SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis

Abstract

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...

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