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. 1999 Oct 26;96(22):12356-61.
doi: 10.1073/pnas.96.22.12356.

Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity

A A Carmen  1 , P R Griffin, J R Calaycay, S E Rundlett, Y Suka, M Grunstein
Affiliations

Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity

A A Carmen et al. Proc Natl Acad Sci U S A. .

Abstract

Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function. In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and Escherichia coli, and has intrinsic activity when produced in the bacterium. Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification. We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity in vitro for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11. We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity.

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Figures

Figure 1
Figure 1
HOS3 disruption results in histone H4 hyperacetylation. (A) Relative acetylation levels of histone H4 isolated from YDS2 (WT) (lanes 1 and 2) and SRYH3 (hos3Δ) yeast (lanes 3 and 4) were determined by electrophoresing 1 μg (lanes 1 and 3) or 3 μg (lanes 2 and 4) of histones on SDS-polyacrylamide gel. After transfer to PVDF membranes (Millipore) (Experimental Procedures), blots were probed with antibodies specific for H4 acetyl-lysine 5, 8, 12, or 16 (antibodies to be described elsewhere). Protein load was visualized by staining the membrane with amido black. Antibody binding then was detected by using ECL (Amersham) and anti-rabbit Ig HRP-linked antibody (Amersham) as described in Experimental Procedures. (B) Quantitation of increased acetylation levels of histone H4 sites observed for histones isolated from hos3Δ versus wild-type yeast. Western blots in A were reacted with 35S-labeled anti-rabbit Ig, detected by PhosphorImager, and analyzed by using imagequant software as in Experimental Procedures. Quantitation is given as fold increase in acetylation observed for the histones isolated from the SRYH3 strain (hos3Δ) vs. YDS2 (wt).
Figure 2
Figure 2
HOS3 is present in a dimer-sized complex in both yeast and E. coli. (A) E. coli-expressed HOS3 activity containing the CBP-HOS3 chromatographs on Superdex-200 at an apparent molecular mass of 190 kDa. CBP-HOS3 was purified from E. coli by affinity chromatography on calmodulin-Sepharose as described in Experimental Procedures. Twenty-five microliters of each 0.5-ml fraction were used in a 1-hr incubation with 25,000 cpm total of [3H]acetyl-Hela histones. Activity ([3H]acetic acid released) is shown as a function of fraction number. HOS3 activity from yeast strains containing HOS3(2μ) or wild-type HOS3 was purified from nuclear extracts on DEAE-Sepharose, S-Sepharose, Mono S, and Superdex 200 as described in Experimental Procedures. Protein molecular mass standards are indicated above the chromatograph as 669 kDa (thyroglobulin), 443 kDa (apoferritin), 200 kDa (β-amylase), 150 kDa (alcohol dehydrogenase), and 66 kDa (bovine serum albumin). (B) Electrophoretic (SDS/PAGE) and immunological analysis of column fractions in A. Coomassie staining of E. coli CBP-HOS3 reveals the presence of an 84-kDa protein that comigrates with deacetylase activity. This protein reacts with anti-HOS3 antibody as detected by Western blotting (ECL, Amersham). Similar electrophoresis and Western analysis of the column fractions (A) of HOS3 deacetylase from HOS3(2μ) and HOS3 (wt) yeast indicates a 79-kDa protein that comigrates with the peak yeast activities. (C) Deacetylase activities from wild-type (YDS2), hos3Δ (SRYH3), or HOS3(2μ) (SRYH3gal) strains were partially purified on DEAE-Sepharose columns and then directly chromatographed by Superdex 200 gel filtration (1.0 × 46 cm column, 0.5-ml fractions). (D) Western blotting of Superdex 200 fractions from HOS3(2μ) (SRYH3gal), HOS3 wild type (YDS2), and hos3Δ (SRYH3) yeast.
Figure 3
Figure 3
HOS3 is much less sensitive to the deacetylase inhibitor TSA than the related deacetylases HDA1 or RPD3. Activities containing HOS3, HDA1, and RPD3 were obtained by immunoprecipitation from yeast nuclear extracts as described previously (1). HOS3 from E. coli was obtained from the peak fraction in Fig. 2A (fraction 28). [3H]acetyl-Hela histones (50,000 cpm) were incubated with each of the deacetylases for 30 min and assayed as described previously (1). Inhibition of deacetylase activity at various concentrations of inhibitor was calculated as a ratio of enzyme reactions containing TSA vs. no TSA. Maximal conversion of the [3H]acetyl-Hela histones to the release of free acetate was maintained below 2% to reduce the effect of substrate limitation on the apparent inhibition by TSA.
Figure 4
Figure 4
Treatment of acetylated histone peptides with recombinant HOS3 (CBP-HOS3) results in site-specific deacetylation patterns. Fully acetylated peptides for each histone N-terminal tail were synthesized as described in Experimental Procedures. These were treated with recombinant CBP-HOS3 (peak fraction 28, Fig. 2A) for 5, 30, and 240 min with 1.2 μg of enzyme and analyzed by cHPLC-MS/MS as in Experimental Procedures. When applicable, the approximate amount of the detected ion compared with the total peptide is indicated for each site. When the value fell below the threshold of detection (≈5%), nd (not detected) is indicated. If the ion for the deacetylated species could be detected but a value could not be estimated accurately, present is indicated.
Figure 5
Figure 5
MALDI-TOF-MS analysis of H4 and H3 peptides. Deacetylation of fully acetylated H4 (A) and H3 (B) peptides reveals that at least one additional site of deacetylation is present in each peptide as compared with cHPLC-MS/MS analysis. The MALDI-TOF-MS data was calibrated externally using standard peptides. Mass of the deacetylated peptides is as indicated. The presence of mono-, di-, and tri-deacetylated species is indicated for each peptide. Percentage of peptide in each acetylated state is calculated as a ratio of each acetylated form compared with the total input peptide for each reaction.
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