1 Department of Biological Sciences, Columbia University, New York, NY 10027,
USA
2 Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx,
NY 10461, USA
Author for correspondence (e-mail:
mc21{at}columbia.edu)
Accepted 13 January 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report: (1) that ectopic expression of sem-4 in normal touch cells represses mec-3 expression and reduces touch cell function; (2) that egl-5 expression is required for both the fate of normal PLM touch neurons in wild-type animals and the fate of a subset of abnormal touch neurons in sem-4 animals, and (3) that SEM-4 specifically binds a shared motif in the mec-3 and egl-5 promoters that mediates repression of these genes in cells in the tail. We conclude that sem-4 represses egl-5 and mec-3 through direct interaction with regulatory sequences in the promoters of these genes, that sem-4 indirectly modulates mec-3 expression through its repression of egl-5 and that this negative regulation is required for proper determination of neuronal fates. We suggest that the mechanism and targets of regulation by sem-4 are conserved throughout the sal gene family: other sal genes might regulate patterning and cellular identity through direct repression of Hox selector genes and effector genes.
Key words: spalt, Repressor, Hox, LIM homeodomain, AP patterning
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three fundamental questions about the functions of sem-4 and the
other sal genes remain unresolved: (1) do sal genes bind DNA; (2) do they
function as repressors in vivo; and (3) what are their targets? This work
identifies the C. elegans sal gene sem-4 as a regulator of
C. elegans homeobox gene expression and examines the mechanism
through which sal genes control cellular identity. Like other sal genes,
sem-4 is required for multiple aspects of development. The strongest
alleles of sem-4 are egg-laying defective (Egl),
uncoordinated (Unc), partially sterile and constipated, and have
deformed tails (Basson and Horvitz,
1996). Animals with sem-4 mutations exhibit abnormalities
in a range of different cell types, including neurons, muscle cells,
coelomocytes and vulval cells (Basson and
Horvitz, 1996
; Grant et al.,
2000
). In particular, sem-4 animals produce additional
touch-neuron-like cells that express the touch cell effector gene
mec-3 (Basson and Horvitz,
1996
; Mitani et al.,
1993
; Mitani,
1995
).
We report that, in sem-4 animals, the abnormal touch-neuron-like cells and their precursors ectopically express the Hox gene egl-5, an Abdominal-B (Abd-B) homolog. We show that inappropriate expression of egl-5 transforms the fates of these neuroblasts and neurons, that SEM-4 binds to a shared motif in the mec-3 and egl-5 promoters, and that ectopic sem-4 represses mec-3 expression in vivo. Our findings point to three conclusions: first, that sem-4 and other sal genes are repressors that control cellular identity by restricting expression of Hox genes and effector genes; second, that sal genes independently regulate these genes at multiple stages in developmental pathways; and, third, that sal proteins bind directly to a shared motif in regulatory regions of their targets.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phenotypic characterization
Cell lineages were followed as described by Sulston and Horvitz
(Sulston and Horvitz, 1977).
Expression of gfp reporters was observed at 1000x
magnification. The touch sensitivity assay was modified from Hobert et al.
(Hobert et al., 1999
). Each
animal was touched with an eyebrow hair ten times alternately at the head and
tail. At least 100 animals were scored for each stable line or mutant strain.
To ensure that observations of cells in temperature-sensitive sem-4
and Hox mutant strains were not influenced by maternal effects, we scored
worms from at least the third generation grown at each particular temperature.
At least 45 gravid adults were scored for each strain at each temperature.
Expression studies
Expression studies using sem-4 were carried out using a
PCR-amplified fragment of the sem-4 genomic sequence from cosmid
F15C11 that was cloned into the PstI and KpnI sites of Fire
vector pPD 95.75 (Fire et al.,
1990). The fragment contained 5 kb upstream of the sem-4
start site and the entire genomic sequence fused at the 3' end to
gfp. This construct was injected into N2 worms and two stable lines
were obtained. It was difficult to produce stable lines containing this
expression construct because the stable lines exhibited various elements of
the sem-4 phenotype, including sterility and deformed tails. In
addition, in one instance, T.pppp underwent a necrotic death in a
sem-4::gfp transformant, as it did in the sem-4(n1971;
mec-3::gfp) strain. We also used MH1346, containing an integrated
sem-4::gfp reporter, for sem-4 expression studies.
egl-5 expression studies were carried out using the integrated
egl-5::gfp array in strain EM597 (bxIs12), and the
extrachromosomal arrays in strains EM783, EM784 and EM785. bxIs12 was
generated by integration of a transgene (EM#285) that was constructed to
contain 16,027 nucleotides upstream of the egl-5 AUG (beginning at an
NruI site at position 23,448 in cosmid C08C3), the full set of
egl-5 exons and introns, and 2639 nucleotides downstream of the
egl-5 stop codon (ending at position 43,981, the right end of C08C3).
GFP was inserted at an ApaI site in the third egl-5 exon at
position 40,261 and disrupts the homeodomain, so that the expressed protein is
expected to be non-functional. The arrays in EM783, EM784 and EM785 were
constructed by insertion of gfp into the ApaI site in exon 3
of egl-5. EM783 (containing cosmid C08C3 bp 36249-43981), EM784
(C08C3 35986-36293) and EM785 (C08C3 36088-36293) were generated by PCR fusion
(Hobert et al., 1999).
For ectopic sem-4 expression studies,
Pmec-7sem-4 constructs were created by PCR amplification
of portions of cosmid F15C11. For ease of cloning, we omitted the first, small
exon from these clones, which began instead at the second start site (in exon
2) identified by Basson and Horvitz (Basson
and Horvitz, 1996). The resulting DNA fragments were cloned into
the XmaI and KpnI sites of Fire vector pPD52.102
(Fire et al., 1990
). The
Pmec-7sem-4 (n1378) construct contained an
additional point mutation that produced a D506V change and the
Pmec-7sem-4 (n2087) construct contained an
additional point mutation that produced a T301A change. For studies of the
effect of ectopic sem-4 on mec-3 expression,
Pmec-7sem-4 constructs were injected into TU2562. For each
construct, we scored three independent stable lines and, for each stable line,
we scored at least 50 worms. Plasmids were injected with the dominant
rol-6(su1006) marker plasmid pRF4 using standard methods
(Mello et al., 1992
). Injected
DNA forms stable extrachromosomal arrays containing multiple copies of the
injected construct.
Gel-mobility-shift assays
A PCR-amplified full-length sem-4 cDNA was cloned into the
BamHI and NotI sites of pGEX 6P-1 (Amersham Pharmacia
Biotech). The construct produced an N-terminal
glutathione-S-transferase (GST) fusion. This GST::SEM-4 fusion was
overproduced in BL21(DE3)pLysS cells and the fusion protein solubilized as
described in Frangioni and Neel (Frangioni
and Neel, 1993). SEM-4 was cleaved from the glutathione-bound GST
moiety according to the manufacturer's instructions in the following cleavage
buffer: 50 mM Tris-HCl pH 7.0, 150 mM NaCl, 1 mM EDTA, 0.1% bovine serum
albumin (BSA), 10 mM DTT, 2% Triton X-100, 0.5 mM ZnSO4.
The 96 bp mec-3 promoter fragment used as a probe in the
Fig. 4A gel-mobility-shift
assays extended from positions 1714 to 1809 of the mec-3 promoter
(Way and Chalfie, 1988). The
m3-1 sequence extends from 1759 to 1764, m3-2 from 1714 to 1718 and m3-3 from
1648 to 1654. The 100 bp egl-5 promoter fragment used as a probe in
the Fig. 4B gel-mobility-shift
assays extended from position 38515 to 38615 of cosmid C08C3. The e5-1
sequence extends from position 38525 to 38531, e5-2 from 38551 to 38557 and
e5-3 from 38600 to 38606. The 105 bp egl-5 promoter fragment used as
a probe in the Fig. 4D
gel-mobility-shift assays extended from position 35986 to 36091 of cosmid
C08C3. The e5-4 sequence extends from position 35997 to 36003, e5-5 from 36054
to 36060 and e5-T1 from 36070 to 36087.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Because Lineage 2 produced more ectopic mec-3::gfp-expressing cells than Lineage 1, we hypothesized that animals with more ectopic mec-3::gfp-expressing cells should produce PVM-like cells. Conversely, animals with fewer ectopic mec-3::gfp-expressing cells should produce mostly PLM-like cells. At 25°C, in 51 sem-4 adults with four mec-3::gfp-expressing tail cells, 93% (189/204) of the cells resembled PLM, 3% (6/204) resembled PVM and 4% (9/204) could not be unambiguously classified as either PLM or PVM. The cells in this last category generally had rounded cell bodies, like PVM, but had processes that were difficult to see or did not extend ventrally and then anteriorly. In 52 animals with more than four mec-3::gfp-expressing tail cells, 67% (181/269) resembled PLM, 10% (28/269) resembled PVM and 22% (60/269) could not be unambiguously classified.
sem-4 restricts proliferation of touch-cell fate through
repression of egl-5
We found that loss of sem-4 function caused transformation of the
T.pp lineage into two distinct touch-cell lineages: a PLM-like lineage and a
PVM-like lineage. The transformation of the posterior T.pp lineage into a
mid-body PVM-like lineage is a transformation along the AP axis of the worm.
AP transformations often result from defects in the expression or function of
Hox genes that control AP patterning and several different aspects of cellular
identity, including differentiation, growth and proliferation
(Cillo et al., 2001;
Veraksa et al., 2000
).
The C. elegans Hox gene that controls patterning of posterior
structures is the Abd-B homolog egl-5
(Chisholm, 1991;
Salser and Kenyon, 1994
). In
the tail, egl-5 is normally expressed in two pairs of neurons: the
PLM touch neurons and the PVC interneurons
(Ferreira et al., 1999
).
Although egl-5 animals are touch insensitive in the tail
(Chalfie and Au, 1989
;
Chisholm, 1991
), this defect
has been attributed to abnormal development of the PVC interneurons
(Chisholm, 1991
), which are
part of the neural circuit for touch sensitivity
(Chalfie et al., 1985
). We find
that egl-5 is required for normal PLM development: PLM cells in
egl-5 animals showed substantially reduced expression of
mec-3 and abnormal morphologies
(Table 1). We suggest that
egl-5 activates mec-3 expression in PLM cells.
The findings that egl-5 is required for correct determination of PLM fate in wild-type animals and that the T.pp lineage is sometimes transformed to a PLM-like lineage in sem-4 animals suggest that loss of sem-4 function produces ectopic expression of egl-5 in the T.pp lineage. We observed ectopic expression in sem-4 animals of an egl-5::gfp reporter first in the T.pp neuroblast and then in T.ppa, T.ppp, T.pppa and T.pppaa (which ectopically expresses mec-3 in sem-4 animals). Because we did not observe ectopic egl-5 expression in T.pppap (which does not express mec-3 in sem-4 animals), ectopic egl-5 expression in T.pppaa was probably not residual gfp expression from T.pppa. In wild-type worms, we observed expression of a sem-4::gfp reporter in T, T.a, T.p and all descendants of T.p. Thus, ectopic egl-5 expression in the T lineage in sem-4 animals began two cell divisions (in T.pp) after sem-4 expression would normally begin (in T). This ectopic T lineage expression was observed with two different egl-5::gfp reporters in the sem-4 background: one reporter contained a 12 kb region immediately upstream of the egl-5 translation start site; the second reporter contained only a 3 kb region immediately upstream of the start site (Fig. 3). We propose that sem-4 acts both in neuroblasts and neurons to restrict egl-5 expression.
|
We investigated whether mutation of the other C. elegans Hox genes, mab-5 and lin-39, could affect the number or morphology of the ectopic touch neurons in sem-4 animals. A partial loss-of-function allele of lin-39 and a putative null allele of mab-5 moderately decreased both the total number of ectopic touch neurons (data not shown) and the number of PLM-like cells: 70% (141/200) of mec-3-expressing tail cells in sem-4; mab-5 animals and 74% (147/200) in sem-4; lin-39 animals were PLM-like.
We conclude that sem-4 normally prevents the expression of egl-5 and possibly of mab-5 and lin-39 in the T lineage. Because egl-5 appears to activate mec-3 expression in normal PLM cells, we suggest that sem-4 restricts mec-3 expression in the T lineage indirectly through restriction of egl-5. We found that egl-5 is required for correct expression of the PLM fate in wild-type PLM cells and for ectopic expression of the PLM fate in the abnormal touch cells in sem-4 animals. Negative regulation of egl-5 by sem-4 is therefore necessary to restrict inappropriate proliferation of the PLM fate in wild-type animals.
SEM-4 binds to a shared motif in the mec-3 and
egl-5 promoters
Mutations in the mec-3 promoter at a 6 bp site (m3-1 in
Fig. 4A), about 300 bp upstream
of the translation start produced ectopic expression of a mec-3::lacZ
reporter in additional cells in the tail (Xue, 1993). The m3-1 region is one
of several in the mec-3 promoter that is conserved between C.
elegans and the related nematode C. briggsae. The ectopic
mec-3::lacZ expression suggested that m3-1 might be a SEM-4 binding
site. We found that, although purified SEM-4 protein did not shift a 24 bp
fragment with m3-1 at its center (data not shown), it did shift a 96 bp
fragment with m3-1 at its center (Fig.
4A). Four complexes were produced that were competed away by
unlabeled specific competitor.
Mutation of m3-1 in the unlabeled competitor decreased, but did not eliminate, its ability to compete away complexes 2 and 4 (Fig. 4A), and had no effect on its ability to compete away complexes 1 and 3. An oligonucleotide composed of four tandem copies of the 24 bp fragment with m3-1 at the center was not as effective a competitor as the 96 bp wild-type competitor (data not shown). These results suggest that the 96 bp fragment used as a probe contained more than one SEM-4 binding site. The 5' end of this fragment contains a sequence identical at five out of six positions to m3-1 (m3-2 in Fig. 4A) that we tested for SEM-4 binding. Mutations at both m3-1 and m3-2 substantially reduced the ability of the unlabeled oligonucleotide to compete away complexes 1, 2 and 4 (Fig. 4A). Complex 3 is probably formed by SEM-4 binding to a site other than m3-1 or m3-2.
We also looked for putative SEM-4 binding sites in the egl-5 promoter. We found a cluster of three sites (e5-1, e5-2, e5-3), one identical to m3-1 (although on the opposite strand) and two identical to m3-2 (with one on the opposite strand), within an 81 bp region located about 900 bp upstream of the egl-5 translation start site (Fig. 4B). Similar clusters, of three sites within 125 bp, occur infrequently in the region we analysed. Specifically, in the 31 kb separating the translation starts of egl-5 and mab-5 (these genes are transcribed in opposite directions, with their 5' ends facing one another, separated by one predicted gene), only one other similar cluster occurs. This second cluster, of three sites within a 121 bp region, is located about 5 kb upstream of the mab-5 translation start site. We reexamined the region of the mec-3 promoter that contained the two sites we identified and found a third site (m3-3) about 60 bp upstream of the 5' site (Fig. 4A). Thus, this region contains a cluster of three sites within 117 bp. A 206 bp region in the lin-39 promoter, about 10 kb upstream of the translation start site, also contains a cluster of three SEM-4 sites.
SEM-4 shifted a 100 bp fragment containing e5-1, e5-2 and e5-3 (Fig. 4B). Two complexes were produced that were competed away by unlabeled competitor oligonucleotides. Mutation of all three sites in the wild-type competitor significantly decreased, but did not eliminate, its ability to compete away both complexes.
We identified a second region of the egl-5 promoter to which SEM-4
binds. About 3.5 kb upstream of the egl-5 start site, there is a 300
bp sequence (V6CRE) that mediates expression of egl-5 in the V6
hypodermal lineage. We found that a reporter containing V6CRE fused to
gfp was not expressed in the T lineage. Occasionally, some faint
expression was detected in a couple of T lineage cells. In a sem-4
background, however, expression of PV6CREgfp
increased significantly (Fig.
4C). T lineage expression in wild-type worms of a reporter lacking
the first 100 bp of PV6CREgfp
(PV6CRE100gfp) was similarly strong
(Fig. 4C).
The region deleted in PV6CRE100gfp
contains a consensus TRA-1 binding site (e5-T1) and two sites that contain
five out of six bases of SEM-4 binding sites (e5-4 and e5-5). We tested
binding of SEM-4 to a probe composed of the first 105 bp of V6CRE. SEM-4
shifted this probe (Fig. 4D),
forming four complexes that were competed away by unlabeled competitor
oligonucleotides. Mutation of e5-4 and e5-5 in the wild-type competitor
produced a small but consistent reduction in competition
(Fig. 4D). Mutation of e5-T1
had no effect on the ability of the competitor to compete away the complexes
(Fig. 4D).
We conclude that SEM-4 binds to a shared motif in the mec-3 and egl-5 promoters and suggest that these interactions repress mec-3 and egl-5 expression in the T lineage. We propose that sem-4 restricts mec-3 expression both directly, by binding to the mec-3 promoter, and indirectly, through repression of egl-5.
Ectopic sem-4 represses mec-3 expression in
vivo
The mec-3 gene is normally expressed in only ten cells: the six
touch neurons, a pair of neurons in the head (the FLP cells) and a pair of
mid-body neurons (the PVD cells). To test whether ectopic sem-4 could
repress mec-3 in vivo, we expressed sem-4 in the touch cells
under the control of the mec-7 promoter
(Pmec-7sem-4). mec-7 encodes a ß-tubulin
that is expressed strongly in all six touch neurons during their terminal
differentiation and less strongly in several other cells
(Hamelin et al., 1992;
Mitani et al., 1993
;
Savage et al., 1989
). We
analysed the effect of this ectopic sem-4 activity on mec-3
expression by transforming Pmec-7sem-4 into worms
containing an integrated mec-3::gfp reporter.
Transformation with Pmec-7sem-4 decreased the proportion of mec-3::gfp-fluorescent PLM cells from 100% (102/102) in the control line to 48±20% in three transformed lines (350 cells) (Fig. 5A). (In C. elegans, transformed DNAs form extrachromosomal arrays that are often not present in all cells.) We also tested the ability of several truncated versions of SEM-4 to decrease expression of the mec-3::gfp reporter (Fig. 5B-F). The N-terminal half of SEM-4, truncated after zinc finger 3, was a relatively effective repressor: transformation with this construct decreased the proportion of fluorescent PLM cells to 74±18% in three transformed lines (312 cells) (Fig. 5B). When SEM-4 was truncated after zinc finger 2, however, the resulting fragment did not decrease mec-3::gfp expression (Fig. 5C).
|
We tested the effect on touch-cell function of ectopic expression of two
partial loss-of-function sem-4 alleles
(Fig. 5G). The n2654
`neuronal' allele (containing a mis-sense mutation that changes one of the
zinc-chelating histidines in zinc finger 2 to a tyrosine) exhibits more
defects in neurons than in mesoderm (Basson
and Horvitz, 1996); the n1378 `mesodermal' allele
(containing a nonsense mutation, Q569ocher, which truncates zinc fingers 5, 6
and 7) exhibits more defects in mesoderm than in neurons
(Basson and Horvitz, 1996
). We
found that transformation with Pmec-7sem-4(n1378), the
Q569ocher `mesodermal' allele, produced touch insensitivity, although not as
effectively as wild-type sem-4
(Fig. 5G). By contrast,
transformation with Pmec-7sem-4(n2654), the `neuronal'
allele, did not produce touch insensitivity
(Fig. 5G). We also found that
the neuronal allele was more touch insensitive than the mesodermal allele
(Fig. 6). These results are
consistent with the hypothesis that sem-4(n2654) does not function
properly in neurons.
|
Mutant versions of sem-4 display a gain-of-function
phenotype
Production of three mutant versions of SEM-4 under the control of the
mec-7 promoter produced mec-3::gfp expression in additional
cells in the tail (Fig. 5C-E).
Because mec-7 is expressed in cells other than the touch cells
(Hamelin et al., 1992;
Mitani et al., 1993
), the
Pmec-7 constructs probably expressed the truncated SEM-4
proteins in these additional cells. These gain-of-function mutant proteins
contained C2H2 zinc finger 1 and various portions of zinc finger 2
(Fig. 5C-E). These truncated
SEM-4 fragments might have interfered either with endogenous SEM-4 or with
other SEM-4 interacting partners in these additional cells.
We observed a gain-of-function phenotype in the sem-4(n2087)
allele, which contains a nonsense mutation in zinc finger 2
(Basson and Horvitz, 1996). We
found that n2087 worms were more touch insensitive than
n1971 worms, which contain an early splice-site mutation N-terminal
to zinc finger 1 (Fig. 6)
(Basson and Horvitz, 1996
). The
SEM-4 protein fragment encoded by n2087 is the same as that encoded
by the Pmec-7sem-4 construct (shown in
Fig. 5D). Worms transformed
with this construct had the most ectopic mec-3::gfp-expressing cells
(Fig. 5D). The fact that
homozygous n2087 worms exhibit a gain-of-function phenotype suggests
that truncated SEM-4 proteins interfere with the function of a protein other
than SEM-4.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hox genes appear to be targets not only of sem-4 but also of other
sal genes. Drosophila sal might negatively regulate Sex combs
reduced (Scr) and other Drosophila Hox genes. Loss of
sal function in Drosophila BX-C- embryos produced
some limited ectopic expression of the Hox gene Scr
(Casanova, 1989). Mutations in
sal enhanced the phenotypes of Polycomb group (PcG) mutants.
These genes are known to be negative regulators of Hox genes
(Landecker et al., 1994
). Loss
of sal function affects AP patterning in Drosophila.
Mutations in sal incompletely transform both head and tail structures
into trunk-like structures; sal activity has been shown to promote
head development (Jurgens,
1988
). Hox genes in mammals might also be targets of sal family
genes. Patients with TBS, which is caused by mutations in SALL1,
display characteristic features of syndromes associated with mutations in HOX
genes (Powell and Michaelis,
1999
; Surka et al.,
2001
; Veraksa et al.,
2000
).
LIM homeobox genes, such as mec-3, might also be conserved targets
of sal genes. The closest mammalian homolog to mec-3 is the human LIM
homeobox gene Lhx5 (Zhao et al.,
2000). Lhx5 and the human SALL1 gene appear to
be expressed in different sets of cells in the developing thalamus, which
constitutes a very small portion of the entire brain
(Kohlhase et al., 1996
;
Nakagawa and O'Leary, 2001
).
SALL1 and Lhx5 are not expressed in most other regions of
the fetal brain. Their expression in separate thalamic cells could indicate
that SALL1 restricts Lhx5 expression in the thalamus.
The mechanism through which sem-4 negatively regulates its targets
is probably conserved. SEM-4, SALL1 and mouse sall1 are transcriptional
repressors. SALL1 and mouse sall1, fused to heterologous DNA binding domains,
behaved as repressors in mammalian cell culture assays
(Kiefer et al., 2002;
Netzer et al., 2001
). We
suggest that these genes bind directly to regulatory regions of their
targets.
Particular mutant versions of SALL1, like certain SEM-4 truncations, might
act as gain-of-function proteins. TBS is an autosomal dominant disorder caused
by mutations in SALL1 (Kohlhase,
2000). No deletions of the entire gene or mutations that truncate
the protein upstream of the first zinc finger have been detected. Thus, all 21
SALL1 mutant alleles encode truncated protein products that contain
at least the first zinc finger. We found that truncations of SEM-4 containing
the first zinc finger acted as gain-of-function proteins. We suggest that TBS
could result, at least in part, from interference by these truncated proteins
with wild-type SALL1 or other proteins.
SEM-4 negatively regulates genes at multiple levels of a
developmental hierarchy
Very little is known about the pathways that lead from Hox proteins to
determination of the fates of particular structures or individual cells.
Recent evidence has suggested that Hox proteins can act independently on genes
that function at different points along a particular developmental pathway
(Veraksa et al., 2000). For
example, the Drosophila Hox gene Ultrabithorax
(Ubx) negatively regulates diverse genes throughout haltere
development (Weatherbee et al.,
1998
). These genes encode signaling molecules, their immediate
targets (including sal and salr) and proteins further
downstream, including transcription factors. We propose that negative
regulators of Hox genes, like the Hox genes themselves, also function at
different levels in a given developmental hierarchy. We found that
sem-4 negatively regulates both the Hox gene egl-5 in
precursors and differentiating cells and the LIM homeobox gene mec-3
in differentiating cells. Furthermore, we discovered that egl-5
positively regulates mec-3 in normal PLM cells. Because SEM-4 binds
to a shared motif in the promoters of both mec-3 and egl-5,
we conclude that sem-4 negatively regulates each gene independently
and also inhibits mec-3 expression through inhibition of
egl-5.
No evidence for a global repression system for Hox genes in C.
elegans has been reported. Polycomb group (PcG) and
trithorax group (trxG) genes were originally identified in
Drosophila as repressors and activators, respectively, of Hox gene
expression (Brock and van Lohuizen,
2001). PcG genes are now known to function together in a chromatin
repressive complex (Francis and Kingston,
2001
). Although the roles of Hox and trxG genes in patterning are
conserved in C. elegans, a role for PcG genes has not yet been
established. We speculate that sem-4 might function as a member of a
general repressive complex akin to the PcG complex.
Drosophila and mammalian studies have suggested that sal genes
might function as PcG genes. Casanova
(Casanova, 1989) found that
sal mutations caused limited ectopic expression of the Hox genes
Ubx and Scr, and Landecker et al.
(Landecker et al., 1994
) found
that sal mutations enhanced mutations in the PcG genes
polyhomeotic and Polycomb-like. Human SALL1 localizes to
chromocenters in mammalian cells (Netzer
et al., 2001
) and mouse sall1 interacts with components of
chromatin remodeling complexes (Kiefer et
al., 2002
). One additional speculation is that Drosophila
sal might bind to a 138 bp silencing sequence in the Polycomb
response element in Abd-B, the egl-5 ortholog
(Busturia et al., 2001
). We
have identified two sites that match the SEM-4 binding sequence in this
Drosophila silencing element.
PcG genes might play a role in positive, in addition to negative,
regulation of Hox genes (Brock and van
Lohuizen, 2001): mutations in some PcG genes enhance trxG mutant
phenotypes. sem-4 also appears to have a positive regulatory role in
Hox gene expression in certain tissues: sem-4 might activate
lin-39 in vulval lineages (Grant
et al., 2000
) and egl-5 in hypodermal lineages (Y. Teng
et al., unpublished).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Basson, M. and Horvitz, H. R. (1996). The Caenorhabditis elegans gene sem-4 controls neuronal and mesodermal cell development and encodes a zinc finger protein. Genes Dev. 10,1953 -1965.[Abstract]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Brock, H. W. and van Lohuizen, M. (2001). The Polycomb group no longer an exclusive club? Curr. Opin. Genet. Dev. 11,175 -181.[CrossRef][Medline]
Buck, A., Kispert, A. and Kohlhase, J. (2001). Embryonic expression of the murine homologue of SALL1, the gene mutated in Townes-Brocks syndrome. Mech. Dev. 104,143 -146.[CrossRef][Medline]
Busturia, A., Lloyd, A., Bejarano, F., Zavortink, M., Xin, H.
and Sakonju, S. (2001). The MCP silencer of the
Drosophila Abd-B gene requires both Pleiohomeotic and GAGA factor for
the maintenance of repression. Development
128,2163
-2173.
Casanova, J. (1989). Mutations in the spalt gene of Drosophila cause ectopic expression of Ultrabithorax and Sex combs reduced. Roux's Arch. Dev. Biol. 198,137 -140.
Chalfie, M. and Au, M. (1989). Genetic control of differentiation of the Caenorhabditis elegans touch receptor neurons. Science 243,1027 -1033.[Medline]
Chalfie, M. and Wolinsky, E. (1990). The identification and suppression of inherited neurodegeneration in Caenorhabditis elegans. Nature 345,410 -416.[CrossRef][Medline]
Chalfie, M., Sulston, J. E., White, J. G., Southgate, E., Thomson, J. N. and Brenner, S. (1985). The neural circuit for touch sensitivity in Caenorhabditis elegans. J. Neurosci. 5,956 -964.[Abstract]
Chisholm, A. (1991). Control of cell fate in the tail region of C. elegans by the gene egl-5. Development 111,921 -932.[Abstract]
Cillo, C., Cantile, M., Faiella, A. and Boncinelli, E. (2001). Homeobox genes in normal and malignant cells. J. Cell. Physiol. 188,161 -169.[CrossRef][Medline]
Desai, C., Garriga, G., McIntire, S. L. and Horvitz, H. R. (1988). A genetic pathway for the development of the Caenorhabditis elegans HSN motor neurons. Nature 336,638 -646.[CrossRef][Medline]
Duggan, A., Ma, C. and Chalfie, M. (1998).
Regulation of touch receptor differentiation by the Caenorhabditis elegans
mec-3 and unc-86 genes. Development
125,4107
-4119.
Ferreira, H. B., Zhang, Y., Zhao, C. and Emmons, S. W. (1999). Patterning of Caenorhabditis elegans posterior structures by the Abdominal-B homolog, egl-5. Dev. Biol. 207,215 -228.[CrossRef][Medline]
Fire, A., Harrison, S. W. and Dixon, D. (1990). A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans. Gene 93,189 -198.[CrossRef][Medline]
Francis, N. J. and Kingston, R. E. (2001). Mechanisms of transcriptional memory. Nat. Rev. Mol. Cell Biol. 2,409 -421.[CrossRef][Medline]
Frangioni, J. V. and Neel, B. G. (1993). Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal. Biochem. 210,179 -187.[CrossRef][Medline]
Grant, K., Hanna-Rose, W. and Han, M. (2000). sem-4 promotes vulval cell-fate determination in Caenorhabditis elegans through regulation of lin-39 Hox. Dev. Biol. 224,496 -506.[CrossRef][Medline]
Hall, D. H., Gu, G., Garcia-Anoveros, J., Gong, L., Chalfie, M.
and Driscoll, M. (1997). Neuropathology of degenerative cell
death in Caenorhabditis elegans. J. Neurosci.
17,1033
-1045.
Hamelin, M., Scott, I. M., Way, J. C. and Culotti, J. G. (1992). The Mec7 ß-tubulin gene of Caenorhabditis elegans is expressed primarily in the touch receptor neurons. EMBO J. 11,2885 -2893.[Abstract]
Hobert, O., Moerman, D. G., Clark, K. A., Beckerle, M. C. and
Ruvkun, G. (1999). A conserved LIM protein that affects
muscular adherens junction integrity and mechanosensory function in
Caenorhabditis elegans. J. Cell Biol.
144, 45-57.
Jurgens, G. (1988). Head and tail development of the Drosophila embryo involves spalt, a novel homeotic gene. EMBO J. 7,189 -196.
Kenyon, C. (1986). A gene involved in the development of the posterior body region of C. elegans. Cell 46,477 -487.[Medline]
Kiefer, S. M., McDill, B. W., Yang, J. and Rauchman, M.
(2002). Murine sall1 represses transcription by recruiting a
histone deacetylase complex. J. Biol. Chem.
277,14869
-14876.
Kohlhase, J. (2000). SALL1 mutations in Townes-Brocks syndrome and related disorders. Hum. Mutat. 16,460 -466.[CrossRef][Medline]
Kohlhase, J., Schuh, R., Dowe, G., Kuhnlein, R. P., Jackle, H., Schroeder, B., Schulz-Schaeffer, W., Kretzschmar, H. A., Kohler, A., Muller, U. et al. (1996). Isolation, characterization and organ-specific expression of two novel human zinc finger genes related to the Drosophila gene spalt. Genomics 38,291 -298.[CrossRef][Medline]
Landecker, H. L., Sinclair, D. A. R. and Brock, H. W. (1994). Screen for enhancers of Polycomb and Polycomblike in Drosophila melanogaster. Dev. Genet. 15,425 -443.[Medline]
Li, C. and Chalfie, M. (1990). Organogenesis in C. elegans: positioning of neurons and muscles in the egg-laying system. Neuron 4,681 -695.[Medline]
Li, D., Dower, K., Ma, Y., Tian, Y. and Benjamin, T. L.
(2001). A tumor host range selection procedure identifies
p150sal2 as a target of polyoma virus large T antigen.
Proc. Natl. Acad. Sci. USA
98,14619
-14624.
Ma, Y., Dawei, L., Chai, L., Luciani, A., Ford, D., Morgan, J.
and Maizel, A. L. (2001a). Cloning and characterization of
two promoters for the human HSAL2 gene and their transcriptional
repression by the Wilms tumor suppressor gene product. J. Biol.
Chem. 276,48223
-48230.
Ma, Y., Singer, D. B., Gozman, A., Ford, D., Chai, L., Steinhoff, M. M., Hansen, K. and Maizel, A. L. (2001b). Hsal1 is related to kidney and gonad development and is expressed in Wilms tumor. Pediatr. Nephrol. 16,701 -709.[CrossRef][Medline]
Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1992). Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10,3959 -3970.[Medline]
Mitani, S. (1995). Genetic regulation of mec-3 gene expression implicated in the specification of the mechanosensory neuron cell types in Caenorhabditis elegans. Dev. Growth Differ. 37,551 -557.
Mitani, S., Du, H., Hall, D., Driscoll, M. and Chalfie, M.
(1993). Combinatorial control of touch receptor neuron expression
in Caenorhabditis elegans. Development
119,773
-783.
Mollereau, B., Dominguez, M., Webel, R., Colley, N. J., Keung, B., de Celis, J. F. and Desplan, C. (2001). Two-step process for photoreceptor formation in Drosophila. Nature 412,911 -913.[CrossRef][Medline]
Nakagawa, Y. and O'Leary, D. D. (2001).
Combinatorial expression patterns of LIM-homeodomain and other regulatory
genes parcellate developing thalamus. J. Neurosci.
21,2711
-2725.
Netzer, C., Rieger, L., Brero, A., Zhang, C. D., Hinzke, M.,
Kohlhase, J. and Bohlander, S. K. (2001). SALL1, the
gene mutated in Townes-Brocks syndrome, encodes a transcriptional repressor
which interacts with TRF1/PIN2 and localizes to pericentromeric
heterochromatin. Hum. Mol. Genet.
10,3017
-3024.
Powell, C. M. and Michaelis, R. C. (1999).
Townes-Brocks syndrome. J. Med. Genet.
36, 89-93.
Raaphorst, F. M., Otte, A. P. and Meijer, C. J. L. M. (2001). Polycomb-group genes as regulators of mammalian lymphopoiesis. Trends Immunol. 22,682 -690.[CrossRef][Medline]
Rusten, E., Cantera, R., Urban, J., Technau, G., Kafatos, F. C.
and Barrio, R. (2001). spalt modifies EGFR-mediated
induction of chordotonal precursors in the embryonic PNS of
Drosophila promoting the development of oenocytes.
Development 128,711
-722.
Salser, S. J. and Kenyon, C. (1994). Patterning C. elegans: homeotic cluster genes, cell fates and cell migrations. Trends Genet. 10,159 -164.[CrossRef][Medline]
Savage, C., Hamelin, M., Culotti, J. G., Coulson, A., Albertson, D. G. and Chalfie, M. (1989). mec-7 is a ß-tubulin gene required for the production of 15-protofilament microtubules in Caenorhabditis elegans. Genes Dev. 3,870 -881.[Abstract]
Sulston, J. E. and Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev. Biol. 56,110 -156.[Medline]
Surka, W. S., Kohlhase, J., Neunert, C. E., Schneider, D. S. and Proud, V. K. (2001). Unique family with Townes-Brocks syndrome, SALL1 mutation and cardiac defects. Am. J. Med. Genet. 102,250 -257.[CrossRef][Medline]
Trent, C., Tsung, N. and Horvitz, H. R. (1983).
Egg-laying defective mutants of the nematode C. elegans.
Genetics 104,619
-647.
Veraksa, A., Del Campo, M. and McGinnis, W. (2000). Developmental patterning genes and their conserved functions: from model organisms to humans. Mol. Genet. Metab. 69,85 -100.[CrossRef][Medline]
Way, J. C. and Chalfie, M. (1988). mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans. Cell 54,5 -16.[Medline]
Way, J. C. and Chalfie, M. (1989). The mec-3 gene of Caenorhabditis elegans requires its own product for maintained expression and is expressed in three neuronal cell types. Genes Dev. 3,1823 -1833.[Abstract]
Weatherbee, S. D., Halder, G., Kim, J., Hudson, A. and Carroll,
S. (1998). Ultrabithorax regulates genes at several levels of
the wing-patterning hierarchy to shape the development of the
Drosophila haltere. Genes Dev.
12,1474
-1482.
Wu, J., Duggan, A. and Chalfie, M. (2001).
Inhibition of touch cell fate by Egl44 and Egl46 in C. elegans.
Genes Dev. 15,789
-802.
Xue, D., Tu, Y. and Chalfie, M. (1993). Cooperative interactions between the Caenorhabditis elegans homeoproteins UNC-86 and MEC-3. Science 261,1324 -1328.[Medline]
Zhao, Y., Hermesz, E., Yarolin, M. C. and Westphal, H. (2000). Genomic structure, chromosomal localization and expression of the human LIM-homeobox gene LHX5. Gene 260,95 -101.[CrossRef][Medline]