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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02698
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Author for correspondence (e-mail:
tih1{at}columbia.edu)
Accepted 16 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Histone, Methylation, Oogenesis, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
best studied of these histone modifications include the methylation of
arginine (R) and lysine (K) residues, acetylation of K residues and
phosphorylation of Serine (S) and Threonine (T) residues. The nucleosomal
diversity created by these modifications forms the basis of the `histone code'
hypothesis, which proposes that chromatin domains, defined by local
combinatorial signatures of histone modifications, have distinct outcomes for
chromatin structure and gene expression
(Strahl and Allis, 2000;
Jenuwein and Allis, 2001).
Of the various modifications made to histones, methylation is one of the
most complex, both in terms of the nature of the signal and its biological
consequences (Lachner and Jenuwein,
2002). Both the site of methylation and the degree of methylation
affect the biological outcome of methylation. For instance, histone H3
methylation at H3K4 (lysine at residue 4 in histone H3), H3K36 and H3K79 is
usually associated with gene activation, whereas methylation at H3K9 and H3K27
leads to gene repression, although exceptions to these rules exist
(Martin and Zhang, 2005).
Furthermore, K residues can be subject to different degrees of methylation
(mono-, di- or trimethylation), with different consequences for the cell
(Rice et al., 2003). These
methyl marks serve as binding sites for proteins that assemble complexes that
in turn regulate chromatin structure and gene transcription
(Daniel et al., 2005).
In 2000, the first histone lysine methyltransferase (HMT) was identified,
Suv39h1, and its enzymatic activity was mapped to its SET domain
(Rea et al., 2000). The SET
domain takes its name from the three Drosophila genes in which it was
first recognized: Su(Var)3-9, Enhancer of zeste (E(z)), and
trithorax (trx). SET domain proteins have been identified
and studied in yeast, plants, worms, flies and mammals. These proteins play
important roles in development (Marguerron et al., 2005), and misexpression of
HMTs occurs in some human cancers
(Santos-Rosa and Caldas,
2005).
Gene silencing in germ cells is a widespread phenomenon, and recent studies
have begun to examine the contributions of histone modifications to germ cell
gene regulation (Pirrotta,
2002). In Caenorhabditis elegans and Drosophila,
formation of the embryonic primordial germ cells is accompanied by changes in
histone acetylation and methylation
(Schaner et al., 2003), and
two maternally expressed SET domain proteins, MES-2 and MES-4, are required in
C. elegans for the viability and proliferation of the germline
(Capowski et al., 1991;
Kelly and Fire, 1998;
Fong et al., 2002;
Bender et al., 2004). Less is
known about histone modifications in the adult gonad. We show here that a SET
domain protein, the product of a Drosophila gene that we have named
eggless (egg), is required for oogenesis at early stages of
egg chamber formation.
Egg is very similar to the human protein SETDB1 and its mouse ortholog ESET
(Schultz et al., 2002;
Yang et al., 2002). These
proteins belong to a small subfamily of HMTs that contain bifurcated SET
domains - that is, SET domains interrupted by insertions of novel stretches of
amino acids (Harte et al.,
1999; Alvarez-Venegas and
Avramova, 2002). The egg mutations we isolated have
provided us with the opportunity to study the role of this subfamily of HMTs
in a developmental context. We show in vivo that Egg catalyzes trimethylation
of histone H3 at its K9 residue (H3K9), and that this modification is present
in both germ and somatic cells during oogenesis. In the absence of
Eggcatalyzed histone methylation, oogenesis arrests at early stages. Egg
chamber formation is defective, and egg chambers never fully bud off from the
germarium. We show that apoptotic cell death and reduced somatic cell
proliferation are likely underlying causes of this early arrest.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EMS mutagenesis screen
A standard ethyl methanesulfonate (EMS) mutagenesis screen was performed to
isolate female-sterile and lethal mutations uncovered by the chromosome 2R
deficiency Df(2R)Dll-Mp(60E1-2;60E6). Males from a cn bw
isoline were fed EMS (0.025 mol/l) in a 1% sucrose solution, and mated en
masse to Tft/CyO virgin females. cn bw/Tft
F1 males were mated singly to
Df(2R)Dll-Mp/SM6 virgins, and the cn
bw/Df(2R)Dll-Mp F2 progeny were screened for
lethality or female sterility. In total 3500 mutagenized second chromosomes
were screened, and 45 lethal or female sterile mutations were recovered. Inter
se crosses placed these 45 mutations into seven complementation groups. One
complementation group consisted of 13 mutations in the gene we named
eggless, described in this report.
Genomic rescue experiments to identify the egg gene
A Drosophila genomic library (courtesy of Jym Mohler, Barnard
College, New York, NY, USA) was screened using a cDNA for the
Exu-associated protein (Eap) gene, which maps near
egg. A 9 kb genomic fragment (GC64N), from one of the clones obtained
in this screen, detected three mRNAs (3.9, 1.1 and 1.4 kb) on northern blots
of ovary RNA. These mRNAs were mapped within GC64N as shown in Fig. S1 in the
supplementary material. The entire GC64N 9 kb fragment, and internally deleted
or truncated DNA fragments (N
1-N4), were subcloned into pCaSpeR4
and transformed into flies. Each genomic fragment was then crossed into
egg/Df(2R)Dll-Mp backgrounds and tested for its
ability to rescue egg-associated female sterility. The rescue pattern
of these constructs (see Fig. S1 in the supplementary material) mapped
egg to the region defined by N2 and N4.
Sequencing and northern blot analysis
Genomic DNA was prepared from hemizygous egg235 or
egg1473 flies as described in Mansfield et al.
(Mansfield et al., 2002).
Amplified DNA from four to six independent PCR reactions using
egg-specific primers was pooled, purified (Qiagen), and used as
template for sequencing with 15 primers designed at intervals of 200-300 bp
encompassing the entire egg coding sequence. (The sequence of these
primers is available on request.) Sequencing was performed on an ABI 3100
capillary sequencer at the Herbert Irving Comprehensive Cancer Center's DNA
Analysis and Sequencing Facility (Columbia University). The breakpoints of the
deletion identified in egg1473 were confirmed by
sequencing a cloned 350 bp DNA fragment spanning the deletion breakpoints. Two
embryo cDNAs, LD15023 (3.9 kb) and LD09692 (3.5 kb) were obtained from the
Drosophila Genome Research Center (DGRC; Bloomington, Indiana), and sequenced
in full. LD15023 is the longest cDNA, and contains a poly(A) tail as well as
an inframe AUG start codon. LD09692 is identical to LD15023, except that it is
285 bp shorter at its 5' end.
Northern analysis was performed following the methods of Mansfield et al.
(Mansfield et al., 2002), with
poly(A+)RNA isolated from hand-dissected ovaries using Ambion's
MicroPoly(A)Pure small scale mRNA purification kit. One microgram of RNA was
electrophoresed through a 1% agaroseformaldehyde gel and transferred to a
Magna NT nylon filter. Probes consisted of 32P-labeled PCR
fragments from either the 5' end (covering exons 1-3) or 3' end
(covering exons 6 and 7) of egg.
Egg antibody production and western analysis
Anti-Egg polyclonal antiserum was raised in rats (Cocalico Biological)
against a bacterially expressed GST fusion protein containing Egg residues
79-315. The DNA encoding these Egg residues was PCR-amplified from an
egg cDNA, LD09692, with primers containing overhanging restriction
sites (EcoRI or XhoI) and cloned into the pGEX-4T-1 vector.
The GST-Egg fusion protein was expressed in Escherichia coli BL21
cells and purified using a MicroSpin GST Purification Module (Amersham). The
identity and purity of the fusion protein were determined by western blot
using an anti-GST antibody. The antibody was affinity purified by incubating
Egg antisera with Cyanogen Bromide (CNBR) Sepharose beads (Amersham) coupled
to purified GST-Egg and eluting the affinity purified Egg antibodies with 100
mmol/l glycine, pH 2.5, followed by dialysis into PBS pH 7.2 and addition of
an equal volume of glycerol. The specificity of the Egg antisera and affinity
purified Egg antibodies were confirmed by the altered levels or patterns of
Egg signals observed by antibody labeling of egg mutant ovaries (data
not shown) and the recognition of a truncated Egg mutant protein (from
egg235) on western blots.
To prepare ovary extracts for western blots, hand-dissected ovaries were homogenized in RIPA Buffer (150 mmol/l NaCl, 1% NP-40, 0.5% Deoxycholate, 0.1% SDS, 50 mmol/l Tris pH 8.0, 5 mmol/l EDTA) with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche), followed by the addition of an equal volume of 2x sample buffer (120 mmol/l Tris pH 6.8, 4% SDS, 20% glycerol, 0.006% Bromophenol Blue, 50 mmol/l DTT). Samples were boiled for 15 minutes, loaded and electrophoresed through a 9% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (MSI) in transfer buffer (39 mmol/l glycine, 48 mmol/l Tris Base, 0.04% SDS, 20% methanol) using a TE42 transfer apparatus (Hoefer Scientific). The membrane was blocked for several hours in 5% non-fat dry milk in PBST (0.1% Tween-20 in PBS). Affinity-purified Egg antibody, 1:170 in 3% BSA in PBST, was incubated with the membrane overnight at 4°C. The secondary antibody, HRP-conjugated goat anti-rat, was hybridized at 1:5000 in 3% BSA in PBST, for 2 hours at room temperature. All washes were done using PBST. Enhanced chemiluminescence (ECL) was used for detection, following company protocols (Amersham).
Whole-mount antibody labeling and microscopy
Ovaries were dissected from flies held on yeasted food, within 1-2 days
after eclosion. The fixation and antibody-labeling protocol follows methods
reported in Moon and Hazelrigg (Moon and
Hazelrigg, 2004). Primary and secondary antibodies were diluted in
0.1% BSA in PBTx (1x PBS, 0.1% Triton X-100) and before DNA staining
with propidium iodide, ovaries were RNaseA treated (1 mg/ml) for 2 hours at
37°C.
The following antibodies were used to label ovaries: rat anti-Egg (1:1000) and anti-Vasa (1:500, A. Ephrussi); rabbit anti-Vasa (1:1000, R. Lehmann), anti-H3K9me2 (1:1000, Upstate), anti-H3K9me3 (1:1000, Upstate), anti-phosphorylated Histone H3 (Ser10) (1:100, Upstate) and anti-cleaved Caspase-3 (1:500, Cell Signaling Technology). Mouse monoclonal antibodies obtained from the Developmental Studies Hybridoma Bank included anti-Orb (1:10, P. Schedl) and anti-Fas III (1:10; C. Goodman). FITC- or Rhodamine-conjugated secondary antibodies included goat anti-rat, rabbit or mouse F(ab')2 fragments, and were used at 1:500 or 1:1000 (Jackson ImmunoResearch).
Microscopy was performed on an inverted Olympus IX71 confocal microscope with Fluoview software. Images were analyzed using Image J software (W. Rasband, NIH, Bethesda, MD, http://rsb.info.nih.gov/IJ/).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (reviewed
by Lachner and Jenuwein,
2002). Egg also contains characteristic cysteine-rich Pre- and
Post-SET domain regions that are necessary for enzymatic activity
(Xiao et al., 2003). Egg is
the sole Drosophila protein to contain a bifurcated SET domain
(Alvarez-Venegas and Avramova,
2002; Mis et al.,
2006), an unusual disruption of the SET domain that characterizes
its closest relative, human/murine SETDB1/ESET
(Schultz et al., 2002;
Yang et al., 2002). C.
elegans also has a bifurcated SET domain protein, MET-2, of unknown
function. Based on sequence comparisons, the SET domain of Egg falls into the
Su(var)3-9 family of SET domains, which have specificity for methylating the
K9 residue of histone H3 (Alvarez-Venegas
and Avramova, 2002; Mis et
al., 2006). The predicted length of Egg (1262 amino acids) is close in size to SETDB1 (1291 amino acids) and ESET (1307 amino acids). A comparison of Egg and SETDB1 revealed that these proteins are 44% identical in the pre-SET plus first half of the SET domain, and 76% identical in the second half of the SET domain plus the post-SET domain. The MBD domains are less conserved (29% identical). While the putative Tudor domains in Egg were predicted with subthreshold E-values by the SMART protein domain identification program [http://smart.embl.de/], the sequence similarity with SETDB1 in this region is high (44% identical). The amino acid segment that divides the SET domain of Egg is much shorter than the segment that interrupts the SET domain of SETDB1 or ESET (91 amino acids in Egg compared to 338 in SETDB1 and 337 in ESET) but is close in size to the corresponding segment of MET-2 (103 amino acids).
We sequenced two egg alleles that are members of the strong class of alleles. The first, egg235, contained two nucleotide substitutions at the 5' end of intron 4 (Fig. 1E). Failure to splice out this intron would yield an mRNA with an in-frame stop codon just within the intron, encoding a truncated protein lacking any of the identifiable domains of Egg. The second allele, egg1473, contained an internal 856 bp inframe deletion. The predicted protein product of egg1473 lacks the entire SET domain (Fig. 1F).
Egg is expressed during oogenesis
We raised a polyclonal antibody to a unique N-terminal segment of Egg (see
Materials and methods). This antibody labels two protein bands on ovary
western blots, 
170 kDa and 140 kDa
(Fig. 1B). Both proteins were
absent or strongly reduced in egg235 ovaries, replaced by
a smaller truncated protein, indicating that both are egg products.
We used this antibody to label ovaries to determine the expression pattern and
subcellular distribution of Egg during oogenesis
(Fig. 2).
Drosophila ovaries consist of 15-20 ovarioles that hold egg
chambers in progressive stages of development
(Spradling, 1993). These egg
chambers contain germ cells derived from germ stem cells located in the
germarium, a structure at the tip of each ovariole. At their anterior ends
(region 1), each germarium houses two to three germ stem cells (GSCs) that
divide asymmetrically to produce another GSC, which replenishes the GSC
population, and a cystoblast (CB). The CB undergoes four incomplete mitotic
divisions (the dividing cells are called cystocytes) to yield a 16-cell
germline cyst; one of these 16 germ cells becomes the oocyte, and the other 15
germ cells form the nurse cells. After germline cysts are formed, they become
encapsulated (in region 2 of the germarium) by somatic prefollicular cells to
form an egg chamber. At the posterior end of the germarium (region 3) lies a
single stage 1 egg chamber, ready to bud off from the germarium and proceed
through the rest of oogenesis.
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Loss of the Egg SET domain causes early oogenesis arrest
Both of the strong alleles with sequences we report here,
egg235 and egg1473, had similar
oogenesis phenotypes. As egg235 may express low levels of
Egg (Fig. 1B), by inefficient
splicing of the fourth intron, it may provide some egg function.
However, as egg1473 lacks the entire SET domain
(Fig. 1F), it provided us with
the opportunity to determine the consequences to oogenesis of eliminating the
HMT function of egg.
Oogenesis arrested very early in egg1473 females (Fig. 3). (In all cases, egg1473 refers to the genotype egg1473/Df(2R)Dll-Mp.) egg1473 ovaries were tiny, consisting of germaria that did not bud off normal egg chambers. Many egg1473 germaria lacked recognizable regions and instead appeared teardrop-shaped, full of replicating germ cells (see below) and germline cysts that formed no apparent egg chambers (Fig. 3A, middle panels). Disc-shaped germline cysts, which form as germ cells, are encapsulated by prefollicular cells in region 2b of the germarium, were absent in egg germaria. In some cases, egg germaria had an incompletely budded egg chamber at their posterior ends (Fig. 3A, bottom panel).
Labeling with an antibody to Fasciclin III (Fas III), a membraneassociated protein present in prefollicle and follicle cells, demonstrated that these somatic cell types were present in egg1473 germaria (Fig. 3B). However, these cells were reduced in number and failed to completely encapsulate germline cysts. In the partially encapsulated cysts that did exist, germ cells showed at least partial differentiation as nurse cells and oocytes, as judged by the chromosome morphology of the nurse cells, and the accumulation of Orb protein in the oocyte (not shown). In most cases the nurse cells at the posterior end of the mutant germaria had endoreplicated DNA, typical of later stage nurse cell nuclei (Fig. 3A,B).
Egg mediates trimethylation of histone H3 at its K9 residue
As ESET/SETDB1 methylates histone H3 at lysine 9 (H3K9)
(Schultz et al., 2002;
Yang et al., 2002;
Wang et al., 2003;
Yang et al., 2003), we
examined histone methylation in egg1473 ovaries to
determine if Egg also methylates H3K9. Histone methylation patterns in
Drosophila oogenesis have not previously been described, so we first
determined the patterns of H3K9 methylation in wild-type ovaries using
antibodies that specifically recognize either the dimethylated or
trimethylated forms of H3K9 (Fig.
4). We found that both dimethyl H3K9 (H3K9me2) and trimethyl H3K9
(H3K9me3) were present in germ cell and somatic cell nuclei during oogenesis,
and occurred with distinct patterns. In the germarium the H3K9me2 signal was
strong in both the somatic and germ cells, and as oogenesis proceeded the
signal remained high in the somatic cells of egg chambers, while germ cell
signal decreased (Fig. 4A). By
contrast, while H3K9me3 could be detected in both the germ cells and somatic
cells of the ovary, it was always more abundant in the germ cells
(Fig. 4B). Not every somatic
cell showed signal, and no clear pattern of H3K9me3 was present in the soma.
The H3K9me3 signal was strongest in the germarium, and decreased as oogenesis
proceeded. In the oocyte, strong H3K9me2 and H3K9me3 signals were present in
the germinal vesicle, the oocyte nucleus, throughout most of oogenesis.
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Reduced somatic cell proliferation and apoptotic cell death in egg ovaries
The failure of egg1473 egg chambers to bud off the
germarium suggests that encapsulation of germline cysts by somatic cells is
not normal in egg ovaries. This was confirmed by Fas III antibody
labeling, which showed that the prefollicle and follicle cell populations were
reduced in egg1473 germaria
(Fig. 3B). To determine the
proliferative capacities of the germ and somatic cells of egg
ovaries, we labeled wild-type or egg1473 germaria with an
antibody to phosphorylated H3 (Ser 10), a marker of mitotic cells
(Fig. 6)
(Sauve et al., 1999). Ovaries
were simultaneously labeled with an antibody to Vasa, to distinguish whether
observed mitoses were occurring in germ cells or somatic cells.
Germ cell mitoses were observed as single cells dividing (GSC or CB divisions) or as multiple nuclei dividing together (cystocytes): two, four or eight nuclei in a group. Examples of all categories of germ cell divisions were observed in egg1473 germaria in a frequency not significantly different from control germ cells (Fig. 6C). Although there was a slight increase in the number of mutant germaria with no germ cell mitoses, this increase was not statistically significant.
By contrast, mitotic frequencies in the somatic cells (identified by lack
of Vasa labeling) of egg1473 germaria showed a marked and
statistically significant (P<0.001; n=21) decrease
compared with wild-type somatic cells (Fig.
6D). Of 21 control germaria counted, 18 contained at least one
mitotic somatic cell and frequently had multiple somatic cells simultaneously
undergoing mitosis (we observed a maximum of four). In wild-type germaria,
somatic cell mitoses were observed in regions 2b and 3 of the germarium, where
dividing prefollicle and follicle cells lie, and also in single somatic cells
about midway through the germarium, near the 2a/b border, where the somatic
stem cells reside (Zhang and Kalderon,
2001). By contrast, only two of 21 egg1473
germaria contained a single mitotic somatic cell. In both cases, the mitotic
cells were located at the posterior end of the germarium. These observations
indicate that the proliferation of prefollicle and follicle cells, and
possibly also somatic stem cells, is strongly reduced in
egg1473 ovaries.
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). We
observed abundant levels of apoptosis in egg1473 germaria:
of 24 egg1473 germaria, 21 showed positive labeling with
the antibody to cleaved Caspase-3, whereas only 1 of 27 control germaria was
labeled. While apoptosis was observed in both the germ and somatic cells of
egg1473 germaria, it was predominantly seen within the
somatic cells (Fig. 7). Similar
results were obtained using the TUNEL method for detecting apoptosis (not
shown). The majority of somatic apoptotic cells reside in the posterior region
of egg1473 germaria, in cells found between incompletely
budded egg chambers and anterior germ cells. The follicle cells associated
with the incompletely budded egg chamber were also frequently undergoing
apoptosis. Apoptotic somatic cells at the anterior of
egg1473 germaria (including terminal filament and cap
cells) were never observed (n=24). The death and reduced
proliferation of somatic cells that give rise to follicle cells and stalk
cells provides an explanation for the failure of egg mutants to bud
off fully encapsulated egg chambers. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mis et al.,
2006), and is most similar to the human protein SETDB1 and its
mouse ortholog ESET (Schultz et al.,
2002; Yang et al.,
2002). While functional rescue experiments have not yet shown if
the mammalian proteins can substitute for Egg in vivo, the similarity in size,
overall domain architecture and high sequence similarity within their shared
domains suggests that they are likely to be functional orthologs.
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;
Yang et al., 2003). Despite
these important studies, the biological roles of SETDB1 and ESET in
development are poorly understood. RNAi knockdown of SETDB1 in tissue culture
cells is cell lethal (Wang et al.,
2003; Sarraf and Stancheva,
2004), and the very early embryonic lethality of a mouse ESET
insertion allele, along with possible maternal contributions of its gene
product, has complicated the analysis of its in vivo functions
(Dodge et al., 2004). The
genetic analysis of egg reported here has allowed us to examine the
in vivo contributions of this HMT in a developmental context, and to show that
Egg plays an essential role in oogenesis. The strong expression of ESET in
testes suggests that the mammalian proteins may also play similar roles in
gametogenesis (Yang et al.,
2002).
Biochemical studies demonstrated that ESET and SETDB1 methylate histone H3
at its K9 residue (Schultz et al.,
2002; Yang et al.,
2002; Wang et al.,
2003; Yang et al.,
2003), and we show here that in vivo Egg also has H3K9 HMT
activity. Specifically, we found that trimethylation of histone H3K9 occurred
during oogenesis, in both the germ cells and somatic cells, in an
egg-dependent manner (Fig.
5). Egg does not appear to be required for dimethylation of H3K9,
as the H3K9me2 signal remained strong in egg1473 ovaries.
These observations suggest a pathway for H3K9 methylation, with Egg catalyzing
the addition of a terminal methyl group to H3K9me2, previously established by
a separate HMT.
Egg is present in germ cells at the earliest stages of oogenesis, including germ stem cells, cystoblasts, dividing cystocytes and newly formed germline cysts (Fig. 2A). While we have not detected Egg in anterior somatic cells, including terminal filament, cap and interstitial cells, low levels of Egg were present in more posterior somatic cells, including prefollicular cells and follicle cells of stage 1 egg chambers. However, Egg was also expressed in postgermarial egg chambers (Fig. 2B), and is therefore likely to have functions at later stages of oogenesis as well. Of particular interest is the strong accumulation of Egg in the oocyte nucleus, in distinct subnuclear foci. The oocyte nucleus is arrested at prophase of meiosis I, and is generally transcriptionally quiescent, raising the possibility that Egg could contribute to transcriptional repression in the oocyte and/or meiotic cell cycle control.
Strong egg alleles, including egg1473, which deletes the entire SET-domain-coding region, caused very early arrest of oogenesis. Mutant ovaries consisted of germaria in which the early stages of egg chamber formation were not clearly demarcated. While proliferating germ cells were present, the existing germline cysts were not fully encapsulated by somatic follicle cells, and did not bud off normally from the germarium.
We have shown that egg is required for the proliferation and viability of somatic cells in the germarium, and that a reduction in somatic cell populations is likely to be the cause of the encapsulation and budding defects observed in mutant germaria (Figs 6 and 7). In wild-type germaria, we observed mitosis in prefollicular and follicle cell populations, as well as in single cells located near the 2a/b border, where somatic stem cells reside. As the only examples of somatic cells undergoing mitosis in egg1473 ovaries were cells positioned at the posterior end of germaria, we think it is likely that egg affects proliferation of at least three populations of somatic cells: the somatic stem cells, the prefollicular cells and the follicle cells that surround newly formed egg chambers. egg is also required for the viability of both the germ and somatic cells, as apoptotic cell death occurred in both cell types in egg1473 ovaries.
Several HMTs play roles in cell proliferation, and aberrant HMT expression
is in some cases oncogenic (reviewed by
Santos-Rosa and Caldas, 2005).
Histone methylation can impinge on cell proliferation by either of two routes:
by regulating the expression of genes that in turn regulate the cell cycle
(Nielsen et al., 2001), or by
promoting structural changes in chromosomes necessary for mitosis
(Melcher et al., 2000). SETDB1
has recently been shown to function at promoters that are silenced in human
cancers, suggesting that it too may normally play a role in regulating cell
proliferation (Li et al.,
2006).
There are important questions that remain to be answered. We have shown
that Egg mediates H3K9 methylation in early oogenesis, and that loss of
H3K9me3 has striking biological consequences for oogenesis, but we do not yet
know the exact genomic effects of this methylation program. Methylation of
H3K9 plays an important role in the formation of heterochromatin domains, and
also regulates the expression of individual euchromatic genes
(Lachner and Jenuwein, 2002).
Our analysis of Egg localization in whole-mount ovaries indicates that it is
associated with distinct foci within germ and somatic cell nuclei
(Fig. 2A), but the small size
of these chromosomes has not allowed us to precisely map these sites. A goal
of future work will be to identify the genomic targets of Egg, a necessary and
important first step in determining whether Egg regulates euchromatic gene
expression or plays a role in establishing heterochromatic domains.
Another important goal of future work is to determine which cell types
require egg activity during early oogenesis. While we have shown that
Egg is expressed in both germ cell and somatic cell populations in the ovary,
and mediates H3K9 methylation in both cell types, the fact that numerous germ
cell-somatic cell interactions contribute to early oogenesis
(Roth, 2001) implies that the
functional consequences of perturbations in histone methylation patterns in
one cell type could impact on the development of other cells. Thus somatic
cell defects could arise from loss of H3K9me3 in germ cells, and vice versa.
Future experiments, using clonal analysis and the expression of egg
transgenes in specific cell types, should allow us to determine unequivocally
which cells require the HMT activity of egg.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/1/02698/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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