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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go; 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.

 
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. 2Go). 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. 2Go). 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.

 
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{Delta}-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. 3Go). 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.

 
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. 4AGo). The resultant mutants were ligated to TK-CAT and assayed by transfection into Rcho-1 cells. Four mutations (m1, m7, m8, and m9—the 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. 4BGo). 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 (m1–m10) 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.

 
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. 5Go). 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.

 
The M7–M9 region is also recognized by a factor that is present in trophoblast cells but not in fibroblasts (Fig. 6BGo). The center of the M7–M9 region contains the sequence GATAA (Fig. 6AGo), 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 M7–M9 probe is not competed by a double-stranded oligonucleotide corresponding to a functional GATA element in the PL-I promoter (Fig. 6BGo). Similar to what was detected with M1 protein binding, differentiation of the Rcho-1 cells also results in increased protein binding to the M7–M9 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. 7Go).



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Figure 6. Trophoblast-Specific Factor Binding to the M7–M9 Element

A, Sequence of the M7–M9 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 M7–M9 double-stranded oligonucleotide. Binding reactions were supplemented with a 100-fold excess of unlabeled M7–M9 (S), nonspecific (NS), or PL-I GATA element (GATA) double-stranded oligonucleotides as competitors. The arrowhead indicates the specific M7–M9 protein-DNA complex.

 


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Figure 7. Distinct Factor Binding to the M1 and M7–M9 Elements

Whole cell extracts from undifferentiated (UD) or differentiated (D) Rcho-1 trophoblasts were incubated with end-labeled M1 or M7–M9 double-stranded oligonucleotides. Binding reactions were supplemented with a 100-fold excess of unlabeled M1, M7–M9, or nonspecific double-stranded oligonucleotide as competitors. The arrow marks the M1 complex, and the arrowheads point to the major, specific M7–M9 protein-DNA complexes.

 
Both the M1 and the M7–M9 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 M7–M9. However, three observations indicate that the M1 and the M7–M9 binding proteins are distinct: the M7–M9 oligonucleotide does not compete for factor binding to M1, radiolabeled M7–M9 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 M7–M9 protein-DNA complexes (Fig. 7Go). Thus, despite their similarities in sequence, the M1 and the M7–M9 regions contain distinct positive regulatory elements for trophoblast gene expression.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have identified two elements within the PL-II promoter from -1469 to -1458 (designated as M1) and from -1391 to -1354 (designated as M7–M9) 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 M7–M9 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 M7–M9 binding activities being trophoblast-specific factors, an extensive search of other cell types for M1 and M7–M9 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 M7–M9 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 {alpha}-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 M7–M9 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 M7–M9 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 M7–M9 region to account for a higher affinity of this factor. The length of the M7–M9 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 M7–M9 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{alpha}-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 {alpha}-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 M7–M9 elements have G+C contents of only 25% and 16%, respectively.

Finally, even though the 132-bp region spanning the M1 and M7–M9 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 2–4 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 manufacturer’s 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 {alpha}-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.


    ACKNOWLEDGMENTS
 
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).


    FOOTNOTES
 
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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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