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Cell Growth & Differentiation Vol. 11, 71-82, February 2000
© 2000 American Association for Cancer Research

Articles

Transcriptional Regulation of the Cellular Retinoic Acid Binding Protein I Gene in F9 Teratocarcinoma Cells1

Anna L. Means2, James R. Thompson and Lorraine J. Gudas3

Department of Pharmacology, Weill Medical College, Cornell University, New York, New York 10021

Abstract

Retinoic acid (RA) induces the differentiation of many murine teratocarcinoma cell lines such as F9 and P19. In F9 cells, the level of the cellular retinoic acid binding protein I (CRABP I) mRNA is greatly reduced after exposure of the cultured cells to exogenous RA. In P19 cells, the level of CRABP I mRNA is greatly increased after RA exposure. We have identified a 176-bp region in the murine CRABP I promoter, between -2.9 and -2.7 kb 5' of the start site of transcription, which acts as an enhancer in undifferentiated F9 stem cells and through which RA effects inhibition of CRABP I transcription. Within this region are two footprinted sites at -2763 and -2834. This 176-bp regulatory region does not function to enhance CRABP I transcription in P19 stem cells. Several DNA sequences within these two footprinted regions bind proteins from F9 nuclear extracts but not from P19 nuclear extracts (e.g., FP1B, FP1A, and FP2B), as assessed by gel shift assays. This 176-bp CRABP I genomic region has not been sequenced previously and functionally analyzed in cultured cells because it was not present in the murine CRABP I clones used for the promoter analyses reported earlier by another laboratory. The function of this enhancer may be to reduce the expression of the CRABP I gene in specific embryonic cell types in order to regulate the amount of RA to which the cells are exposed.

Introduction

Retinoids are important mediators of cellular differentiation in many transformed cell lines (1) and in many cells of the developing embryo (2) . RA4 is one of the most biologically potent of the naturally occurring retinoids (3) . Vertebrate embryos exposed to RA undergo extensive teratogenesis, the affected tissues depending on the time of RA exposure (4, 5, 6, 7) .

The presence and activity of RA are thought to depend on a number of protein products (8) : (a) there are the metabolic enzymes that regulate the synthesis and stability of RA (9, 10, 11, 12, 13, 14, 15, 16) ; (b) in addition, the nuclear receptors, retinoic acid receptors and retinoid X receptors, become active transcription factors upon binding all-trans or 9-cis RA, respectively (17) ; and (c) the cytoplasmic binding proteins are thought to mediate the availability of RA to the receptors (18) . Two of these proteins, the CRABP I and CRABP II, are thought to regulate the availability of RA to the receptors by either enhancing the metabolism of RA or by sequestering RA in the cytoplasm, away from the receptors (19, 20, 21) . Conversely, these proteins may increase the intracellular concentration of RA, which would increase its availability to the nuclear receptors (22, 23, 24) . Although some of these proposed functions seem contradictory, it is possible that different cell types make use of CRABPs in different ways. For example, the presence or absence of metabolizing enzymes as well as the relative affinities of the metabolic enzymes and the nuclear receptors for RA will determine whether RA bound to CRABPs is likely to be metabolized or to activate transcription via the nuclear receptors.

Of the two CRABPs defined in mammals, CRABP I has a 10-fold higher affinity for RA than does CRABP II (25) , suggesting a greater ability of CRABP I to regulate RA availability. The role of CRABP I in controlling RA availability is further supported by its expression pattern. During embryogenesis, CRABP I is expressed in many of the tissues that are susceptible to RA-induced teratogenesis (26, 27, 28, 29, 30, 31) . This correlation between a high level of CRABP I expression and the susceptibility of cells to teratogenesis by exogenous RA is much greater than that for any other retinoid binding protein or any of the nuclear receptors.

In the murine teratocarcinoma F9 cell line, RA induces differentiation into primitive endoderm-like cells (32) . The level of CRABP I expression in these cells influences the concentration of exogenous RA required to regulate the genes that are the markers of this differentiation pathway (19) . Overexpression of CRABP I reduces the biological response to a given concentration of RA by increasing RA metabolism into more polar, presumably inactive byproducts (20) . Lowered CRABP I expression, achieved by expressing an antisense copy of the CRABP I coding region, makes F9 cells more sensitive to RA-induced transcription of reporter genes (19) .

Because the function of CRABP I has been characterized in these F9 cells and the cells respond to RA by differentiating, we examined the transcriptional regulation of the CRABP I gene in F9 cells. We were also interested in the regulation of CRABP I in the F9 cells because the CRABP I gene is expressed in undifferentiated F9 cells but is expressed at a much lower level after RA exposure (33) . Thus, F9 cells appear to have a feedback mechanism whereby CRABP I can inhibit the action of RA, which itself can down-regulate expression of the CRABP I gene. We also compared the regulation of the CRABP I promoter in other cell lines and found differences in promoter regulation in different cell types.

Results

CRABP I RNA Expression in Embryo-derived Cell Lines.
The CRABP I gene is expressed in a number of tissues in the developing fetus. Analysis of different established cell lines reflects this diversity of expression (Fig. 1)Citation . P19 teratocarcinoma cells, which are morphologically similar to the pluripotent embryonic cells of the blastocyst-stage inner cell mass, differentiate into fibroblast-like cells, neural cell types, and muscle upon addition of RA or DMSO (34, 35, 36) . Before differentiation, a low level of CRABP I transcript was detected (Fig. 1Citation , Lane 1). As reported previously (37) , after RA treatment, the level of the CRABP I transcripts increased >20-fold (Fig. 1Citation , Lane 2). A similar response to RA was observed in the J1 ES cell line, which had been treated with RA (Fig. 1Citation , Lanes 3 and 4). Like P19, ES cells are morphologically similar to cells of the inner cell mass and differentiate into many different cell types (38) . In Balb/3T3 embryonic fibroblastic cells, the level of CRABP I RNA declined severalfold (>4-fold) after RA treatment (Fig. 1Citation , Lanes 5 and 6). The level of CRABP I RNA in both Balb/3T3 and NIH/3T3 fibroblasts also depends on the concentration of fibroblast growth factor 2 and bone morphogenic proteins 2 and 4 present in the culture medium (39) .



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  		Fig. 1. CRABP I RNA expression in different cell lines. The indicated cells were cultured as described in 1 µM RA (+) or in vehicle alone (-). Northern analysis was performed using 15 µg of total cellular RNA per sample. The Northern blot was probed with the CRABP I cDNA (top panel) and then with a fragment of the GAPDH cDNA (bottom panel).

 
With respect to F9 teratocarcinoma cells, CRABP I RNA was detected in the untreated stem cells, and treatment with RA decreased CRABP I RNA to an undetectable level (Fig. 1Citation , Lanes 7 and 8; Ref. 33 ). This reduction in CRABP I RNA in F9 cells was particularly significant because the level of CRABP I in these cells influences the rate of RA metabolism to more polar metabolites (20) . Thus, it appears that RA and CRABP I are involved in a pathway, direct or indirect, of cross regulation in which RA treatment leads to a reduction in CRABP I expression, and CRABP I expression leads to a reduction in the bioactive RA in the cells.

CRABP I Transcriptional Regulatory Elements in F9 and in P19 Cells.
Because of the reciprocal regulation observed for RA activity and CRABP I expression in F9 cells, we examined the mechanism whereby RA reduces transcription of the CRABP I gene in these cells. We ligated progressively larger regions of the CRABP I 5' region to the CAT reporter gene and transfected these constructs into F9 cells that had been treated with RA (final concentration, 1 µM) or with vehicle alone (Fig. 2B)Citation . CRABP I 5' sequences from -7.8 kb to the first 40 nucleotides of exon 1 were sufficient for a high level of CAT activity in vehicle-treated F9 stem cells (Fig. 2BCitation , 7.8ex1/CAT). This activity was 13-fold higher than the activity of the same construct in RA-treated F9 cells. Thus, this 5' promoter region of the CRABP I gene recapitulates the regulation seen for the endogenous CRABP I gene in F9 cells. Deletion of the 5'-most end of this active promoter region, including the removal of sequence from -7.8 to -3.3 kb, did not alter CAT activity in either the vehicle-treated or RA-treated cells (Fig. 2BCitation , 3.3ex1/CAT). However, a further deletion, which removed sequence 5' of -2.7 kb, abolished much of this difference; a 4-fold difference in activity between the vehicle-treated and RA-treated F9 cells was observed for the 2.7ex1/CAT construct. A number of small deletions were made in this region between -2.7 kb and the transcription initiation site. Many of the serial deletions through this region reduced CAT activity but always <2-fold, suggesting that the many weak transcriptional regulatory sites in this region together exert an additive effect (data not shown).



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  		Fig. 2. Panel A, structure of the murine CRABP I gene. Solid rectangles, exons. B, BamHI; E, EcoRI; M, MscI; S, SpeI. Panel B, transient transfection results from F9 cells cultured in the presence of 1 µM RA or in vehicle alone for 4.5 days. Panel C, transient transfection results from P19 cells cultured in 1 µM RA or in vehicle alone for 3 days. For panels B and C, CRABP I/CAT constructs are diagrammed to the left, and their resulting CAT activities are shown in the graph to the right. In panels B and C, the CAT activity is PhosphorImager-quantitated data in arbitrary units. In each experiment, the activity of the vector (pBLCAT3) was defined as 1, and the other constructs were measured as a percentage of that value. 7.8, 3.3, and 2.7 indicate the 5' end of the promoter in each construct relative to the transcription initiation site. Bars, SD; the absence of error bars for some CRABP I promoter constructs signifies SDs that were too small to be seen in this graphing format.

 
These CRABP I promoter/CAT fusion genes were also assayed for their activity in P19 teratocarcinoma cells (Fig. 2C)Citation . P19 cells differ from F9 cells in that P19 cells differentiate in response to RA into many embryonic cell types, whereas F9 cells differentiate only to extraembryonic endoderm. The cell lines also vary in that RA increases CRABP I expression in P19 cells but decreases it in F9 cells. A CRABP I construct, which extended from -7.8 kb to exon 1, when tested in P19 cells, increased CAT activity ~2-fold above the vector containing no promoter (Fig. 2CCitation , 7.8ex1/CAT). No difference in activity was observed between the RA-treated and vehicle-treated P19 cells. CRABP I promoter sequences from -2.7 to exon 1 were unable to drive any expression above that seen for no promoter (Fig. 2CCitation , 2.7ex1/CAT). Thus, the CRABP I promoter is regulated very differently in F9 cells as compared with P19 cells.

To define more precisely the regions of the CRABP I promoter that activated transcription in F9 stem cells, small regions of the CRABP I promoter were fused to the TK minimal promoter driving expression of the CAT reporter gene (Fig. 3)Citation . We determined whether the 5' regulatory regions of the CRABP I promoter were sufficient to direct transcription of a heterologous promoter. We ligated a 4.8-kb SpeI fragment spanning CRABP I sequence from -5.0 to -0.2 kb to the TK minimal promoter (Fig. 3Citation , SPE/TK/CAT). This construct yielded a high level of activity in F9 vehicle-treated stem cells and a much lower level in F9 RA-treated cells. In stem cells, CAT activity for this construct was 24-fold higher than that of the TK minimal promoter alone, and a 6-fold difference in CAT activity between stem and RA-treated cells was observed for this Spe/TK/CAT construct. When we ligated a 3.6-kb MscI fragment including CRABP I sequence from -5.3 to -1.7 kb to the TK minimal promoter, a difference in activity between RA-treated and control cells was again observed (Fig. 3Citation , MSC/TK/CAT). However, this CAT activity was not as high as that of the -5.0/-0.2-kb region in F9 stem cells. This decrease probably reflects the loss of promoter elements mapped in Fig. 2BCitation to the region between -2.7 kb and exon 1.



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  		Fig. 3. Transient transfection results from regions of the CRABP I promoter ligated to the TK minimal promoter. In each experiment, the activity of the corresponding vector (pBLCAT2 or pGTKCAT) was defined as one, and activity from the constructs shown was measured as a percentage of that value. F9 cells were cultured with or without RA, as described in Fig. 2Citation . Bars, SD; the absence of error bars for some CRABP I promoter constructs signifies SDs that were too small to be seen in this graphing format. The CAT activity is PhosphorImager-quantitated data in arbitrary units.

 
To map the regulatory region determined from the deletional analysis shown in Fig. 2BCitation to reside between -3.3 and -2.7 kb, we ligated two different DNA fragments from this region 5' of the TK minimal promoter and the CAT reporter gene. This region between -3.3 and -2.7 kb can be easily divided into two restriction fragments, a 0.4-kb EcoRI/BamHI fragment, from -3.3 to -2.9, and a 0.2-kb BamHI/BamHI fragment, from -2.9 to -2.7 kb. In F9 stem cells, the 0.4-kb region, whether present in either orientation or as two tandem copies (Fig. 3Citation , EcoRI-BAM/TK/CAT constructs), had no activity above that seen for the TK promoter alone. The 0.2-kb region from -2.9 to -2.7 kb increased CAT activity 6-fold above that seen for TK alone in F9 stem cells, whereas no activity from this region was observed after RA treatment (Fig. 3Citation , BAM-BAM/TK/CAT constructs). Thus, the regulation by RA seen in the much larger MscI/MscI promoter fragment is completely recapitulated by the 0.2-kb BamHI fragment located between -2.9 and -2.7 kb in the CRABP I promoter. This 0.2-kb regulatory region is orientation dependent when ligated immediately 5' of the TK promoter. The sense orientation did not have transcriptional activity, whereas the antisense orientation (relative to the CRABP I coding sequence) did (Fig. 3Citation , BAM-BAM(+)/TK/CAT versus BAM-BAM(-)/TK/CAT). However, this orientation specificity was not observed when the region was farther from the TK promoter, in the MSC/TK/CAT and SPE/TK/CAT constructs (Fig. 3Citation ; data not shown). It is possible that the close proximity of the CRABP I and TK promoter elements in the BAM-BAM/TK/CAT constructs puts constraints on protein-protein interactions that are not observed when the protein binding sites are farther apart, as in the endogenous promoter.

In summary, the 0.2-kb region between -2.9 and -2.7 kb 5' of the CRABP I promoter contains an enhancer element that activates CRABP I transcription in F9 undifferentiated stem cells. It is through this enhancer that RA negatively regulates the transcription of the CRABP I gene in F9 cells (Fig. 3)Citation .

DNaseI Footprint Analysis of the CRABP I Promoter.
One method to define more precisely the sequences that activate transcription is the analysis of transcription factor binding sites. Thus, we examined both strands of the CRABP I promoter between -2.9 and -2.7 kb for sequences that were protected from DNase I cleavage by factors in nuclear extracts from F9 stem or RA-treated cells. Initial experiments using conventional binding conditions (40) did not identify any binding sites in this region, although strong binding was seen to other regions of the CRABP I promoter and to the TK minimal promoter (data not shown). Therefore, different conditions under which factors might bind to this region were tested. The most important variable tested was the MgCl2 concentration. In the presence of MgCl2 as low as 3 mM, no binding was detected between -2.9 and -2.7 kb of the CRABP I promoter (Fig. 4Citation , Lane 6, and data not shown). However, in the absence of MgCl2, binding was detected to a region centered at -2834 kb. Varying the incubation temperature from on ice (~4°C) to room temperature (~22°C) or the incubation time from 10 to 30 min did not affect the binding seen (Fig. 4Citation , Lanes 3, 4, and 5).



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  		Fig. 4. DNaseI footprinting under different binding conditions. The CRABP I promoter region from -2.9 to -2.7 kb was incubated with nuclear extract from F9 stem (F9 ST) cells (Lanes 3–6) or without nuclear extract (Lanes 2 and 7). Binding of DNA to proteins in the nuclear extract was done in 6 mM MgCl2 (Lane 6) or with no MgCl2 added (Lanes 3–5). Incubation times were 10 min (Lanes 2–4) or 30 min (Lanes 5–7), at room temperature (Lanes 2 and 3) or on ice (Lanes 4–7). Lane G, location of G nucleotides for each probe. Lane 0, DNase I cleavage pattern in the absence of nuclear extract.

 
On both the sense and the antisense strands, nuclear extract from F9 stem cells protected two sites within the -2.9 to -2.7-kb region, defined above as having transcriptional activity (Fig. 5)Citation . One site, at -2763, gave a strong footprint on the antisense strand and a weaker footprint on the sense strand. The other site, at -2834, gave a stronger footprint on the sense strand than on the antisense strand. This difference in the amount of protection from DNase I cleavage on the two different strands is particularly interesting in light of the orientation dependence of this region when ligated immediately 5' of the TK minimal promoter. (The sequences of these protected sites are shown in Fig. 8Citation by dark lines above and below the sequences, indicating the protection of the top and bottom strands, respectively.)



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  		Fig. 5. Two DNase I protected sites present in the transcriptionally active region of the CRABP I promoter between -2.7 and -2.9 kb. Panel on the right, sense or coding strand; panel on the left, antisense or noncoding strand. Lane G, location of G nucleotides for each probe. Lane 0, DNase I cleavage pattern in the absence of nuclear extract. Lanes ST, patterns of DNase I cleavage in the presence of F9 stem cell extract. Lanes RA, patterns observed in nuclear extract from RA-treated F9 cells. Open boxes to either side, locations on each strand of the DNase I protected regions. Arrows, sites of DNase I hypersensitive sites. Numbers next to the open boxes, location in the promoter. TK, DNase I protection of the TK minimal promoter.

 


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  		Fig. 8. Sequence of a transcriptionally active region of the CRABP I promoter. The newly defined transcriptionally active 176-bp BamHI-BamHI fragment is shown in bold. [This is the region missing from some previously described CRABP I genomic clones (41 , 66 67 68 69 70 71) .] The DNase I footprinted sites determined in Figs. 4Citation and 5Citation are shown for each DNA strand with dark lines. Dashed lines, regions where the border of the protected regions was difficult to define because of lack of DNase I cleavage sites. Arrowheads, sites of DNase I-hypersensitive cleavage. Location of CRI primers used for PCR reactions shown in Fig. 7Citation are indicated by thin lines.

 
Gel Shift Analysis of the Protected Regions of the CRABP I Promoter at -2834 and -2763.
To define the footprinted regions of the CRABP I promoter, mobility shift assays were performed. The probes used are summarized in Table 1Citation . A 36-bp double-stranded probe, called FP1, which encompasses the entire footprinted region at -2834 of the CRABP I promoter, was radiolabeled and incubated with nuclear extracts prepared from F9 and P19 stem cells and F9 and P19 cells that had been treated with RA for 72 h. After separation on a nondenaturing gel, three DNA/protein complexes, FP1A, FP1B, and FP1C, were detected in both the F9 stem and RA-treated cells (Fig. 6A)Citation . In the P19 stem and RA-treated cells, FP1C is present, FP1B is barely detectable, and FP1A is absent. In addition, there appears to be another complex present in P19 cells but absent in the F9 cells, FP1D, which gives a slightly higher band than the FP1C complex (Fig. 6A)Citation .


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  		Table 1 Summary of binding sites used for mobility shift assaysa

 


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  		Fig. 6. Gel shift analysis of the -2834 footprinted region of the CRABP I promoter. A, the FP1 double-stranded oligonucleotide shown in Table 1Citation was 32P end-labeled and incubated with 10 µg of nuclear extract from: F9 stem cells (Lane 2), F9 cells that had been treated with RA for 72 h (Lane 3), P19 stem cells (Lane 4), and P19 cells that had been treated with RA for 72 h (Lane 5). Lane 1 contains probe only. Samples were separated on a 5.2% nondenaturing polyacrylamide gel. Arrows, binding complexes and free probe. B, the FP1 double-stranded oligonucleotide shown in Table 1Citation was 32P end-labeled and incubated with 10 µg of nuclear extract from F9 stem for all samples in Lanes 2–11. Lane 1 contains probe only. Competitors were added in molar excess to the binding reactions, as indicated above each lane: Lane 2, no competitor; Lane 3, 5x molar excess FP1; Lane 4, 200x molar excess FP1; Lane 5, 5x molar excess FP1 Mu1; Lane 6, 50x molar excess FP1 Mu1; Lane 7, 200x molar excess FP1 Mu1; Lane 8, 5x molar excess FP1 Mu2; Lane 9, 50x molar excess FP1 Mu2; Lane 10, 200x molar excess FP1 Mu1; Lane 11, 200x molar excess nonspecific oligonucleotide (NS). Samples were separated on a 5.2% nondenaturing polyacrylamide gel. Arrows, binding complexes and free probe. This experiment was performed three times with two different nuclear extract preparations; one experiment is shown here.

 
To examine the specificity of the F9 cell-specific complexes, a competition assay was performed using F9 stem cell nuclear extract (Fig. 6B)Citation . Although 5x molar excess failed to compete, complexes FP1A and FP1B were effectively competed when 200-fold molar excess of unlabeled FP1 (Table 1)Citation was added as unlabeled competitor to the binding reactions (Fig. 6BCitation , Lanes 3 and 4). FP1C, which failed to be competed, appears to be a nonspecific complex. FP1 Mu1 is a double-stranded oligonucleotide with mutations in the 7-bp region that in Fig. 5Citation was shown to footprint on only the antisense strand; FP1 Mu1 failed to compete for the binding complexes at 5-, 50-, and 200-fold molar excess, indicating that these 7 bases are essential for FP1A and FP1B binding (Fig. 6BCitation , Lanes 5–7). FP1 Mu2, a nucleotide with mutations in the 7-bp region that in Fig. 5Citation was shown to footprint only on the sense strand, effectively competed for the FP1A and FP1B binding complexes at 200-fold molar excess. These data indicate that these bases are dispensable for binding (Fig. 6BCitation , Lane 10). Finally, a 36-bp unrelated binding site (Table 1)Citation did not compete for either of the two complexes (Fig. 6BCitation , Lane 11). These results indicate that FP1A and FP1B are specific DNA binding complexes.

For the -2763 footprinted region, a 36-bp double-stranded probe, called FP2, which encompasses the entire footprinted region at -2763 of the CRABP I promoter, was radiolabeled and incubated with nuclear extracts prepared from F9 and P19 stem cells and F9 and P19 cells that had been treated with RA for 72 h. Three DNA/protein complexes, FP2A, FP2B, and FP2C, were detected in both the F9 stem and RA-treated cells (Fig. 7A)Citation . In the P19 stem and RA-treated cells, FP2B and FP2C are present, but FP2A is not detected (Fig. 7A)Citation .



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  		Fig. 7. Gel shift analysis of the -2834 footprinted region of the CRABP I promoter. A, the FP2 double-stranded oligonucleotide shown in Table 1Citation was 32P end-labeled and incubated with 10 µg of nuclear extract from: F9 stem cells (Lane 2), F9 cells that had been treated with RA for 72 h (Lane 3), P19 stem cells (Lane 4), and P19 cells that had been treated with RA for 72 h (Lane 5). Lane 1 contains probe only. Samples were separated on a 5.2% nondenaturing polyacrylamide gel. Arrows, binding complexes and free probe. B, the FP2 double-stranded oligonucleotide shown in Table 1Citation was 32P end-labeled and incubated with 10 µg of nuclear extract from F9 stem cells (Lanes 2–4). Lane 1 contains probe only. One hundred-fold molar excess of the indicated unlabeled competitor is shown above each lane, and the sequence of each is shown in Table 1Citation . The competitors used are FP2 (Lane 3) and FP2 Mu (Lane 4). Samples were separated on a 5.2% nondenaturing polyacrylamide gel. Arrows, binding complexes and free probe. This experiment was performed three times with similar results; one experiment is shown here.

 
To examine the specificity of these complexes, competition assays were performed using nuclear extracts from F9 stem cells and oligonucleotides from the enhancer region (Fig. 7B)Citation . Complexes FP2A and FP2B were effectively competed when 100-fold molar excess of unlabeled FP2 (Table 1)Citation was added to the binding reactions as unlabeled competitor (Fig. 7BCitation , Lane 3). FP2C, which failed to be competed, appears to be a nonspecific complex. FP2 Mu, a double-stranded site in which the 10 protected nucleotides of the -2763 footprinted region were mutated, failed to compete for the binding complexes, indicating that these bases are essential for FP2A and FP2B binding (Fig. 7BCitation , Lane 4). Thus, the CRABP I transcriptional enhancer defined here contains two sites, at -2834 and at -2763, that each bind two distinct and specific complexes in F9 nuclear extracts.

Sequence Determination of the CRABP I Promoter.
On the basis of the published sequence of the CRABP I promoter, we expected there to be only one BamHI site between -3.3 kb and the transcription initiation site. However, we found that two independent genomic clones of this region each contained two BamHI sites as described above, one at -2.7 kb and one at -2.9 kb. We sequenced through this region of the CRABP I promoter using the CRI-2.9 primer shown in Fig. 8Citation . The sequencing results (Fig. 8)Citation confirmed that this region contained two BamHI sites separated by 176 bp. The difference between our sequence and the published murine CRABP I promoter sequence (41) corresponded exactly to this BamHI fragment; no other variations were detected. Although allelic sequence differences are possible, the fact that the sequence difference corresponds to a precise BamHI restriction fragment suggests that either the previously published sequence (41) contained a deletion or that the genomic clones we had isolated contained an insertion. We show below that the previously published genomic sequence (41) most likely contains a deletion of this region.

To distinguish between these two possibilities, we performed PCR analysis using a primer that anneals within the newly described BamHI fragment and one that anneals 5' of that, within the previously published sequence. If genomic DNA does indeed contain this sequence in the mouse CRABP I promoter, then a PCR product of 219 bp is expected. If this sequence is not in the endogenous CRABP I gene or is in a different location, then no product or a larger product would be expected. We found that genomic DNA isolated directly from mice produced a 219-bp product (Fig. 9Citation , Lane 4), indicating that this 0.2-kb BamHI fragment is indeed in the endogenous CRABP I gene. We also used primers that annealed to the previously published sequence, which flanks this newly defined BamHI fragment, to determine by the size of the PCR product whether either allele of the CRABP I gene lacked this 0.2-kb region. Pairing the CRI-2.9 primer with either a primer at -2.66 or at -2.61 kb (primer sequences are shown in Fig. 8Citation ) yielded a 269-bp band or a 321-bp band, respectively (Fig. 9Citation , Lanes 6 and 2, respectively). These are the sizes expected if both alleles of the mouse genomic CRABP I locus contain the BamHI fragment we have described here. The band sizes expected if the genomic CRABP I promoter did not contain this region would be 176 bp shorter for each of them. In all cases, the absence of genomic DNA resulted in no product, indicating that there was no contamination of our solutions with our genomic clones (Fig. 9Citation , Lanes 3, 5, and 7).



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  		Fig. 9. PCR analysis of the CRABP I promoter region from -2.9 to -2.6 kb with 100 ng of mouse genomic DNA added (Lanes 2, 4, and 6) or with no genomic DNA added (Lanes 3, 5, and 7). Lanes 2 and 3, primers used were CRI-2.9 and CRI-2.61. Lanes 4 and 5, primers used were CRI-2.9 and the internal primer (int.). Lanes 6 and 7, primers used were CRI-2.9 and CRI-2.66. Lanes 1 and 8, 1-kb ladder (Life Technologies).

 
Thus, the CRABP I promoter in C57Bl6 mice contains a 176-bp BamHI fragment from -2.9 to -2.7 kb that is capable of regulating transcription in some but not all cell types. In F9 stem cells, this fragment acts as an enhancer, and it is through this region that RA treatment leads to inhibition of CRABP I transcription.

Discussion

We have defined a region in the CRABP I promoter that is responsible for regulation of transcription in the F9 teratocarcinoma cell line. In untreated F9 cells, this site between -2.7 kb and -2.9 kb contributes greatly to the positive activation of transcription of the CRABP I promoter. After RA treatment, when the endogenous CRABP I gene is down-regulated, this transcriptional regulatory site is no longer active, and no activity is seen from the CRABP I promoter. Thus, we have defined a transcriptional regulatory region whose activity is down-regulated by RA. Previous studies have defined a number of transcription factors down-regulated by RA (1) . These include c-myb (42) , myc family members (43, 44, 45, 46, 47) , oct3/4 (48, 49, 50) , sox2 (51) , PEA3 (52) , and p53 (53 , 54) . Other putative transcription factors with unknown binding sites that are down-regulated by RA include REX-1 (55, 56, 57) and evx-1 (58) .

Within the 176-bp region that we defined as containing this RA-sensitive regulatory element(s), two sequences are protected from DNase I cleavage by F9 nuclear extract. Neither sequence matches an oct 3/4 or an AP-1 site, sites which have been shown previously to mediate inhibition of transcription by RA in other genes (56 , 57) . However, by gel shift assays, two specific DNA complexes that bind to the -2834 footprinted region and two specific DNA complexes that bind the -2763 footprinted region were detected in nuclear extracts prepared from F9 stem cells but were absent in nuclear extracts prepared from P19 stem cells (Figs. 6Citation and 7)Citation . This is consistent with the role of these sequences as enhancers in F9 stem cells and their lack of function in P19 cells. For the -2834 region, DNase I footprinting revealed protected nucleotides that were staggered on the sense and antisense strands. The 10 bp that were protected on the antisense strand were essential for complex formation, whereas the 7 bp protected on the sense strand were not required. Therefore, the 10 antisense strand nucleotides are likely contact bases for the binding proteins. The 7 sense strand nucleotides are likely protected from DNase I digestion by steric interference from the binding proteins. The 10 nucleotides of the -2834 region that are critical for binding do not show homology to any known DNA binding sites.

For the -2763 region, the 10 nucleotides essential for binding contain a consensus binding site, 5'-NGNGGGGA-3', for the DNA binding protein MZF-1 (59, 60, 61, 62) . MZF-1 is a zinc finger protein, originally isolated from myeloid cells, that was shown to be up-regulated by retinoic acid in the HL-60 cell line. Whether MZF-1 is expressed in F9 cells and is involved in regulating CRABP I gene transcription is currently under investigation.

Transcriptional coactivators are also regulated by RA treatment of F9 cells. One, called UTF1, is down-regulated after RA treatment (63) . UTF1 has been shown to act through the transcription factor ATF2. Although the CRABP I binding sites reported here do not contain ATF2 consensus sequences, it is possible that UTF1 may act through other transcription factors as well. Regulation of a coactivator may explain the presence of DNA binding complexes at the CRABP I transcriptional enhancer in both stem and RA-treated F9 cells.

The CRABP I gene appears to have a complex regulatory network that encompasses many different and widely spaced cis-regulatory elements. This complexity has been underscored by assays of CRABP I regulation in tissue culture cells. We have assayed our longest CRABP I/reporter constructs that contain up to 8 kb 5' of the first exon to 3 kb downstream (exon 3) by both transient and stable transfection, and have been unable to detect expression which is more than twice that of vector alone in several embryonic stem cell lines, in Balb 3T3 fibroblasts, and in the P19 teratocarcinoma line demonstrated here (Fig. 2Citation and data not shown). This is in contrast to our data using similar CRABP I promoter constructs for transgenic animal analyses, in which much of the expression of the endogenous CRABP I gene was recapitulated by the longer promoter constructs such as 7.8ex1/lacZ (Fig. 2BCitation ; Ref. 64 ). Similarly, Kleinjan et al. (65) reported being unable to drive expression using CRABP I reporter constructs in the highly expressing MES1 and Tera2 cell lines.

One laboratory has done extensive promoter mapping of the CRABP I gene. Wei and Chang (66) defined a region of the CRABP I promoter from -3.3 kb to -2.3 kb as increasing transcription of a reporter gene 10-fold in P19 stem cells. This region overlaps the region we have defined as active in F9 cells but not in P19 cells. The difference in activity in P19 cells may result from a difference in DNA sequence. The CRABP I promoter region cloned by Wei et al. (41) did not contain the 176-bp BamHI regulatory fragment identified here. Further, Wei et al. (67) found that this CRABP I construct extending to -3.3 kb was activated 2-fold after RA treatment of P19 cells in one study but was repressed by RA treatment 2-fold in another study (68) . These are both in contrast to the endogenous CRABP I gene, which is activated 10–20-fold after RA treatment of P19 cells (37) . Our results differ in that we did not observe any influence of the region of the CRABP I promoter from -3.3 kb to -2.3 kb on reporter activity in P19 cells in transient transfection assays, but we did observe activity of this region of the promoter in F9 cells (Figs. 2Citation and 3)Citation . Again, this difference between our data and that of Wei et al. (67 , 68) most likely results from the lack of one segment of promoter DNA in their experiments but could possibly be related to potential differences in the P19 cell lines maintained in the separate laboratories. Both laboratories did observe the 10–20-fold increase in expression of the endogenous CRABP I gene in response to RA (Fig. 1Citation ; Ref. 37 ).

Wei and colleagues (37 , 66 , 68 , 69) have defined a number of other transcriptionally active regions in the CRABP I promoter between -1.2 kb and -150 bp in P19 and in 3T6 cells. These regions contained a complex array of negative and positive regulatory elements. Many of these regulatory regions were context dependent in that 5' promoter deletions yielded different results than internal deletions. For example, Wei and Chang (66) reported that a CRABP I promoter fragment extending to -1046 had dramatically less reporter activity than a promoter that extended to -993, suggesting the presence of a transcriptional repressor site between -1046 and -993. However, an internal deletion of this exact region in the context of the -3.3 kb promoter resulted in a dramatic decrease in transcription, suggesting that a transcriptional activator site resided between -1046 and -993 (66) . Obviously, this complexity makes interpretation of CRABP I regulatory regions difficult. In an extensive series of 5' CRABP I promoter deletion experiments, we did not observe any negative or positive regulatory sequences between -3.3 kb and -150 bp in P19 cells (Fig. 2CCitation and data not shown. Because promoter context appears to be crucial for transcriptional activity, the lack of the -2.7/-2.9-kb BamHI site may have influenced the interpretation of many of the CRABP I promoter activity data (37 , 41 , 66, 67, 68, 69) .

In summary, the experiments presented here have defined a region of the CRABP I promoter that was not included in many prior studies. This genomic fragment, which has a regulatory function in F9 cells, was apparently not included in all of the genomic clones of CRABP I used previously for promoter analyses in cell culture and in transgenic animal analyses by some others (41 , 66, 67, 68, 69, 70, 71) ; because their library was made from BamHI cut DNA, this 176-bp BamHI fragment was most likely lost from their library. This region, 2.7 kb 5' of the transcription initiation site, confers expression to the CRABP I promoter or to a heterologous promoter in F9 stem cells but not in RA-treated F9 cells. Although several protein binding sites have been defined in this region, future studies are needed to determine the transcription factors acting through this region.

Materials and Methods

Cell Culture.
F9 cells and Balb/3T3 (ATCC clone 31) cells were cultured in DMEM plus 10% calf serum (Irvine Scientific) and 2 mM glutamine as described (19) . P19 teratocarcinoma cells were cultured in DMEM plus 7.5% calf serum and 2.5% fetal bovine serum (Life Technologies) and 2 mM glutamine. J1 ES cells were cultured in DMEM plus 10% fetal bovine serum, 0.1 mM nonessential amino acids (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 0.1 mM 2-mercaptoethanol, 1000 units/ml leukemia inhibitory factor (Life Technologies), and a 1x concentration of penicillin/streptomycin (Life Technologies). Six to 8 h after plating, RA or an equivalent volume of ethanol (vehicle) was added to a final concentration of 1 x 10-6 M.

Northern Blotting.
RNA was prepared from the cell lines indicated by the guanidine isothiocyanate method (72) . Fifteen µg of total RNA from each sample were electrophoresed, blotted, and hybridized as described (33) . Probes used for hybridization were the CRABP I full-length cDNA (33) isolated from the pMT64AA vector (73) by restriction digestion with EcoRI (19) and the glyceraldehyde-3-phosphate dehydrogenase PstI fragment cloned in the pGem4Z vector (74) .

PCR.
PCR was performed using the primers whose sequences are indicated in Fig. 6Citation . PCR was done in 50 mM KCl, 10 mM Tris (pH 8.8), 2.5 mM MgCl2, 0.1% gelatin, 2.4% DMSO, 50 pmol of each primer, and 1 unit of Amplitaq polymerase (Perkin-Elmer) in a volume of 50 µl with or without 100 ng of mouse genomic DNA from mouse strain C57Bl6 (Taconic). After denaturation at 95°C for 5 min, 25 cycles were performed, each cycle consisting of 95°C for 30 s, 68°C for 1 min, and 72°C for 1 min.

DNA Constructs.
CRABP I genomic sequence was originally isolated from a Charon 4A phage library of spleen genomic DNA from mouse strain BALB/c (a kind gift of Dr. R. M. Perlmutter). The EcoRI fragments from this library were then subcloned into pBluescript KS or pGEM4Z vectors. To clone murine CRABP I promoter sequences upstream of the CAT reporter gene, a BamHI site was introduced into the first exon of the CRABP I gene after nucleotide +40. (For all nucleotide numbering, +1 is defined as the nucleotide where transcription initiates.) 2.7ex1/CAT consists of the BamHI fragment of CRABP I 5' sequence from -2693 to +40 ligated into the BamHI site of pBLCAT3 (75) . 3.3ex1/CAT contains CRABP I sequence from the EcoRI site at -3.3 to +40 bp. 7.8ex1/CAT contains CRABP I sequence from the EcoRI site at -7.8 to +40 bp. To clone fragments of the CRABP I promoter upstream of the TK minimal promoter, CRABP I fragments were cloned into either the pBLCAT2 (75) or the pGTKCAT vector. The most significant difference between these two vectors is that the pGTKCAT vector contains three polyadenylation signals in front of the polylinker cloning site, thus resulting in less background expression from vector sequences. Also, the pGTKCAT plasmid affords different cloning sites for promoter insertion. pGTKCAT was made by replacing the HindIII/BamHI fragment containing the luciferase gene of pGL2 (Promega), with the HindIII/BamHI fragment from pBCO (76) containing the CAT gene, to make the plasmid pGCAT0. The TK minimal promoter was then excised from pBLCAT2 with BamHI/BglII digestion and ligated into the BglII site of pGCAT0. Msc/TK/CAT was made by ligating the MscI/MscI fragment (CRABP I nucleotides -5.3 kb/-1.7 kb) into the SmaI site of pGTKCAT. Spe/TK/CAT was made by ligating the SpeI/SpeI fragment (CRABP I nucleotides -5.0 kb/-0.2 kb) into the NheI site of pGTKCAT. The 0.4-kb EcoRI/BamHI and 0.2-kb BamHI fragments (CRABP I nucleotides -3.3 kb/-2.9 kb and -2.9 kb and -2.7 kb, respectively) were excised with BamHI using a BamHI site from the vector that cut in proximity to the EcoRI site of the CRABP I promoter and the two BamHI sites present in the CRABP I promoter. These fragments were ligated into the BamHI site of pBLCAT2 in the orientations indicated.

Transfections.
F9 and P19 cells were transiently transfected by calcium chloride precipitation (77) . At least two different DNA preparations were used for each construct tested. The ß-actin-lacZ reporter construct was cotransfected with CRABP I/CAT reporter constructs to normalize for transfection efficiency. The activity of this ß-actin promoter-lacZ vector is not affected by the addition of RA (78) . The dose of RA used (1 µM) is growth inhibitory in F9 and P19 cells after 2–3 days but is not cytotoxic (79) . Five to 10% of the resulting cell extracts was used to determine ß-galactosidase activity (80) . Equivalent ß-galactosidase units were then used to perform CAT assays (80 , 81) . For quantitation, the activity detected for the vector alone was defined as 1, and the relative activity of promoter constructs was measured accordingly. Because different vectors were used for different promoter analyses, the appropriately matched vector is always set as 1.

DNase I Footprinting.
For nuclear extracts, 30 150-mm tissue culture dishes were plated at 1 x 106 F9 cells/dish, and RA dissolved in ethanol was added 6–8 h later to a final concentration of 1 x 10-6 M for RA-treated cells or an equivalent volume of ethanol alone for untreated cells. Cells were harvested at day 5 after treatment. Nuclear extracts were made according to Dignam et al. (82) with minor modifications described previously (40) . Seventy µg of nuclear extract were used for each footprinting reaction. Unless specifically stated, protein binding and DNase I (Worthington) cleavage were performed as described (40) , except that the MgCl2 concentration varied in the binding reaction and 1.5 mM MgCl2 was added with the DNaseI. Products were electrophoresed on 6% denaturing polyacrylamide gels (83) .

Electrophoretic Mobility Shift Assays.
Complementary oligonucleotides, described in Table 1Citation , were annealed by heating at 85°C for 2 min, followed by successive 15-min incubations at 65°C, 37°C, 22°C, and 4°C. The double-stranded oligonucleotides contained two guanine residue overhangs on each end and were end-labeled by filling in with {alpha}-[32P] and Klenow enzyme. Binding reactions were carried out in a total of 20 µl containing: 0.5 ng (50,000 cpm) of probe, 10 µg of nuclear extract, 1x binding buffer [10 mM HEPES (pH 7.9), 60 mM KCl, 0.5 mM EDTA, 1 mM DTT, and 10% glycerol], and 5 µg of poly(deoxyinosinic-deoxycytidylic acid). Some samples also contained 50 ng of unlabeled double-stranded competitor DNA. Reactions were incubated for 20 min at 22°C and separated on a 5.2% nondenaturing polyacrylamide gel. Gels were run at 100 V in 0.5x TBE running buffer (44.5 mM Tris, 44.5 mM boric acid, and 1 mM EDTA, pH 8.0). Gels were dried and visualized by autoradiography.

Acknowledgments

We thank Drs. Dan Rosen and Alex Langston for the CRABP I genomic clones and Taryn Resnick for editorial assistance.

Footnotes

1 This research was funded by NIH Grant RO1CA43796 (to L. J. G.). Back

2 Present address: Division of Surgical Oncology, Vanderbilt University Medical College, T-2104 Medical Center North, Nashville, TN 37232-2736. Back

3 To whom requests for reprints should be addressed, at Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021. Phone: (212) 746-6250; Fax: (212) 746-8858; E-mail: ljgudas{at}mail.med.cornell.edu Back

4 The abbreviations used are: RA, all-trans retinoic acid; CRABP I, cellular retinoic acid binding protein; ES, embryonic stem; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase. Back

Received for publication 7/14/99. Revision received 12/14/99. Accepted for publication 1/ 5/00.

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