The USF Proteins Regulate Transcription of the Follicle-Stimulating Hormone Receptor but Are Insufficient for Cell- Specific Expression
Leslie L. Heckert,
Michele Sawadogo,
Melissa A. F. Daggett and
Jiang kai Chen
Department of Molecular and Integrative Physiology (L.L.H.,
M.A.F.D., J.k.C.) The University of Kansas Medical Center
Kansas City, Kansas 66160
The Department of Molecular
Genetics (M.S.) The University of Texas M.D. Anderson Cancer
Center Houston, Texas 77030
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ABSTRACT
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Expression of the FSH receptor (FSHR) is limited
to granulosa cells of the ovary and Sertoli cells of the testis.
Previous studies showed that an E box in the proximal promoter of the
FSHR gene is required for transcription and that the predominant E box
binding proteins are the ubiquitous transcription factors, upstream
stimulatory factor 1 (USF1) and USF2. Through cotransfection
analysis, we have shown that both wild-type and dominant negative forms
of the USF proteins regulate the rat FSHR promoter and that
transcriptional activation of FSHR required several domains within the
amino-terminal portion of the USF proteins. Analysis of the FSHR
promoter region using in vivo genomic footprinting
indicated that the E box is occupied by proteins in Sertoli cells but
not in cells that fail to express the receptor, despite the presence of
the USF proteins. To help delineate the regions of the rat FSHR gene
required for correct spatial and temporal expression, transgenic mice
harboring two constructs containing variable amounts of 5'-flanking
sequence (5,000 bp and 100 bp) were generated. Examination of 16
different transgenic lines revealed varied transgene expression
profiles with multiple lines having different amounts of ectopic
expression and two lines failing to express the transgene. In addition,
little or no expression was observed in Sertoli cells. These studies
indicate that additional regulatory sequences outside the region from
-5,000 to +123 bp are needed for proper expression in Sertoli cells.
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INTRODUCTION
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In the testis, germ cell development occurs within a highly
specialized compartment called the seminiferous epithelium. Within this
structure, Sertoli cells form the major somatic component and function
to provide an environment that protects and nurtures the germ cells,
assisting their development into viable sperm (1, 2, 3, 4). Sertoli cells
also play a central role in testicular development, where under the
direction of Sry, the sex-determining gene on the Y chromosome, they
are the first somatic cell to differentiate in the embryonic gonad and
are thought to orchestrate subsequent events in testis formation and
sex determination (5, 6, 7, 8, 9). Sertoli cells provide many functions that are
essential to testis activity and fertility; most notable are the
formation of a support system to house the developing germ cells,
endocytosis of eliminated waste products from 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).
Sertoli cells are regulated by a variety of signaling molecules that
assist their differentiation and development. One of these, the
pituitary hormone FSH, is an integral component of the regulatory
network that forms the hypothalamic-pituitary-gonadal axis. This
hormone provides important stimulus to Sertoli cells that induces
proliferation, final maturation events, and synthesis of specific
protein products (10, 11, 12). FSH recognizes and binds a cell surface
receptor (FSHR) that serves as the communicative link between the
pituitary FSH signal and gonadal response (11, 13, 14). The expression
of this receptor is remarkably cell specific: it is located
exclusively on Sertoli cells of the testis and granulosa cells of the
ovary (11, 13, 15, 16). Because of its cell-specific properties and its
role in FSH signaling, determining the mechanisms that regulate FSHR
gene expression will provide important insight into the regulation of
FSH response and mechanisms that govern cell-specific expression in
granulosa and Sertoli cells.
Studies with transgenic mice and transient transfection have been used
to examine the transcriptional mechanisms that regulate FSHR (17, 18, 19, 20).
Experiments using transgenic mice revealed that 5,000 bp of 5'-flanking
sequence could direct expression of a reporter gene to the gonads,
implicating this region in the cell-specific expression of FSHR (18).
Moreover, transient transfection analysis of deletion and block
replacement mutants indicated that, within this 5,000-bp gene fragment,
a single E box element, located approximately 30 bp upstream of the
transcriptional start site, is the major control site for FSHR
transcription (17, 18, 19). Together, these studies suggest that a promoter
region that includes the E box would be sufficient for cell-specific
expression and that this element is an important contributor to this
process. However, identification of the upstream stimulatory factor
(USF) proteins, USF1 and USF2, as the major Sertoli cell E
box-binding complex obscured this interpretation, as these proteins are
ubiquitous and therefore insufficient, by themselves, to direct
cell-specific expression (17, 19). Thus, if the E box and the USF
proteins are involved, other mechanisms, such as cell-specific
modifications or the use of specific coactivators, need to be invoked.
Alternatively, proteins other than USF1 or USF2, not observed in the
binding assays, may interact with this element and help direct
cell-specific expression. To address these possible mechanisms, more
direct evidence for USF regulation of FSHR is needed as well as a
better-defined region that directs cell-specific expression. Through
cotransfection analysis of wild-type and mutant USF proteins, we
provide data that showed USF1 and USF2 activated FSHR transcription and
that this activation required several domains within the amino-terminal
portion of the proteins. In addition, we show that occupancy of the E
box in vivo was cell specific, but likely requires
assistance of sequences outside of -5,000/+123 bp of the FSHR
gene.
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RESULTS
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USF1 and USF2 Regulate FSHR Transcription through the E Box
USF1 and USF2 are members of the basic helix-loop-helix
(bHLH)-ZIP family of transcription factors and bind DNA as a
dimer, where they are known to form both homodimers and heterodimers
between themselves (21, 22, 23). Binding and transfection studies have
implicated these proteins in the regulation of FSHR transcription (17, 19). To derive more direct evidence for a role of the USF proteins in
the regulation of FSHR, we examined the ability of both wild-type and
mutant USF proteins to regulate FSHR promoter function. The rat FSHR
promoter (-220/+123) driving expression of a luciferase reporter gene
was cotransfected into the mouse Sertoli cell line, MSC-1, with vectors
that express either wild-type or mutant USF proteins. The mutant USF
proteins, U1
N and U2
N, efficiently bind DNA but are unable to
transactivate due to the absence of the amino-terminal transactivation
domains (24, 25). Cotransfection with increasing amounts of vectors
expressing the wild-type USF proteins showed little impact on FSHR
promoter activity (USF1 and USF2, Fig. 1A
). However, cotransfection with
increasing amounts of vector DNA expressing the amino-terminal
transactivation mutant proteins (U1
N or U2
N) resulted in a
dramatic reduction in FSHR promoter activity (Fig. 1B
). The effects of
the
N mutants were promoter specific and E box dependent, as
luciferase production directed by either the SV40 promoter (in
pGL3-Control) or a mutant FSHR promoter lacking the E box (FSHR
Ebox)
was insensitive to cotransfection with the
N mutants (Fig. 1C
). In
addition, no specific effects of the USF mutants were observed on
either the thymidine kinase or Rous sarcoma virus promoters (data not
shown). The results therefore indicate that the USF
N mutants
regulate the FSHR promoter in a manner that is promoter specific and
dependent on the E box.
Although little effect of the wild-type USF proteins was observed, the
impact of the
N mutants suggested that USF regulates FSHR expression
and that the results observed with the wild-type proteins were due to
high levels of endogenous USF proteins. The inability of the wild-type
USF proteins to regulate FSHR promoter activity did not appear to be
due to the level of protein expressed from the vectors, as both USF1
and USF2 expression was comparable to expression of the mutant proteins
as observed by Western blot analysis (Fig. 3B
). However, if the E box
in the transfected promoter was already saturated with USF due to high
levels of endogenous proteins, the ability to influence promoter
activity would be compromised. To address this possibility, we used two
different FSHR promoter mutants that contained subtle changes in the E
box sequence (µ9.3 and µ9.6, Fig. 2A
). As demonstrated by their diminished
ability to compete for the USF proteins in an electrophoretic mobility
shift assay (EMSA), these mutant E boxes bind the USF proteins with
lower affinity than the wild-type sequence (Fig. 2A
). In addition,
basal activity of the mutant promoters was significantly less than the
wild-type (Fig. 2B
and Ref. 17), revealing that the mutant promoters
are not saturated with regulatory proteins when transfected into MSC-1
cells. When these mutant constructs were cotransfected with expression
vectors for either USF1 or USF2, promoter activity increased 2 to 3
times as compared with empty vector alone (Fig. 2B
). In contrast, a
mutant promoter with the entire E box mutated (µ9.2) was only
modestly influenced by cotransfection with vectors expressing the
wild-type USF proteins (Fig. 2B
). Thus, under conditions where the E
box has a lower affinity for the USF proteins and the element is not
saturated with endogenous USF, USF1 and USF2 activated FSHR promoter
activity. The results in Fig. 1
further indicated that this
transcriptional activation requires a domain or domains within the
amino-terminal portion of these proteins.

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Figure 3. Multiple Domains in the Amino-Terminal Regions of
USF1 and USF2 Are Implicated in Transactivation of FSHR
A, Transient transfection analysis of wild-type and mutant USF
proteins. One microgram of FSHR(-220/+123)Luc was transiently
transfected into MSC-1 cells together with 50 ng pRL-TK and either 500
ng of empty expression vector (pSG5) or expression vectors for
wild-type or various mutant USF1 or USF2 proteins. The relative
activity represents the firefly/Renilla luciferase
activities of each transfected sample normalized to the
firefly/Renilla luciferase activities of the promoter
transfected with empty vector alone. Error bars represent
the SEM. A schematic diagram of the various
domains and exons that make up the USF proteins is shown at the
top. B, Western blot analysis of USF expressed from various
expression vectors. Each expression vector was transiently transfected
into MSC-1 cells and examined for protein expression by Western blot
analysis as described in Materials and Methods. Lanes are:
1) pSG5, 2) USF2, 3) U2 640, 4) U2 7123, 5) U2 7148, 6)
U2 7157, 7) U2 7186, 8) U2 1199, 9) U2 N, 10) U2 USR,
11) U2 E4, 12) U2 E5, 13) pSG5, 14) USF1, 15) U1 1130, 16)
U1 N, 17) pSG5.
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Figure 2. Overexpression of USF1 and USF2 Activates FSHR
Promoters Having E box Sequences with Lower Binding Affinity for the
USF Proteins
A, EMSA assay of the FSHR E box. A radiolabeled probe corresponding to
the FSHR E box (5'-TCTTGGTGGGTCACGTGACTTTGCCCGT-3') was
used in an EMSA with nuclear extracts from primary rat Sertoli cells.
Twenty-five femtomoles of radiolabeled probe were incubated with
nuclear extract and resolved on a 4% polyacrylamide gel, as described
in Materials and Methods. Competitor DNAs were added to
the reactions at a concentration equal to 15-fold that of the probe.
Arrows indicate the USF proteins as previously
described. Sequences of each competitor are given below
the EMSA. B, Transient transfection analysis of various mutant E box
promoters. One microgram of either FSHR(-220/+123)Luc (wild-type),
FSHR(-220/+123)µ9.3, FSHR(-220/+123)µ9.6, or
FSHR(-220/+123)µ9.2 was transfected into MSC-1 cells together with
50 ng pRL-TK and 500 ng of either empty expression vector (pSG) or
expression vectors for wild-type USF1 or USF2 (U1 and U2,
respectively). The relative activity represents the
firefly/Renilla luciferase activities of each
transfected sample normalized to the firefly/Renilla
luciferase activities of the wild type promoter transfected with empty
vector alone. Error bars represent the
SEM.
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USF Transactivation of the FSHR Gene Involves Multiple
Amino-Terminal Domains in the Proteins
Recently, domains important to transcriptional activity of the USF
proteins have been localized to their amino-terminal regions (24, 25, 26).
We used a cotransfection paradigm to delineate domains within the amino
termini of USF1 and USF2 that are important for FSHR transcription. A
variety of expression vectors containing cDNAs for various mutant USF
proteins were examined for their ability to influence FSHR
transcription. Each of the proteins examined had a functional
DNA-binding domain consisting of a basic region (BR) and
helix-loop-helix-leucine zipper region (HLH-LZ) and has been shown to
bind to DNA and translocate into the nucleus (25). Since the E box is
already saturated with endogenous USF, mutant USF proteins
that lack important transactivation domains should inhibit promoter
activity by displacing the active proteins at the E box. Comparison of
the promoter effects elicited by mutant and wild-type USF proteins
revealed several transactivation domains important for regulation of
FSHR transcription (Fig. 3A
). The
wild-type USF did not inhibit promoter activity, indicating that
inhibition is not due to the sequestration of important factors away
from the promoter. Deletion of amino acids 640 of USF2
(U2
640) reduced promoter activity 3040%, revealing a
transactivation domain within this region of the protein (Fig. 3A
).
Removal of an additional 83 amino acids that encompasses most of exon 4
(U2
7123) had no further influence on promoter activity. Deletion
of exon 4 alone also had little impact on promoter function (Fig. 3A
, U2
E4), while progressively larger deletions through exon 5
(constructs U2
7148 through U2
1199) resulted in sequentially
greater impact on promoter activity. This suggested that multiple
regions within exon 5 act to support transcriptional activity of USF2.
Removal of the USF-specific region (USR) domain, a region of
high sequence conservation among USF proteins, further decreased
promoter activity. However, removal of either exon 5 or the USR alone
(U2
E5 and U2
USR) failed to inhibit promoter function, indicating
that redundant transactivation domains act within the amino terminus of
USF2. Western blot analysis of cells transfected with each of the
mutants showed that the observed results were not due to differences in
expressed protein (Fig. 3B
).
An important transactivation domain within the first 130 amino acids of
USF1 was also identified (Fig. 3A
, U1
1130), and further deletion
through the USR resulted in a modest reduction in FSHR promoter
activity. Thus, for both USF1 and USF2, multiple domains appear to
contribute to transactivation of the FSHR gene and, at least for USF2,
there is redundancy in the ability of some of these (exon 5 and USR) to
transactivate the promoter.
The E Box of the Endogenous FSHR Gene Is Occupied in Expressing
Cells and Vacant in Nonexpressing Cells
The central role of the E box in FSHR promoter
function, as assessed by transient transfection, prompted examination
of this site on the endogenous FSHR gene using in vivo
genomic footprinting. Primary cultures of rat Sertoli cells and a rat
choriocarcinoma cell line, RCHO, that does not express FSHR were
treated in vivo with dimethyl sulfate (DMS), genomic DNA was
isolated, and the methylation pattern of guanines (and to a lesser
extent adenines) was determined after piperidine cleavage and
ligation-mediated PCR. Genomic footprints generated from the gene
in vivo were compared with footprints generated from DNA of
the same source isolated before treatment with DMS (in vitro
footprint). Comparison of the in vivo and in
vitro footprints revealed that guanines within the FSHR E box
(marked by arrows in Fig. 4
, A
and B) were protected from methylation in cultured Sertoli cells
treated with DMS in vivo (T lanes, Fig. 4A
) when compared
with naked DNA treated with DMS in vitro (N lanes, Fig. 4A
).
Densitometric analysis revealed that, in Sertoli cells, the intensity
of the E box bands was 65% lower in the in vivo DMS-treated
DNA compared with naked DNA treated with DMS. In contrast, the
intensity of the E box bands in the RCHO cells was slightly higher
(127%) with in vivo DMS treatment than with in
vitro treatment, indicating that the E box is not protected from
methylation in RCHO cells (Fig. 4A
). These studies indicate that the E
box within the endogenous FSHR gene is occupied by regulatory
proteins in FSHR-expressing cells (Sertoli) but not in cells that fail
to express the receptor (RCHO). The inability to detect a footprint
over the E box in RCHO cells was not due to the lack of USF proteins,
as EMSA and antibody supershifts revealed the presence of these
proteins in this cell line (Fig. 4C
).

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Figure 4. The E Box in the FSHR Promoter Is Protected from
Methylation in Sertoli Cells but Not in RCHO Cells
A, In vivo genomic footprint analysis of the FSHR
promoter. DNA from rat Sertoli or RCHO cells treated with DMS either
in vitro (N) or in vivo (T) was examined
by ligation-mediated PCR using primers specific to the promoter region
of the rat FSHR gene. An arrow indicates the position of
the E box. B, Sequence of the rat FSHR promoter in the region of the
footprint. The E box is shaded, and the protected
guanines are indicated with arrows. C, EMSA of the FSHR
E box with nuclear extracts from RCHO cells. EMSA was done as described
in the legend of Fig. 2 . Where indicated, competitors were added at a
concentration equal to 100-fold that of the probe or antibodies against
the bHLH proteins USF1 and USF2 were added.
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Sequences outside of -5,000 to +123 bp of the Rat FSHR Gene Are
Required for Expression in Sertoli Cells
The expression of FSHR shows exquisite specificity, being
found only in granulosa cells of the ovary and Sertoli cells of the
testis (11, 13, 15, 16). To help determine whether the E box is
sufficient to direct cell-specific expression of the FSHR gene, two
reporter vectors containing different amounts of FSHR 5'-flanking
sequence were used to generate transgenic mice. Both 5,000 bp and 100
bp of rat FSHR 5'-flanking sequence were placed upstream of the Cre
recombinase gene fused to a portion of the bovine GH (bGH)
gene (Fig. 5A
; -5,000/+123Cre,
-100/+123Cre). Sixteen different lines of animals were obtained (eight
lines from each construct), and the F1 progeny were examined for Cre
recombinase expression by RT-PCR analysis of RNA isolated from 10
different adult tissues using primers that span intron D of the bGH
gene (Cre6 and PolyA5; Fig. 5A
). Expression of Cre mRNA was determined
by the presence of a 334-bp amplified product as compared with a 609-bp
product derived from genomic DNA, as shown for animal 527, a progeny of
founder animal 254 (Fig. 5B
).

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Figure 5. Expression of FSHR-Cre Recombinase in Transgenic
Mice
A, Schematic diagram of the FSHR transgene. Either 5,000 bp or 100 bp
of FSHR 5'-flanking sequence (FSHR Promoter) was used to direct
expression of a gene encoding Cre recombinase fused to a portion of the
bGH gene that includes part of exon 4, intron D, and part of exon 5.
The approximate binding sites for the oligodeoxynucleotides used to
examine expression of the transgene (Cre6 and PolyA5) are shown. B,
Examination of Cre recombinase expression in transgenic mice. RT-PCR
was used to examine Cre recombinase expression in various tissues from
transgenic mouse no. 527 as described in Materials and
Methods. Reactions for cDNA synthesis were done either in the
presence (+) or absence (-) of added reverse transcriptase. Tissues
examined were testis (T), heart (H), liver (L), lung (Ln), kidney (K),
spleen (Sp), stomach (St), brain (B), bladder (Bl), and eyes (E).
Controls that contain no DNA (Neg.), plasmid containing the transgene
(Pos.), or cDNA from a positive expressing animal are also shown. An
arrow indicates the correctly processed Cre transgene
product of 334 bp. M refers to DNA markers.
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Table 1
gives the results obtained from
animals that harbor the -5,000/+123Cre transgene. The results show
that each transgenic animal expressed Cre recombinase in the testis as
well as the brain (expression is indicated as +). While expression was
restricted to these two tissues in most of the animals, various levels
of ectopic expression were observed in some lines, most notably lines
240 and 207. Table 2
gives the results
obtained from animals containing the -100/+123Cre transgene. In two
lines, 252 and 303, no transgene expression was observed. However,
three lines, 254, 289, and 296, expressed Cre only in the testis, while
the remaining three lines showed various levels of ectopic expression.
In female mice, transgene expression was observed in the ovary of each
FSHR(-5,000)Cre transgenic line examined, except for line 232, while
only lines 256, 323, and 347 of the FSHR(-100)Cre animals exhibited
ovarian transgene expression (Tables 1
and 2
). Thus, despite the rather
high level of ectopic expression, especially with the -5,000-bp
construct, the results with lines 254, 289, and 296 suggested that
sequences within the first 100 bp of the promoter are sufficient for
testis-specific expression but lacked information necessary for
ovary-specific expression.
To help evaluate which cells in the testis expressed Cre recombinase,
we examined testis expression in different aged animals. Both 10- and
30-day-old animals were selected, as significantly different
populations of germ cells are present at these two ages. At the earlier
age, only spermatogonia are present, while at the later age, germ cells
have begun to proliferate and differentiate, greatly expanding the
population of these cells in the testis. Expression of the FSHR gene in
Sertoli cells is maintained throughout postnatal development into
adulthood (11), and therefore, if the transgene is properly expressed
in Sertoli cells it should be present at both 10 and 30 days of age. In
contrast, expression only in the older animals would favor germ cells
as the site of expression, since this population of cells is greatly
increased by this age. As indicated in Tables 1
and 2
, all animals
examined expressed Cre recombinase in the testis at 30 days of age.
However, with the exception of animal 347, none of the animals examined
expressed Cre recombinase in the testis at 10 days of age, while FSHR
expression was observed in the testis of all animals (data not
shown).
The different temporal expression patterns of FSHR and Cre strongly
suggested that Cre expression in the testis was within the germ cell
population rather than in Sertoli cells. To test this possibility, Cre
recombinase expression was examined in Sertoli cells and germ cells
isolated from the testis of lines 254 and 347. After 35 rounds of
amplification in a RT-PCR reaction, a very modest signal for expressed
Cre recombinase was observed when Sertoli cell RNA from line 347 was
used as template. However, a robust signal was detected when germ cell
RNA from the same animal was used as template (Fig. 6
, SC and GC lanes, respectively). This
difference was not attributed to variation in the cDNA synthesis as no
obvious difference was observed for amplification of the control mRNA
L7 (Fig. 6
, bottom). No amplified product for Cre
recombinase mRNA was observed in cell preparations from negative
littermates (Fig. 6
, Neg SC and GC samples). Similar results were
obtained for transgenic line 254 (Table 2
). Since germ cells, in small
amounts, often contaminate Sertoli cell preparation, the weak Cre
signal is Sertoli cells most likely represents a germ cell contribution
to this RNA pool. Thus, the high degree of ectopic Cre expression and
temporal misregulation in the testis indicate that the region from
-5,000 to +123 bp of the rat FSHR gene was insufficient to properly
restrict or direct expression to Sertoli cells.

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Figure 6. Expression of FSHR(-100)Cre in Sertoli Cells and
Germ Cells
Sertoli cells and germ cells were isolated from testes of male
transgenic animals from line 347. RT-PCR was used to examine Cre
recombinase expression in Sertoli cells (SC) and germ cells (GC)
isolated from male transgenic mice (347) and negative littermates
(Neg). Reactions for cDNA synthesis were done either in the presence
(+) or absence (-) of reverse transcriptase, and each reaction was
examined for the presence of L7 (ribosomal protein) cDNA to confirm
cDNA synthesis (bottom). Other samples include
H2O (negative control) and intact testis (T) from animal
347 (positive control). Arrows indicate amplified
product from correctly processed Cre transgene (334 bp,
top) and amplified product for L7
(bottom). M refers to DNA markers.
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DISCUSSION
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Characterization of the transcriptional mechanisms regulating FSHR
expression has, to date, been limited to the proximal promoter region
and 5'-flanking sequences extending to -5,000 bp (18, 19, 20, 27, 28).
Previous studies using transient transfection analysis have shown that
all detectable regulatory sequences reside in the first 100 bp of this
genomic segment and that, within this region, the E box plays a
significant regulatory role (17, 19). In addition, in vitro
binding assays suggest that the USF proteins regulate FSHR
transcription through the E box (17, 19). To further elucidate
mechanisms important to FSHR transcription, we have examined the
ability of the USF proteins to modulate FSHR promoter function and the
contribution of the E box to in vivo regulation of the
gene.
Studies reported herein have yielded several important findings
regarding the mechanisms governing FSHR expression. Through
cotransfection analysis, we have shown that both USF1 and USF2 activate
FSHR transcription. Although the wild-type proteins failed to
significantly activate the FSHR promoter over the level due to
endogenous transcription factors, the effect of mutant USF proteins
strongly supported the conclusion that USF is involved in FSHR
transcriptional regulation. Mutants lacking the amino- terminal
transactivation domain (
N mutants) greatly inhibited FSHR promoter
function, and this inhibition was promoter specific and required the E
box (Fig. 1
), demonstrating that USF binds specifically to the FSHR
promoter in vivo. The inhibitory activity of the
N
mutants was attributed to a competition between the mutants and
endogenous USF proteins for binding to the E box. Thus, if proteins
other than USF were responsible for activation through the E box,
cotransfection of the wild-type USF proteins should have inhibited as
well. This conclusion is further supported by the observation that
cotransfection with expression vectors for wild-type c-Myc, another
bHLH-Zip protein that shares the same binding site requirements as USF,
inhibited FSHR promoter activity (data not shown). In addition, more
direct evidence for USF activation of the FSHR promoter was provided by
our finding that cotransfected USF1 and USF2 increased transcription
from promoters containing E box sequences having lower affinities for
the USF proteins. This also suggested that the inability of wild-type
USF to regulate the wild-type promoter was due to saturation of the E
box with endogenous USF proteins.
The transactivation domains of these important regulatory proteins were
mapped using a variety of USF1 and USF2 mutants. By evaluating the
ability of the mutants to inhibit promoter activity, three main
transactivation domains of USF2 and two for USF1 were identified. For
USF1, the first 130 amino acids appeared to be most critical for
transactivation, while modest affects were observed with a sequential
deletion through exon 5 and the USR. Similar to our findings, Roy
et al. (24) showed that deletion of the first 130 amino
acids significantly diminished transactivation by USF1, while further
deletion through the USR had little effect. Luo and Sawadogo (25) also
identified the amino-terminal portion of USF1 as critical for
transactivation, but their data support a more prominent role for the
USR. This difference may reflect the use of distinct promoters, which
has been shown to influence the mechanism of USF transactivation
(24, 25).
Activation of the FSHR promoter by USF2 required domains within the
first 40 amino acids of the protein, within the region encoded by exon
5, and within the USR. However, both the exon 5 region and the USR
appeared redundant with other sequences in the amino-terminal
domain, as deletion of either one alone did not alter FSHR promoter
function. Although previous studies also identified exon 5 and the USR
as containing important transcriptional activation domains, several
notable differences are apparent when comparing these earlier results
with those observed on the FSHR promoter (25). Thus, in contrast with
our results, two additional studies failed to reveal a significant role
for the first 40 amino acids of USF2 in transactivation (25, 26).
Interestingly, studies with USF1 identified this domain as important
but found that its activity depended on the context of the promoter
examined (24). Furthermore, Luo and Sawadogo found that deletion of the
USR alone abolished transcriptional activation by USF2, while in our
studies, function of the USR was only revealed when other
amino-terminal sequences were absent (25).
Our studies also examined a USF2 construct (U2
E4) that represents a
natural splice variant that is expressed at various levels relative to
the wild-type protein in different cell types (29, 30). Although no
transcriptional effect was observed with this mutant on either the FSHR
or the adenovirus major late promoter, studies on the major
histocompatibility complex (MHC) class I gene clearly identify exon 4
as encoding a critical activation domain (26). Thus, comparison of our
studies with others already published on USF transactivation domains
show an emergence of data implicating differences in the functional
activity of these proteins. For the FSHR promoter in MSC-1 cells, the
USF proteins appear to function somewhat differently than what has been
reported for the activation of either the adenovirus major late or
MHC promoters. Interestingly, earlier studies have shown that
the transactivation properties of the USF proteins are both cell type
and promoter context dependent, suggesting that these contribute to the
differences observed (24, 25, 31, 32). Currently, information on the
domains of USF proteins is limited, and a more thorough comparison on
mechanisms of USF transactivation awaits additional studies that employ
a variety of cell types and promoters.
The promoter region of the FSHR gene was examined in Sertoli cells
using in vivo genomic footprinting. Like in vitro
footprinting, this approach predicts that protected regions represent
sites of protein/DNA interaction and are important sites of
transcriptional regulation. Importantly, though, in vivo
footprinting has the added advantage of examining interactions within
living cells where the chromatin conformation and concentrations of
regulatory proteins have not been altered. The signal obtained from the
footprinted region depends on the endogenous occupancy level of the
site as well as the number of expressing cells in the preparation.
Analysis of the FSHR promoter region showed that the signal associated
with the two guanines within the E box was significantly lower in DNA
treated in vivo with DMS compared with DNA treated in
vitro. Moreover, in RCHO cells, no apparent difference was
observed between in vivo and in vitro footprints
with respect to the extent of methylation of this site. Examination of
other nonexpressing cell lines rendered the same results as the RCHO
cells (data not shown). Thus, despite the presence of the USF proteins
in these nonexpressing cells, they do not bind to the E box of the
endogenous FSHR gene.
To delineate the region of the FSHR gene needed for cell-specific
expression, we generated transgenic mice harboring two constructs
containing variable amounts of 5'-flanking sequence (5,000 bp and 100
bp). Eight different lines of animals carrying the 5,000-bp promoter
were examined, but none showed selective expression in testis or the
correct temporal profile for the FSHR gene. These results suggested
that sites outside this region are needed to appropriately restrict
expression of FSHR to the gonads and to activate the gene at the
appropriate time during development. Surprisingly, upon first
observation, a smaller region (-100) of gene appeared to better
restrict transgene expression, as three lines showed expression only in
the testis. However, in these same three lines, no transgene expression
was observed in the ovary, and closer inspection of the males revealed
that testis expression was due to inappropriate expression in the germ
cells.
In apparent contrast to our findings, previous studies had shown that
5,000 bp of the rat FSHR promoter could direct expression of a reporter
gene (ß-galactosidase) to the gonads in transgenic mice (18).
However, these earlier studies did not determine the cell types that
expressed ß-galactosidase in testis or ovary (histochemical and
immunohistochemical analysis for ß-galactosidase was inconclusive)
suggesting that, like our findings, expression in the germ cells may
have contributed to the majority of the signal in the gonads. In
addition, the fewer number of transgenic lines (2 vs. 16)
and tissues examined (6 vs. 11) and variation in the assay
sensitivity (Northern blot analysis vs. RT-PCR) may likely
have contributed to differences in the amount of ectopic expression
observed in the two studies. Thus, although these earlier studies
implicated the region from -5,000/+69 in cell-specific expression, the
studies presented here suggest that earlier conclusions may be
incorrect and that sequences outside this region are needed for cell-
specific expression. Nonetheless, it is important to note that
differences in the transgenic constructs (-5,000/+69 vs.
-5,000/+123 of promoter and different reporters) may also have
contributed to the differences in expression profiles.
Both FSHR and LH receptor (LHR) are members of the glycoprotein hormone
receptor subfamily within the superfamily of G protein-coupled
receptors (33, 34). Comparison of the gene structures for these
proteins suggested that they evolved from a common ancestral gene and
revealed that the promoters of each are TATA-less (28, 35, 36). LHR,
like FSHR, is expressed predominantly in cells of the ovary and testis,
where it is found in testicular Leydig cells and ovarian theca and
granulosa cells (13). Interestingly, recent studies with transgenic
mice carrying 2 kb of the murine LHR promoter revealed some remarkable
similarities with our FSHR transgenic studies (37). Thus, similar to
the FSHR transgenes, three of five LHR transgenic lines exhibited
expression within the testis but failed to express in the ovary, while
all lines showed ectopic expression in the brain. Also of notable
similarity were the observations that testis expression of the LHR
transgene was not seen in earlier stages of postnatal development
(before 5 weeks) and that it later appeared in the germ cell population
(elongating spermatids and spermatogonia). However, in contrast to our
studies, the investigators did observe transgene expression within the
appropriate cells (Leydig cells) of the testis, indicating that the LHR
promoter could recapitulate some of the regulatory features of the
endogenous gene. Thus, while both the LHR and FSHR promoters failed to
properly restrict expression of the transgenes, only the LHR promoter
was able to direct expression to one of the correct gonadal cells
(Leydig).
The studies in transgenic mice revealed several important findings.
First, the extensive amount of ectopic expression (predominantly brain
and germ cell) as well as two silent transgenes (nos. 252 and 303)
indicated that the FSHR transgenes were strongly influenced by adjacent
DNA sequences at the position of transgene integration. In addition,
since little or no Sertoli cell expression was detected, the elements
needed to confer and/or enhance cell-specific expression were absent
from the transgene construct. Thus, additional regulatory sequences are
needed to overcome chromatin influences at the site of integration and
to enhance transcription in Sertoli cells. The transgenic mouse
studies, together with the cotransfection and in vivo
genomic footprinting data, suggest a mechanism whereby sequences
outside the region from -5,000 to +123 alter the transcriptional
capabilities of the FSHR gene and permit or assist the occupancy of the
E box by the USF proteins (Fig. 7
). Thus,
in FSHR-expressing cells, proteins bound to distal regions of the gene
will likely be required for transactivation, while in nonexpressing
cells these proteins are either absent or nonfunctional on the FSHR
gene. The mechanisms involved in these transcriptional changes remain
to be determined, but may involve alterations in chromatin structure
that permit the USF proteins to bind the E box. Alternatively, proteins
bound to distal regulatory elements may directly interact with the USF
proteins and assist their interactions with the E box in the proximal
promoter. Identification of these regulatory sequences and their
binding proteins will be critical to our understanding of the
mechanisms regulating FSHR.

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|
Figure 7. Cell-Specific Expression of FSHR Requires Sequences
Outside of -5,000/+123
In FSHR-expressing cells (top), important regulatory
elements (distal elements) that reside outside of the region from
-5,000 to +123 bp are bound by proteins that alter the transcriptional
capabilities of the FSHR gene and permit access of the USF proteins to
the E box in the proximal promoter region. In nonexpressing cells
(bottom), proteins that bind these distal elements are
absent, the USF proteins are unable to interact with the E box, and the
gene is inactive.
|
|
 |
MATERIALS AND METHODS
|
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DNA Constructs
All rat FSHR promoter/luciferase constructs are described
elsewhere (17). USF expression vectors are described elsewhere (25).
Two Cre recombinase transgenes were constructed using different
portions of the rat FSHR gene. BSK-Cre-PolyA was generated by
subcloning an 1,100-bp fragment containing the Cre recombinase gene
with a nuclear localization signal (pBS317, a kind gift from Brian
Sauer) 5' to the PstI/EcoRI fragment of the bGH
gene containing part of exon 4, intron D, and exon 5 (polyadenylation
site) into pBluescript SK- (Stratagene, La Jolla, CA). The
FSHR -5,000-bp and -100-bp promoters were isolated by digestion with
SstI/XbaI of either FSHR(-5,000/+123)Luc or
FSHR(-100)Luc (described in Ref. 17) and cloned into the
XbaI/SstI sites of pBSK-Cre-PolyA 5' to Cre
recombinase/bGH sequences generating FSHR(-5,000)Cre and
FSHR(-100)Cre, respectively. Plasmid DNAs were prepared from overnight
bacterial cultures using DNA plasmid columns according to the
suppliers protocol (QIAGEN, Chatsworth, CA).
Transgenic Mouse Production
The 7,333- and 2,433-bp inserts of FSHR(-5,000)Cre and
FSHR(-100)Cre were excised from plasmid sequence by digestion with
SstI and XhoI and resolved by agarose gel
electrophoresis, and the fragments were isolated using Prep-A-Gene
matrix according to the manufacturers instructions (Bio-Rad Laboratories, Inc. Hercules, CA). DNA was eluted in 10
mM Tris, pH 7.5, 0.25 mM
EDTA. Transgenic founders were produced by pronuclear injection into
B6/SJL F1 zygotes performed by the Center for Reproductive Sciences
transgenic core at the University of Kansas Medical Center. The Cre
founders were back-crossed into a CD1 background for production of
transgenic offspring. All animal studies were conducted in accordance
with the principles and procedures outlined in "Guideline for Care
and Use of Experimental Animals."
Genotyping of Transgenic Mice
The genotypes of all offspring were analyzed by PCR of mouse
tail DNAs. Mouse tail cuts (
3 mm) were incubated in 20 µl lysis
buffer (50 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1
mM EDTA, 1% SDS) with 40 µg proteinase-K at 55 C for
3040 min. The mixture was briefly vortexed after 15 min. After
incubation, 178 µl of distilled water were added, and the mixture was
heated in a boiling water bath for 5 min. The sample was cooled to room
temperature, and 1.5 µl of the tail lysate were amplified by PCR (28
cycles for 40 sec at 94 C; 40 sec at 56 C; and 40 sec at 72 C) using
Cre-specific primers Cre1 5'-CTGGTCGAAATCAGTGCGTTC-3' and Cre2
5'-TTACCGGTCGATGCAACGAGT-3'. Positive animals were identified by the
presence of a 393-bp amplified product as observed by agarose gel
electrophoresis.
Analysis of Transgene Expression
F1 transgenic mice positive for either FSHR(-5,000)Cre or
FSHR(-100)Cre transgenes were killed and total RNA was isolated from
various tissues using TRIZOL reagent according to manufacturers
procedures (Life Technologies, Inc., Gaithersburg, MD).
Tissue-specific expression of the transgenes was evaluated using
RT-PCR. Complementary DNA was generated using Superscript reverse
transcriptase (Life Technologies, Inc.), 2 µg total RNA,
and 0.25 µg oligo-dT. Complementary DNA synthesis was performed in
both the presence and absence of reverse transcriptase. The cDNA was
used as a template in a PCR with intron-spanning primers specific for
the Cre recombinase and exon 5 of the bGH gene (upstream primer: Cre6,
5'-CTGGATAGTGAAACAG-GG-3'; and downstream primer: polyA5,
5'-GTACGTCTC-CGT-CTTAT-3'). This primer set was
used to distinguish between genomic DNA (a contaminant in the RNA
preparation) and the expressed transgene. In addition, each cDNA was
examined for the presence of the ribosomal protein L7 to confirm cDNA
synthesis (upstream primer: L7.1,
5'-GGAAAG-GCAAGGAGGAAGCA-3'; downstream primer: L7.2,
TCCTCCATGCAGATGATGC). Amplified products were examined by agarose gel
electrophoresis in which amplification of unprocessed transgene from
genomic DNA resulted in the presence of a 609-bp DNA fragment, while
cDNA generated from processed mRNA resulted in the amplification of a
334-bp fragment. In studies examining Cre recombinase expression in
mouse Sertoli cells and germ cells, samples were also examined for the
expression of FSHR using mouse-specific primers to the FSHR (mFSHR2
5'-GGGGAAGCTTTTGGAGGTAATAGAGGCAGAT-3' and mFSHR3
5'-GGGGTCTAGAGCCTTAAAATAGACTTGTTGCA-3'). In these samples, FSHR
was detected only in the Sertoli cell preparations.
Isolation of Mouse Sertoli and Germ Cells
Seminiferous tubules were prepared from mouse testes (from
either 27- or 50-day- old animals) as described elsewhere but with
slight modification (38). For isolation of mouse seminiferous tubules,
the order of collagenase and trypsin treatments was reversed. Tubules
were cultured in Hams F12 media containing 5% FBS, 1.47
mM L-glutamine, 1.5 mM HEPES, 1%
penicillin/streptomycin, and 3 µg/ml cytosine arabinoside at 37 C in
5% CO2. After 5 days, germ cells were collected
by dislodging the loosely adherent germ cells into the media with
several rounds of pipeting. The collected media were then subjected to
centrifugation (200 x g for 6 min) to pellet the germ
cells. Sertoli cells were collected 24 days later.
Transfection and Enzyme Analysis
The mouse Sertoli cell line MSC-1 (39) was seeded onto six-well
plates (35-mm/well) at a density of 250,000 cells per well. Unless
otherwise stated, cells were transfected with 1 µg luciferase
reporter, 50 ng pRL-TK, and 0.5 µg expression vector using 5 µl
lipofectamine reagent (Life Technologies, Inc.), as
described previously (17). pRL-TK expresses Renilla
luciferase from the herpes simplex virus thymidine kinase promoter and
was included to control for transfection efficiency (Promega Corp. Madison, WI). Sixty hours after transfection, the cells
were lysed and assayed for both firefly and Renilla
luciferase activities using the Dual-Luciferase Reporter Assay System
(Promega Corp.). Specifics of the transfection procedure
are described elsewhere (17, 40). Data were averaged over a minimum of
three independent experiments.
In Vivo Footprinting
Primary rat Sertoli cells were cultured on 150-mm culture dishes
as described previously (38). RCHO cells were grown as described (41).
Cells were treated with a concentration of 0.1% DMS
(Sigma-Aldrich Corp., St. Louis, MO) in prewarmed media
for 2 min, washed three times with PBS, and lysed to isolate genomic
DNA. Genomic DNA was isolated and extracted as described elsewhere
(42). Both in vivo DMS-treated DNA and control in
vitro treated DNA were prepared simultaneously and subsequently
cleaved at methylated guanines using piperidine (Sigma-Aldrich Corp.) diluted 1:10 in water. Piperidine was removed by repeated
DNA precipitation and resuspension in H2O. DNA
samples were resuspended in water and the concentration determined by
reading the absorbency at 260 nm. Isolated genomic DNA (2 µg) was
analyzed by ligand-mediated (LM)-PCR as described elsewhere
(42). Oligodeoxynucleotides used for the ligated linker are published
elsewhere (42). LM-PCR primers to the FSHR gene, designed according to
parameters and specification described in Ref. 42 are as follows:
+168 bp to +143 bp, Tm=60.7 C,
(AS) 5'-d (GACACAGCCAGTGATGACATCCAGAT)-3'; +151 bp to +123 bp,
Tm = 64.3 C, (AS)
5'-d(CATCCAGATCCCGTGCCCAAGAATGC)-3'; +151 bp to +118 bp,
Tm = 70.1 C, (AS)
5'-d(CATCCAGATCCCGTGCCCAAGAATGCCAGCAAGG)-3'. The first primer
extends a FSHR-specific promoter fragment. The second primer, in
combination with the linker primer (described in Ref. 42), amplifies
the fragments. The last primer is labeled to a high specific activity
with P32-ATP using T4 kinase (New England Biolabs, Inc., Beverly, MA) and used to label amplified
fragments. Amplified products were fractionated on a 8% denaturing
gel, dried, and analyzed by autoradiography. Optical densities were
quantified for autoradiography bands using Gel-Pro Analyzer image
analysis software (Media Cybernetics, Silver Spring, MD). To adjust for
loading differences across the gel, optical densities of seven
different bands were normalized, and the relative intensity of the E
box was calculated for each. The average of the seven values was then
used to determine the change in the optical density of the E box bands
between treated and naked DNA samples.
Preparation of Nuclear Extracts and EMSA
To prepare nuclear extracts, cells were washed with ice-cold
buffer (25 mM HEPES, pH 7.4, 1 mM
dithiothreitol (DTT), 1.5 mM EDTA, 10% glycerol) and
scraped from plates into the above buffer with 0.5 mM
phenylmethylsulfonyl fluoride (PMSF) added. Cells were lysed using 30
strokes of a Dounce homogenizer (B pestle), and the nuclei were
pelleted by centrifugation (16,000 x g) for 1 min. The
supernatant was removed and nuclei resuspended in extraction buffer (25
mM HEPES, pH 7.9, 1 mM DTT,
1.5 mM EDTA, 10% glycerol, and 0.5
M KCl). Nuclei were extracted on ice for 10
min, and then frozen on dry ice. Nuclei were then thawed and
centrifuged (85,000 x g) for 6 min. Supernatants were
immediately aliquoted and placed at -80 C. Protein concentration was
determined by the BCA method (Pierce Chemical Co.,
Rockford, IL) using BSA as a standard.
For EMSAs, nuclear extracts (610 µg protein) were incubated with 25
fmol of radiolabeled double-stranded oligonucleotide in the presence of
10 mM HEPES, pH 7.9, 3 mM MgCl2, 30
mM KCl, 0.5 mM DTT, 12% glycerol, 0.6
mM EDTA, 0.2 mM PMSF, 50 ng salmon sperm DNA, 1
µg dIdC, 10 µM ZnCl, and 1 µg/ml BSA in a 20 µl
reaction volume as described elsewhere (17). Addition of competitors or
antibodies to the reaction immediately preceded the addition of
extract. Reactions were incubated on ice for 10 min before addition of
probe, and then an additional 30 min on ice before being loaded onto
the gel unless otherwise noted. Protein-DNA complexes were resolved on
a 4% nondenaturing polyacrylamide gel (acrylamide:bis-acrylamide
= 40:1) run in 25 mM Tris (pH 8.5), 190 mM
glycine at 250 V for 1.5 h at 4 C. Gels were dried and analyzed by
autoradiography. Antibodies for USF1 (C-20)X, USF2 (C-20)X, and c-Myc
(N-262)X were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). These antibodies are supplied as rabbit
polyclonal IgG and were used directly as supplied by the manufacturer
at 1 µg IgG per binding reaction.
Western Blot Analysis
MSC-1 cells were transfected with 1.5 58 g of each USF
mutant as described above. Whole cell extracts were resolved on 10%
SDS-polyacrylamide gels (acrylamide:bis-acrylamide = 30:0.8) with
the discontinuous buffer formulation of Laemmli (43) and transferred to
nitrocellulose membranes (Bio-Rad Laboratories, Inc.
Hercules, CA) using a Mini Trans-Blot Electrophoretic Transfer Cell
(Bio-Rad Laboratories, Inc. Hercules, CA). Membrane-bound
proteins were probed overnight at 4 C with anti-USF1 or anti-USF2
antibodies, 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 Pharmacia Biotech, Arlington Heights, IL).
 |
ACKNOWLEDGMENTS
|
---|
We thank the Center of Reproductive Sciences at the University
of Kansas Medical Center and Wen-ge Ma for the generation of the
transgenic mice.
 |
FOOTNOTES
|
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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, Kansas 66160. E-mail:
lheckert{at}kumc.edu
This work was supported in part by NIH Grants R29HD-3521701A1 and
R03HD-35871 (to L.L.H.), RO1CA-79578 (to M. S.), and National
Research Service Award F32 HD-08500 (to M.F.D.).
Received for publication March 13, 2000.
Revision received June 23, 2000.
Accepted for publication August 9, 2000.
 |
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