3.1. The C terminus of SIRT6 is essential for proper nuclear localization
To study potential functions of the N- and C-terminal extensions of human SIRT6, we generated a panel of mutants with progressive deletions from the N and C termini (). We also generated mutants consisting of the N-terminal extension alone (NTE), the C-terminal extension alone (CTE), or full-length SIRT6 lacking the catalytic core (Δcore). For comparison, we included the mutant SIRT6-H133Y protein, which harbors a mutation in a highly conserved histidine within the core sirtuin domain of SIRT6; the corresponding mutation abrogates catalytic activity in all sirtuins tested thus far.
Figure 1 Schematic of SIRT6 deletion and point mutants used in this study. WT, wild-type SIRT6; H133Y, full-length SIRT6 with a catalytic mutation (histidine to tyrosine) at residue 133 (*); mutNLS, K/R-to-A mutations at four residues (••••) (more ...)
As expected, transiently transfected GFP-tagged wild-type SIRT6 was exclusively nuclear in live 293T cells, as was the catalytically inactive H133Y mutant, whereas the unfused GFP control was detected throughout the nucleus and cytoplasm (). Deleting the NTE of SIRT6 (ΔN) did not significantly disrupt the nuclear localization pattern seen for wild-type SIRT6. Conversely, progressive deletion of the CTE of SIRT6 (ΔC2 and ΔC) resulted in a partial or dramatic mislocalization of SIRT6 from the nucleus to the cytoplasm (). A similar mislocalization of C-terminally truncated SIRT6 proteins was also observed when FLAG-tagged SIRT6 proteins were analyzed by indirect immunofluorescence (data not shown). Intriguingly, the core domain alone (lacking both the N- and C-terminal domains) had a more profound mislocalization than the ΔC mutants (), appearing more substantially excluded from the nucleus. These observations suggest that although the NTE alone is dispensable for proper nuclear localization, it may have a partially synergistic function with the CTE in maintaining proper SIRT6 nuclear localization.
Figure 2 Sub-cellular localization of wild-type and mutant SIRT6 proteins. Epifluorescence images of live 293T cells transiently transfected with the indicated EGFP-FLAG-tagged SIRT6 deletion and point mutants. Nuclei are co-stained with Hoechst 33342 (40×). (more ...)
Inspection of the C-terminal sequence of SIRT6 identified a seven-amino acid sequence starting at residue 345—345PKRVKAK351—that resembles a canonical nuclear localization signal (NLS). We mutated the four basic residues (K346, R347, K349, K351) of this putative NLS to alanine in the context of the full-length protein (to generate SIRT6-mutNLS) and examined its localization. GFP-tagged SIRT6-mutNLS was partially mislocalized to the cytoplasm (), and this pattern was indistinguishable from that generated by deleting ~80 amino acids of the C terminus (ΔC2). These data suggest that the 345PKRVKAK351 sequence at the C terminus of SIRT6 is an NLS essential for proper nuclear localization.
Finally, we analyzed the cellular localization of the isolated CTE and NTE sequences of SIRT6 fused to GFP. The CTE (and Δcore) were sufficient to direct nuclear localization of GFP, generating a pattern indistinguishable from that of full-length SIRT6 fused to GFP (). In contrast, the localization pattern of the NTE was indistinguishable from that of GFP alone ().
Together, our results indicate that the CTE of SIRT6 is necessary and sufficient for proper nuclear localization of SIRT6 and identify an NLS at the very C terminus of SIRT6.
3.2. The N terminus of SIRT6 is required for H3K9 and H3K56 deacetylation in cells
SIRT6 is a relatively site-specific histone deacetylase, for which two substrates have so far been identified, lysines 9 and 56 of histone H3 (H3K9 and H3K56). To determine whether the core sirtuin domain of SIRT6 is sufficient for its deacetylase activity in cells or whether the N and C termini contribute to catalytic activity, we transiently overexpressed a panel of FLAG-tagged SIRT6 deletion mutants (see ) and assessed global levels of H3K9 and H3K56 acetylation by western analysis. Similar results were obtained for both SIRT6 substrates. As previously shown (Michishita et al., 2008
; Michishita et al., 2009
; Yang et al., 2009
), overexpression of wild-type SIRT6 reduced global H3K9 and H3K56 acetylation levels (but not several other acetylation marks on H3 and H4 [data not shown]), whereas the catalytically inactive H133Y mutant did not (; lanes 1–3).
Figure 3 Histone deacetylation activity of wild-type and mutant SIRT6 proteins. A–B. Western analysis of H3K9 and H3K56 acetylation levels in 293T cells transiently transfected with FLAG-tagged SIRT6 deletion and point mutants. Quantities of transfected (more ...)
Deleting nearly the entire C terminus of SIRT6 (ΔC) did not abrogate the ability of SIRT6 to reduce H3K9 and H3K56 acetylation in cells (, compare lanes 2 and 5). The efficient deacetylation of H3K9 and H3K56 that was observed upon expression of this protein initially seemed surprising, because this ΔC mutant was predominantly cytoplasmic () and was expressed at lower levels than the exogenous wild-type SIRT6 (). However, in these overexpression experiments, levels of the ΔC protein were substantially higher than endogenous SIRT6, and a considerable fraction of the mutant protein was still present in the nucleus (). In this context, our data suggest that the fraction of the ΔC protein that accesses the nucleus is fully active enzymatically. By deleting fewer amino acids from the C terminus (ΔC2, ΔC3, ΔC4; see ), we were able to achieve better expression (and less severe mislocalization, and data not shown), and these mutants were as proficient as wild-type SIRT6 in reducing global H3K9 and H3K56 acetylation (, lanes 8–10). Together, these data indicate that when SIRT6 is overexpressed in cells, the CTE is not essential for H3K9 or H3K56 deacetylation, providing evidence that this sequence is dispensable for the intrinsic enzymatic activity of SIRT6.
In contrast to the C-terminal deletion mutants, a SIRT6 mutant lacking the NTE (ΔN)— which was appropriately localized to the nucleus ()—was severely compromised in its ability to reduce global H3K9 and H3K56 acetylation (, lane 4). This held true for more conservative deletions of the N terminus as well (data not shown). Although SIRT6-ΔN is expressed at lower levels than wild-type SIRT6 in this experiment, its expression is comparable to that of the ΔC mutant, which is proficient in deacetylating H3K9 and H3K56. Furthermore, wild-type SIRT6 is able to deacetylate H3K9 when expressed at much lower levels than the ΔN mutant (), arguing that the inability of SIRT6-ΔN to deacetylate H3K9 and H3K56 in cells cannot be attributed to lower levels of expression. Similar to the SIRT6-ΔN mutant, the core of SIRT6 (lacking both the N and C termini) lacked catalytic activity in cells (, lanes 6 and 7). This observation is expected, given that the NTE deletion alone abrogates SIRT6 activity, although the more extensive exclusion of this mutant core protein from the nucleus may also contribute to the observed lack of deacetylase activity in cells. Together, our data indicate that the N-terminal extension of SIRT6 is important for its ability to modulate global levels of H3K9 and H3K56 acetylation in cells.
3.3. The N terminus of SIRT6 is essential for the intrinsic catalytic activity of SIRT6
The inability of the SIRT6 N-terminal deletion mutant (SIRT6-ΔN) to reduce cellular H3K9 and H3K56 acetylation levels could be due either to an intrinsic defect in enzymatic activity or to a failure to properly localize to chromatin substrates or interact with critical binding partners. To directly test whether the NTE of SIRT6 is required for the intrinsic deacetylase activity of SIRT6, we purified GST-tagged wild-type SIRT6, the catalytically inactive H133Y SIRT6, and N-terminally deleted SIRT6 from E. coli and assessed their catalytic activity on purified histone H3 in vitro (). As expected, wild-type SIRT6 deacetylated H3K9 and H3K56 (lane 4), whereas the H133Y mutant lacked activity (lane 6). Because all ΔN mutants were partially degraded to GST during purification, we added an excess of GST to the wild-type reaction to control for any effect of the ΔN degradation products. Wild-type SIRT6 still showed robust deacetylase activity in the presence of excess GST (lane 5). In contrast, the ΔN2 mutant appeared to lack activity in vitro (lane 7), similar to the HY mutant, suggesting that the NTE of SIRT6 is required for the intrinsic H3K9 and H3K56 deacetylation activity of SIRT6. More conservative N-terminal deletion mutants also showed impaired catalytic activity in vitro (; see ). As expected from the results of overexpression in cells, the ΔC mutants were proficient in deacetylating H3K9 and H3K56 in vitro (, lanes 8–9). From these data, we conclude that sequences in the NTE are essential for the intrinsic deacetylase activity of SIRT6.
3.4. Catalytic and non-core SIRT6 sequences contribute to cellular chromatin association
We next used several different assays to assess the roles of the NTE and CTE of SIRT6 in the interaction of SIRT6 with chromatin. First, we transiently overexpressed FLAG-tagged wild-type, H133Y, and N- and C-terminally truncated SIRT6 and assayed for the presence of core histones in FLAG immunoprecipitates. As previously shown, wild-type SIRT6 readily associates with the core histones in cells, as detected by Coomassie () and western analysis (); the fact that the four core histones are present in approximately equimolar amounts in the SIRT6 IP argues that this interaction occurs in the context of intact nucleosomes at chromatin (McCord et al., 2009
). Intriguingly, co-immunoprecipitation of the core histones with the catalytically inactive H133Y mutant was dramatically reduced compared to wild-type SIRT6, as observed by both Coomassie stain and western blot for histone H3 (, respectively). This observation suggests that the H133Y mutation not only abrogates the catalytic activity of SIRT6 but also profoundly impacts on the dynamics of SIRT6 association with chromatin in cells.
We next assessed the N- and C-terminal deletion mutants in this chromatin association assay using western analysis for H3, because this method was more sensitive and these mutant proteins were expressed at lower levels than the WT and H133Y SIRT6 proteins. Strikingly, deleting the N terminus of SIRT6 nearly abrogated the ability of SIRT6 to bind histones, an effect even more profound than that observed for the H133Y mutation (). In contrast, deleting the C terminus of SIRT6 had little effect on the interaction with histones in this assay (). Thus, both the NTE and the catalytic activity of SIRT6 appear to be required for its stable association with histones in cells. Consistent with these findings, neither the NTE alone nor the Δcore mutant (containing the NTE and CTE and lacking the core domain; see ) was able to interact with histones in cells (data not shown), suggesting that multiple regions of SIRT6 are required for its interaction with chromatin.
To assay chromatin association using an independent method, we carried out biochemical fractionation of U2OS cells stably expressing the different FLAG-tagged SIRT6 proteins, according to the protocol of Mendez and Stillman (2000)
. Fractionation into soluble cytosolic, soluble nuclear, and chromatin-enriched fractions () revealed that wild-type SIRT6 is largely chromatin-enriched under these conditions, as previously shown (Mostoslavsky et al., 2006
) (). In contrast, both the catalytically inactive H133Y mutant and the ΔN mutant showed a reduction in the relative amount of SIRT6 present in the chromatin-enriched fraction, further supporting the conclusion that both the NTE and the catalytic activity of SIRT6 contribute to its ability to associate with chromatin in cells.
3.5. The NTE and CTE of SIRT6 are important for nucleosome binding
The inability of the H133Y and ΔN mutants to bind chromatin in cells could be due to an intrinsic defect in nucleosome binding, an inability to interact with chromatin-associated binding partners that propagate SIRT6 along chromatin, or another defect in chromatin targeting. To investigate these models, we assessed in vitro
nucleosome binding by incubating the wild-type and mutant GST-SIRT6 proteins with purified HeLa mononucleosomes and measured binding using a gel-shift assay (). As previously shown (McCord et al., 2009
), wild-type SIRT6 binds nucleosomes efficiently, as evidenced by the dramatic shift of nucleosomes to a higher molecular weight. The H133Y mutant induced a similar upward shift of nucleosomes ( and Figure S1
), suggesting that this mutant is largely proficient at binding nucleosomes in vitro
. Although there may be a very subtle difference in the electrophoretic mobility shift for the H133Y mutant compared to wild-type SIRT6, this effect is much less substantial than the defective chromatin association of SIRT6-H133Y observed in cells (). Therefore, the impaired chromatin association of the SIRT6-H133Y mutant protein observed in cells is likely not attributable to an intrinsic defect in nucleosome binding. In contrast, the ΔN mutant was completely impaired in the in vitro
nucleosome binding assay (, lane 4), suggesting that the NTE of SIRT6 is important for direct binding of SIRT6 to nucleosomes. Surprisingly, although the CTE was dispensable for binding histones in an overexpression context in cells (), deleting the CTE of SIRT6 abrogated nucleosome binding in vitro
(, lanes 5 and 6), suggesting that this domain may also impact on the chromatin association of SIRT6 under physiologic conditions.