Functional dissection of SIRT6: identification of domains that regulate histone deacetylase activity and chromatin localization

Abstract

The mammalian sirtuin SIRT6 is a site-specific histone deacetylase that regulates chromatin structure. SIRT6 is implicated in fundamental biological processes in aging, including maintaining telomere integrity, fine-tuning aging-associated gene expression programs, preventing genomic instability, and maintaining metabolic homeostasis. Despite these important functions, the basic molecular determinants of SIRT6 enzymatic function--including the mechanistic and regulatory roles of specific domains of SIRT6--are not well understood. Sirtuin proteins consist of a conserved central 'sirtuin domain'--thought to comprise an enzymatic core--flanked by variable N- and C-terminal extensions. Here, we report the identification of novel functions for the N- and C-terminal domains of the human SIRT6 protein. We show that the C-terminal extension (CTE) of SIRT6 contributes to proper nuclear localization but is dispensable for enzymatic activity. In contrast, the N-terminal extension (NTE) of SIRT6 is critical for chromatin association and intrinsic catalytic activity. Surprisingly, mutation of a conserved catalytic histidine residue in the core sirtuin domain not only abrogates SIRT6 enzymatic activity but also leads to impaired chromatin association in cells. Together, our observations define important biochemical and cellular roles of specific SIRT6 domains, and provide mechanistic insight into the potential role of these domains as targets for physiologic and pharmacologic modulation.

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 (••••) in the C terminus of SIRT6. ΔN, ΔC, and ‘core’ mutants are missing the N-terminal extension (NTE), C-terminal extension (CTE), or both the NTE and CTE, respectively. See text for further details.

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×).

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 DNA were adjusted to achieve the expression levels shown in (B). Results are representative of at least four (A) or two (B) independent experiments. C–D. Western analysis of H3K9 and H3K56 acetylation levels in in vitro NAD⁺-dependent deacetylation reactions, using purified GST-SIRT6 deletion and point mutants and purified calf thymus histone H3. (NAD⁺ was omitted in lanes 3 and 2 of (C) and (D), respectively.) In lane 5, excess GST protein was added to control for the GST degradation product in the ΔN2 protein sample (lane 7). Note the impaired deacetylation for the HY mutant and N-terminally deleted proteins (ΔN’, ΔN, ΔN2, ΔN3), but not the isolated C-terminal deletions (ΔC, ΔC2, ΔC3, ΔC4). We were unable to purify sufficient quantities of the core-1 and core-2 mutants to test their deacetylase activity in vitro. β-tubulin and total histone H3 levels are shown as controls.

Figure 4

Impaired chromatin association of SIRT6 deletion and point mutants. A. Coomassie stain showing co-immunoprecipitation of core histones with wild-type (WT) but not catalytically inactive (H133Y) SIRT6 protein, from 293T cells transiently transfected with FLAG-tagged SIRT6 proteins or empty vector control (-). HC and LC indicate antibody heavy and light chains present in the IPs. B. Western analysis showing relative co-immunoprecipitation of histone H3 with the indicated mutant SIRT6 proteins compared to WT SIRT6, as in (A). Results are representative of at least 3 independent experiments. C. Schematic of cellular fractionation protocol, based on (Mendez and Stillman, 2000). D. Western analysis showing the relative chromatin association of endogenous SIRT6, or of FLAG-tagged wild-type or mutant SIRT6 proteins stably expressed in U2OS cells, using the biochemical fractionation outlined in (C). β-tubulin and histone H3 westerns provide controls for the cytosolic and chromatin-enriched fractions, respectively. E. In vitro gel-shift assay showing similar nucleosome binding efficiency of the wild-type and catalytically inactive H133Y mutant SIRT6. Top: Coomassie stain of the purified GST-SIRT6 proteins used for the gel-shift assay. Bottom: migration of free or SIRT6-bound mononucleosomes (MN) in nucleosome binding reactions, fractionated on a non-denaturing gel and visualized by ethidium bromide staining of nucleosomal DNA. Incubation with GST alone is shown as a negative control. F. In vitro gel-shift assay as in (E) showing impaired nucleosome binding of N- and C- terminal SIRT6 deletion mutants compared to WT SIRT6. In lane 3, excess GST protein was added to control for the GST degradation product in the ΔN protein sample (lane 4). The slight difference in the nucleosome shift may be due to imperfections in the gel, as this was not observed in other experiments (see Figure S1).