©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Mouse Bone Morphogenetic Protein-4 Gene
ANALYSIS OF PROMOTER UTILIZATION IN FETAL RAT CALVARIAL OSTEOBLASTS AND REGULATION BY COUP-TFI ORPHAN RECEPTOR (*)

(Received for publication, March 13, 1995; and in revised form, July 7, 1995)

Jian Q. Feng Di Chen Austin J. Cooney (1) Ming-Jer Tsai (1) Marie A. Harris Sophia Y. Tsai (1) Mei Feng Gregory R. Mundy Stephen E. Harris (§)

From the Department of Medicine/Endocrinology and Metabolism, University of Texas Health Science Center, San Antonio, Texas 78284 Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Bone morphogenetic protein-4 (BMP-4) is one of a member of related polypeptides that are important in bone formation and other developmental processes. We isolated the BMP-4 gene from a mouse genomic library and characterized the exon-intron structure and one of the candidate promoters. Two alternative 5`-noncoding exons, 1A and 1B, were identified by reverse transcription polymerase chain reaction assays. Quantitative competitive polymerase chain reaction using Exon 1A, Exon 1B, and Exon 3 primers indicate the 1A-containing transcript is the primary BMP-4 mRNA expressed in bone cell cultures. Primer extension analysis supports that 1A is the major promoter utilized in bone cell cultures as well as in 9.5-day mouse embryos. 1A promoter activity indicate selective DNA regions functional in bone cells. We found potential regulatory response regions in the 1A 5`-flanking region of the BMP-4 gene for the chicken ovalbumin upstream-transcription factor I (COUP-TFI). Specific binding to the COUP-TFI response regions in the BMP-4 1A promoter was demonstrated. By co-transfection of a COUP-TFI expression plasmid with the BMP-4 1A promoter in fetal rat calvarial osteoblasts, we demonstrated that COUP-TFI inhibits the BMP-4 promoter activity. This suggests that COUP-TFI could act as a silencer for BMP-4 transcription in vivo.


INTRODUCTION

The bone morphogenetic protein (BMP)^1 family comprises a group of closely related polypeptides in the transforming growth factor beta superfamily, which were identified initially by their capacity to stimulate ectopic bone formation in vivo (Wozney et al., 1988; Celeste et al., 1990). Recently, we have found that mRNAs for BMP-2 and BMP-4 are expressed in FRC osteoblasts as they differentiate in primary culture prior to forming mineralized bone nodules (Harris et al., 1994a). Expression of BMP-2 and BMP-4 mRNA coincides with expression of several osteoblast differentiation markers by these cells, including osteocalcin, osteopontin, and alkaline phosphatase (Harris et al., 1994a). BMP-4 is synthesized as a large precursor, as are other transforming growth factor beta members, and is processed to a mature dimer form, which is then secreted into the extracellular matrix (Suzuki et al., 1993). Recombinant BMP-4 stimulates new cartilage and bone formation when implanted near long bones (Shimizu et al., 1994). Expression patterns of BMP-4 mRNA in the apical ectodermal ridge and limb suggest important roles during limb development (Wozney, 1992). BMP-4 has also been shown to be an important factor in dorsal region specification in mouse embryos and high levels of BMP-4 expression are seen at the gastrula stage of Xenopus embryos (Nishimatsu et al., 1993; Jones et al., 1992).

The physiological factors regulating BMP-4 gene expression and function are still largely unknown. Recently, we and others have found that there are at least two different BMP-4 noncoding first exons (Exon 1A and 1B) (Chen et al., 1993; Kurihara et al., 1993). This suggests the BMP-4 gene may have at least two promoters. Analysis of the BMP-4 gene structure and functional studies in bone and other cells should provide a foundation for understanding the environmental and cellular factors which regulate BMP-4 gene transcription and alternate promoter usage.

In this study, we have isolated the mouse BMP-4 gene and determined its primary DNA sequence. We have demonstrated, with the limits of the constructs tested, that the promoter 1A is active in bone cells by transfection studies. With Exon 1A and 1B probes and poly(A) RNA from primary FRC cell cultures, Northern analysis supports this conclusion. These data were confirmed by quantitative competitive RT-PCR using Exon 1A or 1B primers and an Exon 3 primer with a common competitor containing Exon 1A, Exon 1B, and Exon 3 priming sites.

We have also demonstrated that COUP-TFI, a member of the steroid/thyroid hormone receptor superfamily, negatively modulates BMP-4 gene transcription in transient transfection assays using primary fetal rat calvariae cells. We have shown that two elements in the 1A promoter bind to the COUP-TFI protein and that COUP-TFI mRNA is expressed in these cultures during bone cell differentiation.


MATERIALS AND METHODS

Library Screening and Southern Blotting and Hybridization

A mouse genomic Fix II spleen library (Stratagene, La Jolla, CA) was screened with a mouse embryo BMP-4 cDNA probe (1.6 kb) kindly provided by Dr. B. Hogan (Vanderbilt University, Nashville, TN). The probe was labeled with [alpha-P]dCTP using a random primer labeling kit from Boehringer Mannheim. Plaque lift filters were hybridized overnight in hydridization buffer containing 6 times SSC (1 times SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 5 times Denhardt's, 0.5% sodium dodecyl sulfate (SDS), 200 µg/ml sonicated salmon sperm, 10 µg/ml poly(A), and 10 µg/ml tRNA at 68 °C. The filters were washed at 55 °C for 20 min, twice in 2 times SSC, 0.1% SDS buffer, once in 0.5 times SSC, 0.1% SDS buffer. The isolated phage DNA clones were mapped according to standard procedures (Sambrook et al., 1989).

DNA Sequence of BMP-4 Genomic Clone

Fragments from positive clones were subcloned into pBluescript vectors (Stratagene, La Jolla, CA) and sequenced in both directions using Sequenase and a sequencing kit (U.S. Biochemical Corp.).

RT-PCR

First-strand cDNA was synthesized from 1 µg of FRC cell poly(A) RNA with an 18 mer dT primer using Superscript(TM) reverse transcriptase (Life Technologies, Inc.) in a total volume of 20 µl. The cDNA was then used as a template for PCR with two sets of synthesized primers as discussed below. The primer 1 (5`-GAAGGCAAGAGCGCGAGG-3`), corresponding to a region of Exon 1A and primer 3 (5`-CCCGGTCTCAGGTATCA-3`) corresponding to a 5` region of Exon 3 were used to generate Exon 1A-2-3 spliced PCR product. Primer 2 (5`-CAGGCCGAAAGCTGTTC-3`), corresponding to a region of Exon 1B, and the same primer 3 were used to generate Exon 1B-2-3 spliced PCR products. The positions of primer 1, 2, and 3 are indicated on Fig. 1B. GeneAmp PCR kit was used according to the manufacturer's procedure (Perkin-Elmer). Each cycle consisted of a denaturation step (94 °C for 1 min), an annealing step (56 °C for 2 min), and an elongation step (72 °C for 1 min). The PCR products were analyzed by agarose gel electrophoresis for size determination. The products were subcloned into pCR(TM) II vector using TA cloning kit (InVitrogen, San Diego, CA). The inserts were sequenced in both directions with a sequencing kit from U. S. Biochemical Corp.


Figure 1: A, partial restriction enzyme map of mouse genomic BMP-4 and diagram of two transcripts. The mouse BMP-4 gene transcription unit is 7 kb and contains 2 coding exons (filled boxes) and 3 noncoding exons labeled Exon 1A, Exon 1B, and Exon 2. The original 19-kb clone has 6-kb 5`-flanking region and an 7-kb 3`-flanking region. The diagram shows approximately 2.4 kb of 5`-flanking region, and a small region (200 bp) of 3`-flanking area. The lower panel shows two alternative transcripts of BMP-4 gene. Both have the same Exons 2, 3, and 4 but a different Exon 1. Transcript A has Exon 1A, and transcript B has Exon 1B. B, DNA sequence of mouse genomic BMP-4 DNA and predicted amino acid sequences within the coding exons. The sequence of the exons is presented. The numbers on the right show the position of nucleotide sequence, and the bold numbers indicate the location of amino acid sequence of the coding region. The end of the transcription unit was estimated based on a 1.8-kb mRNA transcript size. Primer 1 in Exon 1A, primer 2 in Exon 1B, and primer 3 in Exon 3 were used in RT-PCR analysis. Primer B1 and B2 were used in primer extension reactions. C, BMP-4 1A 5`-flanking region and potential response elements in mouse BMP-4 1A promoter. The sequence of 2678-bp mouse BMP-4 gene are shown. Nucleotides are numbered on the left with +1 corresponding to the major transcription start site of the 1A promoter. The response elements of DR-1A Proximal and DR-1A Distal sequences are indicated. The other potential response DNA elements in the boxes are p53, retinoblastoma (RB), SP-1, AP-1, and AP-2. The primer A, indicated as the dotted line above the DNA sequence at +114/+96, was used for primer extension analysis for Exon 1A-containing transcripts.



Generation of BMP-4 Competitor and Quantitative Competitive RT-PCR for 1A-2-3 and 1B-2-3 Transcripts (Siebert and Larrick 1993)

To generate a BMP-4 competitor for both A and B transcripts (Fig. 1A), a deer BMP-4B cDNA which was isolated in our laboratory (Feng et al., 1995) was used as the template for the PCR reaction. This deer BMP-4B cDNA has an extended 1B exon due to an alternate 5` splice site, spliced to Exon 2. The upper priming site of the competitor contains the complement sequences of primer 1 and primer 2, corresponding to 18 nucleotides of Exon 1A and 18 nucleotides of of Exon 1B. The lower priming site corresponds to the 5` region of Exon 3 (primer 3, Fig. 1B). The final PCR products of the BMP-4 competitor are 952 bp for Exon 1A and 935 bp for Exon 1B. Thus, the BMP-4 competitor contains both priming sites for Exon 1A and Exon 1B next to each other on the 5` end and a 3` priming site for Exon 3 on the 3` end (Fig. 4A). The concentration of BMP-4 competitor is determined by spectrophotometry after purification with Magic PCR purification system (Promega, Madison, WI).


Figure 4: Quantitation of BMP-4 A and B transcript levels in FRC cells. A, generation of a BMP-4 competitor to quantitate relative BMP-4 A and B transcript levels in FRC bone cells. The BMP-4 competitor is used to compete for PCR of Exon 1A-2-3-containing transcript when primer 1 (Exon 1A primer) is utilized with primer 3 (Exon 3 primer). The same BMP-4 competitor is used to compete for PCR of Exon 1B-2-3-containing transcripts when primer 2 (Exon 1B primer) is utilized with primer 3 (Exon 3 primer). B, quantitative competitive RT-PCR results for BMP-4 transcript A. C, quantitative competitive RT-PCR results of BMP-4 transcripts B and B`. The upper panel of B and C shows electrophoretic pattern of the BMP-4A and 4B products. The primer 1 (Exon 1A), primer 2 (Exon 1B), and primer 3 (Exon 3) were utilized. The ethidium bromide-stained PCR products were separated on a 2% agarose gel. The 952-bp BMP-4 competitor template has both Exon 1A and 1B priming sites on the 5` end and Exon 3 priming site on the 3` end. The first lane shows the PCR products without BMP-4 competitor in the PCR reaction. The remaining lanes show the PCR products with increasing amounts of BMP-4 competitor (0.025-250 ng). [alpha-P]dCTP was added to the PCR reaction for quantitation of the PCR products. The relative amounts of BMP-4A and 4B products to the competitor products were calculated after size correction. The logarithm of the ratios of BMP-4A products (455 bp) and the sum of the two different size BMP-4B products (495 and 245 bp) to the BMP-4 competitor products (952 and 935 bp) was graphed as a function of the logarithm of the amounts of the BMP-4 competitor added to the PCR reaction. These calculations are shown in the lower panel of B and C. The calculated amounts of the A and B transcripts are marked by the arrows, where the ratios of BMP-4A or 4B transcripts to corresponding competitor products are equal 1 (log 1 = 0).



The PCR reaction conditions used for the quantitative PCR are as described above. 1 µCi of [alpha-P]dCTP was added into each PCR reaction. Reverse-transcribed FRC cell cDNA, 2 mM MgCl(2), 10-fold serial dilutions of the BMP-4 competitor (0.025-250 pg), 0.25 mM dNTP, Taq polymerase, primer 1 or primer 2, and primer 3 (thus, the same lower primer for transcripts A and B) were placed into a master mixture. Primer 1 was used for BMP-4 A transcript quantification, and primer 2 was used for BMP-4 B transcript quantification. After amplification, a 10-µl sample from each 50-µl reaction was resolved by agarose gel electrophoresis. Gels were stained with ethidium bromide (50 µg/ml), photographed, dried, and scanned with the AMBIS Image Acquisition and Analysis System (AMBIS, San Diego, CA). The logarithm of the ratio of rat BMP-4 products (containing Exon 1A or Exon 1B) to BMP-4 competitor PCR products was graphed as a function of the logarithm of the amount of BMP-4 competitor added after correction for the size difference between the rat BMP-4 PCR products and BMP-4 competitor PCR products.

BMP-4 Promoter 1A Plasmid Construction and Transfection

Three BMP-4 1A promoter/plasmids were constructed by excising fragments from the 5`-flanking region of mouse BMP-4 gene and were then cloned into the pBL3CAT expression vector (Luckow and Schutz, 1987). The pCAT-2.6 plasmid consisted of pBL3CAT vector with a 2.6-kb EcoRI and XbaI fragment (-2372/+258) of the BMP-4 gene. The pCAT-1.3 plasmid was similarly generated from a 1.3-kb PstI fragment (-1144/+212). The pCAT-0.5 plasmid was made from a 0.5-kb SphI and PstI fragment (-260/+212). The pCAT-2.6, the pCAT-1.3 and the pCAT-0.5 plasmids all contain part of Exon 1A noncoding region. An additional promoter/plasmid was created from a PCR amplified product, corresponding to the 0.24-kb sequence between nucleotides -25 and +212, and referred to as the pCAT-0.24. The amplified fragment was first cloned into pCRII(TM) vector using TA cloning kit (InVitrogen, San Diego, CA) and then released with HindIII and XhoI, and recloned into pBL3CAT vector. Correct orientation of all inserts with respect to the pBL3CAT vector was verified by DNA sequencing.

The isolated FRC cells, enriched for the osteoblast phenotype, were used as recipient cells for transient transfection assays. The technique of electroporation was used for DNA transfection (Potter, 1988; van den Hoff et al., 1992). After electroporation, the cells were divided into aliquots, replated in 100-mm diameter culture dishes and cultured for 48 h in alpha-minimal essential medium (Life Technologies, Inc.) with 10% fetal calf serum. The cell lysates were assayed for CAT activity according to the method described by Gorman (1988), and CAT activity was normalized by beta-galactosidase assay from a co-transfected Rous sarcoma virus-beta-galactosidase at one-tenth the concentration of the CAT plasmids, according to the method of Rouet et al.(1992).

Gel Retardation Assay/Supershift Assay

Preparation of antibodies against COUP-TFI and gel retardation and supershift assays were carried out as described previously (Cooney et al., 1992). COUP-TFI protein was prepared by transcription from a SP6 promoter-COUP-TFI plasmid and the RNA was then translated in a rabbit reticulocyte lysate system (Promega, Madison, WI). The relative amounts of the translated COUP-TFI protein were determined by [S]methionine-labeled protein analysis on SDS-polyacrylamide gels (Cooney et al., 1992).

Double-stranded oligodeoxynucleotides corresponding to consensus COUP RE (AAGTCA A AAGTCA) (Cooney et al., 1992) was prepared and used in competition assays. BMP-4 DR-1A Proximal (-865 to -878) (GGGCCA A AGGGCA) or BMP-4 DR-1A Distal (-2095 to -2108) (GGGCCA A AGGTCA) binding sites were labeled with [alpha-P]dATP to specific activities of 2 to 8 times 10^8 cpm/µg. 0.1-0.2 ng of labeled DNA probe was incubated with 1-4 µl of in vitro translated COUP-TFI for 15 min at room temperature under the following conditions: 60 mM KCl, 10% glycerol, 3 mM MgCl(2), 1 mM dithiothreitol, 20 mM HEPES (pH 7.9), 0.2-2 µg/ml poly(dI-dC) (Pharmacia Biotech Inc.). The reactions were resolved by electrophoresis in a 5% (w/v) polyacrylamide gel in 0.5 times Tris borate-EDTA running buffer. The gels were dried and then autoradiographed at -70 °C with a DuPont Cronex Lightning Plus intensifying screens with Kodak X-Omat x-ray film. For antibody supershift analysis, the COUP-TFI antiserum was preincubated with the in vitro translation products at room temperature for 5 min prior to the addition of the labeled DNA probes.

Primer Extension Mapping of the Transcriptional Start Sites

The transcriptional start sites were mapped by primer extension using the synthetic oligonucleotide, 5`-CGGATGCCGAACTCACCTA-3`, corresponding to nucleotides +114 to +96 (Primer A, Fig. 1C) in the Exon 1A sequence and an oligonucleotide 5`-CTACAAACCCGAGAACAG-3`, corresponding to nucleotides +29 to +12 of Exon 1B sequences (Primer B1, Fig. 1B). The numbering for Exon 1B is based on the longest 5`-noncoding cDNA of BMP 4 with a 1B exon (Chen et al., 1993). The transcription start site(s) for 1B promoter have not been determined. Total RNA from FRC cells and 9.5-day mouse embryos (gift of B. Hogan, Vanderbilt University, Nashville, TN) was used with both primers. The primer extension assay was carried out using the primer extension kit from Promega (Madison, WI). The annealing reactions were carried out at 60 °C in water bath for 1 h. The products were then electrophoresed on 8% denaturing urea polyacrylamide gels and autoradiographed. One other Exon 1B sequence, +50 to +67 (Primer B2, Fig. 1B), was also tested in primer extension reactions with FRC cell and mouse embryo RNAs. The primer sequences chosen are identical in both rat and mouse gene.^2

Northern Blot Analysis mRNA

The total FRC osteoblast RNA was extracted by the RNAzol B method using the protocol supplied by Cinna Biotecx (Houston, TX). Poly(A) RNA was isolated using small oligo(dT) cellulose PUSH columns (Stratagene, San Diego, CA). 0.1% SDS was added in all buffers. The Northern blot analysis was as described previously (Harris et al., 1994b). Hamster COUP-TFI cDNA probe is a 1.5-kb fragment. The mouse BMP 4 cDNA probe is a 1.6-kb fragment. The filter was later reprobed with a 0.6-kb chicken glyceraldehyde-3-phosphate dehydrogenase cDNA probe. BMP-4 and COUP-TFI mRNA levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase mRNA level. Confluent FRC osteoblast cultures, before medium change, was defined as Day 0 and the relative level of mRNA detected with a given probe to glyceraldehyde-3-phosphate dehydrogenase mRNA was set at 1.0 for Day 0 (Harris et al., 1994b).


RESULTS

Cloning and Sequencing of Mouse BMP-4 Gene

Three clones were isolated from 2 times 10^6 plaques of mouse spleen 129 genomic library using full-length coding region mouse embryo BMP-4 cDNA probe (B. Hogan, Vanderbilt University, Nashville, TN). One 19-kb clone contained 5 exons and an 6-kb 5`-flanking region and an 7-kb 3`-flanking region was utilized for characterization of the BMP-4 gene. A diagram of part of that clone with exons indicated is shown in Fig. 1A. The 7-kb transcription unit and part of the 5`-flanking region of the mouse BMP-4 gene were sequenced. The nucleotide sequence of mouse BMP-4 and deduced amino acid sequence of coding exons (408 residues) are shown in Fig. 1B. Primers used in the quantitative competitive RT-PCR experiments described below are indicated in this figure. Fig. 1C shows the DNA sequence of 2372 bp of the 5`-flanking region and candidate DNA response elements upstream of the Exon 1A. Primers used in primer extension experiments are also shown in Fig. 1(B and C).

Mapping the Transcriptional Start Site

Evidence for Utilization of Two Alternate Exon 1 Sequences for the BMP-4 Gene

We have sequenced a variety of BMP-4 cDNAs from several sources including human prostate cancer cell line PC-3 and primary FRC cells. Four independent FRC cell BMP-4 cDNAs all contained Exon 1A sequences spliced to Exon 2. However, the human prostate PC-3 BMP-4 cDNA contained an apparently unique Exon 1, which we refer to as Exon 1B, spliced to Exon 2 (Chen et al., 1993). A double-stranded oligonucleotide probe (70 bp) to Exon 1B was synthesized based on the human PC-3 Exon 1B sequence. This Exon 1B probe was then used to identify the Exon 1B region in the mouse genomic BMP-4 clone (data not shown). The candidate Exon 1B is 1696 bp downstream from the 3` end of Exon 1A.

Primer Extension Analysis

Primer extension analysis was performed to map the mouse BMP-4 gene transcription start sites. We used an oligonucleotide from Exon 1A, primer A (Fig. 1C), and two oligonucleotides from Exon 1B, primer B1 and B2 (Fig. 1B). Total RNA was utilized from 9.5-day mouse embryo and FRC cells grown in vitro. As shown in Fig. 2, a major extended fragment from the primer A (Fig. 1C and Fig. 2) was obtained in both mouse embryo and FRC cell total RNAs, which migrates at 115 bp. The extended 5` end of the 115-bp fragment represents the major transcription start site for 1A-containing transcripts. The size of this 5`-noncoding Exon 1A is 306 bp. The 67-bp product may represent an alternate transcription start site or incomplete extension. No detectable extended fragment using primer B1 or B2 (Fig. 1B) (Exon 1B) was detected using either mouse embryo or FRC cell total RNAs. This suggests that 1B-containing transcripts are less abundant than 1A-containing transcripts in these mouse embryos and FRC bone cells.


Figure 2: Primer extension assay. The total RNAs prepared from FRC cells (on the left) and 9.5-day mouse embryo (on the right) were used with primer A (Fig. 1C) or primer B1 (Fig. 1B). Two major extended fragments, 67 and 115 bp in lanes marked A are obtained from primer A. No major fragments are detected using the B1 primer. Primer B2 (Fig. 1B) also gave negative results with both FRC and mouse embryo total RNA as templates (data not shown).



Primary FRC Osteoblasts Produce BMP-4 1A and 1B Exon-containing Transcripts

As stated, four FRC BMP-4 cDNAs were sequenced and found to contain Exon 1A sequences spliced to Exon 2. The human U2-0S BMP-4 cDNA sequence also contains Exon 1A (Wozney et al., 1988). This suggests that BMP-4 gene sequences upstream of Exon 1A may contain regulatory sequences utilized in bone cells and 9.5-day mouse embryos.

To test whether the BMP-4 1B exon is expressed in FRC cells, we designed oligonucleotide primers to ascertain whether spliced 1B-2-3 exon products and 1A-2-3 exon products (control) could be obtained by RT-PCR from FRC poly(A) RNA. The 3` primer was in Exon 3 (primer 3) and the 5` primers were either in Exon 1A (primer 1) or 1B (primer 2) (see Fig. 1B).

The RT-PCR products were cloned and sequenced. A photograph and diagram of the products obtained are presented in Fig. 3. Both 1A-2-3 and 1B-2-3 products were obtained. The results indicate that FRC osteoblasts produce transcripts with either a 1A exon or a 1B exon, but not both. This suggests that the intron region between 1A and 1B exon may contain regulatory response elements under certain cellular contexts, resulting in BMP-4 B transcripts. Of the 1B-2-3 RT-PCR products obtained from FRC osteoblasts, we obtained two products with different 3` splice sites for Exon 1B. By comparison with the genomic DNA, both 3` ends of the two Exon 1Bs have reasonable 5` splice consensus sequences, consistent with an alternate splicing pattern obtained for the 1B-2-3 RT-PCR products. Most importantly, no 1A-1B-2-3 RT-PCR-spliced products of the BMP-4 gene were obtained. Thus, 1B does not appear to be an alternatively spliced 5`-noncoding exon and suggests the BMP-4 gene may have at least two promoters.


Figure 3: Gel electrophoresis of Exon 1A-2-3 (lane 2) and Exon 1B-2-3 RT-PCR products (lane 1). RT-PCR was performed with two pairs of primers using reverse transcribed FRC cDNA as the template. The products were verified by the DNA sequence analysis. Schematic diagram of spliced BMP-4 RT-PCR products with 1A and 1B exons are shown for FRC cells (right). The primers and their locations are noted in Fig. 1B. RT-PCR product 1A-2-3 which contains Exon 1A, Exon 2, and the 5` region of Exon 3, is produced with primer 1 and primer 3. Primer 2 (Exon 1B) and primer 3 (Exon 3) generate two RT-PCR products with Exon 1B`-2-3 pattern (245 bp) and the 1B-2-3 pattern (495 bp).



Quantitative Competitive RT-PCR Indicates 1A Transcripts Are on Average 10-fold More Abundant in FRC Cells

Northern analysis demonstrated that a single 1.8-kb BMP-4 transcript can be detected in FRC cells during bone cell differentiation and the 1.8-kb band hybridized to both a 1A exon probe or a 2-4 exons probe. No 1.8-kb BMP-4 transcript was detected with 1B exon probe (data not shown). We then designed a 952 bp competitor with both a priming site for 1A and 1B next to each other on the 5` end and a 3` priming site for Exon 3 on the 3` end. A diagram of construction of the BMP 4 competitor is shown in Fig. 4A. Our single competitor template could then be used for quantitating both 1A and 1B-containing transcripts. Fig. 4B shows the results using increasing concentrations of BMP-4 competitor with the Exon 1A primer and Exon 3 primer pair and a constant amount of reverse transcribed FRC cDNA. We estimate that in the FRC cDNA (10 ng), there is 2.6 pg of BMP-4 1A-2-3 transcripts. Fig. 4C shows the results using increasing concentrations of the BMP-4 competitor with the Exon 1B primer and Exon 3 primer pair and the same constant amount of FRC reverse transcribed cDNA used above. We estimate that in the FRC cDNA (10 ng), there is a total of 0.2 pg of BMP-4 1B-2-3 transcripts (combined 1B and 1B` of Fig. 4C). Therefore, there are over 10 times more 1A-2-3 transcripts than total 1B-2-3 transcripts in FRC cells (summation of B plus B` signals in Fig. 4C). Detailed analysis during differentiation has not been carried out.

Promoter Activity of Mouse BMP-4 Gene in FRC Cells

Primary FRC cells were selected for analysis of mouse BMP-4 promoter activity. BMP-4 mRNA is modulated in a transient fashion during prolonged culture in these cells (Harris et al., 1994b).

After 48 h of transfection with various BMP-4-CAT reporter gene constructs, the cells were harvested and the CAT activity was determined. As indicated in Fig. 5(A and B), pCAT-0.24 plasmid (-25/+212) has measurable CAT activity. The CAT activity of this pCAT-0.24 plasmid was 3-fold lower than that the background parent pBL3CAT plasmid. This suggests the -25 to +212 has transcriptional or translational inhibitory activity. The pCAT-0.5 (-260/+212), pCAT-1.3 (-1144/+212), and pCAT-2.6 (-2372/+258) showed progressive increasing promoter activity when transfected into primary FRC cells. These data are shown in Fig. 5B. With pCAT-0.5 (-260/+212) there is a 10-fold increase in CAT activity relative to pCAT-0.24 (-25/+212). pCAT-1.3 (-1144/+212) shows an additional 6-fold increase in CAT activity over pCAT-0.5, and pCAT-2.6 (-2372/+258) shows an additional 2-fold increase in CAT activity over pCAT-1.3 (-1144/+212). Thus the net increase in CAT activity between the pCAT-0.24 (-25/+212) and the pCAT-2.6 (-2372/+258) in FRC cells is approximately 100-fold.


Figure 5: Map of the BMP-4 1A 5`-flanking-CAT plasmid and promoter activity in FRC cells. A, the 2.6-kb EcoRI and XbaI fragment, 1.3-kb PstI fragment, 0.5-kb SphI and PstI fragment, and 0.24-kb PCR fragment were inserted into pBLCAT3 as described under ``Materials and Methods.'' The closed box indicates the noncoding Exon 1A. The CAT box represents the CAT reporter gene. The values are percentages of CAT activity relative to that of pCAT-2.6 (100%) and represent the average of four independent assays. Similar results were obtained with independent FRC cultures. B, autoradiogram of the CAT assays using the FRC cells transfected with the BMP-4 1A 5`-flanking CAT plasmids in A.



Potential Response Elements in the BMP-4 Promoter 1A

We initially searched the 5`-flanking regions of the Exon 1A and found potential response elements for SP1, p53, Fos-Jun (AP-1), AP-2, SP-1, COUP-TF (DR-1A), and Zif 268. The 1A 5`-flanking region contains several direct repeats (DR-1 and DR-5, direct repeats with 1 or 5 base pair spacing between repeats of 1/2 sites of A/GA/GT/GCA) that potentially might respond to retinoic acid RXR-receptor family members. The DR-1 repeats may also be modulated by the transcriptional factor, COUP-TFI.

Since COUP-TFI has been shown to be an important modulator of transcription of a variety of genes (Ladias and Karathanasis, 1991; Lehmann et al., 1991; Tran et al., 1992; Kliewer et al., 1992a, 1992b), and BMP-4 mRNA is transiently expressed during bone cell differentiation, we examined these potential DR-1 COUP-TFI response region in the 1A promoter.

COUP-TFI Binds the BMP-4 1A Promoter at -2095 bp (DR-1A Distal) and at -865 bp (DR-1A Proximal)

COUP-TFI has the ability to homodimerize with itself and heterodimerize with RXR and negatively modulate transcription from direct repeats with the canonical 1/2 site sequence AGGTCA spaced by 1 nucleotide (Cooney et al., 1992; Kliewer et al., 1992a, 1992b; Tran et al., 1992). Two candidate BMP-4 DR-1 (DR-1A) repeats were tested for specific COUP-TFI binding by gel shift assays(Cooney et al., 1992). The BMP-4 DR-1A Distal sequence at -2095 bp has the sequence GGGCCA A AGGTCA. The DR-1A Proximal sequence at -865 bp has the sequence GGGCCA A AGGGCA.

A consensus COUP-TFI binding oligonucleotide (AGGTCA A AGGTCA), referred to as COUP RE, was used as positive control. The result in Fig. 6A shows that in vitro transcribed/translated COUP-TFI binds both the proximal and distal BMP-4 DR-1A elements (lanes 6 and 11). Non-radioactive COUP-TFI RE oligonucleotide competes the binding of labeled BMP-4 DR-1A oligonucleotides to the COUP-TFI produced in vitro (lanes 7 and 12). In a supershift assay, COUP-TFI antibodies further retarded the COUP-TFI/alpha-P-labeled BMP-4 distal and proximal BMP-4 DR-1A elements (lanes 9 and 14). The COUP-TFI thus forms a high affinity stable complex with the distal and proximal DR-1A BMP-4 elements. An oligonucleotide containing a progesterone response element (PRE) at 25-fold excess over the labeled BMP-4 DR-1A elements could not compete the binding to the COUP-TFI protein (Fig. 6A, lanes 8 and 13). As shown in Fig. 6B, the proximal DR-1A-COUP-TF interaction required more non-radioactive consensus COUP-TF oligonucleotide to compete the COUP-TFI binding. This suggests the proximal BMP-4 DR-1A binds to COUP-TFI with higher affinity than the distal BMP-4 DR-1A element (Fig. 6B).


Figure 6: COUP-TFI specifically binds to a Distal and Proximal DR-1A elements in the BMP-4 1A promoter. A, COUP-TFI obtained from in vitro translation was incubated with the labeled ``wild type'' COUP RE or the BMP-4 DR-1A Proximal or Distal REs. COUP is the non-radioactive wild type COUP RE. PRE is the progesterone response element oligonucleotide (Cooney et al., 1992). COUP-TFI Ab is a polyclonal antibody to COUP-TFI. All reaction products were electrophoresed on nondenaturing 6% polyacrylamide gels. B, the labeled double-stranded oligonucleotide probes of the three COUP RE (wild type, DR-1A proximal, and DR-1A distal) were incubated in the presence of increasing concentrations of cold nonlabeled double-stranded oligonucleotides of wild type COUP RE, AGGTCAAAGGTCA. The BMP-4 DR-1A Proximal RE (middle, GGGCCAAAGGCA) binds COUP-TFI specifically. The wild type COUP RE competes with this DR-1A proximal RE. The BMP-4 DR-1A Distal RE also specifically binds to COUP-TFI (right). The DR-1A Proximal element requires higher concentration of non-radioactive wild type COUP-TF RE to compete the BMP-4 DR-1A binding.



COUP-TFI Is a Negative Regulator of the BMP-4 1A Promoter

Co-transfection of various ratios of a cytomegalovirus enhancer (CMV)-driven human COUP-TFI expression vector with pCAT-2.6 (-2372/+258) BMP-4 1A promoter CAT constructs into primary FRC osteoblasts was performed. A ratio of pCMV-COUP-TFI to pCAT 2.6 (-2372/+258) plasmids of 0.1 is sufficient to suppress the BMP-4 1A promoter by 65%. On the other hand, pCMV-COUP-TFI at equivalent ratios had little effect on the pBL2CAT (tk-CAT) promoter in these primary FRC osteoblasts (Luckow and Schutz, 1987). The pBL2CAT contains no COUP-TF response elements, and thus indicates the effect of pCMV-COUP-TF on the BMP-4 promoter is not due to general squelching. The results are presented in Fig. 7.


Figure 7: BMP-4 1A promoter activity is inhibited by COUP-TFI. Left top panel shows that COUP-TFI has little effect on the pBL2CAT (ptk-CAT) activity. The tk promoter contains no COUP-TF response elements. The right top panel shows that equivalent CMV-driven COUP-TF expression plasmid inhibits BMP4 1A promoter activity. CAT activity is normalized to beta-galactosidase activity. 10 µg of pCAT 2.6 was used per transfection with 1-5 µg of pCMV-COUP-TFI. The normalized data are quantitated and shown in the bottom left (pBL2-CAT) and bottom right (pCAT 2.6).



COUP-TFI mRNA Expression during Differentiation of FRC Osteoblasts

Fig. 8shows that COUP-TFI mRNA and BMP-4 mRNA levels change during long term cultures of FRC osteoblasts in which bone nodules are formed. Both COUP-TFI and BMP4 mRNA expressions are transient in FRC osteoblasts as they differentiate in culture. COUP-TFI is most likely involved in regulating a variety of genes in bone cells as well as in many other tissues (Tsai et al., unpublished). We would not expect a direct correlation of mRNA levels of BMP-4 and COUP-TFI. The net 2-fold change in COUP-TFI mRNA levels, however, does suggest that in vitro differentiating bone cells can modulate COUP-TF mRNA. Whether this change is directly or indirectly related to BMP-4 expression is unknown.


Figure 8: COUP-TFI mRNA expression in FRC cells during in vitro bone cell differentiation and nodule formation. The fold change of COUP-TFI mRNA has been normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels as described under ``Materials and Methods'' and then calculated based on the level at day 0 set at 1.0. The 1.8-kb BMP-4A mRNA levels are shown for comparison. Poly(A) RNA (5 µg) was electrophoresed on denaturing gels, transferred, and hybridized with the appropriate probes as described (Harris et al., 1994a). bullet, BMP-4 mRNA; circle, COUP-TFI mRNA.




DISCUSSION

In this study, we have isolated the murine BMP-4 gene transcription unit and flanking regions and examined its DNA sequence in order to lay the foundation for understanding factors and signaling pathways that may regulate transcription of the BMP 4 gene in the context of primary cultures of rat bone cells and other tissues. Over 6 kb of 5`-flanking region have been isolated, and the exon and intron structure has been defined by DNA sequence analysis. The transcription unit comprises 7030 bp with three 5`-noncoding exons. These results are similar and extend those reported by Kurihara et al.(1993). The first noncoding exon, Exon 1A, defines a transcription start site utilized in bone cells and the 9.5-day mouse embryo. The candidate 1B promoter has not been extensively investigated and the transcription start site is not known. However, by quantitative RT-PCR we demonstrate that 1B transcript are present in bone cells but at a 10-fold lower concentration than 1A transcripts.

A 2360-bp 5`-flanking region of Exon 1A has been sequenced, and several potential regulatory elements have been identified by their sequence homology to known transcription factor binding sites. Four BMP-4 1A 5`-flanking CAT reporter gene plasmid constructs with different size deletions were transfected into primary FRC osteoblasts to establish regions of the BMP-4 1A promoter that are important for transcriptional regulation in bone cells. A region between -23 and -260 shows over a 10-fold stimulation of transcription. Between -23 and -1144 there is over a 60-fold stimulation of transcription.

Two direct repeat elements (DR-1 sites) similar to RXR-COUP-TFI binding sites were found to bind the transcription factor COUP-TFI, and by transfection studies in primary osteoblast cultures, we demonstrated that COUP-TFI is a negative regulator of the BMP-4 1A promoter in vitro. We have also demonstrated that COUP-TFI mRNA is expressed in primary cultures of FRC osteoblasts and undergoes marginal (2-fold) changes in level during differentiation, in parallel with BMP 4 expression.

Mammalian BMP 4 gene structure is very similar to the fruit fly homologous gene, decapentaplegic or dpp (St. Johnston et al., 1990). The dpp gene has two coding exons and at least three 5`-noncoding exons, which define three alternate promoters. In the dpp gene there are other promoters further upstream and regulatory DNA region in the 3`-flanking area (St. Johnston et al., 1990). The dpp mature coding regions share over 75% identity to BMP 4 mature coding region. The position of the splice site between the two coding exons is also conserved (St. Johnston et al., 1990). The dpp gene is critical for dorsal-ventral specification during embryogenesis and imaginal disc differentiation during pupation (Shimell et al., 1991). What is of interest for this study is that different promoters for the dpp gene are used at different developmental stages and in different tissues, presumable with unique sets or combinations of transcription factors for each promoter. This supports the hypothesis that the mammalian BMP 4 gene will utilize different promoters in different developmental context. We believe we have identified at least two BMP 4 promoters and have shown that both are utilized in heterogeneous primary bone cell cultures, which contain isolated areas of rapid bone formation (mineralizing nodules) between areas that do not differentiate (Harris et al., 1994a, 1994b). We are now in a position to identify differential utilization of the 1B and 1A exons using in situ RT-PCR during different stages of our in vitro bone cultures. This analysis is being extended, using mouse embryo sections at different developmental stages. Deer antler tissue and prostate tissue express much higher 1B transcripts and should allow us to develop more specific probes and map the transcription start sites for the 1B promoter (Chen et al., 1993; Feng et al., 1995).

In situ hybridization analysis of BMP 4 expression has shown a wide but selective expression pattern during embryogenesis (Jones et al., 1991). BMP 4 mRNA is detectable in Xenopus oocytes (Nishimatsu et al., 1992). The BMP 4 mRNA expression greatly increases between blastula and gastrulae and subsequently declines in Xenopus during later embryogenesis (Nishimatsu et al., 1992). BMP 4 mRNA expression domains in chick and mammalian embryos are highly focal and specific (compared to BMP 2) during limb bud/limb formation (Francis et al., 1994; Jones et al., 1991; Jones et al., 1992;Wozney et al., 1992). BMP 4 is expressed at high levels in select regions of the developing nervous system, in the developing branchial arches and later, during tooth development (Jones et al., 1991; Vainio et al., 1993). BMP 4 is selectively expressed in rhombomere 3 and 5 at stage 5 in the chick embryo (Graham et al., 1994). In two of the above studies, the biologically active BMP 4 protein was shown to 1) stimulate differentiation of dental mesenchyme and autoregulate BMP 4 mRNA expression, and 2) selectively control apoptosis of neural crest cells in rhombomere 3 and 5 of the developing hindbrain (Vainio et al., 1993; Graham et al., 1994). Thus we propose, that with the large variety of developmental contexts in which BMP 4 is expressed, the use of alternate BMP 4 promoters will allow selected tissue and developmental specific gene networks to operate within a given cell on the BMP 4 gene. The consequence of having BMP 4 transcripts in a cell with alternate 5`-noncoding exons could result in controlling where the BMP 4 mRNA is localized in the cell and selective rates of translation of BMP 4 1B mRNA versus BMP 4 1A mRNA transcripts in these different cellular contexts (for review, see Curtis et al.(1995)). Our analysis of 1B and 1A expression patterns in embryos will help in developing more extensive models.

Analysis of predicted secondary structures of Exon 1A and 1B indicates dramatic differences using the M FOLD program in GCG. The DeltaG levels for all structures were between -92 and -95 kcal/mol. These predicted secondary structures in the 5`-noncoding region could play important roles in BMP 4 cellular localization and translation by binding select sets of RNA binding proteins in different cells or tissues (Curtis et al., 1995).

COUP-TFI negatively modulate RXR-dependent pathways (Cooney et al., 1992; Kliewer et al., 1992a, 1992b). RXR heterodimerizes with thyroid, retinoid, and vitamin D receptors, and the early response gene, NUR77 or NGF 1B (Cooney et al., 1992; Perlmann and Jansson, 1995). Thus COUP-TFI levels to RXR levels in a given cell may allow the balancing of diverse hormonal and growth factor signals during embryogenesis and homeostasis (Lu et al., 1994).

The potential in vivo significance of COUP-TFI levels and expression on the BMP 4 gene can be addressed by first analyzing areas of overlapping expression of COUP-TFI and BMP 4 expression. Several areas in mouse embryos indicate a strong correlation between BMP 4 expression and COUP-TFI expression in both spatial and temporal aspects. First, in 10.5 post coitus embryos, there are high levels of both COUP-TFI and BMP-4 mRNAs in nasal pits and facial processes, and in the otic vesicles (Jones et al., 1991; Jonk et al., 1994; Lu et al., 1994). Several areas of the diencephalon and rhombomeres overlap in expression domains for both genes that may be relevant to the control of brain development and selected apoptosis of neural crest cells by BMP 4 in rhombomere 3 and 5 (Lu et al., 1994; Graham et al., 1994).

There are high levels of both COUP-TFI and BMP 4 mRNA in the mesenchyme surrounding whisker follicles (Jones et al., 1991; Jonk et al., 1994). Targeted overexpression of BMP 4 using a cytokeratin promoter to whisker follicles and other tissues results in some of the surviving mice with a large number of developmental defects, including craniofacial malformations, fusion of dentary and maxillary bones, growth retardation, cleft palate, absence of whiskers, and defects in hair regeneration (Blessing et al., 1993). This indicates that controlling the level and spatial and temporal aspects of BMP 4 expression are extremely important for proper differentiation in a variety of tissues. COUP-TFI could play a role in regulating the level of BMP 4 expression from the 1A promoter.

Finally, both BMP 4 and COUP-TFI expression patterns overlap in an interesting ways in the developing limb bud. In 10.5-day post coitus embryos, there is a distal-to-proximal gradient of BMP 4 expression. COUP-TFI expression shows a proximal-to-distal gradient (Jones et al., 1991; Lu et al., 1994). This type of reciprocal expression pattern would be ideal for controlling the level of BMP 4 expression. In the limbs of later 14.5-day post coitus embryos, there is an overlap in BMP 4 expression and COUP-TFI expression in the mesenchyme surrounding the chondrogenic region of the long bones as well as in the apoptotic region between the developing digits (Jones et al., 1991; Lu et al., 1994). Thus there are now two direct associations between BMP 4 expression and COUP-TFI expression in cell death associated with limb/digit development and in neural crest apoptosis in rhombomeres 3 and 5. Further analysis of BMP 4 expression pattern in which the COUP-TFI gene has been inactivated may help unravel the relationship between COUP-TFI and the BMP 4 gene.

In summary, we have characterized the BMP-4 gene and one of the promoters in the context of primary cultures of fetal rat osteoblasts. Bone cells utilize primarily the upstream 1A promoter. Mouse embryos (9.5 days) also appear to utilize predominantly the 1A promoter. Analysis of the 1A promoter indicates the transcription factor COUP-TFI could modulate BMP-4 gene transcription in vivo.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AR-39529 and CA-40035 and Grant 2947RI from the Council on Tobacco Research (to S. E. H.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L47480[GenBank].

§
To whom correspondence should be addressed: University of Texas Health Science Center at San Antonio, Dept. of Medicine/Endocrinology, 7703 Floyd Curl Dr., San Antonio, TX 78284-7877. Tel.: 210-567-4860; Fax: 210-567-6693.

(^1)
The abbreviations used are: BMP, bone morphogenetic protein; FRC, fetal rat calvarial; TFI, transcription factor I; COUP-TFI, chicken ovalbumin upstream-transcription factor I; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction; RT, reverse transcription; CAT, chloramphenicol acetyltransferase; RXR, retinoid X receptor; RE, response element.

(^2)
S. E. Harris and J. Q. Feng, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to Thelma Barrios and Nancy Garrett for preparation of this manuscript.


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