Molecular Cell 24, 211–220, October 20, 2006 ª2006 Elsevier Inc.DOI 10.1016/j.molcel.2006.09.008
Histone H2B Deacetylation at Lysine 11
Is Required for Yeast Apoptosis Induced
by Phosphorylation of H2B at Serine 10
Sung-Hee Ahn,1Robert L. Diaz,1Michael Grunstein,2
and C. David Allis1,*
1Laboratory of Chromatin Biology
The Rockefeller University
New York, New York 10021
2Department of Biological Chemistry
UCLA School of Medicine and
the Molecular Biology Institute
University of California
Los Angeles, California 90095
Chromatin alterations, induced by covalent histone
modifications, mediate awide rangeof DNA-templated
processes, including apoptosis. Apoptotic chromatin
condensation has been causally linked to the phos-
phorylation of histone H2B (serine 14 in human; serine
10inyeast,H2BS10ph) inhuman andyeast cells.Here,
we extend these studies by demonstrating a unidirec-
tional, crosstalk pathway between H2BS10 phosphor-
ylation and lysine 11 acetylation (H2BK11ac) in yeast.
We demonstrate that the H2BK11 acetyl mark, which
exists in growing yeast, is removed upon H2O2treat-
ment but before H2BS10ph occurs, in a unidirectional
fashion. H2B K11Q mutants are resistant to cell death
elicited by H2O2, while H2B K11R mutants that mimic
deacetylation promote cell death. Our results suggest
thatHos3 HDAC deacetylates H2BK11ac, which inturn
mediates H2BS10ph by Ste20 kinase. Together, these
tions governing histone modifications that promote
a switch from cell proliferation to cell death.
Apoptosis plays important roles in the development and
survival of all metazoans. For example, apoptosis is
required for the removal of autoreactive immune cells,
virus-infected cells, and cells with unrepairable genetic
damage; these cells pose the risk of carcinogenesis
and other human diseases that are critically dependent
upon a balance between cell growth and cell removal
(reviewed in Jin and El-Deiry ). Therefore, the con-
stant turnover of cells, driven by apoptotic mechanisms,
is important for homeostasis in tissues and organs of
Although considerable progress has been made in
understanding fundamental apoptotic pathways, many
details of its regulation and ultimate apoptotic pheno-
types are poorly understood, notably in unicellular or-
ganisms. For example, DNA damage and other stimuli
such as hydrogen peroxide (H2O2) can induce budding
yeast to undergo cell death resembling apoptosis in ver-
tebrates (Madeo et al., 1999), although how similar this
process is to true ‘‘mammalian’’ apoptosis remains con-
troversial. S. cerevisiae displays characteristic hall-
marks of apoptosis, including DNA fragmentation and
chromatin compaction (Madeo et al., 1999). In addition,
a growing list of genes, confirmed as apoptotic regula-
tors in metazoans, has been identified in budding yeast,
including the caspase Yca1, apoptosis-inducing factor
Aif1, histone chaperone Asf1/Cia1, and histone H2B
phosphorylation (Madeo et al., 2002; Wissing et al.,
2004; Yamaki et al., 2001; Ahn et al., 2005a). In the latter
case, H2B phosphorylation at serine 14 (H2BS14ph) in
humans and serine 10 (H2BS10ph) in yeast is mediated
by the conserved enzyme Mst1 in human and Ste20 in
yeast, respectively (Cheung et al., 2003; Ahn et al.,
2005a). Furthermore, yeast H2BS10ph plays a direct
role in mediating apoptotic chromatin compaction. In
keeping, yeast H2B S10A mutants are resistant to cell
death elicited by H2O2; in contrast, H2B S10E phospho-
site mimics promote cell death and induce ‘‘constitu-
tive’’ condensed chromatin (Ahn et al., 2005a). Thus,
conservation of the enzyme systems and choice of the
particular core histone tail substrate and site support
the general view that the basic machinery of apoptosis
is present and functional in unicellular organisms.
Histone proteins are well-known substrates for
numerous covalent posttranslational modifications.
Indeed, each core histone, notably H3 and H4, can be
posttranslationally modified in a remarkably large
number of ways, thus generating the potential for
modification crosstalk, defined as positive or negative
‘‘communication’’ between different covalent modifica-
tions on one or more histone tails. While considerable
evidence suggests that histone crosstalk occurs in vitro
(Fischle et al., 2003), much less supporting evidence
exists in vivo. Phosphorylation of histone H3 at serine
10 (H3S10ph) has been linked, for example, to the acet-
ylation of lysine 14 (H3K14ac) resulting in an open chro-
matin conformation and gene activation (Cheung et al.,
2000; Lo et al., 2001). Indeed, Snf1 and Gcn5, the en-
zymes that phosphorylate H3S10 and acetylate H3K14,
respectively, appear to work synergistically to mediate
these events (Lo et al., 2001; Clements et al., 2003).
Previous studies have documented that H2B lysine 11
is acetylated (H2BK11ac) in logarithmically growing
yeast (Suka et al., 2001). Here, we show that acetylation
of histone H2B at K11 inhibits the phosphorylation of an
adjacent site S10, catalyzed by Ste20 kinase, pointing to
a mutually exclusive existence of the K11ac and S10ph
marks in the tail of H2B. In support, yeast undergo-
ing apoptosis lacked K11 acetylation, but displayed
high levels of Ste20-mediated H2BS10ph. In addition,
K11Q, an acetyl-mimic mutant in H2B, failed to activate
the apoptotic pathway; in contrast, K11R, a mutant
mimicking deacetylation, displayed apoptotic features
including H2BS10ph upon induction of H2O2. Finally,
we identified Hos3 as the HDAC responsible for remov-
strong support to the general view that a regulatory net-
work of acetyl/phos unidirectional crosstalk serves to
integrate input signals to the tail of yeast H2B, control-
ling a switch from cell proliferation to cell death.
H2B K11R Mutants Are Resistant to H2O2-Induced
Previously, we reported that histone H2B is specifically
phosphorylated at S10 in a hydrogen peroxide (H2O2)-
induced cell death pathway in S. cerevisiae (Ahn et al.,
2005a). As shown in Figure 1A, S10 is located adjacent
to K11 in H2B, a site known to be acetylated in growing
yeast (Suka et al., 2001). As phosphorylation of the adja-
cent S10 correlates precisely with the cell death, we
sought to address whether a potential interplay exists
between these two histone modifications in the yeast
H2B tail. To this end, yeast cells were generated
expressing H2B in which K11 was mutated to either
glutamine, to mimic constitutive acetylation (K11Q), or
arginine, to mimic constitutive deacetylation (K11R), in
a four-histone plasmid shuffle strain (Ahn et al., 2005a).
No growth difference was apparent between these
H2BK11 point mutants compared to wild-type (WT), as
indicated by their growth rate (data not shown), sug-
gesting that other acetylation events may compensate
for lack of H2BK11ac during cell growth.
Upon treatment with 1 mM H2O2for 200 min, no differ-
ence was observed between WT and the H2B K11R mu-
tant with regard to survival properties (Figure 1B). Both
strains displayed 20%–30% cell viability as measured
by a plating assay (Figure 1B), followed by 70%–80%
cell death as measured by phloxin B-stained cells
producible resistance to H2O2with 70%–80% cell viabil-
ity and 20%–30% phloxin B-stained cells, comparable
to the S10A mutant (Figures 1B and 1C; also see Ahn
et al. [2005a]). Furthermore, H2BK16, another known
acetylation site in yeast H2B (Suka et al., 2001; see
Figure 1A), displayed a phenotype similar to WT in
both assays when K16 was mutated to R or Q. (Figures
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),apreviouslydescribed apoptotic phosphorylationsite(Ahnetal.,2005a).BothK11 andK16areacetylated inlogarithmicallygrowing yeast
(Sukaetal.,2001).Note that K15,also known tobeacetylated inmammaliancells (Thorne etal.,1990),is also adjacent toamammalian 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 H2O2for
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 H2O2treatment 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 H2O2as WT
cells, the H2B K11Q mutant was resistant to H2O2treatment in either assay.
(D) Exponentially growing WT, H2B K11R, H2B K11Q and H2B K16R strains were treated with 1 mM H2O2for 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 ox-
idative 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.
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