LETTERS SIRT1 regulates the histone methyl-transferase SUV39H1 during heterochromatin formation Alejandro Vaquero 1 {, Michael Scher 1,3 , Hediye Erdjument-Bromage 4 , Paul Tempst 4 , Lourdes Serrano 2 & Danny Reinberg 1,3 In contrast to stably repressive, constitutive heterochromatin and stably active, euchromatin, facultative heterochromatin has the capacity to alternate between repressive and activated states of transcription 1 . As such, it is an instructive source to understand the molecular basis for changes in chromatin structure that corre- late with transcriptional status. Sirtuin 1 (SIRT1) and suppressor of variegation 3–9 homologue 1 (SUV39H1) are amongst the enzymes responsible for chromatin modulations associated with facultative heterochromatin formation. SUV39H1 is the principal enzyme responsible for the accumulation of histone H3 containing a tri- methyl group at its lysine 9 position (H3K9me3) in regions of heterochromatin 2 . SIRT1 is an NAD 1 -dependent deacetylase that targets histone H4 at lysine 16 (refs 3 and 4), and through an un- known mechanism facilitates increased levels of H3K9me3 (ref. 3). Here we show that the mammalian histone methyltransferase SUV39H1 is itself targeted by the histone deacetylase SIRT1 and that SUV39H1 activity is regulated by acetylation at lysine residue 266 in its catalytic SET domain. SIRT1 interacts directly with, recruits and deacetylates SUV39H1, and these activities indepen- dently contribute to elevated levels of SUV39H1 activity resulting in increased levels of the H3K9me3 modification. Loss of SIRT1 greatly affects SUV39H1-dependent H3K9me3 and impairs locali- zation of heterochromatin protein 1. These findings demonstrate a functional link between the heterochromatin-related histone methyltransferase SUV39H1 and the histone deacetylase SIRT1. SIRT1 is a member of the Sir2 family of NAD 1 -dependent histone deacetylases 3,4 and promotes heterochromatin formation through the coordination of several events 3 . SIRT1 deacetylates histones (H4K16 and H3K9), recruits histone H1b, and promotes the loss of a mark associated with transcriptionally active chromatin, H3K79me2, and the establishment of marks associated with repressed chromatin such as H3K9me3 and H4K20me1 (ref. 3). How SIRT1 affects the levels of histone modifications other than deacetylation remains unclear. We first tested whether SIRT1 might recruit an enzyme respon- sible for H3K9me3, during purification of Flag-tagged SIRT1 expressed in human 293 cells. Indeed, immunoprecipitated SIRT1 contained histone H3 lysine methyltransferase (HKMT) activity (Fig. 1a, lanes 5–7), the levels of which were greatly decreased in a similarly purified but catalytically inactive form of SIRT1 (lanes 8–10) 3 . This HKMT activity was specific for H3K9 (Fig. 1b). Histone octamers reconstituted with recombinant histones contain- ing either wild-type or mutant H3, in which Lys 27 was replaced with Ala (K27A), exhibited similar levels of methylation. In contrast, mutant H3, in which Lys 9 was replaced with Ala (K9A), was an inappropriate substrate. Two candidates for this specific HKMT were SUV39H1 and the euchromatic histone-lysine N-methyltransferase 2 G9A (also known as EHMT2), given their specificity for H3K9me formation 5 . Inter- action between SIRT1 and G9A was undetectable (not shown). However, when 293 cells were co-transfected with Flag–SIRT1 and a Myc-tagged version of the major cellular activity for H3K9me3 (SUV39H1), the presence of either resulted in the co-immunoprecipitation of the other in pull-down experiments (Fig. 1c, d). The interaction between SUV39H1 and SIRT1 was specific, because the related NAD 1 - dependent deacetylase SIRT2 failed to pull-down SUV39H1 (Fig. 1c). This is consistent with the functional link found between the homologues of SUV39H1 and SIRT1 in Schizosaccharomyces pombe (Clr4 and Sir2, respectively) 6 . Chromatin immunoprecipita- tion experiments were then performed on cells containing an indu- cible SIRT1 fused to the yeast Gal4 DNA-binding protein domains 3 that were transfected with Myc-tagged SUV39H1 (Fig. 1e). Myc– SUV39H1 was present at the integrated thymidine kinase promoter containing Gal4 sites in these cells only on induction of Gal4–SIRT1 with which it colocalized, demonstrating their interaction in vivo. This correlated with a significant loss in H3K9ac levels and enrichment in H3K9me3 levels at the promoter. Co-immunoprecipitation experiments were performed to identify the interaction domains. A point mutation that renders SIRT1 cataly- tically inactive caused deficiency in interaction (Fig. 1f). The amino terminus of SIRT1 that interacts with histone H1b (ref. 3) also interacts with SUV39H1 (see below). Removal of either the SET domain or the chromo domain of SUV39H1 decreased its interaction with SIRT1, whereas removal of the first 89 N-terminal residues including the chromo domain eliminated interaction (Supplemen- tary Fig. 1a). The chromo domain alone exhibited detectable inter- action (Supplementary Fig. 1b), indicating that the N-terminal domain (1–43) and the chromo domain (44–88) of SUV39H1 were both required for interaction with SIRT1. This is similar to the case reported for SUV39H1 interaction with the polycomb human pro- tein PC2 (ref. 7). To understand how SIRT1 affects global levels of H3K9me3, we tested for its affect on the levels of SUV39H1-specific activity using methyltransferase assays performed in vitro with histone octamers as substrates. Increasing amounts of recombinant SIRT1 led to elevated levels of SUV39H1-mediated methylation of histone H3 (Fig. 2a, compare lane 2 with lanes 4–6), whereas recombinant SIRT2 (Fig. 2a, lanes 8–10) and bovine serum albumin (not shown) were ineffectual. The increased SUV39H1 activity exhibited the expected substrate activity, being unable to methylate recombinant octamers containing mutant H3K9A or mononucleosomes (Supplementary Fig. 2). The SIRT1-mediated stimulation of SUV39H1 activity was 1 Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Jersey 08854, USA. 2 Department of Genetics, Human Genetics Institute, Rutgers University, 145 Bevier Road, Piscataway, New Jersey 08854, USA. 3 Department of Biochemistry, NYU-Medical School, 522 First Avenue, New York, New York 10016, USA. 4 Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA. {Present address: ICREA and IBMB-CSIC/IRB, Parc Cientific de Barcelona, Josep Samitier 1–5, 08028 Barcelona, Spain. Vol 450 | 15 November 2007 | doi:10.1038/nature06268 440 Nature ©2007 Publishing Group