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INTRODUCTION |
Expression of the gene for the common
-subunit of the pituitary
glycoprotein hormones is restricted to two cell types within the
anterior pituitary gland. In combination with the specific
-subunits
produced by thyrotropes and gonadotropes, it forms the biologically
active hormones TSH,1
lutenizing hormone, and follicle-stimulating hormone. Many reports have
described areas within the proximal promoter region of the
-subunit
gene that are important for expression in both pituitary cell types
(1-9) and in the placenta (3, 8, 10, 11), where it is also a component
of chorionic gonadotropin. We have recently discovered a more distal
area of the 5'-flanking region of the mouse gene, located approximately
4 kilobase pairs upstream from the start of transcription, that directs
high levels of expression of a
-galactosidase reporter that was
restricted to the thyrotropes and gonadotropes of transgenic mice (12).
Subsequent studies demonstrated that when an 859-bp
KpnI-BglII fragment derived from the region
between
4600 and
3700 was fused directly to a proximal
-subunit
promoter from
341 to +43, the high level of cell-specific transgene
expression was maintained (13). Furthermore, we also demonstrated that
the 859-bp region was also capable of stimulating proximal promoter
activity in transient transfections of cells derived from thyrotropes
(
-TSH) and gonadotropes (
-T3), both of which express the
-subunit gene endogenously (13). More recently, our laboratory (14)
has shown that the functional interaction between the upstream enhancer
and the proximal promoter in transfections of both cell lines is
dependent on an intact pituitary glycoprotein hormone binding element
(PGBE) located from
337 to
330, an area within the proximal area
that was shown to bind a LIM homeodomain factor (15, 16). In addition,
interaction at the more proximal gonadotrope-specific element (GSE)
from
213 to
200, which binds the steroidogenic factor SF-1 (17), is also required for the enhancer to function in gonadotrope cells but not
in thyrotrope cells (14). To identify the sites of functional interaction within the upstream region, we now report that the most
proximal 125 bp of the previous 859-bp enhancing area are sufficient to
account for all of the proximal promoter stimulatory activity in both
pituitary-derived cell types that express the
-subunit. We
demonstrate that the native orientation of the region with respect to
the proximal promoter is crucial to maintain activation. We also show
that several areas within this region bind proteins present in nuclear
extracts from pituitary-derived cells including somatotrope-derived GH3
cells and that the areas of interaction contain potential binding sites
for known transcription factors that also bind at important functional
areas within the proximal promoter (17-19). Interestingly, we further
show that the same upstream region inhibits proximal
-promoter
activity in GH3 cells. The mechanism whereby the upstream region exerts
its repressive effect differs from activation in that it is not
dependent on interaction at two proximal promoter binding sites and on
the orientation of the upstream region with respect to the proximal region.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructions for Enhancer-Proximal Promoter Analysis and
Transient Transfection Assays--
The construction of luciferase
vectors containing the region from
341 to +43 of the
-subunit
promoter without and with mutated PGBE and GSE sites and the same
plasmids with the 859-bp upstream enhancer fused upstream were
described previously (13, 14). As a first approach, the 859-bp region
was subdivided by digestion at PstI restriction sites. After
separation on agarose gels and purification using Qiaex II resin
(Qiagen), each fragment was inserted into the PstI site of
pGEM5zf+ (Promega). Subsequent excision with EcoRV and
HindII generated a blunt-ended fragment that was ligated
into the SmaI site of a plasmid that contained the region
from
341 to +43 of the
-subunit promoter inserted between the
BamHI and HindIII sites of pSELECT (Promega).
After checking by sequencing for the correct forward orientation of each PstI fragment, the fused enhancer/promoter region was
excised by digestion with KpnI and HindIII and
inserted between the same sites of the promoterless pA3LUC mammalian
expression vector (20, 21). This resulted in luciferase expression
plasmids with the areas from 70 to 246, from 241 to 654, and from 694 to 833 (renumbered as 1-184 in Fig. 6, also see the area between the
underlined PstI sites in Fig. 2) fused upstream of the
proximal
-subunit promoter region from
341 to +43. Progressively
shorter 5' prime truncations of the enhancer region were generated
using a PCR strategy that employed sense oligonucleotides with the
following sequences: (a)
5'-GCCGGTACCCTGCAGGTCTGCACATAAATTC-3', (b)
5'-GCGGGTACCCACTCAGTCAATATCTTATCTCT-3', (c)
5'-GCCGGTACCCAGAAGCAATTAAGCAGTCA-3', (d)
5'-GCCGGTACCCTGCAGAATAAAAGCTCTTTG-3', (e)
5'-GCGGGTACCACAGGTGTTAGGAACTC-3', (f)
5'-GCGGGTACCCAGCCCGTGACCTCAT-3', and (g)
5'-GCGGGTACCCTGCAGTCTAGGAGATTTG-3'.
These oligonucleotides comprise sequences originating at positions 241, 421, 552, 649, 733, 798, and 827, respectively, of the upstream 859-bp
enhancer sequence, respectively (GenBankTM accession number
AFF044976). The last three represent positions 85, 150, and 179 of the
renumbered 210-bp region shown in Fig. 2. They were designed with a 5'
KpnI site (italicized) to facilitate subsequent subcloning.
Each oligonucleotide was used in conjunction with a common antisense
22-nucleotide amplimer that is complementary to the sequence coding for
amino acids 4-10 of luciferase and used to amplify the appropriate
truncated enhancer/promoter fragment from the full-length 859-bp
enhancer/promoter fusion in pA3LUC. The resulting fragments were then
digested with KpnI and HindIII and reinserted
between the same sites of pA3LUC. A 5' deletion plasmid originating at
position 85 and fused to a truncated Rous sarcoma virus promoter was
similarly constructed starting with the previously described 859-bp
enhancer Rous sarcoma virus fusion vector (13). To generate an
enhancer/promoter plasmid containing sequences that extended downstream
of position 859, we first subcloned, into pGEM7zf (Promega), a fragment
that extended from
4600 to
3400 that was derived by KpnI
and SphI digestion from a plasmid containing
-subunit
sequences from
5000 to +43 (12). After sequencing this downstream
300-bp extension, we utilized PCR with this plasmid and a 5'
oligonucleotide that spanned the KpnI site at
4600 along
with an antisense oligonucleotide that originated 237 bp downstream of
the BglII site at
3700 with
5'-GCGGGATCCATAATTCACCTTTAGGGAGG-3'. Incorporation of a
BamHI site (italicized) allowed the amplified product to be
excised with KpnI and BamHI and inserted in the forward orientation between the same sites of the pSELECT plasmid containing the
341 to +43
-subunit promoter sequence. The 3' extended enhancer/promoter fusion region was then excised with KpnI and HindIII and cloned into pA3LUC as
before. An
-subunit proximal promoter luciferase plasmid containing
the 210-bp region inserted in the opposite orientation was constructed
using the following PCR strategy. An oligonucleotide was synthesized
that had the same sequence as the one described above that originated at position 649 of the 859-bp enhancer sequence, except that a BamHI site was incorporated at the 5' end instead of a
KpnI site. When this was used in PCR with an antisense
oligonucleotide that spanned the downstream BglII site (at
859), it allowed the amplified product to be digested with
BamHI and BglII and inserted at the BamHI site upstream of position
341 in the pSELECT plasmid
containing this proximal promoter region. Because BamHI and
BglII overhangs can anneal, it allowed the generation of
enhancer/promoter fusions with the 210-bp region in both orientations.
The reverse orientation was identified by sequencing, and the
enhancer/promoter fragment was recloned into pA3LUC as described above.
Transient transfections using electroporation for
-TSH (3 × 106),
-T3 (4 × 106), and GH3 (4 × 106) cells were performed as described previously (13,
22). 20 µg of
-subunit promoter and enhancer/promoter luciferase
constructs were used for the
-TSH and
-T3 cell transfections, and
2 µg of a cytomegalovirus
-galactosidase plasmid were included as an internal control of transfection efficiency. Transfections were
carried out in duplicate with a Rous sarcoma virus promoter luciferase
vector and a promoterless pA3LUC vector as positive and negative
controls (20). Experiments were performed a minimum of three times with
at least two preparations of each plasmid. After 16-24 h, luciferase
and
-galactosidase activities were measured from duplicate aliquots
of freeze-thawed cytoplasmic lysates. Luciferase activities of the
various enhancer/promoter constructs were normalized to the
corresponding
-galactosidase value and expressed as the fold
stimulation ± S.E. of the normalized activity of the
341 to +43
promoter in the various cell types.
Preparation of Nuclear Extracts and DNase I Protection
Analyses--
Nuclear extracts were prepared from dispersed TtT-97
thyrotropic tumors or
-TSH,
-T3, and GH3 cells as described
previously (23, 24). Cultured cells were placed in medium containing 10% charcoal-stripped fetal calf serum for 48 h before harvesting for extract preparation. Protein concentrations were determined by
Bio-Rad DC Protein Assay (Bio-Rad) using bovine serum albumin (Roche
Molecular Biochemicals) as a standard. The previously described pGEM5zf
plasmid containing the 184-bp PstI fragment from position 649 to position 833 of the 859-bp enhancer region was digested with
NotI and NdeI to generate a fragment for
footprinting analysis that could be labeled uniquely at the upstream
end using [
-32P]dGTP and dCTP to fill in the
NotI overhang (GGCC). To generate a footprinting fragment
that was labeled at the downstream position, we constructed a plasmid
that contained a fragment extending from 649 to a position 100 bp 3' of
the BglII site. This was amplified by PCR from the
previously described KpnI to SphI fragment
containing vector using the KpnI 5' tagged sense primer
originating at 649 and an antisense amplimer with a 5'
HindIII site (italicized) with the following sequence:
5'-GCGAAGCTTGGGGGAAATATCACTGCATG-3'. As before, excision
with EcoRI and MluI enabled unique filling of the
MluI overhang (CGCG) with [
-32P]dGTP and
dCTP. This 3' extension allowed the area immediately 5' of the
BglII site to be displayed in an optimal area of the footprint gel. DNase I protection assays were carried out as described previously (24). Briefly, radiolabeled probes were allowed to interact
with 10 µg of bovine serum albumin (no extract) or 60-70 µg of
nuclear extract protein derived from TtT-97,
-TSH,
-T3, or GH3
cells, subjected to DNase I digestion under defined conditions, and
analyzed on a denaturing 5% polyacrylamide-8 M urea gel.
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RESULTS |
Localization of Proximal Promoter Activation to the Proximal 210 bp
of the Upstream Region--
Previous transgenic and transient
transfection data indicated that sequences required for the enhancement
of the
-subunit proximal promoter specifically in thyrotropes and
gonadotropes were present in a KpnI-BglII
fragment located between
4.6 and
3.7 kilobase pairs upstream of the
transcriptional start site (13). To further localize the sequences
responsible for enhancement within this 859-bp region, subfragments of
the KpnI-BglII fragment were fused to the
341
to +43 proximal promoter region and analyzed independently for their
ability to enhance in both thyrotrope- and gonadotrope-derived cells.
The results of this analysis are shown in Fig.
1. A strategy that involved progressively
deleting sequences from the 5' end demonstrated that the enhancing
effect of the 859-bp area previously reported in both
thyrotrope-derived
-TSH cells and gonadotrope-derived
-T3 cells
(13) could be entirely accounted for by the most proximal 210 bp from
649 to 859. The lack of other areas eliciting any enhancing capability was further confirmed by showing that neither of two PstI
fragments, which comprised all but the terminal 69 bp of the sequence
upstream of position 654, exhibited any capacity to stimulate the
proximal promoter (Fig. 1). Because the sequences responsible for the
stimulatory effect appeared to map to the region immediately 5' of the
BglII site at
3700 (13), we thought it important to
determine if sequences immediately downstream of the BglII
site could further augment the previously observed enhancement.
Therefore, a larger fragment from
4600 to
3400 was generated from a
previously described
-subunit genomic clone (12), and PCR was used
to amplify a fragment that extended more proximally to a position 237 bp downstream of the BglII site. This was inserted upstream
of
341 in the proximal
-subunit promoter, and the stimulatory
effect of this 3' augmented enhancer region was compared with the
previous 859-bp area. The results of this analysis revealed no further
enhancement in either cell type when the enhancer region fused to the
proximal promoter-included sequences 3' of the BglII site
(data not shown). Therefore, the sequences mediating enhancement appear
to be entirely contained within the previously determined 210-bp
area.

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Fig. 1.
Enhancement of
-subunit proximal promoter activity by the upstream
region localizes to the most proximal 210 bp. 20 µg of a
luciferase plasmid containing the indicated subfragments of the 859-bp
upstream region fused to the proximal -subunit region from 341 to
+43 or one with the proximal region alone in conjunction with 2 µg of
a cytomegalovirus -galactosidase vector were transfected by
electroporation into 3 × 106 -TSH cells or 4 × 106 -T3 cells. After 16-20 h of incubation at
37 °C, cell extracts were prepared, and luciferase and
-galactosidase activities were measured. The figure shows the
relative normalized luciferase activities expressed as fold stimulation
of the enhancer/promoter fusion over that of the proximal promoter
alone ± S.E. for n determinations.
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Nuclear Proteins from Pituitary Cells Bind to Areas of the 210-bp
Region Corresponding to Transcription Factor Consensus Binding
Sites--
The nucleotide sequence of the 210-bp region that accounts
for all of the enhancer activity of the upstream 5' region of the mouse
-subunit gene is shown in Fig. 2.
Nucleotides 649-859 of the previously described 859-bp enhancer region
redesignated 1-210 are shown. The sequence shaded in gray
denotes the location of a 60-bp sequence that is 60-70% homologous to
other sequences that occur within several reported rodent genes as
revealed by a GenBankTM search. Boxed and
labeled sequences correspond to consensus binding sites for several transcription factors that have been reported previously to play a role in the activity of the proximal
-subunit promoter (17-19) as well as in other pituitary genes (25-29). These include several binding sites for GATA and ETS factors. Also present are two E-box motifs (CANNTG) and single sites that could potentially interact with Pit-1, Sp1, and nuclear hormone receptor family members
such as the steroidogenic factor SF-1. A proximal promoter area from
213 to
200 that binds SF-1 (the GSE) has been reported by our group
(14) and by others (5, 17) to be critical for both basal and
enhancer-stimulated
-subunit promoter activity in gonadotrope cells.
To begin to investigate which proteins are interacting with the 210-bp
region and could perhaps be playing a role in the enhancement of the
proximal promoter, we performed a comparative DNase I footprinting
analysis using nuclear extracts derived from pituitary cells that
express the endogenous
-subunit gene (TtT-97 thyrotropic tumor,
-TSH and
-T3 cells) as well as those that do not
(somatotrope-derived GH3 cells). Fig. 3
shows the results of this analysis using fragments encompassing the 210-bp region that have been labeled at either the upstream
(A) or downstream (B) position. In Fig.
3A, which utilized a 184-bp PstI fragment that
terminates 26 bp from the proximal end of the fully enhancing region
(see Fig. 2), protection from DNase I digestion can be seen in three
general areas. Using DNA size standards loaded in a parallel lane
(Stds) the approximate location of the footprinted areas are
from 77 to 112, from 132 to 150, and from 152 to 178 on the antisense
strand. No protection by any extract was evident upstream of position
77. The area from 77 to 112 appears to be similarly protected by the
two thyrotrope cell extracts (TtT-97 and
-TSH), but the footprint
generated by
-T3 nuclear extracts, although overlapping the
thyrotrope footprint, appears to be less well protected for the most
proximal 10 bp, suggesting that a different factor(s) present in each
cell type may be interacting at this region. As shown in Fig. 2, this
area contains sequences that correspond to binding sites for both E-box
and Ets factors. When the sense strand labeled at the downstream
position was analyzed, the same overall area was protected (Fig.
3B), but the area from 74 to 129 was subdivided into three
clearly defined footprints from 74 to 86, from 92 to 105, and from 109 to 129. The first two were seen with all of the extracts, including
those from GH3 cells. These encompass the upstream E-box and Ets sites.
The protected area from 109 to 129, which contained a second Ets site,
was observed with both thyrotrope-derived cell nuclear extracts (TtT-97
and
-TSH) but appeared not to be protected by either
-T3 or GH3 nuclear extracts. Two other more downstream footprints from 133 to 147 and from 152 to 178 that were also seen with the other strand were also
protected by all of the extracts, including those from GH3 cells. These
footprints contain a second E-box motif and potential binding sites for
GATA factors, Sp1 and SF1. It is of interest that parts of the area of
the enhancer that interact with the pituitary cell nuclear extracts
colocalize with the 60-bp region that is repeated elsewhere in the
mouse genome, shown in Fig. 2 as a gray box. A surprising
observation resulting from these protein-DNA binding experiments
suggests that non-
-subunit-expressing pituitary GH3 cells may
contain factors that interact with the enhancer region, and thus such
factors are not confined to cell types that express the endogenous
gene, i.e. thyrotropes and gonadotropes.

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Fig. 2.
Nucleotide sequence of the functional 210-bp
region. The nucleotide sequence of the most proximal 210 bp of the
859-bp upstream enhancing area is shown. Positions 1-210 are identical
to those from 649 to 859 of the GenBankTM sequence
AFF044976. Two PstI sites used to generate a 3' deleted
fragment for both functional and DNase footprint assays are
underlined. Sequences homologous to binding sites for
various transcription factors are boxed and identified. The
gray box denotes a 60-bp sequence that is repeated with
65-70% homology in other areas of the mouse genome.
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Fig. 3.
DNase I protection analyses of the 210-bp
region with various pituitary cell nuclear extracts. A,
a 184-bp PstI fragment (from 1 to 184 in Fig. 2) excised
from pGEM5zf with NotI and NdeI was labeled at
the upstream position by specifically end-filling the NotI
GGCC overhang with [ -32P]dGTP and dCTP. B,
a 310-bp fragment extending from position 1 (in Fig. 2) to a position
100 bp downstream of the BglII site (position 206 in Fig. 2)
was excised from pGEM7zf with EcoRI and MluI and
labeled at the downstream position (the CGCG MluI overhang)
with [ -32P]dGTP and dCTP. Both fragments were
subjected to DNase footprinting using bovine serum albumin ( ) and
nuclear extracts from TtT-97, -TSH, and -T3 cells, and the
downstream labeled fragment was also footprinted with extracts from GH3
cells. , areas within the 210-bp region that are protected from
DNase I digestion by proteins present in one or more of the various
nuclear extracts. Numbers define their corresponding
locations within the region calculated from the relative migration of
HpaII-digested pBR322 DNA size markers run in a parallel
lane (Stds in A).
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The 210-bp Region Represses Proximal Promoter Activity in GH3
Somatotrope-derived Cells--
The finding that nuclear proteins from
somatotrope-derived GH3 cells, which do not express the
-subunit,
bind to sequences within a region that is involved in the activation of
proximal
-subunit promoter activity in homologous
-TSH and
-T3
cells prompted us to investigate whether the upstream 210-bp area was also capable of enhancement in GH3 cells. We had previously shown that
in non-pituitary CV-1 monkey kidney cells, the larger 859-bp region
only modestly stimulated the proximal
-subunit promoter, although it
was capable of enhancing the activity of a viral promoter in a cell
type-independent fashion (13). Fig. 4
shows the effect of the 210-bp area on the proximal
-subunit
promoter in GH3 cells compared with that previously seen in
-TSH and
-T3 cells. As shown before, the 210-bp area confers a 25- and 7-fold
stimulation to the proximal promoter in
-TSH and
-T3 cells,
respectively. However, in the non-
-subunit-expressing
pituitary-derived GH3 cells, the upstream 210-bp region dramatically
inhibits the activity of the proximal promoter by 75% (note that the
scale of the x axis in Fig. 4 is not linear). Therefore, the
upstream regulatory region serves a role not only to activate
-subunit expression in
-subunit-expressing cell types but also to
repress it in another pituitary cell type that does not express the
endogenous gene.

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Fig. 4.
The 210-bp region represses proximal promoter
activity in GH3 cells. 20 µg of a construct containing the
210-bp region fused in either the forward or reverse orientation
(denoted by the or , respectively) to the -subunit proximal
promoter region from 341 to +43 or one with the proximal region alone
was transfected as before into -TSH or -T3 cells or into 4 × 106 GH3 cells along with 2 µg of a -galactosidase
transfection control. The figure shows the fold stimulation in -TSH
and -T3 cells and the inhibition of the proximal promoter activity
(set to 1) in GH3 cells derived from the normalized enhancer/promoter
and promoter alone luciferase activities ± S.E. for n
determinations. Note that the x axis below 1 has been
expanded to display both stimulation and inhibition on the same
graph.
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Reversing the Orientation of the 210-bp Region Results in Loss of
Activation but not Repression--
We previously showed that the
upstream region exhibited positional independence by its ability to
confer enhancement when fused directly to the proximal
341
-promoter in transgenic mice as well as in transiently transfected
-subunit-expressing cells (13). To see if the elements responsible
for the stimulatory effect satisfied another criterion of a classical
enhancer, that of orientation independence, the 210-bp fragment was
inserted in the opposite direction and assessed for proximal promoter
enhancement. Fig. 4 shows that in both
-TSH and
-T3 cells, the
enhancing activity was totally abrogated by reversing the direction of
the 210-bp region with respect to the proximal promoter. Thus, the elements responsible for stimulating the proximal promoter are unable
to functionally interact with the proximal promoter when their
orientation, with respect to those elements located proximally such as
the PGBE and GSE sites, were reversed. In fact, in
-T3 cells, the
opposite orientation of the 210-bp area consistently inhibited proximal
promoter activity by 40-50%. In contrast, reversing the orientation
of the 210-bp area had no effect on its ability to inhibit the proximal
-subunit promoter in GH3 cells, suggesting that the mechanism of
repression was orientation-independent and therefore different from
that mediating the activation.
Repression in Somatotrope Cells Is Not Dependent on Intact Proximal
PGBE or GSE Sites--
In a previous report (14), we demonstrated that
in order for the larger 859-bp enhancer area to exert its maximum
stimulatory effect on the proximal
-subunit promoter, interaction of
proteins present in
-subunit-expressing cells with proximal promoter
elements must be preserved. Mutation of the proximal PGBE site, which
leads to the disruption of binding most likely of a LIM homeodomain protein (15, 16), resulted in a dramatic reduction of enhancing capacity in
-TSH cells and complete abrogation of proximal promoter stimulation in
-T3 cells. However, mutation of the more proximal GSE
site, which was shown to affect the binding of a gonadotrope cell
protein as well as the steroidogenic factor SF1, reduced the effect of
the upstream enhancer only in
-T3 cells (14). We now wanted to see
if repression by the upstream region was also dependent on interaction
at these proximal elements. The same constructs with the 859-bp region
fused to the wild type or mutated
341 to +43 proximal promoter were
transfected into GH3 cells, and the results of these studies are shown
in Fig. 5. The larger 859-bp area also
inhibits the proximal promoter to a similar degree as the extreme 3'
210-bp region. The data in Fig. 5 also further demonstrate that
mutation of either the PGBE or GSE site still resulted in the ability
of the upstream region to inhibit proximal promoter activity in GH3
cells. This further supports the notion that to mediate repression of
-subunit promoter activity in GH3 cells, the upstream region
functionally interacts with the proximal promoter through a different
mechanism than does activation in thyrotrope- and gonadotrope-derived
cells. Also presented in Fig. 5 are data that show that although the upstream regulatory region inhibits the
-subunit promoter in GH3
cells, a viral promoter was stimulated 2.4-fold, which was similar to
that seen previously in
-TSH,
-T3, and CV-1 cells (13).

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Fig. 5.
Mutation of either the proximal PGBE or GSE
site has no effect on repression by the upstream region in GH3
cells. 20 µg of a luciferase plasmid containing the -subunit
proximal region from 341 to +43 or one with the same region bearing a
mutated (X), PGBE (P), or GSE (G)
proximal site (all with or without the 859-bp enhancer region fused
upstream) was electroporated into GH3 cells as described in the legend
to Fig. 4. A similar vector containing a truncated Rous sarcoma virus
viral promoter with or without the 859-bp region was also included. The
figure shows the fold stimulation or inhibition of the viral promoter
or proximal promoter activity, respectively, ± S.E. for n
determinations, calculated from the normalized luciferase activities as
described in Fig. 4.
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Both Activation and Repression Are Dependent on Multiple
Sites--
To further dissect the specific elements within the 210-bp
area that might be contributing to both activation and repression, we
devised 5' and 3' deletion strategies that progressively eliminated the
footprinted areas which contained the putative transcription factor
binding sites shown in Fig. 2 and also shown schematically in Fig.
6A. Fig. 6B shows
that the most 5' 85 bp, which contain two GATA sites and a Pit-1 motif,
contribute to neither activation in
-TSH or
-T3 cells or
repression in GH3 cells of the proximal
-subunit promoter. However,
the truncated region still continues to stimulate a viral promoter 2-4
fold in all cell types. This area was also not footprinted by the
pituitary cell nuclear extracts (Fig. 3), except at the most 3' 10 bp.
However, removal of an additional 65 bp (to 150) which harbors two
E-boxes, two Ets sites, and a GATA site dramatically decreased
enhancement to only 2-fold in
-TSH cells and actually inhibited the
proximal promoter by 50% in
-T3 cells. In GH3 cells, an inhibition
of proximal
-subunit promoter activity of only 25% was seen as a
result of removing the 5' 150 bp. Deletion of an additional 28 bp,
leaving only the terminal 32 bp which contains two GATA motifs,
resulted in a fragment that had no activating or repressing effect on
the proximal promoter in any cell type. Although the terminal 32 bp had
no activity alone, when they were deleted in the context of the 210-bp
region (1-184), reduced stimulatory capability in both
-TSH and
-T3 cells (from 20- to 12-fold and from 6- to 3-fold, respectively) was observed. Inhibition in GH3 cells was maintained, but only to 50%.
These data suggest that both activation and repression of the proximal
-subunit promoter can be mediated by only the terminal 125 bp of the
previously described upstream region from
4600 to
3700 and that
within that area, the elements that mediate activation in
-TSH cells
may differ from those in
-T3 cells, and, in turn, these may also
differ from those that confer GH3 cell-dependent
repression.

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Fig. 6.
Multiple areas within the most proximal 125 bp contribute to both activation and repression by the upstream
region. A, a schematic representation of the 210-bp
region with the location of sequences corresponding to transcription
factor binding sites designated as (Pit-1, Sp1, and SF-1), (GATA factors), rectangles (E-box binding factors), and (Ets factors). B, 20 µg of a luciferase plasmid containing
the indicated subfragment of the 210-bp upstream region fused to the
proximal -subunit region from 341 to +43 or one with the proximal
region alone was transfected by electroporation into -TSH, -T3,
or GH3 cells. Fold stimulation and inhibition of the proximal promoter
by the 210-bp region subfragments is calculated from the normalized
luciferase activities ± S.E. for n determinations as
described in Fig. 4.
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DISCUSSION |
In this report, we have further characterized the sequences
present in a 859-bp upstream region of the mouse
-subunit gene that
are capable of directing specific pituitary cell-dependent activation of the proximal promoter. We have localized the enhancer function to the most proximal portion of the 859-bp region and showed
that sequences immediately downstream did not contribute any further
stimulatory effect. Utilizing a DNase footprinting approach, we showed
that this area interacts with proteins present in thyrotrope- and
gonadotrope-derived cells. Some of these protected areas contain
consensus recognition sites for transcription factors that play a role
in the activity of the proximal
-subunit promoter or other pituitary
promoters in transiently transfected pituitary-derived cell lines.
These include binding sites for GATA factors that contribute to
proximal
-subunit promoter activity in gonadotropes (18) and also
synergize with Pit-1 to activate the TSH
promoter in thyrotrope
cells (30). A perfect consensus nuclear hormone response element, which
could potentially bind SF-1, and two E-box sequences are located within
the footprinted areas. Similar motifs in the proximal promoter have
been shown to be important for
-subunit proximal promoter activity
in
-T3 cells (5, 19). Finally, sequences potentially capable of
binding Pit-1, Sp1 and Ets factors, which contribute to growth hormone
and prolactin promoter activity (25-29), are also located in the
functional upstream area. Pit-1 protein is not present in
-TSH cells
(31) and therefore does not appear to be required for
-subunit expression.
The mechanism whereby an enhancer located >3000 bp distant can act on
proximal elements to influence transcriptional efficiency represents an
intriguing problem in understanding gene expression. We previously
showed that the ability of the enhancer to functionally interact with
the proximal promoter was dependent on a proximal site (PGBE) that
interacted with a LIM homeodomain protein (15, 16). Such LIM
domain-containing proteins have been shown to participate in
protein-protein interactions through adaptor molecules referred to as
LIM domain-binding (Ldb) proteins (32, 33). Bach et al. (34)
cloned a similar cofactor, termed clim, from mouse pituitary gland
using a two-hybrid screen with the LIM domain of the pituitary
restricted P-Lim/Lhx3 homeodomain factor and showed that it conferred
transcriptional synergy between P-Lim and P-OTX, another homeodomain
protein on the proximal
-subunit promoter. Interestingly, a
Drosophila homolog of these Ldb cofactors termed
Chip was identified in a genetic screen designed to identify factors that facilitate communication between remote enhancers and
promoters (35). In related studies, Wadman et al. (36) demonstrated that the nucleotide sequence preferred by erythroleukemia cell complexes, which contained Ldb, possessed an arrangement of E-box
and GATA motifs similar to that present between positions 132 and 148 of the upstream enhancer (Fig. 2). It is intriguing to speculate that a
Ldb factor present in thyrotropes and gonadotropes, but not in
somatotropes, mediates the enhancer/promoter functional interaction
that stabilizes the complex of a helix-loop-helix protein and a GATA
factor. GATA-2 has been reported to be expressed early in pituitary
gland development (37) and to cooperate with Pit-1 in the cell-specific
expression of the TSH
subunit in thyrotropes (30). It also
contributes to proximal
-subunit promoter activity in of
-T3
gonadotropes (18).
Although the pattern of protection of the 210 bp fragment by GH3 cell
nuclear extracts qualitatively resembled that generated by extracts
from expressing
-TSH and
-T3 cells, the 210-bp region dramatically repressed the proximal
-subunit promoter in GH3 cells.
Inhibition of
-subunit gene expression by the upstream region in GH3
cells was perhaps not unexpected, as the flanking region between
4600
and
3700 was able to restrict the function of the
341
-promoter,
which was active in all pituitary cells, to only thyrotropes and
gonadotropes of transgenic mice (13). The current transfection data
support our previous notion that both the upstream region and the
proximal area between
381 and
341 (13) can exert pituitary cell
restrictive properties, and this is accomplished in somatotropes
through a functional interaction of the upstream region with the
proximal region to suppress ectopic activity. Because P-Lim, which
interacts at the PGBE site (15, 16) that is required for maximal
enhancer function (14), is present in GH3 cells (38), it is highly
likely that the inhibitory effect is also being mediated through
interference with binding at the PGBE site. However, our data are not
consistent with such a mechanism as repression in GH3 cells by the
upstream regulator is still seen when the PGBE site is mutated,
suggesting that functional interaction at a different proximal site or
sites via a different mechanism is involved in repression.
Another piece of evidence that supports a different mechanism of action
for the repressive effect is the demonstration that fusing the 210-bp
area to the
341
-promoter in the reverse orientation completely
abolishes its ability to behave as an enhancer in
-TSH and
-T3
cells, whereas it was equally effective as a repressor in GH3 cells in
either orientation (Fig. 4). This suggests that the factor(s)
interacting with upstream elements to mediate repression, unlike those
that confer activation, can interact functionally with proximal
elements, irrespective of their physical or spatial geometry.
Interestingly, in
-TSH cells, the reversed enhancer merely abolished
activation, whereas in
-T3 cells, it resulted in the acquisition of
a 50% inhibition of proximal promoter activity. Because the level of
activation in
-T3 cells was consistently less than that in
-TSH
cells (7-fold versus 20-fold), it is tempting to speculate
that
-TSH cells and
-T3 cells both contain an enhancer binding
activator(s), but that
-T3 cells also contain an inhibitory factor(s) that competes with the activator(s) to lower the overall level of enhancement. When the influence of the activator(s) is nullified by reversing the orientation, the inhibitor(s) now becomes dominant, resulting in the repressive effect seen in
-T3 cells.
The results of these studies may have important implications regarding
the emergence of differentiated pituitary cell phenotypes from a common
precursor in Rathke's pouch precursor cell that expresses the
-subunit (37). Our previous transgenic data (12) showed that the
onset of
-enhancer-driven transgene expression occurred by embryonic
day 9.5 in Rathke's pouch, which parallels the temporal and spatial
expression of the endogenous
-subunit gene (39, 40). This suggested
that the factor(s) required for activation is already present and
initiates
-subunit gene expression at this early stage of
development, before any hormone-producing cells have emerged. As the
developmental program progresses, an inhibitory factor(s) is expressed
in cells destined to develop into the somatotrope lineage. However, the
presomatotropes also cease expression of the activator(s), and the
inhibitor(s) is thus able to effectively silence the endogenous
-subunit gene. This idea is supported by the finding that certain
naturally occurring pituitary tumors that probably arise from premature
undifferentiated cells continue to express both the
-subunit and
growth hormone (41, 42).
The deletion studies to further define the elements responsible for
activation and repression within the 210-bp region show that the 5'
most 85 bp are not involved in either effect, but the remaining 125 bp
harbor multiple cell type-specific areas involved in both activation
and repression. In
-T3 cells, activation was mapped to the 65 bp
between 85 and 150, and, similar to what was seen with the reverse
orientation when enhancement was nullified by the removal of this
region, the remaining 60 bp now developed inhibitory properties. In
contrast, the 3' terminal 60-bp region, which lacks the E-box/GATA
composite site described earlier, was still activated by about 2-fold
in
-TSH cells but was only modestly inhibited in GH3 cells. These
data underscore the complexity of this novel upstream regulator of the
-subunit gene which mediates different influences on the cognate
proximal promoter region depending on the nuclear factor content of the
pituitary cell type in which it is being assayed. To our knowledge, no
other gene regulatory region exhibits these opposing cell-specific
influences through functional elements in such close proximity to one
another. There are many reported examples of classical enhancer regions
that are both position- and orientation-independent and interact to stimulate expression from proximal promoter regions of genes such as
Pit-1 (43) and
-globin (44). Other elements such as the neuron
restrictive silencer element have been reported to silence expression
from brain-specific genes in non-neural tissue (45). Since the
-subunit regulatory region appears to carry out both activation and
repression through the same or closely associated elements by different
mechanisms, we believe that this unique regulatory region will
represent a new class of control region important for governing complex
temporal and spatial patterns of gene activation and repression.