1 Laboratoire de Génétique et Physiologie du Développement,
UMR 9943 CNRS-Université, IBDM-INSERM-Université de la
Méditerranée, Campus de Luminy, Case 907, F-13288 Marseille,
Cedex 09, France
2 Laboratoire de Biologie Animale, Université de Provence, 3 place Victor
Hugo, F-13331 Marseille, France
* Author for correspondence (e-mail: fasano{at}lgpd.univ-mrs.fr)
Accepted 11 November 2003
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SUMMARY |
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Key words: teashirt, Homeotic, Mouse, Drosophila, wingless, Wnt
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Introduction |
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In addition to the Hox genes, region-specific homeotic genes have been
identified. Among them, Drosophila teashirt (tsh) encodes a
transcription factor with three atypical zinc finger motifs (Cx2Cx12HMx4H) and
is crucial for patterning the trunk (three thoracic and eight abdominal
segments) during embryogenesis (Fasano et
al., 1991; Röder et al.,
1992
; de Zulueta et al.,
1994
). In addition, tsh is required for midgut
morphogenesis (Mathies et al.,
1994
), the development of the proximal part of the adult
appendages (Erkner et al.,
1999
; Wu and Cohen,
2000
; Soanes et al.,
2001
) and for patterning of the adult eye
(Pan and Rubin, 1998
;
Bessa et al., 2002
;
Singh et al., 2002
).
During Drosophila embryogenesis, segmentation and patterning
activities give rise to distinct head, gnathal, trunk and tail segments. In
the embryo, tsh is expressed in the trunk where Tsh protein is
crucial for several developmental processes. Tsh collaborates with certain Hox
proteins to specify trunk identity
(Röder et al., 1992;
de Zulueta et al., 1994
). In
tsh loss-of-function mutants, the anterior-most trunk segment (T1) is
transformed into a labial head segment
(Fasano et al., 1991
;
Röder et al., 1992
). In
the absence of Tsh and Hox activities, all ventral trunk segment identities
are replaced with those found in the head
(Röder et al., 1992
). Tsh
is required to establish the identity of T1 and to repress head identity in
cooperation with the Hox protein Sex combs reduced (Scr)
(de Zulueta et al., 1994
).
Ectopic expression of tsh results in the transformation of the labial
head segment into T1, while the antennal and maxillary head segments also
acquire a trunk identity in that they develop anterior denticle belts,
alternating with a posterior naked cuticle domain
(de Zulueta et al., 1994
), as
seen in the normal larval trunk.
wingless (wg) is a segment polarity gene required for
intrasegmental patterning. tsh acts as a modulator of Wg signalling
in the trunk during embryogenesis (Gallet
et al., 1998; Gallet et al.,
1999
). Around stage 10/11, Tsh protein accumulates to a high level
in the nuclei of posterior cells receiving Wg signal that will form the naked
cuticle. By contrast, in the anterior part of the segment, where Wg does not
signal, lower levels of nuclear Tsh are detected, which in part contribute to
the patterning of denticles (Gallet et
al., 1998
). Very high-level production of Tsh replaces denticles
with naked cuticle. In addition, the maintenance of wg expression is
controlled by tsh in the ventral part of the trunk
(Gallet et al., 1998
).
tsh is also crucial for eye development, which is controlled by
Drosophila Pax6 eyeless (ey) and twin of eyeless
(toy) genes. Both ey and toy have the ability to
induce ectopic eye formation when ectopically expressed during larval
development (Halder et al.,
1995; Czerny et al.,
1999
). Other transcription factors are involved downstream of
these genes: sine occulis (so), eye absent
(eya) and dachshund (dac). Homologues of the entire
cascade of genes exist in vertebrates where Six, Eya and
Dach appear to play important roles in eye development
(Halder et al., 1995
;
Pignoni et al., 1997
;
Shen and Mardon, 1997
). The
fly eye arises from a larval structure called the eye-antennal imaginal disc
where Tsh, Toy, Ey, Eya and Dac are detected in overlapping domains. It has
been postulated that Tsh is required to prevent the premature expression of
downstream transcription factors So, Eya and Dac
(Bessa et al., 2002
). Depending
on the cellular context, ectopic tsh expression can either induce
ectopic eyes or repress endogenous eye morphogenesis. In addition,
tsh and ey mutually induce each other's expression
(Pan and Rubin, 1998
;
Bessa et al., 2002
;
Singh et al., 2002
).
The molecular mechanisms underlying tsh function are poorly
understood. However, it has been shown that Tsh can repress transcription
(Alexandre et al., 1996;
Waltzer et al., 2001
;
Saller et al., 2002
). Tsh
binds to a specific enhancer of the modulo (mod) gene, a
target of Scr and Ultrabithorax (Ubx)
(Graba et al., 1994
). Tsh
inhibits mod expression in the epidermis of the T1 segment
(Alexandre et al., 1996
). In
the midgut mesoderm, tsh is required for the transcriptional
repression of Ubx that is mediated by high levels of Wg in
collaboration with the co-repressors Brinker (Brk) and C-terminal Binding
Protein (CtBP) (Waltzer et al.,
2001
; Saller et al.,
2002
). In this case, no sequence similar to the mod
enhancer bound by Tsh could be identified and indeed direct binding of Tsh to
the Ubx enhancer could not be detected in vitro. Instead, Tsh
requires Brk to repress Wg signalling for Ubx expression in the
midgut. Brinker binds the Ubx enhancer and then recruits Tsh and CtBP
into a ternary repressor complex.
Three putative Tsh genes (Tsh1, Tsh2 and Tsh3) have been
identified in the mouse. The conservation between Drosophila and
murine Tsh amino acid sequences is very low (35%) and essentially restricted
to the region of the three atypical zinc-finger motifs (Cx2Cx12HMx4H) and an
acidic domain towards the N terminus. The murine Tsh proteins possess in
addition two, more classical (Cx2Cx12Hx3-4H), zinc-finger motifs at their
C-terminal end, which are not found in Tsh. The expression patterns of
Tsh1 and Tsh2 at different stages of mouse embryogenesis are
reminiscent of tsh as they appear in restricted regions of the trunk,
the gut and the limbs (Caubit et al.,
2000). Tsh3 is detected in a temporal and spatial pattern
that is similar, though not identical, to Tsh1 (L.F., N. Coré
and X.C., unpublished).
An important question is whether the proteins encoded by the vertebrate
tsh-like genes are functionally conserved relative to
Drosophila Tsh. To analyse this question, we expressed the mouse and
Drosophila Tsh genes in Drosophila at different times and
places during development. Abnormalities induced in the Drosophila
larvae by ectopic expression of Tsh genes are very similar to those caused by
ectopic expression of tsh. Furthermore, the three Tsh genes are able
to rescue developmental defects of a tsh loss-of-function mutant. We
also provide evidence that, just as tsh, all three Tsh genes
participate in the network of eye determination genes. In addition, mouse Tsh
and Tsh proteins all act as transcriptional repressors. Our results, in
combination with expression data of Tsh genes in Drosophila and mouse
embryos (Fasano et al., 1991;
Caubit et al., 2000
), indicate
that, despite their evolutionary distance and sequence diversity, the three
mouse Tsh genes and Drosophila tsh exert very similar activities in
Drosophila.
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Materials and methods |
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For transfection of mammalian cells, Tsh1, Tsh2 and Tsh3
cDNAs (amino acids 15-stop) were inserted in-frame downstream of the Gal4
DNA-binding domains (DBD; amino acids 1-147) within the pABgal expression
plasmid (Baniahmad et al.,
1992) (a gift from M. Muller, Sart-Tilman, Belgium). Deletion of
the putative CtBP interaction motif (PIDLT) in mouse Tsh1 was performed by
PCR. Drosophila tsh
PLDLS generated by PCR was
inserted into pUAST vector. For GST-pull down assays, Tsh1, Tsh2 and
Tsh3 cDNAs (amino acids 15-stop) were inserted downstream of the GST
coding sequence in pGEX-4T2. For mouse Tsh1 antibody production, the
N-terminal part of mouse Tsh1 (amino acids 31-218) was cloned in-frame
downstream of GST in pGEX-4T1.
Mouse Tsh1 antibody production
Two rats (Lou strain) were initially immunized with 200 µg of protein
suspended in complete Freund's adjuvant and boosted three times with 100 µg
of protein suspended in incomplete Freund's adjuvant. Specificity was tested
on purified full-length fusion mouse Tsh1 protein and on crude mouse embryonic
extracts by western blotting.
Expression of Tsh and tsh genes in Drosophila
To generate the transgenic flies expressing mouse Tsh or TshPLDLS,
embryos from a Drosophila strain carrying the y1
w1118 mutations were injected with pUAST constructs as
described elsewhere (Rubin and Spradling,
1982
). w+ germline transformants were
isolated, and transgene insertions were mapped to individual chromosomes by
standard genetic crosses. The results presented here are based on three
representative homozygous lines for Tsh1 and two for Tsh2
and Tsh3. Mouse Tsh genes, tsh (from P{UAS-tsh.G})
and tsh
PLDLS were expressed ubiquitously in the
epidermis under the control of the P{GawB}69B Gal4 driver (referred
to as 69B-Gal4) (Brand and
Perrimon, 1993
). For transgene expression in the eye,
P{GAL4-dpp.blk1}40C.6
(Staehling-Hampton and Hoffmann,
1994
) and P{GAL4-ey.H}
(Hazelett et al., 1998
)
drivers were used (referred to as dpp-Gal4 and ey-Gal4,
respectively).
All the ectopic expressions in flies were performed at 29°C except when otherwise stated.
Rescue of the tsh null mutant phenotype
For the rescue of tsh8 null mutant
(Fasano et al., 1991) by Tsh
genes, tsh8/CyO; P{UAS-Tsh} were
obtained by standard genetic crosses. The expression of the transgenes was
performed by crossing tsh8/CyO;
P{UAS-Tsh} males with tsh8/CyO; P{GawB}69B
females. Embryos were collected overnight and aged for 24 hours at 22, 25 or
29°C before preparing cuticles, as described by Fasano et al.
(Fasano et al., 1991
).
tsh8/tsh8 cuticles from embryos expressing the
transgenes were compared, on the basis of the respective phenotypes, with
wild-type phenotype (tsh8/CyO) from embryos expressing the
transgenes as a control. Controls without the 69B-Gal4 driver
displayed the expected cuticular phenotype of homozygous
tsh8 individuals (data not shown).
Western blot
69B>Tsh embryos were collected for 18 hours, dechorionated and
lysed in 40 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.02% Triton, protease
inhibitors (Roche Molecular Biochemicals). Protein concentration was
determined using the Bradford assay
(Bradford, 1976), and the
proteins analysed by 8% SDS-PAGE. Expression of mouse Tsh proteins in
69B>Tsh embryos was verified by western blotting with an anti-Myc
antibody (A14, Santa Cruz Biotechnology) with an anti-Modulo antibody (MAb
LA9) used to control the amount of loaded proteins.
Immunostaining and in situ hybridization on whole-mount embryos
Embryos were collected overnight and then fixed
(Röder et al., 1992). In
situ detection of mod transcripts was performed using a digoxigenin
DNA-labelled probe (Krejci et al.,
1989
). Detection of wg mRNA was performed as described
previously (Röder et al.,
1992
).
Immunodetection of mouse Tsh1 was realized using anti-mouse Tsh1 antibody
used at 1/100. Tsh immunodetection was performed as described
(Gallet et al., 1998). The
secondary antibody was a FITC-conjugated goat anti-rat antibody (Jackson
Laboratories). Nuclei were stained by propidium iodide. Embryos were mounted
in Fluoromount-G (Southern Biotechnology Associates) and analyzed by laser
confocal microscopy (Zeiss).
GST pull-down assays
GST fusion proteins were isolated from BL21 E. coli lysates using
standard protocols. The production of GST-mouse Tsh fusion proteins was
favoured by a 1-hour induction with 0.1 M IPTG. Resin-bound GST fusions were
pelleted, washed in PBS containing protease inhibitors and resuspended in the
same buffer containing 0.01% Triton x-100. Mouse Ctbp1 (pcDNA3.1.myc-Ctbp1,
gift from E. Olson, Dallas), mouse Tsh1, Tsh2 and Tsh3 in pcDNA3.myc were
synthesized using a coupled in vitro transcription/translation kit (Promega).
For the binding assays, 10 µg GST fusion in 30 µl Gluthatione
Sepharose-4B beads were incubated for 1 hour at 4°C with 5-15 µl
[35S]methionine labelled proteins in 500 µl 100 mM NaCl, 0.1%
Triton x-100, 10 mM Tris-HCl pH 7.4, 2 mM MgCl2 and protease
inhibitors (Roche Molecular Biochemicals) and were eluted for 3 minutes in
loading buffer. Proteins were analyzed by SDS-PAGE and autoradiographed.
Mammalian cell culture and transfections
MDCK (Madin-Darby Canine Kidney) cells were grown in DMEM (Stratagene)
supplemented with 10% fetal bovine serum (BioWhittaker) and 1%
penicillin/streptomycin (Stratagene) under a humidified atmosphere containing
9% CO2. One day before transfection, 104 cells per well
were seeded in 24-well plates. 18 hours later, cells were co-transfected with
0.25 µg of the reporter vector pGL2-5xUAS-Luc (gift from M. Muller,
Sart-Tilman, Belgium), 0.025-0.1 µg pABgal-mouse Tsh1, 2 or 3, and 0.1
µg pcDNA3.1.myc-Ctbp1 and 3 µl FuGene 6 per µg of DNA according to
the manufacturer's instructions (Roche Molecular Biochemicals). pABgal and
pcDNA3.myc were added to keep the amount of expression plasmids constant.
pSV-ß-Galactosidase (Promega) was used to normalize for variations in
transfection efficiencies. Cells lysis and measure of luciferase activity were
performed (Caccavelli et al.,
1998). For each experiment, at least three independent
transfections in doublets were performed. Results are expressed as the
mean±s.d. of three independent pools of transfected cells.
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Results |
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Tsh genes expression affects the expression of tsh target genes
The ability of Tsh genes to induce the same transformations as ectopic
tsh strongly suggests that mouse and Drosophila Tsh genes
act on the same target genes. One gene controlled by tsh is wg.
wg is expressed in stripes in each segment until stage 10 of development
(Bejsovec and Martinez-Arias,
1991; Dougan and DiNardo,
1992
; Yoffe et al.,
1995
), whereupon expression is lost from ventral regions of the
labial and maxillary head segments but maintained in homologous positions of
the trunk (Fig. 3A).
tsh is required for the maintenance of wg at this stage in
the ventral part of each trunk segment. Consequently, following ectopic
expression of tsh, wg is maintained in the ventral part of the labial
and maxillary head segments (Fig.
3B) (Gallet et al.,
1998
). Similarly, Tsh1, Tsh2 and Tsh3 are able
to maintain wg expression in these head segments. Like tsh,
the maintenance is particularly marked in the labial segment
(Fig. 3C, see figure legend for
quantitative data). The detection of wg in the labial segment
correlates with the ability of Tsh genes to induce labial to T1
transformation.
|
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Ectopic expression of Tsh genes in the eye |
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Mouse Tsh proteins are transcriptional repressors
Repression of mod by Tsh indicates that, like Tsh, mouse Tsh
proteins can act as transcriptional repressors. In addition, the proteins are
all nuclear (Fig. 1A)
consistent with the idea that mouse Tsh proteins act as transcriptional
regulators. A transcriptional repressing activity has been previously
attributed to the N-terminal half of Tsh
(Waltzer et al., 2001).
Moreover, Tsh can bind CtBP and in a complex with Brk and CtBP, represses
Ubx transcription in the visceral mesoderm
(Saller et al., 2002
). In the
N-terminal part of Tsh, lies a consensus motif for the interaction with CtBP
(PLDLS). Deletion of this motif prevents the binding of CtBP to Tsh
(Fig. 5A). We made a
UAS-tsh
PLDLS transgene that specifically lacks the
sequence encoding the PLDLS motif in order to test whether this motif is
crucial for repressor activity of Tsh in vivo. Expression of Tsh
PLDLS
protein is unable to repress mod expression in the labium
(Fig. 5A compare with
Fig. 3E). This indicates that
repression of mod transcription by Tsh depends on the PLDLS motif
presumably via its interaction with CtBP.
|
Interestingly, the three mouse Tsh proteins present a putative motif for the interaction with CtBP, PIDLT. We tested whether the mouse Tsh proteins could interact with mouse CtBP1 by performing GST pull-down assays (Fig. 5C). All three mouse Tsh proteins interact with mouse Ctbp1. In mammalian cells, although the in vitro affinity for mouse Ctbp1 and the repressor potential vary between the mouse Tsh proteins, co-expression of mouse Ctbp1 with the three Gal4-mouse Tsh fusions potentiates the repression of the reporter (Fig. 5D). Deletion of the PIDLT motif in Gal4-mouse Tsh1 affects its basal repression activity, but a significant level of inhibition of the reporter persists. Importantly, this deletion leads to loss of the synergistic inhibition by mouse Ctbp1 (Fig. 5D). As mouse Ctbp1 is ubiquitously expressed, it is probable that it is similarly ubiquitously expressed in other mammals and it is thus likely that endogenous CtBP in canine MDCK cells partially contributes to the basal repression by mouse Tsh1 in the absence of transfected mouse Ctbp1. Our results indicate that mouse Tsh proteins present intrinsic, Ctbp-dependent, transcriptional repressing activity. Because, however, residual inhibiting activity is seen upon deletion of the PIDLT motif, mouse Tsh1, and presumably mouse Tsh2 and mouse Tsh3, must also repress transcription in a CtBP-independent manner.
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Discussion |
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The ability of tsh and Tsh genes to induce T1 identity indicates
that both collaborate with the same genes and/or proteins that determine
segment identity. The identity of the T1 segment is specified by the combined
action of tsh and the Hox gene Scr
(Röder et al., 1992;
de Zulueta et al., 1994
),
whereas the labial segment forms where Scr is expressed in the
absence of Tsh. This observation, together with the capacity of Tsh protein to
directly interact with Scr (L.F. and O. Taghli, unpublished), suggest that Tsh
may modify the transcriptional regulatory properties of the Scr protein to
allow the expression of target genes essential for the T1 segment identity.
The fact that ectopic expression of Tsh1, Tsh2 or Tsh3 can
transform the labial segment into T1 provides strong evidence that Tsh can
specify T1 identity in cooperation with Scr and suggests that mouse
Tsh proteins can substitute for Tsh and regulate specific target genes
responsible for morphological features of the trunk.
Several studies have shown that mouse and human Hox genes can carry out
equivalent functions to their Drosophila counterparts when introduced
in flies [Hoxb6/Antp (Malicki et
al., 1990; McGinnis et al.,
1990
), Hoxb4/Deformed
(Malicki et al., 1992
),
Hoxa5/Scr (Zhao et al.,
1993
), Hoxb1/labial
(Lutz et al., 1996
)].
Interestingly, ectopic expression of mouse Hoxa5, which is
functionally homologous to Scr, can activate ectopic expression of a
salivary gland target gene, and induce the homeotic transformation of the
larval thoracic segments T2 and T3 towards T1
(Zhao et al., 1993
). These
data imply that, in addition to regulating specific Scr target genes, the
mammalian protein Hoxa5 is also able to interact with putative Scr
co-factor(s). Although functional analysis of Tsh genes are necessary in
mouse, it is tempting to hypothesize that Tsh, together with the Hox genes,
may be part of common developmental genetic mechanisms for patterning the
invertebrate and vertebrate trunk. The expression patterns of the Tsh genes
(Caubit et al., 2000
) during
mouse embryogenesis are consistent with a function in trunk versus head
boundary specification in vertebrates.
Mouse Tsh proteins, like Drosophila Tsh, act as modulators of Wg signalling in Drosophila
We observed that denticles are replaced with naked cuticle when the dose of
Tsh is increased by combining two insertions. In addition, Tsh genes rescue
the segment polarity phenotype of tsh8 null mutants. These
results indicate that Tsh genes can operate in the gene network involved in
formation of naked cuticle. In tsh8 null mutants,
wg expression is not maintained in the trunk and the reduced naked
cuticle domains resemble late wg loss-of-function phenotype. The
rescue of the tsh8 cuticular phenotype by Tsh genes
suggests that these genes are sufficient to ensure wg transcription
and/or signalling in the posterior part of each segment as seen in wild-type
flies. Although the maintenance of wg in the tsh8
trunk was not assessed upon Tsh and tsh expression, we showed that
Tsh genes, like tsh, are able to maintain its expression in the
gnathal segments. This would suggest that, like tsh, Tsh genes could
control wg expression in the trunk as well as in the head. An
autoregulatory loop involving Tsh in the Wg signalling pathway has been
postulated for the maintenance of wg expression
(Gallet et al., 1998),
suggesting that in addition to regulating wg expression, mouse Tsh
genes, like Drosophila Tsh, might modulate Wg signalling. Given the
striking conservation of the Wg and Wnt signalling components between species,
one could hypothesize that at least some aspects of tsh activity in
Wg/Wnt signalling may be conserved from Drosophila to vertebrates.
However, further investigation is required to assess for a role of Tsh genes
in Wnt signalling in vertebrates.
Mouse Tsh genes, like Drosophila tsh, affect the development of the fly eye
Our data also demonstrate the ability of Tsh genes to operate in the
formation of the adult fly eye. Interestingly, ectopic expression of
ey or its vertebrate homologue Pax6 induce ectopic eyes in
Drosophila (Halder et al.,
1995), demonstrating their equivalence in the development of
complex sensory structures. Pax6 also plays a crucial role in
vertebrate eye formation (Chow et al.,
1999
; Ashery Padan et al.,
2000
). The Pax/Dac/Eya/Six regulatory network first identified in
the context of the Drosophila eye has been shown to be involved in
vertebrate somitogenesis. Indeed, this regulatory relationship extends to
other members of these families: Pax3, Six1, Eya2 and Dach2
(Heanue et al., 1999
;
Kardon et al., 2002
). In the
fly, tsh in cooperation with homothorax (hth), a
negative regulator of eye development, prevents premature expression of the
downstream genes so, eya and dac
(Singh et al., 2002
;
Bessa et al., 2002
). In
addition to a genetic interaction in the developing Drosophila eye, a
direct protein interaction has been described between Tsh and Hth and its
partner Extradenticle (Exd) (Bessa et al.,
2002
). Our results suggest that, like tsh, mouse Tsh
genes can regulate the activity and/or expression of some genes involved in
formation of the fly eye. In vertebrates, numerous hth and
exd homologues are found: Meis1-3 and Prep1 for
Hth, and Pbx1-3 for Exd. One of the aims of future
work will be to investigate whether Tsh proteins are involved in similar
protein/gene networks in vertebrates.
Mouse Tsh proteins have a transcriptional repressor activity
Comparison of the organization of Tsh with Tsh-related proteins in mouse
and humans (Caubit et al.,
2000) (L.F. and X.C., unpublished) suggests that common functional
features are probably defined by the region encompassing the three zinc-finger
motifs and by the presence of a motif known to interact with CtBP.
Interestingly, mouse and Drosophila Tsh proteins display intrinsic
transcriptional repression activity. Our results and those of others
(Saller et al., 2002
) suggest
that the repression ability of Tsh proteins is partly due to their interaction
with the co-repressor CtBP. In the visceral mesoderm, Tsh is recruited to the
Ubx enhancer in a repressor complex containing Brk and CtBP
(Saller et al., 2002
), wherein
Tsh does not seem to bind directly to DNA, but rather Brk is the DNA-binding
partner. In the ectoderm, however, Tsh directly binds to the mod
enhancer and represses transcription in vivo
(Alexandre et al., 1996
). We
show that the association of CtBP with Tsh is dependent on the
CtBP-interacting motif (PLDLS) located in the N-terminal part of Tsh, and this
CtBP/Tsh complex contributes to the observed repression. An analogous motif
(PIDLT) is found in the C-terminal part of the three mouse Tsh proteins.
Despite the different context encompassing the PIDLT motif in the mouse
proteins (C-terminal), we show that this motif is functional and essential for
the repressor function of mouse Tsh1. Although we only directly addressed the
role of this motif in mouse Tsh1, Tsh2 and Tsh3 repression activity is equally
potentiated by mouse Ctbp1, suggesting that mouse Ctbp1 is a co-repressor
acting with all mouse Tsh proteins. Interestingly, the PIDLT motif lies within
a region of the three mouse Tsh proteins where the sequence similarity is low
and thus appears to be a highly conserved functional domain in a variable
region. In addition, it is worth noting that, in mammalian cells, some
repression activity persists in mouse Tsh1 after deletion of the
CtBP-interacting motif, implying that other mechanisms of transcriptional
repression are used by mouse Tsh. In contrast to Tsh, which contains a
repressor domain rich in Ala (L.F. and O. Taghli, unpublished), analysis of
the mouse Tsh protein sequences fail to reveal a comparable feature or any
known motif that could account for the mouse Tsh1
PIDLT repressor
activity.
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ACKNOWLEDGMENTS |
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