Multiple Promoter Elements Contribute to Activity of the Follicle-Stimulating Hormone Receptor (FSHR) Gene in Testicular Sertoli Cells
Leslie L. Heckert,
Melissa A. F. Daggett and
Jiangkai Chen
Department of Molecular and Integrative Physiology The
University of Kansas Medical Center Kansas City, Kansas 66160
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ABSTRACT
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The FSH receptor (FSHR) is expressed only in
granulosa cells of the ovary and Sertoli cells of the testis. This
highly specific pattern of gene expression asserts that transcriptional
events unique to these two cell types are responsible for activation of
the FSHR gene. We have characterized the promoter elements required for
activity of the rat FSHR gene in a Sertoli cell line MSC-1, primary
cultures of rat Sertoli cells, and two non-Sertoli cell lines.
Transient transfection analysis of deletion and block replacement
mutants identified several elements, both 5' and 3' to the
transcriptional start sites, that are essential for full promoter
activity in Sertoli cells. These studies confirmed the use of an
important E box element (CACGTG), which had the single greatest impact
on promoter function. Bases within the core CACGTG of the E box, as
well as flanking sequences, were shown to be essential for its
function. Electrophoretic mobility shift assays identified both
upstream stimulatory factor 1 (USF1) and USF2 as primary
components of the complexes binding the E box. Sequence requirements
for USF binding in vitro modestly diverged from the
sequence requirements for in vivo function of the element.
Comparison of the E box binding proteins in different cell types
revealed that similar proteins bind the E box in Sertoli and
non-Sertoli cell lines. Extracts from primary cultures of rat and mouse
Sertoli cells have a second E box-binding complex that cross-reacts
with USF antibodies that is not present in the cell lines.
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INTRODUCTION
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Sertoli cells are the major somatic cells of the seminiferous
epithelium, the site of germ cell development in the testis (1, 2, 3, 4).
These cells play important roles in both testicular development and
function, as they provide an environment that protects and nurtures the
germs cells, assisting their development into viable sperm (1, 3).
Functions attributed to Sertoli cells include formation of a support
system to house the developing germ cells, endocytosis of eliminated
waste products of the developing sperm, formation of the blood-testis
barrier, assistance in spermiation, delivery of nutrients to germinal
cells, and secretion of proteins, ions, and other substances proposed
to be important for germ cell development and viability (1, 3). Thus,
the cellular relationship between germ cells and Sertoli cells is
critical to testicular performance and fertility. Sertoli cell function
is influenced by a host of regulatory signals, most notably the
pituitary hormone FSH. In prenatal and immature animals, FSH is
important for stimulating the proliferation of Sertoli cells, final
maturation events such as formation of tight junctional complexes,
stimulation of the synthesis of specific Sertoli cell proteins, and the
generation of the first wave of spermatogenesis (5, 6, 7). The presence of
the FSH receptor (FSHR) on Sertoli cells and its regulation is critical
for the hormonal response of the testis to FSH.
Although Sertoli cells are recognized for their importance in testis
development and function, the events that promote or determine Sertoli
cell development and differentiation are poorly understood. Because the
FSHR is expressed only in testicular Sertoli cells, examination of its
gene and the mechanism that activates it provides an opportunity to
identify proteins that will likely be responsible for a host of
transcriptional events critical to Sertoli cell function. Previous
studies have shown that 5000 bp of 5'-flanking sequence of the rat FSHR
gene are sufficient to restrict expression of a reporter gene to the
gonads of transgenic mice (8). This suggests that within this region of
the gene resides the information necessary for Sertoli cell-specific
expression.
The structure of the FSHR gene was first characterized in the rat and
later in the human (9, 10). This single-copy gene is organized into 10
exons and 9 introns and spans more than 50 kbp of DNA (9, 10). RNAse
protection assays and primer extension have mapped the transcriptional
start site in the human to position -99 relative to the start of
translation, while studies on the rat gene identified two start sites
at positions -80 and -98 relative to the translational start codon
(9, 11). Inspection of the 5'-flanking sequence of the gene revealed a
notable lack of canonical TATA or CCAAT promoter elements (9, 10). In
many promoters that lack a TATA motif, an initiator element (Inr) is
critical in positioning RNA polymerase II (12, 13). Several Inr
elements have been described and classified according to sequence
homology and are generally found to span the transcriptional start
sites (12, 13, 14). The Inr directs accurate basal transcription and is
important for core promoter activity in some TATA-containing as well as
TATA-less genes (12, 14, 15). Frequently, these elements work in
conjunction with upstream elements to help position the polymerase.
These elements are often rich in cytosines and guanines (GC rich) and
bind the transcription factor SP1 (12). In the case of the FSHR gene,
however, the promoter lacks such a GC-rich region (9).
Determination of the transcriptional mechanisms that regulate FSHR
expression in the gonads will provide important insight into both
cell-specific transcriptional events that are important for gonadal
function and mechanisms that control the response of the gonads to FSH
through modulation of receptor levels. Presently, little is known about
transcriptional control of the FSHR gene, but deletion analysis of the
rat FSHR promoter identified a region between -220 and +98, relative
to the first transcriptional start site, to be important for full
promoter activity (16). Similarly, in the human promoter, a region
between -126 and +98 gave full promoter response (11).
To date, only a single response element has clearly been identified
(16). As described in a paper by Goetz et al., an E box is
required for full promoter function of the rat FSHR gene (16). The
promoters of the rat, human, sheep, and mouse FSHR genes all contain an
E box consensus sequence, CANNTG, which is known to bind members of the
basic helix-loop-helix (bHLH) family of proteins that consist mostly of
transcriptional regulators involved in control of growth and
differentiation (11, 17, 18, 19, 20, 21, 22, 23). Mutation of the rat FSHR E box
(GTCACGTGAC to TATGAACTCT) reduced promoter activity
approximately 50% in Sertoli cells (22). In vitro binding
analysis revealed that complexes binding the rat FSHR E box
cross-reacted with antibodies generated against the bHLH protein,
upstream stimulatory factor 1 (USF1) (16, 24). USF1 is a member
of a second group of bHLH proteins that have an additional dimerization
domain, the leucine zipper (25). USF1 is expressed ubiquitously and
binds its target DNA as a dimer, where it forms both homodimers and
heterodimers with the closely related protein USF2 (26, 27). Although
USF is expressed in numerous cells and tissues, it is possible that it
may help determine specificity of the receptor by dimerization with a
unique protein partner or through acquisition of a cell-specific
modification. Currently, there are no data that confirm USF1 regulation
of the FSHR gene in vivo.
To extend our understanding of FSHR gene regulation, we have made a
series of block replacement mutations that span the proximal promoter
region of the rat FSHR gene and evaluated their effects on promoter
function. These studies verify the importance of the E box and identify
several additional promoter regions that are required for full
transcriptional activity. To further elucidate the role of the E
box-binding proteins in promoter function, a series of E box mutants
were characterized for their effect on promoter function in
vivo and protein binding in vitro.
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RESULTS
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-100 to +123 bp of the Rat FSHR Gene Yields Highest Promoter
Activity
To determine the minimal portion of the rat FSHR gene required for
full promoter activity, we created a series of constructs containing
different amounts of 5'-flanking sequence. These promoter fragments
were used to drive expression of a luciferase reporter gene and
characterized by transient transfection analysis in the Sertoli cell
line MSC-1 (28). Promoter activity increased when the bases between
-3600 bp and -2700 bp, relative to the first transcriptional start
site, were removed (Fig. 1
). Promoters
containing 2700, 1300, 763, and 220 bp of 5'-flanking sequence were
similar in activity, while removal of the sequences between -220 and
-100 resulted in a second increase in promoter activity. These studies
indicate that, in MSC-1 cells, promoter function is regulated by at
least two negative elements located between -3600 and -2700 and -220
and -100 and that most of the positive elements needed for full
promoter activity are located between -100 and +123 bp of the proximal
promoter region.

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Figure 1. The -100 to +123 bp Promoter Construct Exhibits
the Highest Activity in MSC-1 Cells
Promoter constructs containing 123 bp downstream of the first
transcriptional start site and different amounts of 5'-flanking
sequence of the rat FSHR gene were used to drive expression of the
luciferase reporter gene. One microgram of each promoter construct was
cotransfected with 0.2 µg RSV-ß-galactosidase into MSC-1 cells as
described in Materials and Methods. Luciferase activity
of each was determined and normalized to ß-galactosidase activity to
control for transfection efficiency. The data represent the
luciferase/ß-galactosidase activity of each promoter construct
normalized to the luciferase/ß-galactosidase activity of the
promoter-less control pGL3-basic. Transfections were done a minimum of
three times. Error bars represent the SEM.
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An E Box and Several Weaker Elements Are Required for Promoter
Function in MSC-1 Cells
To identify the elements required for activity of the minimal
promoter region, we made a series of 14 block replacement mutations
that span the region from -70 to +123 and placed them in the context
of the -220/+123 bp promoter (Fig. 2
).
Transient transfection analysis in MSC-1 cells revealed several levels
of regulation by regions of the FSHR promoter (Fig. 3
). The most profound effect was observed
for the mutation through region 9.2 (µ9.2, Fig. 3
). This mutation
disrupts the E box located at approximately -23 bp. A more modest
effect was observed with a second upstream mutation disrupting region 6
(µ6, Fig. 3
). Interestingly, regions downstream of the
transcriptional start sites also significantly influenced promoter
activity. These mutations span regions 13.4, 14, and 15 as well as
those further downstream in regions 17, 18, and 19. Minor, but
statistically significant (P < 0.01), effects were
also observed with mutations through regions 7, 10, and 11.

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Figure 2. Sequence of the Rat FSHR Gene from -70 to +123 and
Block-Replacement Mutants
The sequence of the rat FSHR gene is shown in uppercase letterson the top strand. The sequence is numbered according to the
first transcriptional start site, indicated by the arrow
at +1 in the figure. The second transcriptional start site is indicated
by the arrow at +19. The sequence of each promoter
mutation, numbered sequentially from µ6 to µ19, is indicated in
lowercase letters below that of the replaced wild-type
sequence.
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Figure 3. Multiple Elements Contribute to FSHR Promoter
Activity in MSC-1 Cells
Mutations indicated in Fig. 2 were inserted into the -220/+123 bp
promoter of the rat FSHR gene and used to drive expression of the
luciferase reporter gene. The promoter constructs (1.0 µg) were
cotransfected with RSV-ß-galactosidase (0.2 µg) into MSC-1 cells.
The luciferase activity of each construct was determined and normalized
to ß-galactosidase activity to control for transfection efficiency.
The data represent the luciferase/ß-galactosidase activity of each
mutant normalized to the luciferase/ß-galactosidase activity of the
wild-type construct. Corresponding mutations are indicated belowthe bars. At the top, is a schematic
diagram showing the relative positions of each mutation.
Transfections were done a minimum of three times and include data from
at least two different preparations of DNA. Error bars
represent SEM. Asterisks indicate values
that are statistically different from the wild-type control
(P < 0.01) as determined by Students
t test.
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To confirm the use of the downstream regions, promoter constructs
were created that included different amounts of the 3'-sequence.
Transient transfection of these 3'-deletion constructs into MSC-1 cells
confirmed the importance of this region in regulation of the FSHR gene
(Fig. 4
). Further inspection of these
results suggests that mutations in regions 18 and 19 do not contribute
significantly to promoter activity, as removal of sequence between +123
and +98 had no effect on promoter activity. Removal of sequences
containing region 17 (+98 to +79) diminished promoter activity
slightly, suggesting this region plays a minor role in promoter
function. However, further deletion to +45 significantly diminished
promoter activity, supporting the previous observation that region 15
is important for activity. Additional decreases in promoter activity
were seen as the promoter was further deleted to +21 and then again to
+8. It is interesting to note that the combined effect of the mutations
in the 3'-end dramatically reduced promoter activity to an extent
nearly equal that of the E box mutation, identifying this region of the
gene as a critical component of FSHR regulation.

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Figure 4. Elements 3' to the Transcriptional Start Sites Are
Essential for Promoter Function
FSHR promoter constructs containing 220 bp of 5'-flanking sequence and
different amounts of sequence 3' to the transcriptional start sites
were used to drive expression of the luciferase reporter gene. One
microgram of each promoter construct was cotransfected with 0.2 µg
RSV-ß-galactosidase into MSC-1 cells. Luciferase activity was
normalized to ß-galactosidase activity to control for transfection
efficiency. The luciferase/ß-galactosidase activity of each promoter
construct was normalized to the luciferase/ß-galactosidase activity
of the -220/+123 FSHR promoter construct. Transfections were done a
minimum of three times. Error bars represent the
SEM. An asterisk indicates that the values
are statistically different from those of the next largest promoter
construct (P < 0.01), as determined by Students
t test.
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A Guanine Cytosine Pair Is Important for Region 15 Activity
Since region 15 was identified as an important 3'-element in the
FSHR promoter, we further characterized this site by determining its
functional sequence requirements in MSC-1 cells. Transient transfection
analysis of additional region 15 mutants identified a guanine and
cytosine (µ15.4) that, when mutated, diminished promoter activity to
the same extent as the full block replacement mutant µ15 (Fig. 5
). This confirmed the importance of
region 15 and identified the guanine and cytosine at the 3'-end of the
site as critical for the elements associated activity.

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Figure 5. A Guanine and Cytosine Are Important for the
Activity in Region 15
Four smaller region 15 mutations (µ15.1, µ15.2, µ15.4, µ15.5)
were introduced into the FSHR (-220/+79) luciferase promoter construct
and cotransfected with RSV-ß-galactosidase into MSC-1 cells as
described in Fig. 1 . The luciferase and ß-galactosidase activities
were determined, and the activity of each smaller mutant was normalized
to the luciferase/ß-galactosidase activity of the FSHR(-220/+79)
wild-type construct (wt2). The original µ15 construct (see Fig. 2 for
the sequence) was also examined, and its activity was normalized to the
FSRH(-220/+123) wild-type construct (wt1). Error bars
represent the SEM. An asterisk indicates
that the values are statistically different from that of the wild-type
control (P < 0.01) as determined by Students
t test.
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Promoter Function Differs in Sertoli Cells and Ectopic Cell
Lines
To help identify potential elements that may provide specificity
to the FSHR promoter and to compare promoter activity in MSC-1 cells
and primary Sertoli cells, we characterized the deletion and block
replacement mutants in two nongonadal cell types and primary cultures
of rat Sertoli cells. Examination of the 5'-deletion mutants in BeWo
cells, a human choriocarcinoma cell line, revealed that the promoter is
nearly silent in these cells (2x greater than the base vector pGL3,
Fig. 6
). However, activity of the
promoter was somewhat higher in
T3 cells, a mouse gonadotrope cell
line, with the -100/+123 construct almost 5-fold greater than pGL3
(Fig. 6
). In primary cultures of rat Sertoli cells, promoter activity
was considerably greater, reaching more than 15-fold that of the base
vector. Comparison between Sertoli cells and the two non-Sertoli cell
lines (BeWo and
T3) revealed that, while promoter activity in
Sertoli cells increased significantly when subsequently larger portions
of the 5'- region were removed, activity of the promoter remained low
in the ectopic cell lines.

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Figure 6. The Activity of the FSHR Promoter Is Higher in
Primary Cultures of Sertoli Cells Than in Ectopic Cell Lines
Each of the indicated promoter constructs was cotransfected with
RSV-ß-galactosidase into either primary Sertoli cell cultures or two
nonexpressing cell lines, T3 and BeWo, as described in
Materials and Methods. Luciferase activity of each was
determined and normalized to ß-galactosidase activity to control for
transfection efficiency. The data represent the
luciferase/ß-galactosidase activity of each promoter construct
normalized to the luciferase/ß-galactosidase activity of the
promoter-less control pGL3-basic. Transfections were done a minimum of
three times. Error bars represent the SEM.
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The block replacement mutants were examined in the ectopic cell
lines to help identify elements that are unique to Sertoli cells and to
reveal possible repressor elements that might be involved in silencing
the FSHR gene in nonexpressing cells. In BeWo cells, mutations in
regions 7, 11, 13, 14, 15, and 18 had modest, but statistically
significant (P < 0.01), effects on promoter activity
(Fig. 7A
). Importantly, no
increase in promoter activity was detected for any of the mutants,
ruling out the involvement of a repressor in silencing promoter
activity in these cells. Similar to the results in MSC-1 cells,
mutations in upstream regions 6 and 9.2 significantly diminished
promoter activity in
T3 cells, while mutations through regions 10
and 11 had a more minor impact (Fig. 7B
).
However, in contrast to expression in MSC-1 cells, there was also a
significant effect observed with a mutation through region 8 in the
T3 cells. In the 3'-end of the promoter, regions 13 and 14
predominated, while a mutation through region 15 had little impact.
Similar to BeWo cells, no elements involved in silencing the gene were
revealed in
T3 cells.

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Figure 7. Promoter Function in Ectopic Cell Lines
Mutations indicated in Fig. 2 were inserted into the -220/+123 bp
promoter of the rat FSHR gene and used to drive expression of the
luciferase reporter gene. The promoter constructs (1.0 µg) were
cotransfected with RSV-ß-galactosidase (0.2 µg) into BeWo cells (A)
or T3 cells (B) as described in Materials and
Methods. The luciferase activity of each was determined and
normalized to the ß-galactosidase activity. The data represent the
luciferase/ß-galactosidase activity of each mutant normalized to the
luciferase/ß-galactosidase activity of the wild-type
FSHR(-220/+123)Luc construct. Corresponding mutations are indicated
below the bars. Transfections were done a minimum of
three times. Error bars represent SEM.
Asterisks indicate values that are statistically
different from the wild-type control (P < 0.01) as
determined by Students t test.
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Since MSC-1 cells are a transformed Sertoli cell line, we sought to
validate their use by comparing FSHR promoter function in these cells
to that in primary cultures of rat Sertoli cells. Transient
transfection analysis of the 3'-deletion mutants in primary Sertoli
cells produced similar results to those observed in MSC-1 cells
(compare Figs. 4
and 8A
). However, minor differences were seen when the
sequences between +123 and +98 were removed. In MSC-1 cells, this had
no significant effect on promoter activity, whereas in primary Sertoli
cells, activity increased. Importantly, in both cell types, subsequent
deletions to +45, +21, and +8 resulted in sequential decreases in
promoter activity (Figs. 4
and 8A
). Analysis of the block replacement
mutants (µ6µ16) in Sertoli cells stressed the importance of
upstream regions 6, 7, and 9.2, regions flanking the start sites (10, 11, 12) and regions downstream of the start sites (13.4, 14, 15,
and 16; Fig. 8B
). Although these results are similar to those observed
in MSC-1 cells (Fig. 3
), several minor differences were noted. Thus,
mutations in regions 7, 12, 13.4, and 16 all had slightly greater
impact on promoter activity in primary cells than in MSC-1 cells. In
addition, a mutation through region 8 resulted in an increase in
promoter activity that was not observed in MSC-1 cells. Together, the
block replacement and deletion studies reveal that in Sertoli cells the
E box has the greatest single influence on promoter function and that,
together with an element in region 6 and 7, is important for the
activity associated with the 5'-region of the promoter. In addition,
FSHR promoter function in Sertoli cells requires several elements 3' to
the transcriptional start sites that together exert a significant
regulatory influence on promoter activity nearly equal to that of the E
box.
Bases within the Core CACGTG of the E Box, as Well as Flanking
Sequences, Are Essential for Function of the Element
The large impact on promoter activity observed with the E box
mutation underscores its importance in FSHR gene regulation. Therefore,
determination of the sequences required for its function is critical to
the evaluation of the promoter and the proteins that bind the element.
Five different mutations were made in the region of the E box and
placed into the context of the -220/+123 bp promoter. Functional
evaluation of these E box mutants showed considerable similarity in
MSC-1 and primary Sertoli cells (Fig. 9
).
Replacement of each base in the E box core, 5'-CACGTG-3', resulted in
significant loss of promoter function (Fig. 9
, µ9.2). Interestingly,
mutation of two bases on each side of the E box core sequence (µ9.4)
also dramatically reduced promoter activity, demonstrating that the
flanking sequences as well as the core of the E box are critical for
transcription factor recognition or function. Changing the E box core
sequence from 5'-CACGTG-3' to 5'-CACaTG-3' (µ9.5), the
same sequence that is found in the human promoter, had a small impact
in primary Sertoli cells (P < 0.05) and was not
different from the wild-type sequence in MSC-1 cells. However, further
alteration of the human sequence from 5'-CACaTG-3' to
5'-CAgaTG-3' (µ9.6) significantly reduced promoter
activity, suggesting that a guanine at position 3 in the E box is not
well tolerated. Interestingly, µ9.3, which also has a guanine at
position 3 but a cytosine at position 4, had slightly higher activity
than µ9.6 in both Sertoli cells and MSC-1 cells.

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Figure 9. Bases within the E Box Core as Well as Its Flanking
Sequence Are Required for Full Promoter Activity
Each of the indicated E box mutations were introduced into the FSHR
(-220/+123) luciferase promoter construct and cotransfected with
RSV-ß-galactosidase into either primary cultures of rat Sertoli cells
or MSC-1 cells. The luciferase and ß-galactosidase activities were
determined and the luciferase/ß-galactosidase activity of each mutant
was normalized to the luciferase/ß-galactosidase activity of the
wild-type construct. Transfections were done a minimum of three times.
Error bars represent SEM. Statistical
significance was determined by Students t test using
two-sample unequal variance. Statistically significant differences
(P < 0.01) are indicated for values different from
µ9.2 (a), µ9.3 (b), µ9.4 (c), µ9.5 (d), and µ9.6 (e).
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E Box-Binding Complexes Cross-React with USF1 and USF2
Antibodies
To identify proteins binding the E box in the different cell lines
and primary Sertoli cells, we employed electrophoretic mobility shift
assays (EMSAs). One major shifted complex was observed with a
radiolabeled E box probe and extracts from MSC-1 cells (Fig. 10A
). This complex bound specifically
to the E box, as indicated by the ability of a homologous sequence
[wild type (wt)] but not a nonspecific (NS) sequence to
compete for complex binding. When extracts from primary rat Sertoli
cells were used, two major shifted complexes were observed, both of
which are specific for the E box sequence (Fig. 10A
). Specifically
bound complexes migrating to position 1 (band 1) on the gel were
observed in both MSC-1 and Sertoli cells, while specific complexes
migrating to position 2 (band 2) were only observed in primary rat
Sertoli cells (Fig. 10A
). These complexes cross-reacted with USF1 and
USF2 antibodies in MSC-1 (band 1) and primary rat Sertoli cells (bands
1 and 2). For each extract, the USF1 antibody cross-reacted with a
greater portion of the binding complexes than did the USF2 antibody. No
cross-reactivity was observed with antibodies against two additional
members of the bHLH family, c-Myc and E2A (E47 and E12).

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Figure 10. Both USF1 and USF2 Bind the FSHR E Box in Sertoli
Cells
A, A radiolabeled probe corresponding to the FSHR E box
(5'-TCTTGGTGGGTCACGTGACTTTGCCCGT-3') was used in EMSA with
whole-cell extracts from MSC-1 cells (MSC-1) and primary rat Sertoli
cells (SC). Radiolabeled probe (8.3 fmol) was incubated with 9.5 µg
of whole-cell extract and resolved on a 4% polyacrylamide gel as
described in Materials and Methods. Where indicated,
unlabeled homologous (WT) or nonspecific (NS;
5'-CTAGAGTCGACCTGCAGGCATGCAAGCTTGGCATTC-3') DNAs were added to the
reaction at a concentration equal to 80x that of the probe. Where
indicated above the lanes, antibodies specific for the
bHLH proteins, USF1, USF2, c-Myc, and E2A, were added to the reactions
before the addition of the probe. The major specific complexes are
marked by arrows and a corresponding number. B, EMSAs
were performed as described in panel A using whole-cell extracts from
either primary rat Sertoli cells (rSC) or primary mouse Sertoli cells
(mSC).
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To explore the differences in E box-binding proteins observed between
MSC-1 cells, a mouse Sertoli cell line, and primary rat Sertoli cells,
we evaluated these complexes in extracts from primary mouse Sertoli
cells. In the presence of these extracts, two major shifted complexes
bound specifically to the E box probe and migrated to similar positions
as the complexes formed with rat Sertoli cell extracts (Fig. 10B
). In
addition, mouse Sertoli cell complexes similarly cross-reacted with
antibodies against USF1 and USF2. The presence of an additional E
box-binding complex in primary rat Sertoli cells (band 2) can therefore
be attributed to differences between the cell line and primary cultures
and not to differences between species.
To determine whether similar or different proteins occupy the E box in
ectopic cell lines, EMSA was used to examine the binding complexes in
extracts from BeWo and
T3 cells. In each cell type, one major
shifted complex (band 1 in Fig. 11
)
bound specifically to the E box and migrated to the same position as
the major band formed from MSC-1 cells (Fig. 11
). Similarly, the
proteins from each extract cross-reacted with both USF1 and USF2
antibodies but not with the c-Myc and E2A antibodies. In
T3 cells
(Fig. 11A
), a slower migrating, specifically bound complex just above
the major band was also observed. In BeWo cells (Fig. 11B
), two faster
migrating nonspecific bands were seen. These data indicate that similar
proteins bind the E box in Sertoli and non-Sertoli cell lines.

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Figure 11. USF1 and USF2 Bind the FSHR E Box in T3 and
BeWo Cells
A, A radiolabeled probe corresponding to the FSHR E box
(5'-TCTTGGTGGGTCACGTGACTTTGCCCGT-3') was used in EMSA with
nuclear extracts from either MSC-1 or T3 cells. Radiolabeled probe
(8.3 fmol) was incubated with 9.5 µg of nuclear extract and resolved
on a 4% polyacrylamide gel as described in Materials and
Methods. Where indicated, unlabeled homologous (WT) or
nonspecific (NS; 5'-CTAGAGTCGACCTGCAGGCATGCAAGCTTGGCATTC-3') DNAs were
added to the reaction at a concentration equal to 80x that of the
probe. Where indicated above the lanes, antibodies
specific for the bHLH proteins, USF1, USF2, c-Myc, and E2A, were added
to the reactions before the addition of the probe. The major specific
complex is marked by an arrow and the number 1. B, EMSAs
were done as described in panel A using nuclear extracts from either
MSC-1 or BeWo cells.
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Binding of USF to the E Box Differs Modestly from the Functional
Requirements of the Element in Vivo
To determine whether the in vitro binding affinities of
USF1 and USF2 correlated with the in vivo function of the
promoter, DNA fragments containing each of the mutant E boxes were used
as competitors in an EMSA. The relative affinities of the DNA sequences
for the USF complexes were determined by comparing the ability of each
DNA fragment to compete for complexes binding the E box in Sertoli cell
extracts (Fig. 12
). This revealed the
following relative binding affinities: wt > µ9.5 >
µ9.3 > µ9.6
µ9.4 > µ9.2. Thus, with the
exception of µ9.4, the relative binding affinities correlated well
with the functional requirements of the element (Fig. 9
: wt >
µ9.5 > µ9.3 > µ9.6 > µ9.2
µ9.4;
P < 0.01). The discrepancy with the µ9.4 mutant
suggests that either the USF proteins are not responsible for
activation of the promoter through the E box or that the mutations not
only affect the binding affinities for the USF proteins but their
transactivation ability as well.

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Figure 12. The USF Complexes Have Different Affinities for
the E Box Mutants
EMSAs were performed as described in Fig. 10 using whole-cell extracts
from primary Sertoli cell cultures and a radiolabeled E box probe
(5'-TCTTGGTGGGTCACGTGACTTTGCCCGT-3'). Competitor DNAs were
generated by DNA amplification of the region between -70 and +8 using
two FSHR promoter-specific primers and each of the indicated mutant
promoters as a template. Competitors were added at the fold excess
indicated above the lanes. Results were confirmed using
three different preparations of extracts and two separate sets of
generated competitors. The major specific complexes are marked as they
were in Fig. 10 .
|
|
 |
DISCUSSION
|
---|
Using deletion analysis and block replacement mutagenesis, we have
characterized the FSHR promoter region spanning from approximately 5000
bp 5' to the first transcriptional start site to 123 bp in the 3'
direction. These studies are the first to extensively evaluate the
promoter using a block replacement approach, which identifies important
promoter regions while maintaining promoter context, and to fully
characterize the promoter in eutopic and ectopic cell lines and primary
cultures of Sertoli cells. Comparison of the results in different cell
types revealed several interesting similarities and differences. All
cells examined showed an increase in promoter activity when the bases
between -5000 and -2700 were removed, suggesting either a general
repressor element is present in this region or a structural constraint
in the larger plasmid construct diminishes its transfection into the
cells. In primary Sertoli cells, there was a second increase when the
region between -2700 and -220 was removed that was not observed in
the other cells, implicating the presence of a negative element within
this region that is active only in the primary cultures. Analysis of
the deletion mutants also revealed the presence of a repressor element
between -220 and -100 that was active only in Sertoli cells. Removal
of this region significantly increased promoter activity in MSC-1
cells, and to a lesser extent in primary Sertoli cells, while little or
no change in promoter activity was observed in BeWo or
T3 cells. It
must be noted that transfections were performed using equal microgram
amounts of DNA rather than equal molar amounts that could account for
differences in promoter activity between constructs of substantially
different sizes. However, among the deletion mutants (Fig. 1
), adjacent
constructs did not differ significantly in the molar amounts of DNA
transfected (<15% in most cases). The relatively small differences in
the molar amounts used between adjacent constructs cannot fully account
for the repressive activity observed between either the -5000 and
-2700 promoters or between the -220 and -100 promoters. However, it
may, in part, explain the increase observed in primary Sertoli cells
when the region between -2700 and -220 was removed. In MSC-1 and
primary Sertoli cells, the -100/+123 bp promoter was the most active
construct, and its activity was substantially higher than that observed
in the ectopic cell lines. Thus, the transcriptional events occurring
within this region of the promoter play an important role in gene
activation and exhibit some cell specificity with regard to promoter
function. For these reasons, the -100/+123 bp promoter was selected
for further analysis by block-replacement mutagenesis.
The block-replacement studies identified elements upstream of the
transcriptional start sites that are important for activity in MSC-1
and Sertoli cells (regions 6, 7, and 9.2; Figs. 3
and 8
). The role of
region 6 in FSHR gene regulation has not previously been described.
Sequence analysis of this region revealed potential binding sites for
members of the ets family of transcription factors, suggesting that a
member of this family may be important for activation of the FSHR gene
(29). Currently, our understanding of the role of this element is
limited and awaits further characterization of the element and its
binding proteins.
In addition to the upstream elements, several regions 3' to the
transcriptional start sites (regions 13.4, 14, and 15) were also
important for promoter function in MSC-1 and Sertoli cells. Although
there seem to be some differences in specific sites of the 3'-end that
are important, in each cell type investigated, there were multiple
3'-regions that contributed to promoter activity. In MSC-1 and Sertoli
cells, deletion analysis of the 3'-end confirmed its importance in FSHR
promoter function. Studies using the human FSHR promoter showed that
deletion of bases corresponding to +98 to +66 in the rat reduced
promoter activity approximately 80% (11). Although we observed
promoter effects in this region, they were milder than those observed
by Gromoll et al. (11) with the human FSHR promoter. In
addition, a large internal deletion in the human promoter that
corresponds to the rat sequences from -9 to +43, reduced promoter
activity 60%, suggesting the presence of weaker promoter elements
spanning the transcriptional start site (11).
Our studies and the studies with the human promoter both support a role
for the 3'-region in FSHR promoter activity. A functional role for
cis-acting elements located downstream of the
transcriptional start site has been reported for several genes
(30, 31, 32, 33, 34). It is interesting to note that many TATA-less genes have been
reported to have a common element, MED-1, in their 3'-ends (34). A
similar sequence was not detected within the 3'- region of the rat FSHR
gene. The molecular mechanism by which these elements enhance
transcription is largely unknown, but they may act in a manner similar
to 5'-promoter elements to help recruit components of the general
transcription machinery or may be important in start site selection or
elongation. In the rat FSHR 3'-region, a mutation through region 15 had
the greatest impact on promoter activity. Further inspection of this
element revealed a perfect consensus sequence (5'-TTTSGCGC-3') for the
transcription factor E2F (35). Additional mutagenesis revealed that the
last two bases of this site were critical for activity, while mutation
of bases elsewhere in the E2F site had only mild effects on promoter
function (Fig. 5
). Preliminary data in primary rat Sertoli cells
indicated that mutation of the last two bases in this site (µ15.4)
reduced promoter activity to 53% of wild type. Although these studies
do not rule out a role for E2F, the minor effects observed with
mutations through other conserved bases in the E2F site underscores the
possibility that a factor other than E2F regulates this element and
points to the GC-rich region as the site important for protein
interaction with the promoter. Further characterization of region 15
will be critical to our understanding of the FSHR gene and the role of
3'- elements in regulation of gene transcription.
In contrast to a previous report, we did not find that mutations
between the transcriptional start sites had a large impact on promoter
activity (16). To address the discrepancy, additional mutations were
made in region 11 (µ11c altered the wild-type sequence from
5'-CAGATCTCTCT-3' to 5'-tgGATCcagag-3' and
µ11.1 changed the sequence to 5'-CAGATCTaaaa-3') and
examined by transient transfection analysis. Both of these retained
80% or greater of the wild-type activity in MSC-1 cells (our
unpublished results). However, this region did appear to be more
important for promoter activity in primary Sertoli cell cultures than
in MSC-1 cells. Interestingly, a significant drop in promoter activity
was observed when the 3'-end was truncated from +21 to +8 (part of
region 11 and 12). This supports a role for this region but suggests
that it may be more important in promoter function when the downstream
region is absent or nonfunctional. Since the rat FSHR gene has two
major transcriptional start sites (9), it is possible that there are
two Inr-like regions, and loss of one can be compensated for by the
other. Interestingly, we observed that a mutation just past the second
transcriptional start site (region 13.4) has more of an effect on
promoter activity than mutations between the start sites (µ11 and
µ12). Additional studies are clearly required to determine whether
these regions are acting as Inrs, but previous studies found that the
region encompassing the rat FSHR transcriptional start sites (-19/+65)
binds to proteins that can bind the Inr of the terminal
deoxynucleotidyltransferase gene (13), suggesting an Inr function
within this region.
The block-replacement studies revealed both similarities and
differences in promoter function in MSC-1, BeWo, and
T3 cells. In
BeWo cells, except for the modest influence of region 7, there is no
significant impact of mutations 5' to the transcriptional start sites.
Notably, a mutation through the E box (region 9.2) had only a minor
impact on promoter function in BeWo cells. This is in contrast to
expression in MSC-1 and
T3 cells, where mutation of the E box had
the greatest impact on promoter activity. However, similar to MSC-1
cells, regions downstream of the transcriptional start sites (regions
13.4, 14, 15, and 18) influenced promoter activity in BeWo cells. In
T3 cells, the use of promoter elements in regions 6, 9.2, 13.4, and
14 underscore the similarities with FSHR expression in MSC-1 cells.
However, these studies also revealed important differences between
promoter function in MSC-1 and
T3 cells. Thus, a mutation in region
8 impacts promoter activity in
T3 cells but not MSC-1 cells and a
mutation in region 15 had little consequence in
T3 cells in contrast
to its impact in MSC-1 cells.
Mutation of the E box in region 9.2 had the greatest influence on
promoter activity when compared with other mutations spanning the -70
to +123 region. The importance of the E box was previously demonstrated
for the rat FSHR promoter, and its major binding proteins were shown to
cross-react with USF1 antibody (16). Our studies confirm these findings
and further build upon them by extending the functional
characterization of the E box and correlating the sequence specificity
for promoter activity with the sequence specificity required for
DNA/protein interaction. We also examined the complexes using
additional antibodies to members of the bHLH family and showed that in
MSC-1, primary rat and mouse Sertoli cells,
T3, and BeWo cells, the
major binding complexes cross-react with both USF1 and USF2 antibodies
but not with c-Myc or E2A antibodies (Figs. 10
and 11
).
Cross-reactivity of the complexes with the USF1 antibody was more
extensive than with the USF2 antibody, suggesting that a majority of
the complexes are either USF1 homodimers or heterodimers of USF1 and
some other protein, while a more minor component is USF1-USF2
heterodimers.
The observation that the major E box-binding complex in BeWo cells was
similar, if not identical, to that observed in MSC-1 and
T3 cells
offers some important insight into the mechanism by which this promoter
is regulated. Thus, despite the presence of the USF proteins, the E box
and promoter remained largely inactive in BeWo cells, suggesting that
the ability of the USF proteins to transactivate the promoter in these
cells is impaired. This altered function may be due to differences in
either modification of the USF proteins, proteins with which they
interact, or other regulatory features of the promoter. Another
possibility is that a protein(s) other than USF is important for
regulation of the FSHR promoter through the E box.
In mammalian cells, there are substantial numbers of transcriptional
regulators that can bind to the CACGTG sequence, all of which tend to
be in the bHLH-Zip class of transcription factors (36, 37, 38, 39, 40, 41, 42). These
include USF, TFEB, TFE3, c-Myc, Max, and Mad. Thus, resolving the
specificity of these transcriptional regulators has been a formidable
task. Studies on the binding of partially purified USF from HeLa cells
reported the importance of both core E box sequences and flanking
sequences and derived a consensus sequence for USF binding
(RYCACGTGRY) (43). This sequence fits well with the
sequence of the FSHR E box (43). Unfortunately, reports matching the
binding site requirements with the functional activity of USF are
lacking. It is important to point out that the binding of different
bHLH-Zip proteins to the CACGTG E box is sensitive to the conditions of
the EMSA (43). Thus both the proteins that bind the element and their
sequence requirements are sensitive to the in vitro binding
conditions. Under the conditions of our EMSA, the predominant binding
complex consisted of USF1 and USF2.
To help corroborate in vitro binding data with in
vivo functional data, we analyzed both the functional requirements
of the element in vivo and the binding requirements for the
proteins in vitro. These studies revealed a discrepancy
between the role for the flanking sequence (µ9.4, Fig. 9
) in promoter
function and its role in USF binding. One explanation of these results
is that the major in vitro binding complex is not
responsible for activity associated with the E box in vivo.
Although other binding proteins are not readily observed in the
in vitro binding assays, our preliminary data indicate that
additional E box-binding complexes are present in Sertoli cells that
are more readily observed when extracts are depleted of USF. A second
explanation is that mutation of the flanking sequences not only alters
the binding of USF to the DNA element but also influences its function.
Thus, USF may bind slightly to the µ9.4 element, but the mutation
alters USF conformation such that it cannot transactivate. However, to
definitively establish USF as the key regulator of the FSHR gene, more
direct approaches are required. Thus determining the effects of
wild-type and mutant forms of the USF proteins on promoter function or
characterizing FSHR expression in cells or animals that lack USF
proteins will be important to confirm or refute the role of USF in FSHR
gene regulation. In addition, further characterization of the FSHR E
box-binding proteins will be important in determining the proteins that
dimerized and/or interact with USF as well as other proteins that bind
the FSHR E box.
Although the FSHR promoter did exhibit some specificity with respect to
its activity in Sertoli cells vs. nonexpressing cells, the
fact that some expression did occur in
T3 cells suggests that not
all the regulatory components required for cell specificity are
represented in our assay system. Analysis of block-replacement mutants
indicated that transcription factors in Sertoli cells and
T3 cells
occupy several of the same regions on the promoter. However, it is not
known whether the same proteins activate transcription in both cell
types or whether different proteins are involved. Such "leaky"
expression from the promoter may be due to the presence of abnormally
expressed transcription factors or to the uncharacteristic
accessibility of the promoter in transiently transfected DNA. Thus,
these same transcription factors that activate the promoter in
T3
cells may not function when the promoter is integrated into chromatin.
It seems likely that elements within the -100/+123 promoter together
with other control regions within the gene will generate the
specificity observed for the FSHR. Further characterization of both the
FSHR gene and its promoter region is required to complete our
understanding of the transcriptional mechanisms activating FSHR in
Sertoli cells. Identification of the proteins involved will be critical
to our understanding of FSHR gene regulation and, as suggested by the
exquisite specificity of the gene, will likely provide important
insight into transcriptional events critical for Sertoli cell
function.
 |
MATERIALS AND METHODS
|
---|
Reagents
Reagents and venders include the following: Tris-HCl, NaCl,
MgCl2, glycerol, bis-acrylamide, potassium acetate, sodium
hydroxide, Triton X-100, SDS, acrylamide, and boric acid (Fisher,
Pittsburgh, PA); phenylmethyl sulfonyl fluoride, EDTA, and
Nonidet P 40 (NP-40), Tween 20 from Sigma Chemical Co. (St. Louis MO);
dithiothreitol from Boehringer Mannheim (Indianapolis, IN);
lipofectamine, HEPES, DMEM, horse serum, FBS, Penicillin/Streptomycin,
and Waymouths media from Life Technologies (Gaithersburg, MD);
nucleotides and poly(dI-dC)·poly(dI-dC) from Pharmacia (Piscataway,
NJ); ethanol from Aaper (Shelbyville, KY); restriction enzymes from
Life Technologies or Promega (Madison, WI); and T4 polynucleotide
kinase from New England BioLabs (Beverly, MA). Radionucleotides were
purchased from DuPont/NEN (Boston, Mass). USF1, USF2 and
goat-antirabbit horseradish peroxidase antibodies were purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cell Lines
MSC-1 cells (28) were grown in DMEM supplemented with 5% FBS,
penicillin, and streptomycin.
T3 (44) cells were grown in DMEM
supplemented with 5% FBS, 5% horse serum, penicillin, and
streptomycin. BeWo cells (ATCC, Rockville, MD) were grown in
Waymouths media supplemented with 10% FBS, 2 mM
glutamine, penicillin, and streptomycin. Primary Sertoli cells were
prepared and cultured as described elsewhere (45) with slight
modification. The cells are initially plated in the presence of 5% FBS
and 3 µg/ml Cytosine arabinoside. After 40 h in culture
the cells were treated with a hypotonic solution of 10 mM
Tris (pH 7.4) for 2 min to remove germ cells and then fed Hams F12
supplemented with 0.215 g glutamine per liter, 1.5 mM
HEPES, and antibiotics.
DNA Preparation
All plasmid DNAs were prepared from overnight bacterial cultures
using Qiagen DNA plasmid columns according to the suppliers protocol
(Qiagen, Chatsworth, CA). DNAs used for transfection were prepared with
Midi-prep or Mega-prep Qiagen columns. Oligodeoxynucleotides were
purchased from Life Technologies. Double-stranded oligodeoxynucleotides
were generated by heating complementary oligodeoxynucleotides to 95 C
in the presence of 10 mM Tris (8.0), 100 mM
NaCl, and 1 mM EDTA and allowing them to cool slowly to
room temperature. Radiolabeled oligodeoxynucleotides were generated
using T4 polynucleotide kinase and
-32P-ATP.
FSHR Promoter Clones
The FSHR promoter was isolated from rat genomic clone 54.111
(9). A 6000-bp SstI fragment of 54.111 containing
approximately 5000 bp 5' to the first transcriptional start site and
1000 bp 3' to this site was subcloned into pGEM 4Z [clone 54.111(3)].
A fragment containing sequences from -220 to +1000 (relative to the
first transcriptional start site) was removed from 54.111(3) by
digestion with EcoRV and XbaI, and the remaining
promoter region was ligated to promoter sequences spanning -220 to
+123. The -220 to +123 fragment was generated by PCR amplification
using FSHR-specific primers (5'-primer:
5'-AATGTGAATCTGCTGCTATAGACTGAT-3' and 3'-primer:
5'-AGCAAGGAGACCAGGAGCAAGGCCACCCTTATTTATCCAT-3')
and clone 54.111(3) as template. Reaction conditions are described
elsewhere (46). The PCR fragment was digested with EcoRV (a
restriction endonuclease site naturally found in the promoter at
position -220) and XbaI (a restriction endonuclease site
introduced into the 5'-end of the 3'-primer). The 3'-primer has a
single base pair change (indicated in bold and underlined)
that alters the natural translational start site of FSHR from ATG to
GTG to minimize potential problems from altered luciferase translation
products. Ligation of the upstream promoter region (-5000 to -220) to
the downstream region (-220 to +123) generated the clone
FSHR(-5000/+123)GEM. An SstI/XbaI fragment of
FHSR(-5000/+123)GEM was subcloned into the
SstI/NheI sites of the luciferase vector
pGL3-Basic (Promega) to generate FSHR(-5000/+123)Luc. To construct
FSHR(-3600/+123)Luc, FSHR(-5000/+123)Luc was digested fully with
SstI and partially with EcoRI, promoter sequences
upstream of -3600 were removed, and the ends were filled in with
Klenow and then ligated together. To generate FSHR(-2700/+123)Luc,
FSHR(-5000/+123)Luc was digested with SstI and
PstI, the 2300-bp fragment containing sequences from -5000
to -2700 was removed, overhangs were filled in with Klenow, and the
ends were ligated. To create FSHR(-1300/+123)Luc, FSHR(-5000/+123)Luc
was digested with SstI and EcoRI, upstream
sequences were removed, and the ends were filled in with Klenow and
then ligated together. FSHR(-5000/+123)GEM was digested with
HindIII and the 886-bp promoter fragment (spanning from
-763 to +123) was subcloned into the HindIII site of
pGL3-Basic, producing the clone FSHR(-763/+123)Luc. A 343-bp
EcoRV/HindIII fragment from FSHR(-5000/+123)GEM
was subcloned into the SmaI/HindIII sites of
pGL3-Basic to generate FSHR(-200/+123)Luc. FSHR(-100/+123) was
generated by PCR amplification using 54.11(3) as template, an upstream
primer, 5'-GATCGGAGCTCATATAATCACTATTGACAC-3'
(underlined sequences are homologous to the FSHR
promoter from -100 to -82) with an engineered SstI site
near the 5'-end, and the same downstream primer used to generate the
-220/+123 fragment. The amplified fragment was digested with
SstI and XbaI and subcloned into the
SstI/NheI sites of pGL3-Basic.
Generation of FSHR(-220/+123) mutant constructs was accomplished by
bidirectional primer extension using Deep Vent DNA polymerase (New
England Biolabs). The reaction conditions and strategy are described
elsewhere (46). The upstream standard 5'-primer for each mutant was
RVprimer3, a primer with matching sequences upstream from the
polylinker in pGL3-Basic. The standard downstream primer was Luc.1, a
primer that anneals to specific sequences within the coding region of
the luciferase gene. The template for each mutant was
FSHR(-220/+123)Luc. The cloning sites for the mutants were
MluI and HindIII in the multiple cloning site of
FSHR(-220/+123)Luc. Mutations introduced into each clone are indicated
by lower case letters in Fig. 2
. For mutations in regions 9,
10, 12, and 13, only a single mutant primer was used and extension was
only in one direction. These mutants were cloned into the
BglII (at the +1 site of the promoter) and MluI
sites (mutants 9 and 10) or into the BglII and
HindIII sites (mutants 12 and 13). All clones were confirmed
by sequencing using either the Sequenase 2.0 sequencing kit from USB
(Cleveland, OH) or the ABI Prism dRhodamine Terminator Cycle Sequence
Ready Reaction Kit (PE Applied Biosystems, Foster City, CA).
Transfection and Enzyme Analysis
MSC-1 cells were seeded onto 35-mm plates at a density of
250,000 cells per well. Twenty to 24 h after plating the cells
were transfected using 5 µl of lipofectamine, 1 µg of luciferase
reporter, and 0.20 µg of Rous sarcoma virus (RSV)
ß-galactosidase. For all mutant promoter constructs, at least two
independent clones were examined. Details of the transfection procedure
are described elsewhere (46). Cells were harvested and assayed 60
h after transfection according to previously described procedures (46).
For each transfection, the luciferase/ß-galactosidase activity of
each construct was normalized to the luciferase/ß-galactosidase
activity of the wild-type promoter. The values were then averaged over
a minimum of three independent experiments. Transfection and enzyme
assays for
T3 and BeWo cells are as described elsewhere (46).
Primary cultures of Sertoli cells were seeded onto 35-mm plates at a
density equal to 0.08 testis/well. Approximately 72 h after the
initial plating, the media was change to DMEM without any additives.
Twelve hours later, the cells were transfected using 5 µl of
lipofectamine, 0.5 µg of luciferase reporter, and 0.50 µg of
RSV-ß-galactosidase. Twelve to 16 h later, the transfection
media was removed and replaced with fresh DMEM. Forty eight hours
later, cells were harvested and assayed as described above. Statistical
analysis was performed using a two-sample Students t test
(two tailed) assuming unequal variances.
EMSA
Whole-cell extracts were prepared as described elsewhere (47).
Whole-cell extracts (610 µg protein) were incubated with 12.5 fmol
of radiolabeled double-stranded oligonucleotide in the presence of 12
mM HEPES, pH 7.9, 5 mM MgCl2, 80
mM KCl, 0.6 mM dithiothreitol, 12% glycerol,
0.6 mM EDTA, 100 µg/ml BSA, 0.1 mM
phenylmethylsulfonyl fluoride, and 200 ng of salmon sperm DNA in a 20
µl reaction volume. Addition of antibodies to the reaction
immediately preceded the addition of extract. Reactions were incubated
on ice for 5 min before addition of probe and then an additional 30 min
after unless otherwise noted. Protein/DNA complexes were resolved on a
4% polyacrylamide gel (acrylamide-bis-acrylamide, 40:1) polymerized,
and run in a buffer containing 25 mM Tris (pH 8.5), 190
mM glycine, and 0.5 mM EDTA at 250 V for
1.5 h at 4 C. The gels were dried and analyzed by autoradiography.
Nuclear extracts, when used, were prepared as described elsewhere
(46).
Western Blot Analysis
Whole-cell extracts were resolved on 10% SDS-polyacrylamide
gels (acrylamide-bis-acrylamide, 30:0.8) with the discontinuous buffer
formulation of Laemmli (48) and transferred to Nitrocellulose membranes
(Bio-Rad Laboratories, Hercules, CA) using a Mini Trans-Blot
Electrophoretic Transfer Cell (Bio-Rad). Membrane proteins were probed
overnight at 4 C with anti-USF1 antibody, diluted 1:10,000 in TBST (15
mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween
20), and then subsequently incubated with goat antirabbit horseradish
peroxidase-conjugated antibody for 90 min at room temperature. Specific
protein complexes were visualized with the enhanced chemiluminescence
(ECL) system (Amersham Life Sciences, Arlington Heights, IL).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Leslie L. Heckert, Department of Molecular and Integrative Physiology, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansa 66160. E-mail:
lheckert{at}kumc.edu
This work was supported in part by NIH Grant R29HD-3521701A1 (to
L.L.H.) and by an NICHD-supported Center of Reproductive Sciences Grant
(HD-33994). M.F.D. was supported by a training grant in reproductive
biology (HD-07455).
Received for publication March 16, 1998.
Revision received June 19, 1998.
Accepted for publication July 15, 1998.
 |
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