B R I E F
R E P O R T
A Human FSHB Promoter SNP Associated With Low
FSH Levels in Men Impairs LHX3 Binding and Basal
FSHB Transcription
Courtney A. Benson, Troy L. Kurz, and Varykina G. Thackray
Department of Reproductive Medicine and the Center for Reproductive Science and Medicine, University
of California, San Diego, La Jolla, California 92093
FSH production is important for human gametogenesis. In addition to inactivating mutations in the
FSHB gene, which result in infertility in both sexes, a G/T single-nucleotide polymorphism (SNP) at
⫺211 relative to the transcription start site of the 5⬘ untranslated region of FSHB has been reported
to be associated with reduced serum FSH levels in men. In this study, we sought to identify the
potential mechanism by which the ⫺211 SNP reduces FSH levels. Although the SNP resides in a
putative hormone response element, we showed that, unlike the murine gene, human FSHB was
not induced by androgens or progestins in gonadotropes. On the other hand, we found that the
LHX3 homeodomain transcription factor bound to an 11-bp element in the human FSHB promoter
that includes the ⫺211 nucleotide. Furthermore, we also demonstrated that LHX3 bound with
greater affinity to the wild-type human FSHB promoter compared with the ⫺211 G/T mutation and
that LHX3 binding was more effectively competed with excess wild-type oligonucleotide than with
the SNP. Finally, we showed that FSHB transcription was decreased in gonadotrope cells with the
⫺211 G/T mutation compared with the wild-type FSHB promoter. Altogether, our results suggest
that decreased serum FSH levels in men with the SNP likely result from reduced LHX3 binding and
induction of FSHB transcription. (Endocrinology 154: 3016 –3021, 2013)
F
ollicle-stimulating hormone is produced specifically by
gonadotrope cells in the anterior pituitary and plays a
critical role in mammalian fertility. FSH is required for
ovarian folliculogenesis in females, whereas in males it
promotes spermatogenesis in conjunction with testosterone (1, 2). FSH is a heterodimeric glycoprotein composed
of an ␣-subunit that is shared in common with LH and
TSH, and a unique ␤-subunit specific to FSH that confers
biological specificity (3). Transcription of FSHB appears
to be a rate-limiting step in the production of mature FSH
and is regulated by several hormones, including GnRH
and activin (4, 5).
Mutations that inactivate the FSHB gene, resulting in
infertility, have been identified in both men and women,
including 3 men with azoospermia, small testes, and decreased serum FSH levels and 6 women with delayed puberty and isolated FSH deficiency (6 –12). In addition to
these inactivating mutations, a G/T single-nucleotide
polymorphism (SNP) at ⫺211 relative to the transcription
start site in the 5⬘ untranslated region of FSHB was associated with reduced serum FSH in European men from the
Baltics, Italy, and Germany (13–17). Serum FSH levels
were reduced 15.7% in G/T heterozygotes and 40% in TT
homozygotes when compared with GG homozygotes in a
cohort of Estonian men (13). Additionally, a significant
increase of G/T heterozygotes (25.1% vs 22.4%) and TT
homozygotes (2.4% vs 1.1%) was reported in patients
with male factor infertility compared with normal men
(18). A significant increase of TT homozygotes (2.5%) in
oligozoospermic versus normozoospermic men was also
found in an Italian study (16).
Because the ⫺211 G/T SNP is associated with reduced
serum FSH levels in men, we investigated the molecular
mechanisms responsible for the decreased FSH levels. Pre-
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
Received March 29, 2013. Accepted June 5, 2013.
First Published Online June 13, 2013
Abbreviations: AR, androgen receptor; ␤-gal, ␤-galactosidase; FOXL2, forkhead box L2;
HRE, hormone response element; luc, luciferase; SNP, single-nucleotide polymorphism.
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Endocrinology, September 2013, 154(9):3016 –3021
doi: 10.1210/en.2013-1294
doi: 10.1210/en.2013-1294
vious studies have demonstrated that the nucleotide in the
murine Fshb promoter corresponding to the ⫺211 nucleotide in the human promoter reduces steroid and activin
responsiveness (19 –22). Because the ⫺211 G/T SNP is
located in the midst of a putative hormone response element (HRE) that is conserved in mammals (19), it has been
suggested that the reduced FSH levels in men with the
⫺211 SNP are due to decreased steroid hormone responsiveness. However, unlike the murine promoter, we recently showed that the human FSHB promoter was not
significantly induced by progestins or activin in immortalized gonadotrope cells (22). Although insertion of a
Sma/mothers against decapentaplegic-binding element
into the human FSHB promoter was sufficient to increase
promoter responsiveness to activin, insertion of an HRE
into the promoter did not result in progestin responsiveness. These data suggest that another mechanism is responsible for the reduced FSH levels observed in men with
the ⫺211 SNP. In this study, we investigated what factor
regulates FSHB transcription via the FSHB promoter region containing the ⫺211 nucleotide. We also determined
how the SNP affects binding of the relevant factor to the
FSHB promoter and whether the SNP alters FSHB transcription in gonadotropes.
Materials and Methods
Plasmid constructs
Construction of the murine ⫺1000 Fshb-luciferase (luc) reporter plasmid was described previously (21). Insertion of the
⫺381 HRE into the human FSHB promoter was also described
previously (22). The human ⫺1028/⫹7 FSHB-luc plasmid was
provided by Dr Daniel Bernard. The QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, California) was used to
generate the ⫺211 G/T mutation in the human FSHB-luc plasmid. The rat androgen receptor (AR) expression vector, pSG5rAR was provided by Dr Jorma Palvimo (23), whereas the
pcDNA3-human LHX3 expression vector was provided by Dr
Simon Rhodes.
Cell culture
Cell culture was performed using the L␤T2 cell line, which has
many characteristics of mature, differentiated gonadotropes (24,
25). Cells were maintained in 10-cm plates in DMEM (Mediatech Inc, Herndon, Virginia) with 10% fetal bovine serum
(Omega Scientific Inc, Tarzana, California) and penicillin/streptomycin antibiotics (Gibco/Invitrogen, Grand Island, New
York) at 37°C and 5% CO2.
Transient transfection
L␤T2 cells were seeded at 4.5 ⫻ 105 cells per well on 12-well
plates and transfected 18 hours later, using Polyjet (SignaGen
Laboratories, Rockville, Maryland), following the manufacturer’s instructions. For all experiments, the cells were transfected
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with 400 ng of the indicated luc reporter plasmid and 200 ng of
a ␤-galactosidase (␤-gal) reporter plasmid driven by the herpes
virus thymidine kinase promoter to control for transfection efficiency. L␤T2 cells were switched to serum-free media containing 0.1% BSA, 5 mg/L transferrin, and 50mM sodium selenite 6
hours after transfection. After overnight incubation in serumfree media, the cells were treated with vehicle (0.1% ethanol),
100nM methyltrienolone (R1881) (NEN Life Sciences, Boston,
Massachusetts), or 100nM DHT (Sigma-Aldrich Co, St Louis,
Missouri) for 24 hours.
Luciferase and ␤-gal assays
The cells were washed with 1⫻ PBS and lysed with 0.1M
potassium phosphate buffer (pH 7.8) containing 0.2% Triton
X-100. Lysed cells were assayed for luc activity using a buffer
containing 100mM Tris-HCl (pH 7.8), 15mM MgSO4, 10mM
ATP, and 65␮M luciferin. The ␤-gal activity was assayed using
the Tropix Galacto-light assay (Applied Biosystems, Foster City,
California), according to the manufacturer’s protocol. A Veritas
Microplate Luminometer (Promega, Madison, Wisconsin) was
used for both assays.
Statistical analyses
Transient transfections were performed in triplicate, and each
experiment was repeated at least 3 times. The data were normalized for transfection efficiency by expressing luc activity relative to ␤-gal and relative to the empty pGL3 reporter plasmid to
control for hormone effects on the vector DNA. The data were
analyzed by Student’s t test for independent samples or 1-way
ANOVA followed by post hoc comparisons with the TukeyKramer honestly significant difference test using the statistical
package JMP version 10.0 (SAS, Cary, North Carolina). Significant differences were designated as P ⬍ .05.
Electrophoretic mobility shift assay
Human LHX3 was transcribed and translated using a TnT
coupled reticulolysate system (Promega). The oligonucleotides
were end-labeled with T4 polynucleotide kinase and [␥-32P]ATP,
and 2 ␮L L␤T2 nuclear extract or 4 ␮L TnT lysate was incubated
with 1 fmol 32P-labeled oligonucleotide at 4°C for 30 minutes in
a DNA-binding buffer [10mM HEPES [pH 7.8], 50mM KCl,
5mM MgCl2, 0.1% Nonidet P-40, 1mM dithiothreitol, 2 ␮g
poly(deoxyinosinic-deoxycytidylic) acid, and 10% glycerol]. After 30 minutes incubation, the DNA binding reactions were run
on a 5% polyacrylamide gel (30:1 acrylamide/bisacrylamide)
containing 2.5% glycerol in a 0.25⫻ Tris/Borate/EDTA buffer.
A rabbit Forkhead box L2 (FOXL2) (H-43) antibody (Santa
Cruz Biotechnology, Santa Cruz, California) and rabbit LHX3
(Lim3) antibody (U.S. Biological, Swampscott, Massachusetts)
were used to supershift specific protein-DNA complexes; rabbit
IgG was used as a control for nonspecific binding. The following
oligonucleotides were used for EMSA: ⫺360/⫺331 murine Fshb
5-AATTAAGACATATTTTGGTTTACCTTCGCA-3⬘, ⫺213/
⫺184 murine Fshb 5⬘-CATATCAGATTCGGTTTGTACAGAAACCAT-3⬘, ⫺229/⫺200 human FSHB 5⬘-CTGTATCAAATTTAATTTGTACAAAATCAT-3⬘, and ⫺229/⫺200
human FSHB with the ⫺211 G/T mutation 5⬘-CTGTATCAAATTTAATTTTTACAAAATCAT-3⬘.
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Benson et al
SNP Reduces LHX3 Binding and FSHB Transcription
Results and Discussion
Transcription of the human FSHB gene is not
regulated by androgens in gonadotrope cells
Although the human FSHB promoter does not appear
to be responsive to progestins (22), it is possible that it can
be induced by androgens. Because the ⫺211 nucleotide is
in the middle of a putative HRE, we tested whether human
FSHB transcription was altered by treatment with a synthetic androgen, R1881, in immortalized L␤T2 gonadotrope cells transfected with AR. As shown in Figure 1A, the
⫺1000 murine Fshb-luc was induced 4-fold by 100nM
R1881, whereas the ⫺1028 human FSHB-luc was not
induced. In addition, insertion of an HRE into the human
FSHB promoter did not increase androgen responsiveness. We also determined that the human FSHB promoter
was unresponsive to treatment with 100nM DHT (Figure
1B). In combination with our previous study demonstrating a lack of progestin responsiveness (22), our results
Endocrinology, September 2013, 154(9):3016 –3021
indicate that, unlike murine Fshb, human FSHB transcription is not induced by progestins or androgens in gonadotrope cells. Our results are also in agreement with several
studies that reported that testosterone treatment of
GnRH-deficient men did not result in increased FSH levels
(26 –28). Thus, it is unlikely that reduction of FSH levels
in men with the ⫺211 G/T SNP is due to a change in steroid
responsiveness of the FSHB promoter.
A
A
B
B
Figure 1. The human FSHB promoter is not induced by androgen
treatment in immortalized gonadotropes. The murine ⫺1000 Fshb-luc,
the human ⫺1028 FSHB-luc, or the human ⫺1028 FSHB-luc with a
consensus HRE (⫹ HRE) was transiently transfected into L␤T2 cells
along with 200 ng of AR. A, After overnight starvation in serum-free
media, cells were treated for 24 hours with 0.1% ethanol or 100nM
R1881. B, The cells were treated for 24 hours with 0.1% ethanol or
100nM DHT. The results represent the mean ⫾ SEM of 3 (A) or 4 (B)
experiments performed in triplicate and are presented as fold
androgen induction relative to vehicle. The different uppercase letters
indicate that mFshb-luc transcription is significantly different from
hFSHB-luc or hFSHB-luc ⫹ HRE using a 1-way ANOVA followed by
Tukey’s honestly significant difference post hoc test.
Figure 2. The LHX3 homeodomain protein binds to a ⫺221/⫺211
element in the human FSHB promoter. A, L␤T2 nuclear extracts were
incubated with a ⫺360/⫺331 murine Fshb oligonucleotide (oligo)
(lanes 1– 4), a ⫺213/⫺184 murine Fshb oligo (lanes 5– 8), or a ⫺229/
⫺200 human FSHB oligo (lanes 9 –12) and tested for complex
formation in EMSA. Protein-DNA complexes are shown, with IgG
control (lanes 2, 6, and 10), anti-FOXL2 antibody (lanes 3, 7, and 11),
and anti-LHX3 antibody (lanes 4, 8, and 12). Antibody supershifts (ss)
are indicated on the left and right. B, L␤T2 nuclear extracts were
incubated with a ⫺229/⫺200 human FSHB oligo (lane 15) and tested
for complex formation with competition from excess cold oligos
containing single base pair mutations ranging from ⫺222 to ⫺209
(lanes 1–14). Abbreviations: WT, wild type; mut, mutant; mut comp,
mutant competitor.
doi: 10.1210/en.2013-1294
A
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β
α
β
Figure 4. FSHB transcription is reduced with the ⫺211 G/T mutation
in the human FSHB promoter. The wild-type (WT) human ⫺1028
FSHB-luc and the ⫺211 G/T mutation (mut) were transiently
transfected into L␤T2 cells, as indicated. The results represent the
mean ⫾ SEM of 6 experiments performed in triplicate and are
presented as luc/␤-gal. *, FSHB-luc transcription is significantly
decreased with the ⫺211 G/T mutation compared with the wild-type
promoter using Student’s t test.
α
B
Figure 3. LHX3 binding to the human FSHB promoter is reduced with
the ⫺211 G/T SNP. Nuclear extracts from murine L␤T2 cells (A) or in
vitro transcribed and translated (TnT) human LHX3 (B) were incubated
with a ⫺229/⫺200 human FSHB oligonucleotide (oligo) (lanes 1–3 and
5–14) or a ⫺211 G/T mutant oligo (lane 4) and tested for complex
formation in EMSA. LHX3-DNA complex is shown in lane 1, with the
IgG control in lane 2 and the LHX3 supershift (ss) with an anti-LHX3
antibody in lane 3. Competition with 0.125 to 2 pmol of cold, wildtype ⫺229/⫺200 FSHB oligo (WT comp) or the ⫺211 G/T mutated
oligo is shown in lanes 5 to 9 and 10 to 14, respectively.
Abbreviations: WT comp, wild-type competitor; comp, competitor.
LHX3 binds to an element in the human FSHB
promoter that encompasses the ⴚ211 G/T SNP
Because the human FSHB promoter is not regulated by
androgens or progestins, we sought to identify factors necessary for FSHB transcription that are altered by the ⫺211
G/T mutation. In addition to steroid responsiveness, the
⫺196 nucleotide in the murine Fshb promoter, corresponding to the human ⫺211 nucleotide, is involved in
activin induction of Fshb (22). Because this region was
suggested to contain a FOXL2 binding element (29), we
tested whether this region binds the FOXL2 transcription
factor in EMSA. We used a 30-mer oligonucleotide from
⫺360/⫺331 of the murine Fshb promoter as a positive
control for FOXL2 binding because it contains a characterized FOXL2 binding element (29). As previously re-
ported, we showed that L␤T2 nuclear extracts formed a
protein-DNA complex with the ⫺360/⫺331 region of the
murine Fshb promoter (Figure 2A, lane 1) and that the
complex was diminished and supershifted with a FOXL2
antibody (Figure 2A, lane 3), indicating that this complex
contains FOXL2. A protein-DNA complex also formed
with the ⫺213/⫺184 region of the murine Fshb promoter
(Figure 2A, lane 5) but this complex was not supershifted
by the FOXL2 antibody (Figure 2A, lane 7). The proteinDNA complexes formed with the ⫺229/⫺200 region of
the human FSHB promoter were also not shifted with the
FOXL2 antibody (Figure 2A, lane 11), indicating that
FOXL2 does not bind to this region of the human or murine FSHB promoter. Because the LHX3 homeodomain
protein was reported to bind the porcine and human FSHB
promoters in this region and regulate basal transcription
of FSHB in gonadotropes (30), we tested whether the
LHX3 antibody could supershift the protein-DNA complexes. Interestingly, one of the complexes formed with
the ⫺229/⫺200 region of the human FSHB promoter was
supershifted by the LHX3 antibody, confirming that
LHX3 can bind to this region of the human FSHB promoter. The identity of the protein that binds to the corresponding region of the murine promoter (⫺213/⫺184)
remains unknown because the protein-DNA complex was
not supershifted by the LHX3 antibody.
Having demonstrated LHX3 binding to the ⫺229/
⫺200 region of the human FSHB, we then determined
whether the LHX3 binding element overlaps with the
⫺211 nucleotide using competition with single-nucleotide
mutations ranging from ⫺222 to ⫺209. Excess mutant
oligonucleotides from ⫺221 to ⫺211 were unable to completely compete with LHX3 binding to the wild-type
⫺229/⫺200 oligonucleotide (Figure 2B, lanes 2–12).
These results indicate that the LHX3 binding site at ⫺221/
⫺211 extends 3 base pairs (bp) past the previously pub-
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Benson et al
SNP Reduces LHX3 Binding and FSHB Transcription
lished 8-bp binding element (30) on the 3⬘ end to include
the ⫺211 nucleotide. The additional 3 bp makes the
LHX3 binding element in the human FSHB promoter the
same length as the LHX3-binding consensus sequence that
was defined in vitro by Bridwell et al (31).
LHX3 binding to the human FSHB promoter is
reduced with the ⴚ211 G/T SNP
Because the LHX3 binding element on the human
FSHB promoter encompasses the ⫺211 SNP, we tested
whether binding of LHX3 to the human promoter with the
⫺211 G/T mutation was reduced compared with the wildtype promoter. For these experiments, we used murine
LHX3 from L␤T2 nuclear extracts as well as a pcDNA3human LHX3 expression vector to generate in vitro transcribed and translated human LHX3 to determine
whether LHX3 binding to the human FSHB promoter was
altered by the ⫺211 G/T SNP. The human LHX3 expression vector results in the production of full-length LHX3a
as well as a truncated M2 form (32). As shown in Figure
3, murine LHX3, human LHX3a, and M2 bind to the
⫺229/⫺200 region of the human FSHB promoter (Figure
3, A and B, lane 1). To confirm that the protein-DNA
complexes contained LHX3, we supershifted the complexes with the LHX3 antibody (Figure 3, A and B, lane 3)
but not with the IgG negative control (Figure 3, A and B,
lane 2). Our results demonstrated that LHX3 bound with
greater affinity to the wild-type FSHB promoter compared
with an oligonucleotide containing the ⫺211 G/T mutation (Figure 3, A and B, lane 1 vs 4). Additionally, we
showed that LHX3 binding to the ⫺229/⫺200 oligonucleotide was more effectively competed with excess wildtype oligonucleotide than with the ⫺211 G/T mutant oligonucleotide (Figure 3A, lane 7 vs 12; Figure 3B, lane 8 vs
13). Collectively, our results support the hypothesis that
binding of LHX3 to the human FSHB promoter with the
⫺211 G/T SNP is reduced compared with the wild-type
promoter and that decreased LHX3 binding results in reduced FSHB transcription and serum FSH levels. These
results also provide supporting evidence that the LHX3
binding element includes the ⫺211 residue because competition with the ⫺211 G/T oligonucleotide is less effective
than the wild-type oligonucleotide.
Human FSHB transcription is impaired with the
ⴚ211 G/T SNP
Although our EMSA data indicated that LHX3 binding
to the ⫺211 G/T mutation is reduced compared with the
wild-type human FSHB promoter, a connection between
decreased LHX3 binding and FSHB transcription has yet
to be demonstrated in gonadotrope cells. To test this, we
performed transient transfection assays in L␤T2 cells to
Endocrinology, September 2013, 154(9):3016 –3021
determine whether basal transcription of human FSHB
was altered by the ⫺211 G/T mutation compared with the
wild-type promoter. As indicated in Figure 4, basal transcription of human FSHB was significantly reduced by
approximately 50%. This is a similar amount to what was
observed previously in 2 heterologous human cell lines,
JEG-3 and TE671 (33). Unlike basal transcription, GnRH
induction of FSHB was not altered by the ⫺211 G/T mutation (data not shown).
Because a decrease in basal FSHB transcription is observed with the ⫺211 G/T mutation compared with the
wild-type human promoter, our studies indicate that the
decreased serum FSH levels observed in men with the
⫺211 SNP are probably due to reduced LHX3 binding
and regulation of basal FSHB transcription. In one study,
men who were homozygotes for the T allele accounted for
approximately 25% of infertile men with low FSH levels
(16), suggesting that a genetic screen for the ⫺211 G/T
SNP may be a useful diagnostic tool for identifying a subset of infertile men who are good candidates for recombinant FSH therapy. Recombinant FSH has been shown to
increase sperm counts in infertile men with isolated FSH
deficiency (34, 35). It will also be interesting to determine
whether there is any association between the ⫺211 G/T
SNP and reduced FSH levels and infertility in women,
because FSH also plays a critical role in female
reproduction.
Acknowledgments
We thank Kellie Breen, Danalea Skarra, and Scott Kelley for their
suggestions and critical reading of the manuscript.
Address all correspondence and requests for reprints to: Varykina G. Thackray, PhD, Department of Reproductive Medicine,
University of California, San Diego, 9500 Gilman Drive, MC
0674, La Jolla, California 92093. E-mail: vthackray@ucsd.edu.
This work was supported by National Institutes of Health
(NIH) Grant R01 HD067448 to V.G.T. This work was also
supported by Eunice Kennedy Shriver National Institute of Child
Health and Human Development/NIH through a cooperative
agreement (U54 HD012303) as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research
and by a University of California, San Diego, Academic Senate
Health Sciences Research Grant.
Disclosure Summary: The authors have nothing to disclose.
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