A Requirement for the POU Transcription Factor, Brn-2, in Corticotropin-Releasing Hormone Expression in a Neuronal Cell Line

Thiruvamoor Ramkumar and Stuart Adler

Departments of Obstetrics and Gynecology and Cell Biology and Physiology (S.A.) Division of Biology and Biomedical Sciences (T.R.) Program in Molecular Genetics (T.R., S.A.) Washington University Medical School Saint Louis, Missouri 63110


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The expression of the endogenous CRF gene was examined in the human neuroblastoma cell line, BE(2)-M17. In this cell line, treatment with retinoic acid induces CRF mRNA transcription. We examined the requirement for the POU transcription factor, Brn-2, for this response. We confirmed that Brn-2 is expressed in retinoic acid-induced BE(2)-M17 cells. Expression of antisense Brn-2 message aborted the retinoic acid-mediated induction of CRF transcription. However, overexpression of Brn-2 was not sufficient for CRF expression in the absence of retinoic acid. These experiments support the hypothesis that Brn-2 is an intermediary for retinoic acid-induced CRF expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CRF is a neuropeptide, synthesized primarily by the paraventricular nucleus of the hypothalamus (PVN) in the mammalian brain (1). Previous studies involving the biosynthesis and secretion of CRF have provided substantial information regarding the role of this molecule in maintaining and modulating neuroendocrine homeostasis (2, 3). Although the downstream effects of secreted CRF have been studied at length, the upstream molecular events that affect its temporal and spatial expression patterns are yet to be fully elucidated. Studies in vivo or using nonneuronal tissue culture models have implicated several ubiquitous transcription factors, including CREB, glucocorticoid receptor, and estrogen receptor, in modulating CRF gene expression (4, 5, 6, 7, 8, 9, 10, 11, 12). In situ studies with the AP-1 family of transcription factors show circumstantial evidence for a possible role in CRF gene regulation (13, 14).

The POU family of transcription factors is expressed in a cell lineage-specific pattern and is known to be involved in neuroendocrine pathways. For example, Pit-1 regulates the expression of PRL and GH (15), and Unc-86 is required for sensory neuron development in Caenorhabditis elegans (16). Brn-2, another POU domain transcription factor, has been shown to be involved in retinoic acid-mediated neural differentiation of pluripotent embryonal carcinomas (17). In situ hybridization studies demonstrate the presence of Brn-2 protein in the PVN (18). Furthermore, transgenic mouse experiments have also shown that null mutants of Brn-2 fail to properly develop neurons that make up the CRF-synthesizing population of the PVN (19, 20). In vitro experiments have shown the presence of Brn-2-binding sites in the 5'-promoter region of the CRF gene (21). In the CV-1 monkey kidney cell line, these sites have been demonstrated to activate the expression of an artificial reporter gene driven by the CRF promoter.

Although a number of studies have given us information on the candidate factors controlling CRF gene expression, detailed molecular analyses are hampered by the lack of a suitable neuronal system in which the endogenous CRF gene is expressed. Recently, a human neuroblastoma-derived cell line, BE(2)-M17, has been shown to express the endogenous CRF gene upon retinoic acid induction (22, 23). In the following report, we present experiments performed in this neuronal cell line. Using antisense Brn-2 constructs, we demonstrate the requirement for Brn-2 in retinoic acid-induced expression of CRF. In addition to its demonstrated role in hypothalamic development, this suggests that Brn-2 may be required for the expression of the CRF gene in terminally differentiated neurons.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Brn-2 Site Binding Activity Is Expressed in BE(2)-M17 Cells upon Induction with Retinoic Acid
The BE(2)-M17 cell line, derived from a human neuroblastoma, has been shown to express the endogenous CRF peptide in a time-dependent manner upon induction with retinoic acid (23). To ensure this was not a result of gross genomic rearrangements involving the sequences in and around the CRF gene, we performed Southern blots on the genomic DNA from the BE(2)-M17 cell line. The blots were probed with a DNA fragment containing the second exon of the human CRF gene. We did not detect any differences between genomic DNA from BE(2)-M17 and control human leukocyte DNA (data not shown).

In a mouse embryonal carcinoma cell line, P-19, retinoic acid has also been shown to induce the expression of Brn-2, a POU domain transcription factor, while progressing toward a neuronal fate (17). In the same study, the absolute requirement for Brn-2 in neuronal differentiation was illustrated by the use of antisense Brn-2 message.

We hypothesized that retinoic acid also induces the expression of Brn-2 in the BE(2)-M17 cell line as a required step in the pathway for expression of the CRF gene. Nuclear extracts were prepared from untreated cells and cells treated with retinoic acid for 3 days. The nuclear extracts were then used for specific gel shift assays in reactions that were normalized for the total amount of protein. An oligonucleotide with a Brn-2-binding site was used as a probe. Parallel experiments were performed with unlabeled Brn-2-binding site oligonucleotides as specific competitors or with an oligonucleotide containing the CREB-binding site as nonspecific competitor.

Consistent with the neuronal origin of these cells, endogenous Brn-2 site binding activity is present, even in untreated BE(2)-M17 cells, as evidenced by the gel shift (Fig. 1aGo). This shift is specific, as it is competed by excess unlabeled oligonucleotides (Fig. 1aGo, lanes 2 and 3). Moreover, a similar shift can be obtained using in vitro translated Brn-2 protein (Fig. 1aGo, lane 7) and hypothalamic nuclear extracts (data not shown). A similar but more robust shift is seen when extracts from retinoic acid-treated cells are used. Since the reactions were standardized for the total amount of protein, this suggests that there is more active Brn-2 protein in the induced cells. The shift from the induced cell extracts is also specific as it is competed away by a specific unlabeled competitor, while the shifts are unaffected when a nonspecific competitor is used (Fig. 1aGo, lanes 4–6). To control for nonspecific changes in the activity of the nuclear extract preparations, gel shifts with labeled CREB oligonucleotide were also performed. While the shifts on the Brn-2 oligonucleotide were induced by the retinoic acid treatment, induced and uninduced extracts had similar binding activity on the CREB oligonucleotide (data not shown).



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Figure 1. Brn-2 Is Expressed in BE(2 )-M17 Cells upon Induction with Retinoic Acid

A, The presence of Brn-2 site binding activity was assayed by electromobility shifts. The arrow indicates the specific shifts, which are similar to those observed with hypothalamic tissue extracts. The probe used to obtain the shifts was a Brn-2-binding site from hCRF. Lane 1, Probe alone; lane 2, untreated BE(2 )-M17 cells; lane 3, with 125-fold excess specific unlabeled competitor; lane 4, 3-day retinoic acid-treated cm;0>BE(2 )-M17 cells; lane 5, with 125-fold excess specific unlabeled competitor; lane 6, with 100-fold excess nonspecific unlabeled competitor with a CREB-binding site; lane 7, translated Brn-2 protein. (+C, with specific unlabeled competitor; +nsC, with nonspecific unlabeled competitor) B, The presence of Brn-2 message was confirmed by reverse transcriptase-PCR. The arrow indicates the Brn-2 RT-PCR product. Lane 1, Molecular size ladder; lane 2, BE(2 )-M17 cells induced with retinoic acid; lane 3, untreated BE(2 )-M17 cells; lane 4, retinoic acid-treated, low-copy BE(2 )-M17 stable transformant of PGK-{alpha} Brn-2; lane 5, retinoic acid-treated, high-copy BE(2 )-M17 stable transformant of PGK-{alpha} Brn-2.

 
The expression of Brn-2 was also studied using an RT-PCR method (Fig. 1bGo). We were able to detect low levels of the Brn-2 mRNA in the untreated parental cells (Fig. 1bGo, lane 3). It should be noted that this observation was rather inconsistent. The parental cells that were treated with retinoic acid showed visible amounts of Brn-2 transcript (Fig. 1bGo, lane 2).

Brn-2 Affects Expression of CRF Reporter Genes in Retinoic Acid-Treated and Untreated Cells
Using the same sense and antisense Brn-2 expression plasmids used by Fujii and Hamada (17), we studied the effect of Brn-2 on transcription from a full-length CRF promoter. The reporter plasmid used was constructed from a 8-kb human CRF genomic (24), in which the coding region of the second exon was replaced in-frame by a sequences encoding the firefly luciferase gene (25). This construct was cotransfected with either cytomegalovirus (CMV)-Brn-2 (Fig. 2aGo) or PGK-{alpha} Brn2 (Fig. 2bGo) into BE(2)-M17 cells and either treated with retinoic acid or left untreated. Various amounts of the Brn-2 expression plasmids were transfected, and fusion gene expression was assayed.



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Figure 2. Brn-2 Affects Expression of CRF-Luciferase Reporter in Retinoic Acid-Treated and Untreated Cells

Transient transfections were performed with CRF reporter plasmid along with either CMV Brn-2 (A) or PGK-{alpha} Brn-2 (B) in varying amounts as indicated. The cells were then harvested and assayed for luciferase as described. The standardized luciferase values are plotted along with the amount of transfected plasmid. {square}, No retinoic acid; {circ}, treated with retinoic acid. Results are means ± SD of at least three experiments.

 
In agreement with previous studies demonstrating an increase in CRF mRNA and protein in response to retinoic acid treatment (23, 26), we observed an increase in CRF reporter gene expression. There is a 6.5-fold increase in expression levels upon retinoic acid treatment (Fig. 2aGo). Furthermore, the retinoic acid-treated cells respond rather robustly to the presence of exogenous Brn-2. A 8- to 10-fold increase in expression was observed when the amount of transfected CMV Brn-2 was increased to 0.5 µg/well. Further increases in the amount of Brn-2 did not affect fusion gene expression. We postulate this to be due to a limiting factor and/or competition for cellular transcription factors. On the other hand, the untreated cells did not show any marked increase in expression from the CRF-luciferase reporter gene, even after addition of a substantial amount of CMV Brn-2. This suggests that the presence of Brn-2 alone is not sufficient to mediate the increase in CRF expression seen with retinoic acid treatment.

Participation of Brn-2 in the expression of the transfected CRF reporter gene was demonstrated using an antisense construct. The retinoic acid-treated and untreated cells both show reproducible decreases in luciferase expression on cotransfection of a plasmid that expresses the antisense Brn-2 mRNA (Fig. 2bGo). Very small amounts of antisense plasmid were sufficient to produce this change, which is dramatic in the treated cells. This is striking when compared with the levels of CMV Brn-2 sense plasmid construct, considering that the strength of CMV promoters is much higher than PGK promoters.

The relative expression values with cotransfected antisense Brn-2 in the untreated cells were similar to background levels. The expression of CRF/luciferase for the treated cells was always higher than that of untreated cells, over the entire range of antisense plasmid concentration. This suggests that in treated cells, either small quantities of functional Brn-2 are present, or there is a Brn-2-independent pathway for retinoic acid-mediated expression.

Brn-2 Binding Sites Can Mediate Both Brn-2 and Retinoic Acid Responses
To determine whether the induction observed with retinoic acid is specific and to assess the role of Brn-2 and its binding sites in this induction, we evaluated the responses of artificial promoters and compared these results to a CRF promoter (Fig. 3Go). We used a 5-kb hCRF reporter plasmid (CRF L{Delta} 2S), a minimal 36-bp promoter (P36 L{Delta} 2S), and the corresponding promoter containing three tandem repeats of the native Brn-2 binding site (Multi-3 P36). The P36 plasmid was not responsive to retinoic acid induction. Similarly, this P36 plasmid was not induced by expression of Brn-2 by cotransfection of the Brn-2 expression vector. In addition, the cotransfection of the Brn-2 antisense expression vector did not result in a lower signal for the P36 plasmid. These data serve to exclude the possibility of cryptic or unrecognized retinoic acid response elements or Brn-2 response elements in the plasmid backbone or reporter gene. The Multi-3 plasmid containing Brn-2-binding sites was not only induced by Brn-2 cotransfection and repressed by expression of antisense Brn-2, but this plasmid was also induced by retinoic acid. These data indicate that Brn-2-binding sites can mediate transcriptional responses dependent on Brn-2, and, in addition, also confer responsiveness to retinoic acid, even in the absence of sequences identified as canonical retinoic acid receptor-binding sites. In contrast, the 5-kb CRF promoter, as shown with the CRF fusion plasmid, is induced by retinoic acid and exhibits an inhibition of basal expression with antisense Brn-2, but is not induced by Brn-2 expression in the absence of retinoic acid. These data demonstrate that isolated Brn-2-binding sites can transfer retinoic acid responses and Brn-2 responses to a minimal promoter. Yet, in the context of the CRF promoter, while antisense Brn-2 produces inhibition of the basal expression level, increased responses to Brn-2 are not seen in the absence of retinoic acid.



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Figure 3. Brn-2 Binding Sites in Isolation Are Both Retinoic Acid and Brn-2 Responsive

Transient transfections were carried out in the BE(2 )-M17 cell line. Luciferase reporter constructs used were either a minimal P36 promoter, a P36 promoter with three tandem Brn-2 binding sites, or a 5-kb hCRF promoter. Each of the reporters was examined under three different conditions, and results are shown as fold expression, determined as the ratio of luciferase activity from each treatment vs. its corresponding control. Open bars, Activity in the absence (control) or presence of retinoic acid; hatched bars, activity with cotransfection of either CMV-Neo (control) or CMV-Brn-2 plasmids; solid bars, activity with cotransfection of either PGK-MT (control) or PGK-{alpha} Brn-2. Results are means ± SD of three experiments.

 
The above data demonstrate that Brn-2-binding sites are sufficient to transfer a retinoic acid response to a minimal heterologous promoter in BE(2)M-17 cells. We next created a mutated -532 CRF promoter in which six of the multiple Brn-2 binding sites present in the native promoter were altered to sequences with low affinity for Brn-2. This mutated promoter has markedly decreased expression, both basally and in response to cotransfected Brn-2 in Hela cells (Fig. 4Go). Quite unexpectedly and in contrast to their effect in Hela cells, in BE(2)M-17 cells these mutations of the Brn-2-binding sites resulted in a marked increase in promoter activity of 6.6 ± 1.4-fold compared with the parental construct (Fig. 4Go). In addition, the mutated promoter had a decreased induction of expression in response to either retinoic acid (not shown) or 9-cis-retinoic acid (Fig. 4Go). These data suggest two potential roles for the Brn-2-binding sites in the CRF promoter. First, in the context of the native promoter and in the neuronal BE(2)M-17 cell line, but not in Hela cells, the Brn-2 sites appear to be associated with repression of the promoter, an effect that can be partially relieved by mutation. That is, activity of the mutated promoter is higher than the parental promoter in BE(2)M-17 cells but is lower in activity in Hela cells. Second, treatment with retinoids, either retinoic acid or 9-cis-retinoic acid, causes increased expression via the Brn-2 sites, and this induced expression is diminished by these mutations.



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Figure 4. Brn-2 Binding Sites in the CRF Promoter Mediate Both Cell Type-Specific Repression and Responsiveness to Retinoids

Transient transfections were performed in Hela cells and in the BE(2 )-M17 cell line. Luciferase reporters were the -532 CRF promoter and the same promoter mutated as indicated in the six Brn-2 binding sites. Luciferase activity was determined in Hela cells with either cotransfected CMV Neo plasmid (0.4 µg/well) as control, or the CMV Brn-2 expression plasmid (0.4 µg/well). In BE(2 )M-17 cells, cells were unstimulated or treated with 9-cis-retinoic acid. Results shown are the means ± SEM from three experiments.

 
Stable Transformation of Sense and Antisense Brn-2 Constructs Affect Inducibility by Retinoic Acid
Three lines of stable transformants of the BE(2)-M17 cell line were made for further analysis. One stably transformed line containing the CMV Brn-2 plasmid, and two transformed cell lines containing either a high-copy (MAM-19) or a low-copy (MAM-3) of PGK-{alpha} Brn-2 plasmid were isolated. These transformants, along with the parental BE(2)-M17 cell line, were treated with retinoic acid and were transiently transfected with CRF reporter plasmid and assayed for luciferase expression.

As seen in the dose-response profile (Fig. 2Go), the parental BE(2)-M17 shows a 6.5-fold increase in CRF expression with retinoic acid (Fig. 5Go). No major differences were seen in the CMV Brn-2 stable line. The antisense transformants, MAM-3, which has 3 copies of PGK-{alpha} Brn-2, and MAM-19, which has 19 copies of PGK-{alpha} Brn-2, show a marked and reproducible drop in activity both in treated and untreated cells. MAM-19 shows virtually no CRF luciferase expression, with only background levels of luciferase activity. This suggests that both basal and retinoic acid-induced transcription of CRF are inhibited by the presence of antisense Brn-2 and suggests a possible requirement for Brn-2 transcription factor for even basal expression of CRF. In the MAM-3 and MAM-19 stable transformant cells expressing the antisense Brn-2 construct we did not detect endogenous Brn-2 mRNA (Fig. 1bGo, lanes 4 and 5). This could be either due to antisense mechanisms resulting in the degradation of Brn-2 mRNA within the cells, or, alternatively, due to interference of the antisense messages with the reverse transcriptase-PCR assay.



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Figure 5. Stable Transformation of Sense and Antisense Brn-2 Constructs Affect Inducibility by Retinoic Acid

Stable transformants of the indicated plasmids were made in the BE(2 )-M17 cell. MAM-3 and MAM-19 are low- and high-copy transformants with PGK-{alpha} Brn-2. The transformants were then transiently transfected with a CRF reporter plasmid and then either left untreated or treated with 5 µM retinoic acid. The cells were then assayed for luciferase activity. The luciferase values obtained were then standardized to that of the untreated wild-type BE(2 )-M17 cells. Results are means ± SD of at least three experiments.

 
Endogenous CRF Expression in BE(2)-M17 Cell Lines Is Affected by Brn-2
To document the effect of sense and antisense Brn-2 expression on the levels of the endogenous CRF mRNA levels, we evaluated CRF mRNA by reverse transcriptase-PCR. Total RNA was isolated from parental BE(2)-M17 cells, which were either left untreated or treated with retinoic acid. RNA was also isolated from stable transformants MAM-3 and MAM-19, after they were grown in the presence of retinoic acid for 3 days. After RT of the RNA, the resulting cDNA was spiked with the addition of 0.1 ng of plasmid that had the CRF primer-binding sites, but generated a distinctly shorter fragment. This was used as an internal control and for quantitation. The samples were then subjected to PCR. An external control for the total amount of RNA included in the reactions was also performed using cyclophilin mRNA as the target sequence (data not shown).

As previously observed (23), we note that the endogenous CRF mRNA is transcribed after induction with retinoic acid. (Fig. 6Go, lanes 1 and 2). When MAM-3 and MAM-19 are treated with retinoic acid, we still detect the endogenous CRF message but at markedly lower levels, more so for MAM-19 than for MAM-3 (Fig. 6Go, lanes 3 and 4). It is interesting to note that complete inhibition of the endogenous CRF mRNA is not seen even in the high-copy stable line MAM-19. Neither of the stable lines express their endogenous CRF gene when not treated with retinoic acid (data not shown). The quantitative data from Fig. 5Go and multiple repetitions of the experiment in Fig. 6Go are presented in Table 1Go.



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Figure 6. Endogenous CRF Expression in BE(2 )-M17 Cell Lines Is Affected by Brn-2

Total RNA isolated from the indicated cells was subjected to RT-PCR. After RT, the reactions were spiked with 0.1 ng of a plasmid containing complementary sequences to the PCR primers, to serve as an internal control. The reactions were resolved by electrophoresis. Lane 1, Untreated BE(2 )-M17 cells; lane 2, retinoic acid-treated BE(2 )-M17 cells; lane 3, retinoic acid-treated, low-copy BE(2 )-M17 stable tansformant of PGK-{alpha} Brn-2; lane 4, retinoic acid-treated, high-copy BE(2 )-M17 stable transformant of PGK-{alpha} Brn-2.

 

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Table 1. Quantitation of Expression Levels of CRF

 
Retinoic Acid Inducibility Is Not Mediated by the CRE
To test the hypothesis that the retinoic acid induction of CRF transcription is mediated by the previously identified cAMP response element (CRE), we used a CRF reporter construct with a mutation in the CRE (Fig. 7Go). Transient transfections were performed in the BE(2)-M17 cell line, both in the absence and presence of retinoic acid. We used a 532-bp proximal hCRF promoter construct as our control (CRF-532), while the mutant had a nonfunctional CRE in the same context (-532 X-CRE). The mutation alters the CRE at -220 to an EcoRI site. As shown in Fig. 7Go, both the wild-type and mutant reporter plasmids are equally responsive to retinoic acid, although we did notice a slight difference in the basal expression levels between the two constructs. Further, to ascertain a role in retinoic acid response for protein kinase A (PKA), a key factor in the cAMP second messenger pathway, we cotransfected an expression vector [rous sarcoma virus (RSV)-protein kinase inhibitor (PKI)] for PKI along with CRF-532. PKI is a peptide inhibitor of PKA and has been shown to be active in transient transfection assays. The presence of PKI did not affect retinoic acid inducibility but dropped the basal expression levels noticeably (Fig. 7Go).



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Figure 7. Retinoic Acid Induction Is Not Mediated by the CRE

Transient transfections were carried out in the BE(2 )-M17 cell line as described. Luciferase reporter constructs used were either a 532-bp CRF promoter (CRF-532) or a similar construct but, with its CRE at -220 mutated to an EcoRI site (-532 X-CRE). CRF-532 was also cotransfected with PKI, a peptide inhibitor of PKA. All the transfections were done, both in the presence (open bars) and absence (solid bars) of retinoic acid. Results are calculated means ± SD of at least three experiments, and the mean of the luciferase values of CRF-532 in the absence of retinoic acid was arbitrarily set to 1.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CRF plays a key role in regulating the daily excursions of glucocorticoid production and in the mammalian response to stress (1, 2, 3). The primary site of CRF synthesis is the PVN, where it initiates the regulatory cascade of the HPA axis by controlling the expression and secretion of ACTH from the anterior pituitary. Studies to date have indicated two distinct mechanisms for the control of CRF expression. In the first case, in which CRF expression is steroid sensitive, glucocorticoids act via a negative feedback loop. This sensitivity is linked to the circadian rhythm of CRF gene expression and secretion. The second mechanism, in which CRF is insensitive to plasma glucocorticoid levels, is apparent during conditions of stress. In spite of elevated levels of glucocorticoids, CRF expression remains high. The biochemical mechanisms for both of these regulatory pathways remain to be elucidated.

CRF is a single-copy gene, and the primary sequences of the gene and the peptide product have been determined (24). Detailed molecular studies of CRF expression, however, have been hampered by difficulties involved in obtaining experimental amounts of hypothalamic explants and the lack of a suitable cell line of hypothalamic origin. In this study we present experiments in a cell line of neuronal origin, which expresses the endogenous CRF mRNA and peptide upon stimulation with retinoic acid.

Previous studies on CRF gene regulation have examined the effects of various cellular stimuli on the mRNA and peptide expression from exogenously transfected gene constructs in nonnative and often nonneuronal cell lines. In one such study, a monkey kidney CV-1 cell line was used with transfected CRF reporter plasmids (21). That study demonstrated the presence of binding sites for Brn-2 in the 5'-promoter-proximal region (21). The identified binding sites mediated transcriptional activation when a plasmid expressing Brn-2 was cotransfected (21), a result we have confirmed in Hela cells.

Transgenic mice with targeted deletions of the Brn-2 gene have further implicated Brn-2 as a candidate transcription factor in the control of CRF expression (19, 20). The Brn-2 null mice fail to develop and differentiate the hypothalamic region where the CRF neurons reside. This strongly suggests that Brn-2 is required for the differentiation of progenitor neuronal cells in the PVN into CRF-expressing, postmitotic neurons. Unfortunately, this type of experimental model is unable to determine the role of Brn-2 in the modulation of CRF gene expression in the mature and differentiated, CRF-producing cells.

Brn-2 has previously been shown to participate in the differentiation of P-19 embryonal cells, a model system for neuronal development (17, 27). In these cells, retinoic acid induces differentiation toward a neuronal fate, an effect conclusively shown to be mediated through Brn-2. The cellular system that we have used in our study is the BE(2)-M17 cell line. The BE(2)-M17 cells are derived from a human neuroblastoma and have previously been shown to express the endogenous CRF peptide upon induction with retinoic acid. Our experiments were designed to test the hypothesis that Brn-2 does indeed affect the expression of CRF in the BE(2)-M17 cell line.

Consistent with this hypothesis, retinoic acid induces the expression of Brn-2. This was demonstrated at both the protein level and mRNA level using electromobility gel shift assays and reverse transcriptase-PCR, respectively. These experiments suggest that, although there is a noticeable increase in the amount of Brn-2 binding activity upon retinoic acid treatment, a constant basal level of Brn-2 activity is present in the untreated cells. Explanations consistent with these observations include the possibility that there is a requirement for Brn-2 above a critical threshold of cellular concentration for CRF expression, or that other factor(s) are needed to complete the pathway to CRF expression. Data obtained from promoters mutated in the Brn-2-binding sites provide evidence for an additional mechanistic role for Brn-2 and its binding sites. Surprisingly, in the neuronal cell line, but not Hela cells, mutation of Brn-2-binding sites results in increased promoter activity, yet with decreased responsiveness to retinoid induction. While the mechanism of this cell type-specific repression is not clear, these data are consistent with our observations that in BE(2)M-17 cells in the absence of retinoid treatment, expression of Brn-2 is not sufficient to induce CRF expression. Yet, in the context of a minimal heterologous promoter, tandem Brn-2 sites confer responsiveness to either Brn-2 or retinoids in this cell line. For appropriate expression of the CRF promoter, both retinoid treatment and expression of Brn-2 appear to be necessary for full promoter activity.

Experiments with antisense Brn-2 constructs provide the strongest case for Brn-2’s role in CRF regulation. Effects of antisense Brn-2 were observed with both the endogenous CRF gene and transfected CRF-reporter genes. Dose-response profiles with the antisense Brn-2 in the presence of a CRF-reporter plasmid indicate that even low levels of antisense constructs can markedly inhibit transcription from the CRF promoter. The observed inhibition is apparent even in the presence of retinoic acid. A similar pattern was observed when the endogenous CRF mRNA was studied in cell lines stably transfected with the antisense Brn-2 construct. CRF mRNA induction, as measured by RT-PCR, was markedly lower in the Brn-2 antisense transfected cell lines when compared with controls. Moreover, the inhibition was dependent on the number of stable insertion events of the antisense construct. These experiments suggest a role for Brn-2 in CRF expression. Another series of dose-response experiments evaluating CRF-reporter gene expression in the presence of overexpressed Brn-2 protein indicates that the Brn-2 protein alone cannot account for the increase in expression seen upon induction with retinoic acid.

Numerous recent studies have shown that many transcription factors, including the POU proteins, have the ability to interact with other transcription regulators to effect additional control over gene expression (28). These interactions, although specific, may be facilitated (or inhibited) by the DNA sequences that constitute the factor-binding sites (29). Moreover, sequences flanking the binding site may also contribute to these interactions (30). In our study, we noticed a difference in the ability of a Brn-2-binding site to activate transcription dependent on the promoter context. Brn-2-binding sites present in the intact CRF promoter are unable to respond to expressed Brn-2 in the absence of retinoic acid. The same Brn-2-binding sequence is transcriptionally active when present in tandem repeats, in the context of a minimal promoter. This hints at the possible influence of the surrounding sequences in the native CRF promoter context on the ability of Brn-2 to bind and/or activate transcription. A possible explanation for this modulation of activity is the binding of other transcription factors to flanking sequences. One such known site is the CRE at -220 bp, which has been shown to be active. We tested this hypothesis by mutating the CRE. We also suppressed the activity of CREB by expressing an inhibitor for PKA, a critical component in the cAMP second messenger pathway. In both the cases, the retinoic acid response was not inhibited, indicating that the CRE and any transcription factors binding to it are not required for the observed retinoic acid response.

Our results suggest several regulatory hypotheses. One possibility is that retinoic acid activates Brn-2 expression, which in turn activates another transcription factor, a direct regulator of CRF. We regard this scenario as unlikely, since it does not explain the requirement for retinoic acid even in the presence of Brn-2. Alternatively, retinoic acid could activate both Brn-2 and another factor concurrently, with CRF expression being dependent on the presence of both factors. Of course, one candidate for the second transcription factor is a retinoic acid receptor/retinoid X receptor (RAR/RXR). Since the nuclear events associated with ligand-activated nuclear receptors are rather rapid, this possibility may be tested by evaluating the time course of CRF induction. Normally, retinoic acid induction of Brn-2 proceeds over 3–7 days. If the cells transfected with a Brn-2 overexpressing plasmid (CMV Brn-2) were induced by retinoic acid, the time required to see the first traces of CRF mRNA should be noticeably reduced compared with controls. However, this predicted change in induction time with expression of Brn-2 has not been observed (S. Adler, unpublished data). A third hypothesis is that retinoid treatment is required for chromatin remodeling to activate the CRF gene. This process would have to be slow to account for the 3–7 days required for the retinoid-dependent response, and these proposed chromosomal structural changes would not necessarily explain the same effects observed with transiently transfected reporter gene plasmids. One final hypothesis is that both retinoid treatment and the presence of Brn-2 are required to turn off the expression of an inhibitor of CRF transcription that acts via binding to one or more of the Brn-2 site(s) or to sequences overlapping one or more of the Brn-2 site(s). The binding of inhibitor would be in direct competition for these sites, with Brn-2 itself acting as an activator. This hypothesis is most consistent with the data presented showing activation of the promoter associated with mutation of Brn-2 binding sites and with our previous studies of placental CRF expression, which identified a candidate repressor of CRF expression in nonplacental cells (25). The extended time course required for retinoid induction would reflect the nuclear half-life of the inhibitor, and/or time required for dilution of the nuclear inhibitor protein due to continuing cell division. The requirement for Brn-2 expression would reflect its direct role along with retinoid receptors in turning off expression of the inhibitor coupled with direct activation of the CRF promoter as Brn-2 successfully competes for binding to the promoter sites.

The requirement for Brn-2 in CRF expression may be similar to the requirement for Pit-1, another POU factor, in the expression of PRL. Pit-1 has been shown to be necessary for both the differentiation and the maintenance of PRL- and GH-secreting cells, while playing a direct role in the cell type- specific transcription of these genes (15). The interactions of Pit-1 and estrogen receptor in pituitary lactotrophs (31, 32) may serve as a model and suggest that a similar interaction may exist between Brn-2 and a retinoic acid receptor family member in the regulation of CRF (Fig. 7Go). Furthermore, the negative regulation of PRL by glucocorticoid receptor (33) may similarly be a model for the negative regulation of CRF by glucocorticoid receptor. It was also noted in preliminary observations that reporter constructs with deleted proximal promoter sequences containing the identified Brn-2-binding sites, retained the ability to be induced by retinoic acid. This observation, that distal sequences may play a role in the nuclear receptor response, parallels a similar requirement for regulation of PRL. The potential existence of all of these types of interactions between POU factors and nuclear receptors in CRF regulation remains an intriguing possibility for future studies.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Lines
The BE(2)-M17 cell line (22) was a generous gift from Dr. June Beidler (Memorial Sloan-Kettering Cancer Center, New York, NY). The cell line was initially grown at 37 C in a 5% CO2 incubator and in MEM/F12 medium supplemented with 15% FBS (Intergen, Purchase, NY), 1x nonessential amino acids (Mediatech, Washington DC) and penicillin-streptomycin and fungizone (Tissue Culture Support Center, Washington University, St. Louis, MO). The cells were then adapted to a similar media containing 10% FBS and 5% enriched calf serum (ECS, Gemini Biological Products, Calabasas, CA) instead of 15% FBS. Cells grown in this medium were used for all experiments. The Hela cell line was obtained from American Type Culture Collection (Manassas, VA). Hela cells were grown in 10% CO2 in DMEM with 5% FBS and 5% ECS and penicillin-streptomycin. All cells are routinely surveyed for mycoplasma using a PCR method from Stratagene (La Jolla, CA).

Transfections
Transient transfections of the BE(2)-M17 cells were performed using calcium phosphate (34) in six-well 35-mm plates. Typically, the cells were seeded at 1.25 x 105 cells per well in 2 ml of growth media on day 1. The actual transfection on day 3 was carried out by replacing the growth medium with DMEM containing 5% FBS + 5% ECS and incubated in 10% CO2 for 4 h. Later, 125 µl of transfection mixture containing 2.5 µg of total DNA in N,N-bis (2-hydroxyethyl)-2-aminoethane-sulfonic acid (BES)-buffered saline (BBS)-CaCl2 were dripped onto the medium, and the cells were incubated overnight in a 5% CO2 environment. The following day, the cells were washed with DMEM and fed with growth medium. The transfected cells were then harvested on day 5 in 150 µl of lysis buffer containing 50 mM Tris-HCl, 50 mM 2(N-morpholino)ethane sulfonic acid (pH 7.8), 1 mM dithiothreitol, and 1% Triton X-100. The lysate was then assayed for luciferase and ß-galactosidase activity. Cells were treated with 5 µM retinoic acid or 0.5 µM 9-cis-retinoic acid (Sigma Chemical Co., St. Louis, MO) using solutions in 95% ethanol, on day 1 after seeding. All subsequent manipulations were carried out in the presence of retinoid. Alternatively, BE(2)-M17 cells were transfected and analyzed using an 8-day schedule. Cells were seeded on day 1 at a density of 5 x 104 cells per well and fed on day 4, transfected on day 6, and harvested on day 8. Transient transfections of Hela cells in six-well plates were performed in DMEM containing 5% FBS/5% ECS using a total of 2.5 µg DNA as previously described (25).

Stable transfections used essentially the same protocol as for transient transfections. The target plasmid DNA was mixed with RSV-Hygromycin DNA (35) in a 10:1 ratio during transfection. Growth medium containing 100 µg/ml of hygromycin B (Sigma Chemical Co.) was used for selection. Three days post transfection, the wells were washed to remove the dead, untransfected cells. The remaining transformants were trypsinized, transferred to four 150-mm dishes, and maintained under selective pressure. The selective medium was replaced every 3 days until the appearance of foci, apparently from a single stably transfected cell.

In experiments cotransfecting varying amounts of Brn-2 or {alpha} Brn-2 plasmids, the effects of promoter competition were controlled by transfecting constant total amounts of CMV and PGK plasmids. CMV Neo was used as the compensatory plasmid in the case of CMV Brn-2 and PGK-MT, an empty vector, with PGK-{alpha} Brn-2. In all transfection experiments the total amount of expression plasmids was kept at 1.25 µg/well.

Plasmids and Luciferase Assays
The human CRF-luciferase reporter plasmids used were as described previously (25). The sense (CMV Brn-2) and anti-sense (PGK-{alpha} Brn-2) constructs that were used in these experiments were a precious gift from Dr. H. Fujii (Osaka University, Osaka, Japan). The plasmids, CMV-Neo and PGK-MT, were used as controls for dose-response experiments. PGK-MT was constructed by deleting the {alpha} Brn-2 fragment from PGK-{alpha} Brn-2 plasmid. CRF-532 and -532 X-CRE plasmids used in this study were as previously described (25). A plasmid for expressing the specific inhibitory peptide for PKA, RSV-PKI (36), was obtained from Dr. Richard Maurer (Oregon Health Sciences University, Portland, OR). To create a reporter containing three tandem repeats of the Brn-2-binding site, a partially kinased oligonucleotide duplex from -146 to -107 bp of the hCRF proximal promoter was multimerized in three copies using DNA ligase. These multimerized fragments were subcloned in front of the minimal 36-bp promoter, P36 (37). RSV-ß-Gal (25) was used for standardization of transfection efficiency. RSV-Hygromycin was a gift from Dr. H. Elsholtz (University of Toronto, Ontario, Canada).

Luciferase assays were done as previously described (38) in a MonoLight 2010 luminometer from Analytical Luminescence Laboratory (San Diego, CA). ß-Galactosidase assays were performed using chlorophenol red ß-galactopyranoside (Boeringher Mannheim, Indianapolis, IN) as substrate (39) and read on an Anthos Plate Reader (Anthos Labtec Instruments, Salzburg, Austria).

Determination of the Number of Stable Insertion Events
The hygromycin-resistant colonies were picked and individually transferred to 96-well plates, 24-well plates, and 6-well plates sequentially. Representative stocks of 20 resistant picks were frozen in liquid nitrogen and/or were grown in 100-mm dishes for DNA isolation. Genomic DNA was prepared as described (40) and was slot blotted onto Magna nylon membranes (Micron Separations, Inc., Westborough, MA). A 400-bp PstI fragment from the Brn-2-coding region was isolated and radiolabeled by random priming with minor modifications to the manufacturer’s instructions (Prime It, Stratagene). The membrane was probed and washed, before exposure to storage phosphor screens. A parallel control experiment was performed using human {gamma}-actin as probe. The phosphor storage screens were developed on a PhosphorImager 425B (Molecular Dynamics, Inc., Sunnyvale, CA). The signal intensities were quantitated using ImageQuant 2.0 software (Molecular Dynamics, Inc.), and the number of stable insertion events was estimated by comparison with the {gamma}-actin signal. A transformed line with a high copy number (MAM-19) of transfected plasmid and another line with a low copy number (MAM-3) were identified for further experiments.

Electromobility Shifts
Nuclear extracts from retinoic acid-treated and untreated cells were prepared as previously described (25). B6/SJL mice (The Jackson Laboratory, Bar Harbor, ME) were used as a source for hypothalamic nuclear extracts using a protocol suitable for small amounts of tissue (41). The extracts were prepared from tissue isolated and pooled from the median hypothalamic region of three mice. Brn-2 protein was expressed by in vitro translation using the TNT T7 reticulocyte lysate system (Promega Corp., Madison, WI). Complimentary DNA oligomers were synthesized, annealed, and 3'-end filled with radioactive deoxynucleotides and used as the probe in the assay. The final filled-in sequence was dTCTGCTCCTGCATAAATCATAGGGCC and has been shown to be a strong Brn-2 binding site (21). The nonspecific oligonucleotide used in the competition reactions contained the CREB-binding site with the final filled sequence, which read dGATCGGATCCGATTGCCTGA-CGTCAGAGAGCAGATCTATCG. The activity of the different nuclear extracts was also checked using radiolabeled CREB oligonucleotide as probe. Equal amounts of total protein, as determined by the method of Lowry et al. (42), were used in the binding reactions, which were performed as described (43). Unlabeled and filled, duplex oligonucleotides at 125-fold molar excess were used as competitors for protein binding. The binding reactions were resolved on a 4% polyacrylamide gel in Tris-taurine-EDTA buffer. The gels were then fixed, dried, and exposed to phosphor storage screens, and the resulting images were processed on a Molecular Dynamics, Inc. PhosphorImager.

Reverse-Transcriptase PCR
RNA was isolated from treated cells as described previously (44). Total RNA was then used to generate cDNA utilizing the following primers:

Brn-2: Forward, dCGCCGACCTCGGACGACCTG

Reverse, dCCCCAGCTTGAGTTCACTGGACG

CRF: Forward, dCCAAGT-A/C-C-A/G-TTGAGAGACTGA

Reverse, dTTCCCCAGGCGGAGGAAGT

Cyclophilin: Forward, dTTCATCTGCACTGCCAAGAC

Reverse, dAACCCAAAGGGAACTGCAG

SuperScript reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) was used for cDNA synthesis as per manufacturer’s instructions. As an internal control and for quantification, the subsequent PCR reaction was spiked with a constant amount of plasmid DNA containing the complementary sites for the two primers. The PCR product from the spiked plasmid DNA is of a different length when compared with that obtained from the cDNA. An external control was also performed using the cyclophilin transcript as the target sequence. The PCR reactions were performed for 25 cycles as follows:

Brn-2: 95 C for 30 sec, 64 C for 30 sec, and 72 C for 2 min

CRF: 95 C for 30 sec, 55 C for 30 sec, and 72 C for 2 min

Cyclophilin: 95 C for 60 sec, 52 C for 60 sec, and 72 C for 2 min

The PCR reactions were resolved on a 2% agarose gel and photographed with ethidium bromide UV fluorescence using Polaroid 557 film (Polaroid Corp., Cambridge, MA) The resulting negative was digitized on a Personal Densitometer (Molecular Dynamics, Inc.) and quantitated using ImageQuant software. A parallel experiment with known amounts of DNA was photographed and digitized for use as a standard.

Southern Blots
Genomic DNA was isolated from BE(2)-M17 cells. The DNA was subjected to restriction enzyme digestion and resolved on a 0.7% agarose gel. The blotting, hybridization, and washing were carried out as previously described (39). A 400-bp PstI fragment from the Brn-2-coding region was isolated and radiolabeled by random priming with minor modifications to the manufacturer’s instructions (Prime It Kit, Stratagene). Magna nylon membranes (MSI) were used as the hybridization medium. The membranes were exposed to a phosphor storage screen and visualized on the PhosphorImager. Normal human DNA was used as control and was a kind gift of Cris Welling (Internal Medicine, Washington University).

Site-Directed Mutagenesis
Oligonucleotide-directed mutagenesis was performed in phagemid vectors using minor modifications of the method of Kunkel (45). Deoxyuracil containing phagemid DNA was prepared initially from CRF -532 pBKSII(-), and new template was prepared from confirmed mutants for each successive round of mutagenesis (25). Individual identified Brn-2-binding sites CRF II–V (21) and additional distal sites, CRF VI and CRF VII, were modified using the following antisense oligonucleotides:

CRF II (-128 nt), dCTC CTG CAT GCG GCG CAG GGC GC

CRF III–IV (-213 nt), dCTC ACA TCC AAT GCT ATC AAC AGA TAT GCA TCG CCT CTT G

CRF V (-302 nt), dCTT GAA TGA GAT GCC CCA AGT GTG

CRF VI (-342 nt), dGAA AGG CCA TAT GCG GGG TGT GC

CRF VII (-511 nt), dCAG TAT CTG GGC ATA TCC CTT TG

All mutations were confirmed by dideoxy DNA sequencing, and corresponding reporter plasmids were made by subcloning the mutated sequences using conventional techniques. In agreement with the previous report of Li et al. (21), labeled duplex oligonucleotides containing modified Brn-2 binding sites corresponding to this pattern of mutational disruption of the CRF II site showed decreased or absent binding using in vitro translated Brn-2 in electromobility shift assays (data not shown).



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Figure 8. Model for a Parallel Role for Pit-1 and Brn-2 in Development

Based on the experiments described in this study, we present a possible model for the role of Brn-2 in CRF neuron development and CRF gene expression. This model reflects the established role of Pit-1 in the development of lactotrophs and expression of the PRL gene. We further suggest parallel roles for the nuclear receptors, estrogen receptor (ER) and a member of the retinoic acid receptor (RAR/RXR) family, along with the documented negative effect of glucocorticoid receptor (GR), on gene transcription in both cases.

 

    ACKNOWLEDGMENTS
 
The authors wish to thank Dr. J. Thomas, Dr. C. D. Scatena, and M. A. Mallon for critical review and comments. The technical assistance of Mary Ann Mallon and Molly Adler is gratefully acknowledged.


    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@medicine.wustl.edu.

This study was supported by NIH Grant DK-45506 to S.A. T.R. was supported by a predoctoral fellowship, DAMD17–94-J-4240, from the United States Army Medical Research and Development Command.

Received for publication May 7, 1997. Revision received May 7, 1999. Accepted for publication May 13, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Reichlin S 1998 Neuroendrocrinology. In: Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds) William’s Textbook of Endocrinology. W.B. Saunders Co., Philadelphia, pp 165–248
  2. Vale W, Spiess J, Rivier C, Rivier J 1981 Characterization of a 41-residue ovine hypothalamic peptide that stimulates the secretion of corticotropin and ß-endorphin. Science 213:1394–1397[Medline]
  3. Rivier C, Brownstein M, Spiess J, Rivier J, Vale W 1982 In vivo corticotropin releasing factor induced secretion of adrenocorticotropin, ß-endorphin and corticosterone. Endocrinology 110:272–278[Abstract]
  4. Spengler D, Rupperecht R, Phi Van 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]
  5. 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]
  6. 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]
  7. Owens MJ, Bartolome J, Schanberg SM, Nemeroff CB 1990 Corticotropin releasing factor concentrations exhibit an apparent diurnal rhythm in hypothalamic and extrahypothalamic brain regions: differential sensitivity to corticosterone. Neuroendocrinology 52:626–631[Medline]
  8. 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]
  9. Guardiola-Diaz HM, Kolinske JS, Gates LH, Seasholtz AF 1996 Negative glucocorticoid regulation of cyclic adenosine 3', 5'-monophosphate-stimulated corticotropin-releasing hormone-reporter expression in AtT-20 cells. Mol Endocrinol 10:317–329[Abstract]
  10. Malkoski SP, Handanos CM, Dorin RI 1997 Localization of a negative glucocorticoid response element of the human corticotropin releasing hormone gene. Mol Cell Endocrinol 127:189–199[CrossRef][Medline]
  11. Haas AD, George S 1989 Estradiol or ovariectomy decreases CRF synthesis in hypothalamus. Brain Res Bull 23:215–218[CrossRef][Medline]
  12. Dibbs KI, Anteby E, Mallon MA, Sadovsky Y, Adler S 1997 Transcriptional regulation of human placental corticotropin-releasing factor by prostaglandins and estradiol. Biol Reprod 57:1285–1292[Abstract]
  13. Aronin N, Sagar SM, Sharp FR, Schwartz W 1990 Light regulates expression of Fos-related protein in the rat suprachiasmatic nuclei. Proc Natl Acad Sci USA 87:5959–5962[Abstract]
  14. Jacobson L, Sharp FR, Dallman MF 1990 Induction of fos-like immunoreactivity in hypothalamic corticotropin releasing hormone neurons after adrenalectomy in the rat. Endocrinology 126:1709–1719[Abstract]
  15. Rosenfeld MG 1991 POU domain transcription factors: powerful development regulators. Genes Dev 5:897–907[CrossRef][Medline]
  16. Finney M, Ruvkun G 1990 The unc-86 gene product couples cell lineage and cell identity in C. elegans. Cell 63:895–905[Medline]
  17. Fujii H, Hamada H 1993 A CNS-specific POU transcription factor, Brn-2 is required for establishing mammalian neural cell lineages. Neuron 11:1197–1206[Medline]
  18. 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 (erratum 340:662)
  19. Nakai S, Kawano H, Yudate T, Nishi M, Kuno J, Nagata A, Jishage K, Fujii H, Hamada H, Kawamura K, Shiba K, Noda T 1995 The POU domain transcription factor Brn-2 is required for the determination of specific neuronal lineages in the hypothalamus of the mouse. Genes Dev 9:3109–3121[Abstract]
  20. 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]
  21. 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]
  22. Ross RA, Beidler JL, Spengler BA, Reis DJ 1981 Neurotransmitter synthesizing enzymes in 14 human neuroblastoma cell lines. Cell Mol Biol 3:301–312
  23. Stephanou A, Melino G, Knight RA, Annicchiarico-Petruzzellli M, Sarlis NJ, Finazzi-Agro A, Lightman SL 1992 Interleukin-6 and corticotropin releasing hormone mRNA are modulated during differentiation of human neuroblastoma cells. Neuropeptides 23:45–49[Medline]
  24. Shibihara S, Morimoto Y, Furutani Y, Notake M, Takahashi H, Shimizu S, Horikawa S, Numa S 1983 Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene. EMBO J 2:775–779[Medline]
  25. 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]
  26. Kasckow JW, Parkes DG, Owens MJ, Stipetic MD, Han JH, Nemeroff CB, Vale WW 1994 The BE (2)-M17 neuroblastoma cell line synthesizes and secretes corticotropin-releasing factor. Brain Res 654:159–162[CrossRef][Medline]
  27. Jones-Villeneuve EMV, Rudnicki MA, Harris JK, McBurney MW 1983 Retinoic acid-induced neural differentiation of embryonal carcinoma cells. Mol Cell Biol 3:2271–2279[Medline]
  28. Ryan AK, Rosenfeld MG 1997 POU domain family values: flexibility, partnerships, and developmental codes. Genes Dev 11:1207–1225[CrossRef][Medline]
  29. Cepek KL, Chasman DI, Sharp PA 1996 Sequence specific DNA binding of the B-cell-specific coactivator OCA-B. Genes Dev 10:2079–2088[Abstract]
  30. Walker S, Hayes S, O’Hare P 1994 Site-specific conformational alteration of the Oct-1 POU domain-DNA complex as the basis for differential recognition by Vmw65 (VP16). Cell 79:841–852[Medline]
  31. Simmons DM, Voss JW, Ingraham HA, Holloway JM, Broide RS, Rosenfeld MG, Swanson LW 1990 Pituitary cell phenotypes involve cell-specific Pit-1 mRNA and synergistic interactions with other classes of transcription factors. Genes Dev 4:695–711[Abstract]
  32. Adler S, Waterman ML, He x, Rosenfeld MG 1988 Steroid Receptor-mediated inhibition of rat prolactin gene expression does not require the receptor DNA-binding domain. Cell 52:685–695[Medline]
  33. Camper SA, Yao YAS, Rothman FM 1985 Hormonal regulation of the bovine prolactin promoter in rat pituitary tumor cells. J Biol Chem 260:12246–12251[Abstract/Free Full Text]
  34. Chen C, Okyama H 1987 High Efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752[Medline]
  35. Kaster KR, Burgett SG, Rao RN, Ingolia TD 1983 Analysis of a bacterial hygromycin B resistance gene by transcriptional and translational fusions and by DNA sequencing. Nucleic Acids Res 11:6895–6911[Abstract]
  36. Day RN, Walder JA, Maurer RA 1989 A protein kinase inhibitor gene reduces both basal and multihormone-stimulated prolactin gene transcription. J Biol Chem 264:431–436[Abstract/Free Full Text]
  37. 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]
  38. 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]
  39. 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]
  40. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K 1994 Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York, vol 1
  41. Deryckere F, Gannon F 1994 A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques 16:405[Medline]
  42. Lowry OH, Rosebrough NJ, Farr A, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275[Free Full Text]
  43. Scatena CD, Adler S 1998 Characterization of a human-specific regulator of placental corticotropin-releasing hormone. Mol Endocrinol 12:1228–1240[Abstract/Free Full Text]
  44. Chomczynski P, Sacchi N 1987 Single step method of RNA isolation by acid guanidine thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[CrossRef][Medline]
  45. Kunkel TA 1985 Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492[Abstract]