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highly coordinated process of programmed cell death that has been extensively studied in
multicellular organisms. Chromatin condensation and DNA fragmentation, hallmark
features of apoptosis, have been firmly linked to phosphorylation of serine 14 (Ser14) of
the amino-terminal tail of mammalian H2B.2Interestingly, H2B (Ser14) phosphorylation
was found to be catalyzed by Mst1 (for mammalian sterile20-like kinase), a pro-apoptotic
kinase whose physiological (nuclear) substrates were not clear.
[Cell Cycle 4:6, 780-783; June 2005]; ©2005 Landes Bioscience
2005; Vol. 4 Issue 6
Kiersten A. Henderson2,†
C. David Allis1,*
1Laboratory of Chromatin Biology; The Rockefeller University; New York, New York
2Molecular Biology Program; Memorial Sloan-Kettering Cancer Center and Weill
Graduate School of Medical Sciences of Cornell University; New York, New York USA
†These authors contributed equally to this work.
*Correspondence to: C. David Allis; Laboratory of Chromatin Biology; The
Rockefeller University; Box 78; New York, New York 10021 USA; Email: alliscd@
Received 04/12/05; Accepted 04/14/05
Previously published online as a Cell Cycle E-publication:
histones, H2B, phosphorylation, yeast apoptosis,
meiosis, pachytene, chromatin condensation
We thank Upstate Biotechnology Inc. (UBI;
Lake Placid, New York) for assistance in the
development of the H2B Ser10 phospho-specific
antibody. We also thank Robert Diaz, Edwin
Smith, and Judith Recht for critical reading of the
This research is supported by NIH grant
(GM40922) and funds from the Rockefeller
H2B (Ser10) Phosphorylation is Induced during Apoptosis and Meiosis
in S. cerevisiae
The nucleosome, composed of an octamer of highly conserved histone proteins and
associated DNA, is the fundamental unit of eukaryotic chromatin. How arrays of nucleo-
somes are folded into higher-order structures, and how the dynamics of such compaction
are regulated, are questions that remain largely unanswered. Our recent studies demon-
strated that phosphorylation of histone H2B is necessary to induce cell death that exhibits
phenotypic hallmarks of apoptosis including DNA fragmentation and chromatin condensa-
tion in yeast (serine 10)1and in mammalian cells (serine 14).2In this article, we extend
these findings by uncovering a role for H2B phosphorylation at serine 10 (Ser10) in
another biological event that is associated with dramatic alterations in higher-order chromatin
structure, meiosis. Our data show strong staining, indicative of H2B (Ser10) phosphory-
lation, during the pachytene stage of yeast meiotic prophase. These data broaden the use
of this phosphorylation mark in chromatin remodeling that closely correlates with chromatin
compaction. How phosphorylation marks are translated into meaningful downstream
events during processes as diverse as apoptosis and meiosis remain a challenge for
APOPTOTIC H2B PHOSPHORYLATION
The faithful packaging of chromatin, the repeating polymer of DNA and associated
histone proteins (H2A, H2B, H3 and H4), is an essential step in the accurate execution
of proliferation, differentiation and apoptosis in eukaryotic cells. Each of these biological
processes is characterized by elaborate mechanisms that involve different, but poorly
understood, levels of chromatin compaction. One well-studied mechanism for introduc-
ing variation into the chromatin polymer is the addition of post-translational histone
modifications (e.g., acetylation, phosphorylation, methylation, etc.). Indeed, the distinct
patterns of covalently modified histones can serve as a signaling platform to mediate
downstream events leading to compaction of chromatin fibers (reviewed in ref. 3).
Considerable progress has been made in identifying histone modifications that are asso-
ciated with chromatin condensation during mitosis and meiosis, although the functional
significance of these changes remains unclear. Perhaps the best example of core histone
modification is the phosphorylation of histone H3 at serine 10 (Ser10), which is detected
during mitotic and meiotic prophases, when chromosomes reach maximal condensation.4-7
While this phosphorylation event is necessary for the initiation of chromosome condensation
during mitosis in Tetrahymena thermophila,4its functional significance is not clear in budding
yeast as H3 Ser10 point mutants (H3 S10A) progress through the cell cycle normally.6
Failure to uncover a causal relationship between H3 phosphorylation and expected chromo-
some dynamics in yeast could conceivably result from a redundant phosphorylation event
on another histone residue, such as histone H2B. In support, Ipl1, the H3 Ser10 kinase,
has been found to phosphorylate H2B in vitro,6suggesting that H2B phosphorylation
may play a role during mitotic and/or meiotic chromatin compaction.
To date, histone H2B phosphorylation has been best characterized during apoptosis, a
Increasing evidence suggests that
some features of apoptosis may extend
to unicellular organisms such as S. cere-
visiae.8,9An apoptotic-like phenotype,
characterized by DNA fragmentation
and modest chromatin changes, has
been demonstrated in S. cerevisiae
when treated with various agents
including acetic acid, osmotin, oxygen
radical and pheromone.8-10Recently,
we have shown that a similar phospho-
rylation event on the amino-terminal
tail of H2B from yeast is required for
an apoptotic-like cell death in budding
yeast.1Our findings suggesting that an
analogous serine (Ser10 in yeast H2B)
has a “death” function that is catalyzed
by Sterile 20 kinase provide evidence
for a chromatin-mediated apoptotic
pathway that is remarkably well con-
served between yeast and humans. As
shown in Figure 1, a precise positive
correlation is observed between TUNEL-
positive and phosphorylated (Ser10)
H2B-positive yeast upon treatment
with hydrogen peroxide (Fig. 1A);
essentially all of this phosphorylation is
dependent upon the presence of func-
tional Ste20 kinase (Fig. 1B).1
In S. cerevisiae, apoptotic DNA
fragmentation correlates precisely with
H2B phosphorylation at Ser10.
Intriguingly, a H2B S10A point
mutant exhibits increased cell survival accompanied by a loss of both
DNA fragmentation and chromatin condensation, whereas in con-
trast, a phospho-mimic H2B S10E mutant displays phenotypic
markers of apoptosis, including a striking induction of “constitutive”
compacted chromatin.1Hence, H2B Ser10 phosphorylation might
be directly involved in regulating apoptotic chromatin condensation
by directly affecting internucleosomal contacts and histone-DNA
interaction (“cis” effects). In addition, double-strand DNA breaks
(DSBs) occur earlier than H2B phosphorylation in the apoptotic
pathway as judged by phospho Ser129 H2A antibody staining (the
phosphorylation site in yeast that is analogous to Ser139 in mammalian
H2A.X; also known as γ-H2A.X), as is the case with mammalian
H2B (Ser14) phosphorylation.1,2H2B Ser14 phosphorylation may
enhance caspase-activated nuclease activities (CAD) on chromatin
and facilitate their recruitment to chromatin.2In a caspase-independent
apoptotic pathway such as H2B phosphorylation in S. cerevisiae, an
unknown nuclease, instead of CAD could be recruited to the chromatin
or unknown specific factors could dock at phosphorylated H2B to
regulate higher-order chromatin structure (“trans” effects). Whether
chromatin changes are brought about by “cis” or “trans” effects, the
H2B tail-selective phosphorylation serves an important role in
mediating large-scale changes in chromatin structure, characteristic
of late events in apoptosis (see below).
MEIOTIC H2B PHOSPHORYLATION
In mammalian cells, phosphorylated (Ser14) H2B colocalized
with γ-H2A.X in DNA-damage foci induced by γ-irradiation (IR).11
Thus, we were interested in the possibility that the role of H2B
(Ser10) phosphorylation may extend to meiosis, and in particular, to
the stage of meiotic prophase when DSBs are known to occur
(reviewed in ref. 12).
To test this hypothesis, nuclear extracts were prepared from
diploid yeast cells derived from rapidly-sporulating SK1 strains that
were induced for synchronous sporulation.13Extracts from different
meiotic stages were then resolved on an SDS-PAGE gel and examined
by Western blotting using mitotic H3 (Ser10) phospho-specific
(α-Phos (Ser10) H3) as well as apoptotic H2B (Ser10) phospho-spe-
cific antibodies (α-Phos (Ser10) H2B). Interestingly, both H2B (Ser10)
and H3 (Ser10) phosphorylation events were enhanced at 2 hr and
declined after 7 hr following induction into meiosis (Fig. 2A),
suggesting that similar to H3 (Ser10) phosphorylation,6H2B
(Ser10) phosphorylation occurs during meiotic prophase. Not all
phosphorylation marks on core histones follow the above pattern
suggesting some specificity. For example, serine 1 phosphorylation
of histone H4, a mark known to correlate with mitosis14and damaged
DNA (MMS-induced),15is induced at much later stages (9–11 hr)
of meiosis (data not shown).
To confirm the above results and to better define the precise
stage(s) of meiotic prophase when H2B (Ser10) phosphorylation is
maximal, we performed double immunostaining of surface spread
H2B (Ser10) Phosphorylation is Induced during Apoptosis and Meiosis in S. Cerevisiae
Figure 1. Histone H2B is specifically phosphorylated at Ser10 by Ste20 kinase in “dying” yeast. (A) Wild
type (WT) cells, treated with or without 1 mM hydrogen peroxide for 200 minutes, were stained with TUNEL
and α-Phos (Ser10) H2B. Note the perfect agreement between TUNEL and α-Phos (Ser10) H2B staining in
all cells, demonstrating that apoptotic DNA fragmentation correlates precisely with H2B phosphorylation at
Ser10. (B) Total nuclear protein, isolated from cells treated with or without hydrogen peroxide, was fractionated
by SDS-PAGE before immunoblots were probed with α-Phos (Ser10) H2B. While H2B from the S33A H2B
mutant reacted similarly to WT H2B, Ser10 phosphorylation signal is lost in H2B S10A or ste20 deletion
mutants (see red box).1
782 Cell Cycle 2005; Vol. 4 Issue 6
meiotic nuclei for Phos H2B (Ser10) and Zip1, a component of the
synaptonemal complex. The pattern of Zip1 localization indicates
the stage in meiotic prophase, with extensive linear Zip1 staining
denoting fully synapsed chromosomes,16the hallmark of the pachytene
stage of prophase where chromosomes are condensed.17As shown in
Figure 3, prior to entry into meiosis (premeiotic), when Zip1 was
absent, Phos H2B (Ser10) was largely absent (93%; 63/70 nuclei).
Consistent with the kinetics of H3 (Ser10) phosphorylation,6H2B
(Ser10) phosphorylation gradually increased as cells progressed
through prophase and by pachytene the
vast majority of nuclei (98%; 52/53)
stained brightly with H2B (Ser10) phospho-
specific antibody. The Phos H2B (Ser10)
signal was specific as it was eliminated
from pachytene nuclei upon competition
with phosphorylated H2B (Ser10) peptide
(Fig. 3); this staining is not diminished
when unmodified (control) H2B peptides
were used in parallel competition experi-
ments (data not shown). Also consistent
with the kinetics of H3 (Ser10) phospho-
rylation, H2B phosphorylation, as detected
by Western blot (Fig. 2A), declined rapidly
around the first meiotic division, which
occurred at 8 hr in this culture. We further
tested the correlation between high levels
of H2B (Ser10) phosphorylation and the
pachytene stage of meiosis by examining
H2B phosphorylation in a ndt80 mutant,
which arrests at pachytene with condensed
chromosomes.18The ndt80 mutant failed
to undergo meiotic divisions and main-
tained phosphorylated H2B at high levels
(Fig. 2B), suggesting that, like Phos H3, maximal
phosphorylation of H2B (Ser10) occurs at the
pachytene stage of meiotic prophase.
Contrary to our initial expectation, H2B
(Ser10) phosphorylation was not induced in
response to the formation of the DSBs that
initiate meiotic recombination. The wild type
profile of H2B phosphorylation was observed in
a mutant lacking Spo11, the protein that forms
meiotic DSBs (Fig. 2B; reviewed in ref. 19).
These results demonstrate that H2B (Ser10)
phosphorylation correlates with progression
through meiotic prophase and perhaps with
meiotic chromosome condensation, rather than
other events associated with meiotic DSB repair.
Our findings indicate that H2B (Ser10)
phosphorylation is a common feature of both
apoptosis and meiosis, and is thus a mark used
more broadly in biological pathways that involve
large-scale changes in chromatin structure. Is
there a common theme linking these different
physiological processes? One possible link may
be through effects of histone phosphorylation
on chromatin condensation. In the case of apop-
tosis, chromatin becomes compacted and this
compaction is associated with processes that
ultimately lead to permanent elimination of the
genome through cell death. In prophase I of meiosis, in contrast,
there are cyclical changes in chromatin compaction that are thought
to coordinate and/or promote chromosome dynamics [e.g., recombi-
nation; higher-order chromosome structures such as the synaptonemal
complex and axial elements (specialized meiotic chromosome axes);
Another possible link may involve the enzymes responsible for
adding and/or removing the H2B (Ser10) phosphorylation mark
during apoptosis and meiosis. Although Ste20 has no known role in
H2B (Ser10) Phosphorylation is Induced during Apoptosis and Meiosis in S. Cerevisiae
Figure 2. H2B (Ser10) phosphorylation is induced during meiotic prophase. Western analysis of a time
course of synchronous meiotic yeast cultures (diploid SK1 strains). (A) H2B Ser10 phosphorylation is
strongly induced during meiotic prophase. α-Phos (Ser10) H3 demonstrates the unique timing and robustness
of the signal obtained from α-Phos (Ser10) H2B. A general H3 antibody is used as a loading control. (B)
The wild type-like H2B Ser10 phosphorylation pattern is observed in spo11 deletion mutants, whereas
this phosphorylation level is maintained in ndt80 deletion mutants.
Figure 3. α-Phos (Ser10) H2B stains pachytene stage of meiotic prophase. Chromosomes from a
time course through meiotic prophase of WT diploid SK1 strains were spread onto glass slides and
examined for Zip1 and H2B Ser10 phosphorylation signal by double immunostaining. Bright staining
of Phos (Ser10) H2B is detected in nuclei in the pachytene stage of meiosis (indicated by extended
Zip1 structure). This H2B signal is competed away with the Phos (Ser10) H2B peptide (bottom row),
but not with the unmodified H2B peptide (not shown).
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meiosis, Ste20 could be a candidate kinase for meiotic H2B (Ser10)
phosphorylation. Considering that Ipl1 can phosphorylate both H3
and H2B in vitro,6H2B (Ser10) phosphorylation mediated by Ipl1
and/or Ste20, may be functionally redundant with meiotic H3
(Ser10) phosphorylation, a possibility that remains to be addressed.
Although phosphorylation of H2B (Ser10) correlates with con-
densed chromatin, the precise role of this phosphorylation mark is
unknown. For example, it is not clear whether H2B phosphorylation
exerts its effects solely through cis structural effects on chromatin
folding alone, or if instead it allows the binding of different trans
effectors that in turn bring about diverse downstream effects.
Following the paradigm now well established for histone acetylation
and methylation, we predict that a focused attack on H2B phospho-
rylation, the enzyme systems responsible for its steady-state balance,
and the “effectors” that “read” this mark, if they exist, will lead to
valuable insights into how the genome is condensed during meiosis,
and in other settings, such as apoptosis.
Yeast strains and culture conditions. Yeast strains used for the
apoptotic study are derivatives of S288C (BY4741; Research
Genetics) background. Genotypes of these yeast strains include:
JHY311 (MATa his3∆1 leu2∆0 met15∆0 ura3∆0 hht1-hhf1::KAN
hhf-2hht2::NAT hta1-htb1::HPH hta2-htb2::NAT pQQ18[CEN
LEU2 HTA1-HTB1 HHT2-HHF2]). SAY2 (htb1-S10A), SAY3
(htb1-S33A), and SAY148 (ste20∆::HIS3MX6) were derived from
JHY311.1H2O2treatments were carried out as described in Ref. 1.
SK1 diploid strains include: SKY165 (MATa/MATα ho::LYS2/
ho::LYS2 lys2/lys2 ura3/ura3 leu2::hisG/leu2::hisG). SKY169
(ndt80∆::LEU2 nuc1∆::LEU2 his4X::LEU2 arg4-nsp) and SKY10
(spo11∆::hisG-URA3-hisG his4x::LEU2) were derived from SKY165.
Yeast nuclei and histone extraction, Western blot and immuno-
fluorescence staining. Yeast nuclei and histones were extracted, and
used for Western analysis as described.6Antibodies were diluted as
follows: α-Phos (Ser10) H2B: 1:5000, α-H4: 1:5000, α-Phos
(Ser10) H3: 1:1000, α-H3: 1:1000. TUNEL staining was carried
out as previously described.1For synaptonemal complex and Phos
H2B (Ser10) immunofluorescence analysis, chromosomes were surface
spread and stained as described.21,22Guinea pig α-Zip1 was used at
1:1000 dilution and α-guinea pig-Alexa 546 at 1:1000 (Molecular
Probes). Rabbit α-Phos H2B (Ser10) was used at 1:100, and
α-Rabbit Alexa 488 at 1:1000 (Molecular Probes). For peptide
competition, either unmodified (amino acids 4–14 of yeast H2B) or
Ser10-phosphorylated H2B peptide was added to the diluted α-Phos
H2B (Ser10) (1.4 µg/µL final concentration). Slides were mounted
with cover slips in Prolong antifade (Molecular Probes). Images were
captured on a Zeiss Axiophot microscope with a 100x objective
using a Cooke Sensicam cooled CCD camera. Data capture and
image processing were performed using the Slidebook software pack-
age (Intelligent Imaging Innovations).
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H2B (Ser10) Phosphorylation is Induced during Apoptosis and Meiosis in S. Cerevisiae