CREB Binding Protein (CBP) Activation Is
Required for Luteinizing Hormone Beta
Expression and Normal Fertility in Mice
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Ryan S. Miller, Andrew Wolfe, Ling He, Sally Radovick and
Fredric E. Wondisford
Mol. Cell. Biol. 2012, 32(13):2349. DOI:
10.1128/MCB.00394-12.
Published Ahead of Print 16 April 2012.
CREB Binding Protein (CBP) Activation Is Required for Luteinizing
Hormone Beta Expression and Normal Fertility in Mice
Ryan S. Miller,a Andrew Wolfe,a,c Ling He,b Sally Radovick,a and Fredric E. Wondisfordb,c
Division of Pediatric Endocrinology, Department of Pediatrics,a Division of Metabolism, Department of Pediatrics,b and Department of Physiology,c Johns Hopkins
University School of Medicine, Baltimore, Maryland
R
eproductive viability in humans is dependent on normal function of the hypothalamic-pituitary-gonadal axis. Gonadotropin-releasing hormone (GNRH) is produced by hypothalamic
neurosecretory cells and released in a pulsatile manner into the
hypothalamo-hypophyseal portal circulation, through which the
hormone is transported to the anterior pituitary gland. GNRH
bound to its receptor on pituitary gonadotrophs results in an increase in GNRH receptor density and stimulates the synthesis and
secretion of luteinizing hormone (LH) and follicle-stimulating
hormone (FSH) into the circulation (14, 30, 41). LH and FSH are
heterodimeric glycoproteins consisting of a common ␣-subunit
and hormone-specific ␤-subunit encoded by the Lhb and Fshb
genes, respectively. Lhb and Fshb gene expression in the anterior
pituitary is dependent on pulsatile GNRH secretion. Rapid, highamplitude GNRH pulses stimulate an increase in Lhb mRNA levels, leading to an increase in LH synthesis and release from the
gonadotroph (11, 18, 45, 51).
At least three major transcription factors are important for Lhb
gene expression: steroidogenic factor 1 (SF-1), pituitary homeobox factor 1 (Pitx1), and early growth response factor 1 (Egr-1).
GNRH stimulates the expression of Egr-1, resulting in Egr-1 binding to two conserved cis elements of the proximal Lhb promoter
(5, 43, 49). Egr-1 is rapidly and markedly induced by GNRH, while
SF-1 and Pitx1 expression levels are unchanged following GNRH
administration (43, 49). In rat, Egr-1 expression increases in
proestrous, suggesting that it may be an important signal for ovulation (39). Loss of Egr-1 in the gonadotroph, unlike loss of SF-1
or Pitx1, renders the cell unable to respond to GNRH, and Egr-1
knockout mice have a selective defect in Lhb expression that is not
responsive to gonadectomy (13, 29). In contrast, pituitary glandspecific SF-1 knockout mice have markedly decreased expression
of LH and FSH, but they are able to produce LH in the pituitary
gland in response to exogenous GNRH (55). In the L␤T2 gonadotroph cell line, mutation in the PITX binding site of the human
Lhb promoter decreased basal, but not GNRH1-induced, transcriptional activity (13). These data indicate that while SF-1 and
July 2012 Volume 32 Number 13
Pitx1 enhance the transcriptional response, Egr-1 is the major
mediator of GNRH-stimulated Lhb gene expression.
CREB binding protein (CBP; Crebbp is the mouse gene
transcript [mRNA] for CBP) and the closely related protein
p300 have been identified as essential cofactors for many nuclear transcription factors. CBP was first identified as a transcriptional coactivator of CREB (7, 28). Protein kinase A (PKA)
phosphorylation of CREB at Ser133 promotes the formation of
a transcriptional complex on the cyclic AMP (cAMP) response
element (CRE) containing CREB, CBP, and CREB-regulated
transcription coactivator 2, resulting in activation of gluconeogenic genes in hepatocytes (36, 50). CBP is subsequently able to
activate gene transcription via intrinsic histone acetyltransferase activity and recruitment of other transcription factors
(7, 15, 16, 28, 33, 46). Conversely, insulin is able to induce PKC
phosphorylation of CBP at Ser436, a residue located near the
CREB binding domain of CBP (53). CBP phosphorylation at
Ser436 disrupts the complex on the CRE, freeing CBP to interact with other transcription factors (21).
We chose to study CBP action in the gonadotroph because
CBP can be phosphorylated by mitogen-activated protein kinase
(MAPK) and PKC, both of which are direct pathways for CBP
activation via GNRH and insulin (21, 24). It has been established
that gonadotrophs express insulin receptors on the cell surface
and that L␤T2 cells are able to bind insulin (4, 17). We and others
have demonstrated that insulin augments GNRH-mediated Lhb
expression and LH secretion primarily via induction of Egr-1 (1, 4,
40). Thus, CBP may function to integrate multiple inputs, repro-
Received 22 March 2012 Accepted 4 April 2012
Published ahead of print 19 April 2012
Address correspondence to Ryan S. Miller, rsmiller@jhmi.edu.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/MCB.00394-12
Molecular and Cellular Biology
p. 2349 –2358
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Normal function of the hypothalamic-pituitary-gonadal axis is dependent on gonadotropin-releasing hormone (GNRH)-stimulated synthesis and secretion of luteinizing hormone (LH) from the pituitary gonadotroph. While the transcriptional coactivator
CREB binding protein (CBP) is known to interact with Egr-1, the major mediator of GNRH action on the Lhb gene, the role of
CBP in Lhb gene expression has yet to be characterized. We show that in the L␤T2 gonadotroph cell line, overexpression of CBP
augmented the response to GNRH and that knockdown of CBP eliminated GNRH responsiveness. While GNRH-mediated phosphorylation of CBP at Ser436 increased the interaction with Egr-1 on the Lhb promoter, loss of this phosphorylation site eliminated GNRH-mediated Lhb expression in L␤T2 cells. In vivo, loss of CBP phosphorylation at Ser436 rendered female mice subfertile. S436A knock-in mice had disrupted estrous cyclicity and reduced responsiveness to GNRH. Our results show that GNRHmediated phosphorylation of CBP at Ser436 is required for Egr-1 to activate Lhb expression and is a requirement for normal
fertility in female mice. As CBP can be phosphorylated by other factors, such as insulin, our studies suggest that CBP may act as a
key regulator of Lhb expression in the gonadotroph by integrating homeostatic information with GNRH signaling.
Miller et al.
MATERIALS AND METHODS
Constructs. The Lhb promoter-luciferase reporter contains the ⫺140 to
⫹1 fragment of the mouse Lhb gene promoter cloned into pA3Luc proximal to luciferase as previously described (4). The wild-type CBP and
S436A mutant vectors were generated using the QuikChange site-directed
mutagenesis kit (Stratagene) as previously described (56). The PathDetect
in vivo signal transduction pathway trans-reporting systems (Agilent
Technologies) was used to generate a trans-activator protein consisting of
Egr-1 fused to the Saccharomyces cerevisiae GAL4 DNA binding domain
(DBD). The primers used to generate the Egr-1 insert were designed to
achieve in-frame translation of the Gal4 –Egr-1 fusion protein and to
avoid the Egr-1 start codon. The primer sequences were 5=-GTGTGGAT
CCGGGGCAGCGGCCAAGGCCGAGA (sense) and 5=-GTGTGAATTC
GGGTTAGCAAATTTCAATTGTC (antisense). Sequencing confirmed
the correct orientation of the insert in relationship to the Gal4 DBD.
Transfection. L␤T2 cells were maintained in Dulbecco modified Eagle
medium (DMEM; Cellgro) supplemented with 10% fetal calf serum. Cells
were plated at approximately 1 ⫻ 106 cells per well in a 6-well plate the
night prior to transfection. For experiments that did not require viral
transduction, cells were transfected using Lipofectamine 2000 reagent
(Invitrogen) the morning after being split. Medium was changed the following day to DMEM with 0.56 mmol glucose and 10% charcoal-resinstripped fetal bovine serum (FBS). The following morning, cells were
treated with 30 nM GNRH for the times indicated below prior to harvest.
Cells were harvested in either lysis buffer for luciferase activity, cell lysis
buffer (Cell Signaling) for protein assay, or TRIzol (Invitrogen) for RNA
isolation. For measurement of luciferase activity, 100 ␮l of cell lysate was
mixed with luciferin substrate and luminescence was measured with a
Lumat LB luminometer (Berthold Technologies).
Viral transduction. For CBP knockdown, cells were transduced with
adenovirus expressing short hairpin RNA (Ad-shRNA) against the 5= untranscribed region (5=UTR) of Crebbp (sequence 1, 5=-CACCGTTGCTGAGG
CTGAGATTTGGCGAACCAAATCTCAGCCTCAGCAAC; sequence 2,
5=-AAAAGTTGCTGAGGCTGAGATTTGGTTCGCCAAATCTCAGCC
TCAGCAAC). For Egr-1 knockdown, cells were transduced with AdshRNA against a region of the coding sequence of Egr-1 (sequence 1,
5=-CACCGGACTTAAAGGCTCTTAATACCGAAGTATTAAGAGCCT
TTAAGTCC; sequence 2, 5=-AAAAGGACTTAAAGGCTCTTAATACTT
CGGTATTAAGAGCCTTTAAGTCC). Virus was generated using the U6
entry vector system (Invitrogen) and verified by sequencing. Cells were
transduced either 24 h after being plated or 24 h after transfection. Medium was changed 24 h later, and assays were performed 48 h after transduction.
One-hybrid assay. L␤T2 cells were maintained as described above.
The morning after being plated on 6-well plates, cells under all conditions
were transduced with Ad-shRNA directed against the 5= UTR of CBP. The
following day, cells were transfected with the plasmids indicated below
using Lipofectamine 2000 according to the manufacturer’s protocol. All
cells were cotransfected with pFA-CMV expressing the GAL4 DBD–Egr-1
fusion protein (GAL4 –Egr-1) and the pFR-Luc reporter (Agilent). Cells
were also transfected with either an empty vector, wild-type (WT) CBP, or
S436A CBP, as indicated below. A subset of cells was transfected with the
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pFR-Luc reporter only and treated with GNRH to demonstrate a lack of
nonspecific activation of the reporter. Six hours after transfection, cell
culture medium was changed to DMEM containing 10% charcoal-resinstripped FBS. The following morning, cells were treated with 30 nM
GNRH for 4 h prior to being harvested in lysis buffer for the luciferase
assay performed as described above.
Quantitative PCR (Q-PCR). Following treatment with GNRH, cells
were harvested in TRIzol reagent and RNA was isolated according to the
manufacturer’s protocol. RNA was quantitated by spectrophotometry
and reverse transcribed into cDNA with the iScript cDNA synthesis kit
(Bio-Rad). Reverse transcription-PCR (RT-PCR) was performed using the
iQ SYBR green supermix on the MyiQ single-color real-time PCR detection
system module for the iCycler (Bio-Rad). RT-PCR was performed for Lhb,
Egr-1, and Crebbp mRNA using 36b4 RNA as a control. The following primers were used for RT-PCR: the Lhb forward primer 5=-AACTCTGGCCGC
AGAGAATG, the reverse primer 5=-GCAGTACTCGGACCATGCTA, the
Egr-1 forward primer 5=-CAGCGCCTTCAATCCTCAAG, the reverse
primer 5=-TCCACCATCGCCTTCTCATT, the Crebbp forward primer
5=-CACAGAACCAGTTTCCATCATCCAGT, the reverse primer 5=-CAT
GTTCAGAGGGTTAGGGAGAGCA, the 36b4 forward primer 5=-TGTT
TGACAACGGCATTT, and the reverse primer 5=-CCGAGGCAACAGTT
GGGTA.
ChIP. Chromatin immunoprecipitation (ChIP) was performed using
the ChIP-IT Express kit (Active Motif) according to manufacturer’s protocol. Following treatment with GNRH for the times indicated below,
cross-linking was performed with formaldehyde and cells were harvested
in lysis buffer. DNA was sheared by sonication, and chromatin was incubated with CBP antibody, Egr-1 antibody, or nonspecific IgG (Santa
Cruz). After washing of cells and reverse cross-linking, PCR was performed using Taq polymerase (Denville) for the proximal Lhb promoter
(positions ⫺307 to ⫺6) using the following primers: 5=-CTGAAAGCTT
CAGATCCTCAGAGCCT (sense) and 5=-TCCAAAGCTTATACCTTCC
CTACCTTC (antisense). The distal promoter (⫺1189 to ⫺893) primers
were 5=-AACAAGATGGGCATCCTGGATCGG (sense) and 5=-GATATC
CAGACACGTTCCTGTGGG (antisense). PCR products were detected
following electrophoresis on a 1% agarose gel. Q-PCR was performed as
described above, using primers for the proximal Lhb promoter. Q-PCR
data are corrected for input chromatin.
Western blot analysis. Proteins were resolved by SDS-PAGE on a 4
to 12% bis-Tris gradient gel (CBP) or 10% bis-Tris gel (Egr-1) and
transferred to an Immobilon P membrane. Immunoblotting was conducted using commercially available polyclonal CBP and Egr-1 antibody (Santa Cruz) or affinity-purified rabbit anti-phospho-serine
436-CBP generated by Invitrogen (Carlsbad, CA) against the peptide
PVCLPLKNA(pS)DKRNQQTIL (pS indicates phosphoserine) as previously reported (23). Blots were incubated with antibody overnight at 4°C,
and antibody binding was visualized using the ECL Plus reagent (Amersham Pharmacia, Piscataway, NJ).
Animal experiments and procedures. Animal protocols were approved by the Institutional Animal Care and Use Committee at Johns
Hopkins University. The generation of CBP S436A mice has been described previously (56). For breeding studies, 2- to 3-month-old mice
were observed over 60 days. Pups were removed and counted the day after
delivery. Vaginal cytology was assessed daily between 9 and 10 a.m. for the
numbers of days indicated below in cycling female mice that were individually housed starting 7 to 10 days prior to study. Vaginal cells were
collected with calcium alginate swabs soaked in 0.9% saline and dried on
a slide. Cells were fixed in methanol and stained with the Diff-Quick kit
(IMEB, Inc.) according to the manufacturer’s protocol. Stages were assessed according to predominant cell type (32). Blood was collected via
mandibular bleed in the morning for basal samples or 8 to 9 p.m. for surge
values. Serum LH and FSH levels were analyzed on the Luminex 2000IS
platform using the Milliplex rat pituitary panel (Millipore). Serum testosterone levels were analyzed by the Ligand Assay Core at the University of
Virginia. Ovaries and testes were collected from mice following CO2 as-
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ductive and metabolic, to regulate Lhb gene expression (26). As
prior studies have shown that Egr-1 and CBP can interact directly
via the transactivation domain of Egr-1 and the N terminus of
CBP to increase gene transcription, we hypothesized that CBP
interacts with Egr-1 in the gonadotroph to promote Lhb gene
expression (38). In current studies, we test the hypothesis that
CBP activation via phosphorylation is required for synthesis of
LH␤ and for normal gonadotroph function. Our results indicate
that GNRH-mediated phosphorylation of CBP is required for
Egr-1-mediated Lhb gene expression and is necessary for normal
function of the central reproductive axis.
CBP Activation is Required for Lhb Expression
an Lhb promoter-luciferase reporter and either pcDNA3.1 only or pcDNA containing full-length CBP. RLU, relative light units. (B) Relative levels of Lhb mRNA
expression were determined in L␤T2 cells that were transfected with either scrambled Ad-shRNA or CBP Ad-shRNA and then treated with GNRH 48 h later. (C)
Immunoblot of total CBP from L␤T2 cells transduced with either scrambled (Scr) or CBP Ad-shRNA. (D to F) Changes in transcript levels of Lhb, Egr-1, and
Crebbp were determined in L␤T2 cells following 4 h of GNRH treatment. ns, not significant. (G) Total CBP and phospho-CBP (p-CBP) (S436) protein levels
were detected by immunoblotting following GNRH treatment for the indicated periods of time. (H) Immunoblot of phospho-CBP with and without alkaline
phosphatase. Means at points without a common letter differ (P ⬍ 0.05). *, P ⬍ 0.01. Bars represent means ⫾ standard errors of the means (SEM).
phyxiation and perfusion with Bouin’s solution (Sigma). Ovaries were
sectioned every 100 ␮m and stained with hematoxylin and eosin by the
Johns Hopkins University Molecular and Comparative Pathobiology
Phenotyping Core. Gonadectomies were performed on 3- to 4-month-old
male mice under ketamine-xylazine anesthesia. Blood was collected 10
days later for LH and FSH.
Data analysis. Results of RT-PCR are expressed as fold changes in
gene expression from that of an untreated control. ChIP Q-PCR data are
displayed as fold enrichment versus an untreated control. Statistical analyses were performed using the Student t test for comparisons between two
groups. One-way analysis of variance (ANOVA) with the Student-Newman-Keuls post test was performed for all other comparisons. Statistical
analyses were performed using GraphPad InStat version 3.0 for Windows
NT (GraphPad Software, San Diego, CA). Differences were considered
significant at a P of ⬍0.05.
RESULTS
The transcriptional coactivator CBP is required for GNRHstimulated Lhb promoter activity in L␤T2 cells. We utilized the
L␤T2 mouse-derived gonadotroph cell line to determine if CBP was
required for GNRH-mediated activation of the Lhb promoter. Cells
were cotransfected with a mouse Lhb promoter-luciferase reporter
construct and either an empty vector (pcDNA3.1) or pcDNA3.1 containing full-length CBP. Overexpression of CBP did not significantly
change basal reporter activity but did result in greater GNRH-stimu-
July 2012 Volume 32 Number 13
lated activity than the empty vector (Fig. 1A). Fold change in Lhb
promoter activity was affected as well, as overexpression of CBP resulted in a 3.9-fold increase in reporter activity following treatment
with GNRH, versus a 2.5-fold increase in reporter activity in cells
transfected with the empty vector (P ⬍ 0.05).
As CBP overexpression was able to enhance the response to
GNRH, we next eliminated endogenous CBP to determine if this
would decrease responsiveness to GNRH. L␤T2 cells were transduced with adenovirus expressing either a scrambled shRNA or
shRNA against the 5= untranslated region of CBP. Cells that were
transduced with shRNA against CBP had no response to GNRH,
while cells transduced with scrambled shRNA demonstrated a 2.5fold increase in LH␤ mRNA following treatment with GNRH
(Fig. 1B). Western blotting demonstrated a nearly total knockdown of endogenous CBP in cells transduced with viral shRNA
against the 5=UTR of CBP (Fig. 1C).
CBP activation of the Lhb promoter is dependent on CBP
phosphorylation at Ser436. The above-described experiments
demonstrate that in the L␤T2 gonadotroph cell line, CBP is required for GNRH-mediated stimulation of Lhb expression. To
determine the mechanism by which GNRH activates CBP,
changes in the quantities of Lhb, Egr-1, and Crebbp mRNA were
measured by real-time RT-PCR following treatment of L␤T2 cells
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FIG 1 CBP mediates GNRH induction of Lhb expression in L␤T2 cells. (A) Luciferase activity was measured following GNRH treatment in cells transfected with
Miller et al.
Ser436. (A) Total CBP protein levels from whole-cell lysates after L␤T2 cells
were transduced with either scrambled shRNA or CBP 5= UTR Ad-shRNA,
followed by transfection with pcDNA3.1 only, pcDNA expressing WT CBP
(CBP-wt), or pcDNA expressing S436A mutant CBP (CBP-mut). (B) Relative
Lhb transcript levels in L␤T2 cells following transduction with adenovirus
expressing CBP 5=UTR shRNA and transfection with pcDNA3.1, WT CBP, or
S436A mutant CBP. Cells were treated with GNRH 4 h prior to the harvest of
RNA. *, P ⬍ 0.01 versus values for WT untreated, pcDNA-transfected, and
mutant untreated cells; #, P ⬍ 0.05. Bars represent means ⫾ SEM.
with GNRH. We found that while Egr-1 mRNA increased over
50-fold and Lhb mRNA increased nearly 3-fold, there was no
change in the level of Crebbp mRNA following treatment with
GNRH (Fig. 1D to F).
We then sought to determine if GNRH activated CBP by phosphorylation. Phosphorylation of CBP at Ser436 has been shown to
play an important role as a regulator of metabolism. We previously reported that insulin regulates hepatic gluconeogenesis
through CBP phosphorylation at Ser436 (21, 56). To determine
the role of CBP phosphorylation in the gonadotroph, we isolated
protein from L␤T2 cells at various time points following treatment with GNRH. By probing with an antibody specific to phospho-Ser436 of CBP, we were able to demonstrate that GNRH
stimulates CBP phosphorylation within 15 min without changing
total CBP protein (Fig. 1G). Treatment with alkaline phosphatase
eliminated the signal (Fig. 1H), demonstrating the specificity of
the antibody for the site phosphorylated by GNRH.
To determine if CBP phosphorylation at Ser436 is required for
GNRH stimulation of LH␤ promoter activity, L␤T2 cells were
transfected with a vector expressing either full-length wild-type
CBP or the mutant S436A CBP. To eliminate the possibility that
endogenous CBP could activate the promoter, cells were also
transduced with adenovirus expressing shRNA against the 5= UTR
of CBP. CBP protein was detected in cells transduced with scrambled Ad-shRNA and cells that were subjected to CBP knockdown
and reconstitution of either WT or S436A mutant CBP but not in
cells transduced with CBP shRNA and transfected with an empty
vector (Fig. 2A). Following knockdown of endogenous CBP and
overexpression of WT or S436A CBP, cells were treated with
GNRH and mRNA was harvested for real-time RT-PCR. Cells
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FIG 2 GNRH induction of Lhb expression requires CBP phosphorylation at
transfected with WT CBP showed a 2-fold increase in LH␤ mRNA
following treatment with GNRH, while cells transfected with the
empty vector or S436A CBP showed no response to GNRH (Fig.
2B). This shows that in L␤T2 cells, GNRH-mediated Lhb expression is dependent on phosphorylation of CBP at Ser436.
CBP interactions with Egr-1 on the proximal Lhb promoter
following GNRH treatment are phosphorylation dependent.
Previous studies have shown direct interactions between CBP and
Egr-1 in other systems (38, 44). As Egr-1 is critical to Lhb expression, we performed a chromatin immunoprecipitation (ChIP) assay to investigate the hypothesis that GNRH activation of CBP
results in recruitment of CBP to the proximal region of the Lhb
promoter containing Egr-1 binding elements. L␤T2 cells were
treated with GNRH for 30, 60, or 90 min followed by proteinDNA cross-linking. Following shearing and immunoprecipitation
with antibodies against Egr-1 or CBP, PCR was performed on
chromatin using a series of overlapping primers to detect binding
across the proximal 1,500 bp of the promoter. After 30 min of
treatment with GNRH, there was increased binding of CBP and
Egr-1 to the region of the promoter containing the proximal
GNRH-responsive elements (Fig. 3A). The distal region of the
promoter does not contain Egr-1 binding sites and showed no
increase in levels of bound CBP or Egr-1 following GNRH administration (Fig. 3A). This was also demonstrated by performing Q-PCR on chromatin samples using primers that span
the Lhb promoter region containing the proximal Egr-1 binding sites. We detected an increase in Egr-1 binding at 60 and 90
min and an increase in CBP binding at 90 min after GNRH
treatment (Fig. 3B).
We then sought to determine if the increase in CBP binding to
the Lhb promoter reflected a CBP–Egr-1 interaction occurring in
response to CBP phosphorylation but independent of changes in
Egr-1 level. To detect GNRH-mediated interactions between CBP
and Egr-1, we performed a modified mammalian two-hybrid assay with the full-length Egr-1 cDNA fused to the Gal4 DNA binding domain and a reporter construct containing the Gal4 consensus binding site (pFR-Luc). After knockdown of endogenous CBP
with shRNA, cells were transfected with vectors expressing either
WT or S436A mutant CBP. Following treatment with GNRH, cells
transfected with WT CBP stimulated greater reporter activity than
cells transfected with the empty vector or a vector expressing
S436A mutant CBP (Fig. 3C). Cells transfected with only the pFRLuc reporter showed only background-level activity. These results
indicate that CBP activation of Egr-1 requires CBP phosphorylation at Ser436 but does not require an increase in Egr-1 synthesis
(Fig. 3D).
The proximal Lhb promoter contains response elements to
other transcriptional promoters that could also interact with CBP.
We conducted a ChIP experiment after depleting L␤T2 cells of
Egr-1 to determine if CBP binding to the proximal Lhb promoter
is lost in the absence of Egr-1. L␤T2 cells were first transduced
with Ad-shRNA against Egr-1 and then treated with GNRH prior
to being cross-linked. Q-PCR was then performed using primers
to amplify the proximal Lhb promoter as described above. In cells
transduced with scrambled shRNA, Egr-1 and CBP binding to the
proximal promoter increased following GNRH treatment. In contrast, CBP binding to the proximal promoter did not increase in
cells transduced with Egr-1 Ad-shRNA (Fig. 3E). Immunoblotting
was performed on whole-cell lysate protein samples obtained
CBP Activation is Required for Lhb Expression
from L␤T2 cells 48 h after transduction with Egr-1 Ad-shRNA to
demonstrate target knockdown (Fig. 3F). These results indicate
that GNRH-mediated recruitment of CBP to the proximal promoter requires Egr-1 and suggest that CBP interacts directly with
Egr-1 on the Lhb promoter to promote Lhb transcription.
The S436A mutation in vivo results in impaired fertility in
female mice. To determine the importance of CBP phosphorylation in vivo, breeding studies were performed with mice containing a generalized S436A knock-in mutation. Breeding pairs
were established between WT and S436A mice to assess litter
size and intervals between litters. While results of pairings between WT female and S436A male mice did not differ from
those of WT ⫻ WT pairings, pairings consisting of a WT male
and a S436A female resulted in significantly fewer pups per
litter and longer times between litters than WT ⫻ WT pairings
(Fig. 4A and B). Pairings with an S436A female had fewer litters
over the course of 60 days, as demonstrated in Fig. 4C. This
indicates that loss of CBP phosphorylation affects fertility in
female mice but not in male mice.
July 2012 Volume 32 Number 13
S436A mice have abnormal cycling and a reduction in peak
LH levels. To assess further the reproductive defect due to the loss
of CBP phosphorylation, estrous cyclicity was evaluated in female
WT and S436A mice. Vaginal swabs were collected on 16 consecutive days and stained for cells indicative of cycle phase. Representative cycling data are shown for two WT and two S436A female
mice (Fig. 5A). Evaluation of ovarian histology by hematoxylin
and eosin staining showed significantly fewer corpora lutea and
more atretic follicles in S436A mice, indicating a disturbance in
cycling and ovulation as a consequence of the mutation in CBP
(Fig. 5B). S436A mice did not cycle normally; they spent a significantly greater proportion of time in diestrus and less time in
estrus than WT mice (Fig. 5C). Cycling data were then collected in
the morning on 5 consecutive days, and blood was collected in the
evening for LH and FSH levels during diestrus and proestrus to
capture gonadotropin levels during the LH surge in proestrus.
While LH levels in diestrus were similar among the two groups,
S436A mice did not have a significant increase in LH levels in
proestrus versus diestrus, and the mean LH value in proestrus was
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FIG 3 CBP–Egr-1 interactions on the proximal Lhb promoter in response to GNRH. (A, B) CBP and Egr-1 occupancy of the Lhb promoter in L␤T2 cells was
detected by chromatin immunoprecipitation following GNRH treatment for the indicated periods of time. PCR (A) and Q-PCR (B) were performed using
primers against both the proximal (⫺126 to ⫺26) and the distal (⫺1189 to ⫺893) Lhb promoter. *, P ⬍ 0.01 versus untreated cells. (C) L␤T2 cells were
transduced with Ad-shRNA against the 5=UTR of CBP, followed by transfection with the pFR-Luc reporter. Cells were also transfected as indicated with pcDNA
only, WT CBP, or S436A CBP. Luciferase (Luc) activity was then measured after treatment with GNRH. *, P ⬍ 0.01 versus values for WT untreated cells and other
conditions; #, P ⬍ 0.05. (D) Model of postulated CBP–Egr-1 interactions in the modified two-hybrid system. (E) CBP and Egr-1 occupancy of the Lhb promoter
in response to GNRH was detected by chromatin immunoprecipitation in L␤T2 cells 48 h after Egr-1 knockdown with Ad-shRNA. Real-time quantitative PCR
was performed using primers against the proximal (⫺126 to ⫺26) Lhb promoter. *, P ⬍ 0.01. (F) Immunoblot showing Egr-1 protein following transduction of
L␤T2 cells with scrambled or Egr-1 Ad-shRNA. Bars represent means ⫾ SEM.
Miller et al.
per litter was observed over 60 days. Means without a common letter differ (P ⬍ 0.01). (B) Lengths of time to the first litter and between the first and second litters
were measured for each breeding pair. Means without a common letter differ (P ⬍ 0.05). Bars represent means ⫾ SEM. (C) Total number of litters and pups
produced over 60 days for each breeding pair category. Each line represents values for one breeding pair, and each “⫻” represents a litter. M, male; F, female.
There were 10 breeding pairs per group.
significantly higher in WT mice than in S436A mice (15.7 ng/ml
versus 3.4 ng/ml; P ⬍ 0.01) (Fig. 5D). In contrast, FSH levels in
diestrus were similar among WT and S436A mice, and both
groups demonstrated similar increases in FSH on the day of proestrus (Fig. 5E).
Our investigations of the S436A mutation in mice also demonstrated a defect in GNRH responsiveness in males, despite apparent normal fertility. In the basal state, male S436A mice did not
exhibit abnormalities. Morning LH, FSH, and testosterone levels
did not differ between WT and S436A male mice (Fig. 6A), and in
S436A mice, testicular histology appeared grossly normal (Fig.
6B). To determine if male S436A mice respond normally to
GNRH, we measured basal serum LH and FSH levels in mice that
had been gonadectomized 10 days prior. In gonadectomized mice,
FSH levels were not different, but LH levels were significantly
higher in WT than in S436A mice (Fig. 6C). LH was then measured before and 10 min after subcutaneous administration of
GNRH to intact males. While there was a 6.6-fold increase in LH
in WT mice, we detected only a 1.3-fold increase in LH in S436A
mice (Fig. 6D). FSH did not increase following GNRH administration in either WT or S436A mice. These data indicate that loss
of CBP phosphorylation results in a defect at the pituitary level
that cannot be overcome by GNRH stimulation.
DISCUSSION
In this report, we show that the transcriptional coactivator CBP is
critical to Egr-1-mediated activation of Lhb expression in pituitary gonadotrophs and plays a critical role in reproductive function in female mice. In vitro studies were conducted in L␤T2 gonadotrophs, as these cells have been used extensively to determine
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mechanisms of Lhb gene regulation (12, 27, 34, 37, 42, 45, 48). The
proximal Lhb promoter contains an enhancer region with two
Egr-1 binding sites and two SF-1 binding sites separated by a Pitx1
binding site (19, 25, 47). In L␤T2 cells, one transcriptional coactivator, small nuclear RING finger protein (SNURF), has been
shown to interact with Sp1 (found in the distal enhancer region)
and SF-1 but not Egr-1 (8).
CBP functions as a coactivator for many transcription factors
and can be phosphorylated by a number of different signaling
pathways (21, 24, 46). While CBP and the related protein p300
have overlapping functions and high sequence homology, p300
lacks the consensus PKC phosphorylation site, conferring unique
functions to the two proteins. Mice heterozygous for either a CBP
or p300 knockout are viable, but homozygotes are embryonic lethal, as are CBP p300 compound heterozygotes (16). There is considerable evidence that CBP and p300 are present in cells at limiting concentrations, providing a potential mechanism for tight
regulation of Lhb expression (22, 35). In gonadotrophs, GNRH
signaling via PKA phosphorylation results in CREB phosphorylation. This event may recruit CBP to the CRE on the Cga gene
promoter in rodents but not in humans, as the CRE is not present
in the proximal human Cga promoter (37). According to our
model, GNRH acting via PKC signaling pathways activates CBP
via phosphorylation, allowing CBP to interact with Egr-1 on the
Lhb promoter, thus permitting expression of Lhb (Fig. 7).
While our studies have not determined the exact mechanism
by which CBP phosphorylation promotes interaction with Egr-1,
a previous study using CBP protein fragments fused to GAL4 in a
two-hybrid system showed that the N terminus of Egr-1 was able
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FIG 4 Assessment of fertility in S436A mice. Breeding pairs were established and observed for numbers of litters produced over time. (A) The number of pups
CBP Activation is Required for Lhb Expression
to physically interact with both the N and C termini of CBP, including the region containing Ser436 (38). Nuclear magnetic resonance (NMR) structure studies suggest a potential mechanism
by which CBP phosphorylation allows the protein to interact with
transcription factors. The TAZ1 domains of CBP and p300, which
share 92% sequence identity, contain a serine at position 436 in
CBP, versus a glycine at position 422 in p300. The fourth ␣-helix
of the TAZ1 domain was found to be longer in a CBP-transcription factor complex, owing to the presence of the helix-destabilizing glycine residue at the end of the p300 TAZ1 domain (9). Thus,
CBP phosphorylation in this domain may alter the protein conformation in a way that would allow it to interact with transcription factors such as Egr-1.
Using complementary methods of CBP overexpression and
knockdown via shRNA, we demonstrated that CBP is required for
GNRH-mediated Lhb expression in the L␤T2 cell line. This is
consistent with the results of other studies that have identified
CBP as an important regulator of pituitary glycoprotein hormone
subunit genes. In GH3 cells, CBP was shown to mediate thyroidreleasing hormone stimulation of both the alpha glycoprotein
(Cga) and thyroid-stimulating hormone beta (Tshb) promoters,
with distinct CBP domains required for activation of each gene
July 2012 Volume 32 Number 13
(20). CBP was also shown to mediate T3-dependent repression of
human Cga expression in a heterologous cell line (54). In each
case, CBP was shown to interact with promoter-specific transcription factors (P-Lim, Pit-1, and p53) to regulate expression of both
alpha and beta glycoprotein subunits via distinct domains.
While we showed that CBP is required for GNRH-mediated
Lhb expression, treating L␤T2 cells with GNRH did not change
levels of CBP mRNA or protein but did result in rapid phosphorylation at Ser436. In order to test the functional significance of the
serine phosphorylation site, we studied GNRH responsiveness in
L␤T2 cells expressing either WT or S436A mutant CBP. Initial
experiments did not indicate a difference in GNRH responsiveness between WT protein and phosphorylation mutant protein.
This is possibly because in cells transfected with mutant CBP,
enough endogenous CBP was present to permit normal functioning of the cell. This is also consistent with results of prior studies
that suggest that CBP can signal efficiently in limited concentrations (7, 26). By transducing cells with viral shRNA directed
against the 5=UTR of CBP prior to overexpression, we were able to
eliminate endogenous CBP while preserving WT and S436A transcripts produced by the pcDNA vector. Using this approach, we
were able to demonstrate that while basal levels were unaffected by
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FIG 5 Assessment of the reproductive phenotype of S436A mice. (A) Vaginal cytology was performed at 24-h intervals by microscopic evaluation of stained
vaginal smears, and the predominant cell type was recorded for each day. Data are shown for two representative animals from each group. (B) Hematoxylin- and
eosin-stained histological sections of ovaries are shown from wild-type and S436A knock-in mice. Scale bars, 200 mm. (C) The proportion of time in diestrus
versus estrus was recorded for each group. There were 7 to 8 mice per group. (D, E) Evening serum LH (D) and FSH (E) levels were measured in diestrus or
proestrus for WT or S436A mice. There were 4 to 6 mice per group. *, P ⬍ 0.01; #, P ⬍ 0.05. Bars represent means ⫾ SEM.
Miller et al.
were 13 to 16 mice per group. No significant differences were detected. (B) Hematoxylin- and eosin-stained histological sections of testes are shown from
wild-type and S436A knock-in mice. Scale bars, 200 mm. (C) Basal morning serum LH and FSH levels were measured in WT and S436A male mice 10 days
following gonadectomy. There were 9 to 10 mice per group. (D) The fold change in serum LH was measured following subcutaneous administration of GNRH
to WT and S436A male mice. GNRH stim, GNRH stimulation. There were 7 mice per group. *, P ⬍ 0.05 versus WT. Bars represent means ⫾ SEM.
the loss of WT CBP, cells expressing the S436A mutant CBP are
unable to increase Lhb transcription in response to GNRH.
Our studies also demonstrate that CBP likely promotes Lhb
expression via interactions with Egr-1, the most critical transcription factor for Lhb. We identified Egr-1 as a target of CBP coactivation for several reasons. First, CBP has been shown to directly
interact with Egr-1. CBP overexpression increases Egr-1-mediated activation of a 5-lipooxygenase reporter construct in COS
cells; conversely, Egr-1 can also activate CBP, resulting in acetylation and stabilization of Egr-1 (38, 52). Additionally, both CBP
and Egr-1 are activated by PKC. PKC activation has been shown to
increase Egr-1 expression levels in gonadotrophs following
GNRH administration (5, 19), while atypical PKC has been shown
to phosphorylate CBP in hepatocytes following administration of
insulin (21). Consistent with studies showing CBP–Egr-1 interactions, we found that treatment of L␤T2 cells with GNRH increases
binding of CBP to the proximal Lhb promoter in the region con-
FIG 7 Model of proposed CBP interactions on the cAMP response element
(CRE) and proximal Lhb promoter.
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taining Egr-1 binding sites. This promoter region also contains
binding sites for other transcription factors, including a response
element for SF-1, which is known to interact with CBP. However,
we showed that CBP was not able to bind the proximal Lhb promoter after Egr-1 knockdown. Thus, while it is possible that CBP
interacts with other transcription factors on the Lhb promoter,
CBP is not able to bind the promoter without Egr-1. This underscores the critical role for Egr-1–CBP interaction on the proximal
Lhb promoter in response to GNRH.
We also found that phosphorylation of CBP at Ser436 was critical
for Egr-1 activation, as we showed in a modified two-hybrid system
that treatment of cells with GNRH resulted in activation of Egr-1 in
cells expressing WT CBP, but not in cells expressing S436A mutant
CBP. This experiment showed that CBP activated Egr-1 via interactions on the promoter, rather than by increasing Egr-1 mRNA levels,
as the quantity of Egr-1 in this system remains fixed.
In the present study, we extended our findings from L␤T2 cells
and demonstrated the requirement of CBP for gonadotroph function in vivo. While the S436A mutation impairs fertility only in
female mice, we detected a lack of GNRH responsiveness in both
males and females. Breeding pairs containing an S436A mouse
had both fewer litters over time and fewer pups per litter. In contrast, breeding pairs containing an S436A male exhibited normal
fertility. This was not entirely surprising, as Lee et al. previously
demonstrated female infertility but not male infertility in Egr-1
knockout mice (29). The reduction in litter size seen with S436A
mice paired with normal fertility in male mutant mice suggests
that there is a threshold of Lhb expression required for normal
gonadal function.
We then tested peak LH responses in males and females and
measured estrous cycling in females to determine if there was ad-
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FIG 6 Assessment of phenotype in male mice. (A) Basal morning serum LH, serum FSH, and testosterone levels were measured in WT and S436A mice. There
CBP Activation is Required for Lhb Expression
July 2012 Volume 32 Number 13
and metformin act directly on gonadotrophs to alter reproductive
function in obesity-related infertility.
ACKNOWLEDGMENTS
This work was supported by Eunice Kennedy Shriver NICHD/NIH
grant U54 HD 58820 (F.E.W., S.R., and A.W.) and NIDDK/NIH grant
K08DK078644 (R.S.M.), Baltimore DRTC NIH grant P60 DK79637
(F.E.W., S.R., and A.W.), and NIH grant R2479637 (F.E.W.).
We thank Katie Moore for her work on the one-hybrid assay and
pCBP Western blots, Pamela Mellon for providing the L␤T2 cell line,
Cory Brayton and Nadine Forbes for mouse dissection, slide preparation,
and tissue staining, Kristen L. Lecksell for slide scanning, and the Ligand
Assay and Analysis Core of the University of Virginia Center for Research
in Reproduction for performing the testosterone assay.
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equate LH secretion to produce a proestrus surge. While S436A
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S436A mice were unable to respond normally to GNRH.
Our findings are the first to identify the critical role of CBP in
gonadotroph function. As a transcriptional coactivator, CBP can
be activated by a number of different signaling pathways, including MAPK and PKC. As such, CBP may act as an integrator of
signals in the gonadotroph, providing an additional level of responsiveness in the pituitary gland. Our group has previously
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