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First published online February 24, 2006
doi: 10.1242/10.1242/dev.02275
National Institute of Biological Sciences, Beijing, People's Republic of China.
Author for correspondence (e-mail:
zhanghong{at}nibs.ac.cn)
Accepted 5 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: sor-1, PcG, Nuclear bodies, SAM domain, C. elegans
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lippman and Martienssen,
2004). The RNA components can be a non-coding structural RNA, such
as Xist RNA, which functions in mammalian X chromosome inactivation
(Pannings and Jaenisch, 1998),
or a small interfering RNA (siRNA) derived from the RNAi machinery, which is
employed in the silencing of genes within the heterochromatic domains in
S. pombe and Drosophila
(Volpe et al., 2002;
Pal-Bhadra et al., 2004).
These RNA components could be responsible for targeting the interacting
protein complex to specific loci and/or in later maintaining the repressed
state of the genes (Akhtar,
2003; Lippman and Martienssen,
2004).
The epigenetic silencing of Hox genes mediated by Polycomb group (PcG)
proteins is also thought to involve the formation of localized repressive
chromatin structures (Levine et al.,
2004; Lund and van Lohuizen,
2004). Hox genes encode homeodomain-containing transcription
factors that specify the positional identities of cells along the
anteroposterior axis during multicellular organism development
(Gellon and McGinnis, 1998).
In PcG mutants, Hox genes are ectopically expressed in regions
outside their normal expression domains, resulting in homeotic
transformations, in which the body structures are duplicated or lost. Two
conserved PcG complexes have been identified, the ESC/E(Z) complex, containing
Extra sex combs and Enhancer of zeste, and the PRC1 complex, the core
components of which include Polycomb (PC), Posterior sex combs (PSC),
Polyhomeotic (PH) and RING1 (Levine et
al., 2004). The ESC/E(Z) complex has been shown to associate with
histone deacetylases and also contains an intrinsic histone methyltransferase
activity that specifically methylates H3 at lysine 9 (K9) and lysine 27 (K27)
(van der Vlag and Otte, 1999;
Muller et al., 2002). The PRC1
complex functions as an E3 ubiquitin ligase that is specific for H2A
(de Napoles et al., 2004;
Wang et al., 2004). Recent
observations suggest that, like other forms of epigenetic gene silencing,
PcG-mediated Hox gene repression might also involve RNA
(Sun and Zhang, 2004). For
example, the ESC/E(Z) complex and the PRC1 complex are required for X
chromosome inactivation in mammals and they are recruited to the X chromosome
in a Xist structural RNA-dependent manner, although their direct interaction
has not been established (Silva et al.,
2003; Plath et al.,
2003; Plath et al.,
2004).
PcG-mediated Hox gene repression is an ancient mechanism, conserved from
flies to mammals. However, the nematode C. elegans apparently lacks
PRC1 complex genes. Furthermore, although the ESC/E(Z) complex is found (e.g.
mes-2 and mes-6), mutations in these genes result in only
mild homeotic defects (Korf et al.,
1998; Holdeman et al.,
1998; Ross and Zarkower,
2003). Nevertheless, C. elegans Hox genes are subject to
global repression. This repression is mediated by a novel gene,
sop-2. In sop-2 mutants, the onset of Hox gene expression is
normal but is subsequently expressed ectopically in diverse body regions. Like
the components of the PRC1 complex, SOP-2 forms nuclear bodies, called SOP-2
bodies (Zhang et al., 2003).
The formation of SOP-2 bodies is tightly correlated with its function and its
formation appears to require the RNA-binding activity of SOP-2
(Zhang et al., 2003;
Zhang et al., 2004a;
Zhang et al., 2004b), although
the nature of the RNA under physiological conditions remains to be elucidated
(Zhang et al., 2004b).
Identification of additional components in the SOP-2 nuclear bodies will
provide insight into the role of SOP-2 in Hox gene repression. Although SOP-2
bears little sequence similarity to PRC1 complex proteins, components of the
PRC1 complex are also localized into distinct nuclear bodies, called PcG
bodies, and also have RNA-binding activity
(Netter et al., 2001;
Zhang et al., 2004b). The
study of the SOP-2/RNA complex is likely to shed light on the salient features
of PcG-mediated gene repression. In this study, we describe the identification
and characterization of a PcG-like gene in C. elegans,
sor-1, that encodes a novel RNA-binding protein with limited regions of
similarity to the mouse PcG protein Rae28. SOR-1 and SOP-2 colocalize in SOP-2
nuclear bodies and direct interact with each other. Our studies reveal that
SOR-1 and SOP-2 define a putative PcG-like complex in global repression of Hox
genes in C. elegans. Remarkably, neither SOR-1 nor SOP-2 is conserved
in other organisms, not even in the congeneric species C. briggsae,
suggesting a surprising lack of evolutionary constraint on an ancient
regulatory system.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The bx91 mutation is temperature sensitive. At 15°C, animals have no obvious defects. At 25°C, animals arrest at L1 and L2 stages. Adult sop-2(bx91) animals were shifted from 15°C to 25°C and the arrested early larvae were examined for the expression pattern of Hox genes.
Isolation, mapping and cloning of sor-1
sor-1 mutations were isolated in screens for mutants with ectopic
expression of mab-5::gfp and egl-5::gfp reporters.
Approximately 17,000 haploid genomes were screened. bp1, bp2 and
bp3 are located at the same genetic locus by genetic mapping and
non-complementation experiments. Three factor mapping placed sor-1
between unc-32(0.00) and sma-3(–0.93), about
–0.23, on LGIII. Six out of eight Sma nonUnc and seven out of 28 Unc
nonSma recombinants carried sor-1 mutation. PCR products from
ZK1236.3 were injected into + sor-1(bp2) +/unc-32 + sma-3 together
with transformation marker pRF4(rol-6(su1006)), and its ability of
rescuing the early larval lethality of sor-1(bp2) was assessed. The
heterozygotes transformants gave rise to adult sor-1(bp2) males and
hermaphrodites, while 100% of sor-1(bp2) animals without the
transgene arrested at L1 and L2 stages.
cDNA clones of sor-1, yk526e9 and yk336g5, were kindly provided by Dr Kohara (NIG, Japan) and were sequenced to confirm the predicted exon/intron junctions. The 5' end of the sor-1 cDNA was obtained by RT-PCR using SL1 sense primer and an antisense primer that is specific to sor-1. SL1 is a trans-spliced leader that is present at the 5' end of many C. elegans cDNAs. The sor-1 mutations identified in our genetic screens were determined by sequencing the corresponding sequence of the sor-1 locus.
RNA interference
Single-stranded RNA (ssRNA) was transcribed from the T7 and T3-flanked PCR
templates (ZK1236 nucleotides 6709-7498) with MEGAscript T3 and T7 kits
(Ambion). The ssRNAs were then annealed, and injected into muIs16, bxIs13,
bxIs14, mab-5 egl-5; bxIs14 and sop-2(bx91); bxIs13 animals. F1
progenies generated 4 hours after injection were scored for larval lethality,
ectopic expression of Hox genes, or generation of anterior rays, as shown by
the expression of pkd-2::gfp reporter.
sor-1::gfp reporter gene
The sor-1::gfp reporter was constructed by PCR fusion based
approach (Hobert, 2002). The
fused PCR products were derived from two overlapping PCR DNA fragments. One
contained the DNA derived from ZK1236 (nucleotides 3272 to 9530), which
includes a 3 kb promoter region and the entire ORF of sor-1. Another
one contained the gfp and the unc-54 3'UTR from
pPD95.67. The PCR products were co-injected with pBX-1(pha-1+) into
pha-1 mutant worms and the transformants were analyzed.
Preparation of antibody to SOR-1 protein
The sor-1 cDNA corresponding to the N terminus of SOR-1 (amino
acid 230 to 390) was cloned into the pET28 expression vector. This his-tagged
fusion protein produced by E. coli BL21 was purified to be used as an
immunogen in rabbits. The antisera were first absorbed with bacterial acetone
powder, followed by NAB protein A spin purification kit (Pierce).
Indirect immunofluorescence
Embryos were obtained from well-fed adult hermaphrodites. The
permeabilization of embryos and young larvae was performed by Freeze-Cracking
methods (Albertson, 1984). The
freeze-cracked slides were fixed, blocked and incubated with anti-SOR-1
antibody at a final dilution of 1:400 at room temperature for 2-4 hours. The
worms were then washed three times and incubated with Rhodamine
Red-X-conjugated goat anti-rabbit IgG. The specificity of SOR-1 antibody was
demonstrated by lack of staining in control experiments with pre-immune sera,
with antisera that were preincubated with 0.25 mg of purified SOR-1 fusion
protein, or in sor-1(RNAi) embryos. sop-2(bx91) L2 larvae
were shifted from 20°C to 25°C, and the embryos derived form these
animals were used for immunostaining.
GST pull-down experiments
Constructs encoding GST-SOR-1 and GST-SOP-2 were made by subcloning
portions of the cDNAs of SOR-1 and SOP-2 into pGEX-4T-1. Fusion proteins were
expressed in BL21 cells and purified with glutathione sepharose resin
according to the manufacturer's recommendation (Pharmacia). For in vitro
systems of 35S-labelled proteins, the corresponding sor-1
and sop-2 were cloned into pcDNA3 as templates for transcription and
translation (TNT Coupled Reticulocyte Lysate System, Promega). The GST fusion
proteins (200 ng) were incubated with 35S-labelled protein and 10
µl glutathione sepharose beads in binding buffer [25 mM Tris.Cl (pH 7.6),
150 mM NaCl, 1 mM DTT, 0.5% Triton X-100, 10% Glycerol, 1 mM PMSF] for 2 hours
at 4°C. The reactions were then washed for four times with 1 ml binding
buffer. Bound proteins were analyzed by SDS-PAGE and analyzed by
autoradiograph.
For in vivo GST pull-down assay, SOP-2 (residues 58-140) and SOR-1 (48-200) were cloned into the vectors of pGEX-6P-1 and PET30a, respectively, and co-expressed in E. coli strain BL21(DE3). GST-4B resin was first used for purification of the complex. GST was removed by thrombin and then the digested complex was subjected to ion exchange column (Amersham-Pharmacia) for further purification.
Gel filtration assay
The complex was purified to more than 95% homogeneity in assay buffer (10
mM Tris, pH 8.0, 3 mM DTT, 100 mM NaCl). The complex (1.0 ml, about 2.0 mg
complex) was subjected to gel filtration analysis (Superdex200,
Amersham-Pharmacia). Samples from the peak corresponding to the complex were
visualized by SDS-PADE and stained with Coomassie Blue.
EMSA and RNA-binding assays
RNA-binding reactions contained 20 mM HEPES (pH 7.6), 100 mM KCl, 2 mM
EDTA, 0.01% NP40, 1 mM DTT, labeled RNA fragment (20,000 cpm). Ten times cold
irrelevant ssRNA, dsRNA and 100 times yeast tRNA (mass excess) were used for
competition experiments. Reactions (25 µl) were incubated on ice for 20
minutes and electrophoresed on 4% native TBE PAGE gel and analyzed by
autoradiograph.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In
sor-1 mutants, egl-5 is ectopically expressed in head
region, and sometimes in the anterior seam cells
(Fig. 1B). Similarly, the
expression domain of mab-5 is greatly expanded. During early
wild-type larval development, expression of mab-5 is limited to the
cells of posterior body region, including P9-P12, V5, and V6
(Fig. 1C) (Kenyon et al., 1997). In
sor-1 mutants, mab-5::gfp is ectopically expressed in the
head, and in the syncytial hypodermal cell 7 (hpy7)
(Fig. 1D). Taken together,
these observations suggest that wild-type sor-1 is required for
global repression of Hox genes.
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sor-1 mutations cause anterior to posterior homeotic transformations
To determine whether the endogenous Hox genes are ectopically expressed in
sor-1 mutants (like their reporter genes), we examined whether
homeotic transformations, phenotypic readouts of mis-expression of Hox genes,
occur in sor-1 mutants. We studied the development of a row of
epidermal seam cells in males, in which the role of Hox genes mab-5
and egl-5 in cell fate specification is well characterized
(Emmons, 1999). During
wild-type male development, the three most-posterior seam cells, V5, V6 and T,
give rise to nine sensory rays. mab-5 is expressed in the lineages of
V5 and V6, and is required for the generation of rays from these cells
(Salser and Kenyon, 1996).
egl-5 is expressed in the posterior branches of the V6 lineage where
it specifies the identities of the V6 rays
(Ferreira et al., 1999). The
anterior seam cells, from V1 to V4, which do not express mab-5 and
egl-5, generate three parallel longitudinal cuticular ridges, known
as alae. Ectopic expression or loss of function of Hox genes mab-5
and egl-5 results in homeotic transformation of the fates adopted by
seam cells V1 to V6. The early larval lethality of sor-1 mutants
identified in our screens prevents us from analyzing the role of
sor-1 in male ray development, an event that occurs later in larval
development. We therefore took advantage of sor-1(RNAi), which at the
appropriate concentration causes weaker loss-of-function defects in the
animals developed from the eggs laid in the first few hours after injection.
sor-1 dsRNAs (gene identification see below) were injected into worms
carrying a pkd-2::gfp reporter, which is specifically expressed in
one of the two ray neurons and serves as a ray marker
(Fig. 1E) (Barr and Sternberg,
2000). We found that in sor-1(RNAi) males, pkd-2::gfp is
ectopically expressed in the anterior body region instead of being confined to
the posterior (89%, n=18) (Fig.
1F), indicating that ectopic rays are generated from anterior seam
cells. By contrast, ectopic anterior rays are completely absent in
sor-1(RNAi) mab-5 egl-5 males (n=13). Thus, loss of function
of sor-1 causes anterior to posterior cell fate transformation in a
Hox gene-dependant manner.
sor-1 mutants exhibit pleiotropic defects
In addition to ectopic expression of Hox genes, sor-1 mutant
animals display many other defects. All sor-1 mutants identified in
our genetic screens arrest as homozygotes at L1 and L2 larval stages. In
sor-1(RNAi) hermaphrodites, vulva defects are apparent, such as
bursting vulva and protruding vulva (Fig.
1G). In addition, 20% of sor-1(RNAi) mutants
(n=41) show partial hermaphrodite-to-male sexual transformation,
revealed by inappropriate expression of pkd-2::gfp, a marker of
male-specific neurons fates (Fig.
1H). sor-1(RNAi) hermaphrodites are also sterile because
of defects in gonads and germline development. These defects cannot be readily
attributed to misregulated expression of Hox genes and suggest that
sor-1 may have other targets in addition to Hox genes.
sor-1 and sop-2 act synergistically in Hox gene repression
The defects of sor-1 mutants are similar to those previously
described for sop-2 mutants
(Zhang et al., 2003). Both
sor-1 and sop-2 are involved in global repression of Hox
genes and in regulating the expression of non-homeotic genes. To assess the
interactions between sop-2 and sor-1, we generated
sop-2; sor-1 double mutants by performing sor-1(RNAi) in
sop-2(bx91) animals. sor-1(RNAi) is likely to recapitulate a
null or strong loss-of-function phenotype in the animals developed from the
eggs laid in 4-48 hours after injection, as the same extent of ectopic
expression of Hox genes was observed in sor-1 (RNAi) and sor-1
(bp1) (in which about two-thirds of SOR-1 is deleted) mutant animals.
Shifitng sop-2(bx91ts) to non-permissive temperature (25°C) at L4
stage appears to cause null defects in the next generation, as the same extent
of defects were observed as those in sop-2(bp7) animals, a putative
null allele of sop-2, in which a stop codon mutation occurs at
position 80, leading to a deletion of the 655 C-terminal residues of SOP-2. We
chose RNAi because both sop-2(bp7) and sor-1(bp1) homozygous
animals arrest at L1 to L2 larval stages and can be maintained only as
heterozygous. We examined the genetic interactions between sor-1 and
sop-2 by comparing the detailed expression pattern of egl-5
in sor-1(RNAi); sop-2(bx91) double mutants with that observed in
sor-1(RNAi) and sop-2(bx91) single mutant animals. First, we
determined the onset of ectopic expression of Hox genes. In sor-1 and
sop-2 single mutants, Hox genes are not ectopically expressed until
the developing embryos reach the threefold stage. By contrast, ectopic
expression of egl-5 in the head region was observed earlier, at the
`pretzel' stage in sop-2(bx91); sor-1(RNAi) embryos (n=15)
(Fig. 2E,F), during which
neither sor-1(RNAi) (n=6) nor sop-2(bx91)
(n=3) shows ectopic expression of egl-5
(Fig. 2A-D). Thus,
sor-1 and sop-2 function synergistically in maintaining the
repressed state of Hox genes in early embryogenesis.
Next, we examined the spatial limits of ectopic expression domains of Hox genes in sop-2; sor-1 animals. In sor-1 and sop-2 single mutants, egl-5 is ectopically expressed in head neurons and in tail cells. The number of cells expressing the egl-5 reporter, however, is dramatically increased in sop-2(bx91); sor-1(RNAi) double mutants. In sop-2(bx91); sor-1(RNAi) mutants, the average number of cells expressing egl-5 is 47 in the head and 28 in the tail, compared with 13 in the head and eight in the tail in sor-1(RNAi) mutants, and with 32 in the head and 12 in the tail in sop-2 mutants. The increased number of egl-5-positive cells in sop-2; sor-1(RNAi) mutants is not an additive effect of sor-1 and sop-2, as the expression domains in the head and tail are dramatically expanded (Table 1; Fig. 2G-I). Moreover, in 89% (n=19) of the sop-2(bx91); sor-1(RNAi) animals, egl-5 is seen in the mid-body region, including the ventral cord and seam cells (Fig. 2J), whereas it is observed in only 13% (n=39) of sor-1 mutants and 24% (n=37) of sop-2 mutants. Therefore, sor-1 and sop-2 act synergistically in some body regions to repress Hox gene expression.
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sor-1 encodes a novel protein
The sor-1 locus was mapped by three-factor mapping between
unc-32 and sma-3, a small genetic interval on chromosome III
(Fig. 3A). It was cloned by
transformation rescue experiments (see Materials and methods). The
sor-1 rescuing DNA fragment contains a single predicted gene
ZK1236.3. Most of the intron/exon boundaries were confirmed by sequencing the
cDNAs of this region (Fig. 3A). We found that a 22 nucleotide leader sequence, the spliced leader 1 (SL1), is
trans-spliced onto the 5' end of sor-1 transcripts. SL1
trans-splicing occurs in about 57% of the genes in C. elegans
(Blumenthal and Steward, 1997).
SOR-1 encodes a protein with 810 amino acids
(Fig. 3B). In
sor-1(bp1) mutants, the CAG glutamine codon at position 304 is
mutated to TAG amber stop codon, resulting in the truncation of 506 amino
acids in the C terminus. In sor-1(bp2) and sor-1(bp3), an
identical nonsense mutation occurs at position 634, leading to a deletion of
the 176 C-terminal residues (Fig.
3B). Existence of these mutations in sor-1 mutant alleles
further confirms the identification of sor-1. Treatment of wild-type
animals with ZK1236.3 dsRNA phenocopies sor-1 mutants, causing the
ectopic expression of mab-5 and egl-5 as described above.
Therefore, the ectopic expression of Hox genes is due to the loss of function
of sor-1.
SOR-1 is an RNA-binding protein
RNA has been postulated to play an important role in PcG-mediated Hox gene
repression (Sun and Zhang,
2004). We therefore tested whether SOR-1 directly binds to RNA.
GST-SOR-1 was purified and incubated with a radiolabeled single- or
double-stranded RNA derived from the 5'UTR of the Hox gene
egl-5, followed by electrophoretic mobility shift analysis (EMSA).
The results showed that a fragment of SOR-1 binds efficiently to the RNA
probes (Fig. 3C). Binding of
SOR-1 to RNA is effectively competed by adding cold unrelated single- or
double-stranded RNA, but not by adding tRNA or DNA
(Fig. 3D). This rules out the
possibility that SOR-1 is a general nucleic acid-binding protein or sticks to
the charged phosphate backbone of nucleic acids non-specifically. RNA binding
by SOR-1 in vitro does not appear to be sequence specific, as several
unrelated RNA templates bind to SOR-1 with comparable efficiency (data not
shown). However, this apparent lack of sequence specificity in vitro may not
reflect the differential affinity for physiologically relevant RNA
targets.
Using a series of nine overlapping SOR-1 fragments, the specific RNA-binding region of SOR-1 was mapped to an 87 amino acid region (amino acid 443 to 530). This region does not contain a recognizable RNA-binding motif, but is rich in proline and glutamine. A transgene deleted for the RNA binding region of SOR-1 cannot rescue the sor-1 mutant phenotypes, indicating the importance of this region for sor-1 function.
To gain further insight into the functional properties of SOR-1, we searched for putative domains in SOR-1. SOR-1 contains a region that shows weak similarity to the mouse PcG protein Rae28 (35% similarity over 381 amino acids from amino acids 115 to 496 of SOR-1) and a second region that has weak similarity to extensin 2 domain (amino acids 422 to 659 of SOR-1, 6.1e–01), which, notably, is also present in SOP-2 (amino acids 300 to 648, 5.9e–01). The extensin motif exhibits a high degree of post-translational modification, including hydroxylation, glycosylation and crosslinking, suggesting that the function of SOR-1 and SOP-2 may be regulated by post-translational modification.
SOR-1 colocalizes to the same nuclear bodies as SOP-2
To examine the expression pattern of sor-1, we generated
transgenic lines expressing sor-1::gfp. The reporter gene contains
the entire coding sequence and the promoter region of sor-1, with
gfp inserted in the C terminus of sor-1. Fluorescence can be
observed in all nuclei in developing embryos. At larval stages,
sor-1::gfp becomes weaker in all of the somatic cells. Interestingly,
SOR-1::GFP forms distinct nuclear bodies besides its homogenous distribution
in the nucleoplasm (Fig.
4A).
To further determine the expression pattern and level of endogenous SOR-1 protein, we raised rabbit polyclonal antibodies against the N-terminal 160 amino acids of SOR-1 (see Materials and methods). The SOR-1 antibody staining pattern was consistent with those shown by the gfp reporter. SOR-1 is nuclear localized and expressed in all cells from the one-cell stage onwards. As development proceeds, the expression level of SOR-1 appears to decline. From the two cell-stage onwards, SOR-1 is found to be inhomogenously expressed in the nuclei with obvious accumulations in distinct nuclear speckles (Fig. 4B). During larval development, SOR-1 is present in all somatic cell nuclei. Although at lower levels of expression, the nuclear bodies in which it localizes can still be formed.
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).
Transgenic animals expressing a functional sop-2::gfp reporter were
immunostained with anti-SOR-1 antibodies. The superimposable confocal images
of SOR-1 (Fig. 4C) and
SOP-2::GFP (Fig. 4D) in
distinct nuclear bodies provide evidence that they are colocalized in SOP-2
nuclear bodies (Fig. 4E).
SOP-2 is required for the localization of SOR-1 into nuclear bodies
To determine the role of SOP-2 and SOR-1 in formation of the SOP-2 nuclear
bodies, we examined the localization of these proteins in sop-2 and
sor-1 mutants using anti-SOP-2 and anti-SOR-1 antibodies. Absence of
SOR-1 does not affect the localization of SOP-2
(Fig. 4F,G). No obvious changes
in SOP-2 localization were observed in sor-1(RNAi) embryos
(Fig. 4F). In
sop-2(bx91) mutant embryos, both SOP-2(bx91) (46/46 cells) and SOR-1
(45/45 cells) are localized in the cytoplasm
(Fig. 4H-M), while the
cytoplasmic localization of SOP-2 and SOR-1 is not evident in wild-type
animals (>50 cells examined). In sop-2(bx91) mutant embryos, the
nuclear localization of SOP-2(bx91) and SOR-1 is also seen in some cells
(25/46 cells for SOP-2, and 30/45 cells for SOR-1). Thus, SOP-2 function is
required for the proper localization of both SOP-2 and SOR-1.
SOR-1 and SOP-2 interact directly
Colocalization of SOR-1 and SOP-2 in SOP-2 bodies and dependence of the
localization of SOR-1 on the function of SOP-2 prompted us to test whether
these proteins directly interact. Glutathione-S-transferase (GST)-SOP-2
carried on glutathione sepharose beads was incubated with S35
labeled SOR-1. The retained proteins after several washes were examined by gel
electrophoresis. We found that SOP-2 specifically binds to the N-terminal 503
amino acids of SOR-1 (Fig. 5A).
And, vice versa, GST-SOR-1 interacts with the 35S-labeled SOP-2
(Fig. 5B). As SOP-2 contains an
RNA-binding activity and reactions of in vitro translated proteins contain
many RNAs, we further tested whether the interaction between SOR-1 and SOP-2
was RNA dependent. We found the interaction was not significantly reduced
after the reaction was treated with RNase (data not shown), supporting the
notion that SOP-2 and SOR-1 interact directly.
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Interaction between the N terminus of SOP-2 and SOR-1 was further demonstrated by the co-purification of SOP-2 and SOR-1 expressed in bacterial. GST-SOP-2 (amino acids 58-140) and non-tagged SOR-1 (amino acids 48-200) were co-expressed in E. coli strain BL21. SOP-2 proteins were purified by the GST affinity column followed by ion exchange and Superdex columns. The elute was subjected to SDS-PAGE and visualized by Coomassie staining. We found that SOR-1 was specifically co-purified with SOP-2 (Fig. 5D).
SOP-2 synergistically interacts with the MES-2/MES-6 complex in regulating non-Hox genes
In addition to a role in germline development
(Fong et al., 2002), the
MES-2/MES-6 complex is also involved in transcriptional repression of Hox
genes (Ross and Zarkower,
2003). To determine whether the MES-2/MES-6 complex and the
putative SOP-2/SOR-1 complex function together in repression of Hox genes, we
compared the expression pattern of Hox reporter genes in sop-2(bx91);
mes-2/mes-6(RNAi) mutants with those in single mutants. As previously
reported, egl-5::gfp is weakly ectopically expressed in
mes-2/mes-6(RNAi) mutants (Table
1). However, no synergistic interactions are observed between
sop-2(bx91) and mes-2/mes-6 (RNAi)
(Table 1). The onset of ectopic
expression and the expression domains of egl-5::gfp in
sop-2(bx91); mes-2/mes-6(RNAi) is similar to those in sop-2
single mutants. Lack of synergy between mutations in these two complexes and
the weaker effects of mes-2 and mes-6 on Hox genes suggest
that the MES-2/MES-6 complex is unlikely to be a prerequisite to recruit the
SOP-2/SOR-1 complex to Hox genes.
However, sop-2(bx91) and a mes-6 mutation cause synthetic lethality and a bursting vulva phenotype. Shifting sop-2; mes-6 L1/L2 larvae from 15°C to the semi-permissive temperature 20°C, 69.3% of animals (n=322) died of bursting vulva, whereas none of the single mutants (n>100) has a bursting vulva phenotype. Shifting sop-2; mes-6 mutant eggs laid in the first 8 hours from 15°C to non-permissive temperature 23°C, 100% animals arrested at L1 stage, while sop-2(bx91) mutants alone arrested at L2 to L4 stages and all mes-6 mutant animals developed into adults. No such synergistic interactions have been observed between sop-2 and a mes-4 mutation, consistent with the fact that MES-4 is not a component of the MES-2/MES-6 complex. These synergistic interactions suggest that SOP-2/SOR-1 and MES-2/MES-6 could act in a single pathway or their effects in separate pathways may be cumulative in regulating non-Hox genes.
SOR-1 and SOP-2 orthologs are absent from other organisms
SOR-1 and SOP-2 do not appear to be homologs of known PcG proteins in other
organisms, although several properties of the SOR-1/SOP-2 complex, including
localization into nuclear bodies and binding to RNA, are reminiscent of the
ones of the PRC1 complex in other organisms. To determine whether SOR-1/SOP-2
defines a new global Hox gene repression system, we examined whether other
organisms contain homologs of SOR-1 and SOP-2. Surprisingly, no recognizable
SOR-1 or SOP-2 homologs were identified in fly, mouse and human by searching
available databases.
To determine whether SOR-1 and SOP-2 are conserved among nematodes, we
searched the sequence of C. briggsae, which is the closest known
species to C. elegans and has almost identical morphology. The C.
briggsae sequence is currently more than 98% complete and a genome-wide
comparison of C. elegans and C. briggsae reveals that 89% of
the C. briggsae genes have either orthologs (62%) or multiple matches
(27%) in the C. elegans genome
(Stein et al., 2003). As in
C. elegans, the ESC/E(Z) complex is present in C. briggsae.
However, C. briggsae lacks PRC1 complex genes. Surprisingly,
orthologs of SOR-1 or SOP-2 could not be identified in C. briggsae
genome. The most closely related protein to SOR-1 in C. briggsae is
CBP23695 (encoded by CBG18142) (2.6e–10, 45.3%) and to SOP-2
is CBP06294 (encoded by CBG02561) (1.1e–19, 52.4%). However,
these two proteins do not appear to be the orthologs of SOR-1 and SOP-2.
Regions of homology are restricted to the low complexity parts of SOR-1 or
SOP-2. More importantly, functionally significant domains in SOR-1 (e.g. the
RNA binding domain) and SOP-2 (e.g. the SAM domain and the RNA-binding
domains) are not present in these proteins. In addition, CBP06294 has a clear
ortholog in C. elegans, the gene ZK84.1 (7.4e–175,
82.1%), and CBP23695 is closer to the C. elegans gene H20J18.1a
(3.7e–16, 54.6%). Interestingly, orthologs of the genes
adjacent to both sor-1 and sop-2 do exist in C.
briggsae and the organization of these neighboring genes in C.
briggsae is comparably co-linear with the ones in C. elegans
(Fig. 6A,B). For example, as in
C. elegans, there are two genes between CBG18138 and CBG18143, and
one gene between CBG20942 and CBG20938 in C. briggsae. This further
supports the assertion that SOR-1 and SOP-2 orthologs are absent from C.
briggsae. In conclusion, the putative SOR-1/SOP-2 complex, the function
of which is similar to that of the PcG complex in other organisms in
maintaining the repressed state of Hox genes, may have undergone specific
evolution in C. elegans, even though the underlying mechanisms appear
to be conserved.
|
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). These
synergistic effects could be due to the disruption of the function or
integrity of the complex by a cumulative effect of two simultaneously
mutations.
|
). Homeotic defects are more severe in the loss of function of
Pc, Ph and Scm than those in loss of function of other
PcG components. This could be due to their distinct roles in
establishing repressive chromatin structures. For example, PSC is essential
for compacting the nucleosomal arrays, while RING1 mediates the ubiquitination
of H2A (Francis et al., 2004;
de Napoles et al., 2004;
Wang et al., 2004).
Other possible integral components of the SOP-2/SOR-1 complex are RNAs.
Both SOR-1 and SOP-2 contain RNA-binding activity. Having two RNA-binding
proteins involved in the same process supports the role of RNA in epigenetic
silencing of Hox genes, although the nature of the RNA components in the
SOP-2/SOR-1 complex has yet to be identified. siRNAs and structural
roX1 and roX2 RNAs are important for the formation and
targeting of the RITS complex and the Drosophila dose compensation
complex (DCC), respectively (Verdel et
al., 2004; Motamedi et al.,
2004; Akhtar,
2003). Analogous to other RNA-mediated epigenetic gene regulatory
phenomena, the RNA components could be involved in maintaining the integrity
of the PcG complex and/or targeting the PcG complex to specific loci
(Fig. 6C).
Transcriptional repression mediated by the SOP-2/SOR-1 complex and the PRC1 complex
In other organisms, the ESC/E(Z) complex and the PRC1 complex function
together in the maintenance of the Hox gene expression. The ESC/E(Z) complex
may be involved in recruiting the PRC1 complex to the target loci. Unlike in
sop-2 and sor-1 mutants, which have widespread ectopic
expression of Hox genes, the effect of mes-2 and mes-6
mutations on Hox gene repression is subtle and is only apparent in sensitized
genetic backgrounds (Ross and Zarkower,
2003). Lack of synergy between sop-2(bx91) and
mes-2/mes-6(RNAi) further supports the conclusion that the
MES-2/MES-6 complex may not be involved in recruiting the SOP-2/SOR-1 complex
in Hox gene repression.
In spite of a lack of obvious sequence similarity, several conserved
properties of the PRC1 and putative SOP-2/SOR-1 complexes suggest that they
appear to employ a conserved mechanism in Hox gene repression. First,
components of the PRC1 complex and the SOP-2/SOR-1 complex are localized into
distinct nuclear bodies, although the mechanistic role of the bodies in Hox
gene repression remains unknown. Second, the protein-protein interaction SAM
domain is present in both complexes. Domain-swapping experiments indicate that
only the SAM domains of PcG proteins, including PH and SCM, but not the ones
of non-PcG proteins, including TEL and L(3)MBT, can functionally substitute
for the SAM domain of SOP-2 in terms of repression of Hox genes and proper
formation of SOP-2 nuclear bodies (Zhang
et al., 2004a). Overexpression of the SAM domain behaves as a
dominant-negative regulator that compromises the function of the SOP-2/SOR-1
complex and the PRC1 complex in Hox gene repression (Y.S. and H.Z.,
unpublished) (Peterson et al.,
2004). These observations indicate an important role of the SAM
domain in PcG-mediated Hox gene repression. Third, both of the SOP-2/SOR-1
complex and the PRC1 complex contain RNA-binding activity
(Zhang et al., 2004b).
Although a role for RNA in PcG-mediated gene silencing of Hox genes in other
organisms has not been demonstrated, PcG proteins are involved in some
RNA-dependent silencing processes, such as X chromosome inactivation and
silencing of transgenes in fly (Plath et
al., 2003; Plath et al.,
2004; Pal-Bhadra et al.,
2002). The evolutionary constraint on the PRC1 complex is, thus,
likely to be conferred at the mechanistic level but not at the protein level.
The reason for the maintenance of the PRC1 complex over very large
evolutionary distance encompassing insects and vertebrates, while in nematodes
a similar complex appears to be evolutionarily volatile, remains to be
explained.
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ACKNOWLEDGMENTS |
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Footnotes |
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