1 Howard Hughes Medical Institute, Department of Human Genetics, University of
Utah School of Medicine, Salt Lake City, UT 84112-5331 USA
2 Instituto de Neurociencias, Universidad Miguel Hernández-CSIC, Apdo.18,
Ctra. Valencia Km 87, 03550-San Juan (Alicante), Spain
3 IGBMC, Dépt de Génétique Physiologique, BP 10142, 67404
Illkirch Cedex, CU Strasbourg, France
Author for correspondence (e-mail:
carl.thummel{at}genetics.utah.edu)
Accepted 8 October 2003
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SUMMARY |
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Key words: Nuclear receptor, Hormone, Cofactor, Ecdysone, Molting
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Introduction |
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The fruit fly, Drosophila melanogaster, provides an ideal model
system for defining the molecular mechanisms of hormone action in the context
of an intact developing organism. Progression through the Drosophila
life cycle is dependent on pulses of the steroid hormone ecdysone that direct
the major developmental transitions, including molting and puparium formation
(Riddiford, 1993). Ecdysone
exerts its effects through a heterodimer of two members of the nuclear
receptor superfamily, the EcR ecdysone receptor (NR1H1) and the fly RXR
ortholog USP (Ultraspiracle, NR2B4)
(Riddiford et al., 2001
).
These receptors are widely expressed, allowing the transduction of the
systemic ecdysone signal throughout development
(Talbot et al., 1993
;
Henrich et al., 1994
). Upon
binding ecdysone, the EcR/USP heterodimer directly induces target gene
expression, including a small set of early regulatory genes that were
originally identified as ecdysone-inducible puffs in the larval salivary gland
polytene chromosomes (Ashburner et al.,
1974
; Thummel,
1996
; Richards,
1997
; Henrich et al.,
1999
; Riddiford et al.,
2001
). These include E74, which encodes two related
proteins that share a C-terminal ETS DNA binding domain, E74A and E74B
(Burtis et al., 1990
), and
E75, which encodes three orphan members of the nuclear receptor
superfamily, E75A, E75B and E75C (NR1D3)
(Segraves and Hogness, 1990
).
The early ecdysone-inducible transcription factors coordinate the expression
of numerous secondary-response late genes that are responsible for directing
appropriate stage- and tissue-specific biological responses during development
(Thummel, 1996
;
Richards, 1997
;
Henrich et al., 1999
;
Riddiford et al., 2001
).
Mutations in EcR and USP lead to a range of phenotypes that reflect the
requirements for ecdysone signaling during development. EcR mutants
display defects and delays in larval molting, with some animals arresting
development at the molts (Bender et al.,
1997; Schubiger et al.,
1998
; Li and Bender,
2000
). Many of these mutants retain the cuticle and mouthhooks
from the preceding instar. Animals that survive to the third instar are
significantly delayed and form elongated prepupae that retain their larval
morphology. Most of these phenotypes are shared by usp mutants,
consistent with the idea that EcR and USP act together as a heterodimeric
ecdysone receptor (Perrimon et al.,
1985
; Oro et al.,
1992
; Hall and Thummel,
1998
). Molting defects are also seen in animals that carry
mutations in the E75A and ßFTZ-F1 (NR5A3)
ecdysone-regulated orphan nuclear receptor genes
(Yamada et al., 2000
;
Bialecki et al., 2002
).
E75A and ßFTZ-F1 mutants die with duplicated cuticles,
mouth parts and anterior spiracles, indicating an inability to complete the
molting cycle. For E75A mutants, these defects are caused by a
reduced ecdysone titer, implicating a key role for this receptor in hormone
biosynthesis or release (Bialecki et al.,
2002
).
We describe a novel nuclear receptor interacting protein encoded by rigor mortis (rig) that is required for the ecdysone-regulated processes of molting and puparium formation. Rig acts downstream from ecdysone biosynthesis and release to control the expression of specific ecdysone-regulated genes and it interacts directly with several Drosophila nuclear receptors, including EcR, USP and ßFTZ-F1. Antibody stains reveal that Rig is localized to the cytoplasm of imaginal cells and neuroblasts, but shuttles in and out of the nuclei in the larval salivary glands and midguts in a spatially and temporally restricted manner. Taken together, these data suggest that Rig is an integral part of the ecdysone signaling cascade, acting as a novel cofactor for one or more Drosophila nuclear receptors.
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Materials and methods |
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Ecdysone feeding experiments
Embryos from the cross y w; l(2)k07839/CyO
y+ x y w;
Df(2R)exu1/CyO y+ were collected for 3
to 4 hours. As controls, embryos from the crosses y w;
Df(2R)exu1/CyO y+ x y w
or E75A81/TM3, GFP x
E7551/TM3, GFP were collected for 3 to 4
hours. The embryos were maintained at 25°C and allowed to hatch.
l(2)k07839/Df(2R)exu1 and
+/Df(2R)exu1 first instar larvae were selected by the
yellow phenotype of their mouthhooks and denticle belts.
E75A81/E75
51 first
instar larvae were selected by their lack of GFP expression. First instar
larvae were placed on fresh yeast paste in a petri dish lined with damp black
Whatman paper at 25°C. Larvae were allowed to develop until 66 hours after
the beginning of the egg lay, a time point corresponding to 6 hours before the
molt to the third instar. To make yeast paste containing 0.5 mg/ml
20-hydroxyecdysone (Sigma), 4.2 µl of a 12 mg/ml stock solution (in
ethanol) was diluted in 95.8 µl water and added to 0.05 g of dry yeast. As
a negative control, 4.2 µl ethanol was diluted in 95.8 µl water and
added to 0.05 g of dry yeast. The staged second instar larvae were transferred
to these yeast samples, allowed to feed for 6 hours, and then returned to
regular yeast paste. The animals were scored 18-24 hours later, a time
corresponding to the middle of the third instar in wild-type animals.
Northern blot hybridizations
RNA was isolated from staged +/Df(2R)exu1 control or
l(2)k07839/Df(2R)exu1 mutant second and third
instar larvae by direct phenol extraction as described previously
(Andres and Thummel, 1994).
Animals were staged at 6-hour intervals from the first-to-second instar molt,
and again at the second-to-third instar molt for two time points. Equal
amounts of RNA from each stage were fractionated by formaldehyde agarose gel
electrophoresis and transferred to a nylon membrane (Genescreen; Dupont) as
described previously (Karim and Thummel,
1991
). A probe to detect rig transcription was generated
by PCR amplification of bases 910-1400 from the cDNA LD12835. The remaining
probes were generated as described previously
(Karim and Thummel, 1991
;
Andres et al., 1993
). All DNA
fragments were gel purified (Geneclean; Bio101) and labeled by random priming
(Prime-it Kit; Stratagene). The blots were hybridized, washed and stripped as
described previously (Karim and Thummel,
1991
).
In vitro protein binding assays
cDNAs for EcR, FTZ-F1, DHR3, USP and SVP were fused to the coding region
for GST in the pGEX-2T plasmid vector (Pharmacia) as described previously
(Beckstead et al., 2001) and
expressed at high levels in bacteria. For expression of
35S-labelled Rig, the complete coding sequence was inserted into
the pSG5 vector (Stratagene) and coupled transcription/translation was
performed using T7 RNA polymerase with the TNT lysate system (Promega). GST
and GST fusion proteins were expressed in E. coli and purified on
gluthathione-Sepharose beads (Pharmacia), as described by the manufacturer.
Purified proteins were quantified by Coomassie Blue staining after SDS-PAGE
separation and Bradford protein assay. All proteins were of an appropriate
length for the corresponding constructs. Glutathione-Sepharose beads were
equilibrated with binding buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.3 mM
DTT, 1 mM PMSF, 10 mM MgCl2, 5% glycerol, 0.5% NP40, and protease
inhibitor mixture), loaded with equimolar amounts of GST or GST fusions and
washed as described by Beckstead et al.
(Beckstead et al., 2001
). For
binding assays, 5 µl of 35S-labelled Rig protein was incubated
with 20 µl of bead-bound GST-fusion proteins for 1 hour at 20°C with
gentle agitation in a final volume of 200 µl binding buffer. After three
washes with 200 µl binding buffer, the beads were resuspended in 15 µl
SDS-loading buffer, boiled for 5 minutes, and the bound proteins were analyzed
by SDS-PAGE. Gels were dried, and radiolabeled Rig was detected by
autoradiography.
Antibody stains
A region of rig encoding the C-terminal amino acids 862-1235 was
amplified by PCR using the following primers:
5'-CAGAATTCCGACATTAAGGACGCGCTGGA-3' and
5'-ACGGTCGACTTAATGCTCGGCAGTAGATC-3'. This fragment was purified,
cut with EcoRI and SalI and inserted into pGEX-5X-3
(Amersham). Purified GST-Rig was injected into three rabbits (Cocalico
Biologicals Inc.) and antisera was screened at a 1:500 dilution by western
blotting (ECL, Amersham Biosciences) using transformants that overexpress Rig
under the control of the hsp70 promoter (hs-rig). Antiserum
was centrifuged and the supernatant was passed over two columns to
affinity-purify anti-Rig antibodies, as described by Carroll and Laughon
(Carroll and Laughon, 1987).
The first column contained a protein extract from pGEX-5X-3 bacteria and the
second column contained purified GST-Rig. Specific anti-Rig antibodies were
eluted from the second column and dialyzed into PBS. Each fraction (
0.5
ml) was tested at a 1:500 dilution by western blot analysis using protein
isolated from pGST-Rig bacteria and Rig overexpressed from hs-rig
transformants. For detecting Rig protein in larval and prepupal organs, second
and third instar w1118 larvae were staged from the molt.
Animals were dissected and stained with affinity-purified anti-Rig antibody at
a dilution of 1:100, using 1:200 Cy3-labeled anti-rabbit secondary antibodies
(Jackson). Nuclei were detected by staining with a 1:1000 dilution of mouse
anti-histone antibody (mAb052, Chemicon) and 1:1000 Cy2-labeled anti-mouse
secondary antibodies (Jackson). The stains were imaged on a BioRad MRC1024
confocal laser scanning microscope using dual detector channels to
independently visualize the Cy2 and Cy3 signals.
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Results |
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A lethal phase analysis was performed as a first step toward defining the functions of rig during development. Crosses were set up between y w; l(2)k07917/CyO y+ or y w; l(2)k07839/CyO y+ and a deficiency for the region, y w; Df(2R)exu1/CyO y+, to test for embryonic lethality among the offspring. Progeny from the stock y w; +/CyO y+ were used as a control. These experiments revealed that 19-28% of the embryos from rig mutant crosses died (Table 1), a number attributable to lethality from the homozygous CyO balancer chromosome (Table 1, control), indicating that zygotic rig function is not required during embryonic development.
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Ecdysone feeding fails to rescue rig mutant animals
The defects in molting and puparium formation seen in rig mutants
could result from either a decrease in the ecdysone titer or a decrease in the
ability of the ecdysone signal to be transduced. To distinguish between these
possibilities, we examined the effects of feeding ecdysone to rig
mutant larvae. This method has been shown to effectively rescue phenotypes
associated with ecdysone-deficient mutations
(Garen et al., 1977;
Venkatesh and Hasan, 1997
;
Freeman et al., 1999
;
Bialecki et al., 2002
).
Mid-second instar larvae were transferred to food either with or without 0.5
mg/ml 20-hydroxyecdysone (20E, the active form of the hormone) for 6 hours and
scored 18-24 hours later, a time corresponding to the middle of the third
instar in wild-type animals. Feeding ecdysone to control larvae
(+/Df(2R)exu1) reduces their viability as the number of
active third instar larvae is decreased and the number of animals dying as
second instar larvae or while molting to the third instar is increased
(Table 2). This detrimental
effect is most likely due to the excessive levels of hormone present in these
animals. In contrast, little effect is seen when rig mutant larvae
are exposed to the same hormone feeding regime. The number of animals dying
during the second-to-third instar molt increased in rig mutants, but
the overall range of lethal phenotypes was not significantly affected
(Table 2). Feeding ecdysone to
E75A mutant second instar larvae, however, had a dramatic effect on
their development, rescuing most of them to the third instar stage, consistent
with the results of earlier work, and the identification of E75A
mutants as ecdysone deficient (Table
2) (Bialecki et al.,
2002
). We conclude that ecdysone is not limiting in rig
mutants and that rig functions downstream of ecdysone biosynthesis
and release.
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|
rig encodes a WD-40 repeat protein with an LXXLL motif
Plasmid rescue was used to obtain 1,161 bp of DNA sequence flanking the
l(2)k07839 P-element insertion. This sequence overlapped with 692 bp
of sequence flanking the l(2)k07917 P-element insertion, reported by
the Berkeley Drosophila Genome Project (BDGP)
(Spradling et al., 1999), as
well as a family of 21 overlapping cDNAs in the BDGP expressed sequence tag
database (CG30149 on FlyBase,
2003
). A representative of this family, LD12835, was sequenced.
This cDNA is 4,120 bp long and contains a predicted open reading frame
encoding a 1,235 amino acid protein. The coding region is preceded by an
in-frame stop codon and followed by a poly(A) sequence, suggesting that it is
full length. The length of the cDNA is also similar to the 4.5 kb rig
mRNA detected by northern blot hybridization
(Fig. 3). The P-element in both
l(2)k07917 and l(2)k07839 is inserted into the 5'
untranslated region of the gene, 70 bp upstream from the predicted start
codon. Analysis of the predicted Rig protein sequence reveals the presence of
7-12 WD-40 repeats, depending on the protein motif search engine used. For
example, the Simple Modular Architecture Research Tool (SMART) predicts seven
WD-40 repeats (Schultz et al.,
2000
), the Rep-V1.1 search tool identifies eight WD-40 repeats
(Andrade et al., 2000
) and the
Protein Sequence Analysis tool recognizes 12 WD-40 repeats
(http://BMERC-www.bu.edu/wdrepeat).
The positions of nine of these WD-40 repeats are predicted by two or more of
the above search engines and are highlighted in
Fig. 4. BLAST searches using
the Rig protein sequence did not reveal significant similarity to previously
identified proteins outside of the WD-40 repeats. The Rig protein sequence
also contains an LXXLL motif, a signature sequence found in many nuclear
receptor cofactors (box, Fig.
4) (Heery et al.,
1997
; Torchia et al.,
1997
; Voegel et al.,
1998
; Glass and Rosenfeld,
2000
).
|
|
Rig protein shows dynamic subcellular localization in larval cells
Antibodies raised against the C-terminal region of Rig were used to stain
organs dissected from staged larvae and prepupae. Rig was detected in the
brain (Fig. 6D-F) and salivary
glands (Fig. 6G-I) of early and
late second instar larvae, consistent with the presence of rig mRNA
at these stages (Fig. 3) and
the known essential roles for rig during larval development
(Table 1). No Rig was detected
in tissues isolated from l(2)k07839/Df(2R)exu1
mutant larvae, indicating that the antibody is specific for Rig protein
(Fig. 6A-C). At all stages
examined during second and third instar larval development, Rig protein
appears to be restricted primarily to the cytoplasm of cells in the brain and
imaginal discs tissues that are fated to form specific parts of the
adult fly during metamorphosis (Fig.
7). Rig is also localized to the cytoplasm of larval salivary
gland cells during the second (Fig.
6) and early third instar (Fig.
8A-C) stages. Rig protein, however, begins to shift into the
nucleus of these cells in the mid-third instar (24-30 hours after the molt,
Fig. 8D-F) maintaining this
localization through the end of larval development (36-42 hours after the
molt, Fig. 8G-I). At puparium
formation, Rig protein shuttles out of the nucleus of salivary gland cells to
become more abundant in the cytoplasm (Fig.
8J-L). A similar dynamic movement of Rig can be seen in cells of
the larval midgut during third instar development. Rig protein is relatively
abundant in imaginal cells of the larval midgut, and remains restricted to the
cytoplasm (Fig. 9, arrows),
similar to its cytoplasmic localization in other imaginal cells
(Fig. 7). In contrast, Rig
localization within the larval midgut cells is more dynamic. Rig is primarily
excluded from the nucleus of larval midgut cells in the early third instar
(12-18 hours after the molt, Fig.
9A-C), is approximately equally distributed between the nucleus
and cytoplasm of larval midgut cells at 18-24 hours after the molt
(Fig. 9D-F) and is nuclear in
the larval cells by 24-30 hours after the molt
(Fig. 9G-I). Unlike the larval
salivary gland expression pattern, however, Rig shuttling into the nucleus of
larval midgut cells is not uniform throughout the tissue and thus appears to
be spatially regulated. Nuclear localization was seen within distinct clusters
of adjacent midgut cells, although it was difficult to determine whether this
localization occurred in the same region of the midgut in other animals at
that stage (data not shown).
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Discussion |
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Five Drosophila nuclear receptor cofactors have been identified to
date: Alien (Dressel et al.,
1999), SMRTER (Tsai et al.,
1999
), MBF1 (Takemaru et al.,
1997
), Taiman (Bai et al.,
2000
) and Bonus (Beckstead et
al., 2001
). Of these, only bonus appears to have
activities in common with rig, although relatively limited genetic
studies have been undertaken for most of these cofactors. No mutants have been
characterized for SMRTER or Alien, which act as co-repressors in tissue
culture transfection assays (Dressel et
al., 1999
; Tsai et al.,
1999
). MBF1 null mutants are viable and display a strong genetic
interaction with tdf/apontic mutants that indicate a role in
tracheal and nervous system development
(Liu et al., 2003
). Somatic
clones of taiman mutants reveal a role in border cell migration
during oogenesis (Bai et al.,
2000
). In contrast, bonus mutants display first instar
larval lethality as well as defects in salivary gland cell death and cuticle
and bristle development, implicating a role for bonus in ecdysone
responses during development (Beckstead et
al., 2001
). Also like rig, bonus mutations result in
gene-specific defects in ecdysone-regulated transcription, and Bonus protein
can interact with a range of Drosophila nuclear receptors, including
EcR, USP, SVP, DHR3 and FTZ-F1. Bonus, however, interacts with these receptors
in an AF-2-dependent manner, unlike Rig
(Fig. 5). Moreover, the larval
lethal phenotypes of rig mutants do not resemble those reported for
bonus mutants and, unlike Rig, Bonus protein appears to be
exclusively nuclear in both larval and imaginal tissues. Further work is
required to determine whether bonus and rig might act
together to regulate ecdysone response pathways.
Rig is distinct from all known Drosophila nuclear receptor
cofactors in that it is not part of an evolutionarily conserved protein
family. Alien, SMRTER, MBF1, Taiman and Bonus all have vertebrate homologs,
and Taiman and Bonus are the fly orthologs of the well characterized
vertebrate nuclear receptor cofactors AIB1 and TIF1, respectively
(Bai et al., 2000;
Beckstead et al., 2001
). In
contrast, Rig does not contain identifiable enzymatic activities nor the
conserved functional domains that define most nuclear receptor cofactors.
BLAST searches with the Rig protein sequence did not reveal any closely
related sequences in other organisms, although the top hits, which show
limited homology in the WD-40 repeats (25-33% identity), are in factors known
to modify chromatin, including human histone acetyltransferase type B subunit
2 (RBBP-7) and chromatin assembly factor 1 (CAF-1).
The WD-40 repeats that comprise about half of the Rig protein sequence are
likely to play an important role in its activity. Consistent with this
proposal, an N-terminal fragment of Rig, containing two WD-40 repeats but
missing the LXXLL motif (amino acids 1-300), is capable of interacting with
GST-DHR3 and GST-USP, suggesting that these repeats are sufficient for
Rig-nuclear receptor interactions (data not shown). WD-40 repeats provide
multiple surfaces for protein-protein interactions and have been identified in
over 150 proteins that function in a wide range of processes, including
cytoskeleton assembly, transcriptional regulation, and pre-mRNA processing
(reviewed by Smith et al.,
1999). In Drosophila, WD-40 repeats are associated with
several transcriptional regulators, including the p85 subunit of TFIID
(Kokubo et al., 1993
), the
Polycomb group protein encoded by extra sex combs
(Gutjahr et al., 1995
), and
the Groucho corepressor (Stifani et al.,
1992
). In addition, a WD-40 repeat protein, TBL1, has been
identified as part of a multiprotein complex with thyroid hormone receptor
that contains the SMRT nuclear receptor corepressor and HDAC-3
(Li et al., 2000
). The
presence of these sequences in Rig may thus provide a scaffold for
protein-protein interactions that could mediate the formation of multiprotein
transcriptional complexes on ecdysone-regulated promoters. Further biochemical
studies of Rig should provide insights into the significance of its WD-40
repeats as well as a foundation for understanding how Rig exerts its effects
on transcription.
Rig may contribute to the spatial and temporal control of ecdysone signaling through its subcellular localization
It is not clear how Rig expression in the brain, imaginal discs and
salivary glands of second and third instar larvae is related to the lethal
phenotypes of rig mutants, although neuroendocrine signaling is
clearly required for molting, a process that is defective in rig
mutant larvae (Riddiford,
1993). The subcellular localization of Rig protein at later
stages, however, correlates with the distinct fates of larval and imaginal
cells during metamorphosis. Rig protein appears to be restricted to the
cytoplasm of cells that are fated to form parts of the adult fly, including
neuroblasts, imaginal discs, and the imaginal islands of the larval midgut
(Figs 7,
8). In contrast, Rig shows
dynamic changes in its subcellular distribution in larval salivary gland and
midgut cells, both of which undergo steroid-triggered programmed cell death
during metamorphosis. It is possible that these differences in subcellular
localization could contribute to the distinct fates of these tissues in
response to ecdysone signaling.
In addition to this spatial correlation, there is also a temporal
correlation between the times at which Rig protein shuttles between the
cytoplasm and nucleus in larval tissues and the coordinated changes in
ecdysone-regulated gene expression that occur during the third instar. The
switch from cytoplasmic to nuclear localization in larval salivary glands and
midguts occurs at approximately the same time, 24-30 hours after the
second-to-third instar larval molt (Figs
8,
9), suggesting that Rig may be
responding to a common temporal signal. Cell type-specific factors, however,
must also contribute to this regulation as Rig is localized to the nucleus of
only a subset of cells in the larval midgut (data not shown). Interestingly,
this protein redistribution correlates with a poorly understood event that is
represented by widespread changes in ecdysone-regulated gene expression,
called the `mid-third instar transition'
(Andres and Cherbas, 1992;
Andres et al., 1993
). It is
possible that the cytoplasmic-to-nuclear transport of Rig in larval tissues
contributes to the regulation of this response, which prepares the animal for
metamorphosis one day later. Similarly, Rig returns to the cytoplasm of
salivary gland cells at puparium formation, in synchrony with the widespread
changes in ecdysone-regulated gene expression associated with the onset of
metamorphosis. This translocation, however, is not seen in the larval midgut,
where Rig protein remains in the nucleus of some cells (data not shown). Rig
shuttling thus appears to be differentially controlled in both a temporally
and spatially restricted manner, correlating with major switches in
ecdysone-regulated transcription. The observation that the first of these
shifts in subcellular distribution occurs during the major lethal phase of
rig mutants the mid-third instar
(Table 1) suggests that
these intracellular movements contribute to the critical functions of Rig
during development.
Interestingly, several recent reports have described the subcellular
redistribution of nuclear receptor cofactors in both vertebrate and
Drosophila cells. The p/CIP vertebrate nuclear receptor coactivator
is differentially distributed within the cells of the mouse female
reproductive organs (Qutob et al.,
2002). For example, p/CIP is detected primarily in the nuclei of
highly proliferative follicular cells while it is most abundant in the
cytoplasm of terminally differentiated cells of the corpus luteum. p/CIP
displays active nucleocytoplasmic shuttling in response to growth factors in
cell culture, and interacts directly with the microtubule network in the
cytoplasm. Similarly, MEK-1 kinase-mediated phosphorylation of the SMRT
mammalian corepressor leads to the translocation of this factor from the
nucleus to the cytoplasm in cell culture transfection assays
(Hong and Privalsky, 2000
).
The functional homolog of this protein in flies, SMRTER, also shows active
redistribution from the nucleus to the cytoplasm in response to a MAP kinase
pathway, in this case mediated by EGFR/Sno/Ebi in the Drosophila eye
(Tsuda et al., 2002
). In both
of these systems, regulated phosphorylation of SMRT/SMRTER results in
dissociation of a repressor complex and derepression of target gene
transcription.
These observations raise the possibility that the subcellular location of
Rig could determine its regulatory function in different cell types. For
example, by analogy with SMRT/SMRTER, loss of Rig from the nucleus of larval
cells might disrupt a corepressor complex on specific promoters, leading to
coordinate target gene derepression. This is consistent with the proposal that
the ecdysone receptor exerts critical repressive functions during larval
development (Tsai et al.,
1999; Schubiger and Truman,
2000
). Alternatively, Rig protein in the cytoplasm may tether one
or more nuclear receptors, preventing them from acting on their cognate target
genes in the nucleus. We do not favor this model, however, because antibody
stains reveal an exclusively nuclear localization for EcR, USP and
ßFTZ-F1 at the onset of metamorphosis
(Talbot et al., 1993
;
Henrich et al., 1994
;
Yamada et al., 2000
). It is
also interesting to note that Rig protein appears to localize to discrete
regions within the nuclei of larval midgut cells that do not contain
chromosomes (Fig. 9G-I) while
Rig co-localizes with the giant polytene chromosomes in larval salivary gland
cells (Fig. 8D-F). Rig may thus
exert some functions in the nucleus that are independent of chromatin binding.
Further biochemical studies of Rig, including the identification of additional
proteins that interact with this factor, should provide insights into the
significance of the subcellular localization of Rig protein as well as a
mechanistic understanding of how Rig contributes to ecdysone responses during
Drosophila larval development.
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ACKNOWLEDGMENTS |
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Footnotes |
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