1 Department of Molecular and Cell Biology, Institute of Medical Sciences,
University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK
2 Institute of Cell Biology, University of Bern, 3012 Bern, Switzerland
* Author for correspondence (e-mail:b.mueller{at}abdn.ac.uk )
Accepted 13 November 2001
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Summary |
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Key words: Development, Histone gene expression, Mitosis
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Introduction |
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The presence of the histone hairpin structure in the C. elegans
replication-dependent histone genes indicates that the post-transcriptional
regulation of these histone genes is also dependent upon HBP activity. We
previously identified the C. elegans HBP homologue on the basis of
its sequence similarity to vertebrate HBP
(Martin et al., 1997).
Sequence conservation between HBPs is highest in the RNA binding domain, and
an alignment of HBP sequences in this region, highlighting the conserved
residues, is shown in Fig. 1A.
Hairpin sequences in the 3' UTRs of the C. elegans core histone
genes deviate at position 11 from the vertebrate hairpin consensus sequence
(Fig. 1B)
(Marzluff, 1992
). At this
position, C. elegans hairpin sequences contain a C, whereas the
vertebrate sequences contain a U. Uniquely, C11 is absolutely essential for
C. elegans HBP RNA binding
(Michel et al., 2000
), which
is indicative of a highly specific interaction between HBP and hairpin RNA.
The binding specificity is intrinsic to the C. elegans RNA-binding
domain (Michel et al.,
2000
).
Here, we analyse HBP expression during C. elegans development,
using a HBP promoter-green fluorescent protein (GFP) fusion construct, and
investigate the role of HBP during C. elegans development, using
RNA-mediated interference (RNAi) (Fire et
al., 1998) to deplete the endogenous levels of HBP during both
embryonic and postembryonic development. Reducing HBP function in this way
results in a decrease in histone protein levels and defects in mitosis
associated with improperly condensed chromosomes, confirming its proposed role
in regulating histone biosynthesis. An identical defect was observed in
embryos depleted of histones H3 and H4 using RNAi. These data demonstrate that
HBP is an essential component required for the correct regulation of histone
biosynthesis during C. elegans development.
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Materials and Methods |
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Nematode strains
Standard C. elegans culturing techniques were used. N2 (Bristol)
and MG152 were used as wild type. MG152 carries an integrated transgene
expressing a GFP-tagged histone H2B
(Kaitna et al., 2000). All
experiments involving this strain were carried out at 25°C.
Construction of R06F6.1::GFP reporter constructs
The genomic region upstream of the R06F6.1 coding region was amplified with
primers ceHBPA (5'-CAATCAGCTGTTCGCGCCGG-3') and ceHBPB
(5'-CTAGAGTCGACCTGCAGGCGTCGGCGAAATCCTCGAC-3') from pFM#6
containing the 7,976 bp PstI fragment of T19E10 encompassing the
R06F6.1 gene, and GFP with a nuclear localisation signal was
amplified from plasmid pPD95.67 (a generous gift from Andy Fire and
co-workers) with primers ceHBPC
(5'-GTCGAGGATTTCGCCGACGCCTGCAGGTCGACTCTAG-3') and GFPD
(5'-GGGAGCTGCATGTGTCAGAG-3') using the Expand High Fidelity PCR
system (Roche). The two PCR products were then combined and the fusion product
was amplified with primers ceHBPD (5'-CGGTGCGAACACACTCACGC-3') and
GFPE (5'-GGCCGACTAGTAGGAAACAG-3') as described
(Hobert et al., 1999). The
fusion product contains 2923 bp upstream of the AUG (and spans the sequence
between R06F6.1 and the preceding gene, T19E10.1), followed
by the sequence coding for the first 41 amino acids of R06F6.1,
spanning 1 intron, fused to the GFP coding region. Products were gel purified
and, after diagnostic restriction digest, used to establish transformed C.
elegans lines employing standard microinjection technique
(Mello and Fire, 1995
).
RNA interference
RNAi by injection was performed essentially as described previously
(Fire et al., 1998). Sense and
antisense transcripts were synthesised separately from linearised pFM#4
(containing a full length HBP cDNA) using appropriate Megascript RNA synthesis
kits (Ambion) and annealed at 70°C for 5 minutes, followed by 20 minutes
at 37°C. Production of double-stranded RNA was verified by non-denaturing
gel electrophoresis. dsRNA was injected at approximately 1 mg/ml into one
gonad arm per animal (this results in RNAi effects in the progeny of both
arms). Injected animals were cultured together for 6-12 hours post-injection.
Progeny laid during this period typically displayed no, or weak, RNAi
phenotypes and were discarded. Single injected animals were then cultured
separately for 24 hours, before being transferred to a fresh plate every 24
hours until no further progeny were produced. The broods from these plates
were then scored for phenotype analyses.
RNAi by feeding was carried out as described previously
(Kamath et al., 2000;
Timmons et al., 2001
). A 580
bp BamHI XhoI R06F6.1 fragment containing HBP
sequence was excised from pFM#4 and inserted into the feeding vector L4440
(Timmons et al., 2001
). The
resultant plasmid, pPE#R7, was then transformed into HT115(DE3) bacteria.
Overnight cultures of the transformed bacteria were used to seed fresh NGM
plates containing 1 mg/ml IPTG and 25 µg/ml carbenicillin. L4 larvae, or
eggs harvested from gravid hermaphrodites, were then added to the plates and
grown at 20°C unless otherwise stated. In the case of L4 larvae, the
animals were transferred to fresh plates 24 hours after the beginning of
egglaying and then transferred again every 24 hours until no more fertilised
oocytes were produced. The progeny laid during the first 24 hours were
discarded since these sometimes showed weaker, incomplete phenotypes. Where
eggs were used to initiate cultures, 10-20 eggs were co-cultured to adulthood
on the same seeded plate, and the postembryonic phenotypes monitored
throughout this period. A 981 bp fragment encompassing the full
his-10 gene (a histone H4 gene) and the his-9 gene (a
histone H3 gene) lacking the most 3' 14 bp was amplified directly from
C. elegans genomic DNA using the primers ceHis34F
(5'-TTATCCTCCGAATCCGTACA-3') and ceHis34R
(5'-CTCGGATACGTCTTGCCAATT-3') using the Expand High Fidelity PCR
System (Roche). The fragment was inserted into pGEM-Teasy (Promega), analysed
by DNA sequencing and excised with EcoRI, and finally inserted into the
feeding vector L4440 to produce plasmid pPE#R11.
Western blotting
Protein extracts were prepared as described previously
(http://info.med.yale.edu/mbb/koelle/protocol_list_page.html). To obtain
R06F6.1(RNAi)-treated animals for preparation of protein extracts,
newly hatched L1 larvae were grown on HT115 bacteria transformed with pPE#R7
until they reached adulthood. Extracts were analysed by 15% SDS-PAGE. Proteins
were transferred onto Hybond-P membrane (Amersham Pharmacia Biotech) using a
semi-dry electroblotting system. Rabbit polyclonal anti-histone H3 antibodies,
goat polyclonal anti-histone H4 antibodies raised against a C-terminal epitope
(Santa Cruz Biotechnology Inc) and mouse monoclonal anti-tubulin antibodies
(Sigma) were used as primary antibodies. Secondary antibodies were anti-rabbit
IgG HRP and anti-goat IgG HRP conjugate (Sigma) and anti-mouse IgG HRP
conjugate (Promega). Antibodies were detected using ECL western blotting
detection reagents (Amersham Pharmacia Biotech)
Immunofluorescent detection of histone H3
Embryos were dissected from gravid hermaphrodites in egg salts
(Edgar, 1995), attached to
poly-L-lysine slides and permeabilised by freeze-cracking
(Miller and Shakes, 1995
). The
embryos were fixed for 5 minutes in methanol at -20°C, followed by 5
minutes in acetone at -20°C. They were then re-hydrated through an
ethanol/PBS wash series, blocked in 30% donkey serum for an hour and incubated
with rabbit anti-histone H3 antibody (Santa Cruz Biotechnology Inc), diluted
1:100 in PBS, overnight at -4°C. After three 10 minute PBS washes, the
slides were incubated for 45 minutes with a rhodamine-conjugated goat
anti-rabbit secondary antibody (TCS Biological), diluted 1:50 in PBS at room
temperature. They were then washed three times in PBS (the first wash
including 1 µg/ml 4',6-diamidino-2-phenolindole dihydrochloride
(DAPI) and mounted in antifade reagent. Fluorescent images of fixed embryos
were obtained as Z-series captured using a Bio-Rad MRC1024 confocal laser
scanning microscope and processed using Confocal Assistant and Adobe
Photoshop.
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Results |
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The C. elegans replication-dependent histone gene clusters lack
genes for the linker histone H1 family
(Roberts et al., 1987), but
there are eight genes coding for histone H1 proteins that have the structure
of replacement variant histone genes, that is they contain introns and lack
the conserved RNA hairpin in the 3' untranslated region
(Jedrusik and Schulze, 2001
).
C. elegans also has a small number of core histone genes with
introns. Replacement variant genes for one histone H2A, H2B and H4 each are
located on chromosome V, two more histone H2A genes are on chromosome IV; and
three further histone H3 genes are on chromosomes III, V and X, respectively.
In addition, two H3-like genes (F58A4.3, F54C8.2) are located on
chromosome V.
The apparent lack of replication-dependent C. elegans histone H1
genes is unusual. Replication-dependent histone H1 genes are found from
Drosophila to man. In Drosophila, the replication-dependent
histone genes are organised as discontinuous arrays of 100 repeats
containing histone H2A, H2B, H3, H4 and H1 genes on chromosome 2
(Pardue et al., 1977
;
Matsuo and Yamazaki, 1989
). In
addition, single copies of replacement variant histone genes, H2A and H4, and
two copies of replacement variant Histone H3 were described
(Adams et al., 2000
). In
humans, genome sequence data indicate that at least 42 replication-dependent
core histone and five replication-dependent H1 histone genes, as well as a
testis-specific H1 gene are clustered on chromosome 6. A further group of at
least three replication-dependent histone genes is located on chromosome 1. At
least another nine histone genes, including replacement variant histone,
testis-specific histone and macroH2A histone genes were found singly on
different chromosomes (Albig and Doenecke,
1997
). Other experimental data suggest that the number of human
histone genes may be higher (Albig et al.,
1993
; Albig et al.,
1997
; Albig and Doenecke,
1997
). In conclusion, the number and organisation of histone genes
in humans and C. elegans is similar: replication-dependent genes are
clustered, and a low number of other histone genes are distributed throughout
the genome. One difference is that C. elegans is apparently lacking
replication-dependent histone H1 genes but has a similar number of replacement
variant H1 genes.
The majority of C. elegans histone genes are of the
replication-dependent type. We have previously described the interaction
between HBP and the RNA hairpin structure in the 3' untranslated region
of these genes (Michel et al.,
2000). Here, we test directly whether the C. elegans HBP
is involved in regulation of histone gene expression.
R06F6.1::GFP reporter construct is expressed in all somatic
cells throughout development
The C. elegans HBP is the gene product of the gene
R06F6.1. To determine where R06F6.1 is expressed, we
generated transgenic lines that expressed a GFP reporter construct under the
control of R06F6.1 upstream sequences. Several lines were generated
and all displayed the same expression pattern. Nuclear GFP expression was
detected in all cells during embryonic and postembryonic development, with
expression appearing strongest in actively dividing cells
(Fig. 2). This was particularly
obvious during postembryonic development, where strong GFP expression was
restricted to proliferating cells and cells undergoing endoreduplication
(Fig. 2B,C). For example, the
cells of the lateral hypodermis divide in a stem-cell-like fashion throughout
postembryonic development and show very strong GFP expression compared to
cells that have left the cell cycle (Fig.
2B,C). Thus, HBP promoter activity is highest in cells undergoing
DNA replication, consistent with a role for HBP in regulating histone gene
expression.
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Depleting HBP function in early embryos results in cell division
arrest
We used RNAi to address the function of the C. elegans HBP.
Depleting HBP in the early embryo, either by growing the parent hermaphrodites
on R06F6.1 dsRNA expressing bacteria or by injecting R06F6.1
dsRNA into the syncitial germline of the parent hermaphrodite, led to the same
embryonic lethal phenotype (Table
2). The first few cell divisions occurred with approximately the
same timing as was observed for untreated, wild-type embryos, but after the
embryos reached approximately 30 cells in size, no further cell divisions were
observed (data not shown).
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In order to better characterise the molecular basis of the
cell-proliferation defects that we observed in R06F6.1(RNAi) embryos,
we used a strain that expresses a histone H2B-GFP fusion protein, which allows
the visualisation of the chromosomes in intact, living embryos
(Kaitna et al., 2000). The
3' untranslated region of this fusion protein is derived from the
unc-54 gene and therefore is not dependent upon HBP for its
expression. Using this strain we followed the chromosomal behaviour during
development in R06F6.1(RNAi) embryos
(Fig. 3). Abnormalities in
chromosomal morphology became apparent during the first mitotic division of
these embryos, with the chromosomes appearing less well condensed during
metaphase and anaphase, than in wild-type embryos; however it was not until
the second mitotic division that more severe defects became apparent.
Chromatin bridges were evident between the two AB daughter cells upon reaching
the end of anaphase, and cytokinesis occurred even though some chromatin
remained at the midline (Fig.
3C,D). Although the nuclear morphology of these AB daughter cells
appeared abnormal as visualised by differential interference contrast (DIC)
optics, they initiated the next round of cell division appropriately. The P1
blastomere division, which occurs 2-3 minutes later, was less abnormal since,
although the chromosomes appeared less well condensed than wild type during
metaphase and anaphase, no chromatin bridges were evident between the P1
daughter cells. The nuclear morphology of the P1 daughter cells under DIC
optics was normal (Fig. 3C,D).
Chromosome condensation during all subsequent cell divisions became
increasingly abnormal, with the chromosomes forming irregular condensations,
rather than tight metaphase plates, accompanied by the presence of chromatin
bridges between daughter cells at the end of anaphase
(Fig. 3G,H).
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R06F6.1 is required for postembryonic divisions
During postembryonic development, the 550 cells present in the newly
hatched larva are added to by the proliferation of multiple cell lineages
(Sulston and Horvitz, 1977).
To determine whether HBP is required during postembryonic cell division we
examined the effect of depleting HBP function by growing newly hatched larvae
on R06F6.1 dsRNA-expressing bacteria. Such R06F6.1(RNAi)
larvae grow up to become sterile adults, which almost invariably show an
abnormally everted vulva (Ev1 phenotype)
(Table 2;
Fig. 4).
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The sterile phenotype suggested possible defects in the proliferation of
the germ cells. The germline in C. elegans originates from two
germline precursor cells, which proliferate mitotically during the first three
larval stages (Kimble and Hirsh,
1979). This phase of germline development is unaffected in
R06F6.1(RNAi) larvae, and most (82%; n=32) animals are able
to produce the first set of mature germ cells, which develop as sperm. In
wild-type animals, the later germ cells mature into oocytes; however, in
R06F6.1(RNAi) animals very few mature oocytes were produced
(n=32), and germline proliferation appeared to cease soon after
reaching adulthood. Thus, it is likely that the sterility reflects a
combination of failure of mitosis in the adult germline coupled with defects
in the maturation of the germ cells into oocytes.
We also examined the development of the vulva and the uterus in
R06F6.1(RNAi) animals, since the Ev1 phenotype arises from defects in
these structures (Seydoux et al.,
1993). The vulva is produced from cells that divide and undergo
morphogenesis during the third and fourth larval stages (L3 and L4,
respectively) (Sulston and Horvitz,
1977
; Sternberg and Horvitz,
1986
). At the same time, the uterus and spermathaecae are
generated from the somatic gonad precursor cells
(Newman et al., 1996
). In
R06F6.1(RNAi) larvae, the three vulval precursor cells, P5.p, P6.p
and P7.p are induced to form vulval cells as in wild type, but fewer than wild
type numbers of vulval cells are generated from these precursors, resulting in
abnormal vulval morphogenesis (Fig.
4). Similarly, the later divisions of the somatic gonad cells,
which generate the uterus and spermathecae, also fail to occur, resulting in
the absence of these structures (Fig.
4).
Postembryonic development is also defective in R06F6.1(RNAi)
males. Like the vulva and uterus, the male copulatory apparatus is also
generated during mid-late postembryonic development, from a limited set of
blast cells that proliferate at around the same time as the hermaphrodite
uterus and vulva (Sulston et al.,
1980). R06F6.1(RNAi) males display defects in the
morphology of this organ (100%, n=24)
(Fig. 4). Although we have not
followed the proliferation of the cell lineages involved in the development of
the copulatory structures in R06F6.1(RNAi) males, the defects we
observed are consistent with failures in the generation of the cells
responsible for producing them.
Thus, R06F6.1(RNAi) results in multiple defects in postembryonic
somatic cell divisions that are consistent with the cell division defects that
we observed in R06F6.1(RNAi) embryos. There are however many cell
lineages that proliferate during early larval development, and it is striking
that we did not observe defects in early cell proliferation events. Mutations
that affect all postembryonic cell divisions result in a sterile, thin,
uncoordinated phenotype reflecting failures in neuroblast and epidermal cell
divisions that generate the mature nervous system and epidermis, respectively
(Albertson et al., 1978;
Sulston and Horvitz, 1981
;
O'Connell et al., 1998
). That
we did not see such defects in R06F6.1(RNAi) larvae and adults may be
attributable to the fact that many of these cells are generated during early
development, before significant depletion of HBP function has been induced by
the ingested dsRNA. However, it has been observed that the nervous system is
generally refractile to RNAi effects (Fire
et al., 1998
), and this may also account for the lack of defects
associated with the generation of this tissue in R06F6.1(RNAi)
animals.
HBP is required for histone gene expression
Taken together, our observations indicate that C. elegans HBP is
required for mitosis during both embryonic and postembryonic development. The
most likely explanation for this is a reduction in histone expression, caused
by decreased HBP-dependent post-transcriptional modification of histone RNA.
In order to confirm this hypothesis, we examined the levels of endogenous H3
and H4 histones in both wild-type and R06F6.1(RNAi) animals. Using
western blot analysis of protein extracts derived from adult worms fed
R06F6.1 dsRNA from hatching, we observed a reduction in the levels of
both H3 and H4 histones when compared with extracts prepared from wild-type
adults, with H4 histone expression being reduced beyond the level of detection
(Fig. 5A). It is not clear why
there are apparent differences in the reduction of histone H3 and H4 levels in
R06F6.1(RNAi) animals. It may be that the histone H3 signal is
stronger because of cross-reactivity of this antibody with the replacement
variants histone H3 proteins and other H3-like proteins
(Table 1). Alternatively, this
may reflect a real difference in histone H3 and H4 protein levels in response
to reduced HBP function.
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The reduction of histone H3 upon treatment with R06F6.1(RNAi) was confirmed by immunostainings in whole, fixed embryos with the anti-histone H3 antibody (Fig. 5B,C) (in our hands, the anti-histone H4 antibody did not recognise its epitope in fixed embryos, using any of the commonly available fixation protocols). Using the anti-H3 antibody, all nuclei were immunostained in 90% of wild-type embryos tested (n=30). In contrast, only 17% of R06F6.1(RNAi) embryos (n=35) showed equivalent levels of immunostaining.
Histone gene expression is required for embryonic and postembryonic
development
As reducing HBP function led to reduced histone expression, it seemed
likely that this was the cause of the RNAi phenotypes we observed. To further
confirm this, we tested whether reducing histone expression directly using
RNAi would reproduce the R06F6.1(RNAi) phenotypes. We therefore
carried out RNAi against his-9 and his-10 (which encode
histone H3 and H4 paralogues, respectively). The nucleotide sequences of both
replication-dependent and replacement histone H4 genes are >85% identical,
and nucleotide sequence identity between histone H3 genes exceeds 77%, with
the replication-dependent H3 genes being >88% identical. Such levels of
identity mean that dsRNA derived from his-9 and his-10 is
likely to inhibit the expression of all H3 and H4 genes, leading to a general
depletion of histone H3 and H4 proteins. his-9/10(RNAi) resulted in
an embryonic-lethal phenotype (Table
2), similar to previous experiments with the his-10 gene
(Maeda et al., 2001), as well
as with a replacement histone H3 gene (Y49E10.6) and a
replication-dependent H2A gene (T23D8.6)
(Gonczy et al., 2000
;
Fraser et al., 2000
). In
contrast, a reduced lethality was observed when another replication-dependent
histone H4 gene was depleted (T23D8.5)
(Fraser et al., 2000
).
his-9/10(RNAi) embryos arrested at the same stage of development as
R06F6.1(RNAi) embryos (Table
2) (data not shown). However, his-9/10(RNAi) additionally
affected the localisation of the H2B::GFP fusion protein, which resulted in
significant decreases in the amount of chromosomal fluorescence. This was
accompanied by increased levels of diffuse cytoplasmic and nuclear
fluorescence (data not shown), suggesting that the H2B fusion protein, while
still expressed, failed to localise to the chromosomes correctly. Assembly of
H2B::GFP into nucleosomes is almost certainly dependent upon the presence of
endogenous histones. Therefore, the greatly reduced incorporation of H2B::GFP
into chromatin is likely to reflect reduced levels of endogenous H3 and H4
core histones caused by his-9/10(RNAi). Since we did not observe the
same reduction in chromosomal fluorescence in R06F6.1(RNAi) embryos,
we take this to mean that his-9/10(RNAi) may be more effective at
depleting H3 and H4 histone levels than R06F6.1(RNAi). This may be
due to reduction in the expression of the replacement histone genes in
addition to the replication-dependent genes. Alternatively, a fraction of the
histone mRNAs may acquire a poly(A) tail in a HBP-depleted background, as is
the case in Drosophila (Sullivan
et al., 2001
), which allows for translation of these mRNAs.
Despite this difference, his-9/10(RNAi) embryos exhibited identical
defects in chromosome behaviour to those we observed in R06F6.1(RNAi)
embryos (Fig. 3F). This
supports our conclusion that the chromosomal defects in embryos depleted for
HBP function are due to reduced levels of histone biosynthesis.
We also examined the effect of depleting histone expression during
postembryonic development, and in this case we observed differences between
the R06F6.1(RNAi) and the his-9/10(RNAi) postembryonic
phenotypes (Table 2). Whereas
R06F6.1(RNAi) larvae displayed a normal growth rate, and appeared
essentially wild type under the dissecting microscope until the late L4/adult
stage, his-9/10(RNAi) larvae grew slowly, were thin and displayed a
translucent appearance under the dissecting microscope. Those animals that
reached adulthood were sterile, as we found for the R06F6.1(RNAi)
animals. In some cases his-9/10(RNAi) adults also displayed a
multivulval (Muv) phenotype (Table
2; Fig. 6),
characteristic of the inappropriate activation of the vulval cell fate in the
vulval precursor cells that normally adopt a non-vulval cell fate
(Fay and Han, 2000). Thus,
although displaying the sterility observed in R06F6.1(RNAi) animals,
his-9/10(RNAi) results in additional postembryonic defects, including
abnormal regulation of cell fate, not observed in R06F6.1(RNAi)
animals.
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Discussion |
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Our observation that HBP promoter activity, as measured by the expression
of GFP fused to the HBP promoter region, is highest in dividing cells is in
apparent contradiction to the observation that in synchronised cell culture,
HBP levels are regulated post-transcriptionally, with mRNA levels being
constant throughout the cell cycle
(Whitfield et al., 2000).
However the situation in C. elegans may be different as during
postembryonic development cells do not continuously go through subsequent cell
cycles, but cell division and DNA replication are programmed and can occur
after long periods of quiescence. Thus, transcriptional regulation of C.
elegans HBP may reflect this difference in the mode of cell division.
However, from our current data we cannot address possible post-transcriptional
regulation of HBP expression.
The phenotype of R06F6.1(RNAi) embryos is similar to the phenotype
of Drosophila embryos lacking the maternal contribution of the
Drosophila HBP homologue, dSLBP
(Sullivan et al., 2001). As in
C. elegans, dSLBP is required for mitotic cell divisions in the early
embryo, and loss of maternal dSLBP function results in defects associated with
chromosome condensation. Similarly, absence of maternal dSLBP, as we found for
R06F6.1(RNAi), does not prevent the first few mitotic divisions, but
results in the gradual accumulation of defects in chromosomal condensation. We
take this to indicate that sufficient processed core histone mRNAs, or core
histone proteins, are provided maternally to support these first few divisions
and that in wild-type embryos these limited supplies of histones are
supplemented by the HBP-dependent expression from maternally loaded,
unprocessed histone mRNA or newly synthesised zygotic histone mRNAs. It is
also possible that in HBP-depleted embryos, as was found for
Drosophila (Sullivan et al.,
2001
), polyadenylation of histone mRNA may occur that would result
in some histone synthesis, thereby allowing a limited number of cell
divisions.
Reducing HBP/SLBP function in the early embryo would thus result in a chromosome condensation defect that grew worse as the limited source of histones became depleted, until insufficient histones were present to allow the packaging of newly synthesised DNA into chromosomes, at which point cell division would cease. This is what was observed in Drosophila embryos with a reduced dSLBP function and is also what we found in the R06F6.1(RNAi) embryos.
Although depletion of histones H3 and H4 caused a similar phenotype to depleting HBP in early embryos, his-9/10(RNAi) larvae exhibited a more severe postembryonic phenotype compared to R06F6.1(RNAi) animals. Some of the differences may be caused by a more severe reduction in histone synthesis in his9/10(RNAi) animals. This was suggested by the finding that the chromosomal localisation of the H2B::GFP fusion protein was significantly impaired by depleting H3 and H4 histones but was not appreciably affected by reducing HBP function. Higher histone synthesis in R06F6.1(RNAi) animals may be due to the expression of the replacement variant histone genes. In addition, it is possible that this is supplemented by replication-dependent histone gene expression brought about by some residual HBP synthesis or by some polyadenylation of histone RNA as discussed above.
More curious is the discrepancy between the effect of
R06F6.1(RNAi) and his-9/10(RNAi) on the fates of the vulva
precursor cells. The Muv phenotype in his-9/10(RNAi) animals may be
caused by a general reduction in histone proteins. In yeast, depletion of
histone H4 protein does not lead to a general change in transcription
(Kim et al., 1988); however in
at least one case this leads to gene activation, presumably due to a change of
the nucleosome structure of the promoter sequence
(Han et al., 1988
). Thus it is
possible that local changes in chromatin structure, brought about by the
depletion of histones, may lead to the aberrant expression of genes involved
in vulval cell fate determination. Alternatively, since the his-9 and
his-10 genes also show high nucleotide identity with replacement
variant histones (i.e. histones not regulated by the HBP-hairpin interaction),
it is possible that the his-9/10(RNAi) inhibits the expression of
replacement variant histone genes that may be essential for vulval cell fate.
Reducing the function of the linker histone H1.1 results in the activation of
otherwise silent transgenes in the germline
(Jedrusik and Schulze, 2001
).
Thus, it is possible that the inhibition of one or more of the replacement
variant histone genes results in the defects in vulval precursor cell fate
that we observed. Further characterisation of the individual role of these
proteins will shed light on their involvement in the regulation of vulval cell
fate.
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Acknowledgments |
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References |
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