©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Promoter and Upstream Regulatory Activities of the Mouse Cellular Retinoic Acid-binding Protein-I Gene (*)

(Received for publication, October 19, 1995; and in revised form, December 15, 1995)

Li-Na Wei (§) Liming Chang

From the Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The promoter and its upstream regulatory region of the mouse cellular retinoic acid-binding protein I (crabp-I) gene were examined in transgenic mouse embryos, a mouse embryonal carcinoma cell line P19, and a mouse embryonic fibroblast cell line 3T6. In transgenic mouse embryos, a beta-galactosidase reporter gene under the control of crabp-I promoter and its upstream regulatory region displayed a very specific pattern of expression characteristic of crabp-I gene expression during developmental stages. In tissue culture systems, the minimal promoter of this gene was identified, and regions containing positive and negative regulatory activities were dissected from the upstream 3-kilobase sequence using assays for transient reporter activity. It is concluded that the minimal promoter of the mouse crabp-I gene is located between 120 and 150 base pairs upstream from the transcription initiation site. Several cell type-specific positive and negative regulatory regions for this promoter have been identified. A region encoding a common negative regulatory activity in both P19 and 3T6 cells is also inhibitory to two heterologous promoters, and specific protein-DNA interactions between this DNA fragment and nuclear extracts of P19 and 3T6 are demonstrated by gel retardation experiments.


INTRODUCTION

Retinoic acid (RA) (^1)exerts pleiotropic effects in animals, and the effects are mediated through various cellular components. The RA receptors and retinoic acid X receptors are transcription factors that regulate gene expression in response to RA (For review, see (1) and (2) ), whereas a group of cellular retinoic acid-binding proteins (CRABPs) are believed to be involved in metabolic pathways of RA (For review, see (3) and (4) ). crabp-I is ubiquitously expressed in adult tissues at a very low basal level and is highly expressed in several RA-sensitive tissues such as the eye and the testis(5, 6) . In embryos, strong expression of this gene is also spatially and temporally specific to tissues that are most sensitive to RA, especially the central nervous system(7, 8, 9, 10) . Based upon the promoter sequence, the mouse crabp-I gene has been characterized as a house keeping gene(11) . However, its upstream region contains numerous inverted repeat sequences and putative binding sites for transcription factors, suggesting that a complex regulatory mechanism may be involved in its cell- and stage-specific expression (12) . The bovine crabp-I gene has also been characterized (13) , and it appears that both the exon/intron junctions and the promoter region of this gene are highly conserved among animal species.

Although crabp-I deficient mice displayed no apparent phenotypes(14, 15) , previous studies in transgenic mice (16) and embryonal carcinoma cells (17) showed an association of elevated crabp-I expression with abnormal cellular differentiation and RA-regulated gene expression. Studies in embryonic palate cells demonstrated that expression of RA receptor-beta, TGF-beta3, and tenascin was altered as a result of introduction of anti-crabp-I oligonucleotides(18) . Recent biochemical studies provided more evidence for a role of crabp-I in RA catabolism(19) . It is suggested that the level of crabp-I expression must be tightly controlled because abnormally high level of expression may disturb RA concentration, thereby affecting gene expression in specific cells at a critical time(16) .

Consistent with the observation of weak crabp-I expression in most adult tissues, its expression is also very weak in most cell lines examined, except in a mouse embryonic fibroblast cell line 3T6 (11, 20) . Significant induction of this gene has only been observed in embryonal carcinoma cell lines, such as P19 and F9, treated with RA (20) . The study of the mouse crabp-I genomic structure has revealed several interesting features within a 3-kb upstream sequence, such as a GC content of greater than 70%, 9 pairs of inverted repeats, 5 copies of GC boxes (Sp-1 sites, GGGCGG), and several potential binding sites for transcription factors(11, 12) . Recently, using pharmacological treatments, we showed that RA induction of this gene could be enhanced by 5-azacytidine (21) and DC-erythro-dihydrosphingosine (sphinganine)(22) . The effect of sphinganine was associated with an 870-bp DNA fragment in the most 5`-end of the upstream region containing a putative AP-1 binding site (TGACTCA). The effect of 5-azacytidine was examined by analyzing the methylation status of the 3-kb upstream sequence, which revealed hypermethylation of this region in cells where crabp-I expression was low. Demethylation was associated with up-regulation of this gene expression(21) . The biological activity of the 3-kb upstream sequence was demonstrated in transgenic mouse embryos using an Escherichia coli beta-galactosidase (lacZ) reporter(12) . However, the transgene expression pattern differs slightly from the endogenous crabp-I expression pattern detected by in situ hybridization(8) , possibly due to the use of a heterologous DNA fragment, the mouse Hox1.3(23) , in the fusion.

In this study, to address both the promoter and the upstream regulatory activities of the mouse crabp-I gene, we constructed a series of lacZ reporter fusion genes by inserting a lacZ structural gene fragment, in frame, into the fifth amino acid codon of the mouse crabp-I gene. The biological activity of the full-length fusion gene was tested in both cultured cells and transgenic mouse embryos, and systematic deletion mutants were made to dissect minimal promoter and cell type-specific regulatory regions. Gel retardation assays were conducted to demonstrate specific protein-DNA interactions between the regulatory DNA fragments and nuclear extracts of P19 and 3T6 cells.


EXPERIMENTAL PROCEDURES

Techniques for Cell Cultures

P19 cells were maintained in alpha-minimal essential medium supplemented with 2.5% fetal calf serum, and 7.5% calf serum and 3T6 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum as described (11, 20) .

lacZ Reporter Constructs

A reporter construct was made by ligating an E. coli lacZ structural gene containing an SV40 poly(A) site (22) to a 3.2-kb crabp-I genomic DNA fragment at the KpnI site within exon I(12) . This fusion gene was first tested in P19 cells as well as transgenic mouse embryos and used as the parental vector for making fusion genes systematically deleted in specific upstream sequences.

Cell Transformation and Quantitation of lacZ and Luciferase Reporter Activity

Cells were plated in 24-well plates (5 times 10^4 cells/well) and transformed with reporter plasmid DNA using the calcium phosphate precipitation method. Lac activity was determined between 24 and 40 h, using orthonitrophenyl-beta-D-galactopyranoside (Sigma) as substrate as described previously(22, 24) . Protein concentrations were determined using a Bio-Rad protein assay kit. Transient lacZ activity was represented as A/µg total protein. For internal deletion and heterologous promoter fusion, the activity of each fusion gene was represented as a percentage of the parental vector activity. Luciferase activity was determined with a Promega luciferase assay system and represented as relative luciferase unit by normalizing to a cotransfected lacZ control activity in each transfection. For all the assays, triplicate cultures were used in each experiment, and three independent experiments were conducted to obtain the means and standard errors of the mean (S.E.).

Transgenic Mice Production and Enzyme Histochemical Analysis of lacZ Expression in Transgenic Mouse Embryos

Microinjection was performed according to a standard procedure (25) in the transgenic mouse center at the University of Minnesota. Transgenic mice were identified by Southern blot analysis of tail DNA. In situ detection of lacZ reporter gene expression was conducted as described (12) . Stained embryos were examined and photographed using a Nikon stereoscope SMZ-10 photo system.

Gel Retardation Assay

Gel retardation experiments were modified from an established protocol(26) . Nuclear extracts from P19 and 3T6 cells were prepared using the method of Standke et al.(27) . Briefly, the cell pellet was resuspended in a hypertonic solution (20 mM HEPES, pH 7.6, 10 mM KCl, 1 mM MgCl(2), 0.5 mM dithiothreitol, 0.1% Triton X-100, 20% glycerol) and lysed by 50 strokes in a glass Dounce homogenizer. The homogenate was centrifuged at 2,000 times g for 5 min, and the pelleted nuclei were extracted with 0.4 M NaCl by gently inverting at 4 °C for 30 min. Nuclear extract was preincubated in a solution containing 10 mM Tris (pH 7.5), 10 mM NaCl(2), 1 mM dithiothreitol, 1 mM EDTA, 1 µg of poly(dI-dC), 1 µg of salmon sperm DNA, and 5% glycerol at room temperature for 10 min. Cold competitor oligonucleotides, when included, were added during this preincubation period. Oligonucleotides were labeled with [alpha-P]dCTP using Klenow fragment; a total radioactivity of 2 times 10^5 cpm (prepared from approximately 0.2 ng of DNA fragment) was added to each reaction, and the mixture was incubated at room temperature for 20 min. The protein-DNA complexes were analyzed on a 5% polyacrylamide gel containing 5% glycerol in a low ionic strength buffer (6.7 mM Tris, pH 7.9, 3.3 mM sodium acetate, 1 mM EDTA).


RESULTS

Reporter Constructs for Generating Systematic Deletion in Mouse crabp-I Gene Upstream Region

The lacZ fusion gene containing the complete 3-kb upstream sequence of the mouse crabp-I gene was made as described under ``Experimental Procedures'' and designated as CRABP-lacZ. From this parental vector, systematic deletions were made using available restriction sites or by polymerase chain reactions as shown in Fig. 1, where the numbering system has been adopted from our previous study (12) for consistency. Within the 3-kb upstream sequence (sequence reported in Fig. 1of (12) ), several potential sequences for regulatory protein binding have been identified by sequence comparison to a transcription factor data base, including an AP-1 site between nucleotide (nt) 758 and 766 (TGACTCA), an imperfect RA response element (RARE) of DR5 type between nt 2113 and 2131 (CCATGAAGGAAAAGTGA), 5 copies of Sp-1 binding sites between nt 2902 and 3075 (GGGCGG), and 9 inverted repeat sequences scattered in a region between nt 85 and 2920(12) . Construct 870 is deleted in the 5`-end 870 bp, deleting the putative AP-1 binding site and the first four repeats. The 1960 construct is deleted for three more inverted repeats, and the 2100 construct is further deleted for one repeat. The 2140 construct is further deleted for a 40-bp fragment containing a putative RARE. The 2400 construct is further deleted for approximately 200 bp, and the 2600 construct contains only the last inverted repeat and 5 copies of Sp-1 sites. The 2990, 3020, and 3110 constructs retain only a minimal DNA fragment, each containing 4, 3, and 0 copies of Sp-1 binding sites, respectively.


Figure 1: Reporter fusion gene constructs for 5`-deletion analysis of mouse crabp-I upstream region. The BamHI fragment of E. coli lacZ structure gene (pMC1871, Pharmacia) was filled-in with Klenow enzyme and fused, in frame, into blunt-ended KpnI site of the mouse crabp-I EcoRI genomic fragment containing exon I (12) . A fragment containing SV 40 poly(A) fragment was added to the 3`-end of this fusion. This generated the parental vector, designated as CRABP-lacZ, which contained 3.2 kb of crabp-I genomic sequence including 3 kb in the upstream region. The constructs 870, 1960, 2990, 3020, and 3110 were made using PCR-amplified fragments, and the constructs of 2100, 2140, 2400, and 2600 were made by HindIII (H), XhoI (X), PstI (P), and SmaI (S) digestion, respectively. The nucleotide position was numbered from 5` to 3`-end, according to the published sequence ( Fig. 1in (12) ) for consistency. A filled triangle indicates the putative AP-1 site, and an arrow under the CRABP-lacZ construct indicates the putative RARE. Above the constructs, detailed features of the promoter and its immediate 5`-flanking region are shown. Five vertical bars indicate the five Sp-I sites, and a horizontal arrow indicates the position of transcription initiation. Translation initiation codon (ATG) is indicated at nt 3233. -, CRABP-I region; , CRABP-I coding region; box, lacZ; &cjs2110;, SV40 poly(A) site.



Promoter Activity of CRABP-lacZ in Transgenic Mouse Embryos

The full-length fusion gene, CRABP-lacZ, was introduced into transgenic mice. In situ lacZ analysis of two independent lines was conducted systematically, which revealed an identical pattern of reporter gene expression (Fig. 2). lacZ expression is detected in the mesencephalon of embryos as early as E9.5 (A) and extends to rhombencephalon and spinal cord at E10.5 (B). By E11.5, the expression diffuses to a wider area in the mesencephalon, rhombencephalon, and spinal cord, but the overall intensity of expression is reduced (C). By E12.5, the expression decreases dramatically with weak stain remained in the roof of the midbrain (D). This pattern agrees very well with the results of several studies using in situ hybridization (7, 8, 9, 10) . Thus, it is concluded that this fusion gene, driven by the mouse crabp-I promoter and the complete 3-kb upstream sequence, contains information needed for its spatially and temporally specific expression in mouse embryos.


Figure 2: Spatial and temporal specific CRABP-lacZ transgene expression in transgenic mouse embryos. Transgenic mouse embryos were dissected at gestation dates of E9.5 (A), E10.5 (B), E11.5 (C), and E12.5 (D) and analyzed for lacZ expression in whole mount embryos as described(12) . The stained (lacZ positive) areas are indicated. m, mesencephalon; r, rhombencephalon; s, spinal cord. The magnification is 30, 20, 15, and 15times, for A, B, C, and D, respectively.



Minimal Promoter Activity and Cell Type-specific Regulatory Activity of crabp-I Gene Upstream Sequence

To determine regulatory activities of various 5`-upstream sequences and to shed light on factors responsible for different levels of expression in high expressing and low expressing cells, specific lacZ activity of each fusion gene was determined in P19 (a low expressing cell line, solid bars) and 3T6 (a high expressing cell line, open bars) cells (11, 20) and represented as A/30 µg of protein as shown in Fig. 3. The construct 2990 represents the shortest fusion gene active in both cell lines, whereas 3020 or 3110 has no activity in either cell line. Thus, it is concluded that the same minimal promoter is used in both high expressor (3T6) and low expressor (P19), and the active promoter region is located between nt 2990 and 3020, approximately 120-150 bp upstream from the transcription initiation site (nt 3140). By adding 390 bp containing one more Sp-1 site (the 2600 construct), the reporter activity increases 2-3-fold in P19 cells, and 8-9-fold in 3T6 cells. Thus, sequence between nt 2600 and 2990 contains strong positive DNA elements for crabp-I expression in the high expressor (3T6). The 2140 and 2400 constructs have a 4- and 2-fold, respectively, higher activity over the construct 2600 in P19 cells, yet they are both much weaker than the 2600 construct in 3T6 cells. Thus, this region contains relatively strong positive regulatory activity in P19 cells yet slightly inhibitory activity in 3T6 cells. Surprisingly, a dramatic decrease in reporter activity has been observed in both cell lines for 2100, which contains an imperfect RARE of DR5 type between nt 2100 and 2140. In 3T6 cells, this negative effect is abolished when more 5`-sequence is added to the reporter (1960, 870, and the full promoter constructs). In P19 cells, 1960 and 870 remain very weak, and a maximal promoter activity is obtained only when an 870-bp fragment of the most 5`-end is added (the full-length construct). Therefore, these 5`-deletion mutants demonstrate both common and cell type-specific regulatory regions of crabp-I gene expression in transient assays. In both cell lines, a common minimal promoter is utilized, between nt 2990 and 3020, and a dramatic reduction in promoter activity is observed for the construct 2100. In the high expressor (3T6), the region responsible for the maximal transient promoter activity is located between 150 and 540 bp upstream from the transcription initiation site (between nt 2600 and 2990). In the low expressor P19, the same region encodes approximately of the maximal transient promoter activity of the full-length construct. Some positive activity is encoded in the region between nt 870 and 2100 for 3T6 cells, whereas a strongly positive element is located in the first 870 bp for P19 cells.


Figure 3: Specific reporter activity of 5`-deletion analysis. Promoter activity of each construct (shown in Fig. 1) was determined as described under ``Experimental Procedures'' and represented as A/30 µg of protein. Triplicate cultures were used in each experiment, and three independent experiments were conducted in P19 (solid bars) and 3T6 (open bars) cells to obtain the means (A/30 µg protein) and S.E. values.



Effects of Deleting Specific DNA Sequence in the Region between nt 2100 and 2600

The study shown in Fig. 3suggested that the sequence between nt 2100 and 2140 had a profound negative regulatory activity. To study this region in more detail, various mutants deleted internally in this region were constructed (Fig. 4A) and tested in P19 and 3T6 cells (Fig. 4B). Specific lacZ activity of each internal deletion was compared to the parental vector activity (CRABP-lacZ) and represented as relative activity (%). In P19 cells (solid bars), deletion of 2100/2140, 2100/2400, or 2100/2600 inhibits the reporter activity dramatically (greater than 90%), and deletion of 2140/2400 or 2140/2600 slightly decreases the reporter activity (approximately 40%). In 3T6 cells (open bars), deletion of 2100/2140 and 2100/2600 also inhibits the reporter activity dramatically, yet all the other deletions show a slightly positive effect. Thus, the two overlapping sequences, 2100/2140 and 2100/2600, are critical for a strong promoter activity in both cell types, as deletion of either region results in dramatic decrease in the reporter activity. Yet, 5`-deletion analysis shows a profound negative effect of the sequence between nt 2100 and 2140 when it is situated 5` to its natural 3`-flanking sequence. This would suggest that this sequence could not be a simple positive or negative element, and the sequence in its vicinity is important for the regulatory activity.


Figure 4: Relative reporter activity of internal deletion analysis. A, the deletion of various sequences in the region between nt 2100 and 2600 was made by restriction enzyme digestion from the parental construct CRABP-lacZ. The constructs 2100/2140, 2100/2400, 2100/2600, 2140/2400, and 2140/2600 were made by using HindIII-XhoI, HindIII-PstI, HindIII-SmaI, XhoI-PstI, and XhoI-SmaI digestion (restriction sites shown in Fig. 1), respectively. A filled triangle indicates the putative RARE between nt 2100 and 2140. B, relative reporter activity of each construct was represented as the percentage of the parental construct CRABP-lacZ activity in P19 (solid bars) and 3T6 (open bars), and three independent experiments were conducted to obtain the means and S.E. values.



Positive and Negative Effects of Regulatory Sequence between nt 2100 and 2600 on Heterologous Promoters

Deletion studies shown in Fig. 3and Fig. 4suggested that the most dramatic regulatory activity of CRABP-I promoter common to both cell types was encoded in a fragment between nt 2100 and 2600. This region was further examined using heterologous promoters. A human Harvey ras promoter(28) , constitutively active in P19 and 3T6 cells, was first fused to the 40 bp (2100/2140). No significant effect was observed in either P19 or 3T6 cells (data not shown). Therefore, various fragments were dissected from region 2100/2600 and fused, in both orientations, upstream to the ras promoter (Fig. 5A) and tested in P19 (solid bars) and 3T6 (open bars) as shown in Fig. 5B. It is clear that 2140/2400 and 2100/2600 fragments are able to repress ras promoter, regardless the orientation of the fusion, in both P19 and 3T6. In contrast, 2400/2600 sequence has a slight enhancing effect on this promoter. To determine if the negative effects on the ras promoter could be reproduced on other heterologous promoters, a luciferase reporter driven by a thymidine kinase promoter (29) was used (Fig. 6A) and tested (Fig. 6B). It appears that both fragments, in either sense or antisense orientation, are also inhibitory to the thymidine kinase promoter in both P19 (solid bars) and 3T6 (open bars). Therefore, it is concluded that the 2100/2600 fragment and a minimal 2140/2400 sequence are inhibitory to heterologous promoters regardless the orientation of the fusion.


Figure 5: Regulatory activity of region 2100/2600 on the ras promoter. A, the 2100/2600, 2400/2600, and 2140/2400 constructs were made by ligating HindIII-SmaI, PstI-SmaI, and XhoI-PstI fragments (restriction sites shown in Fig. 1), respectively, to the 5`-end of a lacZ reporter containing the ras promoter in the sense orientation. Antisense constructs for each region were also made and designated as 2100/2600, 2400/2600, and 2140/2400. B, relative reporter activity of each construct was represented as the percentage of the parental vector ras-lacZ activity in P19 (solid bars) and 3T6 (open bars), and three independent experiments were conducted to obtain the means and S.E. values.




Figure 6: Regulatory activity of regions 2100/2600 and 2140/2400 on the thymidine kinase promoter. A, the 2100/2600 and 2140/2600 constructs were made by ligating HindIII-SmaI and XhoI-PstI fragments (restriction sites shown in Fig. 1), respectively, to the 5`-end of a luciferase reporter containing the thymidine kinase promoter in the sense orientation. The antisense constructs were also made and designated as 2100/2600 and 2140/2400. B, relative reporter activity of each construct was represented as the percentage of the parental vector thymidine kinase-luciferase activity in P19 (solid bars) and 3T6 (open bars), and three independent experiments were conducted to obtain the means and S.E. values.



Gel Retardation Experiment

To determine specific DNA-protein interactions between the common negative regulatory region 2100/2600 and nuclear proteins, gel retardation experiments were performed. Multiple bands were observed when the 2100/2600 fragment was used (data not shown). Using the sequence 2100/2400 as the probe, a single specifically retarded band was observed as shown in Fig. 7, where various amounts of unlabeled probe have been included as the competitors. A specifically retarded band is observed using nuclear extracts from both P19 (lane 2) and 3T6 cells (lane 6). This band, representing protein-bound fragment, diminishes gradually when more competing probes are added (lanes 3-5 for P19 and lanes 7-9 for 3T6). Thus, the 2100/2400 sequence can be bound by specific nuclear proteins present in both P19 and 3T6 nuclei.


Figure 7: Gel retardation. The HindIII-PstI fragment (restriction sites shown in Fig. 1) was labeled with P with Klenow enzyme and tested for binding to specific proteins isolated from P19 and 3T6 nuclei as described in the text. The sample order is as follows: 1) no nuclear extract (probes alone), 2) 10 µg of P19 extract, 3) 10 µg of P19 extract + 10times unlabeled fragment (cold competitor), 4) 10 µg of P19 extract + 100times unlabeled fragment, 5) 10 µg of P19 extract + 500times unlabeled fragment, 6) 7 µg of 3T6 extract, 7) 7 µg of 3T6 extract + 10times unlabeled fragment, 8) 7 µg of 3T6 extract + 100times unlabeled fragment, and 9) 7 µg of 3T6 extract + 500times unlabeled fragment. Arrow head indicates the position of the specifically retarded band.




DISCUSSION

We have demonstrated that the mouse crabp-I gene promoter, including approximately 3 kb of its upstream region, is able to direct a lacZ reporter expression in transgenic mouse embryos. The expression pattern agrees with results generated from several in situ hybridization studies(7, 8, 9, 10) , indicating that this region contains spatial and temporal information for crabp-I gene expression. By systematic deletion analysis in both high expressing (3T6) and low expressing (P19) cells, the minimal promoter is located between nt 2990 and 3020, approximately 120-150 bp upstream from the transcription initiation site. In transient reporter assays, a 390-bp fragment (nt 2600-2990) immediately upstream from the transcription initiation site (nt 3140) encodes the maximal promoter activity in the high expressing cell line 3T6, whereas a complete 3-kb upstream sequence is needed to obtain the maximal promoter activity in the low expressing cell line P19. Additional sequence in the further upstream region of the 3-kb sequence appears to have no effect on this promoter in either cell line (data not shown). The 870-bp fragment in the 5`-end of this 3-kb fragment is important for the maximal activity in P19 cells. This agrees with our previous study(21) , showing the requirement of this 870-bp fragment for optimal crabp-I expression in P19 cells treated with RA and sphinganine, a compound known to increase AP-1 activity(30) .

Common to both P19 and 3T6 cells, a strong negative effect is observed for deletion to nt 2100, approximately 1 kb upstream from the transcription initiation site. A further deletion of 40 bp (deletion to nt 2140) abolishes this negative effect in both cell types. Results from these 5`-deletion studies would suggest that the 40-bp sequence (2100-2140) contains negative regulatory information for crabp-I gene expression. However, studies of internal deletions (Fig. 4) show that the sequence 2100-2140 is critical for a high level of reporter expression in both P19 and 3T6 cells, as deletion of 40 bp results in greater than 94% decrease of the full promoter activity in both cell types. This would argue against a negative activity of this 40-bp sequence because deletion of this presumably negative element should have either little effect or have abolished its negative effect. It is possible that the 2100/2140 sequence is a portion of a complex regulatory unit, which could operate very differently depending upon the sequence in its vicinity and the combination of available regulatory proteins.

The regulatory mechanism under physiological conditions could be much more complicated considering the complexity of DNA structure, modification of DNA, and multiple protein interactions in the cells. Based upon studies of internal deletions (Fig. 4) and heterologous promoters ( Fig. 5and Fig. 6), it is suggested that fragment 2100/2600 may be a complex regulatory unit that can be affected by many factors. It is clear that the 40-bp sequence is critical for the full crabp-I gene promoter activity in the context of the natural crabp-I gene regulatory region (as demonstrated by internal deletion analysis in Fig. 4), and yet, in conjunction with its 3`-flanking sequence, this region becomes a negative regulatory element (Fig. 4Fig. 5Fig. 6). It is interesting that the 40-bp sequence 2100/2140 contains a putative DR5-type RARE. However, by itself, this sequence has little effect on heterologous promoters (data not shown). It would be interesting to determine the protein factors bound to these sequences and how they interact with each other.

Based upon studies using heterologous promoters, the sequences derived from 2140/2400 and 2100/2600 are able to function as strong negative regulatory elements in both cell types. However, 5`-deletion analysis reveals positive activity of 2140/2400 when it is fused to its natural 3`-flanking sequence (the construct 2140) in P19 cells. This also suggests that the sequence 2140/2400 constitutes a portion of a complex regulatory unit. Interaction between this sequence and its neighboring sequences determines the final regulatory activity of the whole unit. Likewise, sequence 2400/2600 has little effect on heterologous promoters, yet, when situated in its natural position, it is able to increase and inhibit crabp-I promoter activity in P19 and 3T6 cells, respectively (Fig. 3). Consistent with these results, gel retardation experiment (Fig. 7) shows that fragment 2100-2400 can be bound by specific nuclear factors that are present in both P19 and 3T6 cells, and the protein-bound fragments migrate at the same position in both cases. In contrast, very different and more complex patterns of band shift have been observed for 2100/2600 fragment (data not shown).

P19 cells, in undifferentiated states, express endogenous crabp-I at a very low level. The expression can be specifically induced by RA, which is prohibited by cyclohexamide, a protein synthesis inhibitor(19) . In contrast, 3T6 cells express endogenous crabp-I constitutively at a much higher level, yet RA has little effect on its expression(11, 20) . Based upon data collected from this and other studies, a model is proposed for the regulatory elements controlling crabp-I gene expression as shown in Fig. 8. It is hypothesized that both positive and negative regulatory mechanisms are needed for the control of crabp-I gene expression. For most cell types, crabp-I gene utilizes the minimal promoter located between nt 2990 and nt 3020, which is constantly demethylated (21) and active. The upstream region of this promoter contains numerous regulatory DNA elements for both positive and negative transcription factors and their associate proteins. The region between nt 2600 and nt 2990 contains sequence for some positive factors that are probably present more abundantly in some highly expressing cells such as 3T6. The 870-bp fragment of the most 5`-end contains a sequence for positive factors that can be induced by certain drugs (such as sphinganine) in some cells (like P19). In contrast, the region 2100/2600 contains a sequence for negative transcription factors (such as repressors). For most cell types, either the lack of these positive factors for sequences 1/870 and 2600/2990 or the presence of negative factors for sequence 2100/2600 prohibited its optimal level of expression. In the presence of RA, this promoter activity is enhanced in certain cell types such as embryonal carcinoma because of diminishing levels of negative factors, induction of positive factors, or a combination of both. A total of nine inverted repeats are present within the 3-kb region, which have the potential to form complex structures, thereby bringing these regulatory elements to a close proximity. This model is being tested by asking specifically if the dissected elements can be associated with known regulatory proteins in terms of physical interaction and biological activity. With this information, it would be possible to begin to address how crabp-I gene expression is regulated in specific cell types and during developmental stages.


Figure 8: A model for mouse crabp-I gene regulatory elements. Nucleotide number starts from the 5`-end to the 3`-end according to previous sequence data (12) for consistency. Putative regulatory elements in the 3-kb upstream region, such as AP-1, RARE, and Sp-1, are indicated above the sequence. Question marks represent unknown factors. Transcription initiation site is indicated with a horizontal arrow under the sequence. Relative regulatory activity of each region in P19 and 3T6, as shown above the sequence, is arbitrarily scaled from -4 to +4 according to relative activity detected in transient transfection studies (Fig. 3Fig. 4Fig. 5Fig. 6). The negative signs for region 2140/2400 shown in the parentheses indicate negative activity of this region fused to the heterologous promoters.




FOOTNOTES

*
This study was supported by National Institutes of Health Public Service Grant DK46866-01 (to L.-N. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology, University of Minnesota Medical School, 3-249 Millard Hall, 435 Delaware St. S.E., Minneapolis, MN 55455. Tel.: 612-625-9402; Fax: 612-625-8408.

(^1)
The abbreviations used are: RA, retinoic acid; RARE, RA response element; bp, base pair(s); kb, kilobase(s); nt, nucleotide(s); CRABP, cellular retinoic acid-binding protein.


ACKNOWLEDGEMENTS

-We thank Dr. K. Roberts for critical reading of this manuscript. We thank C.-H. Lee and Y. Lin for excellent technical help.


REFERENCES

  1. Mangelsdorf, D. J., Umesono, K., and Evans, R. M. (1993) in The Retinoids Vol. 2, pp. 319-350, Academic Press, New York
  2. Sucov, H. M., and Evans, R. M. (1995) Mol. Neurobiol. 10, 169-184 [Medline] [Order article via Infotrieve]
  3. Ong, D. E., Newcomer, M. E., and Chytil, F. (1993) in The Retinoids (Sporn, M. B., Roberts, A. B., and Goodman, D. S., eds) pp. 283-318, Academic Press, New York
  4. Bass, N. M. (1993) Mol. Cell. Biochem. 123, 191-202 [Medline] [Order article via Infotrieve]
  5. Kato, M., Blaner, W. S., Mertz, J. R., Das, K., Kato, K., and Goodman, D. S. (1985) J. Biol. Chem. 260, 4832-4838 [Abstract]
  6. Wei, L.-N., Mertz, J. R., Goodman, D. S., and Nguyen-Huu, M. C. (1987) Mol. Endocrinol. 2, 526-534
  7. Vaessen, M.-J., Kootwijk, E., Mummery, C., Hilkens, J., Bootsma, D., and van Kessel, A. G. (1989) Differentiation 40, 99-105 [Medline] [Order article via Infotrieve]
  8. Perez-Castro, A. V., Toth-Rogler, L. E., Wei, L.-N., and Nguyen-Huu, M. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8813-8817 [Abstract]
  9. Ruberte, E., Friederich, V., Chambon, P., and Morriss-Kay, G. (1993) Development 118, 267-282 [Abstract/Free Full Text]
  10. Lyn, S., and Giguere, V. (1994) Dev. Dynamics 199, 280-291 [Medline] [Order article via Infotrieve]
  11. Wei, L.-N., Tsao, J.-L., Chu, Y.-S., Jeannotte, L. J., and Nguyen-Huu, M. C. (1990) DNA Cell Biol. 9, 471-478 [Medline] [Order article via Infotrieve]
  12. Wei, L.-N., Chen, G. J., Chu, Y.-S., Tsao, L.-J., and Nguyen-Huu, M. C. (1991) Development 112, 847-854 [Abstract]
  13. Shubeita, H. E., Sambrook, J. F., and McCormick, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5645-5649 [Abstract]
  14. Gorry, P., Lufkin, T., Dierich, A., Rochette-Egly, C., Decimo, D., Dolle, P., Mark, M., Durano, B., and Chambon, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9032-9036 [Abstract]
  15. de Bruijn, D. R. H., Oerlemans, F., Hendriks, W., Baats, E., Ploemacher, R., Wieringa, B., and van Kessel, A. G. (1994) Differentiation 58, 141-148 [CrossRef][Medline] [Order article via Infotrieve]
  16. Wei, L.-N., Lee, C.-H., Chang, S.-L., and Chu, Y.-S. (1992) Dev. Growth & Differ. 34, 479-483
  17. Boylan, J. F., and Gudas, L. J. (1991) J. Cell Biol. 112, 1965-1979
  18. Nugent, P., and Greene, R. (1995) In Vitro Cell. Dev. Biol. 31, 553-558
  19. Fiorella, P. D., and Napoli, J. L. (1994) J. Biol. Chem. 269, 10538-10544 [Abstract/Free Full Text]
  20. Wei, L.-N., Blaner, W. S., Goodman, D. S., and Nguyen-Huu, M. C. (1989) Mol. Endocrinol. 3, 454-463 [Abstract]
  21. Wei, L.-N., and Lee, C.-H. (1994) Dev. Dynamics 201, 1-10 [Medline] [Order article via Infotrieve]
  22. Wei, L.-N., Lee, C.-H., and Chang, L. (1995) Mol. Cell. Endocrinol. 111, 207-211 [CrossRef][Medline] [Order article via Infotrieve]
  23. Zakany, J., Tuggle, C., Patel, M. D., and Nguyen-Huu, M. C. (1988) Neuron 1, 679-691 [Medline] [Order article via Infotrieve]
  24. Kress, C., Vogels, R., DeGraaff, W., Bonnerot, C., Meijlink, F., Nicolas, J.-F., and Deschamps, J. (1990) Development 109, 775-786 [Abstract]
  25. Hogan, B. L. M., Costantini, F., and Lacy, E. (1986) Manipulating the Mouse Embryo: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  26. Kumar, V., and Chambon, P. (1988) Cell 55, 145-156 [Medline] [Order article via Infotrieve]
  27. Standke, G. J. R., Meier, V., and Groner, B. (1994) Mol. Endocrinol. 8, 469-477 [Abstract]
  28. Ishii, S., Merlino, G. T., and Pastan, I. (1985) Science 230, 1378-1381 [Medline] [Order article via Infotrieve]
  29. Wagner, M. J., Sharp, J. A., and Summers, W. C. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 1441-1445 [Abstract]
  30. Su, Y., Rosenthal, D., Samulson, M., and Spiegel, S. (1994) J. Biol. Chem. 269, 16512-16517 [Abstract/Free Full Text]

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