DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 1045
Development 137, 1045-1053 (2010) doi:10.1242/dev.042812
© 2010. Published by The Company of Biologists Ltd
Double bromodomain protein BET-1 and MYST HATs
establish and maintain stable cell fates in C. elegans
Yukimasa Shibata1, Hisako Takeshita1, Noriko Sasakawa1 and Hitoshi Sawa1,2,*
SUMMARY
The maintenance of cell fate is important for normal development and tissue homeostasis. Epigenetic mechanisms, including
histone modifications, are likely to play crucial roles in cell-fate maintenance. However, in contrast to the established functions of
histone methylation, which are mediated by the polycomb proteins, the roles of histone acetylation in cell-fate maintenance are
poorly understood. Here, we show that the C. elegans acetylated-histone-binding protein BET-1 is required for the establishment
and maintenance of stable fate in various lineages. In most bet-1 mutants, cells adopted the correct fate initially, but at later stages
they often transformed into a different cell type. By expressing BET-1 at various times in development and examining the rescue of
the Bet-1 phenotype, we showed that BET-1 functions both at the time of fate acquisition, to establish a stable fate, and at later
stages, to maintain the established fate. Furthermore, the disruption of the MYST HATs perturbed the subnuclear localization of
BET-1 and caused bet-1-like phenotypes, suggesting that BET-1 is recruited to its targets through acetylated histones. Our results
therefore indicate that histone acetylation plays a crucial role in cell-fate maintenance.
INTRODUCTION
During animal development, stem cells progressively lose their
multipotency and their progeny’s fates are increasingly restricted.
Moreover, once cell fate begins to be restricted, including in nontotipotent progenitors, cells maintain their differentiated
characteristics and cannot revert to stem cells in vivo, although they
can dedifferentiate into stem cells when forced to express certain
defined factors in vitro (Takahashi and Yamanaka, 2006). For cell
fates to be stably maintained in vivo, the gene expression program
must also be maintained. Epigenetic mechanisms, including DNA
methylation and histone modifications, are believed to play
important roles in maintaining transcriptional programs. Among
histone modifications, the roles of histone methylation in the
maintenance of cell fate are relatively well understood. For example,
in Drosophila, the polycomb genes, which include the genes for the
histone methyltransferase, E(z), and for methylated histone binding
protein (Polycomb), are required for the maintenance of cell fates
through the transcriptional silencing of Hox genes (Ringrose and
Paro, 2004). In mammals, an H3K4 histone methyltransferase,
MLL, is crucial for the maintenance of memory Th2 cell function
(Yamashita et al., 2006). However, unlike histone methylation, the
well-described roles of histone acetylation are in transcriptional
activation per se and in cell differentiation, but its roles in the
maintenance of cell fate are poorly understood.
BET-family proteins are evolutionally conserved and have two
bromodomains, which recognize acetylated histones (Florence and
Faller, 2001; Loyola and Almouzni, 2004). In Saccharomyces
cerevisiae, the BET-family protein Bdf1 has an anti-silencing
function that prevents the spreading of the SIR silencing complex
from the telomere or mating loci (Ladurner et al., 2003). In
Drosophila, a BET-family gene, fs(1)h is required for the
transcriptional activation of a Hox gene, Ubx (Chang et al., 2007;
Florence and Faller, 2001). BET-family proteins, but not other
bromodomain proteins, co-localize with the chromosomes during
mitosis (Loyola and Almouzni, 2004), suggesting that they might
protect the cell transcriptional state from being disrupted by mitosis.
However, it is unknown if BET-family proteins are required for the
maintenance of cell fates in multicellular organisms.
In C. elegans, histone modification enzymes are shown to be
required for the normal development. For example, MYS-1, a member
of the MYST family of acetyltransferases (MYST HATs), represses
the induction of vulval fates (Ceol and Horvitz, 2004). Histone
methyltransferase MES-2 represses the expression of Hox genes in
inappropriate regions (Ross and Zarkower, 2003) and promotes the
loss of developmental plasticity in early embryonic cells (Yuzyuk et
al., 2009). However, the roles of chromatin factors in the maintenance
of cell fate have not been established in C. elegans.
Here, we found that a C. elegans BET-family protein, BET-1, is
required for the maintenance of various cell fates. In bet-1 mutants,
although cell-fate specification can be abnormal, when cells
acquired normal characteristics initially, their fates were not
maintained, and they eventually took on the fates of their relatives
(e.g. cousin or sister cell) at later stages. Rescue experiments
suggested that BET-1 must be expressed both at the time of cell-fate
acquisition and later, when aberrant cell transformation occurred in
the bet-1 mutants. Disruption of the MYST HATs caused
phenotypes similar to those of bet-1 mutants and disturbed the subnuclear localization of BET-1, suggesting that BET-1 is recruited to
its target sites through acetylated histones. Our results indicate that
histone acetylation plays crucial roles in the establishment and
maintenance of stable cell fates.
1
Laboratory for Cell Fate Decision, Riken, Center for Developmental Biology, Kobe,
Hyogo, 650-0047, Japan and 2Department of Biology, Graduate School of Science,
Kobe University, Kobe, Hyogo, 657-8501, Japan.
*Author for correspondence ([email protected])
Accepted 1 February 2010
MATERIALS AND METHODS
Strains and culture
N2 Bristol was used as the wild-type strain. n4075 (Ceol and Horvitz, 2004),
e1338 (Chalfie and Sulston, 1981) and os22 (Sawa et al., 2000) were used
as the mys-1, mec-3 and psa-1 mutations, respectively. Animals were
DEVELOPMENT
KEY WORDS: Fate maintenance, BET, MYST HAT, Acetylated histone, C. elegans
1046 RESEARCH ARTICLE
Development 137 (7)
cultured at 22.5°C. To obtain synchronized animals, newly hatched larvae
were collected for 1 hour, so larvae ‘1 hr after hatching’ were actually 0 to 1
hour post-hatching.
The following GFP or RFP markers were used (TC and TL indicate
transcriptional and translational fusions, respectively): wIs51 [scm::gfp]
(TC) (Zhong et al., 2003), osEx113 [psa-3::gfp] (TL) (Arata et al., 2006),
zdIs5[mec-4::gfp] (TC) (Clark and Chiu, 2003; Lai et al., 1996), qIs56[lag2::gfp] (TC) (Kostic et al., 2003), mnIs17[osm-6::gfp] (TL) (Collet et al.,
1998), vsIs33[dop-3::rfp] (TC) (Chase et al., 2004), muIs32[mec-7::gfp]
(TC) (Pujol et al., 2000), uIs22[mec-3::gfp] (TC) (Toker et al., 2003),
vtIs1[dat-1::gfp] (TC) (Nass et al., 2002), osIs5[scm::wrm-1::gfp] (TL)
(Mizumoto and Sawa, 2007) and qIs74[psys-1::gfp::pop-1] (TL) (Siegfried
et al., 2004).
Mosaic analysis
Cloning of bet-1
The whole-cell extract from pbet-1::flag or pcDNA3.1-transfected COS7
cells was incubated with 4 mg of biotin-labeled peptides (Millipore) in a
buffer containing 20 mM HEPES, 150 mM NaCl, 1 mM MgCl2, 10 mg/ml
proteinase inhibitor cocktail (Nacalai Tesque), 1 mM EDTA, 1% NP40 and
1 mM DTT for 2 hours at 4°C, then incubated with 40 ml of M-280
streptavidin beads (Dynal) for 1 hour at 4°C. The beads were washed three
times, subjected to SDS-PAGE and immunoblotted with an anti-Flag
antibody.
Plasmid construction
pbet-1::bet-1::gfp comprised a 3 kb genomic fragment between the
region 5⬘ upstream of the first exon to the NcoI site in the first exon,
amplified by PCR, and a cDNA fragment from the NcoI site to the codon
corresponding to residue 630, ligated to pPD95.77 (a gift from A. Fire,
Stanford University, CA, USA). For scm::bet-1::gfp, a fragment from
yk1007f09 corresponding to almost the entire BET-1 protein (residues 1630) was inserted between an scm promoter fragment (Koh and Rothman,
2001) and the gfp gene from pPD95.77. For phs::bet-1::gfp, the
yk1007f09 fragment was inserted between the heat-shock promoter from
pPD49.78 (a gift from A. Fire) and the gfp gene from pPD95.77. For
phs::dd::bet-1::gfp, the destabilization domain (DD) amplified from
pPTuner (Clontech) was inserted between the yk1007f09 fragment and
the heat-shock promoter in phs::bet-1::gfp. For dat-1::mCherry, a 1.4 kb
genomic fragment corresponding to the 5⬘ upstream region of dat-1 and
the mCherry gene were inserted into pGEM-T-easy (Promega).
pPD118.17 was used for mec-3::gfp (a gift from M. Chalfie, Columbia
University, NY, USA). dat-1::mCherry (25 mg/ml) or pPD118.17 (25
mg/ml) were injected with herring sperm DNA (100 mg/ml) to make
complex arrays (Kelly et al., 1997). Ectopic expression of the markers and
their increase in adults compared with young larvae was observed using
the complex arrays (see Table S4 in the supplementary material).
Plasmids were injected with pUnc76 (Bloom and Horvitz, 1997), pRF4
(Mello et al., 1991) or lin-44::gfp (Herman et al., 1995). The pbet-1::flag
construct, comprising a yk1007f09 fragment and the FLAG tag was
inserted into pcDNA3.1 (Invitrogen). bet-1, mys-2, mys-3, pcaf-1 and
mys-4 RNAi constructs were, respectively, restriction fragments of
yk1007f09 (NcoI-HindIII), yk1626h11 (BamHI-PstI), yk1438d03
(BamHI-PstI), yk1407g12 (EcoRI-KpnI) and a PCR-amplified fragment
of exon 7 (PstI-SalI) of mys-4 inserted into L4440 (Timmons and Fire,
1998). The cbp-1 feeding RNAi clone was described previously (Kamath
et al., 2003).
Treatment of animals and microscopy
The Psa phenotype was detected as described (Sawa et al., 2000). The
expression of mec-3::gfp in the posterior lateral ganglion (PLG) and psa3::gfp was detected by confocal microscopy (Zeiss LSM510), that of bet1::gfp and dd::bet-1::gfp was detected by spinning-disc confocal
microscopy (CSU10, Yokogawa), and the other gfp or rfp markers was
detected by epifluorescence (Axioskop2plus, Zeiss). To analyze the
V5.pa lineage, we observed the right PLG, as the left PLG contains
progeny of the Q cell. For heat-shock, animals were transferred to plates
pre-heated to 33°C, maintained at 33°C for 30 or 10 minutes, then
transferred to 22.5°C.
Immunostaining
Immunostaining was performed as described (Shibata et al., 2000). Mouse
anti-GFP 3E6 (Molecular Probes) and FITC-labeled donkey anti-mouse IgG
(Santa Cruz) diluted with PBST containing 5% skim milk were used. All of
the antibodies were used at a 1:1000 dilution.
Peptide-binding assay
RESULTS
bet-1 encodes a BET-family protein that colocalizes
with chromosomes
In a screen for the absence of phasmid socket cells, which are
derived from the T cell (Psa phenotype) (Sawa et al., 2000), we
isolated three mutant alleles of bet-1: os46, os88 and os118 (see
Table S1 in the supplementary material). We cloned bet-1 and found
that it corresponds to Y119C1B.8, which encodes a C. elegans BETfamily protein with two bromodomains (see Fig. S1A in the
supplementary material), a motif that binds acetylated histones
(Loyola and Almouzni, 2004).
The os46 bet-1 allele had a nonsense mutation in the region
encoding the first bromodomain (see Fig. S1A in the supplementary
material). The Psa phenotype and the extra-DTC phenotype
(described below) of gk425 (a bet-1 allele from the International C.
elegans Gene Knockout Consortium), which had an N-terminal
deletion, was weaker than that of os46 (see Table S1 and Fig. S2B
in the supplementary material). bet-1 RNAi given at the first larval
stage (L1) enhanced the extra-DTC phenotype of gk425 but not that
of os46 (see Fig. S2B in the supplementary material). The bet-1
cDNA that we isolated from gk425 encoded a truncated BET-1
protein lacking half of the first bromodomain, when the third
methionine (86M) was used as the initiation codon (see Fig. S2A in
the supplementary material). Thus, gk425 is a relatively weak allele,
whereas os46 shows a strong loss-of-function and is likely to be null.
Therefore, we used os46 in all the subsequent experiments.
We analyzed bet-1 expression using gfp fusion genes that encode
the BET-1 protein fused to GFP near its C-terminus (BET-1::GFP).
BET-1::GFP expression using the native promoter rescued the Psa
phenotype (see Table S1 in the supplementary material), indicating
that BET-1::GFP is functional. When we expressed BET-1::GFP
under the endogenous bet-1 promoter, GFP fluorescence was
observed in most, if not all, cells, including the T, V5.pa, Z1/Z4 and
Q cells, and all of their descendants (see Fig. S1Ba-e in the
supplementary material). The BET-1::GFP signal exhibited a
granular distribution in the nuclei at interphase. During division,
BET-1::GFP signals were observed along the metaphase plate,
indicating that BET-1 colocalized with the chromosomes at mitosis
(see Fig. S1Bf,C in the supplementary material), like other BETfamily proteins (Loyola and Almouzni, 2004).
DEVELOPMENT
The bet-1(os46) mutation was located between unc-11 and dpy-5 on LGI by
three-factor mapping and between cosmids T01A4 and D1007 by single
nucleotide polymorphism (SNP) mapping. Feeding RNAi against
Y119C1B.8 in this region caused the Psa phenotype (see Table S1 in the
supplementary material), the production of extra distal tip cells (DTCs) and
PDE neurons and embryonic lethality. scm::bet-1::gfp and bet-1p::bet1::gfp rescued the Psa and extra-PDE phenotypes of os46 mutants.
Therefore, bet-1 is Y119C1B.8.
Mosaic analysis was performed as described (Yochem and Herman, 2003).
In mosaic animals, BET-1::GFP expression was used to monitor loss of the
extrachromosomal array. GFP expression was examined in the V cellderived seam cells and the T.p cell-derived phasmid socket or hypodermal
cells.
BET-1 and MYST HATs maintain cell fates
RESEARCH ARTICLE 1047
To examine whether BET-1 functions cell-autonomously, we
performed a mosaic analysis. Genetic mosaics were generated by the
spontaneous loss of an extrachromosomal array containing
scm::bet-1::gfp during embryogenesis in bet-1 mutants. When BET1::GFP was expressed in the T, but not the neighboring V6, lineage,
the Psa phenotype was usually rescued, but expression in the V6, but
not the T, lineage did not rescue it (see Fig. S3 in the supplementary
material). Therefore, BET-1 functions cell-autonomously.
Aberrant increase in the numbers of specific cells
during the development of bet-1 mutants
In addition to the Psa phenotype, we found that the bet-1 mutation
caused the ectopic expression of markers for particular cell types
(PDE, DTC and AVM/PVM). First, in the adult posterior lateral
ganglion (PLG) of bet-1 mutants, multiple cells expressing dat1::gfp or osm-6::gfp (markers for the PDE neuron) were observed
(Fig. 2B,D,M) compared with the one positive cell (PDE neuron) in
wild type (Collet et al., 1998; Nass et al., 2002). Second, in the
anterior or posterior half of the gonad, bet-1 adults often had more
than one distal tip cell (DTC) that expressed lag-2::gfp, whereas
there was only one DTC in wild type (Fig. 2E,F,M). All the DTCs
in bet-1 mutants, as in wild-type animals, were positioned at the tip
of the gonad arms and had a cup-like shape (see Fig. S5A in the
supplementary material), indicating that extra DTC cells had been
produced rather than the marker simply being expressed ectopically.
Third, mec-4 and mec-7 are normally expressed in the touch
neurons, including AVM and PVM (Fig. 2G,I,M) (Chalfie et al.,
1994; Lai et al., 1996). In bet-1 adults, ectopic mec-4::gfp- or mec7::gfp-positive neurons were observed. Their positions and
morphology indicated that they were ectopic AVM and PVM
neurons (see below for more evidence; Fig. 2H,J,M). As the extra
Fig. 1. Defects in the T lineages of bet-1 mutants. (A) T lineages in
wild type and bet-1 mutants. X indicates cell death. Progeny were
identified as hypodermal (H) or neural (N) cells by their nuclear
morphology. The number of T cells that had the indicated lineages is
shown below the diagrams. Among them, those that showed the
interchange of positions of the T.p daughter cells are indicated in
parenthesis. Asterisks in wild type indicate phasmid socket cells.
(B,C) Nomarski images of the tail of wild type (B) and a bet-1 mutant
(C) at the L2 stage. Arrows and arrowheads indicate the phasmid
socket cells and hypodermal cells derived from the T.p cell, respectively.
Asterisks indicate the T.ap cell. (D,E) psa-3::gfp expression in the T-cell
daughters in wild type (D) and bet-1 mutants (E) at the L1 stage.
Merged GFP and Nomarski images. Anterior is to the left, ventral is to
the bottom. Scale bars: 5 mm.
AVM phenotype was more frequently observed than the extra PVM
one, we mostly analyzed the AVM phenotype in the following
experiments. Because the bet-1 mutation caused the ectopic
induction of multiple characteristics for each cell type (i.e. the
expression of multiple markers or, for DTCs, appropriate
morphology and marker expression), BET-1 might regulate the
expression of cell-fate determinants that are required for multiple
cell-specific characteristics. Although the determinant(s) for the
PDE or DTC is unknown, the LIM homeodomain transcription
factor MEC-3 is expressed in the AVM (Fig. 2K,M) and functions
as its fate determinant (Way and Chalfie, 1988). Consistent with our
hypothesis, we observed ectopic mec-3::gfp-positive neurons in the
anterior right side of bet-1 adults, where the AVM is positioned in
wild type (Fig. 2L,M), and found that the production of all of the
AVM-like cells in bet-1 mutants was completely dependent on mec3 (Fig. 2M). These results suggest that BET-1 regulates the
expression of cell-fate determinants, such as MEC-3, to prevent the
excessive production of certain types of cells.
DEVELOPMENT
Asymmetry of T-cell division is initially normal,
but later disrupted, in bet-1 mutants
A tail hypodermal cell, the T cell, divides asymmetrically to produce
hypodermal cells from the anterior daughter cell (T.a), and neural
cells, including the phasmid socket cells, from the posterior one
(T.p) (Fig. 1A). Analysis of the cell lineages in bet-1 mutants
revealed that the daughters of the T.p cell (T.pa and T.pp) often
adopted a hypodermal fate, as judged by their nuclear morphology
and the expression of scm::gfp (see Fig. S4J in the supplementary
material), consistent with the Psa phenotype of the animals (Fig. 1AC). These results might mean that the asymmetry of the T-cell
division was defective. However, the asymmetric localization of
POP-1 and WRM-1 in the daughters of the T cell was normal (n=10
each for POP-1 and WRM-1; see Fig. S4A-F in the supplementary
material). psa-3, which is a direct target of Wnt signaling and is
required for the neural fate of T.p (Arata et al., 2006), was expressed
asymmetrically (stronger in T.p and its daughter cells than in the
anterior ones) in bet-1 mutants, as in wild type (Fig. 1D,E; see Fig.
S4K in the supplementary material). In addition, the daughters of the
T.p cell, but not those of the T.a cell, migrated to interchange their
positions in most bet-1 mutants (Fig. 1A, bottom), as observed in
wild type (Sulston and Horvitz, 1977). Furthermore, at the time of
the migration, the daughters of the T.p cell in bet-1 mutants, as in
wild type, had a neuroblast-like appearance with smaller nucleoli
than the T.a daughters, which had a hypodermal appearance (21 of
21; see Fig. S4H in the supplementary material). These results
indicate that the T.p cell and its daughters initially acquired the
correct neural fate in most bet-1 mutants but that the fate was
subsequently disturbed in the T.p daughters after their positional
interchange.
1048 RESEARCH ARTICLE
Development 137 (7)
Interestingly, we also found that the number of these ectopic cells
increased during development. In wild-type animals, these cells are
produced at early larval stages (PDE at L2, AVM at mid-L1 and
DTC at L1). We compared the expression patterns of markers (dat1::gfp for PDE, lag-2::gfp for DTC, and mec-3::gfp and mec-4::gfp
for AVM) between the adult stage and middle-larval ones (PDE at
L3, DTC at L2, AVM at late L1), which are the stages occurring
shortly after the production of these cells in wild type (Fig. 2N). At
the larval stages, some bet-1 mutants already expressed the marker
genes in multiple cells but the number of cells expressing the marker
genes was smaller than in adults (Fig. 2N; data for DTCs represent
numbers of DTCs in the anterior or posterior half of the gonad). To
confirm the increases in these cell types, the number of ectopic cells
was first counted in individual animals at the larval stages and then,
after their recovery and growth, recounted at the adult stage. This
experiment showed that the number of PDEs (dat-1::gfp), DTCs
(lag-2::gfp) and AVMs (mec-4::gfp) increased in 38% (n=64), 20%
(n=40) and 35% (n=20) of individual bet-1 mutants, respectively.
We did not observe any cells that lost marker expression in these
experiments. In addition, in contrast to the larval stages, the number
of GFP-positive cells was the same between 1- and 2-day-old adults
(data not shown). These results indicated that these cells are
ectopically produced in the larval, but not the adult, stages.
Transformation of cell fates to those of relatives
in bet-1 mutants
Because the transformation from neuroblast to hypodermal cells was
observed in the T cell lineage, we suspected that a similar fate
transformation might cause the increase in GFP-positive cells in
other lineages in bet-1 mutants. To test this hypothesis, all of the
DEVELOPMENT
Fig. 2. Excess production of PDEs, AVMs and DTCs in bet-1 mutants. (A-L) Merged GFP and Nomarski images showing the expression of
markers in wild type (WT) or bet-1 mutants at the adult stage. Anterior is to the left, ventral is to the bottom. Arrows indicate gfp-positive cells. The
expression of osm-6::gfp (A,B) or dat-1::gfp (C,D) (markers for PDE) in the right PLG, lag-2::gfp (E,F) in the gonad and mec-4::gfp (G,H), mec-7::gfp
(I,J) or mec-3::gfp (K,L) (a marker for AVM) in the anterior right side of the body is shown. Arrowheads in G and H indicate the ALM neurons that
can be clearly distinguished from AVM neurons by their positions. (M) Bar graphs showing the percentage of animals that had the number of
marker-positive cells indicated on the right. dop-3::rfp was used as a marker for the PVD neuron. Data for DTCs represent the number of DTCs in
the anterior or posterior half of the gonad (also in N). (N) Comparisons of the number of marker-positive cells between the middle larval stages and
the adult stage. GFP-positive cells at larval stages were counted 28 hours after hatching for dat-1::gfp, 19 hours for lag-2::gfp, 14 hours for mec4::gfp and 10 hours for mec-3::gfp. ‘Cell type’ in M and N indicates the cell types that express the corresponding markers in wild-type animals.
Scale bars: 10 mm in A-D,G-L; 100 mm in E,F.
GFP-positive cells near the normal position of these cell types were
ablated by laser during the middle-larval stages. After the ablation,
new GFP-positive cells appeared until the adult stage in bet-1
mutants but not in wild type (see Table S2 in the supplementary
material). These results indicated that GFP-negative cells started to
express GFP during the larval stages in bet-1 mutants.
To determine which cells became ectopic PDEs, AVMs and DTCs
in bet-1 mutants, we ablated their normal precursors (V5.pa, QR or
Z1/Z4, respectively). These cell types were not produced in the
ablated animals (see Table S2 in the supplementary material),
indicating that all of the ectopic cells of each cell type in bet-1
mutants were produced from the same precursor that normally
produces these cell types. This experiment also confirmed that the
ectopic mec-4- and mec-7-expressing cells were AVM-like neurons
derived from QR rather than other touch neurons like ALM.
To analyze the fates of the V5.pa progeny, we followed the
lineages of the V5.pa cell in bet-1 mutants carrying osm-6::gfp,
which is expressed earlier than dat-1::gfp. We began our observation
during the L2 stage and followed the cell fates until late L2, when
osm-6 expression begins in wild-type animals. The lineage analyses
showed that the cell-division patterns during the L2 stage were
abnormal in 4 of 11 bet-1 mutants, suggesting that cell fates might
not be correctly specified in these animals (see Fig. S6Bh-k in the
supplementary material). However, in the remaining 7 animals, in
terms of cell-division patterns, the daughters of the V5.paa cell
acquired distinct fates, as in wild type. In 5 of these 7 animals, osm6::gfp expression was observed only in V5.paaa, which is the PDE
neuron in wild-type animals (see Fig. S6A,Ba-e in the
supplementary material). When these five animals were recovered
from the slide glasses and allowed to grow to the adult stage, ectopic
osm-6::gfp expression was observed in cells with the positions of
the nieces of the original PDE (V5.paapa or V5.paapp; see Fig.
S6Ba-e in the supplementary material). Therefore, in these animals,
V5.paap (the sister of PDE and the mother of PVD in wild type) and
its progeny appeared to behave normally in terms of their cell
divisions and their initial non-expression of osm-6::gfp, but they
subsequently adopted the PDE fate. Consistent with the initially
normal behavior of these cells, most of bet-1 mutants (93%, n=30)
had one mec-3::gfp (marker for the PVD neuron)-positive cell,
similar to wild-type animals (97%, n=30) (Way and Chalfie, 1988),
in the right PLG at the early L3 stage (see Fig. S7A,B in the
supplementary material), suggesting that the PVD fate was specified
normally. Our results indicate that cells that initially acquired nonPDE fates (e.g. the PVD fate) subsequently adopted the PDE fate,
similar to the fate transformation observed for the T.p progeny.
Consistent with the possible transformation from the PVD to the
PDE fate, the PVD marker mec-3::gfp was not expressed in 38%
(n=29) of the bet-1 adult animals.
The above results for the V5.pa lineage indicate that cells that
were initially osm-6 (a PDE marker)-negative at the L2 stage
became osm-6-positive by the adult stage. However, unexpectedly,
we found that the number of osm-6-positive cells decreased slightly
between L3 and the adult stage, despite the increase in dat-1 (another
PDE marker)-positive cells (these markers are expressed in
equivalent numbers of cells at the adult stage; see Table S3 in the
supplementary material). Similarly, although the number of mec-3
(a PVD marker)-positive cells decreased after the L3 stage, bet-1
adults had more cells that were positive for dop-3::rfp (another PVD
marker) than did the L3 larvae. This phenotype can be explained by
considering that OSM-6 (intraflagellar transporter required for cilia
formation) (Collet et al., 1998) and MEC-3 (transcription factor) are
early differentiation markers, whereas DAT-1 (dopamine
RESEARCH ARTICLE 1049
Fig. 3. Cell fate transformations in the V5.pa lineage of bet-1
mutants. (A-D) Fluorescence images of WT (A) and three individual
bet-1 mutants (B-D). Changes in marker expression between the L3 and
adult stages in the right PLG. The left and right panels are images of
the same region of the animals. The expression was observed at the L3
stage and then at the adult stage after the recovery and growth of
individual animals. Arrowheads indicate the cells with altered marker
expression between the two stages. The arrows pointing downward
between pairs of images indicate the passage of time. Asterisks indicate
the positions of neuronal nuclei detected in the Nomarski images.
Anterior is to the left, ventral is to the bottom. Scale bars: 10 mm.
transporter) (Nass et al., 2002) and DOP-3 (dopamine transporter)
(Chase et al., 2004) are terminal differentiation markers required for
neuronal function. In fact, mec-3::gfp and osm-6::gfp expression
started earlier than the expression of dop-3::rfp and dat-1::gfp in
wild type. Our lineage analyses indicated that, in bet-1 mutants, cells
that initially differentiated as non-PDE cells, which might have
included PVD-like neurons, tended to be transformed to the PDE
fate; the beginning of the PDE differentiation program was marked
by the expression of osm-6. However, the fates of these cells might
still have been unstable until they fully differentiated to express dat-
DEVELOPMENT
BET-1 and MYST HATs maintain cell fates
1050 RESEARCH ARTICLE
Development 137 (7)
Fig. 4. bet-1 is required during the acquisition and maintenance of cell fates. (A-C) Schematic diagram of the T (A), QR (B) or V5.pa (C)
lineages in wild-type animals is shown in the upper part of each panel. In the lower part, bar graphs show the percentage of animals with the Psa
phenotype at the L2 stage (A), multiple AVM-like cells expressing mec-4::gfp (B) or multiple PDE-like cells expressing dat-1::gfp at the adult stage
after heat-shock treatment (hs; C) given at the indicated time after hatching. * represents P<0.005 (A,C) or P<0.05 (B) and ** represents P>0.2 (A),
P>0.7 (B) or P>0.1 (C) when compared with the results of no heat shock. *** represents P<0.05 (C) when compared with the results of two heatshock treatments of 10 minutes each, 16 and 24 hours after hatching.
adult stage. Although all the cells in the PLG had expressed osm-6,
but not dop-3, after the ablation at the L3 stage, dop-3-positive osm6-negative cells were observed at the adult stage (4 of 29 animals;
see Fig. S7D in the supplementary material). Taken together, our
data show that in bet-1 mutants, the cell fates were unstable and the
cells could be transformed into other cell types even after the
initiation of their differentiation program.
bet-1 functions to establish and maintain stable
cell fates
To determine when bet-1 is required for the neural differentiation of
T.p progeny, BET-1::GFP expression was induced using a heatshock promoter at various times during development. Heat-shock
treatment at 3 hours after hatching caused the initial GFP
fluorescence to be detected 4-5 hours after hatching, which is the
time of T cell division. We found that the heat shock treatment
before or at 3 hours after hatching rescued the defect in the neural
differentiation of T.p progeny in bet-1 mutants (Fig. 4A). By
contrast, heat-shock treatment given at 4 hours or later did not rescue
the defect. This result indicated that BET-1 must be expressed at the
time T.p is produced, when the cells acquire neural fates through the
expression of psa-3. This is much earlier than the stage of the fate
transformation that we observed in bet-1 mutants.
Similarly, the multiple-AVM phenotype of bet-1 mutants was
rescued by heat-shock treatment 4 hours after hatching, which is
before the division of the AVM mother (QR.pa), but not by treatment
given at 6 hours or later (Fig. 4B), again indicating that BET-1 must
be expressed much earlier than the time when the bet-1 phenotype
arose (14 hours after hatching at the earliest). Taken together, our
results suggest that BET-1 functions when cells acquire their fates
to stabilize them. Without BET-1 function at this time, the fates of
the cells are transformed at later stages.
In the experiments described above, we could not determine the
requirement for BET-1 at the later stages, when cell fates are
maintained, because some of the BET-1 protein that was transiently
induced by heat shock was likely to persist in the cells for a long
time. In fact, fluorescence from the scm::BET-1::GFP transgene
DEVELOPMENT
1; that is, between the L3 and adult stages they could be transformed
to other cell types, including PVD-like neurons, causing a loss of
osm-6-positive cells and a gain of dop-3-positive cells.
To observe cell-fate transformation in more animals and the
possible transformation between the PDE and PVD fates, we
analyzed strains carrying both osm-6::gfp and dop-3::rfp and
compared their expression between the L3 and adult stages. To
detect changes in marker expression in individual cells, we only
scored animals in which it was easy to identify the same cells at both
stages by their spatial patterns (30 of 49 bet-1 mutant animals). In
wild-type animals, cells expressing one of these markers at the L3
stage (24 hours after hatching) were found at the same position at
the adult stage and still expressed the marker (Fig. 3A; n=23). In
27% (n=30) of bet-1 mutants, osm-6::gfp-negative cells at the L3
stage became osm-6::gfp-positive by the adult stage (Fig. 3B,
arrowhead).
Consistent with the slight decrease in osm-6-positive cells during
the L3 to adult stages described above, we observed animals (50%)
in which cells that expressed osm-6::gfp at the L3 stage lost the
expression by the adult stage (arrowhead in Fig. 3C). In about half
of these adults, dop-3::rfp-expressing cells were found at the same
position as the osm-6::gfp-expressing cells at the L3 stage (Fig. 3D),
suggesting a transformation from PDE to PVD (23% of the total
animals scored). In addition, in 10% of these animals, we observed
cells that expressed both markers at the L3 stage (see Fig. S7C in the
supplementary material). These might represent cells in transition
from the PDE to PVD fate, because all of them expressed only dop3::rfp at the adult stage (data not shown; GFP fluorescence at the L3
stage in these animals might represent residual GFP protein retained
even after the cells lost the PDE fate.) In terms of dop-3::rfp, 40%
of the animals gained dop-3::rfp-expressing cells by the adult stage.
In addition, in contrast to the observed loss of osm-6 expression in
some bet-1 mutants, no loss of either dat-1 or dop-3 expression was
ever observed between the L3 and adult stages (data not shown).
To confirm the PDE-to-PVD transformation, we ablated the osm6-negative cells and osm-6 dop-3 double-positive cells in the PLG
at the early L3 stage, then observed the marker expression at the
transcribed in the V5.p seam cell (but not in the neural V5.pa
descendants) at early L2 stage could be detected even in adult
animals and the transgene could efficiently rescue the multiple-PDE
phenotype of bet-1(os46) mutants (6% defective, n=50). To
circumvent this problem, we used a BET-1 protein tagged with a
destabilization domain (DD), which induces the proteasomedependent degradation of tagged proteins (Banaszynski et al., 2006).
When DD::BET-1::GFP was induced by a 10-minute heat-shock
treatment given 16 hours after hatching, the percentage of animals
with GFP fluorescence in the V5.pa descendants had greatly
decreased by 8 hours after the heat shock, compared with the
percentage at 3 hours post-heat shock (see Fig. S8 in the
supplementary material). A similar decrease was not observed in
animals expressing BET-1::GFP without the DD. Therefore, the DD
destabilized the tagged protein in C. elegans.
Using this construct, we first confirmed that BET-1 was required
at the time of fate acquisition, by demonstrating that a 30-minute
heat-shock induction of DD::BET-1::GFP significantly rescued the
multiple-PDE phenotype of bet-1 mutants before (16 hours after
hatching), but not after (24 hours after hatching), the production of
V5.pa-derived neural cells, which are born 19-22 hours after
hatching (Fig. 4C). Weak induction by a 10-minute heat-shock
treatment given 16 hours after hatching was not sufficient to provide
significant rescue. However, an additional 10-minute heat-shock
given 24 hours after hatching provided significant rescue of the
phenotype (Fig. 4C; 10 minutes ⫻ 2). These results indicated that
BET-1 is required for cell-fate maintenance after the acquisition of
cell fate.
Disruption of MYST HATs causes the bet-1-like
phenotype
Because the bromodomain is known to bind acetylated histone,
reducing the histone acetylation by disrupting certain histone
acetyltransferases (HATs) might cause a Bet-1-like phenotype.
Therefore, we performed inactivation of the C. elegans HAT genes,
the Gcn5 family gene, pcaf-1, or the MYST family genes mys-1
(Ceol and Horvitz, 2004), mys-2(K03D10.3), mys-3(R07B5.8) and
mys-4(C34B7.4), by mutation (mys-1) or RNAi. We could not
examine the p300/CBP family gene, cbp-1, because its inactivation
by RNAi caused embryonic lethality. The mys-1 mutants showed the
extra-DTCs phenotype, which was enhanced by mys-2(RNAi) (Fig.
5A; see Fig. S5B in the supplementary material). As in bet-1
mutants, the extra-DTCs phenotype was weaker at the larval stage.
Furthermore, when the DTCs were laser-ablated during the L2 stage,
DTCs were still produced, indicating that cells initially fated to be
non-DTCs transformed into DTCs (see Table S2 in the
supplementary material). In addition, mys-2(RNAi) treatment in the
mys-1 mutants caused the Psa phenotype (3.6%) and induced
multiple dat-1::gfp-positive cells in the PLG (7%), as observed in
bet-1 mutants. Finally, mys-1(RNAi) enhanced the extra DTC
phenotype of the weaker bet-1 allele, gk425, but not that of the
putative null allele os46 (see Fig. S9 in the supplementary material),
indicating that the function of mys-1 is dependent on the bet-1
activity. These results indicate that MYS-1 and MYS-2 function in
the BET-1-mediated maintenance of cell fates.
bet-1 subnuclear localization is dependent on
MYST HATs
MYST HATs primarily acetylate histone H4 (Clarke et al., 1999;
Kimura and Horikoshi, 1998). We found that BET-1 bound strongly
to a histone H4 peptide acetylated at four Lys residues (K5, K8, K12
and K16) but only weakly to the unacetylated H4 peptide or to the
RESEARCH ARTICLE 1051
Fig. 5. Functions of MYST HATs in cell-fate regulation and the
localization of BET-1. (A) The percentage of animals with extra DTCs.
‘L2/L3 stage’ for the mys-1(n4075) mys-2(RNAi) animals, which grew
very slowly, indicates stages after PLG formation and before divisions of
vulval precursor cells. * and ** represent P<0.005 when compared with
mock-treated wild-type animals and mys-1(n4075) mys-2(RNAi) at the
L2/L3 stage, respectively. (B-D) BET-1::GFP localization in hypodermal
cells, including seam cells (arrows), at the L3/L4 stage in wild type (B)
and mys-1(n4075) mys-2(RNAi) (C) and psa-1(os22) (D) mutants.
Anterior is to the left, ventral is to the bottom. Scale bars: 5 mm.
acetylated or unacetylated H3 peptide in vitro (see Fig. S10 in the
supplementary material). Interestingly, BET-1 bound much more
weakly to H4 peptide that was singly acetylated at any residue or
diacetylated (K5 and K12 or K8 and K16) than to the tetraacetylated form, suggesting that acetylation had a synergistic effect
on BET-1 binding. These results indicate that BET-1 recognizes
acetylated histone H4.
To determine whether the BET-1 localization depends on MYS1 and MYS-2, the BET-1::GFP distribution was observed in mys-1and mys-2-deficient animals at late larval stages in seam cells, which
have large nuclei and are suitable for observing subnuclear
localization. In wild-type animals, many puncta indicating the BET1::GFP signal were observed in all cases (n=15). By contrast, in the
mys-1- and mys-2-deficient animals, these puncta were greatly
reduced (6 of 21 animals) or absent (15 of 21 animals) (Fig. 5B,C).
We did not observe such effects on BET-1 localization in mutants of
another chromatin regulator, PSA-1/SWI3 (Fig. 5D) (Sawa et al.,
2000). These results indicate that the subnuclear localization of
BET-1 depends on MYS-1 and MYS-2.
DISCUSSION
bet-1 establishes and maintains stable cell fates
In a small percentage of bet-1 animals, cell fates were initially
abnormal, suggesting that bet-1 is required for correct fate
specification. In the majority of bet-1 mutants, however, cells
acquire the correct cell fates initially but cannot maintain them,
DEVELOPMENT
BET-1 and MYST HATs maintain cell fates
1052 RESEARCH ARTICLE
How does BET-1 function in cell-fate maintenance?
Based on our results and observations in other organisms, we can
propose a possible model for how BET-1 and histone acetylation
might maintain cell fates. We suggest that when cells acquire new
fates, cell-fate regulators recruit HAT to the promoters of fatedeterminant genes to create the initial acetylated nucleosome(s).
Because a Brd2 that is most similar to BET-1 in mammals associates
with a histone H4-specific HAT activity, possibly by MYST HAT
(Sinha et al., 2005), the initial acetylation of promoters might recruit
BET-1–MYST HAT complexes, which acetylate the histone H4 of
adjacent nucleosomes. Repeating this process might create larger
and more stable acetylated genomic regions that help to maintain the
transcriptional state for a longer period. A similar mechanism was
proposed for the function of a complex containing SUVAR39H1 and
HP1, which are a histone methyltransferase and a protein that
recognizes methylation on lysine 9 of histone H3, respectively, in
fission yeast (Bannister et al., 2001). During the period of fate
maintenance following fate acquisition, BET-1 might protect
acetylated histones from HDACs, given that yeast Bdf1 protects
histone H4 from deacetylation by Sir2/HDAC both in vitro and in
vivo (Ladurner et al., 2003).
Epigenetic regulation of cell-fate maintenance
through histone acetylation
Although chromatin hypoacetylation is observed during mitosis in
mammalian cells, the level of acetylation on lysines 12 and 16 of H4
(H4K12Ac and H4K16Ac) is preserved, even during mitosis
(Kruhlak et al., 2001), suggesting that these acetylation patterns
function to maintain the transcriptional status, similar to histone
methylation. In fact, in dosage compensation in Drosophila, the
acetylation of H4K16 by a MYST HAT called MOF is required for
the global upregulation of X-linked genes in males (Hilfiker et al.,
1997; Kelley et al., 1995). Although the developmental roles of the
BET-family proteins and MYST HATs remain largely elusive in
vertebrates, the Drosophila BET-family protein FSH is implicated
as an antagonist of polycomb genes that function in cell-fate
maintenance (Chang et al., 2007). Therefore, BET-family proteins
and MYST HATs might also play fundamental roles in maintaining
cell fates and preventing aberrant cell transformation in other
organisms.
Acknowledgements
We thank the Caenorhabditis Genetics Center, which is funded by the National
Institutes of Health National Center for Research Resources, the C. elegans
Gene Knockout Consortium, Yuji Kohara, Martin Chalfie, Randy Blakely and
Cynthia Kenyon for strains and reagents, and members of the Sawa laboratory
and Junichi Nakayama for helpful discussions and comments. This work was
supported by the Special Postdoctoral Researchers Program, Riken (Y.S.) and by
grants from the Japanese Ministry of Education, Culture, Sports, Science and
Technology (Y.S. and H.S.).
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.042812/-/DC1
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