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SIRT6 exhibits nucleosome-dependent deacetylase activity.

Gil R, Barth S, Kanfi Y, Cohen HY - Nucleic Acids Res. (2013)

Bottom Line: Here, we explored the specific conditions that allow SIRT6 to function as a significant deacetylase.In contrast, SIRT1 shows the opposite characteristics.Thus, our results show that SIRT6 activity is nucleosome dependent, and suggest that its binding to the nucleosome might convert it into an active structure.

View Article: PubMed Central - PubMed

Affiliation: The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.

ABSTRACT
The SIRT6 deacetylase is a key regulator of mammalian genome stability, metabolism and lifespan. Previous studies indicated that SIRT6 exhibits poor deacetylase activity in vitro. Here, we explored the specific conditions that allow SIRT6 to function as a significant deacetylase. We show that SIRT6 associates with the nucleosome and deacetylates histones H3 and H4 when they are packaged as nucleosomes, but not as free histones. In contrast, SIRT1 shows the opposite characteristics. Thus, our results show that SIRT6 activity is nucleosome dependent, and suggest that its binding to the nucleosome might convert it into an active structure.

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SIRT6 and SIRT1 deacetylate histone substrates in total cell extract. (A–E) Recombinant SIRT1-Flag and SIRT6-Flag deacetylation of H3K9Ac, H4K5Ac, H4K8Ac, H4K12Ac and H4K16Ac, when added to total cell proteins extracted from HEK 293T cells. The amount of NAD+ in the cell extract was negligible (3–5 µM). (F–G) Deacetylation of H4K5Ac and H4K12Ac by SIRT1-Flag and SIRT6-Flag after the disruption of the nucleosomes into free histones by nuclease cocktail.
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gkt642-F5: SIRT6 and SIRT1 deacetylate histone substrates in total cell extract. (A–E) Recombinant SIRT1-Flag and SIRT6-Flag deacetylation of H3K9Ac, H4K5Ac, H4K8Ac, H4K12Ac and H4K16Ac, when added to total cell proteins extracted from HEK 293T cells. The amount of NAD+ in the cell extract was negligible (3–5 µM). (F–G) Deacetylation of H4K5Ac and H4K12Ac by SIRT1-Flag and SIRT6-Flag after the disruption of the nucleosomes into free histones by nuclease cocktail.

Mentions: Given that the association between SIRT6 and nucleosome significantly improved its enzymatic activity, we next determined if SIRT1 enzymatic activity improved on its binding to free core histones. Surprisingly, SIRT1 deacetylated histone H3 on K9Ac and K14Ac and histone H4 on K5Ac, K8Ac and K12Ac, when present as free core histones but not as part of the nucleosome (Figure 4A–E). Importantly, these properties of SIRT1 were maintained even for its canonical substrate, H4K16Ac (Figure 4F). However, recombinant SIRT1 deacetylated H3K9Ac, H4K5Ac, H4K8Ac, H4K12Ac and H4K16Ac isolated from HEK 293T cells (Figure 5A–E). This deacetylation activity was specifically mediated by SIRT1, as the acetylation status of these sites was not changed in the presence of cell extract and SIRT6 without NAD+ or cell extract with NAD+ without additional enzymes even after 180 min (Figure 5A–E the first two left lanes of each). Thus, SIRT1 requires an as yet unidentified mediator or modification to deacetylate histones on the nucleosome. SIRT6 efficiently deacetylates H3K9Ac (Figure 5A). However, in comparison to reconstituted nucleosomes (Figure 3D–F), SIRT6 shows only weak deacetylase activity on H4K5Ac, H4K8Ac and H4K12Ac (Figure 5B–E), suggesting that other proteins interfere with its activity.Figure 4.


SIRT6 exhibits nucleosome-dependent deacetylase activity.

Gil R, Barth S, Kanfi Y, Cohen HY - Nucleic Acids Res. (2013)

SIRT6 and SIRT1 deacetylate histone substrates in total cell extract. (A–E) Recombinant SIRT1-Flag and SIRT6-Flag deacetylation of H3K9Ac, H4K5Ac, H4K8Ac, H4K12Ac and H4K16Ac, when added to total cell proteins extracted from HEK 293T cells. The amount of NAD+ in the cell extract was negligible (3–5 µM). (F–G) Deacetylation of H4K5Ac and H4K12Ac by SIRT1-Flag and SIRT6-Flag after the disruption of the nucleosomes into free histones by nuclease cocktail.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkt642-F5: SIRT6 and SIRT1 deacetylate histone substrates in total cell extract. (A–E) Recombinant SIRT1-Flag and SIRT6-Flag deacetylation of H3K9Ac, H4K5Ac, H4K8Ac, H4K12Ac and H4K16Ac, when added to total cell proteins extracted from HEK 293T cells. The amount of NAD+ in the cell extract was negligible (3–5 µM). (F–G) Deacetylation of H4K5Ac and H4K12Ac by SIRT1-Flag and SIRT6-Flag after the disruption of the nucleosomes into free histones by nuclease cocktail.
Mentions: Given that the association between SIRT6 and nucleosome significantly improved its enzymatic activity, we next determined if SIRT1 enzymatic activity improved on its binding to free core histones. Surprisingly, SIRT1 deacetylated histone H3 on K9Ac and K14Ac and histone H4 on K5Ac, K8Ac and K12Ac, when present as free core histones but not as part of the nucleosome (Figure 4A–E). Importantly, these properties of SIRT1 were maintained even for its canonical substrate, H4K16Ac (Figure 4F). However, recombinant SIRT1 deacetylated H3K9Ac, H4K5Ac, H4K8Ac, H4K12Ac and H4K16Ac isolated from HEK 293T cells (Figure 5A–E). This deacetylation activity was specifically mediated by SIRT1, as the acetylation status of these sites was not changed in the presence of cell extract and SIRT6 without NAD+ or cell extract with NAD+ without additional enzymes even after 180 min (Figure 5A–E the first two left lanes of each). Thus, SIRT1 requires an as yet unidentified mediator or modification to deacetylate histones on the nucleosome. SIRT6 efficiently deacetylates H3K9Ac (Figure 5A). However, in comparison to reconstituted nucleosomes (Figure 3D–F), SIRT6 shows only weak deacetylase activity on H4K5Ac, H4K8Ac and H4K12Ac (Figure 5B–E), suggesting that other proteins interfere with its activity.Figure 4.

Bottom Line: Here, we explored the specific conditions that allow SIRT6 to function as a significant deacetylase.In contrast, SIRT1 shows the opposite characteristics.Thus, our results show that SIRT6 activity is nucleosome dependent, and suggest that its binding to the nucleosome might convert it into an active structure.

View Article: PubMed Central - PubMed

Affiliation: The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.

ABSTRACT
The SIRT6 deacetylase is a key regulator of mammalian genome stability, metabolism and lifespan. Previous studies indicated that SIRT6 exhibits poor deacetylase activity in vitro. Here, we explored the specific conditions that allow SIRT6 to function as a significant deacetylase. We show that SIRT6 associates with the nucleosome and deacetylates histones H3 and H4 when they are packaged as nucleosomes, but not as free histones. In contrast, SIRT1 shows the opposite characteristics. Thus, our results show that SIRT6 activity is nucleosome dependent, and suggest that its binding to the nucleosome might convert it into an active structure.

Show MeSH