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

https://doi.org/10.1016/j.mad.2010.01.006Get rights and content

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.

Introduction

Saccharomyces cerevisiae Sir2 is the founding member of an evolutionarily conserved family of sirtuin proteins present in organisms ranging from bacteria to humans. As an NAD+-dependent histone deacetylase, Sir2 deacetylates lysines in the amino terminal ‘tails’ of histones H3 and H4, as well as on the globular core of histone H3 (Imai et al., 2000, Landry et al., 2000, Smith et al., 2000, Xu et al., 2007). In this context, Sir2 modulates the assembly and spreading of heterochromatin at telomeres, silent mating type loci, and ribosomal DNA repeats. In turn, these activities of Sir2 impact on genomic stability, gene silencing, and yeast lifespan (Denu, 2003).

In mammalian genomes, there are seven SIR2 family members, dubbed SIRT1-SIRT7 (Frye, 1999, Frye, 2000). SIRT6 has recently emerged as a critical regulator of transcription, genome stability, telomere integrity, DNA repair, and metabolic homeostasis. The first clues to the in vivo function of SIRT6 came from analysis of SIRT6 deficiency in mice. SIRT6 knockout mouse cells exhibit DNA damage hypersensitivity and genomic instability, and SIRT6-deficient mice develop a striking degenerative and metabolic phenotype with symptoms suggestive of premature aging (Mostoslavsky et al., 2006). SIRT6 was also found to fractionate with chromatin biochemically, suggesting that it might have a chromatin-regulatory function (Mostoslavsky et al., 2006).

However, direct evidence for a physiologic enzymatic activity of SIRT6 at chromatin was lacking. Initial studies did not detect NAD+-dependent deacetylase activity for SIRT6 on several histone substrates. Instead, SIRT6 was observed to promote ADP-ribosylation, an alternative NAD+-dependent reaction observed for some sirtuins (Liszt et al., 2005, Mostoslavsky et al., 2006), but the physiological importance of this activity remains to be determined.

Recently, we discovered that SIRT6 is indeed an NAD+-dependent histone deacetylase, but because it is highly site-specific, this activity had been difficult to observe. We showed that SIRT6 has specificity for deacetylating lysine 9 of histone H3 (H3K9Ac),1 and we identified functions for this activity in maintaining telomere integrity (Michishita et al., 2008) and in negatively regulating aging-associated NF-κB-dependent gene expression programs (Kawahara et al., 2009). We also showed that SIRT6 is required for efficient DNA double-strand break repair in the context of chromatin, though the specific role of histone deacetylation by SIRT6 in this context remains to be clarified (McCord et al., 2009). More recently, we and others (Michishita et al., 2009, Yang et al., 2009) have shown that SIRT6 has a second substrate, lysine 56 of histone H3 (H3K56), and our study (Michishita et al., 2009) demonstrated that SIRT6 is critical for maintaining dynamic changes in H3K56 acetylation levels at telomeres over the cell cycle.

Despite these important cellular and physiologic functions, the basic molecular mechanisms of SIRT6 enzymatic activity – including the mechanistic and regulatory roles of specific SIRT6 sequences – remain poorly understood. Sirtuin proteins share a phylogenetically conserved central ‘sirtuin domain,’ generally thought to comprise an enzymatic core. Eukaryotic genomes typically encode multiple Sir2 family members, and these proteins contain variable N- and C-terminal extensions flanking the ∼270-amino acid sirtuin core domain (Frye, 1999, Frye, 2000). S. cerevisiae harbors several Sir2 family members, and studies of yeast Sir2 proteins have shown that, in addition to residues within the central core domain, regions outside the catalytic core play important roles in acetyl-lysine and NAD+ binding (Zhao et al., 2003), sub-nuclear distribution (Cockell et al., 2000), and interaction with cellular binding partners (Cuperus et al., 2000). In mammals, nuclear localization signals, nuclear export signals, and mitochondrial localization signals have been identified on the N-terminal extensions of SIRT1 (Tanno et al., 2007), SIRT2 (North and Verdin, 2007a), and SIRT3 and SIRT4 (Haigis et al., 2006, Onyango et al., 2002, Scher et al., 2007, Schwer et al., 2002), respectively, but evidence for other functions of the N- and C-terminal extensions of the mammalian SIRTs is relatively limited, and in the case of SIRT6, completely lacking.

Here, we have conducted a functional analysis of the N- and C-terminal extensions of SIRT6, as well as the role of a conserved catalytic residue in the central sirtuin domain of SIRT6. We show that the C-terminal extension (CTE) of SIRT6 is critical for proper sub-cellular targeting and that the N-terminal extension (NTE) contributes to efficient chromatin association and intrinsic catalytic activity. Surprisingly, a catalytically inactive SIRT6 point mutant is defective in chromatin localization, providing the first example in which the catalytic activity of a mammalian sirtuin is required for the protein's stable association with chromatin.

Section snippets

Constructs

To generate vectors for transient transfection, a FLAG tag was inserted downstream of EGFP in pEGFP-N2 (Clontech, to generate pEGFP-N2-FLAG) or was inserted in place of EGFP in pEGFP-N2 (to generate pN2-CT-FLAG). Full-length SIRT6 and SIRT6 deletion mutants were generated by PCR and cloned into these modified pEGFP-N2 vectors as EcoRI-BamHI cassettes, generating C-terminally tagged fusion proteins. For stable transduction and bacterial expression, PCR-generated SIRT6 deletion mutants were

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 (Fig. 1). 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

Discussion

SIRT6 is a critical regulator of transcription, genome stability, telomere integrity, and metabolism, but the enzymatic and regulatory roles of specific domains of SIRT6 have not been clearly defined. Here, we describe new functions of the N- and C-terminal extensions of SIRT6 that contribute to sub-cellular localization, catalytic activity, and chromatin binding. Further, we have uncovered an unexpected effect of mutating a highly conserved catalytic residue on the ability of SIRT6 to

Acknowledgements

We thank Steven Artandi for modified pWZL-3FLAG and pGEX-6P3 vectors, Jeff Glenn for use of the Nikon Eclipse TE300 inverted fluorescence microscope, and Or Gozani and members of the Gozani and Chua labs for helpful discussions. This work was supported by grants to K.F.C. from the NIH (R01AG028867 and K08AG028961) and Department of Veterans Affairs Merit Review. R.I.T. is funded by a National Defense Science and Engineering Graduate Fellowship, a National Science Foundation Graduate Research

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