Histone H2B Deacetylation at Lysine 11 Is Required for Yeast Apoptosis Induced by Phosphorylation of H2B at Serine 10  Sung-Hee Ahn, Robert L. Diaz, Michael Grunstein, C. David Allis  Molecular Cell  Volume 24, Issue 2, Pages 211-220 (October 2006) DOI: 10.1016/j.molcel.2006.09.008 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Histone H2B Is Specifically Deacetylated at Lysine 11 in Dying Yeast Cells (A) Primary sequence alignment of the amino-terminal tails of yeast and human H2B. In yeast, lysine 11 (K11) is located adjacent to serine 10 (S10), a previously described apoptotic phosphorylation site (Ahn et al., 2005a). Both K11 and K16 are acetylated in logarithmically growing yeast (Suka et al., 2001). Note that K15, also known to be acetylated in mammalian cells (Thorne et al., 1990), is also adjacent to a mammalian apoptotic H2BS14ph site (Cheung et al., 2003). Other known acetylation marks are highlighted with blue “K”s. (B and C) Exponentially growing WT, H2B S10A, H2B K11R, H2B K11Q, H2B K16R, and H2B K16Q stains were treated with 1 mM H2O2 for 200 min. Cells were then split into two aliquots for cell survival (B) and cell death (C) assays. Briefly, cell-survival percentage was calculated for each strain by counting the number of colonies formed on YPD agar; cell death was measured by counting the number of phloxin B-stained cells following H2O2 treatment relative to untreated cells, as described in Ahn et al. (2005a). Results were averaged from three independent experiments, and error bars represent overall distribution of the data. Note that, while the H2B K11R mutant was as sensitive to H2O2 as WT cells, the H2B K11Q mutant was resistant to H2O2 treatment in either assay. (D) Exponentially growing WT, H2B K11R, H2B K11Q and H2B K16R strains were treated with 1 mM H2O2 for 200 min. Total nuclear protein was then prepared from these cells before being resolved on SDS-PAGE gel for western analysis; blots were then probed with anti-H2BK11ac, anti-H2BS10ph, anti-H4ac, anti-H3K14ac, and anti-H3. As expected (Ahn et al., 2005a), anti-H2BS10ph reacted strongly with WT cells following oxidative stress. Note, in contrast, that anti-H2BK11ac only reacted with nuclear extracts from untreated cultures but did not react with extracts from H2O2-treated cells. Characterization of this antibody suggests that this failure of antibody reactivity is not due to “epitope disruption” (see Figure S1 and text for details). The level of anti-H4ac and anti-H3K14ac stayed constant in all the nuclear extracts tested. Anti-H3 was used as a loading control. Molecular Cell 2006 24, 211-220DOI: (10.1016/j.molcel.2006.09.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Cells Exhibiting Deacetylation of Histone H2B at K11 Display TUNEL-Positive DNA Fragmentation (A) Exponentially growing WT, H2B K11R, and H2B K11Q strains were treated with 1 mM H2O2 for 200 min. Half of the cells were double stained with TUNEL and anti-H2BS10ph, after which cells were counterstained with DAPI for DNA. Cells carrying the H2B K11R mutant displayed immunostaining patterns similar to WT, while the K11Q mutant did not, suggesting that histone H2BK11 is deacetylated during H2O2-induced yeast apoptosis. (B) Remaining cells from (A) were stained with Annexin V to measure the externalization of PS, an early marker of yeast apoptosis (Koopman et al., 1994). Results were averaged from three independent experiments, and error bars represent overall distribution of the data. Seventy-five percent of WT cells and the K11R mutants displayed externalization of PS, further confirming that deacetylation of H2BK11 is required for apoptosis. (C) Nuclei were prepared from logarithmically grown WT, H2B K11R, H2B K11Q, and H2B K16R strains that were treated with or without acetic acid, and resolved on SDS-PAGE for western analysis using anti-H2BK11ac, anti-H2BS10ph, anti-H4ac, and anti-H3. Identical patterns of H2BS10ph and K11ac upon H2O2 treatment (see Figure 1D) were observed with acetic acid. Anti-H3 was used as a loading control. Molecular Cell 2006 24, 211-220DOI: (10.1016/j.molcel.2006.09.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Deacetylation of H2B Lysine 11 Is Upstream of H2B Serine 10 Phosphorylation during Yeast Apoptosis (A) Logarithmically growing WT cells were harvested after treatment with H2O2 at the indicated times, and their nuclear extracts were assayed by western analysis using anti-H2BS10ph or anti-H2BK11ac. After H2O2 treatment, progressive deacetylation of K11 coincides with the progressive increase in S10ph. Anti-H3 was used as a loading control. (B–D) Exponentially growing yeast strains were treated with 1 mM H2O2 for 200 min and tested for cell survival (B) and cell death (C) as in Figure 1 and for histone modifications as probed by modification-selective antibodies (D). Double mutants, carrying either H2B S10A or ste20Δ with K11R or K11Q, displayed cell viability and cell death comparable to single mutants containing H2B S10A or ste20Δ. Together, these results suggest that K11 deacetylation and S10ph act in the same pathway leading to cell death. (D) Immunoblots containing indicated nuclear extracts were probed with anti-H2BS10ph, anti-H2BK11ac, and anti-H3. H2BK11ac was present in logarithmically growing cells, but not in H2O2-treated single mutants carrying H2B S10A or ste20Δ. None of the double mutants carrying either H2B S10A and K11R or K11R and ste20Δ reacted with anti-H2BS10ph or anti-H2BK11ac. (E) WT and H2B mutant yeast strains were grown on YPD agar plates in the absence of H2O2. H2B S10E mutant, and the double mutant carrying both H2B S10E and K11R or K11Q, displayed growth defects, even without H2O2. Molecular Cell 2006 24, 211-220DOI: (10.1016/j.molcel.2006.09.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Hos3 Deacetylates H2B Lysine 11 during H2O2-Induced Yeast Apoptosis Yeast HDAC knockout strains were treated with H2O2 and tested for cell survival (A) and cell death (B) as in Figure 1. Possible HDAC candidates included the following: Rpd3, Hda1, and Hos3, since their knockout mutants displayed cell viability similar to H2B S10A. (C) Yeast nuclear extracts from H2O2-treated or untreated WT, rpd3Δ, hda1Δ, and hos3Δ were probed with anti-H2BS10ph and anti-H2BK11ac. Only the HOS3 deletion strain fails to remove the acetyl mark on K11 upon H2O2 treatment. Moreover, the death-related H2BS10ph signal was not detected, suggesting that deacetylation of K11 is upstream of S10ph during H2O2-induced yeast apoptosis. Molecular Cell 2006 24, 211-220DOI: (10.1016/j.molcel.2006.09.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Hos3 Deacetylates H2B Lysine 11 and Promotes H2B Serine 10 Phosphorylation by Ste20 In Vitro (A) Schematic delineation of in vitro HDAC and kinase reactions. First, an HDAC reaction was carried out by incubating recombinant active Hos3 (Hos3) or inactive Hos3 (Dead) with indicated H2B peptides as substrates. The peptides were then extracted and incubated with the recombinant active Ste20 (Ste20) or inactive Ste20 (KD; Ste20K649R) for a kinase reaction. When γ[32P]ATP was used as a phosphate source, the kinase activity was assayed by measuring incorporation of the γ[32P]ATP label into peptide substrates by scintillation counting (cpm). For nonradioactive reactions, unlabeled ATP was used instead, and the status of peptide was verified by MALDI-TOF mass spectrometry. As controls, the HDAC and kinase reactions were performed with Hos3 or Ste20 enzyme alone. (B) In vitro HDAC reactions were followed by kinase reactions and carried out using indicated H2B peptides as substrates. For the HDAC reaction, peptides were incubated with recombinant Hos3, or Hos3 inactivated by boiling. After quenching the HDAC reaction, a kinase reaction was performed with recombinant Ste20 at 30°C for 1 hr in the presence of γ[32P]ATP. The kinase reaction was stopped with the addition of phosphoric acid and spotted onto the filter paper; incorporation of [32P] on the substrate peptide was measured by scintillation counting. Results were averaged from three independent experiments, and error bars represent overall distribution of the data. When H2BK11ac peptide was used as the substrate, incubation with both Hos3 and Ste20 yielded a significant increase in [32P] count compared to its incubation with Ste20 alone. (C) Assays were performed as in (B), except that the in vitro kinase assay was carried out using a nonradioactive ATP as phosphate source. After HDAC and kinase reactions, the substrate peptides were analyzed by MALDI-TOF mass spectrometry. The appearance of a peak at 2721 Da corresponds to unmodified H2B peptide (H2BS10K11), at 2762 Da corresponds to acetylated K11 (H2BK11ac), at 2801 Da corresponds to phosphorylated S10 (H2BS10ph), and at 2843 Da corresponds to H2B peptide containing dual modification of S10ph and K11ac (H2BS10phK11ac). All peptides tested contain amino acids 1–20 from yeast H2B. The addition of both Hos3 and Ste20 caused loss of peaks corresponding to unmodified K11 and K11ac, but an increase of S10ph when H2BK11ac was used as a peptide substrate. Molecular Cell 2006 24, 211-220DOI: (10.1016/j.molcel.2006.09.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Model for Histone H2B Phos/Acetyl Crosstalk between S10 and K11 during H2O2-Induced Yeast Apoptosis K11 in H2B is acetylated in exponentially growing yeast cells. Upon H2O2 treatment, Hos3 HDAC directly catalyzes the deacetylation of H2BK11. This, in turn, mediates H2BS10 phosphorylation by Ste20 kinase, which then leads to the activation of the yeast apoptotic cascade. We envision that deacetylation of K11 is required for H2BS10 phosphorylation (see text and Discussion for details). Molecular Cell 2006 24, 211-220DOI: (10.1016/j.molcel.2006.09.008) Copyright © 2006 Elsevier Inc. Terms and Conditions