Characterization of a Human-Specific Regulator of Placental Corticotropin-Releasing Hormone

Caroline D. Scatena and Stuart Adler

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hypothalamic hormone CRH is also expressed in the placentas of humans and higher primates and may play an important role in the regulation of labor. In choriocarcinoma cell lines, activation of cAMP-dependent pathways increases human (h)CRH reporter gene expression. A cAMP-responsive region distinct from the cAMP response element at -220 bp, has been identified between -200 and -99 bp, and a candidate transcription factor was identified in nuclear extracts of human, but not rodent, choriocarcinoma cell lines.

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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CRH is a 41-amino acid neuropeptide hormone that regulates the hypothalamic-pituitary-adrenal (HPA) axis. It is highly conserved and is primarily expressed in the parvocellular neurons of the paraventricular nucleus of the hypothalamus. Secretion of CRH into the hypophyseal portal blood system activates the synthesis and secretion of ACTH from the anterior pituitary corticotrophs. ACTH, in turn, activates the synthesis and secretion of cortisol from the adrenal glands. Cortisol modulates this pathway via an inhibitory feedback loop, affecting both CRH and ACTH. Normally, CRH is expressed in a circadian pattern; however, during periods of stress, CRH continues to be expressed, in spite of the high levels of circulating glucocorticoids (1). The regulation of this HPA pathway is primarily transcriptional. Some neuronal transcription factors, including the CREB/ATF and POU-homeo transcription factor families, have been identified as participating in the regulation of this complex neuroendocrine system (2, 3, 4, 5, 6). Yet, we still lack a complete understanding of the molecular mechanisms responsible for circadian expression, the stress response, and glucocorticoid feedback.

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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fine Mapping of the Regulatory Region
Our previous studies in cell culture systems indicated that the expression of CRH in placenta is controlled by species-specific trans-acting factors (10). Transient transfections using deletions of the hCRH promoter identified a cAMP-responsive regulatory region between -200 and -99 bp, distinct from the canonical CRE at -220 bp. Examination of this region revealed that it does not contain a classic CRE. A human-specific factor that bound to this region was present in nuclear extracts from human choriocarcinoma cell lines (10).

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. 1Go. 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. 2Go). 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. 2Go).



View larger version (23K):
[in this window]
[in a new window]
 
Figure 1. Location of the Human-Specific Nuclear Factor DNA Binding Site

The DNA sequence for the human CRH promoter from -200 to -60 bp is shown for reference. Results obtained from transfection and EMSAs are indicated for each fragment. The 20-bp binding site identified by these analyses is from -128 to -109 bp in the hCRH promoter and is dGGCCCTATGATTTATGCAGG.

 


View larger version (63K):
[in this window]
[in a new window]
 
Figure 2. Competition Studies Map the Location of the Human-Specific Nuclear Factor DNA Binding Site

JEG-3 nuclear extract (23 µg) was incubated with the labeled probe, -146 to -73 bp hCRH, in the absence or presence of excess (1 pmol) unlabeled fragments as indicated. The arrow indicates the location of the complex.

 
One of the characteristics of an independent regulatory element is the ability to transfer regulation to a heterologous promoter. The region from -146 to -107 bp was further analyzed to determine whether this 40- bp sequence and the corresponding DNA binding factor identified in vitro retained the cAMP responsiveness originally associated with the larger region of the promoter. The 40-bp oligo duplex from -146 to -107 bp of hCRH was multimerized as three and six copies and inserted in front of a minimal 36-bp promoter, p36 (12). These reporter genes were cotransfected into the JEG-3 cells along with either the control plasmid, Rous sarcoma virus (RSV)-Neo, or with the protein kinase A (PKA) catalytic subunit ß, RSV-PKA. Responsiveness to PKA pathways was determined as the fold increase in activity by comparing the expression with RSV-PKA to that with the RSV-Neo control. As shown in Fig. 3Go, addition of three or six copies of the 40-mer region progressively increased the PKA responsiveness of the p36 minimal promoter. The results from these experiments indicated that the region from -146 to -107 bp of the hCRH promoter was sufficient for cAMP responsiveness and transferred this responsiveness to a heterologous promoter.



View larger version (13K):
[in this window]
[in a new window]
 
Figure 3. Transfer of cAMP Responsiveness to a Heterologous Promoter

Human JEG-3 cells were cotransfected with the indicated reporters and either RSV-Neo or RSV-PKA. Fold expression is the relative increase in luciferase activity due to the expression and activity of the PKA catalytic subunit ß for each promoter construct, compared with the RSV-Neo control. p36 is a minimal promoter. Multi 3 and Multi 6 are p36 minimal promoters with three and six copies of the -146 to -107 hCRH oligonucleotide, respectively. Data shown are means ± SEM from five experiments.

 
Having confirmed that this smaller region of the promoter still retained regulatory activity, additional EMSAs were performed to determine a minimal binding site. The -146 to -73-bp fragment of the hCRH promoter contains DraII sites centered at -130 and -129 bp (Fig. 1Go). Digestion of this region with DraII generated two fragments for use in EMSA, but only the proximal fragment from -129 to -73 bp was bound by nuclear extracts (Fig. 1Go). These results narrowed the potential nuclear factor binding site to a 17-bp region from -128 to -112 within the hCRH promoter. A 17-bp oligonucleotide duplex was created, which corresponds to this potential site. It was used in EMSA as a labeled probe (Fig. 4Go). The candidate nuclear factor bound this fragment, but only weakly. Also, this sequence was a weak and inconsistent competitor for factor binding to the -146 to -73-bp probe (data not shown).



View larger version (74K):
[in this window]
[in a new window]
 
Figure 4. Purified JEG-3 Nuclear Extract and Crude Placental Nuclear Extract Bind to the 20-mer Sequence

EMSAs were performed using either the 17-mer sequence (-128 to -112 hCRH) or the 20-mer sequence (-128 to -109). Reactions contained partially purified JEG-3 nuclear extract (2.6 µg) or crude human term placental nuclear extract (1 µg). When indicated, 1 pmol excess unlabeled 20-mer competitor DNA was present. P, Probe alone; N, nuclear extract; +C, nuclear extract plus competitor. Arrow indicates specific complex.

 
Methylation and Uracil Interference Analysis
Because some of the fragments used in these studies may have split the binding site, the precise boundaries of the binding site were not certain. To more clearly identify the residues involved in binding, interference assays were performed. These assays identify DNA bases that, when modified, interfere with the binding of the nuclear factor to the hCRH promoter fragment (13). The labeled fragment used in these assays was the hCRH proximal promoter fragment from -146 to -73 bp.

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. 5Go).



View larger version (75K):
[in this window]
[in a new window]
 
Figure 5. Uracil Interference and Methylation Interference

Partially purified JEG-3 nuclear extract was incubated with the hCRH fragment from -146 to -73 bp, which had been end labeled on either the sense or antisense strand. For deoxyuracil interference, the fragments were generated to contain partial substitution of deoxyuracil for thymidine residues. For methylation interference, the fragments were partially methylated using dimethyl sulfate. The DNA bases that when modified affected the binding of the protein are noted with arrows, and their location in the hCRH promoter is given. Free, Probe not bound by extract; bound, probe bound by extract in EMSA. The end-labeled strands are either sense or antisense, as indicated.

 
For the methylation interference analysis, end-labeled hCRH fragments were partially methylated using dimethyl sulfate. These experiments identified the guanines at positions -120 and -113 on the sense strand and at -112 on the antisense strand as residues critical for binding (Fig. 5Go).

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. 5Go). 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. 4Go). The 20-mer also effectively competed the bound factor from the -146 to -73 labeled hCRH fragment (Fig. 6BGo). Therefore, the DNA-binding site for the human-specific nuclear factor was defined as -128 to -109 bp within the hCRH proximal promoter.



View larger version (56K):
[in this window]
[in a new window]
 
Figure 6. Mutation of the Human-Specific Factor DNA Binding Site Alters Binding of the Protein in EMSAs

A, The 20-bp binding site sequence is shown with base contacts indicated in bold and underlined. The M1 mutation contains a change in the hCRH nucleotides at positions -121 and -120 from dTG to dCT, shown in bold and underlined. The M2 mutation contains a change in the hCRH nucleotides at positions -113 and -112 from dGC to dAA, shown in bold and underlined. B, Competition with native and mutant 20-mer binding sites from -128 to -109 bp in the hCRH promoter. JEG-3 nuclear extract (20 µg) was incubated with labeled probe (-146 to -73 hCRH) in the absence or presence of excess unlabeled 20-mer fragments. P, Probe only; N, no competitor; Crescendo, increasing quantities (1 pmol, 3 pmol, 6 pmol) of unlabeled wild type (WT) 20-mer fragment, M1 mutant fragment, or M2 mutant fragment. C, Direct binding EMSA of mutant 20-mer sequences. The labeled fragments used in these EMSAs were -146 to -73 of the hCRH promoter (wild-type, M1, or M2) and were incubated with JEG-3 nuclear extract (20 µg), as indicated. The arrow indicates the location of the complex. P, Wild type probe alone; WT, wild-type probe with nuclear extract; M1, probe with M1 mutation with nuclear extract; M2, probe with M2 mutation with nuclear extract.

 
Mutation of the DNA-Binding Site
Our identification of a minimal binding site for the human nuclear factor relied on in vitro binding studies. Before more functional studies, we performed site-directed mutagenesis to alter specific critical residues in the binding site. Two regions identified by interference studies were selected. The dT at -121 and the dG at -120 on the sense strand of hCRH were changed to dC at -121 and to dT at -120 (mutation M1, Fig. 6Go). The dG at -113 and the dC at -112 on the sense strand of hCRH were both changed to dA (mutation M2, Fig. 6Go). These mutant hCRH fragments were used to generate labeled DNA fragments from -146 to -73 of the hCRH proximal promoter. The mutant probes were compared with the wild-type -146 to -73 hCRH fragment in an EMSA (Fig. 6CGo). The human-specific nuclear factor bound to the wild-type fragment and to the fragment with the M1 mutation, although binding was weak when compared with the wild-type fragment. Introducing the M2 mutation into the labeled fragment completely eliminated the ability of the nuclear factor to bind to its site. These results were confirmed by using an oligonucleotide duplex from -128 to -109 of the hCRH promoter that contains the above mutations in EMSA as unlabeled cold competitors (Fig. 6BGo). These analyses confirmed that the M1 mutation was a weak competitor, while the M2 mutation was unable to compete for binding of the nuclear factor. These mutations thus confirm the identification of the binding site and the DNA base contacts critical for nuclear factor binding.

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. 7Go). 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.



View larger version (13K):
[in this window]
[in a new window]
 
Figure 7. Mutation of the Binding Site Reduces Forskolin Responsiveness of the hCRH Promoter

Human BeWo cells were transfected with the CRH -532 promoters, wild type or mutated as indicated, and responsiveness to forskolin was determined. Results are shown as fold expression and are the means ± SEM from three experiments. CRH 532, wild-type promoter; M2, CRH -532 promoter containing the M2 mutation; XCRE, CRH -532 XCRE promoter with mutation of the canonical CRE at -220 bp; XCRE & M2, CRH -532 double mutant.

 
Identification of the Nuclear Factor as a 58-kDa Protein
Our previous experiments identified the DNA-binding site of the nuclear factor within the CRH promoter. In addition, experiments also linked factor binding to PKA-regulated induction of CRH expression. More information about the protein was determined by using UV-cross-linking.

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 protein’s 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. 8Go). 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.



View larger version (74K):
[in this window]
[in a new window]
 
Figure 8. UV Cross-Linking Studies Reveal the Human-Specific Factor to be a 58-kDa DNA Binding Protein

The labeled bromodeoxyuridine (BrdU) fragment used in this assay was from -146 to -73 bp in the hCRH promoter and was incubated with nuclear extracts from Rcho-1, JEG-3, or BeWo cells, with or without 10 pmol unlabeled -146 to -73 bp hCRH competitor, as indicated. The arrow indicates the location of the 58-kDa protein. MW, 14C-labeled protein mol wt standards; None, labeled BrdU fragment without nuclear extract.

 
The Nuclear Factor Does Not Bind to a CRE and Does Not Contain CREB or ATF-2
The data presented above indicate that the factor we identified binds to a sequence unrelated to the canonical CRE and that its estimated subunit molecular mass is distinct from that of CREB, the 43-kDa transcription factor most widely associated with cAMP transcriptional regulation. To further distinguish the identity of the placental factor from CREB, we evaluated the ability of the canonical CRE sequence to compete for binding in EMSA. Figure 9Go shows that while the 20-mer binding site oligonucleotide competes for binding with the -146 to -73 probe, neither the canonical CRE nor a canonical nuclear factor (NF)-{kappa}B binding site competes in the gel shift assay.



View larger version (98K):
[in this window]
[in a new window]
 
Figure 9. The CRE Does Not Compete with Factor Binding

EMSA binding reactions contained JEG-3 nuclear extract (12.6 µg) and labeled -146 to -73 hCRH probe. Excess competitor (3 pmol) was added as indicated. P, Probe alone; NE, JEG-3 nuclear extract without competitor; + 20-mer, excess unlabeled 20-mer fragment; +CRE, excess unlabeled canonical CRE fragment; +NF-KB, excess unlabeled NF-KB binding site.

 
To further exclude the possibility that the placental factor was identical to CREB, or contained a member of the CREB family in a complex, we performed supershift EMSA with antibody preparations either directed against CREB or the related factor, ATF-2. ATF-2 is a transcription factor related to CREB, which at 56 kDa, is similar in size to the placental factor. Figure 10AGo shows that appropriate commercial antibodies produce a distinctive supershift in assays using preparations of ATF-2 and CREB, and a labeled DNA duplex probe containing the canonical CRE. Reticulocyte lysates appropriately programmed to translate ATF-2, but not a mock-translated preparation, produced a robust gel shift that was specific for binding to the CRE. The presence of specific anti-ATF-2 antibody produced a diagnostic supershift. For similar experiments with CREB-specific antibody, nuclear extracts from a neuroblastoma cell line, BE(2)-C, and from Hela cells, were used. Specific binding to the CRE probe was dependent on the presence of nuclear extract and was competed by unlabeled CRE competitior. A diagnostic supershift was obtained upon addition of the anti-CREB antibody for BE(2)-C cell extracts, and for Hela cell extracts. In contrast to the supershift observed with preparations containing ATF-2 and CREB, Fig. 10CGo shows neither supershift nor diminished intensity of placental factor binding to the -146 to -73 DNA probe in the presence of anti-CREB or anti-ATF-2 antibodies.



View larger version (49K):
[in this window]
[in a new window]
 
Figure 10. The Placental Factor Is Not CREB or ATF-2 and Is Absent From Hela Cell Extracts

A, Specific antibodies produce distinct supershifts with ATF-2 and CREB. Supershift EMSAs were performed using a labeled CRE oligonucleotide as probe and antibodies against ATF-2 and CREB. Shift and supershift (*) are indicated by arrows for ATF-2 (left) and CREB (right). The addition of mock-translated lysate, translated ATF-2, BE(2 )-C nuclear extract (15 µg), or Hela nuclear extract (5.3 µg) are shown. Anti-ATF-2 antibody, anti-CREB antibody, or 1 pmol unlabeled CRE oligonucleotide additions are as indicated. B, Hela nuclear extract lacks the placental shift. EMSA was performed with either Hela nuclear extract (12.6 µg) or JEG-3 nuclear extract (12.6 µg) using labeled probe (-146 to -73 hCRH). The arrow indicates the position of the placental-specific shift. C, Antibodies to ATF-2 and CREB do not affect placental factor binding. EMSA was performed with JEG-3 nuclear extract (12.6 µg) using labeled probe (-146 to -73 hCRH). Addition of anti-ATF-2 or anti-CREB antibody is as indicated. The arrow indicates the position of the placental-specific shift.

 
The Factor Is Present in Nuclear Extracts from Human Term Placenta but Not Hela Cells
Our previous studies (10) and the data presented above have demonstrated that the factor binding to the hCRH 20-mer sequence within the -146 to -73 sequence is present in human choriocarcinoma cell lines, but absent from the corresponding rodent choriocarcinoma cell line. To determine whether the factor might be present in another human cell line, EMSA were performed using nuclear extracts prepared from Hela cells, a human cell line that is not derived from placental choriocarcinoma cells. EMSA failed to demonstrate the presence of the distinctive shift seen with similar extracts from JEG-3 cells (Fig. 10BGo). The lack of EMSA activity in this Hela extract is specific for the placental factor, since this same Hela extract demonstrates activity in supershift EMSA for CREB activity (Fig. 10AGo).

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. 4Go, 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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CRH is a single-copy gene that is highly conserved and that displays a unique pattern of expression. Its primary site of expression is the hypothalamus, where it displays a circadian expression pattern, but one that is modulated by stress and by feedback regulation. It is also expressed in several peripheral tissues including placenta (1). The expression in placenta is uniquely species-specific. Only humans and high primates express the gene in this organ (7). This placental expression may reflect the unique changes in gestation and parturition that have occurred in human evolution. Furthermore, the mechanisms controlling the expression of CRH in the placenta must be distinct from those controlling the expression of CRH in the HPA axis, which are highly conserved across many species (1).

One other placental gene that has been extensively studied is the {alpha}- 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 {alpha}-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 {alpha}-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 {alpha}-CG (16, 17, 18). Experiments in transgenic mice have shown that the bovine {alpha}-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 {alpha}-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 {alpha}-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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
EMSA
The preparation of the JEG-3, BeWo, Hela, and Rcho-1 nuclear extracts and the binding and electrophoresis conditions for the human-specific factor were previously described (10, 29). Results were visualized using autoradiography or by storage screen analysis. Duplex DNA fragments were labeled using either direct incorporation of radioactive nucleotides during PCR, 3'-end labeling with Sequenase (USB Corp., Cleveland, OH) or Klenow fragment (Life Technologies, Gaithersburg, MD), or 5'-end labeling with T4 polynucleotide kinase. DNA fragments used included hCRH -196 to -73, hCRH -146 to -73, hCRH -129 to -73, hCRH -196 to -136, hCRH -112 to -73, hCRH -128 to -112, and hCRH -128 to -109. Unlabeled duplex DNA fragments for competition were prepared by annealing complementary pairs of synthetic oligonucleotides and, if necessary, end filling with either Klenow fragment or Sequenase and deoxynucleoside triphosphates. Unlabeled competitors were added at approximately 1 pmol, and included hCRH -146 to -107, hCRH -112 to -73, hCRH -117 to -98, hCRH -128 to -112, and hCRH -128 to -109, as well as duplex oligonucleotides containing canonical binding sites for CREB and NF-{kappa}B purchased from Promega (Madison, WI).

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 L{Delta}2S 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 L{Delta}2S 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 {gamma}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 {alpha}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.


    ACKNOWLEDGMENTS
 
This work was supported by NIH Grant RO1DK-45506 (to S.A.) from the National Institute of Diabetes and Digestive and Kidney Diseases and by the United States Army Medical Research and Material Command (to C.D.S.) predoctoral award DAMD17-94-J-4187. The authors wish to thank T. Ramkumar for assistance and scientific discussions, clinical colleagues in the Department of Obstetrics and Gynecology for access to human placentas, and Mary Ann Mallon for excellent technical support.


    FOOTNOTES
 
Address requests for reprints to: Stuart Adler, Washington University School of Medicine, Department of Obstetrics and Gynecology, 4911 Barnes Hospital Plaza, St. Louis, Missouri 63110-1094. E-mail: adlers{at}medicine.wustl.edu

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. Back

Received for publication July 21, 1997. Revision received February 18, 1998. Accepted for publication April 20, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Wilson JD, Foster DW (eds) 1992 Textbook of Endocrinology. WB Saunders, Philadelphia
  2. Spengler D, Rupprecht R, PhiVan L, Holsboer F 1992 Identification and characterization of a 3',5'-cyclic adenosine monophosphate-responsive element in the human corticotropin releasing hormone gene promoter. Mol Endocrinol 6:1931–1941[Abstract]
  3. Adler GK, Smas CM, Fiandaca M, Frim DM, Majzoub JA 1990 Regulated expression of the human corticotropin releasing hormone gene by cyclic AMP. Mol Cell Endocrinol 70:165–174[CrossRef][Medline]
  4. He X, Treacy MN, Simmons DM, Ingraham HA, Swanson LW, Rosenfeld MG 1989 Expression of a large family of POU-domain regulatory genes in mammalian brain development. Nature 340:35–42[CrossRef][Medline]
  5. Li P, He X, Gerrero MR, Mok M, Aggarwal A, Rosenfeld MG 1993 Spacing and orientation of bipartite DNA-binding motifs as potential functional determinants for POU domain factors. Genes Dev 7:2483–2496[Abstract]
  6. Schonemann MD, Ryan AK, McEvilly RJ, O’Connell SM, Arias CA, Kalla KA, Li P, Sawchenko PE, Rosenfeld MG 1995 Development and survival of the endocrine hypothalamus and posterior pituitary gland requires the neuronal POU domain factor Brn-2. Genes Dev 9:3122–3135[Abstract]
  7. Robinson BG, Arbiser JL, Emanuel RL, Majzoub JA 1989 Species-specific placental corticotropin releasing hormone messenger RNA and peptide expression. Mol Cell Endocrinol 62:337–341[CrossRef][Medline]
  8. Muglia LJ, Jenkins NA, Gilbert DJ, Copeland NG, Majzoub JA 1994 Expression of the mouse corticotropin-releasing hormone gene in vivo and targeted inactivation in embryonic stem cells. J Clin Invest 93:2066–2072[Medline]
  9. McLean M, Bisits A, Davies J, Woods R, Lowry P, Smith R 1995 A placental clock controlling the length of human pregnancy. Nature Med 1:460–463[Medline]
  10. Scatena CD, Adler S 1996 Trans-acting factors dictate the species-specific placental expression of corticotropin releasing factor genes in choriocarcinoma cell lines. Endocrinology 137:3000–3008[Abstract]
  11. Seasholtz AF, Thompson RC, Douglass JO 1988 Identification of a cyclic adenosine monophosphate-responsive element in the rat corticotropin-releasing hormone gene. Mol Endocrinol 2:1311–1319[Abstract]
  12. Mangalam HJ, Albert VR, Ingraham HA, Kapiloff M, Wilson L, Nelson C, Elsholtz H, Rosenfeld MG 1989 A pituitary POU domain protein, Pit-1, activates both growth hormone and prolactin promoters transcriptionally. Genes Dev 3:946–958[Abstract]
  13. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K 1998 Current Protocols in Molecular Biology. John Wiley & Sons, New York
  14. Fenstermaker RA, Farmerie TA, Clay CM, Hamernik DL, Nilson JH 1990 Different combinations of regulatory elements may account for expression of the glycoprotein hormone {alpha}-subunit gene in primate and horse placenta. Mol Endocrinol 4:1480–1487[Abstract]
  15. Bokar JA, Keri RA, Farmerie TA, Fenstermaker RA, Andersen B, Hamernik DL, Yun J, Wagner T, Nilson JH 1989 Expression of the glycoprotein hormone {alpha}-subunit gene in the placenta requires a functional cAMP response element, whereas a different cis-acting element mediates pituitary-specific expression. Mol Cell Biol 9:5113–5122[Medline]
  16. Steger DJ, Altschmied J, Büscher M, Mellon PL 1991 Evolution of placenta-specific gene expression: comparison of the equine and human gonadotropin {alpha}-subunit genes. Mol Endocrinol 5:243–255[Abstract]
  17. Delegeane AM, Ferland LH, Mellon PL 1987 Tissue-specific enhancer of the human glycoprotein hormone {alpha}-subunit gene: dependence on cyclic AMP-inducible elements. Mol Cell Biol 7:3994–4002[Medline]
  18. Steger DJ, Büscher M, Hecht JH, Mellon PL 1993 Coordinate control of the {alpha}- and ß-subunit genes of human chorionic gonadotropin by trophoblast-specific element-binding protein. Mol Endocrinol 7:1579–1588[Abstract]
  19. Wice B, Menton D, Geuze H, Schwartz AL 1990 Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. Exp Cell Res 186:306–316[Medline]
  20. Strauss III JF, Kido S, Sayegh R, Sakuragi N, Gåfvels ME 1992 The cAMP signalling system and human trophoblast function. Placenta 13:389–403[Medline]
  21. Deutsch PJ, Jameson JL, Habener JF 1987 Cyclic AMP Responsiveness of human gonadotropin-{alpha} gene transcription is directed by a repeated 18-base pair enhancer. J Biol Chem 262:12169–12174[Abstract/Free Full Text]
  22. Silver BJ, Bokar JA, Virgin JB, Vallen EA, Milsted A, Nilson JH 1987 Cyclic AMP regulation of the human glycoprotein hormone {alpha}-subunit gene is mediated by an 18-base-pair element. Proc Natl Acad Sci USA 84:2198–2202[Abstract]
  23. Vallejo M 1994 Transcriptional control of gene expression by cAMP-response element binding proteins. J Neuroendocrinol 6:587–596[Medline]
  24. Majzoub JA, Muglia LJ, Martinez C, Jacobson L 1995 Molecular and transgenic studies of the corticotropin-releasing hormone gene. Ann NY Acad Sci 771:293–300[Medline]
  25. Comb M, Birnberg NC, Seasholtz A, Herbert E, Goodman HM 1986 A cyclic AMP- and phorbol ester-inducible DNA element. Nature 323:353–356[Medline]
  26. Perkins AV, Linton EA 1995 Placental corticotrophin-releasing hormone there by accident or design? J Endocrinol 147:377–381[Medline]
  27. Frim DM, Emanuel RL, Robinson BG, Smas CM, Adler GK, Majzoub JA 1988 Characterization and gestational regulation of corticotropin-releasing hormone messenger RNA in human placenta. J Clin Invest 82:287–292[Medline]
  28. Robinson BG, Emanuel RL, Frim DM, Majzoub JA 1988 Glucocorticoid stimulates expression of corticotropin-releasing hormone gene in human placenta. Proc Natl Acad Sci USA 85:5244–5248[Abstract]
  29. Deryckere F, Gannon F 1994 A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques 16:405[Medline]
  30. Faria TN, Soares MJ 1991 Trophoblast cell differentiation: Establishment, characterization, and modulation of a rat trophoblast cell line expressing members of the placental prolactin family. Endocrinology 129:2895–2906[Abstract]
  31. Ross RA, Beidler JL, Spengler BA, Reis DJ 1981 Neurotransmitter synthesizing enzymes in 14 human neuroblastoma cell lines. Cell Mol Biol 3:301–312
  32. Kunkel TA 1985 Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492[Abstract]
  33. Maurer RA 1989 Both isoforms of the cAMP-dependent protein kinase catalytic subunit can activate transcription of the prolactin gene. J Biol Chem 264:6870–6873[Abstract/Free Full Text]
  34. Waterman ML, Adler S, Nelson C, Greene GL, Evans RM, Rosenfeld MG 1988 A single domain of the estrogen receptor confers DNA binding and transcriptional activation of the rat prolactin gene. Mol Endocrinol 2:14–21[Abstract]
  35. Chen C, Okyama H 1987 High efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752[Medline]
  36. Olansky L, Welling C, Giddings S, Adler S, Bourey R, Dowse G, Serjeantson S, Zimmet P, Permutt MA 1992 A variant insulin promoter in non-insulin dependent diabetes (NIDDM). J Clin Invest 89:1596–1602[Medline]
  37. Eustice DC, Feldman PA, Colberg-Poley AM, Buckery RM, Neubauer RH 1991 A sensitive method for the detection of ß-galactosidase in transfected mammalian cells. BioTechniques 11:739–743[Medline]
  38. Kornhauser JM, Nelson DE, Mayo KE, Takahahi JS 1992 Regulation of jun-B messenger RNA and AP-1 activity by light and a circadian clock. Science 255:1581–1584[Medline]