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First published online 19 July 2006
doi: 10.1242/dev.02490
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Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA.
* Author for correspondence (e-mail: valerie.reinke{at}yale.edu)
Accepted 31 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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Key words: C. elegans, Germline, E2F, Microarrays, Gene expression, pRB, DP
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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).
Phosphorylation of pRB by cyclinD/cdk4 releases E2F, which is then free to
activate genes important for DNA synthesis, promoting progression from
G1 into S phase. However, the roles of pRB and E2F are more diverse
than this model indicates, as these proteins modulate apoptosis, senescence
and differentiation, as well as cell cycle progression (reviewed by
Dimova and Dyson, 2005;
Cobrinik, 2005).
Greatly complicating the understanding of this crucial pathway is the fact
that each of these proteins is a member of a gene family. The mouse and human
genomes each encode three pRB-like `pocket' proteins, eight E2F-like proteins
and two DP-like proteins (reviewed by
Dimova and Dyson, 2005).
Family members can exhibit both redundant and independent functions. For
instance, loss of pRB is compensated by the activity of another pocket
protein, p107, in mouse embryo fibroblasts
(Sage et al., 2003). p107 and
the third pocket protein p130 have independent functions as well, as they
typically bind to a different set of E2Fs than does pRB (reviewed by
Trimarchi and Lees, 2002). E2F
proteins are functionally divided into those that activate transcription
(E2F1-3) and those that repress transcription (E2F4-8). Activator E2Fs are
primarily bound by pRB, repressors E2F4 and E2F5 are bound by p107 and p130,
while E2F6-8 lack any pocket protein-binding domain. Moreover, deletion
mutations in any of six endogenous E2F genes in mice do not display the same
spectrum of defects, suggesting that each protein acts at distinct times and
places in development and organ homeostasis (reviewed by
Dimova and Dyson, 2005). In
vivo, different E2Fs appear to be able to both promote and hinder tumor
formation, and the mechanisms underlying these diverse outcomes are largely
unknown.
Recent microarray and chromatin immunoprecipitation experiments in tissue
culture cells have identified E2F target genes that expand the role of E2F/DP
beyond the regulation of G1/S phase transition, including genes that act in
mitosis, DNA repair and recombination
(Ishida et al., 2001;
Muller et al., 2001;
Ren et al., 2002). In
Drosophila S2 cells, the sole activator E2F, dE2F1, primarily activates cell
cycle-regulated genes, while the repressor dE2F2 targets developmentally
regulated genes independent of the cell cycle
(Dimova et al., 2003).
Overall, these and other analyses have led to the concept that E2Fs activate
genes that promote the cell cycle and repress genes required for
differentiation. However, most of these analyses have been performed in cell
culture, and how tissue-specificity and developmental context impact the
regulation of E2F target genes is just beginning to be addressed.
The hermaphrodite nematode Caenorhabditis elegans provides both a
streamlined pathway and the opportunity for in-vivo analysis. Its genome
encodes a single pRB-like protein (LIN-35), a single DP-like protein (DPL-1)
and three E2F-like proteins (EFL-1, EFL-2 and F49E12.6). Of the three E2F-like
proteins in C. elegans, a phenotype has been attributed only to
efl-1, which is most closely related to the mammalian repressor E2F4
(Ceol and Horvitz, 2001;
Page et al., 2001).
RNA-mediated interference studies of efl-2 and F49E12.6 have not
identified any apparent phenotype (Boxem
and van den Heuvel, 2002; Ceol
and Horvitz, 2001; Kamath et
al., 2003; Rual et al.,
2004; Sonnischen et al., 2005).
The C. elegans pRB/E2F pathway is required for multiple aspects of
somatic development. lin-35, efl-1 and dpl-1 are all
components of the SynMuv B pathway, which inhibits ectopic vulval development
redundantly with a second pathway, the SynMuv A pathway
(Lu and Horvitz, 1998;
Ceol and Horvitz, 2001). In
addition to Rb and E2F orthologs, the SynMuv B pathway includes components of
a histone deacetylase complex that probably acts to repress transcription
(Lu and Horvitz, 1998;
Korenjak et al., 2004).
LIN-35, EFL-1 and DPL-1 proteins have been demonstrated to physically
associate in vitro (Ceol and Horvitz,
2001). Together, the common phenotype, physical association and
similarity to mammalian proteins support the argument that LIN-35, EFL-1 and
DPL-1 act in concert to repress gene expression. Additionally, lin-35,
efl-1 and dpl-1 function together in G1 cell cycle control in
other tissues that exhibit postembryonic cell division, including the
intestine and ventral cord (Boxem and van
der Heuvel, 2002). In all of the above cases, lin-35,
efl-1 and dpl-1 act together to negatively regulate cell
division. Notably, lin-35 acts redundantly with other genes such as
ubc-18 and xnp-1 during pharynx and somatic gonad
development, respectively, without regulating cell proliferation
(Fay et al., 2004;
Bender et al., 2004).
Here we investigate the function of lin-35, efl-1 and dpl-1 in the germline. Null phenotypes of efl-1, dpl-1 and lin-35 reveal distinct requirements for these proteins in regulating germline development. While EFL-1 and DPL-1 are essential for fertility, LIN-35 is dispensable. Global gene expression profiling of dissected gonads from lin-35, efl-1 and dpl-1 mutants reveals an extensively overlapping gene expression program for EFL-1 and DPL-1. Loss of efl-1 and dpl-1 decreased expression of a common set of genes that promote specific aspects of a developmental program -oogenesis and early embryogenesis - rather than cell cycle. We identified an over-represented sequence in the 5' regions of these genes that closely resembles the mammalian E2F-binding site. LIN-35 plays only a minor role in the regulation of these genes and instead appears chiefly to repress a distinct set of genes with diverse functions in the germline. Our results demonstrate that, in vivo, E2F can directly initiate a developmental program by activating genes that promote differentiation with only minor effects on the cell cycle.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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).
All strains were grown at 20°C. The following variants were used: N2 (wild
type), lin-35(n745) I (Lu and
Horvitz, 1998), unc-4(e120); dpl-1(n3316) II
(Ceol and Horvitz, 2001),
efl-1(n3639) V (Coel and Horvitz, 2001), rme-2(b1005) IV
(Grant and Hirsh, 1999) and
AZ212 (H2B:GFP) (Praitis et al.,
2001). We examined two different isolates of lin-35(n745)
and found that both isolates exhibited sterility at 25°C in our hands, a
phenotype not previously reported.
Gonad dissection and microarray analysis
Wild-type and mutant worms were staged by bleaching gravid adults to
collect eggs, which were then hatched in S-basal solution in the absence of
food. Starved L1 larvae were cultured with food (bacterial strain OP50) and
harvested 70-72 hours later, the first point at which efl-1(n3639)
mutants could be distinguished from siblings. Adult worms were placed in
dissection buffer (M9 with 0.1% levamisole and 0.001% Tween 20) on a
coverslip. For each strain, a pair of 30 1/2-gauge needles was used to extrude
approximately 50 gonad arms, excising each just proximal to the spermatheca.
Dissected gonads were carefully isolated from carcasses and transferred into
an Eppendorf tube. Total RNA from each sample (
100 ng) was isolated using
Trizol (Invitrogen) and amplified with T7 RNA polymerase using one round of
linear amplification (Baugh et al.,
2001). Three independent dpl-1 and lin-35 mutant
samples and four independent efl-1 mutant samples were collected.
Fluorescence-labeled cDNA probe for DNA microarray hybridization was
prepared from 3 µg of amplified RNA as described
(DeRisi et al., 1997).
lin-35 and efl-1 mutant cDNA was labeled with Cy3 and
compared to wild-type (N2) cDNA labeled with Cy5. Cy3-labeled
unc-4;dpl-1 mutant cDNA was compared to unc-4
cy5-labeled cDNA. Caenorhabditis elegans whole genome microarrays
were used for hybridization as described
(Jiang et al., 2001). Each
slide was scanned using an Axon scanner (Molecular Devices, Sunnyvale, CA),
and the expression levels for each gene in each channel were collected using
GenePix 3.0 software. Cy5/Cy3 ratios were calculated and normalized by setting
the overall median of ratios to one. All data have been deposited in GEO under
Accession Number GSE5071.
For each set of mutant data, the repeats were averaged, and a Z test
[Z=(observed-expected)/SE] was performed in Excel. A moderate correction for
multiple testing (17,600 genes) was performed by multiplying the
calculated P-value by 10,000. After this correction, all genes with
up- or downregulation greater than twofold, P<0.05 in any given
mutant were selected. The hypergeometric probability test
(http://elegans.uky.edu/MA/progs/overlap_stats.html)
was used to calculate the significance of overlap of gene groups. We
determined whether transcripts of Group I-IV genes are bound by GLD-1, based
on a minimum criteria of >1.5x enrichment in GLD-1 immunoprecipitated
samples compared to control immunoprecipitations (P<0.01). Genes
in Groups III and IV had very little overlap with GLD-1-bound transcripts
(1/42 and 1/84, respectively), while genes in Groups I and II had a
significantly enriched overlap with candidate GLD-1 targets (26/74 and 5/43,
respectively).
Regulatory motif analysis
To identify candidate regulatory sequences in the 5' noncoding
regions of target genes, the online program MEME (Mutiple Em for Motif
Elicitation)
http://meme.sdsc.edu/meme/intro.html)
was applied to sequences upstream taken from the start codon of each target
gene to the neighboring gene, up to 1 kb. Each group of genes (Groups I-IV)
was examined separately, as well as two control sets of genes, one with
oogenic germline-enriched expression not regulated by EFL-1/DPL-1, and one
that does not show germline-enriched expression. Manual examination was
performed by taking all variations of the MEME-derived consensus motif [e.g.
TTC(G/C)CGC(C/G)] and searching through each 5' regulatory sequence for
an exact match. Manual examination of upstream regions of those Group I genes
in which MEME failed to find the E2F consensus motif did not uncover any
additional instances of the motif. The motif sequence logo was created using
the online program
(http://weblogo.berkeley.edu/logo.cgi).
Immunofluorescence
RME-2 and MEX-5 localization was performed as described
(Kelly et al., 2002). Briefly,
gonads were dissected as described above, fixed in 3.7% paraformaldehyde, and
mounted on a slide. The slides were frozen on a dry ice block and the
coverslip cracked off before storing the slides in -20°C methanol. The
slides were washed three times in phosphate-buffered saline (PBS), blocked in
PBS containing 0.1% Tween 20 and 0.5 mg/ml BSA, and then incubated at 4°C
overnight with 
-RME-2 (1:100; gift from B. Grant), -MEX-5
(undiluted; gift from J. Priess) and -LIN-3 (1:50; Santa Cruz
Biotechnology, Santa Cruz, CA). The samples were then stained with DAPI,
washed and incubated at room temperature for 3 hours with a fluorescent
secondary antibody (Molecular Probes, Carlsbad, CA). After further washing in
PBS, the slides were mounted with anti-fade solution and viewed using a Zeiss
Axioplan 2 imaging epifluorescence microscope.
RNAi
Group I genes with oogenic germline-enriched expression that were reported
to have embryonic lethal RNAi phenotypes (25) were tested for PIE-1:GFP
mis-localization. Of these, ten gave embryonic lethality in our hands:
T22F3.3, T05G5.7, spn-4, rnr-2, rme-2, R05H5.3, puf-3,
pos-1, C28C12.2 and cyb-3. mex-5 and mex-6 were used as
controls. PCR using gene-specific primers containing T7 sequences was followed
by in-vitro transcription using T7 RNA polymerase. The resulting dsRNA was
ethanol precipitated with Pellet Paint (Novagen) and resuspended to a
concentration of 2 mg/ml. pie-1::GFP worms
(Reese et al., 2000) were
synchronized and grown until the L4 stage. Worms were washed in M9 and soaking
buffer (0.25x Mg++-free M9, 3 mmol/l spermidine, 0.05%
gelatin) twice before soaking. Approximately 30 worms in 2 µl soaking
buffer were added to 2 µl dsRNA for each gene, incubated for 24 hours at
20°C, and then plated to NGM seeded with OP50 bacteria. For each gene
giving embryonic lethality, embryos released from the uterus of 10
mothers were examined using a Zeiss Axioplan 2 microscope. Cross-RNAi between
mex-5 and mex-6 transcripts probably occurred, resulting in
PIE-1 mis-localization (Schubert et al.,
2000).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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)]. During the fourth larval stage, spermatogenesis occurs in
the proximal germline. Upon the onset of adulthood, oogenesis then initiates
in the proximal germline. Early stage oocytes enlarge substantially by
incorporating cytoplasm from the central core of the germline and taking up
yolk proteins that are synthesized by the intestine. Oocyte cytoplasm contains
many maternally provided mRNAs and proteins that are necessary for proper
embryonic development. The most proximal oocyte undergoes maturation and
ovulation, becoming fertilized when it comes into contact with sperm in the
spermatheca. Fertilization triggers rapid eggshell formation and completion of
meiosis. The newly fertized embryos pass from the spermatheca into the uterus,
where they are held briefly before being laid.
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). EFL-1 protein is also present in germline nuclei,
but its expression is detected only in the medial region, when nuclei are in
the pachytene stage of meiosis I (Page et
al., 2001) (Fig.
1). Partial loss-of-function point mutations in either
dpl-1 or efl-1 result in temperature-sensitive
maternal-effect embryonic lethality due to defects in embryonic asymmetry
(Page et al., 2001).
Additionally, Page et al. (Page et al.,
2001) demonstrated using genetic mosaic analysis that loss of
efl-1 activity in the germline, but not the soma, was sufficient to
cause this embryonic lethality. To identify the primary defect upon complete
loss of dpl-1 and efl-1 activity, we examined animals
bearing probable null alleles in dpl-1 or efl-1. The
dpl-1(n3316) allele contains a deletion that removes the first half
of the protein, including the EFL-1-binding domain. The efl-1(n3639)
allele harbors an early stop mutation, truncating the protein upstream of the
predicted DP and pRB-binding domains (Ceol
and Horvitz, 2001). The dpl-1(n3316) and efl-1(n3639) mutant phenotypes are very similar: germ cells of all stages are present and the gonad has essentially normal morphology, but adults display nearly 100% penetrant sterility (i.e. do not lay fertilized embryos) (Coel and Horvitz, 2001) (Fig. 2C,D). Most oocytes either do not enter or do not exit the spermatheca normally, and over time the proximal gonad in efl-1 and dpl-1 mutants fills up with degenerating oocytes that become endomitotic. The occasional oocyte that does pass through the spermatheca frequently fails to develop a complete eggshell, and does not undergo normal cell division. The efl-1(n3639) and dpl-1(n3316) phenotypes persist even when exogenous sperm is provided from wild-type males, indicating that the phenotypes are not due solely to defective spermatogenesis.
Aberrant ovulation and the endomitotic (Emo) phenotype can be caused by
defects in either the oocyte or the somatic gonad
(McCarter et al., 1997;
McCarter et al., 1999). To
test the site of action of dpl-1 and efl-1, we injected
dsRNA corresponding to dpl-1 or efl-1 into wild-type and
rrf-1 mutants, which can carry out RNAi in the germline but not the
soma (Sijen et al., 2001).
Relative to wild-type controls, rrf-1;efl-1(RNAi) and
rrf-1;dpl-1(RNAi) animals displayed a decreased incidence of
the Emo phenotype. This observation suggests that efl-1 and
dpl-1 do play a role in the soma, possibly in the somatic gonad, to
regulate ovulation (data not shown). However,
rrf-1;efl-1(RNAi) and rrf-1;dpl-1(RNAi)
animals were still completely sterile and collected partial, torn oocytes in
the uterus, indicating that defects in ovulation, though more subtle, still
occurred. This observation suggests a role for efl-1 and
dpl-1 in the germline to promote fertility. In sum, the null
phenotype for both efl-1 and dpl-1 in the germline is more
severe than in previously studied point mutants
(Page et al., 2001).
|
). The germline of lin-35(n745) mutants
generally appears morphologically normal
(Fig. 2B). Unlike
dpl-1(n3316) and efl-1(n3639) mutants, lin-35(n745)
animals are fertile and do not display obvious defects in oogenesis or
ovulation. However, the brood size of lin-35(n745) mutants is 41% of
wild type (132±13 versus 320±42; Student's t test:
P<<0.001).
EFL-1 and DPL-1 regulate a common set of genes and LIN-35 regulates a distinct set of genes
Because EFL-1, DPL-1 and LIN-35 are predicted to act as transcriptional
regulators, we used whole-genome C. elegans DNA microarrays to
identify candidate target genes. To focus on the role of EFL-1, DPL-1 and
LIN-35 in the germline, we isolated gonads away from the rest of the animal.
For each mutant or control sample, we collected 50 dissected adult
gonads, carefully discarding the spermatheca and uterus as well as the rest of
the carcass. The only somatic cells retained were the distal tip cell and
gonadal sheath cells, ensuring that the vast majority of the isolated RNA
derived from germ cells. Because the germline morphology of efl-1(n3639),
dpl-1(n3316) and lin-35(n745) mutants are visually similar to
wild type, non-specific effects on gene expression should be minimal.
We used linear amplification to increase the amount of RNA for all samples,
and reverse transcribed the RNA to labeled cDNA. Using microarrays containing
90% of predicted C. elegans genes
(Jiang et al., 2001), we
performed at least three hybridizations directly comparing each mutant to a
control. The resulting data were analyzed using both an average
fold-difference and a Z-test with correction for multiple testing
(see Materials and methods). We have focused on 272 genes whose expression is
either up- or downregulated in efl-1, dpl-1 or lin-35
mutants relative to controls, exceeding a twofold difference at
P<0.05 (see Table S1 in the supplementary material). Throughout
this paper, we will use the term `downregulated' to refer to target genes with
lower expression in dpl-1, efl-1 and/or lin-35 mutants
relative to controls, and `upregulated' to refer to genes with higher
expression in any mutant relative to controls.
We first examined the overlap and independence between the different mutant datasets (Fig. 3). Our results show that DPL-1 and EFL-1 both function primarily to promote gene expression. At our twofold cutoff, 116 genes were downregulated and 30 genes were upregulated in dpl-1 mutants. Similarly, in efl-1 mutants, 114 genes were downregulated and 20 genes were upregulated. Remarkably, the two mutants showed downregulation of 70% of the same target genes (95/135; P<<0.001) (Fig. 3A). This extensive overlap is consistent with these two proteins acting as a heterodimer to regulate transcription. Only a subset (36) of the 95 genes downregulated in efl-1 and dpl-1 mutants were also downregulated in lin-35 mutants. Instead, distinct genes were primarily upregulated in lin-35 mutants; strikingly few of these were also regulated in either efl-1 or dpl-1 mutants (Fig. 3B). The above observations suggest that EFL-1/DPL-1 can function without LIN-35 to regulate expression of many target genes and, conversely, that LIN-35 often acts independently of EFL-1/DPL-1. When LIN-35 does act on genes regulated by EFL-1/DPL-1, it cooperates with, rather than antagonizes, EFL-1/DPL-1 (Fig. 3A).
Examining the overlap among preselected gene groups can exclude genes that
come close but fail to meet selection criteria, partially disguising general
trends. We therefore used hierarchical clustering to place the 272 genes into
groups with similar regulation (Eisen et
al., 1998) (Fig.
4). From this analysis, we defined four expression groups that
encompass 248 of the 272 genes; the remaining 24 genes showed variable
expression and were not included in a group
(Fig. 4; see Table S1 in the
supplementary material). Group I comprised 75 genes with decreased levels in
dpl-1(n3316) and efl-1(n3639) mutants compared with controls
and were termed `downregulated in efl-1 and dpl-1'.
Sixty-five out of 75 genes in this group were regulated more than twofold
(P<0.05) by both EFL-1 and DPL-1. Seven of the remaining ten genes
just missed the twofold cutoff in one mutant and the remaining three lacked
data for one mutant. Thus these genes are almost uniformly under the control
of both EFL-1 and DPL-1, suggesting that they are targets of an EFL-1/DPL-1
heterodimer. Only 16 out of the 75 genes in Group I were significantly
downregulated in lin-35 mutants, although many more were moderately
affected.
Group II genes (43 genes) generally had lower expression in efl-1, dpl-1 and lin-35 mutants than in control gonads. Twenty-three genes were significantly regulated in all three mutants, and 36 were regulated in at least two mutants, so we call Group II genes `commonly downregulated'. Conversely, genes in Group III (42 genes) had higher expression in one or more mutants relative to controls. However, only five were significantly upregulated in all three mutants and only 17 were upregulated in at least two mutants, so we termed this group `variably upregulated'. Group IV contains 88 genes with significantly increased levels only in the lin-35 mutant relative to controls; we referred to Group IV genes as `upregulated in lin-35'. From these data, we conclude that efl-1 and dpl-1 mutants show strong similarity in gene regulation over all four groups, even if a given gene did not surpass the selection criteria in one mutant, reinforcing the notion that EFL-1 and DPL-1 are likely to act as a heterodimer and regulate common targets. lin-35 shared common targets with efl-1 and dpl-1 for one of the four groups (Group II), but showed distinct regulation in the other three groups. Most notably, lin-35 had minimal effect on Group I genes regulated by efl-1 and dpl-1, and instead regulated expression of a large set of genes not affected by loss of efl-1 or dpl-1 (Group IV).
|
). The
most significant hit among any of the groups was the sequence TTC(G/C)CGC(C/G)
(P<<0.001), found upstream of 58 out of 75 Group I
(downregulated in efl-1 and dpl-1) genes
(Fig. 5A; see Table S1 in the
supplementary material). This sequence bears striking similarity to the known
mammalian E2F consensus site TTTCGCGC
(Chittenden et al., 1991),
suggesting that most Group I genes are likely to be direct targets of the
EFL-1/DPL-1 transcription factor. The TTCGCGCC motif was present within the
first 500 base pairs upstream of the translation start in 48 genes. Multiple
copies of the motif were present in 28 genes, and the duplicate sites were
often juxtaposed (Fig. 5B).
MEME did not identify the E2F consensus motif in Groups II, III or IV, in a
control set of oogenic germline-enriched genes not responsive to EFL-1, DPL-1
or LIN-35 (n=38), or in a control set of genes that did not show
germline-enriched expression (n=51). Our ability to find an E2F consensus site in Group I genes but not the other three groups suggests that most genes in Groups II-IV are not regulated by direct binding of an E2F heterodimer, but are either indirect or downstream. To determine whether at least some genes in Groups II, III and IV might be direct targets, we examined their 5' regulatory sequence manually and identified 13 Group II, 8 Group III and 30 Group IV genes with a C. elegans consensus E2F site (see Table S1 in the supplementary material). Thus, it is possible that the regulatory regions upstream of certain genes in these groups are directly bound by EFL-1/DPL-1.
MEME detected a different motif, TTTTCCAG, in the regulatory regions of Group IV `upregulated in lin-35' genes (P<<0.001; Fig. 5C). This consensus sequence was present in 58/88 regulatory sequences, but its location was not biased toward the translation start site (Fig. 5D). We could find no clear match to any known transcription factor consensus sequence for this motif in any database. No significant motifs were identified in Group II or Group III genes.
The germline expression pattern of downregulated genes differs from upregulated genes
We examined the spatial expression patterns of genes in groups I-IV using
data from an online in-situ hybridization database (Y. Kohara, personal
communication;
http://nematode.lab.nig.ac.jp),
which has images of expression patterns available for 151 of the 248 genes
(see Table S1 in the supplementary material). Images are available for 54
Group I `downregulated in efl-1 and dpl-1' genes, and 47 of
these show staining in the germline. Notably, 37 genes have undetectable
levels in the distal germline with an abrupt increase in the medial germline
that persists proximally, often into embryos
(Fig. 6A-D). Onset of
expression coincides with the appearance of EFL-1, which is detectable only in
the mid-pachytene region of the medial germline
(Page et al., 2001) (see
Fig. 1). Fewer Group II
`commonly downregulated' genes show detectable expression in the germline
based on in-situ data (12/26); however, 10 of 12 were expressed in a
medial/proximal restricted expression pattern, similar to Group I genes. Thus,
germline expression of most genes downregulated in the mutants (Groups I and
II) is generally restricted to the medial and proximal gonad, coincident with
peak EFL-1 protein levels.
Conversely, most Group III and IV genes are broadly expressed in the
wild-type germline. Out of 46 Group IV `upregulated in lin-35' genes
with in-situ images, 35 have detectable germline expression, with 32 visible
in both the distal and proximal germline and three restricted to the
medial/proximal germline (Fig.
6E-H). Only 6/16 Group III genes show detectable germline
expression, but four of these have broad expression and two show
medial/proximal restricted expression. Given that our dissected gonad
microarray data indicates that these genes are normally at lower levels in the
wild-type germline than in the lin-35 mutant germline, this broad
germline expression was unexpected. One possibility is that LIN-35 acts
broadly to decrease, but not abrogate, expression of these genes, because
elevated levels are slightly detrimental to germline function. Another
possibility is that LIN-35 acts to repress expression of these genes in a
specific region of the germline, but that this effect is not substantial
enough to be visible by in-situ hybridization methods, which are difficult to
quantify. Finally, we could be detecting LIN-35-mediated repression of these
germline-expressed genes in the few somatic cells that are included in our
analysis (distal tip and sheath cells) rather than in germ cells, which would
be difficult to distinguish by in situ. This last possibility is consistent
with the observation that lin-35 prevents ectopic germline gene
expression in the soma (Wang et al.,
2005).
EFL-1 and DPL-1 are required for normal expression of RME-2 and MEX-5
We independently tested whether two Group I genes, rme-2 and
mex-5, were regulated in a manner consistent with the microarray
results. rme-2, which encodes the yolk receptor
(Grant and Hirsh, 1999), and
mex-5, which encodes a CCCH zinc finger protein required for correct
embryonic polarity (Schubert et al.,
2000), both require efl-1 and dpl-1 activity for
their expression, based on our microarray experiments. lin-35 has a
moderate effect on mex-5 but not rme-2 expression (see Table
S1 in the supplementary material). We performed semi-quantitative RT-PCR of
both genes in wild type, efl-1, dpl-1 and lin-35 mutants and
saw decreased expression of both rme-2 and mex-5 in
efl-1 and dpl-1 mutants relative to wild type, with minimal
effects in lin-35 mutants, consistent with our microarray data (see
Fig. S1 in the supplementary material). We also performed immunohistochemistry
to determine whether localization and expression level of RME-2 and MEX-5
differs in efl-1, dpl-1 and lin-35 mutants compared to wild
type (Fig. 7). In wild-type
gonads, RME-2 localizes to the oocyte membrane. We saw similar levels and
localization in lin-35 mutants, but efl-1 or dpl-1
mutants displayed severely reduced RME-2 levels, even at tenfold higher
exposure times, consistent with a role for EFL-1 and DPL-1 in promoting
expression of rme-2.
|
). Thus, for both Group I genes we tested, we saw decreased
expression in both efl-1 and dpl-1 mutants, but not in
lin-35 mutants, relative to wild type, consistent with the microarray
results. To test whether the reduced expression of RME-2 and MEX-5 seen in efl-1 and dpl-1 mutants was a downstream, non-specific effect of decreased oogenesis or oocyte maturation, we examined LIN-3. lin-3 is expressed during oogenesis, but is not differentially regulated in lin-35, dpl-1 or efl-1 mutants in our microarray experiments. In wild type, LIN-3 is localized to the oocyte membrane (Fig. 7C), while in lin-3(RNAi) animals this staining is absent (see Fig. S2 in the supplementary material). LIN-3 localization was not altered in lin-35, dpl-1 or efl-1 mutants relative to wild type, suggesting that oogenesis genes not identified in our microarray study are probably still expressed normally in dpl-1 and efl-1 mutants.
|
). However, recent global expression studies in
mammalian and Drosophila cell culture have found that various E2Fs
can activate genes that act at other phases of the cell cycle, as well as
repressing genes that act to promote differentiation
(Ren et al., 2002;
Dimova et al., 2003). The
complexity of the Rb/E2F network has made it difficult to perform and
interpret in-vivo studies, leaving a gap in our understanding of how
accurately the transcriptional properties of E2F in culture reflect its role
in vivo.
In our experiments, we found that EFL-1/DPL-1 primarily promotes expression
of pro-differentiation genes in the wild-type germline
(Table 1). Group I
`downregulated in efl-1 and dpl-1' genes include only a few
likely to have direct roles in the meiotic and mitotic cell cycle, such as a
ribonucleotide reductase subunit rnr-2, and two cyclin B orthologs,
cyb-3 and cyb-2.1
(Sonneville and Gonczy, 2004)
(www.wormbase.org).
Four previously studied targets encode proteins with decreased expression that
is likely to contribute to the defects in fertilization and eggshell formation
seen in dpl-1(n3316) and efl-1(n3639) mutants. These include
the yolk receptor RME-2, the Tis11-like oocyte maturation protein OMA-2, the
predicted chitin-binding protein CEJ-1, and an EGF-related protein required
for fertilization, EGG-2 (Grant and Hirsh,
1999; Detwiler et al.,
2001; Lee and Schedl,
2001; Kadandale et al.,
2005). In particular, rme-2 mutants display ovulation
defects and have a reduced brood size as a consequence
(Grant and Hirsh, 1999). Thus,
decreased rme-2 expression is probably a significant component of the
dpl-1 and efl-1 phenotypes.
|
). MEX-5
and MEX-6 are localized to anterior blastomeres and also establish embryonic
asymmetry (Schubert et al.,
2000). Loss of pos-1, mex-5/6 or mex-1 activity
results in ectopic localization of the germline-specific PIE-1 protein in
somatic cells, either through mis-specification of the germ lineage or through
a failure to degrade PIE-1 in somatic blastomeres, and results in embryonic
lethality (Schubert et al.,
2000; Tabara et al.,
1999; Guedes and Priess,
1997).
|
). We
examined a subset of genes with reported RNAi phenotypes that were embryonic
lethal in large-scale screens. Out of ten genes tested, we found one, T22F3.3,
with inactivation by RNAi that results in persistence of PIE-1 in somatic
blastomeres at a frequency similar to the rate of embryonic lethality (9%)
(see Fig. S3 in the supplementary material). T22F3.3 encodes the only obvious
glycogen phosphorylase in the C. elegans genome and presumably plays
a crucial role in modulating glucose availability. Mutations in human glycogen
phosphorylase cause McArdle disease, which impairs muscle function
(Dimaur et al., 2002).
Alterations in available energy levels could conceivably impact some of the
processes required for timely degradation of PIE-1 in the soma.
Although Group II genes share some common expression characteristics with
Group I genes as described above, they generally encode different types of
proteins. For instance, Group II genes include seven that encode histones H2A,
H2B or H4, but none encoding H3 or H1. In human cells, the promoters of
several histone H2 genes are also bound by E2F4
(Ren et al., 2002). RNAi of
several Group II genes produces embryonic lethal phenotypes based on
large-scale studies, including those encoding histones, the translation
elongation factor EFT-2, and chaperone proteins such as HSP-3 and PDI-2.
The predicted protein functions of the genes in Groups III and IV are heterogeneous, and obvious trends are not readily apparent. Five Group III genes show significant upregulation in all three mutants, including akt-2, a kinase that acts downstream of insulin signaling, toh-2, a metalloprotease, and cdr-4, a cadmium-responsive glutathione-S-transferase. Group IV `upregulated in lin-35' genes include several that encode transcriptional regulatory proteins, such as mes-3, dro-1 and ntl-1, as well as four uncharacterized proteins with histone acetyltransferase domains. Genes encoding the DNA synthesis licensing factor CDT-1, the anti-apoptotic factor CED-9 and the Frizzled receptor MOM-5 are also repressed by LIN-35 in the gonad. Thus, the upregulated genes encode proteins of a wider variety of functions than genes downregulated in efl-1 and dpl-1 mutants.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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Limited compensation by Rb/E2F family members
The streamlined nature of the pRb/E2F pathway in C. elegans
reduces the possibility of compensation by other family members. The
dpl-1 locus encodes the only DP-related protein in the C.
elegans genome; thus, loss of dpl-1 should remove all E2F
activity, as most E2Fs require a DP subunit to regulate gene expression. Our
results show that dpl-1 does not regulate many genes in addition to
those regulated by efl-1. This extensive overlap of dpl-1
and efl-1 in regulating germline gene expression strongly suggests
that EFL-2 or F49E12.6 do not compensate for loss of EFL-1 and that EFL-1 is
the major component of E2F activity in the germline.
lin-35 is the only pocket protein encoded in the worm genome and
clearly has a distinct role in regulating a distinct set of genes (Group IV).
However, additional processes in which LIN-35 participates could be buffered
by the action of other proteins. In somatic tissues, genes of diverse
functions have exhibited redundancy with lin-35, including components
of the SynMuv A or C pathways, the cell cycle inhibitor cki-1, and a
regulator of APC activity, fzr-1
(Ferguson and Horvitz, 1989;
Ceol and Horvitz, 2004;
Boxem and van den Heuvel, 2002;
Fay et al., 2002).
Additionally, a mutation of lin-35 enhances the meiotic recombination
defect of him-17 mutants in the germline
(Reddy and Villeneuve, 2004).
Thus LIN-35 could have a role in germ cell division or differentiation that is
not apparent in lin-35 mutants because of redundancy with other
factors.
Relationship between EFL-1, DPL-1 and LIN-35 in the germline
Based on both genetic and biochemical evidence, components of the C.
elegans Rb pathway act together to repress gene expression in somatic
tissues (reviewed by Kipreos,
2005). In both C. elegans and Drosophila, recent
work has demonstrated that genes normally expressed in the germline are
repressed in the soma by pRB and E2F
(Dimova et al., 2003;
Wang et al., 2005). Our study
is the first in-vivo demonstration of distinct roles for E2F and pRB in
regulating gene expression in the germline. We have shown that E2F mainly
functions to activate, rather than repress, genes important for oocyte and
embryo differentiation (Group I genes), and that the role of LIN-35(pRB) in
this process is minimal or dispensable. Possibly, LIN-35 dissociates from the
E2F complex in the germline, freeing E2F to activate target gene expression.
E2F activity could also be controlled by accumulation of EFL-1 protein at the
mid-pachytene stage of meiosis I rather than by association of LIN-35.
Notably, in Drosophila, chromatin immunoprecipitation studies
demonstrate that both pRB and E2F can be found at the promoters of genes whose
expression is not dependent on pRB (Dimova
et al., 2003; Stevaux et al.,
2005). By analogy, in the C. elegans germline, LIN-35
could be present at the promoters of Group I genes but not be rate-limiting
for their expression.
LIN-35 instead acts to downregulate a distinct set of genes (Group IV) that
do not require EFL-1 or DPL-1 for their expression. The absence of canonical
E2F binding sites from the 5' regulatory regions of most Group IV genes
suggests that LIN-35 is targeted to these sites through the activity of a
different DNA-binding factor, one that potentially binds the TTTTCCAG site
that we found highly represented among Group IV genes. Several instances of an
E2F-independent function of pRB have been described
(Sellers et al., 1998;
Thomas et al., 2001;
Gagrica et al., 2004).
Alternatively, LIN-35 could be acting with either EFL-2 or F49E12.6 in a
DPL-1-independent manner that does not require an E2F consensus site. Finally,
the increased expression of these genes upon loss of LIN-35 activity could be
indirect or occurring in the few somatic cells present in our samples.
Additional experiments will be necessary to distinguish among these
possibilities.
Tissue-specific EFL-1/DPL-1 transcriptional program
Our results demonstrate that EFL-1/DPL-1 can display tissue specificity in
both target gene selection and in the manner of gene regulation (activation or
repression). In somatic tissues, EFL-1/DPL-1 represses genes such as
fkh-6, mat-3 or cye-1 in various cell types to control the
timing and nature of cell division (Chang
et al., 2004; Garbe et al.,
2004; Tilmann and Kimble,
2005; Grishok and Sharp,
2005). However, in the germline, EFL-1/DPL-1 does not regulate
these target genes, nor is it crucial for proliferation of the germline stem
cell population. Instead, EFL-1/DPL-1 activates the expression of a distinct
set of genes whose protein products participate in oocyte differentiation and
embryogenesis. Indeed, many EFL-1/DPL-1 target genes such as rme-2
and mex-1 have known roles only in oogenesis and embryogenesis and
are likely to be expressed specifically in the maternal germline. The presence
of canonical E2F-binding sites in these promoters strongly indicates that
their regulation by EFL-1/DPL-1 is direct, although the mechanisms
establishing the EFL-1/DPL-1 germline-specific program are unknown. Possibly,
unknown germline-specific transcriptional coactivators or chromatin
conformation might influence the activity of EFL-1/DPL-1.
Coordination of gene expression with developmental events
Several transcripts expressed in the medial germline are translationally
repressed by the mRNA-binding protein GLD-1, including two that we identified
as EFL-1/DPL-1 targets (rme-2 and cej-1)
(Jones and Schedl, 1995;
Lee and Schedl, 2001). We have
generated an expanded list of candidate GLD-1 targets by identifying mRNAs
that co-immunoprecipitate with GLD-1 and probing microarrays (M.-H. Lee, V.R.
and T. Schedl, unpublished). Candidate GLD-1 target transcripts had
significantly enriched overlap with Group I and II genes (26/74 and 5/43,
respectively; see Materials and methods). Group I transcripts bound by GLD-1
include several with roles in oogenesis and embryogenesis, such as rme-2,
cej-1, oma-2 and egg-2, as well as spn-4, cyb-2.1 and
cyb-3. Thus, the germline has established a multi-tier mechanism for
ensuring that the expression of these target genes is properly coordinated
with oocyte development. First, detectable EFL-1 protein is restricted to the
medial germline, which limits the significant accumulation of the target
transcripts before the pachytene stage of meiosis I. Second, once expressed,
many of these transcripts are probably translationally suppressed by GLD-1
until oogenesis. This dual regulation ensures that crucial components of
oogenesis are available but held inactive until germ cells are at the
appropriate stage of development.
|
Conclusion |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/16/3147/DC1
|
ACKNOWLEDGMENTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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