Exon 1C Research Articles

Developmental Testis-Specific Regulation of mRNA Levels and mRNA Translational Efficiencies for TATA-Binding Protein mRNA Isoforms

Early spermatids contain roughly 1000-fold more TATA-binding protein (TBP) mRNA than do somatic cells. The appearance of TBP-overexpressing spermatids in the developing testis is accompanied by a large increase in whole-organ levels of total RNA and of poly(A)+RNA per cell. Whereas somatic cells initiate transcription of TBP mRNA at a single promoter/first exon (exon 1C), in adult testis, two additional major promoter/first exons (1D and 1E) are used. We have examined the expression of the somatic and testis-specific TBP mRNA isoforms during rodent testis development. In juvenile testes TBP mRNAs containing either exon 1C or exon 1D, but none containing exon 1E, are detected. At 21 days of age, all TBP mRNA isoforms begin to overaccumulate. The onset of TBP mRNA overaccumulation is marked first by an increase in levels of polysomal TBP mRNA, and later by accumulation of mRNP-associated TBP mRNA. In adult testes, only 30% of the total TBP mRNA is engaged by polysomes; the remainder is sequestered as mRNP particles. All of the TBP mRNA isoforms in adults exist both as free mRNP particles and as polysomes; however, the fraction in polysomes varies from 60% (exon 1C) to 10% (exon 1E). This suggests that sequences within the first exons alter the probability that the mRNA will either assemble into polysomes or into translationally inactive mRNP particles.

Aberrant Expression of Aromatase in Breast Cancer Tissues

The expression of aromatase is tissue-specifically regulated through the alternative use of multiple exons 1 and promoters. We analysed expression levels of aromatase mRNA, preferential utilization of multiple exon 1 of the human aromatase gene, and transcriptional regulation of their multiple promoters in breast cancer tissues by newly developed fluorometric methods. The expression levels of aromatase mRNA in breast cancer tissues were significantly higher than those in regions distal to tumours or in non-malignant breast tissues. Aromatase mRNA in these non-malignant tissues was transcribed from skin fibroblast/fetal liver-specific exon 1 (exon 1b) of the aromatase gene. However, in half the cases of breast cancer patients, the utilization of multiple exons 1 in the aromatase mRNA changed from exon 1b to ovary-specific exon 1 (exon 1c) in their breast tissues. Aromatase mRNA in HepG2 cells as well as in non-malignant breast tissues was also transcribed from exon 1b. Then, the promoter region responsible for the exon 1b-specific utilization in HepG2 cells was examined by fluorometric promoter assay using a new reporter containing four major alternative exons 1 and promoters. The results suggested that transcriptional elements determining preferential utilization of exon 1b in the cells was located on the promoter region of exon 1b from −255 to −1145. To investigate further the cause of the elevation of aromatase mRNA and the switching from exon 1b to exon 1c in the transcription of the aromatase gene, the effects of various factors on the expression levels and preference of alternative exons 1 were examined in cultured adipose stromal cells from breast tissues. Aromatase mRNA was transcribed from exon 1b in the stromal cells, cultured in the presence of calf serum. However, removal of the serum or the addition of forskolin or phorbol ester (TPA) induced a rapid elevation of aromatase mRNA and the switching of aromatase transcripts to exon 1c in the cells, whereas TGFβ almost abolished the expression of aromatase mRNA. Because co-culture of cancer cells such as MCF-7 increased aromatase mRNA of the cells cultured in the serum-containing medium, it is possible that cancer cells secret stimulatory factors acting like forskolin or TPA, or consume serum inhibitory factors acting like TGFβ, consequently causing levels of aromatase mRNA to increase.

Aberrant expression of aromatase in breast cancer tissues

The expression of aromatase is tissue-specifically regulated through the alternative use of multiple exons 1 and promoters. We analysed expression levels of aromatase mRNA, preferential utilization of multiple exon 1 of the human aromatase gene, and transcriptional regulation of their multiple promoters in breast cancer tissues by newly developed fluorometric methods. The expression levels of aromatase mRNA in breast cancer tissues were significantly higher than those in regions distal to tumours or in non-malignant breast tissues. Aromatase mRNA in these non-malignant tissues was transcribed from skin fibroblast/fetal liver-specific exon 1 (exon 1b) of the aromatase gene. However, in half the cases of breast cancer patients, the utilization of multiple exons 1 in the aromatase mRNA changed from exon 1b to ovary-specific exon 1 (exon 1c) in their breast tissues. Aromatase mRNA in HepG2 cells as well as in non-malignant breast tissues was also transcribed from exon 1b. Then, the promoter region responsible for the exon 1b-specific utilization in HepG2 cells was examined by fluorometric promoter assay using a new reporter containing four major alternative exons 1 and promoters. The results suggested that transcriptional elements determining preferential utilization of exon 1b in the cells was located on the promoter region of exon 1b from −255 to −1145. To investigate further the cause of the elevation of aromatase mRNA and the switching from exon 1b to exon 1c in the transcription of the aromatase gene, the effects of various factors on the expression levels and preference of alternative exons 1 were examined in cultured adipose stromal cells from breast tissues. Aromatase mRNA was transcribed from exon 1b in the stromal cells, cultured in the presence of calf serum. However, removal of the serum or the addition of forskolin or phorbol ester (TPA) induced a rapid elevation of aromatase mRNA and the switching of aromatase transcripts to exon 1c in the cells, whereas TGFβ almost abolished the expression of aromatase mRNA. Because co-culture of cancer cells such as MCF-7 increased aromatase mRNA of the cells cultured in the serum-containing medium, it is possible that cancer cells secret stimulatory factors acting like forskolin or TPA, or consume serum inhibitory factors acting like TGFβ, consequently causing levels of aromatase mRNA to increase.

Spermatid-specific Overexpression of the TATA-binding Protein Gene Involves Recruitment of Two Potent Testis-specific Promoters

The gene encoding the TATA-binding protein, TBP, is highly overexpressed during the haploid stages of spermatogenesis in rodents. RNase protection analyses for mRNAs containing the previously identified first, second, and eighth exons suggested that most TBP mRNAs in testis did not initiate at the first exon used in somatic cells (here designated exon 1C). Using a sensitive ligation-mediated cDNA amplification method, 5' end variants of TBP mRNA were identified, and the corresponding cDNAs were cloned from liver and testis. In liver, a single promoter/first exon is used to generate a steady-state level of roughly five molecules of TBP mRNA per diploid cell equivalent. In testis, we detect modest up-regulation of the somatic promoter and recruitment of at least five other promoters. Three of the alternative promoter/first exons, including 1C and two of the testis-specific promoter/first exons, 1D and 1E, contribute roughly equivalent amounts of mRNA which, in sum, account for greater than 90% of all TBP mRNA in testis. As a result, round spermatids contain an estimated 1000 TBP mRNA molecules per haploid cell. Testis TBP mRNA also exhibits several low abundance 5' end splicing variants; however, all detected TBP mRNA leader sequences splice onto the common exon 2 and are expected to initiate translation at the same site within exon 2. The precise locations of the three major initiation exons are mapped on the gene. The identification of the strong testis-specific promoter/first exons will be important for understanding spermatid-specific tbp gene regulation.

Organization and Alternate Splicing of the Murine Folylpolyglutamate Synthetase Gene: DIFFERENT SPLICE VARIANTS IN L1210 CELLS ENCODE MITOCHONDRIAL OR CYTOSOLIC FORMS OF THE ENZYME

The organization of the murine folylpolyglutamate synthetase (FPGS) gene has been determined by sequence analysis that also revealed an interesting complexity in the case of exon 1. The entire nucleotide sequence of the L1210 FPGS cDNA, the 3'- and 5'-untranslated regions, the mitochondrial leader sequence, and the coding region were found to be distributed on 15 exons with an overall length of 10.358 kilobases. Two splice variants of exon 1 were identified by screening of an L1210 cell cDNA library. Variant I (exons 1a + 1b plus 2-15) incorporates all of the sequence homologous to the recently reported (Taylor, S. M., Freemantle, S. J., and Moran, R. G. (1995) Cancer Res. 55, 6030-6034) human exon 1, including two ATG start codons at positions +1 and +126, and encodes both mitochondrial and cytosolic form of FPGS. The most prevalent variant, Variant II (exons 1b plus 2 to 15), incorporates only a portion (92 nucleotides at the 3' end) of this sequence, incorporates only one ATG start codon at position +126, and encodes only cytosolic FPGS. The existence of this variant is consistent with the identification of an appropriately situated internal donor/acceptor site in what was believed to be exon 1. A third related variant (Variant III) with a novel 5' termini was originally identified by screening of a mouse liver cDNA library. This variant, which occurs at moderately low frequency in the L1210 cell cDNA library, incorporates an alternate to exon 1a (exon 1c) spliced to exon 1b plus exons 2-15 and encodes a different mitochondrial leader peptide than Variant I. The identification of these variants suggests another possible mechanism, i.e. at the level of precursor mRNA splicing, for regulating synthesis of mitochondrial versus cytosolic forms of FPGS in the cell. Exon 1c is positioned in the gene upstream of exon 1a separated by an intron of 56 nucleotides within a region of DNA sequence that like the homologous human sequence is distinctly promoter-like. However, the sequence of this region differs from the human sequence in terms of the number, position, and type of putative regulatory elements, particularly in regard to the number of SP-1 binding sites and the position of multiple transcription start sites as determined by enzymatic primer extension.

Structure and Sequence of HumanFGF8

Recent evidence indicates thatFgf8is expressed during vertebrate development in multiple locations involved in the patterning and outgrowth of important embryo structures. Cloning and analysis of the murine gene revealed at least eight potential protein isoforms that share a common carboxyl region, encoded by exons 2 and 3, but possess different amino termini, generated by alternative splicing of RNA encoded by multiple 5′ exons (exons 1A, 1B, 1C, and 1D). We now report the cloning and sequence of the humanFGF8gene. Human FGF-8 isoforms are identical to their murine counterparts in the common carboxyl region. Four of the human isoforms are identical to, or very similar to, the murine isoforms in the amino termini. However, four of the potential murine isoforms do not have corresponding human isoforms due to marked sequence divergence, leading to a blocked reading frame in exon 1B ofFGF8.The lack of the four murine isoforms in humans raises the question of their function in murine development.

THREE DISTINCT PROMOTERS DIRECT TRANSCRIPTION OF DIFFERENT 5′ UNTRANSLATED REGIONS OF THE HUMAN INTERLEUKIN 1 TYPE I RECEPTOR: A POSSIBLE MECHANISM FOR CONTROL OF TRANSLATION

The 5′ untranslated regions (UTR) of the human interleukin 1 (IL-1) type I receptor (IL—1RI) are encoded by one common exon (exon 2) but one of three distinct exons 1 (termed exon 1A, 1B, and 1C). These exons span approximately 50 Kb of genomic DNA. Exons 1A and 1B have multiple transcriptional initiation sites, whereas the promoter for exon 1C uses a single start site. There are no ‘TATA’ or ‘CAAT’ boxes, indicating that these promoters belong to the family of housekeeping gene promoters. Computer sequence analysis of exons 1A, 1B, and 1C predicts the potential to form stable secondary structures (ΔG° 1A= −72.2 Kcal/mol, ΔG° 1B= −125.8 Kcal/mol, ΔG° 1C= −255.4 Kcal/mol). Exon 1C appears to be the most stable whereas exon 1A would yield a mRNA species more likely to be translated than those derived from exon 1B or 1C. The 5′ UTR of exon 1C is also rich (75%) in GC which might inhibit expression. Therefore, we studied the effect of exon 1C on chloramphenicol acetyltransferase (CAT) activity. Deletion of 183 or 296 base pairs from this GC rich region was shown to increase CAT activity. In addition, insertion of a GC-rich fragment of exon 1C inhibited CAT activity driven by SV40 promoter. These results suggest that the 5′ UTR exon 1C of the human IL—1RI may exert a suppressive effect on the translation of IL-1RI transcripts.

Differential promoter usage in prolactin receptor gene expression: hepatocyte nuclear factor 4 binds to and activates the promoter preferentially active in the liver.

Characterization of the rat PRL receptor (PRLR) gene has revealed three separate untranslated exon 1 sequences, each associated with a different transcription start site and 5'-flanking sequence. We show by RT-PCR that exon 1A is expressed primarily in liver but is also detectable in ovary and mammary gland. Exon 1B expression is observed exclusively in the ovary, whereas exon 1C is expressed in all three tissues. Transient transfection of luciferase reporter constructs containing parts of the 5'-flanking regions (0.3-1.1 kb) of exon 1A, 1B, and 1C, respectively, showed activity of the 1A promoter in Chinese hamster ovary (CHO) cells, the human hepatoma cell line, HepG2, and the rat hepatoma cell line, H4II, which was 10- to 14-fold increased compared with the activity of the promoter-less luciferase vector. No activity of the 1A promoter was detected in the human mammary cell line, T-47D. Relative to a vector containing the Simian virus 40 (SV40) promoter, the 1A promoter had 20% activity in H4II cells and 1-3% activity in CHO and HepG2 cells. The 1B promoter produced a 6.1-fold increase of luciferase activity in CHO cells (approximately 2% of the SV40 promoter), whereas no significant activity was detected in HepG2, H4II, and T-47D cells. The 1C promoter was strongly active in T-47D cells (approximately 64-fold over control) and moderately active in the other cell lines tested (9- to 13-fold over control). 5'-Deletion analysis of the 1A promoter revealed that a fragment containing -83/ +81 bp, relative to the transcription start site, was sufficient to drive transcription in hepatoma cells, whereas this construct was inactive in CHO cells. Cotransfection of CHO cells with the -83/+81 construct and an expression vector encoding the liver-enriched transcription factor, hepatocyte nuclear factor 4 (HNF4), revealed a dose-dependent transactivation of the proximal 1A promoter with a maximal stimulation of approximately 10-fold. Electrophoretic mobility shift assays showed binding of HNF4 to the sequence -14/+24 of the 1A promoter, and mutational analysis revealed that the sequence GGGCAAAGTCA at position +11/+21 is required for this binding. We conclude that the 1A, 1B, and 1C promoters of the PRLR gene are used in a cell type- dependent way that may play a role in differential hormonal regulation of the gene. In particular, we have shown that HNF4 operates on the proximal 1A promoter and may be responsible, in combination with other factors, for the increased activity of this promoter in adult female liver.

Two Additional 5′ Exons in the Human Vigilin Gene Distinguish it from the Chicken Gene and Provide the Structural Basis for Differential Routes of Gene Expression

Vigilin, a 150-kDa protein, contains 14 tandemly arranged domains, each consisting of a KH RNA-binding motif and a spacer region. Here, we report on the physical structure of the human Vigilin gene with 29 exons, thereby outnumbering the chicken gene by two additional 5' exons. These additional exons, 1A and 1B, are alternatively though concurrently spliced to exon 1C which is homologous to the first exon in the chicken gene. None of the additional human exons code for an amino-terminal extension of Vigilin, due to in-frame stop codons. Structural features of exon 1A, however, would allow the translation of a 13-amino-acid peptide from an upstream open reading frame preceding the vigilin open reading frame. We suggest that exons 1A and 1B have been gained during evolution, allowing alternative routes of expression control of the human Vigilin gene.

Purification and Properties of Human Cytosolic Folylpoly-γ-glutamate Synthetase and Organization, Localization, and Differential Splicing of Its Gene

Human cytosolic folylpolyglutamate synthetase (FPGS) was expressed in Escherichia coli and purified to homogeneity. Tetrahydrofolate and dihydrofolate were the most effective substrates, while 5-substituted folates were poor substrates. Most pteroyldiglutamates were better substrates than monoglutamates. The human FPGS gene spans 12 kilobases and contains 15 exons and 14 introns. A single FPGS gene was located to chromosome region 9q34.1. Four exon 1 variants were identified, each of which was spliced to exon 2. The exon 1 variant corresponding to the isolated cDNA contains two ATG codons and multiple transcription start sites in this region generates mitochondrial and cytosolic FPGS (Freemantle, S. J., Taylor, S. M., Krystal, G., and Moran, R. G. (1995) J. Biol. Chem. 270, 9579-9584). Exons 1B and 1C, generated by alternate splicing in intron 1, and exon 1A, which is 5' to exon 1 and may encode an additional mitochondrial isoform, are preceded by a number of potential promoter sites. Chinese hamster ovary cell transfectants expressing FPGS activity in the mitochondria contained normal mitochondrial and low cytosolic folylpolyglutamate pools. Mitochondrial FPGS activity is required for mitochondrial folate accumulation, while cytosolic FPGS activity is needed for establishment of normal cytosolic folate pools. The reconstructed FPGS gene restored normal cytosolic and mitochondrial folate metabolism in hamster cells.

Characterization of the human AMPD3 gene reveals that 5′ exon useage is subject to transcriptional control by three tandem promoters and alternative splicing

Previous work has identified multiple human AMPD3 transcripts proposed to differ by mutually exclusive alternative splicing of three exons located at, or near, the 5′ end of the gene. In this study, we perform a more comprehensive evaluation of human AMPD3 gene expression. Combined Northern blot and RNase protection analyses show that alternative mRNAs are widely expressed in human tissues and cells, but at variable relative abundances. Sequencing of human genomic clones, together with human-mouse somatic cell hybrid analysis, demonstrates that the entire gene is comprised of seventeen exons spanning approx. 60 kilobases on the short arm of chromosome 11 in the region p13-pter. Together, RT-PCR and additional RNase protection analyses establish that exons 1a, 1b, and 1c are 5′ terminal sequences in alternative transcripts. Transient transfection experiments show fusion constructs containing proximal flanking and 5′ untranslated sequence from each of these exons are able to direct expression of a reporter luciferase gene in mammalian cell lines. These combined results reveal that AMPD3 gene expression is subject to transcriptional control by three tandem promoters. Differential regulation of the exon 1b promoter in skeletal myocytes, as compared to retinal pigment epithelial cells, is proposed to be mediated by skeletal muscle-specific basic helix-loop-helix protein/E-box interactions. Finally, an internal splice acceptor site in exon 1c is shown to be used alternatively to retain the 3′ portion of this exon in mature AMPD3 transcripts initiating upstream in exon 1b.

Molecular basis of the pleiotropic phenotype of mice carrying the hypervariable yellow (Ahvy) mutation at the agouti locus.

The murine agouti locus regulates a switch in pigment synthesis between eumelanin (black/brown pigment) and phaeomelanin (yellow/red pigment) by hair bulb melanocytes. We recently described a spontaneous mutation, hypervariable yellow (Ahvy) and demonstrated that Ahvy is responsible for the largest range of phenotypes yet identified at the agouti locus, producing mice that are obese with yellow coats to mice that are of normal weight with black coats. Here, we show that agouti expression is altered both temporally and spatially in Ahvy mutants. Agouti expression levels are positively correlated with the degree of yellow pigmentation in individual Ahvy mice, consistent with results from other dominant yellow agouti mutations. Sequencing of 5' RACE and genomic PCR products revealed that Ahvy resulted from the integration of an intracisternal A particle (IAP) in an antisense orientation within the 5' untranslated agouti exon 1C. This retrovirus-like element is responsible for deregulating agouti expression in Ahvy mice; agouti expression is correlated with the methylation state of CpG residues in the IAP long terminal repeat as well as in host genomic DNA. In addition, the data suggest that the variable phenotype of Ahvy offspring is influenced in part by the phenotype of their Ahvy female parent.

Molecular and epidemiological analyses of abnormal expression of aromatase in breast cancer.

One-third of human breast cancers exhibit estrogen-dependent proliferation. It appears that estrogen functions as a mitogenic factor in these carcinomas. As aromatase is the rate-limiting enzyme in estrogen biosynthesis. It could play an important role in the pathogenesis of estrogen-dependent breast cancer. The aromatase gene consists of at least six exons 1, each containing a promoter, and the tissue-specific expression is regulated by alternative use of these multiple promoters. The expression of aromatase in the breast and abdominal adipose tissues is regulated by a promoter flanking exon 1b. Molecular and epidemiological analyses of tissue-specific utilization of multiple exons 1 and promoters revealed a switching from use of the adipose-specific exon 1b to exon 1c in adipose tissues adjacent to the carcinomas in most breast cancer patients. Exon 1c has been shown to be specific for the ovary. Aromatase mRNA in adipose tissues distal to the tumour of the same patients was normally transcribed from exon 1b as was breast tissue in healthy controls. It is noteworthy that a switching from exon 1b to exon 1c was often observed in breast cancer patients having metastatic lymph nodes. These data suggest that switching from an adipose-specific exon 1b to exon 1c could cause a deviation from strict regulation of tissue-specific expression of the adipose aromatase leading to over-expression of the adipose aromatase. Consequently overproduction of local estrogen may promote carcinogenesis or proliferation of breast cancer cells.

Cloning and Characterization of Rat Pancreatic β-Cell/Liver Type Glucose Transporter Gene: A Unique Exon/Intron Organization

Four overlapping λ genomic clones encoding rat pan- creatic β-cell/liver type glucose transporter (GLUT2) have been isolated and characterized. The gene is about 35 kb long and contains 14 exons and 13 introns. Contrary to the exon 1 of the human or mouse counterpart, the rat GLUT2 gene has three additional noncoding exons which were identified by 5′-RACE and all four were designated exon 1a, 1b, 1c, and 1d. The intron sequences bordering the splice site junctions generally follow the GT/AG rule except for one intron which begins with GC. The exon sequences determined from genomic DNA sequencing showed some differences when compared to the published rat GLUT2 cDNA. Transcription initiation site was determined by primer extension and located 661 bp upstream of the ATG translation initiation codon. Several potential binding sites for transcription factors such as C/EBP, Sp1, AP1, HNF-5, and UPE were observed and they may be responsible for the regulation of GLUT2 gene expression. The promoter region of rat GLUT2 showed little homology when compared with those of human or mouse. However, striking sequence identity (84%) was found when the adjacent intron regions flanking exon 1c were compared with the −970/−721 region of the mouse GLUT2 promoter. A series of deleted mutant constructs of the putative promoter region linked to the CAT reporter gene showed promoter activity in the primary hepatocyte culture. The region containing −452/+240 showed the highest CAT activity and fur ther deletion of the region showed gradual decrease in CAT activity.

The canine betaine gamma-amino-n-butyric acid transporter gene: diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner.

The Na(+)- and Cl(-)-coupled betaine transporter, designated BGT1, a member of the neurotransmitter transporter gene family, is responsible for accumulation of betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and presumably in the hypertonic renal medulla. The canine gene for the betaine gamma-amino-n-butyric acid transporter has been cloned and analyzed. The gene extends over 28 kb and consists of 18 exons. The 5' end of the gene has three alternative first exons (1A, 1B, and 1C+D). Analysis of BGT1 mRNA revealed that there is considerable divergence in the 5' untranslated sequence resulting from three different 5' end motifs (A, B, and C) followed by an alternative motif (D) as well as two internal acceptor sites for splicing. Eight kinds of BGT1 mRNA were classified into three types (A, B, and C) according to the 5' end sequence. Northern blot analysis using probes specific for the A, B, or C motif revealed that hypertonicity induces all three types in MDCK cells. Reverse transcription and polymerase chain reaction showed that each type was expressed in a tissue-specific manner. Primer extension and/or RNase protection assays as well as transfection assays into MDCK cells demonstrated that exons 1A, 1B, and 1C+D have independent transcription initiation sites under control of independent promoters. Diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner.

Identification and characterization of a GC-rich promoter of the human parathyroid hormone-related peptide gene.

The human PTH-related peptide (PTHrP) gene comprises eight exons spanning more than 15 kilobases of genomic DNA. The gene has a highly complex controlling region, which contains four alternatively spliced, noncoding exons and at least two putative promoters, one 5' of exon 1A (up-stream TATA element) and the other 5' of exon 2 (down-stream TATA element). To define important cis regulatory sequences of this gene, a functional dissection of PTHrP 5'-flanking DNA was initiated, using chimeric PTHrP-chloramphenicol acetyltransferase (CAT) constructs. This analysis was carried out in PTHrP-negative human renal carcinoma cells, so that RNA derived from transfected DNA could be studied without interference from endogenous PTHrP sequences. Of the initial series of constructs prepared, the most active was a 1.1-kilobase BamHI-HindIII PTHrP-CAT plasmid containing 350 basepairs of DNA 5' of exon 1C and extending into exon 3. Analysis of transfection products by RNase protection and primer extension revealed that this region contains a previously unrecognized promoter of the gene. This element is located immediately 5' of exon 1C, is active in transfected cells when cloned in isolation up-stream of the CAT gene, and appears to be functional in a number of cell lines and tissues on the basis of primer extension analysis. Unlike the other two PTHrP gene promoters, this element is GC rich and does not possess canonical TATA or CAAT sequences. The human PTHrP gene is one of a handful of genes that appear to use both TATA and GC-rich promoter elements.

Identification of an Up-Stream Promoter of the Human Parathyroid Hormone-Related Peptide Gene

Previous evidence has suggested that the human PTH-related peptide (PTHRP) gene uses two promoters, one a short down-stream element lying immediately between two 5' exons (1 and 2) and a second lying in an unknown up-stream location. We approached identification of the up-stream element in three steps. First, Northern analysis carried out using progressively 5' fragments of the gene as probes identified a candidate region some 2.5 kilobases up-stream of exon 1. Second, a battery of overlapping 5' cRNA probes was used in RNase protection experiments to identify two previously unrecognized exons, 212 and 93 basepairs in length (termed exons 1A and 1B to distinguish them from the previously designated exon 1, which was termed exon 1C). Third, primer extension experiments were performed with oligonucleotides complementary to each of the 5' exonic sequences. These experiments identified a transcription start site up-stream of exon 1A and also demonstrated that the 5' exons of the PTHRP gene could be spliced together in several combinations. The up-stream promoter element contains a TATA box, but does not otherwise resemble the down-stream PTHRP gene promoter or the PTH gene promoter. We conclude that the human PTHRP gene contains eight exons spanning more than 15 kilobases of genomic DNA, with promoter elements lying immediately up-stream of exons 1A and 2. The identification of these elements will permit functional analysis of their roles in mediating tissue- and tumor-specific PTHRP gene expression.