Msx1 Is Present in Thyrotropic Cells and Binds to a Consensus Site on the Glycoprotein Hormone
-Subunit Promoter
Virginia D. Sarapura,
Heidi L. Strouth,
David F. Gordon,
William M. Wood and
E. Chester Ridgway
University of Colorado Health Sciences Center Denver, Colorado
80262
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ABSTRACT
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Our studies are aimed at identifying the
transcription factors that activate the glycoprotein hormone
-subunit promoter. Therefore, we performed a Southwestern screening
of a thyrotropic (
TSH) cDNA expression library, using the region of
the promoter from -490 to -310 that contains sequences critical for
expression in thyrotrope cells. A clone was isolated corresponding to
part of the coding sequence of Msx1, which is a homeodomain-containing
transcription factor that has been found to play an important role in
the development of limb buds and craniofacial structures. Northern blot
analysis, using the cloned Msx1 cDNA fragment as a probe, demonstrated
that
-subunit-expressing thyrotrope cells (
TSH cells and TtT97
tumors) contained Msx1 RNA transcripts of 2.2 kb, while
somatomammotrope (GH3) cells that do not produce the
-subunit had
barely detectable levels. The presence of Msx1 protein was demonstrated
by Western blot analysis in
TSH cells. We also demonstrated that
transcripts encoding the closely related Msx2 factor were not
detectable by Northern blot analysis in either thyrotrope or
somatomammotrope-derived cells. Subfragments of the region from -490
to -310 of the
-subunit promoter were used in a Southwestern blot
assay using bacterially produced Msx1 and demonstrated that binding was
localized specifically to the region from -449 to -421.
Deoxyribonuclease I protection analysis, using purified Msx1
homeodomain, demonstrated structurally induced differences in DNA
digestion patterns between -436 and -413, and sequence analysis of
this region revealed a direct repeat of the sequence GXAATTG, which is
similar to the Msx1 consensus-binding site. When nucleotides at both
sites were mutated, Msx1 binding was dramatically reduced, and the
activity of an
-subunit promoter construct decreased by
50% in
transfected thyrotrope (
TSH) cells. These studies suggest that Msx1
may play a role in the expression of the
-subunit gene in thyrotrope
cells.
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INTRODUCTION
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Expression of the glycoprotein hormone
-subunit (subsequently
referred to as the
-subunit) gene in the pituitary gland requires
regions of the promoter located upstream of -200 relative to the
transcriptional start site (1, 2, 3, 4), which differs from placental
expression, where sequences downstream of -200 appear to be sufficient
(5, 6, 7, 8, 9, 10). Whereas the factors necessary for placental
-subunit gene
expression have been studied extensively, only a few of those that
determine
-subunit expression in pituitary thyrotropes or
gonadotropes have been described. The first one identified was
steroidogenic factor-1, which is present in gonadotrope-derived
T3
cells, as well as in adrenocortical and gonadal cells, and activates
the region from -221 to -206 of the human
-subunit promoter, which
corresponds to the region from -219 to -204 of the mouse promoter
(11). A LIM-homeodomain factor, present in thyrotropes, gonadotropes,
and somatomammotropes, was found to activate the
-subunit promoter
through its interaction with the region from -337 to -330 (12, 13).
Recently, Ptx1, which was described as an activator of the POMC gene
promoter (14), was also found to activate the
-subunit promoter
(15). Our laboratory has been interested in defining the factors
necessary for expression of the
-subunit promoter particularly in
thyrotrope cells.
Previous transfection studies from our laboratory have determined that
the region of the
-subunit promoter from -480 to -254 contains
sequences critical for expression in two types of thyrotropic cell
models: the TtT97 tumor, a mouse TSH-producing T3-regulated
tumor (16, 17, 18, 19), and
TSH cells (20), a cloned cell line derived from
the MGH101A tumor, a mouse thyrotropic tumor that produces only the
- but not the ß-subunit of TSH and is not regulated by
T3 (21). The current studies were aimed at determining what
transcription factors present in thyrotrope cells are interacting with
this functionally important region of the
-subunit promoter.
A cDNA expression library in
-Exlox, constructed from
TSH mRNA,
was screened with the promoter region extending from -490 to -310.
Among the proteins identified was Msx1, a homeobox protein that plays
an important role during embryogenesis (22, 23, 24, 25). Msx1 was the first
mammalian member of the Msx subfamily of homeobox genes that was
described, and it was isolated from a cDNA library prepared from mouse
8.5-day embryos (22, 23). This clone hybridized at reduced stringency
with a mouse Hox 1.6 cDNA and was initially named Hox 7.1. Analysis of
the homeobox sequence demonstrated that this gene had significant
similarity with the Drosophila Msh (muscle segment homeobox)
gene and diverged from other Hox genes. In addition, its localization
on mouse chromosome 5 is not linked to the other Hox gene clusters (Hox
A, B, C, and D). Revision of the Hox gene nomenclature resulted in
renaming this Hox 7.1 gene Msx1. Expression of Msx1 has been described
during embryogenesis of limb buds, genital ridges, visceral arches,
craniofacial structures, central nervous system, and the developing
pituitary gland (24, 25). Msx1 expression in adult mouse uterus (26)
and mammary gland (27, 28) has also been reported recently. The only
target genes shown to be activated by Msx1 include Wnt-1, a
developmental regulatory gene (29), and Msx1 itself (30). We report the
presence of Msx1 mRNA and protein in pituitary thyrotropic cells but
not in somatomammotropic cells and identify an Msx1- binding region in
the
-subunit promoter. In addition, mutation of this binding region
abrogated binding of Msx1 and decreased
-subunit promoter activity
in thyrotropes.
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RESULTS
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Cloning of Msx1 from an
TSH cDNA Expression Library
To identify which factors interact with the
-promoter regions
critical for expression in thyrotropic cells, we carried out a
Southwestern screening of an unamplified cDNA expression library
prepared from mouse
TSH thyrotrope cell RNA, using a region of the
mouse
-subunit promoter extending from -490 to -310. More than 1
million phage were screened. A 1000-bp cDNA was isolated that was
almost identical to the published sequence of Msx1, extending from the
homeobox into the 3'-untranslated region of the gene (23, 31) (clone A,
Fig. 1
). Sequencing of the clone and of a
Msx1 cDNA provided by Dr. David Sassoon verified that, in agreement
with Monaghan et al. (31), it contained the sequence GGCC,
rather than GC, beginning at nucleotide 877 in the original sequence
published by Hill et al. (23). This change in frame predicts
a protein that is 26 amino acids shorter. We also verified the presence
of an additional 12 bp corresponding to nucleotides 905916 in the
sequence published by Hill et al. (23) that was not present
in the sequence of Monaghan et al. (31).

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Figure 1. Schematic Representation of the Msx1 and Msx2 cDNAs
The upper lines represent the DNA sequences of the Msx1
(A) and Msx2 (B) transcripts, with several restriction endonuclease
sites indicated. The positions of the homeobox (HB), the single intron
(triangle) and translational start ( ) and termination
( ) sites are indicated. A, Msx1 clones obtained by Southwestern
(clone A) and cDNA hybridization (clone B) screening of an TSH
library are indicated, with arrows denoting the extent
and direction of the DNA fragments sequenced. Msx1 probes A and C, used
in Northern blot analysis, and Msx1 probe B, used in cDNA screening,
are indicated. B, Msx2 probes D, E, and F used in Northern blot
analysis are indicated.
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Subsequent rescreening of the same library using a DNA fragment
corresponding to the most upstream 100 bp of the Msx1 clone (probe B,
Fig. 1
) yielded a 1600-bp clone that contained the complete coding
region of Msx1 (clone B, Fig. 1
) and was also found to be identical to
the published sequences (23, 31) except for the discrepancies noted
above. The sequence we obtained predicts a protein containing 300 amino
acids with a molecular mass of 32 kDa.
Detection of Msx1 Transcripts in Thyrotropic Cells
To demonstrate that Msx1 transcripts were present in thyrotropic
cells, poly A (+) RNA from
TSH cells and TtT97 thyrotropic tumors
were examined by Northern blot analysis, using the 1007-bp isolated
clone as a probe. A transcript of 2.2 kb, which is the same as that
described for whole mouse embryonic RNA, was detected in both
thyrotrope cells. A greatly reduced signal was detected in GH3
somatotrope cells (Fig. 2
). Messenger RNA
loading and integrity were verified by the detection of approximately
equal amounts of actin transcripts in GH3 cells when compared with
TSH RNA (Fig. 2
, lower panel). Since the homeobox RNA
sequence is 82% homologous to a related gene, Msx2 (32, 33), which has
been found to express 2.2-kb and 1.4-kb transcripts in mouse embryonic
and newborn tissue (33), we proceeded to analyze whether the transcript
detected might correspond to Msx2. These results are shown in Fig. 3
. Northern blot analysis was performed
with an 800-bp fragment containing the complete coding sequence for
Msx2, and a single band was detected, after prolonged exposure (17
days), in the same location as that detected using the Msx1 probe, with
the same washing conditions. This band was detected in
TSH cells and
very faintly in TtT97 tumors, but not in GH3 cells. When we used a
250-bp SauI fragment corresponding to the Msx2 homeobox,
which has 82% homology with Msx1, the signal was readily detectable
after 6 days of exposure. We then used a 150-bp SauI to
HindIII fragment corresponding to the 3'-end of the coding
region of Msx2, which has less homology with Msx1, and this probe did
not detect a signal in either cell type after 13 days. No transcripts
of 1.4 kb were detected with any of the Msx2 probes. To further
demonstrate that this signal corresponded to Msx1 transcripts, we
hybridized the filter with a 730-bp PstI to
HindIII fragment corresponding to 600 bp of the
3'-untranslated region and 130 bp of the adjacent exon 2 of Msx1 that
has no homology with Msx2. A strong signal was detected within 2 days,
in the same location as described above. Therefore, we verified that
the signal detected corresponded to Msx1 and not Msx2.

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Figure 3. Northern Blot Analysis of Msx Isoform Transcripts
in Thyrotrope Cells
Poly A(+) RNA from TSH cells, TtT97 tumors, and GH3 cells was
analyzed as described in Fig. 2 , using the radiolabeled Msx1 probe C
and Msx2 probes D, E, and F (Fig. 1 ). After each hybridization, the
filter was washed and exposed for at least 14 days to demonstrate that
the signal was eliminated. The autoradiographs were exposed for 17 days
(probe D), 6 days (probe E), 13 days (probe F), 2 days (probe C), and
4 h (actin) at -70 C with intensifying screens.
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Detection of Msx1 Protein in
TSH Cells
To demonstrate whether Msx1 protein was detectable
in thyrotropic cells, total cell lysates from
TSH cells were
examined by Western blot analysis using an antibody to the homeodomain
of Msx1 (34). This antibody is able to cross-react with Msx2 (34). As a
positive control, we used 60 ng purified Msx1-homeodomain protein (34),
and we detected a single band of
14 kDa, appropriate for the size of
the homeodomain fragment. In
TSH cells, we detected a doublet at
30 kDa (Fig. 4
), consistent with the
size of the full-length Msx1 protein. The doublet may result from the
presence of a truncated form of the Msx1 protein, possibly during
processing of the cell sample, from posttranslational modifications,
such as phosphorylation, from a different Msx protein containing a
homologous homeodomain, or from the presence of two translational start
sites in the Msx1 transcript. We also cannot exclude that a homologous
transcript of the same size may be giving rise to a homologous protein
that differs in size.

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Figure 4. Western Blot Analysis of TSH Cells Using an
Antibody to the Msx1 Homeodomain Peptide
Total cell lysates from TSH cells were separated on a 10%
polyacrylamide-0.1% SDS gel and electrotransferred to a nylon
membrane, incubated with a mouse monoclonal antibody to the Msx1
homeodomain at a dilution of 1:2000, and then with horseradish
peroxidase-conjugated goat-anti-mouse IgG diluted 1:5000. An ECL kit
was used to detect proteins binding to the Msx1 antibody. As a control,
60 ng purified homeodomain peptide were used. Molecular weight
standards are shown on the left.
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Binding of the Full-Length Msx1 Protein to the
-Promoter
Msx1 was expressed as a GST fusion and used to localize
the binding region on the
-subunit promoter. The full-length Msx1
was introduced into a pGEX construct, with a hemagglutinin (HA) epitope
appended to the amino terminus of Msx1 to allow detection with an
anti-HA antibody. Protein extracts from bacteria transformed with this
construct, as well as bacteria transformed with the pGEX construct
lacking HA-Msx1, were size-separated on a 10% acrylamide-SDS gel.
Using an anti-HA antibody, we detected a signal of the appropriate size
(58 kDa) for the GST-HA-Msx1 fusion protein (Fig. 5A
). Aliquots from the same bacterial
extracts were again size-separated on a 10% acrylamide-SDS gel and
examined by Southwestern analysis, using a radiolabeled fragment
corresponding to the
-subunit promoter region from -490 to -310,
identical to that used in the
TSH cDNA expression library screening.
No binding was detected in extracts expressing GST alone. A strong
signal was detected, of the same size as the signal detected with the
anti-HA antibody (Fig. 5B
), indicating a strong interaction between the
full-length Msx1 protein and the
-promoter region from -490 to
-310. Aliquots from the same bacterial extracts were subsequently
examined, again by Southwestern blot analysis, using fragments within
the 180 bp
-promoter region from -490 to -310. This analysis
revealed that the region from -453 to -396, and within this, the
region from -449 to -421 were able to bind to Msx1, but that the
regions from -484 to -446 and from -417 to -373 showed no binding
(Fig. 5
, C-F). These results strongly suggest that the region from
-449 to -421 contains the binding region for Msx1.

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Figure 5. Southwestern Blot Analysis of GST-HA-Msx1 Binding
to -Promoter Fragments
A, One hundred micrograms of bacterial protein extracts containing GST
alone (-) or GST-HA-Msx1 (+) were separated on a 10% polyacrylamide
0.1% SDS gel and electrotransferred to a Nytran filter. Binding with
an anti-HA polyclonal antibody was visualized using a
horseradish-peroxidase goat-anti-rabbit antibody, and detected a
prominent signal of the appropriate size for GST-HA-Msx1
(arrow). Molecular weight standards are shown on the
left. BF, A Southwestern blot analysis was performed
with proteins separated and transferred to Nytran filters as described
above. Filters were subjected to denaturation by incubation in a 6
M guanidinium hydrochloride solution followed by decreasing
concentrations down to 187.5 mM guanidinium hydrochloride,
then incubated with radiolabeled regions of the -promoter fragments
as shown.
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Deoxyribonuclease I (DNase I) Protection Analysis of Msx1 Binding
to the
-Promoter
DNase 1 footprinting analysis was performed using 0.7
µM of a purified Msx1-homeodomain peptide (34) to
saturate the binding sites and increase the ability to detect
footprinted regions. This analysis revealed that Msx1 induced
differences in DNA digestion, displaying enhancement of residues at
positions -436, -429, and -413, with protected bands between these
sites, the strongest protection corresponding to position -426 (Fig. 6
). Since DNase I does not generate a
band at each residue in the control lane, several blank regions are
present that make it difficult to define the precise boundaries of the
protected region in the presence of Msx1. Similar findings have been
reported in studies of the origin of replication of the plasmid pSC101,
where blank regions are observed in the control lane, and in the
presence of increasing amounts of a partially purified 37.5 kDa protein
there is increased enhancement of certain residues (35). The region of
Msx1 binding is located within the -447 to -400 region footprinted by
thyrotropic cell nuclear extracts (both
TSH and TtT97) as previously
reported (36). Examination of this sequence revealed a direct repeat of
the nucleotides GXAATTG, extending from -436 to -421, with two
nucleotides (GA) separating the repeated sequences (Fig. 6
). This motif
agrees with the consensus Msx1 binding motif [C/G]TAATTG (37) and is
likely to be the target site for Msx1 binding to the
-subunit
promoter.

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Figure 6. DNase I Protection Analysis of -Promoter Binding
to Msx1
An -promoter fragment extending from -490 to -310 radiolabeled at
the upstream end was incubated in the presence or absence of 300 ng
purified Msx1 homeodomain peptide as described in Materials and
Methods. Positions of hypersensitive sites and protected bands
are indicated by arrows. The sequence corresponding to
the -436 to -413 fragment is indicated on the right,
with the boxed nucleotides corresponding to the putative
Msx1-binding sites.
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Effect of Mutating the Direct Repeat on Msx1 Binding
To determine whether Msx1 binding was dependent on the GXAATTG
sequences, we mutated the bases from -434 to -430 and from -424 to
-421, disrupting both of the direct repeats, within a fragment
extending from -453 to -396 (Fig. 7A
),
and performed Southwestern blot analysis of bacterially expressed Msx1.
The wild type and mutated probes were radiolabeled to the same specific
activity. The wild type probe demonstrated strong binding (Fig. 7B
, left panel) similar to that previously shown (Fig. 5D
),
while equal counts of the mutated fragment showed dramatically reduced
binding to Msx1 (Fig. 7B
, right panel). The filter was then
again bound to the wild type probe, showing no change in the intensity
of binding compared with the first assay (not shown). These results
indicated that the mutated nucleotides were essential for Msx1
binding.
Effect of Mutating the Direct Repeats on
-Promoter Activity
To determine the effect of disrupting the direct repeat sequences
on the function of the
-subunit promoter, we examined the effect of
mutating the same nucleotides (from -434 to -430 and from -424 to
-421) that abrogated binding to Msx1, on the activity of the
-promoter fragment from -480 to +43 fused to a luciferase reporter,
in transiently transfected
TSH cells that express Msx1 and GH3
somatomammotrope cells that do not. The results are shown in Fig. 8
. When thyrotropic
TSH cells were
transfected with the mutated promoter construct, the expression
decreased by
50% compared with the wild type promoter. In contrast,
this same mutation did not affect the activity of the promoter in
somatomammotropic GH3 cells. This correlated with the lack of effect of
a 5'-deletion of the
-subunit promoter from -480 to -254 in
somatomammotrope cells, where a lower but readily measurable level of
activity was detected (36). These results suggest that the direct
GXAATTG repeat is important for Msx1 binding and for
-promoter
activity in thyrotropic cells.
Effect of Msx1 Cotransfection on
-Promoter Activity
Cotransfection of an Msx1 expression construct under the direction
of the cytomegalovirus (CMV) promoter was performed in
TSH cells, as
well as in GH3 cells that do not contain Msx1, with doses ranging from
10 ng to 20 µg. In
TSH cells, no change in the activity of the
-subunit promoter was noted with doses up to 1 µg; thereafter, a
dose-dependent suppression was observed, with 5 µg, 10 µg, and 20
µg resulting in a 70%, 77%, and 90% inhibition, respectively. A
similar effect was seen in GH3 cells, where the same doses resulted in
a 60%, 72%, and 79% inhibition. The
-subunit promoter mutated at
the Msx1 binding site was also inhibited to a similar degree. A
construct containing a 5'-deletion of the
-subunit promoter to
-120, which lacked any identifiable Msx1 binding sites, was inhibited
by 78%, and the Rous sarcoma virus long terminal repeat (RSV) promoter
was inhibited by 60% when cotransfected with 10 µg Msx1 expression
construct. These results are in agreement with other studies that have
demonstrated that Msx1 can interact with components of the core
transcriptional complex and behave as a transcriptional repressor
independent of specific DNA binding (38, 39).
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DISCUSSION
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We report the cloning of the homeobox protein Msx1 from a mouse
TSH cell cDNA expression library, using as a probe the region of the
-subunit promoter that is important for its activity in thyrotropic
cells. The Msx1 cDNAs isolated from this library matched the previously
published sequence of Msx1 (22, 23). We detected Msx1 transcripts not
only in thyrotrope-derived
TSH cells from which the cDNA expression
library was prepared, but also in the TtT97 thyrotropic tumor, which is
a well differentiated hyperplastic tissue that, in contrast to
TSH
cells, produces both TSHß- and
-subunits and is responsive to
thyroid hormone. In contrast, we detected a very low signal for Msx1
transcripts in rat GH3 somatomammotropic cells. Although the
possibility exists that the mouse Msx1 probes used were unable to
hybridize to rat Msx1, this is unlikely due to the high degree of
homology between the Msx1 genes that have been cloned. For example, the
homeoboxes of mouse and human Msx1 share 94% identity (40).
Most significantly, our studies demonstrate that Msx1 is expressed in
highly differentiated pituitary cells. Until recently, Msx1 was thought
to be expressed exclusively during embryogenesis. Studies from the
laboratory of Paul Sharpe (24, 25) have established the temporal and
spatial expression patterns of Msx1. Using in situ
hybridization, gene expression was detected from embryonic day 8 to 16
in brain, spinal cord, visceral arches (including heart),
nasal/maxillary/mandibular processes (including teeth), eye, ear, tail,
and genital ridges (including Mullerian ducts), and limb buds.
Expression of Msx1 was also detected in Rathkes pouch, the developing
pituitary gland, with an intense signal by in situ
hybridization at embryonic day 10 in the mouse (25), closely preceding
expression of the glycoprotein hormone
-subunit gene, which is the
first hormone gene to be detected during pituitary development and was
reported to be detected by in situ hybridization at
embryonic day 11 in the rat (41). By mouse embryonic day 12.5, Msx1
expression was localized to the pars distalis, the region that will
develop into the anterior pituitary gland (24). However, Msx1
expression subsequently decreased and was found to be very low or
absent throughout the mouse embryo by day 18 (42). Nevertheless, sites
of postnatal Msx1 expression have been described in the mouse,
including uterine epithelium, where expression decreases during
pregnancy (26), in nail bed and hair follicle, where its presence may
correlate with the regenerative ability in these tissues (43), and in
mammary gland, where expression decreases during late pregnancy and
lactation (27, 28).
We showed that Msx1, but not Msx2, is expressed in thyrotropic cells.
Msx2, initially designated Hox 8.1 (31, 33), was found to be expressed
during development in a pattern overlapping that of Msx1 (31, 44, 45),
although Msx2 has not been described in the developing pituitary gland.
Msx2 expression has been detected in adult human osteoblastic cells
where up-regulation by 1,25-(OH)2D3 was
demonstrated (46) and in rat osteoblastic cell lines (47), where it
appears to play a role in osteocalcin gene expression (46, 47), as well
as in adult mammary gland stroma, where estrogen up-regulated its
expression (27, 28). Southern analysis of mouse genomic DNA had
suggested three related Msx genes and perhaps others (23). A third
member of the Msx gene family, Msx3, was amplified from mouse DNA by
PCR (32). Recently, Msx3 was cloned by RACE (rapid amplification of
cDNA ends)-PCR from embryonic mouse RNA and was found to be expressed
exclusively in the developing dorsal neural tube, between embryonic
days 8 and 12.5 (48). Whether or not Msx3 is present in pituitary cells
was not examined in the current studies. The homeodomains share >95%
amino acid homology among the three Msx types, suggesting a basis for
functional redundancy (49), although the distinct patterns of
expression favor distinct roles for each Msx subtype. Although the
nature of these roles still remains to be elucidated, several studies
have suggested that Msx1 promotes cell growth and maintains a
de-differentiated state (50, 51, 52, 53, 54), while Msx2 represses proliferation
and promotes cell death (52).
In contrast to the extensive number of studies that have examined the
distribution of Msx1 and Msx2 transcripts, little if any has been
reported regarding the distribution of the corresponding proteins. We
report the presence of Msx protein in
TSH cells, although we did not
prove that this protein was Msx1 because the antibody is known to
cross-react with Msx2. However, in the absence of detectable Msx2
transcripts, it is unlikely that the protein we detected was Msx2. In
one study that used an Msx2-specific antibody, Msx2 transcript and
protein expression were found to correlate precisely (45). Therefore it
is more likely that the protein we detected was Msx1, although the
possibility that Msx3 (which is 98% homologous in the homeodomain
region) (32), or another as yet undescribed Msx isoform, is present in
thyrotropic cells has not been excluded.
The homeodomain structure of Msx1 contains a helix-turn-helix motif,
suggesting specific DNA-binding activity that mediates control of
specific gene expression, and the binding site preference is determined
by the N-terminal arm and helix III of the homeodomain (55). However,
there are few identified target genes for Msx1 interaction. The only
previous report of a naturally ocurring Msx1-binding motif with
demonstrated binding activity for Msx1 is that present on the promoter
for the developmental protein Wnt-1 (29). This promoter contained two
core consensus Msx1-binding motifs in close proximity (Fig. 9
) (29). Putative Msx1-binding sequences
have also been recognized on the Msx1 promoter itself (30) and on the
myoD enhancer (56, 57). In addition, the rat osteocalcin gene promoter
contains a motif that appears to be a target for Msx2 (34, 47), and the
promoter for another bone-specific gene, the collagen type I (COL1A1)
gene, contains the sequence TAATTA, that is similar to the consensus
Msx1-binding motif and is able to bind to Msx2 (58). In the current
studies, we demonstrated high-affinity binding of Msx1 protein to the
-promoter region from -449 to -421 by Southwestern blot analysis,
and DNase I protection assays demonstrated protein-induced changes in
the pattern of DNA digestion in the presence of Msx1 within the region
from -436 to -413. Sequence analysis revealed that this region
contains a direct repeat of the sequence GXAATTG, which is homologous
to the consensus binding motif for Msx1, [C/G]TAATTG, defined by
random oligonucleotide selection (37), and is also similar to the
binding site found on the Wnt-1 promoter (Fig. 9
). A comparison between
the mouse
-subunit promoter and the corresponding regions of the
rhesus (59) and the pig (60)
-subunit promoters reveals that there
is partial conservation of this element, and potential Msx1-binding
elements are identified at positions -430 (CTAAATAG) and -453
(CTAATAG), respectively. Another potential Msx1 binding site is also
identified in the rhesus
-subunit promoter, at position -494
(AATTG), in a region that is not conserved in the mouse or the pig
-subunit promoters. Analysis of the binding and functional
characteristics of these sites would determine whether Msx1 may also be
important for
-subunit promoter activity in other species.

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Figure 9. Comparison of the Sequences of the Msx1-Binding
Sites on the -Subunit and the Wnt-1 Promoters
The sequences shown are those corresponding to the binding sites
identified on the promoters indicated on the left.
Vertical lines indicate conserved nucleotides.
Boxes indicate the nucleotides that match the core
consensus Msx1-binding motif (37).
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There is even less information regarding the function of Msx1 on its
target genes. Other studies have shown that human chromosome 4
(containing the MSX1 locus) repressed MyoD expression in 10T1/2 cell
hybrids, a function that is consistent with the role of Msx1 in
inhibition of myogenesis (54) and that this repression was relieved by
antisense Msx1 (56). In the case of Msx2, the activity of the
osteocalcin promoter was decreased by mutations within the Msx2-binding
motif (34), and mutation of the collagen type 1 (COL1A1) gene motif
capable of binding Msx2 reduced bone-specific expression (58). We
demonstrated that mutation of the Msx1-binding sites decreased
-subunit promoter activity in thyrotropic
TSH cells by
50%
when compared with the wild type promoter. In contrast, we showed no
effect of the same mutation in GH3 cells that lack Msx1. This
correlated with the lack of effect in GH3 cells of deleting the region
of the promoter from -480 to -254, thus eliminating the Msx1-binding
sites, while a similar deletion resulted in a 20-fold decrease in
activity in
TSH cells (36). These results predicted that Msx1 would
stimulate the activity of the
-subunit promoter in thyrotropes.
However, we have observed that when cotransfecting an Msx1 expression
construct in Msx1-containing
TSH cells and in Msx1-lacking GH3
cells, there was an inhibitory effect on
-subunit promoter activity.
In addition, the
-subunit promoter mutated at the Msx1 binding
region, or truncated so that it lacked that region, was inhibited by
cotransfected Msx1 to a similar extent. A general inhibitory effect of
Msx1 was also seen when other promoters were tested in
cotransfection experiments. The phenomenon of promoter
repression by overexpression of cotransfected Msx isoforms has been
reported by others. For example, Msx2 cotransfection suppressed
osteocalcin promoter activity in one type of osteoblastic cell line,
although this effect was not seen in another type of osteoblastic cell
line (47). In addition, when Msx2 was stably overexpressed in
osteosarcoma cells, it decreased COL1A1 promoter activity (58), and
divergent gene expression of Msx2 and COL1A1 was also observed,
suggesting that endogenous Msx2 was also inhibitory to COL1A1 gene
expression (58). These findings may be explained by recent reports that
demonstrate that both Msx1 and Msx2 can behave as transcriptional
repressors, independent of specific DNA binding and apparently mediated
by their ability to interact directly with components of the core
transcriptional complex (38, 39). These observations may indicate that
the net functional activity of Msx1 on a gene promoter may depend on
the balance between a DNA-independent repressive effect and a
stimulatory effect that relies on specific DNA binding. It is possible
that interactions with other nuclear proteins, which may be in place
with the endogenous Msx1 and the endogenous
-subunit promoter, are
not correctly aligned with exogenous cotransfected Msx1 and
-subunit
promoter constructs, thus modifying the balance that results in
activation of target genes.
In summary, we have cloned an Msx1 cDNA from a thyrotropic
library and determined that Msx1 protein binds to a sequence on the
-subunit promoter from -436 to -421, which contains a direct
repeat of the Msx1-binding consensus sequence GXAATTG. Mutation of
nucleotides within both sites disrupted binding to Msx1 and decreased
activity of the
-promoter in thyrotropic cells. We hypothesize that
Msx1, or a similar protein, plays a role in the expression of the
-subunit gene in thyrotropic cells, possibly by interacting with
other transcription factors present in thyrotropes.
 |
MATERIALS AND METHODS
|
---|
TSH cDNA Library Construction
A mouse
TSH cDNA expression library was constructed from
twice purified polyA(+)RNA into
EXlox (61) using the method of
Gubler and Hoffman (62). First-strand synthesis used 4 µg RNA, an
oligo dT primer, 5-methyl-dCTP, and Moloney murine leukemia virus
reverse transcriptase (GIBCO/BRL, Gaithersburg, MD). Directional
EcoRI/HindIII linkers were added to blunt-ended
fragments as described (63). Size-selected cDNA fragments cleaved with
EcoRI and HindIII that were larger than 800 bp
were ligated into
EXlox (Novagen, Madison, WI) containing
EcoRI and HindIII arms, packaged into phage with
Gigapack II Gold extracts (Stratagene, La Jolla, CA), amplified once,
and titered using Escherichia coli ER1647 host cells. The
unamplified library contained approximately 3.8 x 106
independent recombinants.
Southwestern Expression Library Screening
Plating of the library, induction of protein production,
transferring of proteins to filters, protein denaturation, and binding
reactions were performed as previously described (64) with the
following modifications. A mouse
-subunit promoter fragment from
-490 to -310 (65) was subcloned into pGEM7Zf+ and isolated by
digestion with EcoRI and HindIII. Five hundred
nanograms of that fragment were radiolabeled using 20 µCi each of all
four
[32P]deoxynucleoside triphosphates (dNTPs) in an
end-filling reaction using avian myeloblastosis virus (AMV) reverse
transcriptase (Promega Biotec, Madison, WI), under conditions
recommended by the supplier, to a specific activity of 109
cpm/µg. The radiolabeled probe was added to the binding reaction at a
concentration of 1.5 x 106 cpm/ml. Positive phage
were purified by three additional rounds of screening.
DNA Library Screening
A mouse
TSH cDNA library was plated onto 25 150-mm plates at
a density of
55,000 plaque-forming units (pfu)/plate using E.
coli BL21(DE3)pLysE cells and screened for Msx1 sequences by the
plaque hybridization method (66) using a 100-bp probe consisting of an
EcoRI fragment at the 5'-end of the first Msx1 clone
obtained (probe B, Fig. 1
). Positive phage were purified with three
additional rounds of screening.
Complementary DNA Cloning
Recombinant plasmids were produced by autoexcision of
plaque-pure recombinant phage as previously described (64) and analyzed
by restriction endonuclease digestion. The DNA inserts were sequenced
by the chain termination method (67) using Sequenase (United States
Biochemicals, Cleveland, OH) and primers corresponding to SP6
(GATTTAGGTGACACTATA) and T7gene10 (TGAGGTTGTAGAAGTTCCG) that border the
multicloning region of pEXlox. DNA sequences were compared with GenBank
sequences using the BLAST protocol (68).
Northern Blot Analysis
RNA isolation, purification of polyA(+)RNA, and analysis of
steady state mRNA levels were performed using methods that have been
previously described (69), with the following modifications. The mRNA
was separated on a 0.8% agarose/6% formaldehyde denaturing gel,
transferred by diffusion to nylon membranes (Nytran, 0.2-mm pore size,
Schleicher & Schuell, Keene, NH) using the Turboblotter system
(Schleicher & Schuell), UV cross-linked by exposure to UV light (254
nm) for a total dose of 120 mJ/cm2 (70), and hybridized
using the QuickHyb hybridization solution (Stratagene) to DNA probes
that were radiolabeled by nick-translation (71) using
[32P]dCTP to achieve a specific activity of
1
x 109 cpm/µg. The probes used included: a 1007-bp
EcoRI to HindIII fragment (probe A, Fig. 1
), a
730 bp PstI to HindIII fragment corresponding to
130 bp of the 3'-portion of the coding sequence, and 600 bp of the
3'-untranslated region of Msx1 (probe C, Fig. 1
), an 800-bp fragment
corresponding to the complete coding sequence of Msx2 (probe D,
Fig. 1
), a 250-bp SauI to SauI fragment
corresponding to the Msx2 homeobox (probe E, Fig. 1
), and a
150-bp SauI to HindIII fragment corresponding to
the 3'-end of the coding region of Msx2 (probe F, Fig. 1
). The
Msx2 probes were obtained as follows: the region from the
translational start site to the termination codon (31) was
amplified by PCR using Vent DNA polymerase (New England Biolabs,
Beverly, MA) from a plasmid kindly provided by Dr. David Sassoon,
using the sense oligonucleotide
5'-GCCGGATCCGCCACCATGGCTGACTACAAGGACGACGATGACAAGGCGGCTTCTCCGACTAAAGG-3'
and the antisense oligonucleotide
5'-GCCGGATCCTTAGGATACATGGTAGA-3', both containing BamHI
sites at their 5' ends, and subcloned into the BamHI site of
pBluescriptSK(-) (Stratagene). The sense oligonucleotide contains a
start site consensus sequence (72), the sequence for the Flag-1 epitope
(underlined), the three nucleotides coding for the amino
acid alanine, and 17 nucleotides corresponding to the Msx2 gene
immediately downstream from the translational start site (31). The
plasmid pBluescriptMsx2 was sequenced by the chain termination method
(67) using Sequenase (United States Biochemicals) to verify all
junctions. The fragments described above were isolated by restriction
endonuclease digestion from this plasmid.
Western Blot Analysis
This was performed as previously described (69) with the
following modifications. Total cell lysates from
TSH cells were
prepared by suspending 10 million cells in 100 µl PBS-0.1% Triton
X-100 (PBS-0.1%T) containing 0.5 mM
phenylmethylsulfonylfluoride, 1 mM dithiothreitol, and 1
mM benzamidine. The suspension was sonicated, undissolved
material was sedimented by centrifugation at 2000 x g
for 5 min, and the supernatant was adjusted to 10% glycerol and stored
at -70 C. The supernatant (50 µg of protein) was combined with an
equal volume of 2 x loading buffer (0.125 M Tris-HCl,
pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol), boiled for
2 min, separated on a 10% acrylamide-SDS gel, and electrotransferred
for 18 h at 4 C to a nylon membrane (Nytran, 0.2-µm pore size,
Schleicher & Schuell). The filter was incubated for 1 h at room
temperature with a mouse monoclonal antibody to the Msx1 homeodomain
(kindly supplied by Dr. Cory Abate-Shen, University of Medicine and
Dentistry of New Jersey, Piscataway, NJ) (34) at a dilution of 1:3000
in 20 mM sodium phosphate, pH 7.5, 150 mM NaCl
(PBS-0.1%T). The filter was washed and then incubated for 1 h at
room temperature with horseradish peroxidase-conjugated goat-anti-mouse
IgG (GIBCO-BRL) diluted 1:6000 in PBS-0.1%T supplemented with 0.2%
milk (Carnation, non-fat instant milk). After washing the filter, an
enhanced chemiluminescent (ECL) kit (Amersham, Arlington Heights, IL)
was used to detect proteins binding to the Msx1 antibody.
Production of Msx1 in a Bacterial Expression System
Msx1 protein was produced in a bacterial expression system
as a fusion protein with the carboxyl terminus of glutathione
S-transferase as previously described (69) with the
following modifications. To construct the Msx1 expression vector, a
fragment extending from nucleotides 127-1050 of the Msx1 gene (23) was
amplified by PCR using Vent DNA polymerase (New England Biolabs) from a
plasmid containing the complete coding sequence of Msx1, kindly
provided by Dr. David Sassoon, using the sense oligonucleotide
5'-GCCGGATCCGCCACCATGGCTTACCCATACGATGTTCCGGATTACGCTGCGGCCCCGGCT-GCTGCTAT-3'
and the antisense oligonucleotide 5'-GCCGGATCCAGGTGACTCTGGACCCACCTA-3',
both containing BamHI sites at their 5'-ends, and subcloned
into the BamHI site of pGEX-2T (Pharmacia, Piscataway, NJ).
The sense oligonucleotide contains a start site consensus sequence
(72), the sequence for a hemagglutinin (HA) epitope
(underlined), the three nucleotides coding for the amino
acid alanine, and 17 nucleotides corresponding to the Msx1 gene
immediately downstream from the most upstream in-frame ATG, 18
nucleotides upstream from the translation start site previously
indicated by Hill et al. (23), and coinciding with that
reported by Monaghan et al. (31). The plasmid pGEX-HA-Msx1
was sequenced by the chain termination method (67) using Sequenase
(United States Biochemicals) to verify all junctions, then transformed
into E. coli DH5
cells, grown in 100 ml LB-ampicillin to
an optical density of 1.1 at 600 nm, and induced for 2 h with 1
mM isopropyl-ß-D-thiogalactopyranoside
(Boehringer-Mannheim, Indianapolis, IN). Cells were pelleted,
resuspended in 1 ml of PBS-1%T, and sonicated on ice in 0.5-ml
aliquots for 510 sec. The undissolved material was sedimented by
centrifugation at 2000 x g for 5 min, and the
supernatant was adjusted to 10% glycerol and stored at -70 C. Control
bacterial supernatants were produced from E. coli DH5
transformed with pGEX-2T. Protein concentrations were determined by the
method of Bradford (Bio-Rad, Richmond, CA) (73) using BSA (Boehringer
Manheim, Indianapolis, IN) as a standard. A Western blot with 100 µg
bacterial supernatant protein was performed as described above (see
Western Blot Analysis) using a monoclonal anti-HA antibody
(Babco, Richmond, CA) at a dilution of 1:10000 and horseradish
peroxidase-conjugated goat-anti-mouse IgG (GIBCO-BRL) diluted
1:5000.
Southwestern Blot Analysis
Bacterial supernatants (100 µg of protein/lane) stored at -70
C were thawed and combined with an equal volume of 2 x loading
buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol,
and 10% 2-mercaptoethanol), boiled for 2 min, and separated on a 10%
acrylamide-SDS gel and electrotransferred to a nitrocellulose filter
(BA85, Schleicher & Schuell). Filters were allowed to air dry for 20
min at room temperature and then were subjected to denaturing
conditions with incubation in guanidinium hydrochloride as described
above (see Southwestern Expression Library Screening).
Probes used for Southwestern blot analysis were generated as follows:
the fragment from -490 to -310 was generated as described above (see
Southwestern Expression Library Screening), and the
fragments from -484 to -446, from -449 to -421, and from -417 to
-373 were synthesized as single-stranded complementary sequences with
the addition of half-sites for the restriction endonuclease
SalI at each end, then annealed to form double-stranded
oligonucleotides. The fragments from -453 to -396, wild type and
mutant, were generated by PCR amplification from pSelectm
(-480/+43) and pSelectm
(-480/+43 mut) (see below,
Luciferase Expression Constructs) using as primers the sense
oligonucleotide 5'-GCGTCGACGATGCCTGTTAATTTAAG-3' and the
antisense oligonucleotide 5'TAGTCGACTTCAACAGGAAACAG-3',
which contain half-sites for the restriction endonuclease
SalI (underlined), and subcloned into the
SalI site of pGEM5Zf+ (Promega Biotec) for further
isolation. One hundred nanograms of each mouse
-promoter fragment
were radiolabeled using 20 µCi each of all four
[32P]deoxynucleoside triphosphates in a fill-in
reaction using AMV reverse transcriptase under conditions recommended
by the supplier, to a specific activity of 109 cpm/µg.
Aliquots of all probes were separated on a nondenaturing 5%
polyacrylamide gel followed by autoradiography demonstrating that they
were intact. Filters were incubated for 4 h at 4 C in binding
buffer (see Southwestern Expression Library Screening) with
10 µg/ml native salmon sperm DNA, 10 µg/ml boiled and denatured
salmon sperm DNA, and 3 x 106 cpm/ml of the
radiolabeled probe. Filters were then washed twice for 20 min in
binding buffer, dried at room temperature for 20 min, and exposed to
x-ray film.
DNase I Protection Analysis
A mouse
-subunit promoter fragment from -490 to -310 (65),
which was subcloned into pGEM7Zf+ and isolated by digestion with
EcoRI and Mlu II, was uniquely 3'end-labeled
using
[32P]dATP and
[32P]dTTP in a
fill-in reaction using AMV reverse transcriptase (Promega Biotec),
under conditions recommended by the supplier, to a specific activity of
108 cpm/µg. Thirty thousand counts per min of this
labeled fragment were incubated with 300 ng purified Msx1 homeodomain
peptide (kindly supplied by Dr. Cory Abate-Shen) (34) for a final
protein concentration of 0.7 µM and subjected to DNase I
digestion as previously described (36, 74). DNA fragments were
extracted with phenol-chlorophorm and ethanol precipitated, and
separated by electrophoresis on a 7% polyacrylamide-8 M
urea gel, followed by autoradiography for 72 h at -70 C using
intensifying screens.
Luciferase Expression Constructs
pA3m
(-480/+43)LUC, pA3m
(-120 to
+43)LUC, and pA3RSVLUC were generated as previously
described (1). The mutagenesis of the
-promoter was carried out as
follows. A SmaI to HindIII fragment containing
the
-promoter fragment from -480 to +43 was isolated from
pA3m
(-480/+43)LUC and subcloned into pSelect
linearized with SmaI and HindIII, to constitute
pSelectm
(-480/+43). PCR amplification was carried out with Vent
DNA polymerase (New England Biolabs) using the following primers: one
reaction, with the SP6 primer and the sense oligonucleotide
5'-CCTGTTAATTTAAGAGGCCTGAGCAGGCCTTTTATTTTTCTGTTTCC-3',
that corresponds to the fragment from -449 to -403 of the
-promoter, where the nucleotides from -434 to -430 and from -424
to -421 have been replaced by GGCCT and GGCC (underlined),
and another reaction with the T7 primer and with the antisense
oligonucleotide
5'-GGAAACAGAAAAATAAAAGGCCTGCTCAGGCCTCTTAAATTAACAGG-3',
complementary to the sense mutated
-promoter oligonucleotide
described above. Both amplified fragments were then hybridized, and a
third PCR was carried out using the T7 and SP6 primers, resulting in a
mutated
-fragment extending from -480 to +43 (-480/+43 mut)
flanked by the multicloning sites of pSelect. This fragment was
digested with BamHI and HindIII and subcloned
into pSelect linearized with BamHI and HindIII.
pSelect m
(-480/+43 mut) was sequenced by the chain termination
method (67) using Sequenase (United States Biochemicals) to verify the
sequence of the mutated region and all junctions. The fragment
SmaI to HindIII was then isolated and subcloned
into pA3LUC linearized with SmaI and
HindIII to constitute pA3m
(-480/+43
mut)LUC.
Transient Transfections
TSH and GH3 cell cultures were maintained as previously
described (36) and placed in medium containing 10% charcoal-stripped
FCS for 48 h before transfection (36). Three million cells were
mixed with DNA and transfected by electroporation, using 10 µg of a
luciferase expression construct containing either the wild type or
mutated
-subunit promoter, and 1 µg of a ß-galactosidase
expression vector as an internal control. For cotransfection
experiments, 10 ng, 100 ng, 1 µg, 5 µg, 10 µg, or 20 µg of a
CMV-directed Msx1 expression vector, and a similar vector lacking the
Msx1 gene in amounts required to make up for the difference in DNA
added, were mixed with the cells at the time of electroporation. The
Msx1 expression vector was constructed by ligating a fragment
containing the complete coding sequence of Msx1 (produced as described
under Production of Msx1 in a Bacterial Expression Vector),
into a 3700 bp NotI to NotI fragment containing
the sequence of the human cytomegalovirus immediate early
promoter/enhancer obtained from pCMVß (Clontech, Palo Alto, CA),
preceded by a fill-in reaction with AMV reverse transcriptase
(Promega). Cells were then incubated in medium containing 10%
charcoal-stripped FCS for 44 h, and cell lysates were assayed for
luciferase and ß-galactosidase activities as previously described
(69). Results of luciferase activity were expressed relative to
ß-galactosidase activity, except in cotransfection experiments, where
results were expressed relative to the volume assayed, because
ß-galactosidase activity was affected by cotransfected Msx1.
Transfections were performed in quadruplicate, and experiments were
performed using two to four different plasmid preparations of each
luciferase construct.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to the University of Colorado Cancer Center
Tissue Culture Core Laboratory for providing us with cell lines for our
studies.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Virginia D. Sarapura, M.D., University of Colorado Health Sciences Center, Box B-151, 4200 East 9th Avenue, Denver, Colorado 80262.
This work was supported by NIH Grants P30-CA46934 (to the University of
Colorado Cancer Center Tissue Culture Core Laboratory), DK-02169 (to
V.D.S.), and CA-47411 (to E.C.R.) and by a generous gift from the
Lucille P. Markey Charitable Trust.
Received for publication June 13, 1997.
Revision received July 29, 1997.
Accepted for publication July 31, 1997.
 |
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