SP1/SP3-Binding Sites and Adjacent Elements Contribute to Basal and Cyclic Adenosine 3',5'-Monophosphate-Stimulated Transcriptional Activation of the Rhesus Growth Hormone-Variant Gene in Trophoblasts
Judith T. Schanke1,
Maureen Durning,
Kimberly J. Johnson2,
Lindsey K. Bennett and
Thaddeus G. Golos
Wisconsin Regional Primate Research Center and Department of
Obstetrics and Gynecology University of Wisconsin Medical
School University of Wisconsin Madison, Wisconsin
53715-1299
 |
ABSTRACT
|
---|
Transcriptional activation of the rhesus monkey
GH-variant gene in syncytiotrophoblasts is developmentally regulated by
trophoblast-specific and cAMP-responsive mechanisms. Progressive
deletions of 5'-flanking DNA defined the most proximal 140 bp as the
minimal region retaining full cAMP-stimulated mGH-V transcription. To
identify the regions of this promoter critical for transcription,
transient transfections of reporter plasmids containing systematic 10
base mutations throughout this proximal region were performed. Mutation
of the region from -140/-131 decreased transcription in
syncytiotrophoblasts by 50%, and gel mobility-shift analyses
demonstrated that Sp1 and Sp3 bound to a region containing a GGGAGG
motif at -136/-131. Mutation of the -62/-53 region decreased
transcriptional activation by 6699%, and Sp1 and Sp3 bound to a
GGTGGG motif overlapping this region (at -65/-60). Selective mutation
of this Sp1/Sp3 site decreased basal transcription by approximately
80%, and cAMP-stimulated transcription by up to 75% (with the
greatest effect in primary syncytiotrophoblast cultures), indicating
that the Sp1/Sp3 site is critical for transcriptional activation.
Mutations in the regions adjacent to the Sp1/Sp3 sites (-130/-111 and
-52/-43) also dramatically reduced (by 75%) transcriptional
activation in trophoblasts. We conclude that two Sp1/Sp3 sites as well
as additional elements directly adjacent to these sites contribute to
trophoblast-specific cAMP-responsiveness of the mGH-V proximal
promoter.
 |
INTRODUCTION
|
---|
Gene expression is regulated by the interplay of both ubiquitous
and cell-specific transcription factors. The varied expression and
activation of these factors during cell differentiation and development
control the pattern of cellular gene transcription and ultimately
determine cell phenotype. The placental trophoblast is an excellent
model for investigating the developmental and cell-specific regulation
of gene transcription. In primates, the trophoblast is the cellular
compartment responsible for the endocrine activity of the placenta and
secretes a vast array of hormones and regulatory factors, including
steroids, peptides, cytokines, growth factors, and placenta-specific
protein hormones (1). The primate placenta is unique in that it
expresses placenta-specific members of the GH gene locus, the chorionic
somatomammotropins (CS) and placental GH-variant (GH-V) (2, 3, 4). Roles
for both CS and GH-V in the regulation of maternal/fetal energy balance
and metabolism have been proposed (5, 6). The expression of these genes
is related to the state of differentiation of trophoblasts and is
predominantly localized to the terminally differentiated
syncytiotrophoblast (STB) (7, 8, 9). The expression of GH receptors in the
placenta (10, 11), as well as the observation that GH-V is the primary
somatotropin in maternal blood during the second half of pregnancy (5),
underscores the importance of characterizing the regulation of GH-V
expression throughout primate pregnancy. However, the mechanisms
controlling the tissue specificity and developmental regulation of GH-V
expression are not well understood.
We have previously described the transcriptional regulation of the
placental GH-V gene in primary cultures of rhesus monkey STBs (12) and
have demonstrated that transcriptional activation by cAMP is
developmentally regulated as well as trophoblast specific (12). The
gene is not responsive to cAMP in cells from the first trimester of
pregnancy, when endogenous gene expression is low, but is strongly
up-regulated by cAMP in cells from placentas obtained during the second
or third trimester, when expression is maximal in vivo (12, 13). Thus, the activation by cAMP in vitro coincides with
developmental activation of this gene during normal pregnancy.
In the current study we have examined the locus of cAMP activation of
monkey GH-V gene transcription in both trophoblast and nontrophoblast
cell lines to begin to identify the transcription factors responsible
for this activation. Our results define the promoter of the mGH-V gene
and demonstrate a critical role for multiple regions in cAMP-activated
transcription. Two elements were identified that bind the zinc finger
transcription factors Sp1 and Sp3. Mutagenesis studies demonstrated
that the distal and proximal Sp1/Sp3-binding elements contribute to
transcriptional activation of the mGH-V gene. Additionally, elements
immediately adjacent to each Sp1/Sp3 site also are critical for both
basal and cAMP-stimulated transcription in placental cells.
 |
RESULTS
|
---|
The Majority of the cAMP-Responsive mGH-V Promoter Activity Resides
within the Most Proximal 140 Bases of 5'-Flanking DNA
To identify the specific regulatory regions responsible for mGH-V
transcriptional activation by cAMP (12), deletions were introduced
throughout the 492-bp 5'-flanking region of mGH-V. These constructs
were transiently transfected into the choriocarcinoma cell line JEG-3
and primary cultures of rhesus monkey cytotrophoblasts that
differentiate in culture to STBs (14). Figure 1
, A and B, illustrates that the
fold-activation by 8-Br-cAMP of luciferase constructs, containing as
little as 140 bp of 5'-flanking DNA, was equivalent to activation of
the parent 492 bp construct when transfected into JEG-3 cells or second
and third trimester STBs, respectively. However, responsiveness to cAMP
decreased dramatically upon further deletion. Deletions from -140 to
-107 and -107 to -66 resulted in up to 50% and 90% losses in
transcriptional activity, respectively, in STBs (Fig. 1B
), and 30% and
50% losses in activity in JEG-3 cells (Fig. 1A
). Transcription is
equivalent to a promoterless reporter in the absence of the TATA
element (deletion to -19).

View larger version (32K):
[in this window]
[in a new window]
|
Figure 1. Localization of Regions Conferring Transcriptional
Activation by Deletion of the 5'-Flanking DNA of the Rhesus Monkey GH
Variant Gene
Luciferase constructs containing 19492 bp of 5'-flanking mGH-V
DNA were transiently transfected into JEG-3 choriocarcinoma cells (A
and C), primary cultures of rhesus STBs during the second or third
trimesters of pregnancy (B and D), or nonplacental COS-7 cells (E and
F). Panels A, B, and E present the fold induction of each construct by
8-Br-cAMP. Panels C, D, and F present the basal luciferase activity of
each construct, relative to the parental 492-bp construct. The results
presented are the means of five to seven individual experiments, with
each treatment group represented by triplicate dishes in each
experiment.
|
|
Deletions down to 140 bp did not decrease basal transcription; in fact,
the 140-bp construct had higher basal activity than the parent 492-bp
construct in JEG-3 cells (Fig. 1C
). A reproducible increase in all cell
types was seen upon deletion from -295 to -214 (discussed further
below). However, deletions from -140 to -107, and further deletion to
-66, decreased basal transcription by 73% and 88%, respectively, in
STBs, compared with the full-length construct (Fig. 1D
). Similar
results were seen with -140, -107, and -66 constructs in JEG-3 cells
(Fig. 1C
). Thus, basal as well as cAMP-stimulated transcriptional
elements are located within 140 bp of mGH-V 5'-flanking DNA.
To examine the differences in transcriptional activation between
placental and nonplacental cells, we transfected COS-7 monkey kidney
cells with the same deletion constructs. Although there is essentially
no responsiveness to cAMP treatment (Fig. 1E
), basal transactivation in
nonplacental cells is localized to the same region as described for
cAMP-responsive transactivation in trophoblasts (Fig. 1F
).
A notable decrease in cAMP-stimulated transcriptional activity in JEG-3
cells and STBs was seen upon deletion from -338 to -295 (Fig. 1
, A
and B). Basal as well as cAMP-stimulated transcriptional activation
typically rebounded, however, upon subsequent deletion to -214 (Fig. 1
, C, D, and F), suggesting the presence of a negative regulatory
element between -295 and -214. Since this rebound in GH-V
transcription was found in all cells, this is not a cell-specific
phenomenon and may be the result of the previously noted negative
regulatory element in the rat GH gene upstream silencer-1 region
(15).
Scanning Mutagenesis of the 140-bp mGH-V Promoter Identifies
Multiple Functional Regions
To more precisely define the functional elements within the 140-bp
mGH-V promoter that confer transcriptional activation, 10-bp block
mutations were introduced that scan the entire region from -140 to
-33. Reporter gene constructs containing these mutated promoters were
transfected into primary STBs and JEG-3 cells, as well as nonplacental
COS-7 cells. Elements critical for cAMP-stimulated and basal
transcriptional activity were identified by comparison of mutated
promoters with the wild-type 140-bp promoter construct. Mutation of
bases -130/-121 resulted in 91% and 84% losses in cAMP-activated
transcription in primary trophoblasts and JEG-3 cells, respectively
(Fig. 2
, A and B), whereas mutation of
bases -62/-53 resulted in a 66% (JEG-3 cells) and 99% (STBs) loss
in cAMP-stimulated transcriptional activation, in comparison to the
wild-type construct. Although the borders of the regions varied
somewhat among cell types, the bases consistently most critical for
transcriptional activation were -130/-121 and -62/-53 in both JEG-3
cells and primary STBs, confirming the appropriate use of JEG-3 cells
to model mGH-V transcriptional control in STBs.

View larger version (47K):
[in this window]
[in a new window]
|
Figure 2. Localization of Transcriptional Regulatory Elements
by Block Mutagenesis of the 5'-Flanking DNA of the Rhesus Monkey GH
Variant Gene
Luciferase constructs containing 140 bp of 5'-flanking mGH-V DNA were
transiently transfected into primary cultures of rhesus STBs (A), JEG-3
choriocarcinoma cells (B and C), or nonplacental COS-7 cells (D).
Ten-base pair mutations within the 140-bp promoter were created
scanning the region from -140 to -33 as listed in Table 1 . Panels A
and B indicate the fold induction by 8-Br-cAMP for each mutant
construct, over its untreated control in primary STBs and JEG-3 cells,
respectively. Panels C and D indicate the control levels of luciferase
activity of all mutant constructs transfected into JEG-3 and COS-7
cells, respectively, relative to the wild-type 140 construct. Activity
of the promoterless vector (pGL2) is also shown. The results presented
are the means of three to eight individual experiments, with each
treatment group represented by triplicate dishes in each experiment.
|
|
Mutations in regions adjacent to these elements also have
significant effects in either STBs or JEG-3 cells. Mutation in the
-140/-131 region decreased activity by 50% in primary STB cultures
(Fig. 2A
), while cAMP-stimulated transcription was decreased by
approximately 25% in JEG-3 cells (Fig. 2B
). In JEG-3 cell experiments,
regions -120/-111 and -72/-63 appeared to contribute significantly
to basal and cAMP-responsive transcription (Fig. 2
, B and C). However,
these mutations appeared to have wild-type levels of activity in
primary STB cultures (Fig. 2A
). In STB experiments, mutation of the
region -52/-43 essentially eliminates cAMP-stimulated transcriptional
activity from the mGH-V promoter (Fig. 2A
) and reduced induction by
8-Br-cAMP by approximately 40% in JEG-3 cells (Fig. 2B
).
An examination of basal transcriptional activation of these scanning
mutant constructs in JEG cells, as presented in Fig. 2C
, demonstrated
the importance of regions -130/-121, -72/-63, and -62/-53. Basal
activity in STBs was also consistent with an important role for the
-130/-121 and -62/-53 regions in basal transcription (not
shown).
Transfection assays also assessed the effects of these mutations
on basal promoter activity in nonplacental COS-7 cells. The mGH-V
promoter construct has detectable basal activity in COS-7 cells, but
these cells do not significantly respond to cAMP stimulation (Fig. 1E
).
Transfection of COS-7 cells with the scanning mutagenesis constructs
(Fig. 2D
) indicates that the regions -130/-121 and -72/-53 are
important for basal transcription in COS-7 cells, as they were for
basal and cAMP-stimulated transcription in trophoblasts.
There Is Significant Sequence Homology to Recognition Elements for
a Number of Known Transcription Factors within the mGH-V Promoter
Examination of the sequence of the 140-bp mGH-V promoter region
revealed a number of potential recognition elements for DNA-binding
proteins, including Sp1-, ets-, and GATA-binding sites, and a putative
thyroid hormone response element (TRE) (16) (Fig. 3
). There are also elements homologous to
the proximal and distal Pit-1/GHF-1 binding sites and the Z box of the
pituitary GH gene (17). These elements, with the exception of the Z box
and proximal Pit-1 site, are localized within the two regions
containing functional activity, as defined by the scanning mutagenesis
study (-140/-111 and -72/-43). Oligonucleotide probes for
electrophoretic mobility shift assays were designed to investigate
binding of nuclear proteins to these regions of the mGH-V promoter.

View larger version (16K):
[in this window]
[in a new window]
|
Figure 3. Sequence Homology of the Rhesus Monkey GH-V Gene
Promoter Region with Previously Described Elements
Elements homologous to consensus sequences are indicated by
boxes (labeled above) or arrows (labeled
below).
|
|
Sp1 Family Members Bind to Two Elements within the 140-bp mGH-V
Promoter
A radiolabeled oligonucleotide probe spanning -140 to -119
formed three specific DNA-protein complexes when incubated with JEG-3,
COS-7, HeLa, or GH3 cell nuclear extracts (Fig. 4A
). This region contains an Sp1-like
binding site (GGGAGG; GA box) at -136/-131 (Fig. 3
). All three
complexes were specifically competed by excess unlabeled -140/-119
oligonucleotide as well as by a consensus Sp1 oligonucleotide, but not
by an oligonucleotide containing the overlapping region -132/-105,
which does not contain the entire GA box (Fig. 4
B). The GA box region
consensus site also has some homology to an AP-2 motif, but competition
with a consensus AP-2 oligonucleotide had no effect on the bound
complexes (Fig. 4B
).

View larger version (40K):
[in this window]
[in a new window]
|
Figure 4. Mobility Shift Analysis of Factors Binding to the
-140/-119 Region within the mGH-V Promoter
A, Mobility shift assays with the -140/-119 probe and a range of
nuclear extracts including the choriocarcinoma cell line JEG-3, with or
without 48 h stimulation with 8-Br-cAMP, and the nonplacental
lines COS-7, HeLa, and GH3. One microgram per reaction of sheared
salmon sperm DNA was included as a nonspecific competitor.
Arrows on the left indicate specific complexes. B,
Mobility shift assays with labeled -140/-119 mGH-V oligonucleotide
incubated with JEG-3 choriocarcinoma cell line nuclear extract and the
indicated fold-excess of nonradiolabeled competitor oligonucleotides.
C, Supershift analysis of the JEG-3 -140/-119 complex by
preincubation with 2 µg (2 µl) of each indicated antibody. The
lanes with 4 µg (4 µl) of AP-2 antiserum serve as a nonspecific
control for the lanes containing 2 µg anti-Sp1 plus 2 µg anti-Sp3.
Extracts from cells untreated or exposed to 1.5 mM
8-Br-cAMP were used as indicated. Oligonucleotides used in competition
analysis are given in Table 1 .
|
|
The identity of the bands was investigated with antisera to the zinc
finger transcription factors Sp1, Sp3, and Sp4, which all bind Sp1
recognition sites (18, 19). We also explored whether the relative
amounts of these factors bound to the -140/-119 probe was changed in
cells that had been treated for 48 h with 8-Br-cAMP. Supershift
analysis (Fig. 4C
) showed that while no bands were shifted in response
to anti-AP2 or anti-Sp4 serum, the top-most complex was completely
supershifted by antiserum to Sp1. Incubation with antiserum to Sp3
eliminated the lower two bands and in the presence of both antisera,
negligible amounts of the three original DNA-protein complexes were
observed (Fig. 4C
). Thus, Sp1 and Sp3 account for essentially all
detectable DNA-protein interactions with this probe. The indistinct
smear seen below the supershifted Sp1 band in extracts incubated with
antibodies to both Sp1 and Sp3 is likely to represent a supershifted
Sp3 DNA-protein complex, since this appears only in the presence of
anti-Sp3 serum. There was no apparent effect of 8-Br-cAMP treatment on
the amount or relative proportions of these factors bound to the
-140/-119 probe.
Mutation of the region from -62/-53 also dramatically reduced
transcriptional activity in all cell types, and gel mobility-shift
assays with a probe spanning -75/-35 demonstrated the presence of two
specific bands in both JEG-3 extracts and COS-7 cell extracts (Fig. 5A
). Competition with overlapping
unlabeled competitor oligonucleotides localized the DNA-protein
interactions within the -75/-54 region. We performed competitions
with sterol response element-binding protein-1 (SREBP-1) (20) and Sp1
consensus binding site oligonucleotides since the mGH-V motif AGGTGGGG
(-66/-59) is highly homologous to both of these elements (Fig. 5C
),
and with a consensus GATA-binding site oligonucleotide, since a GATAA
motif is seen at -45/-40 (Fig. 5C
). Fig 5A
shows that only the Sp1
consensus oligonucleotide competed effectively for binding. To
determine the identity of the factors that presumably bind to the
GGTGGG element (GT box), we performed supershift assays with antisera
to Sp1 family transcription factors as in Fig. 4
. Figure 5B
demonstrates that while antisera to Sp4 or AP-2 failed to shift the
electrophoretic mobility of any band, formation of the specific complex
of fastest mobility was completely blocked by antiserum to Sp3, and the
intensity of the upper band was diminished. Incubation with an
antiserum to Sp1 did not affect the lower band, but the upper band was
partially shifted by anti-Sp1 serum. As with the -140/-119 probe in
Fig. 4
, incubation with both Sp1 and Sp3 antisera supershifted or
essentially eliminated all specific DNA-protein complexes. Short
exposure times (not shown) clearly demonstrated that the upper portion
of the complex of slowest electrophoretic mobility contained Sp1, while
the lower portion contained Sp3. As with the -140/-119 probe, there
was no detectable effect of 8-Br-cAMP on complexes formed at the
-75/-34 probe. Supershift analysis with COS-7 extracts and both the
-140/-119 and -75/-35 probes demonstrated essentially identical
binding of Sp1 and Sp3 (not shown).

View larger version (35K):
[in this window]
[in a new window]
|
Figure 5. Mobility Shift Analysis of Factors Binding to the
-75/-35 Region within the mGH-V Promoter
A, Mobility shift assays with labeled -75/-35 mGH-V oligonucleotide
incubated with JEG-3 choriocarcinoma cell nuclear extracts and the
indicated fold-excess of nonradiolabeled competitor oligonucleotides.
SRE-1 is an oligonucleotide from the sterol-regulatory element of the
human low-density lipoprotein receptor gene (20). GATA is a consensus
GATA-binding site oligonucleotide, and Sp1 is a consensus Sp1-binding
site oligonucleotide, both from Promega (Madison, WI). One microgram
per reaction of sheared salmon sperm DNA was included as a nonspecific
competitor. Ar-
|
|
The GT Box at -65/-60 Binds Sp1/Sp3 and Has Significant
Transcriptional Activity
We examined the effects of the scanning mutations at -72/-63 and
-62/-53 as well as specific mutation of the GT box at -65/-60, on
binding of Sp1/Sp3 to the -75/-35 mGH-V probe (Fig. 6
). Although wild-type
unlabeled oligonucleotides competed effectively, none of the mutant
oligonucleotide competed for Sp1 or Sp3 binding (Fig. 6A
),
demonstrating that Sp1/Sp3 could not bind to the promoters with
mutations at -72/-63, -62/-53, or -65/-60.

View larger version (59K):
[in this window]
[in a new window]
|
Figure 6. Competitive Mobility Shift Analysis of Sp1/Sp3
Binding to the -75/-35 Region of the mGH-V Promoter
A, Two micrograms of JEG-3 extract were incubated in the absence or
presence of various competitors at the indicated fold-excess.
Arrows on the left indicate complexes containing Sp1 or
Sp3 as determined by supershift analysis in Fig. 5 . B, Sequences of the
-75/-35 region of the mGH-V promoter and mutated oligonucleotide
competitors. The putative Sp1/Sp3 site is highlighted;
m72/63 and m62/53 oligonucleotides are identical to the mutations
introduced into the promoter for scanning mutagenesis analysis of
transcriptional activity rows on the left indicate specific
and nonspecific complexes. B, Supershift analysis of the JEG-3
-75/-35 complex by preincubation with 2 µg (2 µl) of the
indicated antibody. Anti-AP-2 served as a nonspecific control as in
Fig. 4 . Extracts from untreated JEG-3 cells or cells treated for
48 h with 8-Br-cAMP were used. C, Sequences of the -75/-35
region of the mGH-V promoter and oligonucleotides used in competition
analysis. Consensus binding sites for the relevant transcription
factors in competitor oligonucleotides are underlined.
|
|
To directly examine the importance of the GT box at -65/-60 Sp1/Sp3
site in transcription, we prepared a reporter construct with the 140-
bp mGH-V promoter containing the -65/-60 mutation shown to disrupt
Sp1 and Sp3 binding and compared its activity to the wild-type promoter
as well as the overlapping scanning mutations. Figure 7
demonstrates that the -72/-63,
-65/-60, and -62/-53 mutations had similar effects on basal or
cAMP-stimulated transcriptional activation within each cell type. All
mutations resulted in up to a 90% loss of basal transcriptional
activity (Fig. 7B
). As previously shown in Fig. 1
, transcriptional
activity of the promoter constructs in COS-7 cells was unaffected by
treatment with 8-Br-cAMP (Fig. 7D
). Mutation of the GT box at -65/-60
decreased cAMP-stimulated transcription to levels not significantly
different from the -72/-63 and -62/-53 mutations in both primary
STB cultures and JEG-3 cells (
5090% decrease, respectively; Fig. 7
, C and E). These results also indicate that this downstream GT box
may play a more prominent role in transcriptional activation than does
the distal GA box disrupted by the -140/-131 mutation (Fig. 2
), since
the effects of the -65/-60 mutation were more dramatic than the
effects of the -140/-131 mutation in either JEG-3 cells or primary
STBs (see Fig. 2
, B and D, respectively).

View larger version (33K):
[in this window]
[in a new window]
|
Figure 7. Mutational Analysis of the Proximal GT Box of the
mGH-V Promoter
Luciferase constructs containing 140 bp of 5'-flanking mGH-V DNA
were transiently transfected into JEG-3 choriocarcinoma cells (A and
C), primary cultures of rhesus STBs (E), or nonplacental COS-7 cells
(C). The constructs included either the wild-type (wt) promoter or
promoters mutated at nucleotides -72/-63, -65/-60, or -62/-53.
The -65/-60 construct selectively eliminates the GT box shown in Fig. 3 ; the other mutants are as listed in Table 1 . The pGL2 basic plasmid
serves as a promoterless control. Panels A and B present the basal
luciferase activity of each construct, relative to the WT140 construct,
in JEG and COS cells. Panels C and D present the fold-induction of each
construct by 8-Br-cAMP. Because of substantial variation in the basal
activity between experiments with primary STB cultures, we have
presented cAMP-stimulated luciferase activity in panel E relative to
the activity with the wild-type promoter. In Panels C and E,
cAMP-stimulated luciferase activity of the wild-type promoter was
significantly greater than the mutated promoters (P
< 0.05), which were not significantly different from each other. The
results presented are the means of two individual experiments for COS
cells and three individual experiments with trophoblasts, with each
treatment group represented by triplicate dishes in each experiment.
Individual means for each experiment are indicated by + signs in panels
B and D. All dishes were cotransfected with 12 µg of a
cytomegalovirus-lacZ plasmid, and luciferase activity was normalized to
ß-galactosidase activity.
|
|
 |
DISCUSSION
|
---|
cAMP-regulated pathways have been identified and
implicated in the control of the expression of a diverse range of
endocrine genes in the placenta, including CG
and -ß (21, 22), CS
(23, 24, 25), mouse placental lactogen-I, proliferin and proliferin-related
protein (26), CRH (27), and enzymes of the steroid synthesis pathway
(28, 29). We have previously demonstrated developmentally regulated,
trophoblast-specific expression of the rhesus GH-V gene and have
performed a detailed analysis of the rhesus monkey GH-V promoter to
identify the DNA elements involved in its cell-specific function. We
have defined the most proximal 140 bases of the 5'-flanking region to
be sufficient for cAMP-responsive transcription in both choriocarcinoma
cells and primary trophoblast cultures. These are the first studies to
define cAMP-responsive elements controlling transcription of this
member of the GH gene family in the primate placenta. We demonstrated
that Sp1 and Sp3 bind to two elements that contribute to (-136/-131)
or are critical for (-65/-60) mGH-V promoter activation.
Interestingly, elements adjacent to both of these Sp1/Sp3 sites (at
-130/-121 and -52/-43) also are important for basal and
cAMP-stimulated transcription; however, the identity of protein(s) that
may bind in this region remains unknown.
cAMP has long been known to activate GH expression in the pituitary
gland. Responsiveness to cAMP in the human and rat GH genes is
localized to within 145 bp of the start site of transcription (30, 31, 32).
It has recently been reported that cAMP-response element binding
protein (CREB) regulates human pituitary GH (hGH-N) transcription via a
variant recognition element at -98/-95 (33). However, this CREB site
is not conserved within the homologous region of the mGH-V promoter,
and mutation of the region analogous to this hGH-N CREB site in the
mGH-V promoter did not identify a significant involvement in
cAMP-stimulated transcription.
The GH-V, GH, and CS genes contain Sp1-binding sites located
approximately 136 bp from the start site of transcription. Sp1 sites
are important in the placental transcription of the genes for the
steroidogenic enzymes cytochrome P450 cholesterol side-chain cleavage
enzyme (34, 35, 36), cytochrome P450 aromatase (37), and cytochrome P450
11ß-hydroxysteroid dehydrogenase (38). In addition to activity as an
element of the hCS proximal promoter, an Sp1-binding site at
-136/-131 is important for functional cooperation of the hCS promoter
with a 3'-enhancer located downstream of the CS-B gene (39). In our
studies, mutation of the mGH-V region including the -136/-131 Sp1
motif resulted in a 50% reduction in transcription in STBs, similar to
the 2- to 3-fold decreases reported for the hCS and hGH genes (16, 39, 40). Supershift and binding site competition analysis demonstrated that
both Sp1 and Sp3 are bound to this element. However, mutations in other
regions of the promoter (e.g. -62/-53) drastically
decreased transcription, despite the presence of an intact Sp1 site at
-136/-131. This result indicates that while this Sp1 site contributes
to transcriptional activity, it is not sufficient to sustain
transcription after disruption of other DNA-protein interactions.
One of these critical regions for transcription is centered at
-62/-53. Examination of the promoter sequence revealed an Sp1-like
site (GGTGGG) at -65/-60, and subsequent supershift and competition
analysis demonstrated Sp1 and Sp3 binding at this element. With both
the upstream and downstream Sp1 sites, bands of two distinctly
different mobilities contained Sp3, while a single band contained Sp1.
Previous studies of the platelet-derived growth factor-B promoter have
shown that Sp3 variants of different molecular weights account for a
mobility shift pattern remarkably similar to that seen with our
elements (41). Whether the two Sp3-containing bands seen with
trophoblast extracts represent splice variants or Sp3 complexed with
additional proteins remains to be determined.
It is interesting to note that although very similar complexes
are formed at the two Sp1 sites, mutations in the regions containing
these sites have different effects on transcription. One explanation
for the distinct differences in functional importance may be that the
site at -65/-60 is in close proximity to the TATAA element and is
thus more critically positioned to interact with the basal
transcriptional complex (42, 43, 44). In its absence, transcription is
severely limited. Interaction of factors bound to the more distal Sp1
site with the basal transcriptional initiation complex may be of
relatively less importance. Alternatively, Sp1/Sp3 bound to the
proximal but not distal binding site may facilitate other DNA-protein
interactions as yet unidentified. For example, the adjacent -52/-43
region was critical for transcription in primary STB cultures, and
factors bound at this region may have critical interactions with
Sp1/Sp3 bound at -65/-60.
We identified additional elements not previously reported to be
involved in GH-V expression that appear to be critical for
transcriptional activation. Mutations at -130/-121 dramatically
reduced transcriptional activation, despite the presence of intact
upstream and downstream Sp1 sites. Mutations in the adjacent region,
-120/-111, also significantly reduced transcriptional activation in
JEG-3 cells. This -120/-111 region contains a potential Pit-1-binding
site. Pit-1 plays an important role in the cAMP-stimulated
transcription of GH and Pit-1 promoters (45, 46, 47). We and others have
recently detected the expression of Pit-1 in human and monkey
trophoblasts (48, 49); however, competition and supershift analyses
with JEG-3 cell extracts could not detect Pit-1 binding to this region
(J. T. Schanke and T. G. Golos, unpublished). Additionally,
scanning mutations including either the distal or proximal Pit-1 sites
did not reveal a role in mGH-V transcription in rhesus STBs, as has
previously been noted in studies of the hCS promoter by Jiang et
al. (39). Further studies are needed to identify the factor(s)
acting at the -130/-121 region.
Finally, although Sp1 and Sp3 bind to regions important for
trophoblast-specific transcriptional activation by cAMP, they are not
trophoblast-specific factors; likewise, scanning mutagenesis
demonstrated that the cis-elements important for basal and
cAMP-stimulated transcription in STBs also contribute to basal
transcription in COS-7 cells. Sp1 effects transcriptional activation
through interactions with multiple components of the basal
transcriptional complex, including Drosphila TATA-binding
protein-associated factorII110 (dTAFII 110)
(42), human (h)TAFII55 (43), and TATA-binding protein (44).
We do not yet know how cAMP responsiveness involving Sp1/Sp3 is
restricted to trophoblasts. It is possible that placenta-specific
coactivators may interact with Sp1/Sp3 bound to the mGH-V promoter in
the placenta to confer transcriptional regulation. For example, a B
cell-specific coactivator for Oct-1 (50) and a B cell-enriched TAF,
hTAFII105 (51), have recently been identified, and it seems
likely that cell-specific mechanisms will be identified in other
tissues for fine tuning the effects of widely distributed transcription
factors.
Alternatively, the effects of cAMP may include modulating
protein-protein interactions or transcriptional complex assembly rather
than recruitment of new or trophoblast-specific proteins to DNA-binding
sites. Posttranslational modifications of trans-acting
protein activity by the protein kinase A pathway has been widely
recognized (52, 53, 54, 55, 56, 57), and extensive phosphorylation previously noted for
Sp1 (58) provides incentive to investigate the role of this mechanism
in mGH-V transcriptional activation by cAMP in future studies.
 |
MATERIALS AND METHODS
|
---|
Placental Cell Isolation, Culture, and Transfection
Chorionic villi obtained from placentas at cesarean section were
dispersed with trypsin and DNAse as previously described (14). All
procedures involving the use of animals were approved by the University
of Wisconsin Graduate School Animal Care and Use Committee and were
conducted in accordance with the NIH Guide for the Care and Use of
Experimental Animals. A highly purified fraction of cytotrophoblasts
was obtained by Percoll gradient centrifugation, and cells were plated
for transient transfection experiments. Typically, freshly isolated
cytotrophoblasts were plated in 60-mm dishes at 2 x
106 cells per dish in serum-free medium. Plasmid-liposome
complexes formed using Lipofectamine (Life Technologies, Inc.,
Gaithersburg, MD) or freshly prepared liposomes [1 µg/ml each of
dioleylphosphatidyl-ethanolamine and dimethyldioctadecylammonium
bromide (59)] and 10 µg plasmid DNA were immediately added. Four
hours after plasmid addition, FCS was added to a final concentration of
10%. Cells were scraped from dishes approximately 48 h
posttransfection, harvested by centrifugation, and lysed with
Luciferase Assay Buffer (Promega, Madison, WI). Cell debris was
pelleted and supernatants were assayed for luciferase activity.
Relative light units were determined as the integral of 8 sec of light
output after a 2-sec stabilization period, in a EG&G model 9505 tube
luminometer (EG&G, Natua, NH). The data are expressed in most figures
as means with SE relative light units (RLU)/µg protein in
cell extracts, normalized to luciferase activity in untreated controls
or a wild-type or full-length promoter, as is appropriate for the
experiment at hand. Protein concentrations in cell lysates were
determined with Coomassie protein assay reagent from Pierce (Rockford,
IL). Some cultures were treated with 1.5 mM 8-bromo-cAMP
(Sigma, St. Louis, MO) for 48 h. For the experiments in Fig. 7
, all luciferase constructs were cotransfected with a cytomegalovirus
(CMV) immediate early enhancer/promoter-ß-galactosidase reporter
(60), ß-galactosidase activity was determined with the Galacto-light
assay kit (Tropix, Bedford, MA), and luciferase activity was normalized
to ß-galactosidase activity in each dish. The conclusions reached
with normalized data were consistent with studies done without
cotransfection presented in Figs. 1
and 2
.
Luciferase Reporter Gene Constructs
The mGH-V 5'-flanking DNA used in deletion constructs was
subcloned into the vector pZLUC (12) (kindly provided by Dr. Paul
Deutsch, Cornell University Medical Center, New York, NY). 5'-Deletion
fragments, containing the denoted number of base pairs including the
transcriptional start site, were subcloned, using convenient
restriction sites or PCR amplification, just upstream of the luciferase
gene.
A modified version of the three-step PCR mutagenesis strategy described
by Li and Shapiro (61) was used to create six to 10 base block
mutations scanning the 140-bp mGH-V promoter fragment, which spans
-140 to the BamHI site at +1 (the start site of
transcription). The fragment was PCR-amplified with primers that added
BglII and HindIII sites to the 5' and 3' ends,
respectively, and was subcloned into the
BglII/HindIII site of the Luciferase reporter
vector pGL2 (Promega, Madison, WI) and was used as the template for PCR
mutagenesis. Primers used for PCR mutagenesis are depicted in Table 1
. Typically 50-µl reactions contained
200 ng mGH-V wt140-pGL2 template and 1.5 U Tfl DNA polymerase
(Epicentre Technologies, Madison, WI). The first PCR was carried out
with 20 pmol of each mutation primer paired with 20 pmol of the pGL2
downstream primer: 5' CCT TTC TTT ATG TTT TTG GCG TCT 3'. The reactions
were carried out at 95 C for 50 sec, 45 C for 60 sec, and 72 C for 60
sec for 40 cycles. The products from the first round of amplifications
were used directly in the second-round asymmetric amplification with 20
pmol pGL2 downstream primer. The second-round amplification conditions
were 95 C for 50 sec, 55 C for 60 sec, and 72 C for 60 sec for 30
cycles. The products of the second PCR were gel purified and used as
template in a third round of amplifications using 20 pmol of the pGL2
upstream primer: 5' GGT ACT GTA ACT GAG CTA ACA TAA CC 3' and the
original wild-type mGH-V 140 template. After 40 cycles with the
first-round amplification conditions, the products were purified by gel
electrophoresis and were restricted with BglII and
HindIII and ligated into the luciferase reporter plasmid
pGL2. All constructions and mutations were verified by sequencing.
Nuclear Extracts and Electrophoretic Mobility Shift
Analysis
Nuclear extracts were prepared essentially by the method of
Dignam et al. (62) in the presence of 1 µM
leupeptin and 0.5 mM 4-(2-aminoethyl)
benzenesulfonly-flouride, hydrochloride (Boehringer Mannheim Corp.,
Indianapolis, IN). Nuclear extracts were prepared from the human JEG-3
choriocarcinoma cell line, the monkey kidney line COS-7, and the rat
pituitary line GH3. HeLa cell nuclear extracts were purchased from
Promega Corp. (Madison, WI).
Mobility shift binding reactions typically contained 510 µg nuclear
extract, 13 µg nonspecific competitor (indicated in figure
legends), and 20,000 cpm (0.21.0 ng) end-labeled double-stranded
oligonucleotide probe in a final reaction volume of 20 µl [final
buffer composition: 10 mM HEPES (pH 7.9), 50 mM
KCl, 2.5 mM MgCl2, and 10% glycerol].
Reactions were incubated at room temperature for 15 min.
Oligonucleotides for competitive binding studies were preincubated with
nuclear extract reaction mix for 5 min at room temperature before the
addition of the radiolabeled probe. Oligonucleotide sequences are
depicted in Table 1
. The reactions were then loaded onto a 5%
polyacrylamide gel with 5% glycerol in 0.5 x Tris-borate-EDTA
and were run for 2 h at 150 V, dried, and exposed to x-ray film.
For supershifts, extracts were preincubated with 13 µg of antibody
(Sp1, Sp3, Sp4, and AP-2 polyclonal antibodies, Santa Cruz
Biotechnology, Santa Cruz, CA) or 13 µl normal rabbit serum for
1 h on ice before the addition of radiolabeled probe.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. W. D. Houser, Dr. C. ORourke, and Dr. Jan
Ramer for assistance with animal surgery and S. G. Eisele for
coordinating timed matings. We thank Dr. William Fahl for Sp3 antiserum
for preliminary experiments and Peter Schams for assistance with
luciferase and ß-galactosidase assays.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Thaddeus G. Golos, Wisconsin Regional Primate Research Center, 1223 Capitol Court, University of Wisconsin, Madison, Wisconsin 53715-1299.
This work was supported by NIH Grants HD-26458 to Dr. Golos, HD-08069
to Dr. Schanke, and RR-00167 to the Wisconsin Regional Primate Research
Center.
Preliminary reports of portions of this work were presented at the 77th
Annual Meeting of The Endocrine Society, Washington D.C., June, 1995,
and at the 10th International Congress on Endocrinology, San Francisco,
CA, June, 1996.
This is Wisconsin Regional Primate Research Center Publication 37028.
1 Current address: Epicentre Technologies, Inc., Madison, WI. 
2 Current address: Pharmacopeia, Cranbury, NJ. 
Received for publication August 12, 1997.
Revision received February 11, 1997.
Accepted for publication November 26, 1997.
 |
REFERENCES
|
---|
-
Talamantes F, Ogren L 1988 The placenta as an endocrine
organ: polypeptides. In: Knobil E, Neill J (eds) The Physiology of
Reproduction. Raven Press, New York, pp 20932144
-
Chen EY, Liao Y-C, Smith DH, Barrera-Saldana HA, Gelinas RE,
Seeburg PH 1989 The human growth hormone locus: nucleotide sequence,
biology, and evolution. Genomics 4:479497[Medline]
-
Cooke NE, Liebhaber SA 1995 Molecular biology of the growth
horomone-prolactin gene system. Vitam Horm 50:385457[Medline]
-
Cooke NE, Ray J, Emery JG, Liebhaber SA 1988 Two distinct
species of human growth hormone-variant mRNA in the human placenta
predict the expression of novel growth hormone proteins. J Biol
Chem 263:90019006[Abstract/Free Full Text]
-
Frankenne F, Closset J, Gomez F, Scippo M, Smal J, Hennen G 1988 The physiology of growth hormones (GHs) in pregnant women and
partial characterization of the placental GH variant. J Clin
Endocrinol Metab 66:11711180[Abstract]
-
Kaplan S, Grumbach M 1981 Chorionic somatomammotropin in
primates: secretion and physiology. In: Novy M, Resko J (eds) Fetal
Endocrinology. Academic Press, New York, pp 127139
-
Hoshina M, Boothby M, Boime I 1982 Cytological localization
of chorionic gonadotropin and placental lactogen mRNAs during
development of the human placenta. J Cell Biol 93:190198[Abstract]
-
Liebhaber SA, Urbanek M, Ray J, Tuan RS, Cooke NE 1989 Characterization and histologic localization of human growth
hormone-variant gene expression in the placenta. J Clin Invest 83:19851991[Medline]
-
Jara CS, Salud AT, Bryant-Greenwood GD, Pirens G, Hennen G,
Frankenne F 1989 Immunocytochemical localization of the human
growth hormone variant in the human placenta. J Clin
Endocrinol Metab 69:10691072.5[Abstract]
-
Urbanek M, Russell JE, Cooke NE, Liebhaber SA 1993 Functional
characterization of the alternatively spliced, placental human growth
hormone receptor. J Biol Chem 268:1902519032[Abstract/Free Full Text]
-
Frankenne F, Alsat E, Scippo M-L, Igout A, Hennen G,
Evain-Brion D 1992 Evidence for the expression of growth hormone
receptors in human placenta. Biochem Biophys Res Commun 182:481486[Medline]
-
Golos TG, Durning M, Fisher JM, Johnson KJ, Nadeau KJ,
Blackwell RC 1994 Developmentally regulated expression of the rhesus
placental growth hormone-variant gene: cloning and cAMP-stimulated
transcription in primary syncytiotrophoblast cultures. Endocr J 2:537544
-
Golos T, Durning M, Fisher JM, Fowler P 1993 Cloning of four
growth hormone/chorionic somatomammotropin-related complementary
deoxyribonucleic acids differentially expressed during pregnancy in the
rhesus monkey placenta. Endocrinology 133:17441752[Abstract]
-
Golos T, Handrow R, Durning M, Fisher J, Rilling J 1992 Regulation of chorionic gonadotropin- and chorionic somatomammotropin
messenger ribonucleic acid expression by 8-bromo-adenosine
3',5'-monophosphate and dexamethasone in cultured rhesus monkey
syncytiotrophoblasts. Endocrinology 131:89100[Abstract]
-
Roy RJ, Guérin SL 1993 Two distinct nuclear proteins
bind to the rat growth hormone silencer-1 element. Ann NY Acad Sci 684:207210[Medline]
-
Tansey WP, Catanzaro DF 1991 Sp1 and thyroid hormone receptor
differentially activate expression of human growth hormone and
chorionic somatomammotropin genes. J Biol Chem 266:98059813[Abstract/Free Full Text]
-
Lipkin SM, Naar AM, Kalla KA, Sack RA, Rosenfeld MG 1993 Identification of a novel zinc finger protein binding a conserved
element critical for Pit-1-dependent growth hormone gene expression.
Genes Dev 7:16741687[Abstract]
-
Hagen GS, Muller S, Beato M, Suske G 1992 Cloning by
recognition site screening of two novel GT box binding proteins: a
family of Sp1 related genes. Nucleic Acids Res 20:55195525[Abstract]
-
Kingsley C, Winoto A 1992 Cloning of GT box-binding proteins:
a novel Sp1 multigene family regulating T-cell receptor gene
expression. Mol Cell Biol 12:42514261[Abstract]
-
Yokoyama C, Wang X, Briggs MR, Admon A, Wu J, Hua X, Goldstein
JL, Brown MS 1993 SREBP-1, a basic helix-loop-helix-leucine zipper
protein that controls transcription of the low density lipoprotein
receptor gene. Cell 75:187197[Medline]
-
Steger DJ, Buscher M, Hecht JH, Mellon P 1993 Coordinate
control of the
- and ß- subunit genes of human chorionic
gonadotropin by trophoblast-specific element-binding protein. Mol
Endocrinol 7:15791588[Abstract]
-
Jameson JL, Hollenberg AN 1993 Regulation of chorionic
gonadotropin gene expression. Endocr Rev 14:203221[Medline]
-
Harman I, Costello A, Sane A, Handwerger S 1987 Cyclic
adenosine-3',5'-monophosphate stimulates the acute release of placental
lactogen from human trophoblast cells. Endocrinology 121:5963[Abstract]
-
Morrish DW, Bhardwaj D, Dabbagh LK, Marusyk H, Siy O 1987 Epidermal growth factor induces differentiation and secretion of human
chorionic gonadotropin and placental lactogen in normal human placenta.
J Clin Endocrinol Metab 65:12821290[Abstract]
-
Wu Y, Jorgensen E, Handwerger S 1988 High density lipoproteins
stimulate placental lactogen release and adenosine 3',3'-monophosphate
(cAMP) production in human trophoblast cells: evidence for cAMP as a
second messenger in human placental lactogen release. Endocrinology 123:18791884[Abstract]
-
Yamaguchi M, Imai T, Maeda T, Sakata M, Miyake A, Linzer DIH 1994 Cyclic adenosine 3',5'-monophosphate stimulation of placental
proliferin and proliferin-related protein secretion. Endocrinology 136:20402046[Abstract]
-
Scatena CD, Adler S 1996 Trans-acting factors dictate the
species-specific placental expression of co-corticotropin-releasing
factor genes in choriocarcinoma cell lines. Endocrinology 137:30003008[Abstract]
-
Knoll BJ 1992 Gene expression in the human placental
trophoblast: a model for developmental gene regulation. Placenta 13:311327[Medline]
-
Strauss JF, Kido S, Sayegh R, Sakuragi N, Gafvels M 1992 The
cAMP signalling system and human trophoblast function. Placenta 13:389403[Medline]
-
Brent GA, Harney JW, Moore DD, Larsen PR 1988 Multihormonal
regulation of the human, rat, and bovine growth hormone promoters:
differential effects of 3',5'-cyclic adenosine monophosphate, thyroid
hormone, and glucocorticoids. Mol Endocrinol 2:792798[Abstract]
-
Copp RP, Samuels HH 1989 Identification of an adenosine
3',5'-monophosphate (cAMP)-responsive region in the rat growth hormone
gene: evidence for independent and synergistic effects of cAMP and
thyroid hormone on gene expression. Mol Endocrinol 3:790796[Abstract]
-
Dana S, Karin M 1989 Induction of human growth hormone
promoter activity by the adenosine 3',5'-monophosphate pathway involves
a novel responsive element. Mol Endocrinol 3:815821[Abstract]
-
Shepard AR, Zhang W, Eberhardt NL 1994 Two CGTCA motifs and a
GHF/Pit1 binding site mediate cAMP-dependent protein kinase A
regulation of human growth hormone gene expression in rat anterior
pituitary GC cells. J Biol Chem 269:18041814[Abstract/Free Full Text]
-
Guo I-C, Tsai H-M, Chung B-C 1994 Actions of two different
cAMP-responsive sequences and an enhancer of the human
CYP11A1 (P450scc) gene in adrenal Y1 and placental JEG-3
cells. J Biol Chem 269:63626369[Abstract/Free Full Text]
-
Momoi K, Waterman MR, Simpson ER, Zanger UM 1992 3',5'-cyclic
adenosine monophosphate-dependent transcription of the CYP11A
(cholesterol side chain cleavage cytochrome P450) gene involves a DNA
response element containing a putative binding site for transcription
factor Sp1. Mol Endocrinol 6:16821690[Abstract]
-
Moore CCD, Hum DW, Miller WL 1992 Identification of positive
and negative placenta-specific basal elements and a cyclic adenosine
3',5'-monophosphate response element in the human gene for P450scc. Mol
Endocrinol 6:20452058[Abstract]
-
Kamat A, Alcorn JL, Kunczt CJ, Simpson ER, Mendelson CR,
Characterization of the regulatory regions of human aromatase P450 gene
(CYP19) involved in placenta-specific expression. Program of the
10th International Congress of Endocrinology, San Francisco, CA, 1996 (Abstract P3437)
-
Agarwal AK, White PC 1996 Analysis of the promoter of the NAD
+ dependent 11ß-hydroxysteroid dehydrogenase (HSD11K) gene in JEG-3
human choriocarcinoma cells. Mol Cell Endocrinol 121:9399[CrossRef][Medline]
-
Jiang S-W, Shepard AR, Eberhardt NL 1995 An initiator element
is required for maximal human chorionic somatomammotropin gene promoter
and enhancer function. J Biol Chem 270:36833692[Abstract/Free Full Text]
-
Fitzpatrick SL, Walker WH, Saunders GF 1990 DNA sequences
involved in the transcriptional activation of a human placental
lactogen gene. Mol Endocrinol 4:18151826[Abstract]
-
Liang Y, Robinson DF, Dennig J, Suske G, Fahl WE 1996 Transcriptional regulation of the SIS/PDGF-B gene in human
osteosarcoma cells by the Sp family of transcripton factors. J
Biol Chem 271:1179211797[Abstract/Free Full Text]
-
Gill G, Pascal E, Tseng ZH, Tjian R 1994 A glutamine-rich
hydrophobic patch in transcription factor Sp1 contacts the
dTAFII110 component of the Drosophila TFIID
complex and mediates transcriptional activation. Proc Natl Acad Sci USA 91:192196[Abstract]
-
Emili A, Greenblatt J, Ingles CJ 1994 Species-specific
interaction of the glutamine-rich activation domains of Sp1 with the
TATA box-binding protein. Mol Cell Biol 14:15821593[Abstract]
-
Chiang C-M, Roeder RG 1995 Cloning of an intrinsic human TFIID
subunit that interacts with multiple transcriptional activators.
Science 267:531536[Medline]
-
Chen R, Ingraham HA, Treacy MN, Albert VR, Wilson L, Rosenfeld
MG 1990 Autoregulation of pit-1 gene expression mediated by
two cis-active promoter elements. Nature 346:583586[CrossRef][Medline]
-
Gaidon C, Tian J, Loeffler JP, Bancroft C 1996 Constitutively
active GS
-subunits stimulate Pit-1 promoter activity
via a protein kinase A-mediated pathway acting through deoxyribonucleic
acid binding sites both for Pit-1 and for adenosine 3',5'-monophosphate
response element-binding protein. Endocrinology 137:12861291[Abstract]
-
McCormick A, Brady H, Lars LE, Karin M 1990 Regulation of the
pituitary-specific homeobox gene GHF1 by cell-autonomous and
environmental cues. Nature 345:829832[CrossRef][Medline]
-
Bamberger A-M, Bamberger CM, Pu L-P, Puy LA, Loh P, Asa SL 1995 Expression of pit-1 messenger ribonucleic acid and protein in the
human placenta. J Clin Endocrinol Metab 80:20212026[Abstract]
-
Schanke JT, Conwell CM, Durning M, Fisher JM, Golos TG 1997 Pit-1/GHF-1 splice variant expression in the rhesus monkey pituitary
gland and the rhesus and human placenta. J Clin Endocrinol Metab 82:800807[Abstract/Free Full Text]
-
Strubin M, Newell JW, Matthias P 1995 OBF-1, a novel B
cell-specific coactivator that stimulates immunoglobulin promoter
activity through association with octamer-binding proteins. Cell 80:497506[Medline]
-
Dikstein R, Zhou S, Tjian R 1996 Human TAFII 105
is a cell type-specific TFIID subunit related to hTAFII
130. Cell 87:137146[Medline]
-
Brown K, Gerstberger S, Carlson L, Franzoso G, Siebenlist U 1995 Control of I
B-
proteolysis by site-specific, signal-induced
phosphorylation. Science 267:14851488[Medline]
-
Fagan R, Flint KJ, Jones N 1994 Phosphorylation of E2F-1
modulates its interaction with the retinoblastoma gene product and the
adenoviral E4 19 kDa protein. Cell 78:799811[Medline]
-
Lamph WW, Dwarki VJ, Ofir R, Montminy M, Verma IM 1990 Negative and positive regulation by transcription factor cAMP response
element-binding protein is modulated by phosphorylation. Proc Natl Acad
Sci USA 87:43204324[Abstract]
-
OPrey J, Ramsay S, Chambers I, Harrison PR 1993 Transcriptional up-regulation of the mouse cytosolic glutathione
peroxidase gene in erythroid cells is due to a tissue-specific 3'
enhancer containing functionally important CACC/GT motifs and binding
sites for GATA and Ets transcription factors. Mol Cell Biol 13:62906303[Abstract]
-
Tanaka M, Herr W 1990 Differential transcriptional activation
by Oct-1 and Oct-2: interdependent activation domains induce Oct-2
phosphorylation. Cell 60:375386[Medline]
-
Wang C-Y, Petryniak B, Thompson CB, Kaelin WG, Leiden JM 1993 Regulation of the Ets-related transcription factor Elf-1 by binding to
the retinoblastoma protein. Science 260:13301335[Medline]
-
Jackson SP, MacDonald JJ, Lees-Miller S, Tjian R 1990 GC box
binding induces phosphorylation of Sp1 by a DNA-dependent protein
kinase. Cell 63:155165[Medline]
-
Rose JK, Buonocore L, Whitt MA 1991 A new cationic liposome
reagent mediating nearly quantitative transfection of animal cells.
BioTechniques 10:520525[Medline]
-
MacGregor GR, Caskey CT 1989 Construction of plasmids that
express E coli beta-galactosidase in mammalian cells.
Nucleic Acids Res 17:2365[Medline]
-
Li XM, Shapiro LJ 1993 Three-step PCR mutagenesis for linker
scanning. Nucleic Acids Res 21:37453748[Abstract]
-
Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription
initiation by RNA polymerase II in a soluble extract from isolated
mammalian nuclei. Nucleic Acids Res 11:14751489[Abstract]