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Molecular and Cellular Biology, March 2000, p. 2285-2295, Vol. 20, No. 6
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Structure and Function Analysis of LIN-14, a Temporal Regulator
of Postembryonic Developmental Events in Caenorhabditis
elegans
Yang
Hong,
Rosalind C.
Lee, and
Victor
Ambros*
Department of Biological Sciences, Dartmouth
College, Hanover, New Hampshire 03755
Received 6 July 1999/Returned for modification 5 October
1999/Accepted 27 December 1999
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ABSTRACT |
During postembryonic development of Caenorhabditis
elegans, the heterochronic gene lin-14 controls the
timing of developmental events in diverse cell types. Three alternative
lin-14 transcripts are predicted to encode isoforms of a
novel nuclear protein that differ in their amino-terminal domains. In
this paper, we report that the alternative amino-terminal domains of
LIN-14 are dispensable and that a carboxy-terminal region within exons
9 to 13 is necessary and sufficient for in vivo LIN-14 function. A
transgene capable of expressing only one of the three alternative
lin-14 gene products rescues a lin-14 null
mutation and is developmentally regulated by lin-4. This
shows that the deployment of alternative lin-14 gene
products is not critical for the ability of LIN-14 to regulate downstream genes in diverse cell types or for the in vivo regulation of
LIN-14 level by lin-4. The carboxy-terminal region of
LIN-14 contains an unusual expanded nuclear localization domain which is essential for LIN-14 function. These results support the view that
LIN-14 controls developmental timing in C. elegans by
regulating gene expression in the nucleus.
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INTRODUCTION |
Development of multicellular
organisms requires the precise spatial and temporal control of
developmental events, including cell fate determination and cell
division and differentiation. In Caenorhabditis elegans, the
temporal control of stage-specific developmental events in the larva is
controlled by heterochronic genes (2; reviewed in
references 1, 4, 25). Mutations in heterochronic
genes cause cells to adopt fates that are characteristic of earlier or
later larval stages. For example, loss-of-function (lf)
mutations of lin-14 cause so-called "precocious"
phenotypes in which animals skip their L1-specific developmental
programs and precociously express L2-specific programs. By contrast,
strong lin-14 gain-of-function (gf) mutations
cause "retarded" phenotypes wherein animals fail to execute
later-stage developmental programs and instead reiterate L1
developmental programs (2, 3). Consistent with these mutant
phenotypes, lin-14 activity progressively decreases during
development due to a decrease in LIN-14 protein between the L1 and L2
stages. This temporal gradient of LIN-14 governs the proper sequence of
stage-specific programs for the developing larva (17, 28).
The analysis of lin-14(lf) mutant phenotypes revealed that
lin-14 controls diverse developmental events in a range of
different cell types and cell lineages. These events include
stage-specific division patterns in lateral hypodermal cell lineages
(2), cell cycle progression and developmental commitment in
vulva precursor cells (VPCs) (10), cell divisions in the
intestine (30), lin-29-dependent lateral
hypodermal seam cell differentiation (22), neuronal remodeling (11), and the stage-specific initiation and
expression of the dauer larva developmental program (18). It
is not known how LIN-14 carries out these various developmental roles.
LIN-14 protein is widely expressed (23) and presumably
interacts with cell-specific factors to regulate the timing of target
gene activation and repression.
Molecular analysis of the lin-14 locus reveals a relatively
complex genomic structure with 13 exons spanning over 20 kilobases (kb)
producing at least three different transcripts, lin-14A, lin-14B1, and lin-14B2 (28). The
predicted proteins encoded by these three transcripts are all novel
proteins of approximately 540 amino acids that differ in their
N-terminal sequences. Except for a putative amphipathic helix domain
near its carboxy terminus, there are no recognized functional motifs in
LIN-14. In addition, although LIN-14 is nuclear localized
(23), which would be consistent with the hypothesis that
LIN-14 may be a transcriptional regulator, there is as yet no direct
evidence for a specific biochemical activity for LIN-14.
The existence of alternative LIN-14 isoforms raises the question of
whether these different isoforms carry out different and perhaps
cell-type-specific functions. To further understand how lin-14 controls developmental timing, we performed an in
vivo structure and function analysis of LIN-14 protein. In this report, we show that the alternative amino-terminal lin-14 exons
(exons 2 and 4) are dispensable for LIN-14 activity. We also show that domains necessary and sufficient for LIN-14 in vivo function lie in the
carboxy-terminal half of the protein in a region well conserved among
different nematode species. LIN-14 has an unusual expanded nuclear
localization domain also contained in the carboxy-terminal region,
supporting the idea that LIN-14 controls developmental timing by
regulating gene activity in the nucleus. Our results suggest that
LIN-14 carboxy-terminal domains carry out the primary function of
controlling downstream gene activity, while amino-terminal lin-14 sequences play secondary roles, perhaps in modulating
lin-14 activity in different developmental contexts.
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MATERIALS AND METHODS |
Worm strains.
Nematode strains were grown and maintained as
previously described (31). All animals were grown at 20°C
unless otherwise indicated. The following strains were used (items
separated by commas): VT284 lin-14(ma135)/szT1, VT573
lin-4(e912); lin-14(n179ts), MT1397
lin-14(n179ts), VT499
lin-4(e912)vab-9(e1744)/mnC1, VT885 lin-14(n179ts); maEx166, VT886
lin-14(ma135); maEx167, VT887
lin-14(n179ts); maEx168, VT888
lin-14(ma135); maEx168, VT889 lin-4(e912)
vab-9(e1744); lin-14(n179ts); maEx166, VT889
lin-4(e912) vab-9(e1744); lin-14(ma135); maEx166, and VT890 lin-4(e912) vab-9(e1744);
lin-14(ma135); maEx168.
Germ line transformation.
Worms were transformed by
microinjection as previously described (9).
maEx166 was generated by coinjecting p14GFP (pVT333G, 25 µg/ml) and rol-6(su1006) (pRF4, 150 µg/ml
[20]). maEx167 was generated by coinjecting
p14GFP (pVT333G, 25 µg/ml) and
col-19::GFP (pVT301, 125 µg/ml). maEx168 was generated by coinjecting p14B2GFP (pVT389, 25 µg/ml) and
col-19::GFP (pVT301, 125 µg/ml). The
col-19 promoter is expressed only in adult hypodermal cells
(19). All the deletion constructs D1 to D10 were coinjected
at the concentrations indicated in Table 3, with
col-19::GFP (pVT301, 150 µg/ml) as a
transgenic marker. When lin-14(n179ts) was used as the host strain, transgenic lines were established at 15°C.
Plasmid constructions.
A 5.7-kb EcoRI fragment
from cosmid KE7 containing lin-14 exons 4 to 13 plus about
1.7 kb of the lin-14 3' untranslated region (3'UTR) was
first cloned into the EcoRI site of pBluescript to make
pB14R. An internal EcoRI site within this fragment was
eliminated by partial digestion and end-filling during the cloning. The
green fluorescent protein (GFP) coding sequence was cut from pPD95.02 with AgeI and SmaI and then blunted and cloned
into a blunted EcoNI site of pB14R (with GFP sequences in
frame with the carboxy end of lin-14 coding sequence) to
make pB14RGFP. p14GFP was made by inserting a 12.8-kb
KpnI-SalI fragment from cosmid KE7 into the
corresponding sites of pB14RGFP (see Fig.
1). p14B2GFP was made in two steps.
First, the 1.89-kb BsiWI-AgeI sequence which contains exon 2 was replaced with a 760-bp PCR fragment from
BsiWI to a downstream sequence near a PacI site,
which is about 400 bp upstream of exon 2. Second, exon 4 was then
deleted in p14B2GFP by deleting a 514-bp
PacI-ClaI fragment that contains exon 4.
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FIG. 1.
Genomic organization of wild-type lin-14 and
lin-14 deletion constructs. (A) lin-14
intron-exon structure of the three identified lin-14
transcripts, lin-14B1, lin-14B2, and
lin-14A (adapted from reference 28).
Restriction enzyme map and intron-exon spacing are drawn to scale. Open
box in exon 4 indicates that the 5' end of the exon 4 open reading
frame is undetermined. A, AgeI; B, BglII; E,
EcoRI; K, KpnI; S, SalI. (B and C)
LIN-14 deletion-GFP fusion constructs p14GFP and p14B2GFP. A single
line represents a region that is deleted. E*, EcoRI site
that was eliminated (see Materials and Methods). (D) Truncated
lin-14 constructs expressed from the col-10
promoter. col-10 promoter sequences and 3'UTR sequence of
lin-14 are not shown. Open boxes indicate introns and filled
boxes indicate exons. GFP sequence is represented by the hatched box
(artificial introns in GFP are not shown). Amino acids are numbered
according to Wightman et al. (28). D10 contains the
lin-14(n179ts) mutation (R303G in exon 9; B. Reinhart and G. Ruvkun, personal communication; see Materials and Methods).
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A 1.2-kb
BglII-
BamHI
col-10 sequence
without an ATG start codon was inserted into the
BamHI site
of pB14RGFP to make deletion
construct D1. For constructs D2 to D9, the
same
col-10 sequence,
but with an in-frame ATG codon, was
used. Deletion constructs
D2 to D10 were generated by PCR amplification
of portions of the
lin-14 coding sequence by using pB14R as
a template. To make D10,
exon 9 in D5 was replaced with DNA carrying
the
n179 point mutation
which was PCR amplified from
lin-14(n179ts) genomic DNA, subcloned,
and verified by
sequencing.
Microscopy and photography.
Images of live animals
anesthetized with 1 mM levamisole were captured with an Optronics
DE1750 integrating color CCD video camera and a Scion CG-7 RGB video
capture board on a Power Macintosh 8500AV. GFP fluorescence images were
taken with exposures ranging from 1/4 to 2 s, unless stated
otherwise. The digital images were processed with Adobe Photoshop.
Scoring the rescuing activity of lin-14
constructs.
For each construct, multiple transgenic lines were
generated and one or two of the lines that displayed the most improved general appearance and the highest level of GFP expression were analyzed in detail for the rescue of heterochronic phenotypes. To score
the rescue of lin-14(n179ts), animals were grown at 25°C. The number of seam cells at the end of the L1, the timing of adult alae
formation, and the timing of VPC divisions were scored as previously
described (2). For scoring the formation of precocious dauer
larvae, plates were inoculated with 100 or more transgenic adults and
their progeny were allowed to exhaust the food supply. Dauer larvae
were collected by 1% sodium dodecyl sulfate selection (18),
and L1 dauer larvae were identified by the presence of dauer alae on
animals with body size and a number (n = 12) of gonadal nuclei consistent with developmental arrest at the L1 molt
(18).
Efficiency of LIN-14::GFP nuclear localization.
CCD images were captured in RGB format, and the green channel was
selected with Adobe Photoshop and transferred to an NIH Image file as a
grayscale image. Using NIH Image, the area of each nucleus and the area
of each cytoplasm were separately circumscribed and their mean light
intensity was measured. The data of mean intensity were further
normalized to a 1-s exposure according to the camera's measured
exposure calibration. Background light intensity was also measured and
normalized similarly and subtracted from the nuclear and cytoplasmic
intensities. For any given deletion construct, the GFP fluorescence
intensity of at least 20 nuclei and their surrounding cytoplasm were measured.
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RESULTS |
A LIN-14::GFP fusion construct that can fully rescue a
lin-14 null mutation.
lin-14 sequences
span approximately 20 kb of genomic DNA (28) (Fig. 1A). To
analyze the functional organization of LIN-14 protein, a modified
lin-14 genomic construct, p14GFP, was constructed (Fig. 1B).
p14GFP encompasses lin-14 genomic DNA from 5.2 kb
upstream of exon 1 to about 500 bp downstream of the polyadenylation
signal, with a deletion of approximately 7 kb of intron sequence
between exon 3 and exon 4 (Fig. 1B; see Materials and Methods). p14GFP also contains a GFP sequence inserted in frame at the LIN-14 carboxy terminus (see Materials and Methods) to enable the detection of LIN-14::GFP fusion protein in transgenic worms.
p14GFP was transformed into
lin-14(ma135) animals, a
lin-14 genetic null (
18; V. Ambros,
unpublished data) and stable transgenic
lines were established. The
anatomical pattern of expression of
p14GFP from the transgenic
extrachromosomal array
maEx167 is consistent
with that of
the endogenous LIN-14 protein as previously determined
by LIN-14
antibody staining (
23) (see below). LIN-14::GFP
was
nuclear localized, as is the case for endogenous LIN-14
(
23)
(see
below).
As shown in Tables
1 and
2,
lin-14(ma135);
maEx167 animals were fully rescued for all defects scored,
including precocious
L1 cell lineage patterns in the V lineage,
precocious seam cell
differentiation, precocious vulva development, and
precocious
initiation of dauer larval development (Tables
1 and
2).
Alternative exons 2 and 4 are not required for lin-14
activity.
p14GFP should be capable of making all three
lin-14 transcripts (lin-14A, -B1, and
-B2) since it contains the alternative exons 2 (which is
B1 specific) and 4 (which is A specific). To test
whether lin-14 products containing those alternative exons are required for lin-14 function, we examined the rescuing
activity of a construct (p14B2GFP) that is missing exons 2 and 4. p14B2GFP was generated by the deletion from p14GFP of an approximately 1.0-kb DNA sequence around (and including) exon 2 and the deletion of a
500-bp DNA sequence containing exon 4 (Fig. 1C). While it is impossible
for p14B2GFP to make either lin-14A or lin-14B1 mRNA, GeneFinder (9, 27) predicts that this construct should produce the lin-14B2 transcript (data not shown).
The overall expression pattern of GFP from
maEx168 is
similar to that from
maEx166 (see below) and
maEx167, indicating that
no tissue-specific promoter
sequences were affected by the p14B2GFP
deletions (data not shown).
maEx168, an extrachromosomal array
carrying p14B2GFP, was
tested for its ability to rescue
lin-14(n179ts). n179 is a temperature-sensitive (ts) allele of
lin-14, and at
25°C,
lin-14(n179ts) animals
exhibit the
lin-14(lf) phenotype
(
3), though the
phenotype is somewhat less severe than that
of
lin-14(ma135)
(see Table
1). As summarized in Table
1,
maEx168 fully
rescues
lin-14(n179ts) for all the phenotypes
tested.
The temperature-sensitive
lin-14(n179ts) mutation does not
completely eliminate
lin-14 activity at 25°C. To exclude
the possibility
that p14B2GFP might rescue
lin-14(n179ts) by
boosting the weak
residual activity of the temperature-sensitive LIN-14
protein,
we crossed
maEx168 into animals carrying the
non-temperature-sensitive
allele
lin-14(ma135) and found
efficient rescue of
lin-14(ma135) precocious defects (Table
1). These results provide strong evidence
that neither exon 2 or exon 4 is required for
lin-14 activity
and that a single predicted
product of
lin-14 is sufficient for
LIN-14 function in
different cell
types.
lin-4-dependent developmental down regulation of
LIN-14::GFP expression from transgenes.
The successive
execution of larval stage-specific developmental programs requires the
temporal down regulation of lin-14 activity between the L1
and L2 stages. We found that the effective level of LIN-14::GFP
expressed from maEx167 is within the normal range (in terms
of lin-14 activity) and appears to be down regulated satisfactorily to achieve the normal sequence of larval
developmental events. The phenotype of maEx167 animals
showed no significant evidence of LIN-14 overexpression, as seam cells
made adult cuticle at the L4 molt and the timing of vulva development
was normal (Table 1).
The temporal down regulation of
lin-14 activity is executed
at the posttranscriptional level. This down regulation requires
the
product of the heterochronic gene
lin-4, which encodes a
small
22-nucleotide RNA with sequence complementarity to seven elements
in
lin-14 3'UTR (
17,
29). To confirm that
LIN-14::GFP expression
from transgenic p14GFP is down regulated by
lin-4, LIN-14::GFP
expression was monitored in wild-type
and
lin-4(e912) backgrounds,
maEx166, a
transgenic array similar to
maEx167, was generated
by
coinjecting p14GFP along with pRF4 (
rol-6(su1006))
(
20).
In wild-type
maEx166 animals, the temporal
profile of LIN-14::GFP
expression faithfully reflects the results
previously obtained
by LIN-14 antibody staining of the wild type
(
23).
maEx166 animals
displayed the highest
LIN-14::GFP expression in the nuclei of
late embryos just before
hatching and of newly hatched L1 animals
(Fig.
2A). This high level of LIN-14::GFP
expression starts to
decrease in hypodermal and intestinal nuclei and
in the nuclei
of ventral nerve neurons during the L1 stage and becomes
undetectable
in the L2 stage (Fig.
2B). The only exception is a subset
of neurons
in the head in which LIN-14::GFP remains detectable as
late as
the L3 or L4 stage (Fig.
2C). The expression of
LIN-14B2::GFP
from
maEx168 showed a temporal down
regulation essentially indistinguishable
from that of LIN-14::GFP
expressed from
maEx166 or
maEx167 (data
not
shown).
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FIG. 2.
lin-4-dependent down regulation of
LIN-14::GFP from maEx166. (A) Newly hatched transgenic
L1 larva carrying maEx166, which was generated by injection
of p14GFP (Fig. 1). High levels of green LIN-14::GFP fluorescence
is evident in hypodermal cells, muscle cells, intestinal cells, and
neurons. Arrowheads point to nuclei that show punctuate LIN-14::GFP
fluorescence typical of this construct. The yellow fluorescence seen in
these images is due to the autofluorescence from the intestine. (B)
LIN-14::GFP expression in maEx166 animals becomes
virtually undetectable in hypodermal cells (hyp) by the L3 stage.
Yellow signal is (non-GFP) intestinal autofluorescence. (C) In
maEx166 animals, LIN-14::GFP expression in the head
neurons remains detectable in some L3 animals. (D)
lin-4(e912); maEx166 L1 larvae display
fluorescence levels approximately equal to that of lin-4(+);
maEx166 L1 larvae. (E) At the L3 stage, high levels of
LIN-14::GFP expression persists in hypodermal cells and in
intestinal cells of lin4(e912); maEx166 L3
larvae. (F) Expression of LIN-14::GFP in VPC (Pn.p) cells is easily
detectable in this lin-4(e912); maEx166 L2 larva
[but is undetectable in similarly staged lin-4(+);
maEx166 L2 larvae; not shown]. Animals in B, E, and F are
all oriented anterior down and ventral side to the right. Bar, 3 µm.
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To test whether the down regulation of LIN-14::GFP from
maEx166 or LIN-14B2::GFP from
maEx168 is
lin-4 dependent, we crossed
maEx166 and
maEx168 into a
lin-4(e912) background. In
lin-4(e912);
maEx166 animals, LIN-14::GFP
expression was poorly down regulated
compared to the wild type and was
easily detectable in hypodermal
and intestinal nuclei in animals at
stages as late as the L3 or
L4 (Fig.
2D to F). This indicates that down
regulation of LIN-14::GFP
from
maEx166 requires
lin-4 activity, as is the case for LIN-14.
Similarly, down
regulation of LIN-14B2 was not evident in
lin-14(e912);
maEx168 animals. These results indicate that the 7 kb of
intron
sequences deleted in p14GFP and the additional sequences deleted
from around exons 2 and 4 in p14B2GFP (Fig.
1) are dispensable
for
LIN-14 expression and temporal regulation by
lin-4.
Interestingly, although wild-type VPCs display no detectable levels of
endogenous LIN-14 (
23,
28) or LIN-14::GFP (data
not
shown), LIN-14::GFP was easily detected in VPCs during the
late L1
to L2 stage in
lin-4(e912) animals (Fig.
2F). The expression
of LIN-14 in
lin-4(e912) VPCs is consistent with the
retarded
vulva phenotype of
lin-14(e912) animals and the
critical role
of LIN-14 down regulation in regulating the competence
and cell
cycle progression of VPCs (
10,
12).
Exons 9 to 13 of lin-14 are sufficient for
lin-14 activity in lateral hypodermal cells.
To
further delineate the parts of LIN-14 protein that are necessary and
sufficient for its in vivo function, a series of deletions of the
LIN-14::GFP fusion protein were constructed (Fig. 1D). In these
experiments, LIN-14 deletion constructs were expressed from a
simplified expression vector under the control of the C. elegans
col-10 promoter. col-10 is specifically expressed in
hypodermal cells (19), and thus the rescuing activity of
LIN-14 deletion constructs was scored in the hypodermis. The assays
were performed in a lin-14(n179ts) background because the
temperature-sensitive phenotype of lin-14(n179ts) allows
efficient transformation at permissive temperature (15°C) and a
convenient assay for rescue simply by transferring the transgenic
animals to nonpermissive temperature (25°C). It should be noted that
although the injection conditions were similar for each construct (see
Materials and Methods), the expression levels of these truncated LIN-14
proteins in general were greater than the LIN-14::GFP levels
observed from maEx167 and maEx168 (see Fig. 2 and
4). In particular, the expression level of LIN-14D3::GFP was very
high (see Fig. 4A).
Surprisingly, although
lin-14 contains a total of 13 exons,
we found that a deletion construct containing only exons 9 to
13 (Fig.
1D, construct D5) rescued
lin-14(n179ts) at 25°C (Table
3 and data not shown). Similarly,
deletion construct D9 (containing
exons 8 through 12) rescued
lin-14(n179ts), though not as strongly
as D5 (Table
3). This
suggests that sequences sufficient for
lin-14 activity are
contained within exons 9 to 12, although some
of exon 13 may also
contribute. Sequences in exon 9 appear to
be critical for
lin-14 activity, as construct D6 (containing exons
10 to 13;
see Fig.
1D) showed no rescuing activity (Table
3).
Construct D8
(containing exons 9 and 10 and part of exon 11; see
Fig.
1D) also
showed no rescuing activity, indicating that sequences
in the exon
11-to-12 interval are essential. These findings indicate
that sequences
contained within a carboxy-terminal region of LIN-14
encoded by exons 9 to 13 are both necessary and sufficient for
lin-14 activity,
as assayed by rescue of
lin-14(n179ts) defects
in the
hypodermis. Exons 9 to 12 corresponds to a part of LIN-14
where the
amino acid sequence is well conserved among the nematode
species
C. elegans,
Caenorhabditis vulgaris (Fig.
3), and
Caenorhabditis briggsae (B. Reinhart and G. Ruvkun, personal communication),
consistent with the functional activity of this region of the
protein.
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FIG. 3.
Amino acid sequence alignment between LIN-14 proteins
from C. elegans (ele) and C. vulgaris
(vul). Identical amino acids are labeled in black boxes, and
dashes indicates gaps generated by the aligning program GeneInspector.
The start position of each exon in C. elegans LIN-14 is
labeled. Black lines highlight the potential consensus sequences for
nuclear localization activity (15). The white line between
the two sequences within exon 11 indicates the putative amphipathic
helix motif (28). Also shown is the n179 point
mutation (R303G) (B. Reinhart and G. Ruvkun, personal communication).
Note that sequences shown here are equivalent to the predicted
lin-14B1 product (see Fig. 1A) and the very amino-terminal
end of the C. vulgaris LIN-14 sequence is undetermined.
C. vulgaris sequence was determined from cDNA.
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To exclude the possibility that the rescuing activity of the above
constructs depends on endogenous residual activity of
LIN-14(
n179ts) protein, for example, by synergizing with or
stabilizing the temperature-sensitive
LIN-14 protein, we tested the
rescuing activity of LIN-14D5::GFP
in
lin-14(ma135)
animals, which carry a non-temperature-sensitive
null allele of
lin-14. In
lin-14(ma135) animals carrying a
transgenic
array of construct D5, 60.4% of seam cells are rescued from
making
precocious adult cuticle at the L3 stage. These data are
comparable
to the rescuing activity of LIN-14D5::GFP protein in
lin-14(n179ts) (Table
3, ~73%), demonstrating that the
product of exons 9 to
13 alone supplies
lin-14 activity.
Truncated LIN-14::GFP fusion proteins, particularly D4 to D10, are
generally not well down regulated during larval development.
Unlike
animals transformed with full-length LIN-14::GFP constructs
(see
above), animals carrying truncated constructs often exhibit
bright
fluorescence after the L1 stage (see Fig.
5). Truncated
rescuing
constructs can also cause retarded phenotypes of varying
strengths,
although we have not quantitatively compared the truncated
constructs
to the full-length constructs with respect to retarded
phenotypes (data
not
shown).
Partial dominant negative activity of truncated LIN-14 products.
lin-14(n179ts) at permissive temperature (15°C) provides a
sensitized genetic background for testing the potential antimorphic ("dominant negative") activity of the truncated LIN-14 proteins produced by these deletion constructs. We have found that at 15°C, approximately 13% of seam cells in n179 animals express
precocious adult cuticle (Table 3), and this weak precocious phenotype
indicates that at 15°C, the level of lin-14 activity in
lin-14(n179ts) animals might be slightly below the
threshold required for wild-type seam cell development. Such a leaky
precocious phenotype of lin-14(n179ts) would be enhanced in
the presence of a truncated LIN-14 protein which is able to interfere
with endogenous lin-14 function.
lin-14 deletion constructs were tested for their ability to
either rescue or enhance the leaky precocious phenotype of
lin-14(n179ts) animals at 15°C. As shown in Table
3,
rescuing constructs clearly
prevent the formation of precocious adult
cuticle in
lin-14(n179ts) animals at 15°C, but one of the
nonrescuing constructs, D6 (which
contains exons 10 to 13), enhances
the leaky precocious phenotype.
In
lin-14(n179ts) animals
carrying the D6 transgenic array, more
seam cells express precocious
adult cuticle at 15°C than in
lin-14(n179ts) animals
(Table
3). This antimorphic, or partial dominant negative,
activity of
LIN-14D6::GFP protein requires exon 10, since a further
truncated
construct (D7) containing only exons 11 to 13 does not
modify the
lin-14(n179ts) phenotype. The antimorphic activity
of
LIN-14D6::GFP seems to require the sensitized
lin-14
hypomorphic
genetic background provided by
lin-14(n179ts), since no such activity
was observed in a
wild-type
background.
LIN-14D3::GFP, which contains LIN-14 sequences from exons 5, 6, and
7 and to the beginning of exon 8 (Fig.
1D), shows very
weak antimorphic
activity in
lin-14(n179ts) animals at 15°C (Table
3). The
relatively low strength of the LIN-14D3::GFP negative
activity,
coupled with the extraordinarily high level of expression
of this
construct relative to that of D6, casts doubt on the specificity
of the
D3
phenotype.
LIN-14 has an extended nuclear localization domain.
To
characterize sequences required for the nuclear localization of LIN-14,
we examined the nuclear localization of truncated LIN-14::GFP
proteins. The ratio of nuclear GFP fluorescence to cytoplasmic GFP
fluorescence was measured in transgenic worms carrying various
LIN-14::GFP constructs (see Materials and Methods). Exons 1 to 7 do
not seem to contain any essential nuclear localization signals (NLS),
since LIN-14D4::GFP which contains exons 8 to 13 is efficiently
nuclear localized (Fig. 4B and J).
Further NLS sequences seem to be contained between the end of exon 8 and the beginning of 12, since LIN-14D9::GFP is also nuclear
localized (Fig. 4G and J). LIN-14D5::GFP, which contains exons 9 to
13, is still nuclear localized, but its localization efficiency is less
than that of LIN-14D::GFP (Fig. 4C and J), suggesting that nuclear
localization of LIN-14 is influenced by sequences in exon 8. Further
deletion of sequences from either the N terminus or C terminus
diminishes the nuclear localization of LIN-14 significantly (Fig. 4D,
E, F, and J).
View larger version (48K):
[in this window]
[in a new window]
|
FIG. 4.
Nuclear localization efficiency of various truncated
LIN-14::GFP proteins. (A to I) Hypodermal cells expressing
LIN-14D3::GFP (A) LIN-14D4::GFP (B), LIN-14D5::GFP (C),
LIN-14D6::GFP (D), LIN-14D7::GFP (E), LIN-14D8::GFP (F),
LIN-14D9::GFP (G), LIN-14D10::GFP (at 15°C) (H), and
LIN-14D10::GFP (at 25°C) (I). Animals were at developmental
stages ranging from L1 to L3. (E and F) Unlocalized LIN-14D7::GFP
and LIN-14D8::GFP appear as a uniform green fluorescence, while in
all other panels except A; the green fluorescent spots are nuclei in
which LIN-14::GFP is localized. Nucleoli are relatively free of
LIN-14::GFP staining and appear as darker spots within the nuclei.
The nuclear localization patterns of LIN-14D1::GFP and
LIN-14D2::GFP (data not shown) are similar to that of
LIN-14D4::GFP. (A) Lateral hypodermal seam cells (arrow) are filled
with unlocalized LIN-14D3::GFP and show a higher level of GFP
fluorescence than surrounding hypodermal cells, and the inset shows the
unidentified GFP-containing inclusions that are frequently evident in
animals expressing LIN-14D3::GFP. We have not determined whether
the structures are extracellular or intracellular. The image was
captured with relatively short exposures (1/30 or 1/60 s) so the actual
level of GFP fluorescence is much higher than in the rest of panels,
which were taken at exposures ranging from 1/4 to 2 s. (J)
Quantitative assay of the nuclear localization efficiency of truncated
LIN-14::GFP proteins. In the diagram on the left, black regions in
exon boxes represent the amino acid sequences that are relatively well
conserved among LIN-14 proteins from different nematode species (see
Fig. 3). GFP sequences are not shown, and the intron spaces in D3 are
not drawn in scale. N/C, nuclear-to-cytoplasmic ratio of GFP
fluorescence plotted in a log scale (see Materials and Methods).
LIN-28::GFP is a cytoplasmically localized protein (4) used here as
a control. Bar, 5 µm.
|
|
These results suggest that LIN-14 nuclear localization requires
sequences at approximately the exon 8 and 9 border and also
requires
sequences in the exon 11 and 12 region. Thus, the LIN-14
NLS is either
bipartite or extends over a region of LIN-14 from
exon 8 to exon 12. Basic Arg-Lys clusters similar to a typical
NLS consensus sequence are
found at both ends of this region (Fig.
3), but the above results show
that neither of these regions alone
is sufficient to bring LIN-14 to
the nucleus. Interestingly, LIN-14D10::GFP,
which contains exons 9 to 13 with the
n179 point mutation (B.
Reinhart and G. Ruvkun, personal communication), is localized
to nuclei (Fig.
4),
despite have in no rescuing activity or antimorphic
activity (Table
3).
This suggests that the
n179 mutation impairs
a component of
LIN-14 function other than nuclear localization.
LIN-14 sequences
required for nuclear localization could function
by interacting
directly with the nuclear localization machinery,
or indirectly, via a
nuclear-transported
partner.
The behavior of LIN-14::GFP fusion proteins during cell division
suggests that LIN-14 nuclear localization is rapid and efficient.
For
example, after premitotic nuclear envelope breakdown, the
truncated LIN-14 fusion protein LIN-14D4::GFP appears uniformly
distributed in the cytoplasm and then becomes reconcentrated in
the
daughter nuclear material during a brief period shortly after
metaphase
(Fig.
5). This observation suggests that
the process
of LIN-14 nuclear localization is very efficient and may
involve
interaction with some component of the mitotic apparatus. A
small
amount of the LIN-14D4::GFP fluorescence during metaphase
appears
to be associated with chromosomes, suggesting a possible
chromatin
binding activity of the truncated protein (Fig.
5). However,
we
did not observe a similar mitotic chromatin association of the
full-length LIN-14::GFP and LIN-14B2::GFP proteins (data
not shown).
The relatively lower level of overall expression of
LIN-14::GFP
and LIN-14B2::GFP compared to that of the truncated
LIN-14D4::GFP
protein could account for the difficulty in detecting
chromosome-associated
fluorescence for the full-length fusion proteins.
However, it
is noteworthy that LIN-14::GFP and LIN-14B2::GFP
fluorescence
was not apparent anywhere within dividing hypodermal
cells, yet
was easily detectable before cell division and in daughter
cell
nuclei (data not shown). This suggests that full-length LIN-14
may
be preferentially decreased in level in association with mitosis,
while
in contrast, the truncated LIN-14 proteins may be relatively
stable
during mitosis.
View larger version (82K):
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|
FIG. 5.
LIN-14D4::GFP is efficiently localized to nuclear
material during mitosis. A dividing cell in a transgenic
lin-14(n179ts) at the late L2 stage expressing
LIN-14D4::GFP at 20°C. Small arrow in 0 min image points to a
hypodermal cell undergoing mitosis, and the arrowheads in this and
subsequent frames highlight the mitotic chromosomes. Larger arrows
point to new sister nuclei. LIN-14D4::GFP is completely nuclear
localized in surrounding interphase hypodermal cells. Bar, 3 µm.
|
|
| |
DISCUSSION |
In this work, we report that two of the three previously
identified alternative lin-14 gene products (LIN-14A and
LIN-14B1) are dispensable for lin-14 activity. Although
p14B2GFP is not capable of producing either LIN-14A or LIN-14B1, the
rescuing activity of lin-14(ma135) by p14B2GFP is
indistinguishable from that of p14GFP, which is predicted to produce
all three alternative transcripts. Our deletion analysis identified
domains of LIN-14 in the carboxy-terminal region that are necessary and
sufficient for lin-14 activity in hypodermal cells.
The observed antimorphic, or partial dominant negative, activity
of truncated LIN-14D6::GFP is consistent with the hypothesis that
protein-protein interactions are involved in LIN-14 activity.
Finally, we characterized the nuclear localization domain of LIN-14 and
found that it spans the essential functional domain of LIN-14. This
carboxy-terminal region, comprising exons 9 to 13, includes exons that
are well conserved among LIN-14 proteins from different nematode
species, indicating that this region interacts with relatively
well-conserved cellular components. These interacting components likely
including nuclear localization machinery, other nuclear proteins, and
regulatory partners acting with LIN-14 in the control of gene expression.
Alternative lin-14 products are not required for
lin-14 function in diverse cell lineages.
Earlier
observations suggested that at least one of the three LIN-14 isoforms
may perform tissue-specific functions. A genomic DNA fragment
containing only exons 4 to 13 specifically rescues a precocious
intestinal cell lineage phenotype (30), and overexpression of LIN-14A in intestinal cells causes an apparent cell autonomous gain-of-function lin-14 phenotype (Y. Hong, unpublished
data). During our analysis of LIN-14 deletion constructs, we observed intestinal specific promoter/enhancer elements in the region between exons 4 and 8 (Y. Hong, unpublished data). These observations suggested
that lin-14A might be an intestine-specific product and
raised the question as to whether different lin-14 isoforms perform distinct tissue-specific functions. However, those experiments did not test for whether other LIN-14 isoforms also function in intestinal lineages or, more generally, whether the multiple LIN-14 isoforms are required for full lin-14 function.
Our results suggest that the ability to produce LIN-14 isoforms with
alternative amino-terminal exons is not of developmental
significance,
at least under standard culture conditions. p14B2GFP,
which cannot make
either LIN-14A or LIN-14B1 due to the deletion
of the alternative exons
2 and 4, fully rescues a
lin-14 null
mutant for all defects
we tested, including precocious intestinal
cell lineages. These results
show that exons 2 and 4 are dispensable
for
lin-14 function
in diverse cell types. Although LIN-14A may
be expressed specifically,
or predominantly, in intestinal cells,
apparently the LIN-14A-specific
exon (exon 4) is not required
for LIN-14 function, even in intestinal
cells.
Our experiments did not assay for the rescue of the timing of DD neuron
remodeling. However, Hallam and Jin (
11) have shown
that
exons 4 to 13 expressed under the neuron-specific
unc-25 promoter can rescue the DD neuron remodeling defects of
lin-14(ma135) larvae. This result is consistent with our
finding that exon 2
is dispensable for
lin-14 activity, but
since a construct consisting
of only exons 9 to 13 has not been tested
for effects of DD remodeling,
the possibility remains that exons 4 to 8 could be involved in
the regulation of DD
remodeling.
It is not uncommon for genes involved in developmental regulation to
encode multiple isoforms. In some cases, these isoforms
carry out
distinct functions, and in other cases, genetic isoforms
are
functionally equivalent. For example, the
Drosophila sex
determination
gene
doublesex (
dsx) encodes two
protein isoforms (generated by
alternative splicing) that have opposite
effects on sexual phenotype
(
6). The
Drosophila
homeotic genes
Abdominal-B (
abd-B),
Antennapedia (
Antp),
labial
(
lab),
proboscipedia (
pb), and
Ultrabithorax (
Ubx)
each encode multiple
evolutionarily conserved protein isoforms,
(references
5 and
26 and references within),
although the
functional differences among the isoforms varies from gene
to
gene. The two alternative products of
Abd-B (Abd-Bm and
Abd-Br)
are less distinct in function than are the
dsx
isoforms; nonetheless,
Abd-Bm and Abd-Br exhibit distinguishable
expression patterns
and developmental activities (
14,
16).
The developmental roles
of the six
Ubx protein isoforms
produced via alternative splicing
are less distinct. Although the
various
Ubx isoforms are expressed
in different patterns
(
13,
21), a Ubx mutant that expresses
a single isoform is
essentially wild type except for minor abnormalities
(
7,
26). Thus, individual
Ubx protein isoforms do not
apparently
encode functionally distinct tissue-specific products and
the
existence of the various isoforms seems to mainly serve the purpose
of fine tuning
Ubx activity in different tissues
(
26; reviewed
in reference
8).
Like the
Ubx isoforms of
Drosophila, the
alternative
lin-14 transcripts,
lin-14B1 and
lin-14A, encode products that
are not substantially distinct
in
function.
Carboxy-terminal domains of LIN-14 are sufficient for
lin-14 activity.
Functional domains of LIN-14
sufficient for in vivo lin-14 activity are contained in the
carboxy-terminal exons 9 to 13, a region approximately half the length
of the full LIN-14 protein. Because we wished to test LIN-14 constructs
that lacked all amino-terminal protein sequences and because of
uncertainty about the position of the LIN-14 initiation codon, we used
a hypodermal-specific col-10 promoter to drive expression of
amino-terminal LIN-14 deletion constructs. Thus, our experiments
involving the rescuing activity of LIN-14 deletions D1 to D10 (Fig. 1)
were restricted to hypodermal cells. Consistent with our conclusion
that a product of exons 9 to 13 is sufficient for in vivo
lin-14 function, the region including exons 9 to 12 is
highly conserved in amino acid sequence between different nematode
species C. elegans and C. vulgaris (Fig. 3) and
between C. elegans and C. briggsae (B. Reinhart
and G. Ruvkun, personal communication). Eight of 10 sequenced
loss-of-function mutations are within exons 9 and 12 (B. Reinhart and
G. Ruvkun, personal communication; R. Lee and V. Ambros, unpublished
data), further supporting the conclusion that this region contains
essential LIN-14 functional domains. Since we did not test the rescuing activity of LIN-14 protein derivatives with deletions internal to exons
9 to 13, we do not know the relative importance of all these sequences,
particularly exons 10 and 11. The amino acid sequence of exon 13 and
part of exon 12 is not well conserved evolutionarily (Fig. 3),
suggesting that these unconserved carboxy-terminal sequences correspond
to less critical LIN-14 protein sequences. Indeed, deletion construct
D9, which lacks these unconserved sequences, displayed significant
rescuing activity (Table 3).
The function of
lin-14 exons 1 to 8 is unclear from our
experiments but could be significant under certain circumstances.
Relatively high amino acid sequence conservation between the
C. elegans and
C. vulgaris LIN-14 proteins is not confined
only to
the carboxy-terminal region sufficient for
lin-14
activity (exons
9 to 12). Exon 3, parts of exons 5 and 6, and all of
exon 7 are
also well conserved, suggesting that these sequences perform
conserved,
and hence significant, functions. Although we observed that
deletion
of exons 2 and 4 had no detectable effect on the rescuing
activity
of a LIN-14 transgene under the conditions of our assay, it is
possible that under other culture conditions, these exons may
have
significant roles in obtaining optimal LIN-14 levels in all
the
relevant cell types throughout development. Furthermore, it
is
noteworthy that in lateral hypodermal cells, the rescuing activity
of
LIN-14D5::GFP, which lacks exons 1 to 8, is somewhat lower
than
that of LIN-14::GFP or LIN-14B2::GFP, which contain additional
N-terminal exons, even though the expression level of
LIN-14D5::GFP,
driven by
col-10 promoter, appears at
least as high as that of
LIN-14::GFP or LIN-14B2::GFP in the
hypodermis. Thus, in lateral
hypodermal cells, sequences contained
within the N-terminal domains
of LIN-14, although relatively
dispensable, may be required for
optimum
lin-14 activity.
The N-terminal domains of LIN-14 could affect LIN-14 stability and
hence might play a role in the developmental down regulation
of LIN-14
protein levels. Although
col-10::
lacZ
reporter genes
containing a
lin-14 3'UTR shows
lin-4-dependent down regulation
(
29), other
reporter constructs containing
lin-14 3'UTR, such
as
col-10::
GFP,
col-10::
luciferase (which contain no LIN-14
coding
sequences; Y. Hong, unpublished data), or the amino-truncated
LIN-14::GFP constructs (particularly D4 to D10) are not efficiently
down regulated. In contrast, full-length LIN-14::GFP and
LIN-14B2::GFP
display
lin-4-dependent down regulation.
We have not examined
in detail what LIN-14 coding sequences predispose
a LIN-14::GFP
fusion to accurate down regulation or whether such
sequences act
on the level of the protein or the mRNA. It is possible
that certain
structural features of the LIN-14 amino-terminal region
may significantly
influence LIN-14 stability. These sequences could
include PEST
sequences contained in exons 6 and 7 (
28)
and/or other sequences
in exons 2 to
5.
Finally, unlike the truncated LIN-14::GFP proteins that we tested,
endogenous LIN-14 proteins (visualized by antibody staining),
as well
as the full-length transgenes LIN-14::GFP and LIN-14B2::GFP
(visualized by GFP fluorescence), appear to decrease in quantity
rapidly in conjunction with mitosis and then reappear in daughter
cell
nuclei (M. Hristova, Y. Hong, and V. Ambros, unpublished
data). Further
experiments are required to determine whether this
dynamic LIN-14
behavior is cell cycle triggered and is mediated
by specific N-terminal
sequences of LIN-14.
LIN-14 has an unconventional nuclear localization domain.
LIN-14 nuclear localization appears to require sequences contained in
an approximately 200-amino-acid region between the end of exon 8 and
the beginning of exon 12 (Fig. 4). Since we did not test the nuclear
localization activity of deletions internal to this region, our data do
not distinguish between an NLS extending throughout the region and a
bipartite LIN-14 NLS located at each end. Near the ends of the exon
8-to-12 region are clusters of basic amino acids (RKPRK, amino acids
288 to 292 near the exon 8 and 9 border, and RCRRVR, amino acids 420 to
425 in exon 11) that strongly resemble typical NLS sequences
(15). Neither of these particular elements is sufficient to
localize LIN-14 to the nucleus, but they could act together as a
bipartite NLS. A split or extended nuclear localization signal in
LIN-14 would be unusual, since the nuclear localization signals of DNA
or RNA-binding nuclear proteins are usually confined to a single region
of fewer than approximately 50 amino acids (reviewed in reference
15). LIN-14 could be transported to the nuclei by
direct association with the nuclear transport machinery and/or by
association with other nuclear proteins.
The nuclear localization of LIN-14 does not require the rescuing
activity of LIN-14. LIN-14D10::GFP with the
n179 point
mutation
is efficiently concentrated in nuclei, but does not rescue
lin-14(lf) phenotypes. This result suggests that although
the nuclear localization
domain of LIN-14 physically overlaps with the
essential functional
domain of LIN-14, the NLS activity is genetically
separable from
other essential LIN-14
activities.
LIN-14 may function in a protein complex.
A nonfunctional
truncation of LIN-14, LIN-14D6::GFP, which contains only exons 10 to 13, has antimorphic, or partial dominant negative, activity in a
lin-14 hypomorphic background. This observation suggests
that LIN-14 may function in a protein complex, perhaps as a homodimer,
or as a heterodimer or heteromultimer with other proteins. Although it
cannot be excluded that LIN-14D6::GFP may have acquired novel
function that is unrelated to endogenous lin-14 activity, we
interpret its antimorphic activity to indicate that the truncated
LIN-14 product, LIN-14D6::GFP, lacks certain essential function(s)
but still retains a protein-protein interaction domain that mediates
the association of LIN-14 with itself or with other proteins. Within
exon 11 there is a predicted amphipathic helix sequence (28)
which could mediate protein-protein interactions via coil-coil binding
(reviewed in reference 24). LIN-14D6::GFP contains this amphipathic domain and could interfere with the endogenous lin-14 activity by competing with functional
LIN-14 protein for binding to partners. LIN-14D7::GFP, containing
exons 11 to 13, does not show any detectable dominant negative
phenotype, suggesting that sequences in exon 10 are also critical for
the dominant negative activity of LIN-14D6::GFP.
The antimorphic activity of LIN-14D6::GFP was detected in
lin-14(n179ts) animals, but not in a wild-type background.
Other
hypomorphic alleles, besides
n179, were not tested as
sensitized
genetic backgrounds, so it is possible that
LIN-14D6::GFP acts
allele specifically. However, it is also
plausible that
n179 simply
supplies a partially reduced
level of
lin-14 activity suitable
for detecting the further
reduction in
lin-14 function resulting
from the antimorphic
activity of LIN-14D6::GFP.
These observations are consistent with the hypothesis that LIN-14 may
function in conjunction with other, as-yet-unidentified
proteins to
regulate the expression of
lin-14 target genes. LIN-14
controls, directly or indirectly, diverse developmental events
in
numerous cell types, suggesting that it may have many genetic
targets.
One such candidate target gene is the
C. elegans
cyclin-dependent
kinase inhibitor
cki-1, which is
responsible for mediating the
temporal control of cell cycle
progression by
lin-14 in VPCs (
12).
Presumably,
cell type-specific responses to
lin-14 activity, such
as the
VPC-specific activation of
cki-1, would originate from
the
interaction of LIN-14 with cell-type-specific cofactors, such
as
proteins involved in pre-mRNA synthesis or processing. The
hypothetical
LIN-14 partners may be discovered by identifying
mutations that modify
the phenotypes of either
lin-14(lf) or
lin-14(gf) or by direct biochemical screening for interacting
proteins.
| |
ACKNOWLEDGMENTS |
We thank Eric Moss for first testing the rescuing activity of
lin-14 exons 4 to 13 and his generous help on this project. We are grateful to the Sanger Center for providing cosmid KE7 and to
Brenda Reinhart and Gary Ruvkun for sharing their data prior to
publication. We also thank Eric Moss, Philip Olsen, and Richard Roy for
helpful discussion. pPD series plasmids were kindly provided by Andrew
Fire (personal communication), and their sequences can be obtained at
ftp://ciw1.ciwemb.edu.
This work was supported by U.S. Public Health Service research grant
GM34208 (V.A.). Some nematode strains used in this work were provided
by the Caenorhabditis Genetics Center, which is funded by NIH National
Center for Research Resources (NCRR).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, Dartmouth College, Gilman Laboratories HB 6044, Maynard St., Hanover, NH 03755-3576. Phone: (603) 646-2525. Fax: (603)
646-1347. E-mail: vambros{at}dartmouth.edu.
Present address: Howard Hughes Medical Institute, University of
California San Francisco, San Francisco, CA 94143.
| |
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Molecular and Cellular Biology, March 2000, p. 2285-2295, Vol. 20, No. 6
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
