Departments of Obstetrics and Gynecology and Cell Biology and Physiology (S.A.) Washington University School of Medicine and the Division of Biology and Biomedical Sciences Program in Molecular and Cellular Biology (C.D.S., S.A.) Washington University St. Louis, Missouri 63110
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ABSTRACT |
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This region, which does not contain a canonical cAMP response element (CRE), transfers protein kinase A responsiveness to a heterologous promoter. Electromobility shift assays and methylation and uracil interference studies localized factor binding to a 20-bp region from -128 to -109 bp of the hCRH promoter. This 20-bp fragment exhibited a similar shift in nuclear extracts from both human term placenta and from human JEG-3 cells. Base contacts, identified in interference studies, were confirmed as critical for binding, as a mutation of these bases abolished factor binding. Furthermore, a CRH promoter containing this mutation exhibited a diminished response to forskolin. UV cross-linking demonstrated the protein in nuclear extracts from human, but not rodent, choriocarcinoma cell lines and estimated its size as 58 kDa. Although this factor participates in cAMP-regulated gene expression, competition electrophoretic mobility assays demonstrated that the factor does not bind to a CRE. Furthermore, neither anti-CREB nor anti-ATF2 antibodies alter factor binding. These data identify this 58-kDa protein as the human-specific CRH activator previously identified as a candidate factor contributing to the species-specific expression of CRH in human placenta.
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INTRODUCTION |
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CRH is expressed at other sites in the central nervous system and in peripheral organs. In primates, the placenta produces the highest concentration of CRH outside of the hypothalamus (1), while other animal species, including rats, mice, and guinea pigs fail to express CRH in their placenta (7, 8). Recent studies indicate that placental CRH may serve as a key component in timing the onset of human labor (9). Placental CRH is identical to the peptide synthesized and secreted in the nervous system, and CRH is a single copy gene (1). Thus, the expression of placental CRH in humans and high primates, and not in other species, indicates that unique mechanisms, distinct from those controlling hypothalamic expression, must control expression in placenta.
Our previous studies have investigated the molecular mechanisms controlling this species-specific placental expression of CRH. By comparing the activity of mouse and human CRH promoters in human and rodent trophoblast cell lines, we established that cellular differences, rather than DNA sequence differences, play the dominant role in establishing the species-specific expression pattern (10). Using nuclear extracts from the rodent and human cell lines, we identified three species-specific candidate trans-acting factors (10).
In our earlier studies, the ability to express CRH with both tissue and species specificity appeared to be linked to cellular cAMP responsiveness. In addition to the highly conserved canonical cAMP response element (CRE) located at -220 bp (2, 11), we identified an additional cAMP-responsive region in the human CRH promoter from -200 to -99 bp, which does not contain a canonical cAMP-regulatory site. One of the candidate trans-acting factors binds to this region, and it is present in nuclear extracts from the human, but not rodent, choriocarcinoma cell lines (10). The aim of the current study was to further characterize this protein factor and its DNA binding site and to confirm its participation in placental expression of CRH.
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RESULTS |
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To more precisely define the binding site for this human-specific
factor, nuclear extracts from JEG-3 cells were used in a series of
electrophoretic mobility assays (EMSAs), summarized in Fig. 1. The DNA fragments used in these
analyses included portions of the hCRH proximal promoter spanning the
region from -196 to -73 bp. Initially, two labeled fragments, -196
to -136 bp and -146 to -73 bp, had been used to divide this region
approximately in half. The candidate nuclear factor bound only to the
fragment from -146 to -73 bp (10). Next, to further define the
location of the binding site, unlabeled oligonucleotide pairs were used
as competitors in EMSA with the -146 to -73 labeled hCRH fragment. A
40-bp oligonucleotide duplex from -146 to -107 bp of the hCRH
promoter specifically competed the shifted band created by the
candidate nuclear factor (Fig. 2
). Two
other oligonucleotides, -117 to -98 bp and -112 to -73 bp, did not
compete with the probe for the binding of the candidate nuclear factor
(Fig. 2
).
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For the uracil interference analysis, end-labeled hCRH fragments were
generated to contain partial substitutions of deoxyuracil for thymine
residues. Because crude nuclear preparations contained nuclease
activity that degraded deoxyuracil-substituted DNA (S. Adler,
unpublished results), binding reactions were performed using partially
purified JEG-3 nuclear extracts. The results from these assays revealed
that replacing the thymine with deoxyuracil at positions -121, -118,
-116, and -114 on the sense strand, and at -111 on the antisense
strand, interfered with the ability of the candidate nuclear factor to
bind to the fragment (Fig. 5).
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These results from the interference assays clarified our initial
mapping by EMSA. Interference assays identified the dA/T base pair at
-111 of the hCRH proximal promoter as being important for binding
(Fig. 5). Deletion analysis did not have the resolution to clearly
identify this base pair, and it is not included in the weakly active
17-mer fragment, -128 to -112 bp. Therefore, a 20-mer from -128 to
-109 bp of hCRH was synthesized. The nuclear factor bound this
oligonucleotide (Fig. 4
). The 20-mer also effectively competed the
bound factor from the -146 to -73 labeled hCRH fragment (Fig. 6B
). Therefore, the DNA-binding site for
the human-specific nuclear factor was defined as -128 to -109 bp
within the hCRH proximal promoter.
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Transfection Studies
The -532 CRH promoter was examined for sequences similar to the
20-mer binding site, and no other sites were found (data not shown).
However, a partial homology was found in the luciferase coding region,
and the corresponding 20-mer oligonucleotide exhibited weak binding on
EMSA (data not shown). This site was mutated to a sequence similar to
the M2 mutation, a change of one amino acid, Ser 399 to Lys. This
mutation did not affect the activity of the in vitro
translated luciferase enzyme (data not shown). To eliminate the
possible contributions of the homology in the luciferase gene, this
mutated luciferase gene was used as the reporter in the subsequent
experiments.
The M2 mutation in the binding site of the nuclear factor provided a
way to further characterize the role of the placental nuclear factor by
analyzing the functional consequences of factor binding on the
placental expression of CRH. Transfections in Bewo cells were performed
to compare the activity of the -532 CRH promoter to the activity of a
promoter containing the M2 mutation, a promoter with the canonical CRE
mutated (XCRE) (10), or a promoter containing both XCRE and M2
mutations (Fig. 7). The M2 mutation alone
gave little reproducible change in forskolin responsiveness of the
promoter in the presence of the intact CRE. However, when the CRE was
mutated, the M2 mutation further decreased forskolin responsiveness of
the CRH promoter.
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The molecular mass of a DNA-binding protein can be estimated by using
UV light to cross-link the protein to a labeled DNA fragment that
contains the proteins DNA-binding site. The cross-linking reaction is
facilitated by incorporating bromodeoxyuridine (BrdU) into a labeled
DNA fragment containing the binding site. After UV irradiation and
nuclease digestion, the protein can be resolved on SDS/PAGE and
identified by autoradiography. The migration of the protein relative to
the migration of known mol wt standards allows an approximate molecular
mass of DNA binding subunits to be determined (13). Using this method,
nuclear extracts from the human choriocarcinoma cell lines, JEG-3 and
BeWo, and from the rodent choriocarcinoma cell line Rcho-1, were
incubated with a labeled, BrdU-containing fragment from -146 to -73
of the hCRH promoter. Binding reactions were exposed to UV, and bound
proteins were resolved on SDS/PAGE (Fig. 8). A single band was present with the
JEG-3 and BeWo extracts. Furthermore, this band was specifically
competed by excess cold competitor when included in the binding
reactions. In addition, the band was not present with the Rcho-1
nuclear extract. By comparing the migration of the identified labeled
band to the migration of the known molecular mass standards, we
estimated the molecular mass of the human-specific nuclear factor as 58
kDa.
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In addition, we examined nuclear extracts from human term placentas for
the presence of the factor using EMSA and the specific 20-mer DNA
duplex as probe. As seen in Fig. 4, crude nuclear extracts from human
placenta exhibit the characteristic shift seen with the partially
purified extracts prepared from JEG-3 cells. These data provide further
evidence for the specific expression of this factor in human
placenta.
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DISCUSSION |
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One other placental gene that has been extensively studied is the -
subunit of the glycoprotein CG. It has a central neuroendocrine role as
an anterior pituitary peptide. It also has a species-specific
expression pattern in placenta, with expression in primates and horses.
For
-CG, evolutionary changes in cis-acting sequences
within the promoter of the gene dictate the species-specific expression
in placenta. The presence of one or two copies of a CRE is essential
for
-CG expression, and minor changes in this cis-acting
element result in the loss of expression in placenta (14, 15, 16). In
addition, the cis-acting sequence, TSE (tissue specific
element), contributes to the tissue-specific expression of
-CG
(16, 17, 18). Experiments in transgenic mice have shown that the bovine
-CG promoter is expressed only in pituitary, while a transgene
derived from the human promoter is expressed both in pituitary and
placenta (15). It is therefore a combination and alteration of these
cis-acting elements, and not differences in
trans-acting factors, that play the dominant role in
species-specific expression of
-CG in placenta.
Our studies present a different paradigm to explain the species-specific expression of CRH in placenta. Transfection studies identified three regions of the human CRH promoter that contributed to the expression pattern of CRH in placental trophoblasts. In vitro studies identified candidate nuclear factors binding to the regions targeted by our transfection analyses. Interestingly, these nuclear factors are species-specific. A candidate rodent repressor and activator were identified in rodent trophoblasts, and a candidate human-specific factor was identified in human cells (10). These results demonstrate that differences in trans-acting factors, not cis-acting sequences, are dominant in determining the expression of CRH in placental trophoblasts.
In human trophoblast cells, cAMP plays a critical role in both cell
differentiation and gene expression (19, 20). Many genes in these
cells, including both -CG and CRH, are regulated by cAMP (2, 3, 21, 22). The results from our earlier studies implied that it was the
ability of a cell to respond to cAMP that determines both the tissue
and species-specific expression of CRH (10). CRH has within its
proximal promoter a highly conserved classic CRE where members of the
CREB/ATF family of transcription factors can bind and activate CRH gene
expression (2, 11, 23). Our studies indicated that after mutational
inactivation of the canonical CRE, cAMP responsiveness was retained.
Deletional mapping identified a proximal cAMP-responsive region within
the human CRH promoter from -200 to -99 bp, which does not contain
any characterized classic cAMP-response elements (10). The
human-specific factor binds to hCRH within this region, and fine
mapping studies defined its binding site to be from -128 to -109 bp
within the hCRH promoter. In our current studies, transfection
experiments using human choriocarcinoma cell lines confirmed the
activity of this region and its relationship to cAMP-regulated
pathways.
In transfection studies, the M2 mutation, in combination with the mutation of the CRE, still retained slight activation by PKA pathways, even though the human-specific factor cannot bind to this mutated promoter in vitro. While part of this activity may be due to low levels of binding in vivo, other factors or binding sites might also be contributing to this regulation. Majzoub et al. (24) have also reported PKA responsiveness of the CRH promoter distinct from the canonical CRE. They identified a site between -112 and -98 bp in the hCRH promoter based on its similarity to a sequence found in the human enkephalin promoter (25).1 The site is adjacent to the human-specific factor DNA-binding site, which we identified at -128 to -109 bp in the hCRH promoter, and the two sites overlap by 4 bp. We cannot rule out a combination of these two sites as contributing to the cAMP responsiveness of this region. In our footprinting analysis, we did not detect binding of a factor to the -117 to -103-bp promoter region; however, the extracts used in this analysis were partially purified and selected for enrichment of the human-specific factor binding at -128 to -109 bp. It is possible that using crude nuclear extracts, a footprint might also be detected in the -117 to -103-bp region. In addition, our earlier studies indicated the possibility that other distal PKA-regulated sites exist within the 5-kb human CRH promoter (10). The region from -200 to -99 bp contains the most proximal site we could detect by deletion analysis and may not be the best binding site for the factors mediating the PKA responsiveness of the hCRH promoter. The combination of several cAMP-regulated sites including the CRE, as well as the absence of transcriptional repressors, may ultimately contribute to the tissue and species-specific expression of CRH, as well as the regulated responses of CRH to environmental or developmental signals.
In these studies, we have identified a 58-kDa protein as the human-specific factor that binds to the hCRH cAMP-responsive site from -128 to -109 bp. The factor is specific to humans as we demonstrate its presence in the nuclear extracts of the human choriocarcinoma cell lines, but not in the rodent choriocarcinoma cell line. The lack of expression of the identified DNA binding subunit, along with the expression of a transcriptional repressor, is likely to contribute to the inability of the rodent trophoblast to express CRH. The identification of the 58-kDa protein in human trophoblasts by UV cross-linking does not exclude the possibility of other protein subunits that may be components of this transcription factor. Nor can we exclude a requirement for additional proteins that may be necessary to elicit a response to cAMP.
Additional EMSA and supershift experiments indicate that although the activity of this placental factor is linked to cAMP pathways, it is distinct from CREB and ATF-2. We have also extended our characterization of the specificity of expression of this factor to not only human choriocarcinoma cells, but also to human term placental tissue. Our data also now exclude expression not only from rodent choriocarcinoma cell lines, but also from the human nonplacental Hela cell line which we previously showed lacked cell type-specific expression of CRH reporter genes in transfection studies (10).
The unique placental expression of CRH in higher primates is consistent with a role for CRH in fetal gestation and labor, especially in humans (26). The expression of CRH in human placenta begins around the seventh week of gestation and increases throughout pregnancy. During the last 5 weeks of gestation, there is a significant increase in CRH expression within the placenta (27). Studies have correlated placental expression of CRH with the length of gestation. Elevations of CRH occur earlier in pregnancies complicated by preterm delivery, and the level of CRH is lower in pregnancies extending post term (9). Placental CRH may also cross into the fetal circulation and stimulate the fetal HPA axis, resulting in the increase in cortisol seen within the fetus during the last 5 weeks of gestation. The cortisol surge is necessary for the maturation of fetal organs, and thus may contribute to the fetal signal for initiating parturition (28).
In our model cell culture system, the activity of the 58-kDa protein, in both specific DNA binding in preparations of nuclear extracts and in mediation of a transcriptional response to cAMP, appears to vary with growth conditions and cell density (M. A. Mallon and C. D. Scatena, unpublished results). It is tempting to speculate that these changes in activity may parallel the changes occurring in the trophoblast at term that result in the increased expression of CRH in human placenta.
The changes that occur in human pregnancy at term may also have other implications in the interpretation of our data from cell lines. We have shown that the human choriocarcinoma cell lines express the CRH activator and exhibit appropriate tissue-specific expression of transfected CRH reporter genes. These data do not provide an explanation for the reported absent or inconsistent expression of the endogenous hCRH gene in choriocarcinoma cell lines. It is possible that in these cell lines the endogenous CRH gene is damaged, deleted, or has been rendered inaccessible for cellular transcription. It is also possible that the choriocarcinoma cells are more representative of a preterm trophoblast and that additional factors or activation signals present in placenta at term would result in even greater reporter gene expression or in expression of the endogenous hormone. Also, our preparations of nuclear extracts often display a doublet or split pattern on EMSA, although the presence and intensity of this pattern is variable among our preparations. Possible explanations for this pattern may include differences in protein modification, e.g. the degree of phosphorylation in response to activation of protein kinase A. Alternatively, rather than indicating the consequences of a regulatory event, these forms may be the result of proteolytic cleavage occurring during the preparation of our extracts. The demonstration of the characteristic shift of the CRH activator in a crude nuclear extract from human term placenta may provide both an abundant source of the activator protein and also allow extension of our molecular studies from cultured choriocarcinoma cells to authentic human trophoblasts that reflect the changes occurring at term. Further evaluation of these possibilities remain for future studies.
The role CRH plays in fetal gestation and parturition suggests a requirement for strict regulation of the CRH gene in placenta. Further characterization of the activator protein, including cloning of its gene and determination of the role cAMP plays in its expression and activity, could clarify the mechanisms of regulation and the role of CRH in the human placenta and in parturition.
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MATERIALS AND METHODS |
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Mutated versions of the DNA fragment hCRH -146 to -73 were created by PCR, using plasmids containing the indicated mutation as template, and oligonucleotide primers hCRH -146 to -126 (forward) and hCRH -73 to -93 (reverse). To create the mutated DNA fragment -146 to -73 M1, the plasmid CRH -200 M1 pBKS II (-) was used as template. To create the mutated DNA fragment -146 to -73 M2, the plasmid CRH -200 M2 pBKS II (-) was used as template. Similar reactions were used to generated the wild-type probe hCRH -146 to -73. Mutant plasmids were created using site-directed mutagenesis as described below.
Discarded human placental tissue was obtained at deliveries from pregnant patients with normal uncomplicated term pregnancies and labor, in accordance with the requirements of the local Human Studies Committee. Human tissues were handled in compliance with the requirements of the local Biosafety Committee. Small portions of placental tissue were trimmed of connective tissue and quick-frozen in liquid nitrogen in the delivery suite. This frozen tissue was used to prepare nuclear extracts as described above for cultured cells.
Cell Lines
BeWo, JEG-3, and Hela cell lines were obtained from American
Type Culture Collection (Rockville, MD). The Rcho-1 cell line was the
generous gift of M. Soares (30). The BE(2)-C cell line (31) was a
generous gift from Dr. June Beidler (Memorial Sloan-Kettering Cancer
Center, New York). The BE(2)-C cell line was grown at 37 C in a 5%
CO2 incubator and in MEM/F12 medium supplemented with 10%
FBS (Intergen, Purchase, NY), 5% enriched calf serum (ECS, Gemini
Bioproducts, Calabasas, CA), and 1x nonessential amino acids
(Mediatech, Washington DC). BeWo cells were grown in 5%
CO2 in RPMI 1640 with 5% FBS and 5% ECS. JEG-3 cells were
grown in 5% CO2 in MEM with 5% FBS and 5% ECS. Rcho-1
cells were grown in 5% CO2 in NCTC-135 with 5% FBS, 5%
ECS, 0.4% glucose, 50 µM 2-mercaptoethanol, and 100
µM Na-pyruvate. Hela cells were grown in 10%
CO2 in DMEM with 5% FBS and 5% ECS. All the above growth
media were supplemented with antibiotics. All cells are routinely
surveyed for mycoplasma using a PCR method from Stratagene (La Jolla,
CA).
Site-Directed Mutagenesis
Oligonucleotide-directed mutagenesis was performed in phagemid
vectors using minor modifications of the method of Kunkel (32).
Deoxyuracil containing phagemid DNA was prepared from CRH -200
pBKSII(-), CRH -532 pBKSII(-), and CRH -532 XCRE pBKSII(-) (10).
To create the M1 mutation, the oligonucleotide primer (antisense) used
for the mutagenesis was dCCTGCATAAATAGTAGGGCC, which changes the hCRH
nucleotides at positions -121 and -120 from dTG to dCT. To create the
M2 mutation, the oligonucleotide primer (antisense) used for the
mutagenesis was dCCTCTGCTCCTTTATAAATCATAGGG, which changes the hCRH
nucleotides at positions -113 and -112 from dGC to dAA. In addition,
the luciferase coding region from the L2S reporter plasmid (10) was
mutated to remove a partial homology to the CRH binding region.
Deoxyuracil containing phagemid DNA was prepared from the corresponding
luc XT pBKSII(-) plasmid and the oligonucleotide primer (antisense)
used for mutagenesis was dGTTTACATAACCTTTCATAATCATAGG. This primer
changes the nucleotide codon TCC (ser-399) to AAA (lys), a change in
sequence equivalent to the change introduced by the M2 mutation.
Luciferase activity of the mutant protein was confirmed by in
vitro translation using the TNT reticulocyte lysate system
(Promega, Madison, WI) and assaying the lysate for luciferase activity
similar to our assays of cell extracts described below.
All mutations were confirmed by dideoxy DNA sequencing, and corresponding reporter plasmids were made by subcloning the mutated sequences using conventional techniques.
Expression Vectors and Plasmids
The plasmid for expressing the catalytic subunit of protein
kinase A, RSV-PKA, was obtained from Richard Maurer (33). The RSV-Neo
plasmid expressing the neomycin phosphotransferase II gene is as
previously described (34).
The construction of luciferase reporter genes using L2S plasmids was
previously described (10). To create reporters containing three and six
copies of the oligonucleotide duplex from -146 to -107 bp of the hCRH
proximal promoter, partially kinased duplexes were multimerized in
three or six copies using DNA ligase. These multimerized fragments were
subcloned in front of the minimal 36-bp promoter, p36 (12).
Transfections
Transient transfections were performed using a calcium-phosphate
method (35) in 6- or 12-well plates. The conditions for the 6-well
plates have been previously described (10). For a 12-well plate, minor
changes to the protocol were made. Two days before transfection, 40,000
cells per well were seeded in 1 ml growth media. Transfections used a
total 71 µl of the calcium phosphate solution containing 1.4 µg DNA
prepared in the same proportions as previously detailed (10). Cells
were harvested in 200 µl of a Triton lysis buffer, containing 50
mM Tris/2 (N-morpholino) ethane sulfonic acid
(pH 7.8), 1 mM dithiothreitol (DTT), and 1% Triton X-100.
The lysate was assayed for luciferase activity as previously described
(36), using a Monolight 2010 luminometer (Analytical Luminescence
Laboratories, San Diego, CA). ß-Galactosidase assays were performed
using chlorophenol red ß-galactopyranoside (Boehringer Mannheim,
Indianapolis, IN) as substrate (37) and an Anthos plate reader (Anthos
Labtec Instruments, Salzburg, Austria) with Delta Soft II software (Bio
Metallics, Princeton, NJ). Results (fold expression) are shown as
means ± SEM. For comparisons one-way ANOVA and the
unpaired two-tailed t test were applied using Primer of
Biostatistics software (Windows v. 4.0, MacGraw Hill, St. Louis, MO).
Significance was determined as P < 0.05.
Methylation Interference and Uracil Interference
Nucleotides in the binding site for the human-specific activator
were identified using minor modifications of standard protocols for
methylation interference and uracil interference (13). Specific 5'-end
labeled DNA fragments were prepared using PCR. Before PCR, either the
forward or reverse oligonucleotide primer was end labeled with T4
polynucleotide kinase using 32P-ATP. Subsequent PCR
reactions generated a single end-labeled probe from -146 to -73 of
the hCRH proximal promoter. For the methylation interference assays,
after PCR, the DNA was partially methylated using dimethyl sulfate as
described (13). For uracil interference studies, dUTP was added at a
concentration of 50 µM to the PCR reactions to generate
probes containing partial substitution with deoxyuracil. For generation
of completely deoxyuracil-substituted probes, 400 µM dUTP
replaced TTP in the amplification reactions. Because JEG-3 nuclear
extracts contain nuclease activities that degrade
deoxyuracil-substituted DNA, nuclear extracts used for interference
experiments were partially purified to remove this activity.
Binding reactions for interference assays contained 40,000 cpm of either partially methylated or partially deoxyuracil-substituted end-labeled DNA, and purified JEG-3 nuclear extract as described for EMSA. For each probe, five identical reactions were performed and loaded in adjacent lanes on gels for electrophoresis. After electrophoresis, gels were transferred to diethylaminoethyl-81 paper (Whatman, Maidstone, England) without fixation and exposed to storage screens (Molecular Dynamics, Sunnyvale, CA) for 2 h at -20 C. The locations of the shifted band and free probe for the five lanes were identified using a model 425B PhosphorImager (Molecular Dynamics), and excised in block, electroeluted, and precipitated. Eluted DNA was resuspended in PCR buffer (Life Technologies, Gaithersburg, MD) with 5 mM MgCl2. Deoxyuracil containing DNA was cleaved using uracil DNA glycosylase (Life Technologies). Reactions contained 0.2 U uracil DNA glycosylase and were incubated at 37 C for 30 min, and then 30 min at 90 C. Partially methylated DNA was similarly cleaved by incubation in PCR buffer with 5 mM MgCl2 for 30 min at 90 C. Formamide dyes were added, and equal counts were loaded onto sequencing gels containing 6 M urea, 8% 38:2 (acrylamide-bis), 0.5 x TBE [45 mM Tris-HCl (pH 8.3), 1.4 mM EDTA, 56 mM boric acid, final concentration]. After electrophoresis, fixation, and drying, the results were visualized using storage screens and by autoradiography at -80 C with intensifying screens.
Partial Purification of JEG-3 Nuclear Extract
Crude JEG-3 nuclear extracts were prepared as previously
described (10). All subsequent steps were performed at 4 C. Nuclear
extracts were pooled, diluted 5-fold with column buffer [20
mM Tris-HCl (pH 7.8), 0.1 mM EDTA, 50
mM KCl, 0.1 mM DTT, 10% glycerol], and loaded
onto a 5-ml heparin agarose type 1 H 6508 column (Sigma, St. Louis, MO)
that had been equilibrated with the same buffer. The column was washed
with 5 ml of buffer, and 1 ml fractions were eluted with a 20-ml linear
gradient from 50 mM to 1 M KCl in the same
buffer. Fractions were monitored using conductivity, and selected
fractions were dialyzed for 4 h on ice into column buffer
containing 50 mM KCl, and then dialyzed overnight on ice
into 20 mM HEPES (pH 7.9), 50% vol/vol glycerol, 100
mM KCl, 0.2 mM EDTA, 0.5 mM DTT.
Individual fractions were stored at -20 C and assayed for binding
activity using EMSA. Fractions were also assayed for the presence of
nuclease activity by performing EMSA with the completely
deoxyuracil-substituted probe and determining the preservation of
intact free probe. Fractions with peak binding activity were also free
of nuclease activity.
UV Cross-Linking of Protein to DNA
UV cross-linking and analysis of resulting labeled proteins were
performed using a standard protocol with minor modifications (13). The
BrdU DNA probe, hCRH -146 to -73, was generated using PCR, by
including 200 µM BrdU (Sigma) and 32P-dATP
during the amplification. Each binding reaction combined 40,000 cpm of
this labeled, BrdU-substituted DNA, with either 20 µg BeWo nuclear
extract, 17.4 µg JEG-3 nuclear extract, or 9.4 µg Rcho-1 nuclear
extract. Incubations were performed as described for EMSAs. When
indicated, 10 pmol unlabeled competitor were added. After overnight
incubation at 0 C, binding reactions were exposed for 5 min to UV light
using a Fotodyne UV transilluminator (Hartland, WI). Each sample then
received 2 µl of 50 mM CaCl2, 10 U DNase I
(Boehringer Mannheim), and 1 U micrococcal nuclease (Sigma). Digestion
of DNA was 30 min at 37 C. Samples were precipitated with 25 µl 20%
trichloroacetic acid, resuspended in loading buffer, boiled for 5 min,
and then resolved by electrophoresis on Laemmli discontinuous 10% 38:2
(acrylamide-bis) polyacrylamide gels. Gels were fixed, transferred to
3MM chromatography paper (Whatman), and dried. The results were
visualized using autoradiography at -80 C with an intensifying screen
or by storage screen analysis. The mol wt of the human-specific
activator was determined by comparison to 14C mol wt
standards (Life Technologies) using ImageQuant 2.0 software (Molecular
Dynamics).
EMSA Supershift Assays
Antibody to ATF-2 (SC-187X) was purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Antibody to CREB (catalog no.
9192) was the generous gift of Andreas Nelsbach (New England Biolabs,
Beverly, MA). Oligonucleotides for generating a 41-bp duplex
radioactive probe containing a CRE were
dGATCGGATCCGATTGCCTGACGTCAGAGAGC and
dCGATAGATCTGCTCTCTGACGTCAGGCA. These were annealed and 3'-end
labeled using Klenow fragment. GEM ATF-2 plasmid DNA, a gift from John
J. Keilty (University of Massachusetts, Worcester, MA), was used to
program the T7 TNT reticulocyte lysate system (Promega) for in
vitro translation of ATF-2. Nuclear extracts containing CREB were
prepared from BE(2)-C cells and Hela cells as described above.
Binding reactions for the CREB probe were performed in 25 µl reactions containing 10 mM HEPES (pH 7.8), 1 mM spermidine, 3 mM MgCl2, 7.2% glycerol, 0.6 mg/ml BSA, 0.06% Nonidet P-40, 3 mM DTT, and 150 µg dI·dC (38). When indicated, 1 pmol of unlabeled competitor DNA was included in the reaction. Reactions contained 2 µl of lysate or nuclear extract and were incubated for 10 min at 20 C before addition of specific probe. After addition of probe DNA, incubation continued for an additional 10 min at 20 C. When indicated, 1 µl of antibody was added, and incubation for all reactions was continued for an additional 10 min at 20 C and then at 0 C overnight. Samples were resolved on 20 cm x 0.5 mm nondenaturing 4% 80:1 (acrylamide-bis) gels containing 2.5% glycerol, 0.5x TBE and electrophoresis was in 0.5x TBE buffer.
Supershift binding reactions for the CRH probe were performed in the usual fashion with the inclusion of antibody and an additional 10 min of incubation at 20 C before the incubation at 0 C overnight and resolution by nondenaturing gel electrophoresis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The sequence presented in the publication
differs from the actual CRH sequence by a single nucleotide at -109
The change does not significantly alter the observed homology.
Received for publication July 21, 1997. Revision received February 18, 1998. Accepted for publication April 20, 1998.
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REFERENCES |
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