Cell-Specific Expression of the Mouse Glycoprotein Hormone
-Subunit Gene Requires Multiple Interacting DNA Elements in Transgenic Mice and Cultured Cells
Michelle L. Brinkmeier,
David F. Gordon,
Janet M. Dowding,
Thomas L. Saunders,
Susan K. Kendall,
Virginia D. Sarapura,
William M. Wood,
E. Chester Ridgway and
Sally A. Camper
Department of Human Genetics (M.L.B., T.L.S., S.K.K., S.A.C.)
University of Michigan Ann Arbor, Michigan 48109-0638
Department of Medicine (D.F.G., J.M.D., V.D.S., W.M.W., E.C.R.)
University of Colorado Health Sciences Center Denver, Colorado
80262
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ABSTRACT
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The glycoprotein hormone
-subunit gene is
expressed and differentially regulated in pituitary gonadotropes and
thyrotropes. Previous gene expression studies suggested that cell
specificity may be regulated by distinct DNA elements. We have
identified an enhancer region between -4.6 and -3.7 kb that is
critical for high level expression in both gonadotrope and thyrotrope
cells of transgenic mice. Fusion of the enhancer to -341/+43 mouse
-subunit promoter results in appropriate pituitary cell specificity
and transgene expression levels that are similar to levels observed
with the intact -4.6 kb/+43 construct. Deletion of sequences between
-341 and -297 resulted in a loss of high level expression and cell
specificity, exhibited by ectopic transgene activation in GH-, ACTH-,
and PRL-producing pituitary cells as well as in other peripheral
tissues. Consistent with these results, transient cell transfection
studies demonstrated that the enhancer stimulated activity of a
-341/+43
-promoter in both
TSH and
T3 cells, but it did not
enhance
-promoter activity significantly in CV-1 cells. Removal of
sequences between -341 and -297 allowed the enhancer to function in
heterologous cells. Loss of high level expression and cell specificity
may be due to loss of sequences required for binding of the LIM
homeoproteins or the
-basal element 1. These data demonstrate that
the enhancer requires participation by both proximal and distal
sequences for high level expression and suggests that sequences from
-341 to -297 are critical for restricting expression to the anterior
pituitary.
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INTRODUCTION
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The anterior pituitary gland is composed of five cell types that
are distinguished by the hormones they produce: thyrotropes produce
TSH, gonadotropes produce LH and FSH, somatotropes produce GH,
lactotropes produce PRL, and corticotropes produce ACTH. Three of these
hormones, TSH, LH, and FSH, belong to a pituitary glycoprotein hormone
family that consists of heterodimeric proteins with a common
-subunit and unique ß-subunits. The requirement for regulated
expression of the
-subunit in gonadotropes and thyrotropes in
response to different stimuli suggests that different
cis-acting DNA sequence elements may be involved. Studies
with transgenic mice have supported this idea. Two large human
-subunit transgenes containing more than 5 kb of 5'-sequences were
sufficient for pituitary-specific expression and targeted oncogenesis
of both gonadotrope and thyrotrope lineages (1, 2). In contrast,
1.51.8 kb of the human
-subunit 5'-flanking sequence directed
transgene expression solely to gonadotropes, suggesting the possibility
of a more upstream element necessary for expression in thyrotropes (3, 4). Consistent with this idea, gonadotrope specificity was also
observed in transgenic mice with -315 bp of the bovine
-subunit
promoter driving reporter gene expression (5, 6). However, the
potential for divergence in the location of cell-specific elements was
suggested by the observation that both gonadotrope and thyrotrope
expression can be obtained with only -480 bp of mouse
-subunit
5'-flank (7).
Transfection studies using cell lines representative of thyrotropes
(
TSH) and gonadotropes (
T3), as well as a murine TtT-97 mouse
thyrotropic tumor cell model (8), have implicated individual regions of
the
-subunit gene in cell specificity and hormone regulation. Most
of these studies have focused on defining important
cis-acting elements within the proximal 500 bp upstream of
the transcription initiation site in the mouse and human genes (Fig. 1
) (9). Deoxynuclease I (DNase I)
protection studies using TtT-97 thyrotropic nuclear extracts have
identified cell-specific protein-DNA interactions between -474 and
-419 bp (10). A lin11, Isl1, mec3 (LIM) homeodomain factor-binding
site, functionally important in gonadotropes and thyrotropes, has been
localized to the area between -342 and -329 bp (11, 12) of the mouse
gene and a similar element, pituitary glycoprotein hormone basal
element (PGBE), was identified in the human gene (13). Recently, two
basal elements located just downstream of the PGBE site within the
human
-promoter,
BE1 and
BE2, were demonstrated to be
functionally important for expression in
T3 cells (13). Additional
gonadotrope-specific elements have been localized between positions
-445 to -438 (11), and a GnRE at -406 to -399 (14) of the mouse
gene. These elements were shown to be important for basal activity or
GnRH responsiveness, respectively. Two other elements within the human
gene important for expression in gonadotropes bind the transcriptional
activators SF-1, -225 to -205 bp (15), and GATA2/3, -161 to -146
(16).

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Figure 1. Deletion Constructs Designed to Test Importance of
Sites of Protein-DNA Interaction
A schematic representation of -480 bp of m GSU 5'-flanking sequence
summarizes data identifying thyrotrope-specific (T) and
gonadotrope-specific (G) protein-DNA interaction sites (9). Some
factors that bind these regions have been identified: GnRE, GnRH
responsive element; Ptx-1/P-OTX, pituitary transcription factor; Lhx2,
LIM homeodomain protein; BE1 and 2, -basal element 1 and 2; SF-1,
steroidogenic factor 1; GATA2/3. Transfection studies identifying Ptx1,
BE1, and BE2 were performed with either the rat GSU promoter
(PTX1) or the human GSU promoter ( BE1, BE2) although
consensus binding sites for these factors can be found in mouse GSU
promoter sequence. The location of potential interaction sites with
these factors within the -480 bp is noted. The -381-, -341-, and
-297-bp constructs were designed for transfection and transgenic mouse
assays based on the deletion of binding sites important for gonadotrope
and thyrotrope specificity.
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Transgenic mice provide a rigorous in vivo test of the
relevance of individual sites for cell-specific expression in the
context of chromatin while transient transfection of cell lines allows
the testing of many different constructs and a measurement of their
differential effects on promoter activity in homologous and
heterologous cells. We generated and analyzed transgenic mice bearing
deletion constructs that were designed to test the importance of
specific regions thought to play a role in gonadotrope and thyrotrope
specificity. Previous studies have suggested that an element between
-4.6 and -2.7 kb is important for high-level expression in transgenic
mice (7). We have used transgenic mice and cell transfection to refine
and characterize this enhancer region. The results illustrate that the
region between -4.6 and -3.7 kb functions as an enhancer element and
contributes to the restriction of
-subunit gene expression when
juxtaposed to a proximal
-promoter construct. Our data also indicate
that cell-specific expression in thyrotropes and gonadotropes is due to
interactions between multiple elements. Although neither the LIM- nor
BE1-binding sites are essential for transgene expression in
gonadotropes and thyrotropes, one or both are critical for restriction
of expression to these two pituitary cell types.
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RESULTS
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Deletion Analysis Demonstrates the Importance of Sequences between
-4.6 and -3.7 kb for High-Level Expression
Levels of transgene expression in mice carrying constructs of the
mouse
-subunit promoter (m
) with 5'-termini at -3.7 and -3.1 kb
were compared with previously published data from -4.6 and -0.48
m
-ßgal transgenics to narrow the 5'-flanking region necessary for
high-level expression in the anterior pituitary (7). Fragments (-3.7
and -3.1 kb) of m
were joined within the 5'-untranslated region of
exon 1 to a lac Z reporter gene containing a nuclear localization
signal and used to generate transgenic mice. Twelve and 11 transgenic
mouse lines were generated with the -3.7 and -3.1 kb m
-ßgal
constructs, respectively. The level of transgene expression in each
line was quantitated in pituitary gland homogenates using a
fluorometric assay for ß-galactosidase activity (Materials and
Methods). ß-Galactosidase levels in each transgenic line were
expressed relative to the level in the highest expressing -4.6 kb
m
-ßgal transgenic line, 8365 (7). None of the transgenic mice with
the -3.7 or -3.1 kb m
-ßgal transgenes exhibited high-level
expression (Fig. 2
). In contrast, half of
the lines with the -4.6 m
-ßgal construct expressed at a high
level (7). While the position of integration influences the level of
expression in each founder, the difference between the -4.6 and the
smaller -3.7 and -3.1 m
constructs is statistically significant
(P = 0.0017 and P = 0.0036, respectively)
according to the Fishers Protected Least Significant Difference ANOVA
post hoc test. These data suggest that the sequences between -4.6 and
-3.7 kb are important for high levels of expression in the anterior
pituitary.

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Figure 2. Deletion Analysis in Transgenic Mice Reveals the
Ability of the -4.6- to -3.7-kb Region to Function as an Enhancer
Transgenic mice generated with 3.1 and 3.7 kb of m -subunit 5'-flank
joined to the ßgal reporter gene were used to narrow the enhancer
region to the 859 bp between -4.6 and -3.7 kb. This enhancer region
(E) was tested in transgenic mice in conjunction with various fragments
of m -subunit promoter 5'-flank fused to the ßgal reporter gene
(E/-480, E/-381, E/-341, E/-297). Transgenic mice generated with
enhancerless constructs served as a control for enhancer activity
(-381 and -341). ßgal activity was measured in homogenates of
individual pituitary glands. Each circle represents an
individual transgenic founder. The open circles
represent founders that tested positive for the transgene by PCR but
have no ßgal activity detectable in homogenates or in Xgal-stained
pituitary sections. The ßgal activity of each founder is expressed as
a percentage of the ßgal activity detected in the highest expressing
founder with the -4.6-kb construct (100%) (7). The dashed
line indicates the arbitrary lower limit of high expressing
transgenic mouse lines. Stars next to the constructs
indicate those that are statistically different from the highest
expressing -4.6-kb transgenic line (*, P < 0.05;
**, P < 0.01; ***, P < 0.001;
exact P values are stated in the text). Results of -4.6
kb and -480 bp are reproduced here for ease of comparison (7).
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The 5'-Flanking Region between -4.6 to -3.7 kb Can Function as an
Enhancer in Transgenic Mice
To test whether an 859-bp fragment between -4.6 and -3.7 kb
could function in vivo as a position-independent enhancer,
we fused it 5' of several more proximal m
promoter fragments
containing or lacking sequences thought to be important for gonadotrope
and thyrotrope expression (9). Based on 5'-deletion and mutagenesis
studies in transfected cell lines, we used proximal promoter fragments
of -480, -381, -341, and -297/+43 bp fused to a ßgal reporter
gene containing a nuclear localization signal (Fig. 1A
). These
constructs were termed E/-480, E/-381, E/-341, and E/-297.
Additionally, -480, -381, and -341 m
-ßgal constructs lacking
the 859-bp sequence were tested in transgenic mice. Transgenic founders
were identified by PCR of genomic DNA obtained from tail biopsies. The
initial screening was carried out with primers designed to amplify a
portion of the lac Z gene. The integrity of the transgene was confirmed
by PCR amplification with primers specific for the m
enhancer-promoter fusion region. The identity of these PCR products was
verified by restriction enzyme digestion and DNA sequence analysis. The
pituitary glands from individual transgenic mice were analyzed for
ßgal activity using a chemiluminescent assay on tissue homogenates
and Xgal staining on tissue sections.
Interestingly, the enhancer functioned most effectively when fused
upstream of the -341/+43 bp fragment. The levels of transgene
expression in lines analyzed from the E/-341 m
-ßgal construct
were generally high and exhibited a similar pattern to those observed
with the -4.6 kb m
-ßgal construct (Fig. 2
) (7). In contrast, the
range of expression levels detected in transgenic lines bearing the
-341 m
-ßgal construct was consistently low, similar to that
observed in the -480 bp m
-ßgal transgenics (Fig. 2
) (7). This
suggests that sequences contained within the 859-bp fragment are
required to obtain high levels of expression in the pituitary.
Little or no enhancement was evident when the 859-bp segment was fused
5' of the -480 and -381 bp m
-promoters (Fig. 2
). Although one high
expressing line was generated from each of these constructs, the
majority of the founders did not express the transgene in the anterior
pituitary by two independent assay methods. This suggests that the
enhancer functions optimally when juxtaposed to the -341/+43 proximal
promoter fragment and is inhibited by an additional 40 or 139 bp of
m
-5'-flanking sequence. The lack of enhancement when fused to some
promoter fragments may result from incorrect spacing between elements
within the enhancer and proximal promoter or from differences in
chromatin structure. Transgenics generated from the E/-297 construct
exhibited very poor expression in all 19 animals tested (Fig. 2
). The
range of expression levels was similar to the -480 bp m
-ßgal
transgenics (7). Low transgene activity may result from the loss of an
important LIM-binding site or the adjacent
BE1 site (12, 13). The
differences in ßgal activity between both -4.6 vs.
E/-297 and -4.6 vs. -341 m
-ßgal transgenics were
verified using Fishers Protected Least Significant Difference ANOVA
Post hoc test (P = 0.0008 and 0.0027, respectively). Levels
of ßgal activity in transgenics from m
-4.6 and E/-341 were not
significantly different (P = 0.4). The results indicate that
the 859-bp upstream enhancer can function in vivo in a
position-independent manner and that its effectiveness is influenced by
its context with respect to other proximal promoter sequences.
E/-297 Transgenic Mice Show a Loss of Cell-Type Restriction in the
Pituitary
Previous studies had demonstrated that both -4.6 kb and -0.48 kb
were sufficient for cell-specific expression even though -0.48 kb were
expressed at a much lower level (7). Six of six 4.6-kb transgenics
exhibited appropriate cell specificity and four of four 0.48-kb
transgenics as well. Each of the enhancer-promoter deletion constructs
was assessed for the ability to confer pituitary cell specificity. Half
of the pituitary gland from each transgenic founder was stained with
Xgal, embedded in paraffin, and sectioned. The penetrance of transgene
expression was determined by quantitating the average number of nuclei
stained with Xgal in a set field of approximately 650 cells (n =
12). Founders exhibiting a penetrance of expression of 1% or greater
were examined for cell specificity by immunohistochemistry with
antibodies to the pituitary hormones: TSH, LH, ACTH, GH, and PRL. Cell
specificity was calculated by dividing the number of cells that
exhibited colocalization of the transgene and the endogenous pituitary
hormone gene by the total number of immunostained cells and reporting
it as a percentage. These percentages were compared with those obtained
from -4.6 kb and -480 bp m
-ßgal transgenic lines. The E/-480,
E/-381, -381, and E/-341 constructs all retained appropriate
transgene expression in gonadotropes and thyrotropes (Table 1
). Transgene expression was consistently
seen in a few somatotropes and may reflect GH/TSH double positive cells
that can be found in wild-type pituitary (17). No transgene expression
was detected in corticotropes or lactotropes. In contrast, three of
three transgenics analyzed from the -341 construct exhibited a loss of
pituitary cell specificity. Three lines from the -341 construct and
two from the E/-341 construct were examined by immunohistochemistry.
Each of the E/-341 lines expressed the transgene only in gonadotropes
and thyrotropes (Fig. 3
, A and B). In
contrast, transgene expression was detected in corticotropes and
somatotropes in addition to gonadotropes and thyrotropes in all three
lines from the -341 construct (Fig. 3
, CF). One of these lines also
expressed the transgene in lactotropes (Table 1
). Similarly, the
E/-297 transgene was expressed in pituitary cells that stained
positively for TSH and LH as well as in those cells secreting ACTH, GH,
and PRL (Fig. 4
and Table 1
). These data
suggest that in the absence of the enhancer, sequences between -381
and -341 are important for the restriction of m
-subunit expression
to pituitary gonadotropes and thyrotropes. The enhancer conferred the
ability to restrict expression to the appropriate cell types when fused
to the -341 construct. However, even in the presence of the enhancer
region, cell specificity was lost when the region from -341 to -297
was removed. Thus, both the enhancer and proximal promoter sequences
contribute to restrict expression to the appropriate pituitary
cells.

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Figure 3. Colocalization of the -341 m -ßgal Transgene
to GSU Expressing and Nonexpressing Pituitary Cells
Paraffin sections of Xgal-stained pituitaries from transgenics
generated with either the E/-341 or -341 constructs were
immunostained with hormone-specific antibodies. Cells with
blue nuclei represent those expressing the transgene.
Cells costained with Xgal and the indicated pituitary hormone
(brown cytoplasm) are marked with arrows.
A, E/-341 and TSH; B, E/-341 and LH; C, -341 and TSH; D, -341 and
LH; E, -341 and ACTH; and F, -341 and GH.
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Figure 4. Nonspecific Expression of the E/-297 m -ßgal
Transgene in Anterior Pituitary Cells
Paraffin sections of Xgal-stained pituitaries from transgenic mice
generated with the E/-297 construct were immunostained with antibodies
raised against TSH, LH, ACTH, GH, or PRL. Cells with
blue nuclei represent those expressing the transgene.
Cells with a brown cytoplasm represent those producing
the respective pituitary hormone. Representative cells costained with
Xgal and a particular pituitary hormone are marked with
arrows.
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E/-297 Transgenics Exhibit Inappropriate Transgene Expression in
Peripheral Tissues
For each transgene construct, we examined several lines for
ectopic transgene expression in nonpituitary tissues. Peripheral
tissues from transgenic founders were sectioned and stained with Xgal
to test for inappropriate expression of the transgene. Nontransgenic
mice were used to determine background levels of Xgal staining in the
selected tissues (Fig. 5A
).
Extrapituitary transgene expression was detected in six of the nine
founders with the E/-297 construct but not with other constructs
(Table 2
). The particular tissues, cell
types, and levels of ectopic expression varied between transgenics. For
example, Xgal staining revealed abundant transgene expression in the
kidney (Fig. 5B
) and regional expression in the brain (Fig. 5
, C and
D). Rare scattered cells staining with Xgal were observed in peripheral
tissues of nontransgenic mice and a few transgenic mice with the other
constructs (E/-480, E/-341, -381, and E/-381) (Table 2
). These data
support the importance of the promoter region between -341 and -297
in directing expression of the
-subunit exclusively to the anterior
pituitary.

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Figure 5. Ectopic Expression of the E/-297 m -ßgal
Transgene in Peripheral Mouse Tissues
Frozen sections from nontransgenic kidney (A), E/-297 transgenic line
20087 kidney (B), tg line 19983 brain (C), and tg line 20087 brain (D)
were stained with Xgal to determine transgene expression in peripheral
tissues. A low level of background Xgal stain is evident in panel A
(see Table 2 ).
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The 859-bp Region Can Function as an Enhancer in Transfected
Gonadotrope and Thyrotrope Cell Lines
Constructs containing the m
promoter sequences of -480, -381,
-341, and -297/+43, with or without the 859-bp enhancer, were fused
to the firefly luciferase reporter gene and transiently transfected
into both
T3 and
TSH cell lines. The mean promoter activities
were expressed as light units normalized to the activity of a
cotransfected cytomegalovirus (CMV)-ßgal plasmid to control for
variations in transfection efficiency. In constructs lacking the
enhancer, deletion of sequences from -480 to -381 led to a 20-fold
decrease in promoter activity in
TSH cells and a 5-fold decrease in
T3 cells (Fig. 6
, A and C). The
lowered activity may result from the loss of binding sites for
thyrotropic nuclear proteins, -474 to -419, and elements important
for expression in
T3 cells, -445 to -438 and -406 to -399 (10, 11, 14, 18). Further deletion to position -341 resulted in a slight
increase in promoter activity while deletion to position -297 lowered
activity 5-fold in
TSH cells and 13-fold in
T3 cells (Fig. 6
, A
and C). The latter deletion eliminates the binding site (-342 to
-329) for a LIM homeodomain protein, Lhx2, shown to be important in
both cultured gonadotropes and thyrotropes as well as the adjacent
BE1 element important for full activity in
T3 cells (12, 13).
The fold stimulation achieved using the 859-bp enhancer in both
TSH
and
T3 cells is shown in Fig. 6
, B and D. The highest stimulation
was found with the E/-341 construct where the enhancer increased
promoter activity 35- fold in
TSH cells and 8-fold in
T3 cells.
Differences in the magnitude of the response may be due to higher
promoter activity of the enhancerless constructs in
T3 cells. This
is consistent with the data from transgenic mice that show high
expression levels of the E/-341 construct in pituitary gonadotropes
and thyrotropes. The E/-381 m
-luc construct resulted in a less
robust stimulation of 12-fold and 4-fold in
TSH and
T3 cells,
respectively. The E/-480 and E/-297 constructs showed minimal
stimulation. This may result from differences in spacing of the
enhancer with more proximal elements or from the loss of key basal
response elements. These data demonstrate that the 859-bp enhancer can
strongly stimulate reporter gene activity in cultured gonadotrope and
thyrotrope cells when placed upstream of proximal
-promoter
sequences.
Comparison of Enhancer Activity in Homologous and Heterologous Cell
Types
Transgenic mice containing the E/-297 construct demonstrated
inappropriate expression of the ßgal reporter gene in nonpituitary
tissues such as the kidney and brain (Fig. 5
and Table 2
) while
the E/-341 construct targeted expression solely to the pituitary
gonadotropes and thyrotropes. To test whether such leaky expression
would also occur in cultured nonpituitary cells, we transiently
transfected these constructs and their enhancerless counterparts
into heterologous CV-1 kidney cells (Fig. 7A
). The overall expression of these four
constructs was very low in CV-1 cells relative to that observed in the
pituitary cell lines. This is consistent with the presence of
pituitary-specific elements in both the enhancer and promoter regions.
The E/-341 m
-luc construct resulted in a lower level of stimulation
in CV-1 kidney cells (Fig. 7B
) than that observed in both
T3 and
TSH cells (2-fold vs. 8- to 35-fold). The reduction of
enhancer and promoter function in heterologous cells suggests that
optimal function of the E/-341
-promoter requires the participation
of pituitary-specific factors. The E/-297 construct, which had low
activity (2-fold) in the pituitary cell lines, stimulated promoter
activity by 8-fold in CV-1 cells (Fig. 7B
). These results further
support the importance of the 44 bp between -341 and -297 for
maintenance of pituitary cell specificity. Promoter elements are
necessary to restrict expression to pituitary gonadotropes and
thyrotropes in both cell culture and in vivo assays.

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Figure 7. E/-297 Exhibits an Increased Promoter Activity in
CV-1 Cells Compared with E/-341
The -341, E/-341, -297, and E/-297 luc constructs were transfected
into a heterologous CV-1 cell line to test for promoter activity in a
nonpituitary cell line. Panel A shows activity in light units for each
construct ± SEM, and panel B shows fold stimulation
of the enhancer-promoter constructs compared with the enhancerless
construct. The E/-297 luc construct, which was activated 2-fold over
the -297 luc construct in TSH and T3 cells, illustrated a 7-fold
stimulation in CV-1 cells. The 859-bp region demonstrates enhancer
activity on a heterologous RSV180-promoter in both pituitary and
nonpituitary cells: recombinant constructs with the RSV180 promoter
driving expression of a luciferase reporter gene, in the presence or
absence of the 859-bp distal enhancer, were transfected into TSH,
T3, CV-1 cell lines. The activity of each construct was quantitated
with luciferase assays and normalized to a cotransfected CMV-ßgal
plasmid as an internal control (C), and the fold enhancement was
determined (D). The black bars in panel D represent the
fold stimulation of promoter activity of the E/RSV-luciferase construct
relative to the RSV-luciferase construct. Panel C presents the activity
in light units of each construct ± SEM. At least
three independent experiments were executed in triplicate for each
construct.
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To test whether the 859-bp fragment would stimulate a heterologous
promoter, it was fused to an Rous sarcoma virus (RSV)180 promoter (Fig. 7C
). Figure 7D
shows that the enhancer was able to stimulate the RSV
promoter by about 7- to 10-fold in
TSH,
T3, and CV-1 cells. Thus,
the 859-bp fragment displayed independent enhancer activity when fused
to a heterologous promoter in pituitary and nonpituitary cells. These
data show that the enhancer interacts with ubiquitous and
pituitary-specific factors.
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DISCUSSION
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Previous studies in transgenic mice have suggested that the
sequences between -4.6 and -2.7 kb of the m
-subunit are necessary
for high levels of expression to the anterior pituitary (7). The
current investigations have localized the critical element to 859 bp
between -4.6 and -3.7 kb. This sequence is able to function as an
enhancer in context with the m
-subunit promoter both in the
pituitary gonadotropes and thyrotropes of transgenic mice and in
transfected
TSH and
T3 cell lines. It also directed high levels
of expression of an RSV promoter in the appropriate pituitary-specific
cell lines and in a heterologous CV-1 cell line.
Both cell transfection and transgenic mice were used to analyze a
series of m
-promoter deletion constructs in conjunction with the
859-bp enhancer for expression levels and cell specificity. Previous
transfection studies in
TSH cells showed no difference in expression
of -5-kb m
-ßgal and -0.48-kb m
-ßgal constructs. However,
the 859-bp enhancer region functioned as an enhancer when juxtaposed to
the 480-bp m
promoter sequences in both
TSH and
T3 cells.
While enhancer elements are defined as position independent, reduction
in enhancer strength has been observed as the distance from the
promoter increases (19). This may explain the difference in the -5-kb
m
-ßgal phenomena and E/-480 constructs. The enhancement observed
in cell transfection was not as great as that observed in transgenic
mice (2- to 3-fold vs. 10-fold). This could be attributable
to many factors including alterations in the composition of
transcription factors in cell lines relative to normal pituitary cells
and the influence of higher order chromatin structure and developmental
history on expression. More dramatic enhancement in transgenic mice
than cell transfection has been observed previously. In spite of these
potential problems, the results obtained in cell culture and transgenic
mice are generally consistent.
The ability of the upstream 859-bp sequence to function in a
cell-specific fashion is critically dependent on proximal elements
within the m
-subunit promoter. Both transgenic mouse analysis and
cell transfection studies demonstrated that the enhancer is most
effective in conjunction with a -341 bp fragment. In contrast, when
fused to the -297 m
-promoter, the enhancer exhibited low expression
levels in both cell transfection and transgenic studies. The fact that
the enhancer was not as effective on the -480- and -381-bp fragments
was an unexpected finding. The low level expression of the E/-480 and
E/-381 constructs is not likely to be due to a repressor element
located between -480 and -341 because the -480, -381, and -341
transgenics all have a similar low level of transgene activity (Fig. 2
). Although it is unlikely, an insulator sequence may have been
created at the junction of both the E/-381 and E/-480 constructs that
is not present in the -4.6 or E/-341 transgene. Insulator sequences
may function by blocking communication between an enhancer and more
proximal promoter sequences (20). We cannot rule out the possibility
that a suppressive element is not functional in the context of the
-4.6-kb fragment or that another positive stimulatory element
overrides its influence. The region between -480 and -341 contains
interaction sites for thyrotrope protein(s), several basal gonadotrope
and GnRH-responsive elements, a TRH- responsive element, and
established binding sites for the transcription factors P-OTX/Ptx-1 and
Msx1 (11, 14, 18, 21, 22, 23, 24, 25). We favor the possibility that the spacing
necessary for interaction between these elements and the enhancer is
impaired. The lack of optimal function in the E/-480 and E/-381
constructs could be attributable to unfavorable stereospecific
protein-protein contacts.
In addition to testing regions important for the level of expression in
the anterior pituitary, deletion constructs provided important
information on the role of cis-acting elements on cell
specificity. Immunohistochemical data showed that the E/-341, E/-381,
and -381 transgenics retain restriction of expression to pituitary
gonadotropes and thyrotropes. This is in contrast with the loss of
thyrotrope expression in transgenic mice generated to study portions of
the human and bovine
-subunit genes (5, 6). Species divergence also
exists in the expression of the
-subunit in the placenta. Human and
equine
-subunit genes contain a region, approximately 180 bp from
the initiation start site, with two distinct cis-acting
regulatory elements, a cAMP-responsive element and a
trophoblast-specific element, that confer expression in the placenta
(26, 27). Although transgene expression was detected in the
gonadotropes and thyrotropes in -341 transgenics, a loss of
restriction of specificity resulted in additional transgene detection
in corticotropes, somatotropes, and lactotropes. This suggests that the
sequences between -381 and -341 may contain elements that limit
expression in other pituitary cell types. These data also show that the
enhancer is able to rescue the restriction of cell specificity of the
-341 m
-promoter. The anterior pituitary of E/-297 transgenics
displayed a loss of restriction of specificity, and ectopic transgene
expression was observed in a variety of peripheral tissues. Expression
of the E/-297 construct in nonpituitary cell types was confirmed by
cell transfection data demonstrating E/-341 m
-promoter activity
only in
TSH and
T3 cells while E/-297 m
-promoter activity
occurred in CV-1 cells in addition to the pituitary cell lines. Thus,
the enhancer cannot overcome the loss of tissue specificity acquired by
deletion of the region between -341 to -297.
Transfection data in cultured cells have suggested that the LIM
homeodomain factor-binding site is important for basal levels of
expression in transfected gonadotropes and thyrotropes and, when
deleted, results in a loss of cell-specific enhancer function (Fig. 6
).
We have shown that it may also play a role in vivo in the
restriction of expression to these two cell types (Tables 1
and 2
). The
LIM site at -342 to -329 interacts with closely related homeodomain
factors such as Lhx2 and P-lim (Lhx3) (12, 28, 29). In addition, an
adjacent element has been located in the human
-subunit gene,
BE1, important for high level gonadotrope expression (13). Mutations
in this region at positions -310 to -303 of the mouse gene show a
30% drop in promoter activity in
TSH cells and had no effect in
T3 cells (11). Considering the importance of these factors suggested
by transfection data, one or more may assist in restricting expression
of the
-subunit to the gonadotropes and thyrotropes.
The 859-bp enhancer region facilitates restriction of expression to
gonadotropes and thyrotropes as well as serving as an enhancer. These
multiple functions may be exerted by distinct elements within the
859-bp enhancer region. Sequences similar to those present between
-480 and -341 of the m
-promoter can also be found within the
859-bp enhancer (GenBank accession number AF044976). These include
consensus sites for interaction with thyrotrope and gonadotrope
proteins, SF1, GATA2/3, GnRE, and Ptx1. If these putative elements are
functional, the redundancy could explain the appropriate gonadotrope
and thyrotrope expression in the E/-341 transgenics and the loss of
restriction in the -341 transgenics. Sequences corresponding to the
consensus binding sites for LIM homeodomain proteins and
BE1,
located between -341 and -297 in the m
-promoter, are not evident
within the enhancer. Removal of binding sites for these factors in the
promoter could explain the loss of restriction of expression to the
appropriate anterior pituitary cell types in the E/-297 transgenics.
Therefore, with the exception of the LIM homeodomain proteins and
BE1, it is possible that similar factors interacting with both the
enhancer and proximal promoter sequences contribute to cell
specificity.
Our studies suggest that thyrotrope as well as gonadotrope specificity
of the m
-subunit is a result of interactions between multiple
elements within the promoter and enhancer. A trend toward reduced basal
levels of promoter activity in
TSH cells and
T3 cells (Fig. 6
, A
and C) was seen with the m
-promoter deletion constructs. In both
cell types, the enhancer worked optimally with the -341 promoter (Fig. 6
, B and D). It did not function when fused to the -297 m
-promoter
fragment despite the intact SF-1 site. The importance of SF-1 for
gonadotrope cell specification and gonadotrope transcription is
controversial (30, 31, 32). The significance of SF-1 in gonadotrope
expression was not addressed in these studies; however, we have
evidence that the Lhx2 and
BE1 binding sites in the promoter
are not required for expression in the anterior pituitary. In the
context of the enhancer, one or both of these sites are necessary for
cell-specific restriction of expression.
The loss of pituitary cell-type restriction evident in the m
-subunit
gene is typical of that observed with other pituitary-specific genes
such as PRL, GH, and POMC (33, 34, 35). In each case, constructs tested in
transgenic mice were selected based on regions determined in
transfected cells to be important for specific expression. The PRL, GH,
and POMC transgenics all exhibited position effects. The requirement
for synergistic interactions between multiple elements to achieve
appropriate cell-specific expression was demonstrated in each case.
Our experiments demonstrate the involvement of multiple DNA sequence
elements in high-level expression and cell specificity. No regions
important exclusively for thyrotrope or gonadotrope expression were
identified. Our results also implicate sequences in the
-subunit
promoter (-341 to -297) for organ-specific and cell-specific
expression. These sequences bind one or more LIM homeodomain factors
(12). This is intriguing because two LIM homeodomain genes,
Lhx3 and Lhx4, have been demonstrated to play
important roles in pituitary organogenesis (36). It will be intriguing
to dissect the roles of individual LIM homeodomain genes on activation
and regulation of
GSU transcription.
 |
MATERIALS AND METHODS
|
---|
Generation of the Recombinant Plasmids
The -3.7 kb m
-ßgal transgene was generated by digesting
the 5.0 m
-ßgal plasmid (7) with BglII. The -3.1
m
-ßgal transgene was generated from the same plasmid by digesting
with SpeI and HindIII. Four different fragments
from the mouse
-subunit promoter (-480/+43, -381/+43, -341/+43,
and -297/+43 bp) were isolated by digesting the appropriate fragments
within the pA3luc plasmid (37) with BamHI and
HindIII. These fragments were subcloned into pSELECT1
(pALTER-1, Promega Corporation, Madison, WI) utilizing the
BamHI and HindIII sites. An 859-bp fragment,
-4.6/-3.7 kb, of the mouse
-subunit promoter was isolated by
digesting a -5 kb/+43 m
promoter fragment in pGEM7Zf+ (7) with
KpnI and BglII. This fragment was fused at the
5'-end of the four different mouse
-subunit promoter fragments by
subcloning between the KpnI-BamHI sites of
pSELECT1. The fragment containing the 859-bp region fused to the pieces
of the mouse
-subunit promoter was isolated by cleaving with
EcoRI and HindIII for the transgenic constructs.
The ends were blunted using HMV RT (Promega Corporation), and the
fragments were cloned into the SmaI site of the pnlacF
reporter plasmid (kindly provided by Jacques Peschon and Richard
Palmiter). The transgenes were isolated from plasmid sequences by
digestion with the appropriate restriction enzymes, separated by
agarose gel electrophoresis, and purified for microinjection with the
NucleoSpin Extract Kit (The Nest Group, Inc., South Borough, MA). In a
similar manner, m
-promoter constructs with or without the 859-bp
enhancer sequence were isolated from the appropriate pSELECT1 vector by
digestion with SmaI and HindIII and directionally
reinserted into the pA3luc vector. Plasmid preparations were purified
from bacterial lysates by double CsCl gradient centrifugation. At least
two independent plasmids from each construct were tested.
Generation of Transgenic Mice
The purified inserts were microinjected into F2
hybrid zygotes from (C57BL/6J x SJL/J) F1 parents at
a total concentration of approximately 23 ng/ml. After overnight
incubation, embryos at the two-cell stage were transferred to day 0.5
postcoitum pseudopregnant CD-1 females. (C57BL/6J x SJL/J)
F1 mice were obtained from The Jackson Laboratory (Bar
Harbor, ME), and CD-1 mice were obtained from Charles River (Wilmingon,
MA). All procedures using mice were approved by the University of
Michigan Committee on Use and Care of Animals. All experiments were
conducted in accord with the principles and procedures outlined in the
NIH Guidelines for the Care and Use of Experimental Animals.
Identification of Transgenic Mice
Genomic DNA was prepared from tail biopsies (38) and pituitary
sections and screened for the presence of the transgene by PCR. A
364-bp fragment corresponding to the nucleotides 15379 of the
ß-galactosidase gene was amplified using a 30-bp sense oligo (5'-TTC
ACT GGC CGT CGT TTT ACA ACG TCG TGA-3') and a 30-bp antisense oligo
(5'-ATG TGA GCT AGT AAC AAC CCG TCG GAT TCT-3'). The PCR reactions were
performed under standard conditions using 100200 ng genomic DNA, 0.5
pmol/ml primers, 2.5 mM MgCl2, and 1.7 U
Taq DNA polymerase per reaction. Reactions were carried out
for 30 cycles of denaturation at 94 C for 1 min, annealing at 65 C for
30 sec, extension at 72 C for 30 sec, and a final extension at 72 C for
10 min. The identity of the mouse
-subunit 5'-deletion constructs
was confirmed for each transgenic animal using PCR. Two 20-bp oligos
[forward (5'-CTC ATT TTT TAA GGC ACT GC-3'); and reverse (5'-GAT CAT
ATC ACA TTG CAA CC-3') were used to amplify 430-, 331-, 291-, and
247-bp fragments corresponding to E/-480, E/-381, E/-341, and
E/-297, respectively. The reactions were carried out for 30 cycles of
denaturation at 92 C for 1 min, annealing at 60 C for 1 min, extension
at 70 C for 1 min, and a final extension at 72 C for 10 min. The
identity of the PCR product was further confirmed by digesting with
RsaI to yield fragments of 242 and 188 bp in the E/-480 PCR
product and fragments of 242 and 89 bp in the E/-381 PCR product. In
addition, the identity of the PCR products from all of the constructs
was verified by sequence analysis.
Quantitation of ß-Galactosidase Activity with Fluorimeter and
Luminometer Assays
One lobe of the anterior pituitary was excised using a
scalpel and placed in 250 µl of a lysis buffer containing 100
mM potassium phosphate (pH 7.8) and 0.2% Triton X-100. The
samples were freeze-thawed by incubation on dry ice and 37 C
alternately for three cycles and then sonicated 2 x 20 sec on ice
(550 Sonic Dismembrator, Fisher Scientific, Pittsburgh, PA). The cell
lysates were centrifuged at 12,000 x g for 3 min, and
the supernatant was removed to new tubes. Transgene expression was
quantitated in tissue extracts from -3.7 and -3.1 m
-ßgal
transgenics using a fluorometric assay for ß-galactosidase (7). A
chemiluminescent reporter assay for ß-galactosidase was used to
quantitate transgene expression in tissue extracts from -381, -341,
E/-480, E/-381, E/-341, and E/-297 transgenics. ß-Galactosidase
activity was detected using the Galacto-Light (Tropix, Bedford, MA)
chemiluminescent reporter assay system. Two microliters of the lysate
were incubated for 1 h in a reaction buffer containing Galacton, a
chemiluminescent substrate cleaved by ßgal. The samples were placed
in a luminometer chamber (Auto Lumat LB 953, EG&G Wallac, Gaithersburg,
MD), and 100 µl of a light emission accelerator were added to the
samples. After a 5-sec delay, light production was measured over a
10-sec interval. The level of ßgal activity in each sample was
compared with a standard curve of 0.15, 0.38, 0.96, 2.4, 3.8, 4.8, 6,
and 8 U x 106 ßgal (Sigma, St. Louis, MO).
Reactions were performed in triplicate, luminometer readings were
averaged, and nontransgenic background levels were subtracted.
ß-Galactosidase activity was normalized for the amount of protein in
each homogenate. The Bradford protein assay (39) was performed with the
Coomassie brilliant blue dye reagent on 20 µl of homogenate in
microtiter plates as described (Bio-Rad Laboratories, Hercules,
CA). Absorbance of the samples and the BSA protein standard was
measured at 630 nm using an EL311 sx Auto Reader (Bio-Tek Instruments,
Wihooski, VT).
Histology and Immunohistochemistry on Adult Pituitary and
Peripheral Tissues
Peripheral tissues were removed and frozen immediately in OCT
embedding media (Miles Scientific, Elkhart, IN) on dry ice. Sections
were cut (12 µM) on a cryostat (Bright Instruments
Company, Huntingdon, U.K.) and mounted on
poly-L-lysine-coated slides. Sections were fixed for 5 min
in 0.2% glutaraldehyde, containing 0.1 M
NaH2PO4 (pH 7.3), 5 mM EGTA, and 3
mM MgCl2, washed three times in wash buffer
[0.1 M NaH2PO4 (pH 7.3), 2
mM MgCl2, 0.02% NP-40], and incubated at 37 C
overnight in an Xgal solution containing 1 mg/ml Xgal (Boehringer
Mannheim, Indianapolis, IN), 5 mM
K3Fe(CN)6, and 5 mM
K4Fe(CN)6·3 H2O dissolved in wash
buffer (above). The counterstain was 0.5% neutral red (Sigma).
The cell specificity of transgene expression was evaluated by an Xgal
enzymatic assay followed by immunohistocytochemistry for individual
pituitary hormones. Pituitaries were fixed for 1 h in 4%
paraformaldehyde in pH 7.2 sodium phosphate buffer and incubated at
room temperature overnight in Xgal solution as above, except that Xgal
was reduced to 0.1 mg/ml for pituitaries from the -4.6 m
-ßgal
transgenic lines and the highest expressing E/-341 m
-ßgal
transgenics. After 36 h postfixation in buffered formaldehyde, the
samples were embedded in paraffin and 34 µM sections
were prepared. Immunostaining was performed as previously described
(7).
Cell Culture, Transient Transfection, and Reporter Gene Assay
Transient transfections using calcium phosphate into CV-1 monkey
kidney cells were performed as described previously using 10 µg
reporter plasmid (40). Approximately 750,000 cells were added per 100
mm x 20 mm tissue culture dish 20 h before transfection.
TSH (3 x 106 cells) and
T3 cells (4 x
106 cells) were transfected by electroporation as described
(40). The pituitary cells were transfected with 20 µg of the
indicated luciferase construct and also contained 2 µg of pCMV-ßgal
DNA as an internal transfection control. Each set of transfections was
done in triplicate and also contained a Rous sarcoma virus
promoter-luciferase plasmid transfected in parallel as a positive
control and a promoterless pA3luc vector as a background
control. After 2448 h, luciferase activity was measured in a
Monolight 2010 luminometer from duplicate aliquots of freeze-thaw
cytoplasmic lysates (41) from the cells while ß-galactosidase
activity was measured using a colorimetric assay (42) and were compared
with a standard curve of enzymatic activity. Light units were
normalized to the ß-galactosidase activity.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Mark Berard, Dwayne Petry, and Maggie Van
Keuren from the University of Michigan Transgenic Animal Core for their
work in the generation of transgenic mice. We would like to thank the
University of Michigan Morphology Core, especially Kaye Brabec, for
their assistance in paraffin embedding and sectioning, Amy Young for
her help in typing mice, and Juanita Merchant, Audrey Seasholtz, and
Jeff Chamberlain for the use of their equipment. We acknowledge the
National Hormone and Pituitary Program, the National Institute of
Diabetes and Digestive and Kidney Diseases, the National Institute of
Child Health and Human Development, and the US Department of
Agriculture for providing the PRL, GH, LHß, and TSHß antibodies.
Work done in the laboratory of Dr. Sally Camper is supported by NIH
Grant RO1-HD34283-01. The Transgenic Core Facility is supported by the
University of Michigan Centers for Arthritis, Cancer, and
Organogenesis, and National Institutes of Health/National Science
Foundation grants. Support for the Morphology Core is provided by Grant
P30-HD-18258. Additional support was provided by NIH Grant RO1-DK47407
and a generous gift from the Markey Foundation to E. C. Ridgway.
Cultured cells were grown by the Tissue Culture Core Laboratory at the
University of Colorado Health Sciences Center supported by NIH Grant
CA-46934.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Sally A. Camper, Division of Molecular Medicine and Genetics, University of Michigan Medical School, 4301 MSRB III, Ann Arbor, Michigan 48109-0638.
Received for publication October 31, 1997.
Revision received December 22, 1997.
Accepted for publication January 21, 1998.
 |
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