Identification of Trophoblast-Specific Regulatory Elements in the Mouse Placental Lactogen II Gene
Jiandie Lin and
Daniel I. H. Linzer
Department of Biochemistry, Molecular Biology, and Cell Biology
Northwestern University Evanston, Illinois 60208
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ABSTRACT
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Placental lactogen II, the major ligand for the
PRL receptor during the second half of gestation in rodents, is
synthesized specifically by placental trophoblast giant cells. A
transient transgenic analysis has been used to localize the giant
cell-specific regulatory region within the mouse placental lactogen II
gene to sequences between -1340 and -2019 upstream of the
transcriptional start site. More precise mapping of the regulatory
elements has been accomplished by transfection of promoter constructs
into Rcho-1 trophoblast cells, resulting in the characterization of two
positive regulatory elements in the -1471 to -1340 region; two other
regulatory elements have been implicated but not further characterized,
a negative regulatory element between -2019 and -1778 and another
positive element within the region from -1340 to -569. Both of the
characterized positive regulatory elements are recognized by factors
that are enriched in differentiated giant cells compared with
proliferative trophoblasts, and these factors are either absent or at
low levels in fibroblasts. The complexes that form on the two elements
are distinct and neither element competes with the other for factor
binding, thus implicating at least two different regulatory elements in
late-gestational trophoblast giant cell-specific gene expression.
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INTRODUCTION
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A central feature in the development of the rodent placenta is the
differentiation of the trophoblast giant cells. These cells, which
become nonproliferative and undergo extensive endoreduplication, are
responsible for attaching the embryo to the uterus and are also the
source of a number of pregnancy-specific hormones (1). Although
morphologically identical, giant cells of early to midpregnancy and of
mid- to late pregnancy can be distinguished by their differential
expression of various hormone genes in the PRL/GH family. Giant cells
of early to midpregnancy synthesize placental lactogen I (PL-I),
proliferin (PLF), and PRL-like proteins A and E, whereas these same
cells in mid- to late gestation activate transcription of the placental
lactogen II (PL-II) and proliferin-related protein (PRP) genes while
decreasing expression of the former group of hormones (2, 3, 4, 5, 6, 7, 8, 9). Thus, the
midgestational transition in hormone gene expression reveals at a
molecular level distinct stages of giant cell differentiation.
The switches from PL-I to PL-II and from PLF to PRP synthesis represent
important steps in the physiology of pregnancy. PL-I and PL-II, despite
having only 42% amino acid sequence identity (10, 11), both bind the
PRL receptor with high affinity (12, 13) and display similar
bioactivities in model systems (14); these hormones have been
implicated in the maintenance of the corpus luteum and progesterone
production, the regulation of maternal metabolism, and the development
of the mammary gland for postpartum lactation (14), but it seems likely
that the distinct sequence and structural features of PL-I and PL-II
may result in subtly different activities in vivo. In
contrast to the similar activities of PL-I and PL-II, PLF and PRP have
opposing bioactivities, with PLF stimulating and PRP inhibiting
angiogenesis (15); in this case the genetic switch in hormone synthesis
appears responsible for generating an environment at the implantation
site that initially induces, and later restricts,
neovascularization.
To understand how these genetic switches occur, it is necessary to
characterize the regulation of both early to midpregnancy and mid- to
late pregnancy placental-specific gene expression. We have recently
demonstrated that transcription of the PL-I gene is positively
regulated by the transcription factors GATA-2 and GATA-3 and the
ubiquitous factor AP-1 (16, 17, 18). In addition, the helix-loop-helix
protein Hxt has been implicated in PL-I gene activity (19). Much less
is known about mid- to late gestation giant cell gene expression. Our
initial analysis of PL-II gene transcription identified the region
extending 2.7 kb upstream of the transcription initiation site as
sufficient to drive giant cell-specific expression during mid- to late
pregnancy in transgenic mice, whereas the proximal 569 bp is inactive
(20).
In this study, our goal was to identify the functional elements for
placental-specific expression in the 2.1-kb region of the PL-II gene
from -0.6 to -2.7 kb, using both transient transgenic and transient
transfection analysis. Since placental trophoblast cells derive from
the trophectoderm of the blastocyst (and not from the mother), it is
possible to analyze transgene expression in the placenta in F0 animals
without the need to establish transgenic lines. Transfections have been
carried out in the Rcho-1 cell line, a trophoblast cell line derived
from a rat choriocarcinoma that we have successfully used to identify
regulatory elements in PL-I gene expression (16, 17). The Rcho-1 cell
line recapitulates the in vivo pathways of trophoblast
differentiation and gene expression, including early expression of the
PL-I gene during differentiation and later activation of the PL-II
gene, and therefore provides an excellent model for studying giant cell
gene expression (21, 22).
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RESULTS
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Placental-Specific Transcription from the PL-II Promoter in
Transgenic Mice
To define the regulatory elements involved in the
placental-specific expression of the PL-II gene during the second half
of gestation, different regions of the 2.7-kb promoter were linked to
the bacterial chloramphenicol acetyltransferase (CAT) gene, and the
resultant DNA constructs were introduced into fertilized eggs by
microinjection. The embryos were transferred to pseudopregnant foster
mothers, and pregnancy was terminated on day 13.5 to analyze placental
expression. Based on a PCR analysis of yolk sac DNA, approximately 20%
of the F0 conceptuses were found to be transgenic. A construct
encompassing 1.3 kb upstream of the transcription start site (PL-II
-1.3 CAT) was not active, since seven of eight transgenic placentas
gave no detectable CAT activity (data not shown). However, inclusion of
the next 680 bp up to -2.0 kb (PL-II -2.0 CAT) resulted in
significant CAT expression in all nine transgenic conceptuses that were
analyzed, and in each case CAT activity was found to be much higher in
the placenta compared with the fetus (Fig. 1
; note that the y axis is a log scale).
Variation in the level of transgene expression can be seen, and is most
likely due to the effects of chromosomal location of transgene
integration and to differences in the number of integrated copies of
the transgene. Positional and copy number effects may also explain the
detection of some CAT activity in a few of the transgenic fetuses.

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Figure 1. Placental-Specific Expression from the PL-II
Promoter in Vivo
Placental and fetal extracts were prepared at day 13.5 of gestation
from nine conceptuses containing the PL-II -2.0 CAT transgene and were
assayed for CAT activity. P/F is the ratio of CAT activity in the
placenta to that in the fetus for each transgenic.
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Localization of Trophoblast-Specific Regulatory Elements
The transient transgenic analysis demonstrates that the proximal
2.0-kb 5'-flanking sequences in the PL-II are sufficient, and that
sequences between -2.0 kb and -1.3 kb are required, to direct
trophoblast-specific expression in vivo. However, the
variability in the level of transgene expression makes it extremely
difficult to employ this approach to localize individual regulatory
elements by comparing the activities of different promoter constructs.
Therefore, to identify the important elements within the -2.0 to -1.3
kb flanking region, transcriptional activity was instead assayed in
transiently transfected Rcho-1 trophoblast cells, which differentiate
in culture into PL-II-expressing giant cells.
The PL-II -2.0 CAT construct is equivalent to the PL-II -2.7 CAT
construct in transcriptional activity in transfected trophoblast cells,
whereas the PL-II -1.3 CAT construct shows greatly reduced activity
(Fig. 2
). Since the constructs containing
2.7 or 2.0 kb, but not 1.3 kb, of 5'-flanking sequence are active in
the placenta, these results provide additional support for the
physiological relevance of the Rcho-1 transfection system for the
analysis of trophoblast-specific gene expression. Although the 1.3-kb
promoter is not sufficient to direct a high level of transcription in
trophoblasts, sequences within the region between -0.6 and -1.3 kb
are required for maximal PL-II gene activity, since a deletion of this
region within the context of the PL-II -2.0 CAT construct reduces
transcriptional activity to the low level seen with PL-II -1.3 CAT
(Fig. 2
). Both this internal deletion construct and the PL-II -1.3 CAT
construct are more active than PL-II -0.6 CAT, however, indicating
that positive regulatory elements for giant cell gene expression are
localized both between -0.6 and -1.3 and between -1.3 and -2.0 kb,
and that the combination of these elements synergistically activates
transcription. For all of the promoter constructs extending beyond
-0.6 kb, the effect of these regulatory elements is observed in Rcho-1
cells but not in the mouse L cell fibroblast background, indicating
that these elements function in a trophoblast-specific manner.

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Figure 2. Trophoblast-Specific Activity of the PL-II Promoter
in Transfected Cells
The regions of the PL-II promoter indicated schematically were linked
to the CAT reporter gene and were transiently transfected into Rcho-1
trophoblasts (solid bars) and L cell fibroblasts
(open bars). CAT assays were performed with equal
amounts of protein extract prepared 48 h posttransfection, and CAT
activity was normalized to that obtained with the PL-II - 0.6 CAT
construct. The data shown are from triplicate transfections (mean
± SD), with similar results obtained in two additional
transfection experiments.
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The weak trophoblast-specific regulatory activity conferred by the
-2.0 to -1.3 kb region, which was detected with the PL-II
-2.0
-0.6/-1.3 CAT internal deletion construct, can also be seen
by linking this 680 region from -2019 to -1340 to the minimal
promoter from the herpes virus thymidine kinase (TK) gene (Fig. 3
). Unexpectedly, a reduction of this 680
bp region from the 5' end to -1778 results in a greatly elevated level
of activity in transfected trophoblasts without any increase in
transfected fibroblasts, indicating that the 242-bp region between
-2019 and -1778 contains one or more negative regulatory elements.
Further deletions to -1696, -1609, or -1471 have minimal effects on
promoter activity, thereby localizing one or more positive regulatory
elements for trophoblast-specific transcription to a 132-bp region from
-1471 to -1340.

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Figure 3. Activity of the PL-II Promoter Upstream Regulatory
Region in Trophoblast Cells
A series of 5'-deletions within the -2019 to -1340 region of the
PL-II promoter were ligated to the herpes simplex virus TK minimal
promoter containing the TATA box and to the CAT reporter gene and
transfected into Rcho-1 trophoblasts (solid bars) and L
cell fibroblasts (open bars). CAT activity was
normalized to the basal activity of TK CAT. The data shown are from
triplicate transfections (mean ± SD), with similar
results obtained in two additional transfection experiments.
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The sequence of this 132 bp region was analyzed in greater detail by
generating a set of scanning mutations. The 132-bp region was divided
into ten blocks which were each replaced with a DNA sequence containing
adjacent SstI and ClaI restriction endonuclease
sites (Fig. 4A
). The resultant mutants
were ligated to TK-CAT and assayed by transfection into Rcho-1 cells.
Four mutations (m1, m7, m8, and m9the lower case m will be used for
the mutated sequence and the upper case M for the corresponding
wild-type sequence), which map to two separate patches, were identified
that decrease transcriptional activity (Fig. 4B
). The m1 mutation,
which alters the sequence from -1469 to -1458, had the greatest
effect, reducing transcription to the level obtained with just the
minimal TK promoter. The m7, m8, and m9 mutations each have a smaller
effect than the m1 mutation and define a larger element or cluster of
elements between -1391 and -1354.

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Figure 4. The Upstream Regulatory Region Contains More Than
One Positive Element for Trophoblast-Specific Transcription
A, Sequence of the PL-II promoter from -1471 to -1340. The wild- type
sequence is shown on the top line, and the ten block
mutations (m1m10) are shown below in italics.
Capital letters in italics identify those residues that
remained the same as in the wild-type sequence after mutagenesis. B,
Relative CAT activity of the wild type and mutated -1471 to -1340
PL-II promoter region linked to TK CAT. Constructs were transiently
transfected into Rcho-1 cells, and CAT activity for each PL-II promoter
construct was normalized to the basal activity obtained with the
minimal TK promoter. The data shown are from triplicate transfections
(mean ± SD), with similar results obtained in two
additional transfection experiments.
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Binding of Trophoblast Factors to the PL-II Regulatory Elements
The M1 sequence may represent a novel regulatory element
since searches of transcription factor databases (TRANSFAC and GCG)
failed to identify the sequence as a putative binding site for any
known transcription factor. To determine whether the M1 sequence
interacts with a factor present in trophoblasts, a double-stranded
oligonucleotide encompassing the M1 element was radiolabeled for an
electrophoresis mobility shift assay. Whole cell extracts were prepared
from L cell fibroblasts, proliferating Rcho-1 cells, and differentiated
Rcho-1 giant cells. As predicted, the M1 element forms a specific
complex with a factor present in Rcho-1 cell extracts (Fig. 5
). The abundance of this factor appears
to increase upon cell differentiation, since much more of the specific
protein-DNA complex is detected in extracts from the differentiated
Rcho-1 cells. The mutation in the M1 element that disrupts
transcriptional activation also prevents protein binding, indicating
that the formation of this specific complex is functionally important.
A complex of a similar size is also detected with mouse L cell
extracts; however, unlike the binding of the factor from trophoblasts,
which is competed by excess wild-type M1 oligonucleotide but not by an
unrelated sequence, the binding of proteins from fibroblasts is
effectively competed by the nonspecific oligonucleotide.

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Figure 5. Factor Binding to the PL-II Promoter M1 Regulatory
Element
A, Sequence of the wild-type (M1) and mutant (m1) element
oligonucleotides used in the electrophoretic mobility shift assay.
Oligonucleotides were annealed and labeled by filling in the
5'-overhangs. The underlined region identifies sequences
from the PL-II promoter from -1471 through -1451. B, Whole cell
extracts from undifferentiated Rcho-1 trophoblasts (UD), differentiated
Rcho-1 trophoblasts (D), or L cell fibroblasts (L) were incubated with
end-labeled wild type (M1) or mutant (m1) double-stranded
oligonucleotides. Binding reactions were supplemented with a 100-fold
excess of unlabeled wild-type M1 double-stranded oligonucleotide as a
specific competitor (S) or an unlabeled, double-stranded
oligonucleotide of an unrelated sequence as a nonspecific competitor
(NS). The arrow marks the major protein-DNA complex that
forms with the Rcho-1 extracts and that is blocked by the specific, but
not by the nonspecific, competitor.
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The M7M9 region is also recognized by a factor that is present in
trophoblast cells but not in fibroblasts (Fig. 6B
). The center of the M7M9 region
contains the sequence GATAA (Fig. 6A
), which is disrupted by the m8
mutation, suggesting that GATA-2 and GATA-3 might be important not only
for the early giant cell-specific expression of the PL-I and PLF genes
(17, 18) but also for the late giant cell-specific expression of the
PL-II gene. However, the protein-DNA complex with the M7M9 probe is
not competed by a double-stranded oligonucleotide corresponding to a
functional GATA element in the PL-I promoter (Fig. 6B
). Similar to what
was detected with M1 protein binding, differentiation of the Rcho-1
cells also results in increased protein binding to the M7M9 DNA: two
specific complexes of approximately equal intensity form with
undifferentiated cell extracts, and the amount of higher mobility
complex preferentially increases with the differentiated cell extracts
(Fig. 7
).

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Figure 6. Trophoblast-Specific Factor Binding to the M7M9
Element
A, Sequence of the M7M9 oligonucleotide (residues -1387 to -1355)
used for electrophoretic mobility shift analysis. The consensus
sequence for GATA factor binding is boxed. B, Whole cell
extracts from differentiated Rcho-1 trophoblasts (D) or L cell
fibroblasts (L) were incubated with end-labeled M7M9 double-stranded
oligonucleotide. Binding reactions were supplemented with a 100-fold
excess of unlabeled M7M9 (S), nonspecific (NS), or PL-I GATA element
(GATA) double-stranded oligonucleotides as competitors. The
arrowhead indicates the specific M7M9 protein-DNA
complex.
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Figure 7. Distinct Factor Binding to the M1 and M7M9
Elements
Whole cell extracts from undifferentiated (UD) or differentiated (D)
Rcho-1 trophoblasts were incubated with end-labeled M1 or M7M9
double-stranded oligonucleotides. Binding reactions were supplemented
with a 100-fold excess of unlabeled M1, M7M9, or nonspecific
double-stranded oligonucleotide as competitors. The
arrow marks the M1 complex, and the
arrowheads point to the major, specific M7M9
protein-DNA complexes.
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Both the M1 and the M7M9 elements are A-T-rich, and they are
sufficiently similar in sequence to raise the possibility that the same
factor that binds to M1 might also recognize M7M9. However, three
observations indicate that the M1 and the M7M9 binding proteins are
distinct: the M7M9 oligonucleotide does not compete for factor
binding to M1, radiolabeled M7M9 DNA forms complexes with Rcho-1 cell
proteins that have distinct mobilities compared with the M1 complex,
and the M1 element is unable to block the formation of the M7M9
protein-DNA complexes (Fig. 7
). Thus, despite their similarities in
sequence, the M1 and the M7M9 regions contain distinct positive
regulatory elements for trophoblast gene expression.
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DISCUSSION
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We have identified two elements within the PL-II promoter from
-1469 to -1458 (designated as M1) and from -1391 to -1354
(designated as M7M9) that act together to stimulate gene
transcription in trophoblast giant cells. Mutation of either element
significantly reduces the activity of the -1471 to -1340 region,
which when linked to a minimal promoter is sufficient to drive
transcription specifically in Rcho-1 trophoblasts; this 132-bp region
does not stimulate transcription in fibroblasts. The physiological
relevance of these transfection results is supported by the finding
that a fragment of the PL-II gene extending from -2019 to -1340 is
transcriptionally active in all nine transgenic placentas examined, but
in each case was inactive or active at much reduced levels in the
corresponding fetus.
Consistent with functioning as trophoblast-specific regulatory
elements, the M1 and M7M9 sequences form specific protein-DNA
complexes with proteins from trophoblast cells but not fibroblasts.
Binding to each of these DNA sequences is more pronounced with extracts
from differentiated compared with proliferating trophoblasts,
suggesting that the levels of the binding proteins depend on the
differentiated state of the cell. Only one specific complex is detected
with the M1 element, and this complex does not form with DNA containing
the m1 mutation that eliminates transcriptional activity. A complex of
similar size is detected with fibroblast extracts, but this complex is
nonspecific since its formation is effectively competed by an unrelated
DNA sequence. Nevertheless, this result may indicate that fibroblasts
synthesize a factor related to the M1-binding protein that is found in
trophoblasts, but that the fibroblast factor has lower affinity for
this binding site. Although the data are consistent with the M1 and
M7M9 binding activities being trophoblast-specific factors, an
extensive search of other cell types for M1 and M7M9 binding proteins
has not yet been attempted.
The M1 sequence does not point to a known transcription factor as a
strong candidate for the relevant regulatory protein, but M7M9 does
contain a central GATA factor consensus sequence. Both GATA-2 and
GATA-3 are synthesized in giant cells, can stimulate transcription from
the PL-I gene promoter in transfected trophoblasts, and can activate
the normally silent promoter in transfected fibroblasts (17).
Furthermore, both of these transcription factors are required for PL-I
and PLF gene expression in vivo (18). These two related
factors may even regulate a broader program of placental gene
expression, since they have also been implicated in the
placental-specific expression of the human
-glycoprotein subunit
gene (23). Despite the attractiveness of implicating GATA-2 and GATA-3
in PL-II gene expression, as well, the specific protein-DNA complex
that forms with the M7M9 probe is not competed by a functional GATA
element from the PL-I promoter sequence. Furthermore, cotransfection of
Rcho-1 cells with a GATA-2 or GATA-3 expression construct and PL-II
-2.7 CAT did not result in increased CAT reporter activity (data not
shown), even though GATA factor levels in Rcho-1 cells are limiting for
PL-I promoter activity (17). One way in which a consensus GATA-binding
site may be present in the M7M9 region but have little if any role in
PL-II transcription is if a binding site for another, dominant
factor overlaps and obscures the GATA sequence. More than one
binding site may be present within the M7M9 region to account for a
higher affinity of this factor. The length of the M7M9 region, the
detection of two specific protein-DNA complexes, the inability of any
one of the m7, m8, or m9 mutations to eliminate transcriptional
activity completely, and the presence of short A-T-rich motifs repeated
throughout the M7M9 region would all be consistent with this region
encompassing adjacent binding sites for the trophoblast factor.
Regulatory regions from other genes have also been identified that
confer trophoblast-specific expression in Rcho-1 cells or in transgenic
mice. Regions of the cytochrome P450 side chain cleavage and
17
-hydroxylase gene promoters have been identified that are
transcriptionally activated during Rcho-1 cell differentiation (24, 25), and the PRL-like protein A and decidual/trophoblast PRL-related
protein gene promoters have been shown to be active in Rcho-1 but not
in pituitary cells (26, 27). In these cases, the functional elements
have not yet been identified. In addition to the human
-glycoprotein
subunit gene (28), fragments from the 4311, HLA-G, and adenosine
deaminase genes also direct transcription specifically in the placenta
of transgenic mice, but these transgenes are expressed only or
primarily in spongiotrophoblasts and so might utilize regulatory
factors distinct from the giant cell-expressed PL-II gene (29, 30, 31).
Indeed, a functionally important 30-bp element in the adenosine
deaminase gene-regulatory region that binds a factor present in the
placenta but not in the liver is 70% G+C (31), whereas the PL-II gene
M1 and M7M9 elements have G+C contents of only 25% and 16%,
respectively.
Finally, even though the 132-bp region spanning the M1 and M7M9
elements when linked to a minimal promoter is sufficient for
trophoblast-specific transcriptional activation, other regions also
contribute to the regulation of PL-II gene expression. The transfection
analysis has revealed both a negative regulatory element between -2019
and -1778 and a positive element between -1340 and -569. The
identification of both positive and negative regulatory elements
suggests two potential mechanisms for the late onset of PL-II gene
expression in pregnancy: either one or more of the positive regulatory
factors is present in an active form only in mid- to late gestation, or
the negative regulatory factor is present and effectively represses
gene activity until mid- to late gestation. Thus, at least four
elements will now need to be characterized in terms of the factors with
which they interact and the mechanisms by which they cooperate to
activate transcription of the PL-II gene.
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MATERIALS AND METHODS
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Animal Care and Transgenic Procedures
Mice were purchased from Charles River Laboratory
(Wilmington, MA) and were maintained in a barrier facility on days
of 14-h light, 10-h darkness, with lights on at 0600 h. For
transgenic microinjections, the PL-II promoter CAT cassettes were
excised from the vector and purified by agarose gel electrophoresis.
Purified DNA was passed through an Elutip column (Schleicher & Schuell,
Keene, NH) to remove fine particles before injection into fertilized
eggs using standard methods (32). Pregnancy was terminated on day 13.5,
placentas and embryos were immediately frozen, and DNA was extracted
from dissected yolk sacs and analyzed by PCR using an upstream primer
from the PL-II promoter and a downstream primer from the CAT-coding
region. Placental and embryonic extracts were prepared by
homogenization in 0.25 M Tris-HCl buffer followed by
centrifugation. All procedures were approved by the Northwestern
University Animal Care and Use Committee.
Cell Culture, Transient Transfections, and CAT Assays
Proliferating Rcho-1 cell cultures were maintained in RPMI-1640
medium supplemented with 10% FCS, 50 mM
ß-mercaptoethanol, 1 mM sodium pyruvate, and 100 µg/ml
each of penicillin and streptomycin. To promote differentiation,
confluent Rcho-1 cultures were maintained in 10% horse serum for 1
week at which point the majority of cells appeared to be giant cells.
Mouse L cell fibroblasts were maintained in DMEM supplemented with 10%
FCS, 2 mM L-glutamine, and
penicillin/streptomycin.
For transient transfections, confluent Rcho-1 or L cells were split 1:4
into six-well plates 24 days before trans-fection. Three
micrograms of DNA were mixed with 15 µl of Lipofectamine reagent
in 200 µl OPTI-MEM medium according to the manufacturers
instructions (GIBCO BRL, Gaithersburg, MD). Confluent cell cultures
were incubated with the DNA-lipid mixture for 5 h before feeding
with fresh medium containing calf serum instead of FCS. Cells were
harvested by three freeze-thaw cycles 48 h after the start of
transfection, and protein concentrations were determined using the
Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). For CAT
assays, equal amounts of protein in 125 µl of 0.25 M
Tris-HCl, pH 7.5, were first heated at 60 C for 10 min to inactivate
CAT inhibitors before addition of 1 µl
[14C]chloramphenicol and 2.5 µl 5 mM
N-butyryl Coenzyme A. N-Butyryl
chloramphenicol was extracted from substrates by mixed xylenes followed
by a back extraction with water. The organic mixture was added to 1 ml
of Universol Cocktail (ICN, Irvine, CA), and the amount of
radioactivity was determined using a liquid scintillation counter.
Plasmid Constructions
PL-II promoter sequences were cloned into the pUC-CAT vector
(33), and plasmids were purified by CsCl ultracentrifugation. Nested
deletions of the -2019 bp to -1340 bp region were generated by
partial digestion with BAL31 exonuclease. Individual clones were
sequenced to verify deletions and subcloned into the TK-CAT vector
containing the minimal promoter from the herpes simplex virus TK gene
(34). Site-directed mutagenesis (35) in the -1471 bp to -1340 bp
region was accomplished by PCR with overlapping, complementary 24-mer
oligonucleotide primer pairs spanning this region that each contained a
12-bp sequence of adjacent ClaI and SstI
restriction sites flanked on either side by 12 bp of wild-type promoter
sequence.
Electrophoresis Mobility Shift Assay
Whole cell extracts were prepared by sonication in a lysis
buffer containing 20 mM HEPES, pH 7.4, 0.4 M
KCl, 0.4% Triton X-100, 10% glycerol, 10 mM EGTA, 5
mM EDTA, 1 mM dithiothreitol, and supplemented
with the protease inhibitors benzamidine (1 mM), leupeptin
(10 µg/ml), aprotinin (4 µg/ml), and pefabloc (1 mM).
Cellular debris was removed by centrifugation and supernatants were
stored at -80 C until use. Oligonucleotide primers were annealed and
labeled by end-filling with the Klenow fragment and
-32P-dATP. Approximately 4 µg of each extract were
added to a binding reaction of 25 µl containing 10 mM
Tris-HCl, pH 7.8, 100 mM KCl, 5% glycerol, 0.5 µg poly
(deoxyinosinic-deoxycytidylic)acid, 1 mM EDTA, 1
mM dithiothreitol, 1 mM benzamidine, 10 µg/ml
leupeptin, 4 µg/ml aprotinin, 1 mM pefabloc, and 100 ng
specific or nonspecific competitor oligonucleotides. Reactions were
incubated on ice for 30 min before adding 2 x 104 cpm
of radiolabeled oligonucleotide (1 ng). After incubating for an
additional 30 min, the samples were separated by 4% nondenaturing
polyacrylamide gel electrophoresis at 4 C.
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ACKNOWLEDGMENTS
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We thank Jie Fan for excellent instruction and assistance in
transgenic techniques.
This work was supported by NIH Grant HD-29962, by the Research Center
on Fertility and Infertility at Northwestern University (P30 HD-28048),
and by the Robert H. Lurie Cancer Center (P30 CA-60553).
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FOOTNOTES
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Address requests for reprints to: Daniel I.H. Linzer, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, Illinois 60208.
Received for publication September 9, 1997.
Revision received November 20, 1997.
Accepted for publication December 11, 1997.
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