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
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ABSTRACT |
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INTRODUCTION |
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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.
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RESULTS |
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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. 1a).
This shift is specific, as it is competed by excess unlabeled
oligonucleotides (Fig. 1a
, lanes 2 and 3). Moreover, a similar shift
can be obtained using in vitro translated Brn-2 protein
(Fig. 1a
, 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. 1a
, lanes 46).
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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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. 2a) or PGK-
Brn2 (Fig. 2b
) 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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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. 2b). 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. 3). We used a 5-kb hCRF reporter plasmid
(CRF L
2S), a minimal 36-bp promoter (P36 L
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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As seen in the dose-response profile (Fig. 2), the parental BE(2)-M17
shows a 6.5-fold increase in CRF expression with retinoic acid (Fig. 5
). No major differences were seen in the
CMV Brn-2 stable line. The antisense transformants, MAM-3, which has 3
copies of PGK-
Brn-2, and MAM-19, which has 19 copies of PGK-
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. 1b
, 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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As previously observed (23), we note that the endogenous CRF mRNA is
transcribed after induction with retinoic acid. (Fig. 6, 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. 6
, 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. 5
and multiple
repetitions of the experiment in Fig. 6
are presented in Table 1
.
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DISCUSSION |
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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-2s 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 37 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 37 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. 7). 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.
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MATERIALS AND METHODS |
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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 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-
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- 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
Brn-2 fragment from PGK-
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 manufacturers instructions (Prime It,
Stratagene). The membrane was probed and washed, before
exposure to storage phosphor screens. A parallel control experiment was
performed using human -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
-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 manufacturers 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 manufacturers 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 IIV (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 IIIIV (-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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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This study was supported by NIH Grant DK-45506 to S.A. T.R. was supported by a predoctoral fellowship, DAMD1794-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.
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REFERENCES |
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