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 G2/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.

Figure 1.

SirT2 and Hst2p are NAD⁺-dependent histone deacetylases with a preference for H4K16Ac in vitro and in vivo. (A) TAU gel analysis of hyperacetylated core histones treated with the indicated amounts of SirT2 in the presence and absence of NAD⁺. Levels of acetylation for each core histone are indicated. (B) Hyperacetylated core histones were incubated with the indicated amounts of SirT2 in the presence and absence of NAD⁺ followed by Western blot using antibodies against specific acetylated residues in H3 and H4. (C) Time-course experiment of core histone deacetylation by SirT2 in the presence of NAD⁺ analyzed as in B. Quantifications were determined as indicated previously. (D) RNAi experiments against SirT2 in 293 cells. Whole-cell extracts were probed for the presence of SirT2 and actin by Western blot. Histones were extracted with hydrochloric acid and tested for H4K16Ac levels by Western blot. Levels of total histones were visualized with Coomassie blue staining. (E) Western blot of a time-course experiment for deacetylation performed as in C but with purified Hst2p. (F) Graph of the quantifications of E determined as in C. (G, top) Levels of H4K16Ac and total histones purified from isogenic S. cerevisiae strains (wild type, W303-1b; hst2Δ, YRH45; sir2Δ, YRH15; hst1Δ LNYY315) analyzed by Western blot. The bottom panel shows the histones from the various mutants, stained with Coomassie. The asterisk indicates an H3 breakdown product commonly found in histones purified from yeast (Edmondson et al. 1996).

Figure 2.

H4K16Ac levels peak at S phase and drop dramatically in the G₂/M transition. (A) Mammalian fibroblasts were pulsed with BrdU, stained with H4K16Ac antibodies as indicated, and analyzed by immunofluorescence for colocalization of BrdU and H4K16Ac. Two different samples are shown (Exp1 and Exp2). (B) Same cells as in A were pulsed with BrdU, incubated with H4K16Ac antibody and 7AAD (DNA content marker), and analyzed by FACS for the levels of H4K16Ac at each stage of the cell cycle. (Upper panel) The distribution of the cells through the cell cycle is represented in relation to BrdU incorporation (Sasaki et al. 1986). Distribution of H4K16Ac levels in cells at each stage of the cell cycle is shown graphically and numerically, with 100% representing the levels of H4K16Ac in G₁ phase. (C) Immunofluorescence as in A, but in this case with the mitotic marker H3S28P and H4K16Ac.

Figure 3.

SirT2 is present in the cytoplasm during most of the cell cycle except in the G₂/M transition and in mitosis, where it is found in the nucleus and in contact with chromatin. (A) Immunofluorescence performed as in Figure 2, showing levels of SirT2 and the mitotic marker H3S28P. Samples were derived from three independent experiments. Exp3 shows a cell in metaphase. (B) GFP-SirT2, a stable cell line derived from mammalian 293 cells, was followed during the cell cycle with a live cell microscope system for GFP localization. G₂/M transition and M were identified as previously described and by timing with respect to cell division (A. Vaquero, M.B. Scher, and D. Reinberg, unpubl.; data not shown). The nucleus of the cell is indicated by an arrow during the end of G₂ and beginning of G₂/M transition, when the nucleus is still visually detectable.

Figure 4.

Loss of SirT2 in cells derived from knockout mice produces higher levels of H4K16Ac in mitosis, a delay in S-phase entry, and abnormal levels of H4K16Ac in heterochromatic foci in S phase. (A) Immunofluorescence of primary MEFs derived from SirT1 wild-type or knockout (−/−) mice (Cheng et al. 2003) and SirT2 wild-type or knockout (−/−) mice (H.-L. Cheng and F.W. Alt, unpubl.). Cells were stained with antibodies against H4K16Ac and H3S28P. Two different independent MEFs for SirT2^−/− are shown [(1) and (2)]. (B) FACS analysis of the cell cycle distribution of MEFs wild type (+/+), heterozygote (+/−), and knockout (−/−) in SirT2 stained using propidium iodide. Average values of four different experiments are shown. Proportional levels of G₁, S, and G₂/M are indicated in the table. (C) Immunofluorescence of SirT2 wild-type and −/− cells tested in A using H4K16Ac antibodies. Two different cell lines [(1) and (2)] were used in the experiment. (D) Model showing two different levels of regulation of H4K16Ac by SirTs: SirT1 functions at a local level in the context of genes or loci whereas SirT2 functions at a global level dependent on the G₂/M transition of the cell cycle.