzyme), are essential to maintain telomeric silencing.Simultaneous deletion of SEM1 and UBP6 induces dramatic silencing defect accompanied by significantly increased level of ubiquitinated-histone H2B and markedly reduced levels of acetylated-lysine 14 and 23 on histone H3 at the telomeres.Taken together, our data demonstrate that Sem1p and Ubp6p maintain telomeric heterochromatin structure (and hence silencing) through modulation of histone covalent modifications and association of silencing factors independently of the proteolytic function of the proteasome, thus offering a new regulatory mechanism of telomeric silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Histone covalent modifications and 26S proteasome-mediated proteolysis modulate many regulatory events in eukaryotes. In Saccharomyces cerevisiae, heterochromatin mediates transcriptional silencing at telomeres, HM loci and rDNA array. Here, we show that proteasome-associated Sem1p and its interacting partner, Ubp6p (a deubiquitinating enzyme), are essential to maintain telomeric silencing. Simultaneous deletion of SEM1 and UBP6 induces dramatic silencing defect accompanied by significantly increased level of ubiquitinated-histone H2B and markedly reduced levels of acetylated-lysine 14 and 23 on histone H3 at the telomeres. Further, the loss of Sem1p and Ubp6p triggers relocation of silencing factors (e.g. Sir proteins) from telomere to HM loci and rDNA array. Such relocation of silencing factors enhances gene silencing at HM loci and rDNA array, but diminishes telomeric silencing. Interestingly, both Sem1p and Ubp6p participate in the proteolytic function of the proteasome. However, we find that the telomeric silencing is not influenced by proteolysis. Taken together, our data demonstrate that Sem1p and Ubp6p maintain telomeric heterochromatin structure (and hence silencing) through modulation of histone covalent modifications and association of silencing factors independently of the proteolytic function of the proteasome, thus offering a new regulatory mechanism of telomeric silencing.

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Sem1p and Ubp6p mediate histone H3 acetylation. (A) Loss of Sem1p and Ubp6p results in reduced acetylation of K14 and K23 on histone H3. (B) Deletion of SEM1 and UBP6 causes reduction of H3 K14 and K23 acetylation in the telomere. (C) The ubp6Δ ubp10Δ mutant is able to acetylate K14 and K23. Global steady-state levels of acetylation of different residues were determined using site-specific antibodies. Western blotting analysis and quantitation were performed as described for Figure 3. The ChIP assay was performed as described for Figure 2. Standard deviations were calculated from three replicates. Genotypes of yeast strains are indicated on the top. VIR, right end of chromosome VI; WT, wild-type.
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Figure 4: Sem1p and Ubp6p mediate histone H3 acetylation. (A) Loss of Sem1p and Ubp6p results in reduced acetylation of K14 and K23 on histone H3. (B) Deletion of SEM1 and UBP6 causes reduction of H3 K14 and K23 acetylation in the telomere. (C) The ubp6Δ ubp10Δ mutant is able to acetylate K14 and K23. Global steady-state levels of acetylation of different residues were determined using site-specific antibodies. Western blotting analysis and quantitation were performed as described for Figure 3. The ChIP assay was performed as described for Figure 2. Standard deviations were calculated from three replicates. Genotypes of yeast strains are indicated on the top. VIR, right end of chromosome VI; WT, wild-type.

Mentions: Acetylation of H3K14 and H3K23 is known to be a critical factor in telomeric silencing (17). Therefore, we investigated if the loss of Sem1p and Ubp6p affects acetylation by specific western blot analysis of a battery of acetylated forms of histones H3 and H4. Compared to wild-type cells, >3-fold decrease was seen in the acetylation of H3K14 and H3K23 in the sem1Δ ubp6Δ mutant (Figure 4A). On the other hand, acetylation levels of H3K9 and H3K18 remained unchanged. We next examined H3K14 and H3K23 acetylation in the sem1Δ ubp6Δ mutant carrying a plasmid containing the wild-type gene SEM1 and UBP6, and found that H3K14 and H3K23 are acetylated in the mutant as efficiently as the wild-type (data not shown). Analysis of acetylation of lysine residues in the N-terminal tail of histone H4, which is also known to impact telomeric silencing, did not show any significant changes at K5, K8, K12 and K16 moieties (data not shown). Thus, Sem1p and Ubp6p mediate their effects by altering acetylation at specific sites of histone H3, but not histone H4.Figure 4.


Sem1p and Ubp6p orchestrate telomeric silencing by modulating histone H2B ubiquitination and H3 acetylation.

Qin S, Wang Q, Ray A, Wani G, Zhao Q, Bhaumik SR, Wani AA - Nucleic Acids Res. (2009)

Sem1p and Ubp6p mediate histone H3 acetylation. (A) Loss of Sem1p and Ubp6p results in reduced acetylation of K14 and K23 on histone H3. (B) Deletion of SEM1 and UBP6 causes reduction of H3 K14 and K23 acetylation in the telomere. (C) The ubp6Δ ubp10Δ mutant is able to acetylate K14 and K23. Global steady-state levels of acetylation of different residues were determined using site-specific antibodies. Western blotting analysis and quantitation were performed as described for Figure 3. The ChIP assay was performed as described for Figure 2. Standard deviations were calculated from three replicates. Genotypes of yeast strains are indicated on the top. VIR, right end of chromosome VI; WT, wild-type.
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Figure 4: Sem1p and Ubp6p mediate histone H3 acetylation. (A) Loss of Sem1p and Ubp6p results in reduced acetylation of K14 and K23 on histone H3. (B) Deletion of SEM1 and UBP6 causes reduction of H3 K14 and K23 acetylation in the telomere. (C) The ubp6Δ ubp10Δ mutant is able to acetylate K14 and K23. Global steady-state levels of acetylation of different residues were determined using site-specific antibodies. Western blotting analysis and quantitation were performed as described for Figure 3. The ChIP assay was performed as described for Figure 2. Standard deviations were calculated from three replicates. Genotypes of yeast strains are indicated on the top. VIR, right end of chromosome VI; WT, wild-type.
Mentions: Acetylation of H3K14 and H3K23 is known to be a critical factor in telomeric silencing (17). Therefore, we investigated if the loss of Sem1p and Ubp6p affects acetylation by specific western blot analysis of a battery of acetylated forms of histones H3 and H4. Compared to wild-type cells, >3-fold decrease was seen in the acetylation of H3K14 and H3K23 in the sem1Δ ubp6Δ mutant (Figure 4A). On the other hand, acetylation levels of H3K9 and H3K18 remained unchanged. We next examined H3K14 and H3K23 acetylation in the sem1Δ ubp6Δ mutant carrying a plasmid containing the wild-type gene SEM1 and UBP6, and found that H3K14 and H3K23 are acetylated in the mutant as efficiently as the wild-type (data not shown). Analysis of acetylation of lysine residues in the N-terminal tail of histone H4, which is also known to impact telomeric silencing, did not show any significant changes at K5, K8, K12 and K16 moieties (data not shown). Thus, Sem1p and Ubp6p mediate their effects by altering acetylation at specific sites of histone H3, but not histone H4.Figure 4.

Bottom Line: Here, we show that proteasome-associated Sem1p and its interacting partner, Ubp6p (a deubiquitinating enzyme), are essential to maintain telomeric silencing.Simultaneous deletion of SEM1 and UBP6 induces dramatic silencing defect accompanied by significantly increased level of ubiquitinated-histone H2B and markedly reduced levels of acetylated-lysine 14 and 23 on histone H3 at the telomeres.Taken together, our data demonstrate that Sem1p and Ubp6p maintain telomeric heterochromatin structure (and hence silencing) through modulation of histone covalent modifications and association of silencing factors independently of the proteolytic function of the proteasome, thus offering a new regulatory mechanism of telomeric silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Histone covalent modifications and 26S proteasome-mediated proteolysis modulate many regulatory events in eukaryotes. In Saccharomyces cerevisiae, heterochromatin mediates transcriptional silencing at telomeres, HM loci and rDNA array. Here, we show that proteasome-associated Sem1p and its interacting partner, Ubp6p (a deubiquitinating enzyme), are essential to maintain telomeric silencing. Simultaneous deletion of SEM1 and UBP6 induces dramatic silencing defect accompanied by significantly increased level of ubiquitinated-histone H2B and markedly reduced levels of acetylated-lysine 14 and 23 on histone H3 at the telomeres. Further, the loss of Sem1p and Ubp6p triggers relocation of silencing factors (e.g. Sir proteins) from telomere to HM loci and rDNA array. Such relocation of silencing factors enhances gene silencing at HM loci and rDNA array, but diminishes telomeric silencing. Interestingly, both Sem1p and Ubp6p participate in the proteolytic function of the proteasome. However, we find that the telomeric silencing is not influenced by proteolysis. Taken together, our data demonstrate that Sem1p and Ubp6p maintain telomeric heterochromatin structure (and hence silencing) through modulation of histone covalent modifications and association of silencing factors independently of the proteolytic function of the proteasome, thus offering a new regulatory mechanism of telomeric silencing.

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