We characterized human SirT1, one of the human homologs of the budding yeast Sir2p, an NAD+-dependent histone deacetylase involved in establishing repressive chromatin and increased life span. SirT1 deacetylates histone polypeptides with a preference for histone H4 lysine 16 (H4-K16Ac) and H3 lysine 9 (H3-K9Ac) in vitro. RNAi-mediated decreased expression of SirT1 in human cells causes hyperacetylation of H4-K16 and H3-K9 in vivo. SirT1 interacts with and deacetylates histone H1 at lysine 26. Using an inducible system directing expression of SirT1 fused to the Gal4-DNA binding domain and a Gal4-reporter integrated in euchromatin, Gal4-SirT1 expression resulted in the deacetylation of H4-K16 and H3-K9, recruitment of H1 within the promoter vicinity, drastically reduced reporter expression, and loss of H3-K79 methylation, a mark restricting silenced chromatin. We propose a model for SirT1-mediated heterochromatin formation that includes deacetylation of histone tails, recruitment and deacetylation of histone H1, and spreading of hypomethylated H3-K79 with resultant silencing.

The mammalian cytoplasmic protein SirT2 is a member of the Sir2 family of NAD + -dependent protein deacetylases involved in caloric restriction-dependent life span extension. We found that SirT2 and its yeast counterpart Hst2 have a strong preference for histone H4K16Ac in their deacetylation activity in vitro and in vivo. We have pinpointed the decrease in global levels of H4K16Ac during the mammalian cell cycle to the G 2 /M transition that coincides with SirT2 localization on chromatin. Mouse embryonic fibroblasts (MEFs) deficient for SirT2 show higher levels of H4K16Ac in mitosis, in contrast to the normal levels exhibited by SirT1-deficient MEFs. The enzymatic conversion of H4K16Ac to its deacetylated form may be pivotal to the formation of condensed chromatin. Thus, SirT2 is a major contributor to this enzymatic conversion at the time in the cell's life cycle when condensed chromatin must be generated anew. Yeast Hst2 belongs to the Sir2 family of NAD + -dependent protein deacetylases. This family is defined by the presence of an ∼200-amino-acid sequence responsible for the NAD + -dependent deacetylase activity, also found in a wide variety of proteins from yeast to humans. Sir2 is required for the maintenance of silenced chromatin at the mating type loci, telomeres, and rDNA in yeast (Imai et al. 2000;Landry et al. 2000), and is associated with life span extension in both yeast and worms (Kennedy et al. 1997;Tissenbaum and Guarente 2001). The yeast Sir2 family includes four homologs termed Hst1-4, whose functions are still unclear, with the exception of Hst1, which regulates the expression of middle sporulation genes (Xie et al. 1999). Hst2p localizes to the cytoplasm and can affect nuclear silencing by an unknown mechanism (Perrod et al. 2001). Additionally, Hst2p is involved in caloric restriction (CR) dependent life span extension by a Sir2-independent mechanism (Lamming et al. 2005).Among the seven Sir2 mammalian homologs (SirT1-7), SirT2 is the Hst2p counterpart and also localizes to the cytoplasm (Perrod et al. 2001). SirT2 seems to be overexpressed during mitosis, affecting mitotic exit (Dryden et al. 2003), and has been shown to deacetylate ␣-tubulin (North et al. 2003). However, since tubulin does not seem to be acetylated in yeast (Polevoda and Sherman 2002), the functional conservation of Hst2 (from yeast to human) is intriguing and strongly suggests another, more general function. Here we report that the general cytoplasmic localization of SirT2 is singularly exceptional during the G 2 /M phase when SirT2 is now present in the nucleus on chromatin. This correlates with a global decrease in the levels of acetylated H4K16 in the G 2 /M phase of the cell cycle. Cells deficient in SirT2 reflect this association, exhibiting increased levels as well as mislocalization of H4K16Ac and aberrancies in the integrity of their S phase. Results and Discussion SirT2 and Hst2p are NAD + -dependent histone deacetylases with preference for H4K16Ac in vitro and in vivoThe purification of human SirT2 rendered a h...

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 ∼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. SirT3 is a member of the Sir2 family of NAD + -dependent protein deacetylases (Frye 1999). This family of proteins is implicated in chromatin structure, transcriptional silencing, and aging in organisms ranging from yeast to humans. It has been proposed that human sirtuins generally have nonhistone substrates. Yet the SirT proteins (especially SirT1, SirT2, and SirT3) are highly related to the yeast Sir2 protein, a dedicated histone deacetylase with specificity for Lys 16 of histone H4 (H4-K16), a residue important in attaining a repressed chromatin state in yeast upon its deacetylation (Suka et al. 2002;Shogren-Knaak et al. 2006). Recent speculations suggest a function for SirT1 (and other SirTs) in caloric restriction and life span (Haigis and Guarente 2006). SirT1 initially was found to deacetylate the tumor suppressor p53 (Luo et al. 2001;Vaziri et al. 2001;Langley et al. 2002), as well as other transcription factors (Brunet et al. 2004;Giannakou and Partridge 2004;Haigis and Guarente 2006). However, SirT1 was later found to function in the formation of facultative heterochromatin through its deacetylation of core histones, specifically acetyl-Lys 16 of histone H4 (H4-K16ac) (Vaquero et al. 2004). SirT1 also interacts with histone H1 and deacetylates acetylLys 26 (Vaquero et al. 2004). The ability of sirtuins to deacetylate H4-K16ac is not restricted to SirT1, as SirT2 has been shown to deacetylate H4-K16ac during mitosis (Vaquero et al. 2006).SirT3 is the only Sirtuin with an apparent direct link to extended life span in humans. Mutations in an enhancer region of the SirT3 gene that potentially up-regulate its expression were found at a high frequency in long-lived individuals, suggesting that high expression of SirT3 can be an important marker in life extension (Bellizzi et al. 2005). Overexpression of murine SirT3 also has been shown to increase the expression of genes involved in mitochondrial biogenesis and metabolism in brown fat cells, linking SirT3 concentration to the regulation of nuclear gene expression (Shi et al. 2005). Howe...

Distinct histone lysine methylation marks are involved in transcriptional repression linked to the formation and maintenance of facultative heterochromatin, although the underlying mechanisms remain unclear. We demonstrate that the malignant-brain-tumor (MBT) protein L3MBTL1 is in a complex with core histones, histone H1b, HP1gamma, and Rb. The MBT domain is structurally related to protein domains that directly bind methylated histone residues. Consistent with this, we found that the L3MBTL1 MBT domains compact nucleosomal arrays dependent on mono- and dimethylation of histone H4 lysine 20 and of histone H1b lysine 26. The MBT domains bind at least two nucleosomes simultaneously, linking repression of transcription to recognition of different histone marks by L3MBTL1. Consistently, L3MBTL1 was found to negatively regulate the expression of a subset of genes regulated by E2F, a factor that interacts with Rb.

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. As such, it is an instructive source to understand the molecular basis for changes in chromatin structure that correlate 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. SIRT1 is an NAD+-dependent deacetylase that targets histone H4 at lysine 16 (refs 3 and 4), and through an unknown 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 independently 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 localization of heterochromatin protein 1. These findings demonstrate a functional link between the heterochromatin-related histone methyltransferase SUV39H1 and the histone deacetylase SIRT1.