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, 21 (8), 920-8

SirT3 Is a Nuclear NAD+-dependent Histone Deacetylase That Translocates to the Mitochondria Upon Cellular Stress

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SirT3 Is a Nuclear NAD+-dependent Histone Deacetylase That Translocates to the Mitochondria Upon Cellular Stress

Michael B Scher et al. Genes Dev.

Abstract

In humans, there are at least seven Sir2-like proteins (SirT1-7) with diverse functions, including the regulation of chromatin structure, and metabolism. SirT3 levels have been shown to correlate with extended life span, to localize to the mitochondria, and to be highly expressed in brown adipose tissue. In humans, SirT3 exists in two forms, a full-length protein of approximately 44 kDa and a processed polypeptide lacking 142 amino acids at its N terminus. We found that SirT3 not only localizes to the mitochondria, but also to the nucleus under normal cell growth conditions. Both the full-length and processed forms of SirT3 target H4-K16 for deacetylation in vitro and can deacetylate H4-K16 in vivo when recruited to a gene. Using a highly specific antibody against the N terminus of SirT3, we found that SirT3 is transported from the nucleus to the mitochondria upon cellular stress. This includes DNA damage induced by Etoposide and UV-irradiation, as well as overexpression of SirT3 itself.

Figures

Figure 1.
Figure 1.
Full-length SirT3 is nuclear. (A) Schematic representation of full-length human and mouse SirT3. (B) Immunofluorescence analyses of the cell lines indicated using antibody specific to the N terminus of human SirT3. DAP1 was used to visualize the nuclei. (C) HeLa cells analyzed as in B but in the presence of the competitors indicated.
Figure 2.
Figure 2.
Full-length and truncated SirT3 exhibit similar HDAC activity and substrate specificity. (A, left side) Western blot of affinity-purified SirT3 from baculovirus-derived or human cells as indicated. (Right side) Coomassie blue stain of purified bSirT3 shows that only full-length SirT3 is detectable. (B, top) Nicotinamide exchange reaction in the presence of full-length SirT3 from baculovirus (bSirT3), truncated SirT3 from human cells (hSirT3), or recombinant SirT5 (rSirT5) as indicated. Substrates were acetylated BSA (Ac.B) histones derived from cells treated with histone deacetylase inhibitors (H) and BSA (B). (Bottom) Titration of bSirT3 and hSirT3 with histones as substrate. (C) Comparison of bSirT3 and hSirT3 in histone deacetylation reactions analyzed by Western blot using hyperacetylated histone substrates and with respect to specificity and NAD+ requirement. Antibodies against specific acetylated lysine residues of particular histones are indicated.
Figure 3.
Figure 3.
SirT3 directed to a promoter mediates gene repression and HDAC activity toward H4-K16ac. (A) Results of luciferase assays performed using a stable cell line containing an integrated luciferase reporter under the TK promoter with GAL4 DNA-binding sites. Transient expression of the GAL4 fusion proteins indicated was performed. (B) Western blots of extracts fractionated into nuclear and mitochondrial fractions after transfection with expression vectors for the indicated protein. (C) ChIP experiments performed with a stable cell line containing both an integrated luciferase reporter and an integrated tetracycline-inducible GAL4-SirT3 fusion protein. The experiment was performed in the presence or absence of tetracycline as indicated and with the specific antibodies shown at the top. Primers targeted to the luciferase reporter as well as the actin promoter were used to assay the immunoprecipitations.
Figure 4.
Figure 4.
Constitutively overexpressed SirT3 is actively transported to the mitochondria. (A) Immunofluoresence experiment performed with a stable cell line that constitutively expresses SirT3 with an HA tag using either HA-specific or SirT3 N-terminal-specific antibodies and Mitotracker. (B) Immunofluorescence studies of 293F cells transfected with SirT-HA and then treated with LMB as indicated. Cells were stained with antibodies against the HA tag.
Figure 5.
Figure 5.
SirT3 is expelled from the nucleus upon cellular stress. Immunofluorescence experiment performed in HeLa S3 cells as a function of UV or Etoposide treatment using antibodies specific to the N terminus of hSirT3 and Mitotracker.
Figure 6.
Figure 6.
Full-length endogenous SirT3 is expelled from the nucleus upon SirT3 overexpression. (A) Immunofluoresence experiment showing the localization of HA-tagged SirT3 as a function of induction time with tetracycline in a stable cell line, using antibody specific to the HA tag. (B) Western blot using antibody specific to the SirT3 C terminus and nuclear or mitochondrial fractions derived from either 293F cells or from a 293F stable cell line expressing tetracycline-inducible HA-tagged SirT3 after treatment with tetracycline overnight. Arrows on the right indicate both tagged and endogenous SirT3. Large arrows on the left indicate the sizes of endogenous SirT3. The antibodies used for Western analysis are indicated on the left.

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