©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning of the Promoter-Regulatory Region of the Murine Growth Hormone Receptor Gene
IDENTIFICATION OF A DEVELOPMENTALLY REGULATED ENHANCER ELEMENT (*)

Ram K. Menon (§) , Dietrich A. Stephan , Manbir Singh , Sidney M. MorrisJr. (1), Lanling Zou

From the (1) Departments of Pediatrics and Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15213

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The growth hormone (GH) receptor is essential for the actions of GH on postnatal growth and metabolism. To identify DNA sequences involved in the regulation of transcription of the murine GH receptor gene, a 17-kilobase genomic clone containing the 5`-flanking region, exon 1, and part of intron 1 of the murine GH receptor gene was isolated. Utilizing primer extension and ribonuclease protection assays, two major transcription start sites were identified in RNA from liver of male, female, and pregnant mice. Transient transfection studies using a reporter gene demonstrated promoter activity in a variety of eukaryotic cells. Deletional analysis and DNA-protein binding assays led to the identification of a 30-base pair enhancer element located about 3.4 kilobases upstream of the transcription start sites. Computer analysis of the nucleotide sequence of the enhancer element did not reveal any potential DNA binding motifs for known transcription factors, and this DNA element failed to exhibit binding activity for some common transcription factors. Analysis of both functional activity and DNA-protein binding activity of this enhancer element in adult and fetal hepatocytes suggests that this DNA element may play a role in the developmental expression of the GH receptor gene.


INTRODUCTION

The essential role of growth hormone (GH)() for postnatal growth and for several metabolic processes begins at the cellular level by initial binding to a cell surface protein termed the GH receptor. GH receptor belongs to a newly described gene family that includes the receptors for GH and prolactin, a number of cytokines such as granulocyte colony-stimulating factor, erythropoietin, granulocyte macrophage colony-stimulating factor, and a wide variety of interleukins (1) . Whereas detailed information about the molecular mechanisms involved in the regulation of expression of the GH gene is available (2) , knowledge regarding the factors modulating the expression of the GH receptor gene is scanty (1) .

A variety of factors influence the expression of the GH receptor (3) . Expression is very low in fetal tissues but increases dramatically during postnatal life (4, 5, 6) . In addition, there is a distinct tissue-specific pattern of expression of the receptor with maximum expression in the liver and lesser degrees of expression in the heart, kidney, and intestine (7) . The factors controlling the temporal and tissue-specific expression of the GH receptor are not known, but much of their effect is probably manifested at the level of GH receptor gene transcription. The regulated expression of the GH receptor in various tissues and cell types plays a critical role in numerous physiologic and pathologic processes. Hence, elucidation of the molecular mechanisms involved in the modulation of GH receptor expression requires identification and characterization of the transcriptional regulatory regions of the GH receptor gene.

In this report, we describe the structure and organization of the 5`-flanking region of the murine GH receptor gene, including identification of the transcription initiation sites used in liver. We also demonstrate that the 5`-flanking region of this gene contains a functional promoter as well as at least one enhancer element. Our investigations have also resulted in the identification of sequence-specific protein binding activity within the enhancer element and suggest that the protein(s) binding to the enhancer region are novel.


MATERIALS AND METHODS

Cloning of the 5`-Flanking Region of the GH Receptor Gene

A mouse genomic library (Stratagene) was screened with a mouse cDNA probe. The genomic library was prepared by partial Sau3AI digestion of spleen DNA, with subsequent size fractionation and cloning into the XhoI site of the Lambda FIX II phage vector. Based on the published sequence of the mouse GH receptor cDNA (8) , which will be used as a reference for cDNA nucleotide positions in this report, oligonucleotide primers were designed to encompass a region corresponding to the extracellular portion of the mouse GH receptor. These primers were used in the PCR to amplify a 906-bp fragment from adult mouse liver total RNA. The amplified DNA was then cloned into the plasmid vector PCR1000 (Invitrogen). To select for 5`-sequences, the 191-bp cDNA probe used for screening the genomic library was obtained by EcoRI- HindIII digestion of the 906-bp cDNA. The cDNA was P labeled by random primer extension and used to screen filters by standard techniques (9) . Positive clones were plaque purified, and the purified DNA was used for subsequent analytical and cloning procedures.

Southern Blot Analysis and Restriction Enzyme Mapping

Detailed restriction maps of the genomic clones identified were obtained by the rapid restriction mapping method (10) , which exploits the mapping cassettes included in the Lambda FIX II phage vector (Stratagene).

DNA Sequencing

The DNA clones were partially sequenced directly by PCR sequencing using the dsDNA Cycle System (Life Technologies, Inc.) with synthesized oligonucleotides complementary to the 5`-region of the cDNA. For other DNA sequence analyses, convenient fragments of the clone were subcloned into the plasmid pBluescript II SK+ (Stratagene). Sequencing was carried out by the dideoxynucleotide chain termination method of Sanger et al. (11) using the Seque-nase 2.0 kit (U. S. Biochemical Corp.). Sequencing primers were either complementary to the T3 or T7 sites flanking the multiple cloning site of the vector or were complementary to experimentally established sequences. The sequence data were managed using the sequence analysis program Geneworks (IntelliGenetics, Inc. Mountain View, CA).

RNA Extraction

Total RNA was prepared from tissue or cells using RNAzol as recommended by the supplier (Biotecx, Houston, TX) and quantitated by absorbance at 260 nm. Poly(A)RNA was prepared by oligo(dT) affinity chromatography using the Poly(A)Quik kit (Stratagene).

Primer Extension Reaction

Extension reaction was carried out on 5 µg of adult mouse liver poly(A)RNA with synthetic oligonucleotide complementary to portions of the GH receptor mRNA. The 18-base oligonucleotide designated mGHR1 is defined by complementarity of its 5`-end beginning 49 nucleotides (nt) upstream of the translation start site of the GH receptor mRNA (8) . The primer was P end-labeled using T4 polynucleotide kinase and hybridized with RNA in 50 m M Tris-HCl, 50 m M KCl, 10 m M MgCl, 20 m M dithiothreitol, 1 m M dNTPs, and 0.5 m M spermidine. The extension reaction was initiated by the addition of 2.8 m M sodium pyrophosphate and 1 unit of avian myeloblastosis virus reverse transcriptase (Promega) and allowed to proceed for 30 min at 41.5 °C. After termination of the reaction, the reaction products were size fractionated by denaturing gel electrophoresis. The sizes of the extension products were determined both by including kinased DNA markers on the gel and by counting the number of nucleotides in a concurrently run sequencing ladder.

Ribonuclease Protection Assay

Using experimentally established sequence information, PCR was used to amplify a genomic DNA sequence upstream of mGHR1. This 211-nt fragment was then subcloned into the plasmid pBluescript II SK+ to create pSK(-200). This construct was used to generate P-labeled antisense RNA, which was hybridized to 20 µg of adult mouse liver total RNA at 42 °C for 12 h. Following digestion with RNase A and RNase T, the RNase-resistant radioactivity was size fractionated on 5% urea-polyacrylamide gel electrophoresis and autoradiographed. The sizes of the protected fragments were determined by comparison with kinased DNA markers and a sequencing ladder as described above.

Reporter Gene Constructs

Luciferase reporter gene constructs were engineered to contain various portions of the GH receptor 5`-flanking region in the promoterless pGL2-Basic vector (Promega). Construction of the reporter gene-GH receptor 5`-flanking region hybrids were as follows: a 6.5-kb HindIII fragment cloned into the corresponding site in the polylinker of pGL2-Basic was digested with SacI and religated to obtain pGL2B-3.6. Exonuclease III digestion was performed to delete exon 1-intron 1 junction from pGL2B-3.6 so that the modified fusion construct (pGL2B-3.6[+53]kb) contained 53 base pairs of the first non-coding exon (exon 1). pGL2B-3.6[+53]kb was digested with ApaI and SacI and, following blunt ending, religated to obtain pGL2B-3.0[+53]kb. Progressive unidirectional deletions of the GH receptor 5`-flanking DNA in this pGL2B-3.0[+53]kb construct were engineered by exonuclease III digestion such that all the constructs had identical 3`-ends defined by the inclusion of the first 53 bp of exon 1. All constructs were partially sequenced through their vector-insert junction to verify directionality. Expression of the pGL2-Control plasmid (Promega), which contains the SV40 promoter and enhancer sequences, was measured to monitor transfection efficiency.

The activities of putative enhancer elements were also tested via the ability to exhibit activity in the context of an heterologous promoter. For this purpose, luciferase vector pTK81 (ATCC) containing the thymidine kinase promoter was chosen (12) . The fusion constructs were engineered by exploiting convenient restriction sites in the vector polylinker.

Transient Expression of Reporter Gene

The culture media and the hormones used for tissue culture experiments were obtained from Life Technologies, Inc. and Sigma, respectively, unless otherwise stated. HepG2 cells (ATCC) were maintained in Williams' Medium E containing 2 m M glutamine, 5% fetal calf serum, and penicillin G (100 units/ml) and streptomycin (100 µg/ml); Chinese hamster ovary (CHO) cells (ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin G (100 units/ml), and streptomycin (100 µg/ml). 1 10cells were plated on 60-mm plates 24 h prior to transfection. 15 µg of plasmid DNA was transfected per plate using the calcium phosphate transfection method (Life Technologies, Inc.). After 6 h of incubation, the cells were washed with phosphate-buffered saline and then supplemented with medium for 40 h prior to harvest for luciferase assay.

Adult hepatocytes were obtained from 80-100-g Wistar rats by the in situ perfusion method of Berry and Friend (13) , and fetal hepatocytes were isolated by in vitro collagenase digestion (14) after harvesting the liver from fetuses of 19-day gestation. Following separation on a Percoll gradient, the hepatocytes (>90% viability as assessed by Trypan Blue exclusion) were resuspended in adhesion medium (75% minimum essential medium, 25% Waymouths medium, 10% fetal calf serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone) before plating onto 60-mm Primaria plates (Falcon, Lincoln Park, NJ) at a density of 2 10in 2 ml of adhesion medium per plate. After incubation in 95% O, 5% COat 37 °C for 4-5 h, the medium was replaced by transfecting medium (75% minimum essential medium, 25% Waymouths medium, 20 µg/ml gentamycin, 10 µg/ml transferrin, 30 µ M selenium, 100 microunits/ml hGH, 1.5 µ M porcine insulin, 1 µ M dexamethasone, and 1 µ M 3,5,3`-triiodo- L-thyronine). The transfection was performed according to the method of Graham and Van Der Eb (15) , as modified by Ginot et al. (16) . 18 h after transfection, the plates were rinsed twice with phosphate-buffered saline, and the cells were harvested by the addition of 200 microliters of lysis buffer (25 m M Tris-phosphate, pH 7.8, 2 m M dithiothreitol, 2 m M EDTA, 10% glycerol, 1% Triton X-100). Following a brief freeze-thaw cycle, the insoluble debris was removed by centrifugation at 4 °C for 2-3 min at 12,000 g. The supernatant was then immediately assayed for luciferase activity. All transfections were performed at least in triplicate. Protein concentration of the supernatant was determined using the Bio-Rad protein assay.

Luciferase Activity Assay

Luciferase activity was measured in the cell lysates using reagents from Analytical Luminescence Laboratory (San Diego, CA). Briefly, using the automatic injectors of the Monolight 2010 Luminometer (Analytical Luminescence), 100 microliters (200 µg) of cell extract was mixed sequentially with 100 microliters of buffer (3 m M ATP, 15 m M MgSO, 30 m M Tricine, 10 m M dithiothreitol, pH 7.8) and 100 microliters of 1 m M luciferin; the light output was measured for 30 s. Determinations of protein concentration were used to convert raw activity values to specific activities for all samples. The results of the luciferase assay are expressed as -fold increase over the background specific activity determined by transfecting equimolar amounts of the appropriate vector DNA ( i.e. pGL2-Basic or pTK81) in each experiment.

Electromobility Shift Assay (EMSA)

Nuclear extracts from mouse liver were prepared as described by Gorski et al. (17) . Protease inhibitors (2 µg/ml leupeptin, 1 µg/ml pepstatin, and 1% aprotinin) were included in the buffers used to prepare the nuclear extracts. Double-stranded DNA fragments used as probes were obtained either by restriction enzyme digestion and subsequent gel purification after agarose electrophoresis or by annealing complementary single-stranded oligonucleotides. The DNA was labeled with P using Klenow enzyme. Approximately 6 fmol of DNA was added to 4 µg of nuclear extract in a final volume of 50 microliters containing 1.7 µg of poly(dIdC), 20 m M Hepes, pH 7.9, 100 m M KCl, 5% glycerol, 1 m M EDTA, and 1 m M dithiothreitol. Following incubation at room temperature for 30 min, DNA-protein complexes were resolved on a 6-10% non-denaturing polyacrylamide gel with 90 m M Tris borate, 2 m M EDTA buffer. Competition experiments included the addition of excess unlabeled DNA fragments to the reaction mix.

DNase I Footprinting

The pTK81 vector containing the 552-bp DNA was linearized with HindIII and end labeled with Klenow enzyme. After a secondary digest with KpNI, the purified labeled 552-bp fragment was incubated with nuclear extracts in a final volume of 50 microliters with buffer containing 1.7 µg of poly(dIdC), 20 m M Hepes, pH 7.9, 100 m M KCl, 5% glycerol, 1 m M EDTA, and 1 m M dithiothreitol for 30 min at 4 °C, after which varying concentrations of DNase I were added and allowed to react for 10-30 s. The reactions were stopped with EDTA, phenol-chloroform extraction, and ethanol precipitation. The digested DNA was size fractionated on 6% urea-polyacrylamide gel electrophoresis and autoradiographed. The exact locations of the DNase I footprints were determined by comparison to a Maxam-Gilbert sequencing reaction electrophoresed concurrently.


RESULTS

Cloning of 5`-Flanking Region of the GH Receptor Gene

Six clones were isolated after screening approximately 2 10plaque-forming units with the 5`-end of mouse GH receptor cDNA. As the genomic structure of the mouse GH receptor gene was not known, we synthesized a panel of non-overlapping oligonucleotides that spanned the 131 bp of the most 5`-region of the mouse GH receptor cDNA. These oligonucleotides were then used to screen the clones isolated. One of the clones, designated 167A, hybridized to the oligonucleotide containing the most 5`-nucleotide. Direct PCR sequencing of 167A revealed that this clone contained the first 88 bases (hereafter referred to as exon 1) of the most 5`-sequence of the published cDNA (8) and about 8 kb of the first intron (Fig. 1). Since 167A did not contain exon 2, the exact size of intron 1 remains to be determined.

Identification of Transcription Start Sites

Primer extension was performed using reverse transcriptase after end-labeled antisense oligonucleotide (mGHR1) was hybridized to poly(A)-enriched RNA from pregnant adult mouse liver. The extension of mGHR1 produced two major specific bands of 61 and 87 nucleotides (Fig. 2 A). In addition, three minor bands were noticed in close proximity (within 2 nucleotides) of the 61-nucleotide product. Identical extension products were obtained from RNA extracted from non-pregnant female and male mice.

To demonstrate that the full 5`-untranslated region was contained within the putative first exon (exon 1), ribonuclease protection assays were performed. P-Labeled antisense RNA, transcribed from the appropriate strand of pSK(-200), was hybridized to total cellular RNA from liver of female mice and digested with RNase A and T. The major protected fragments were 61 and 87 nucleotides in length (Fig. 2 A). In a pattern identical to that obtained with the primer extension experiment, three additional protected fragments were detectable clustered around the 61-nucleotide band. Since the 5`-end of the antisense probe is defined by mGHR1, the demonstration that the size of these protected fragments is identical to the length of the primer extension products confirms the location of the transcription start sites at 100 and 126 bp upstream of the exon 1-intron 1 junction (Fig. 1).


Figure 2: A, identification of transcription initiation sites of the GH receptor gene utilized in liver. PE, primer extension. Autoradiograph of the size-fractionated products of a primer extension reaction carried out with 5 µg of adult mouse liver poly(A)RNA is as described under ``Materials and Methods.'' RPA, ribonuclease protection assay. Autoradiograph of RNase-resistant products from analysis of 20 µg of adult mouse liver total RNA is as described under ``Materials and Methods.'' Sequencing reactions were run concurrently in both experiments to determine the sizes of the specific products indicated. B, determination of transcription initiation site usage in luciferase expression plasmids. Autoradiograph of RNase-resistant products from analysis of 20 µg of total RNA from HepG2 cells is as described under ``Materials and Methods.'' The HepG2 cells were transfected with pGL2B-3.6[+53] ( lanes A and B) or promoterless vector (pGL2-Basic) ( lanes C and D). The sizes of the specific products (indicated by arrowheads) are compared with concurrently electrophoresed kinased X174 HinfI DNA markers (shown) and sequencing reactions (not shown). These results represent two separate sets of transfection experiments ( A and C, B and D).




Figure 1: A, the structure of the murine genomic DNA clone 167A. Intron I is indicated by the open bar, exon 1 by the oblique bars, and the 5`-flanking region by the shaded bar. E, EcoRI; C, ClaI; H, HindIII. B, nucleotide sequence of murine GH receptor gene in the vicinity of the transcription initiation sites and exon 1-intron junction. Numbering was arbitrarily established with the most proximal transcription initiation site designated as +1. Major transcription start sites are indicated by asterisks (*) and minor start sites by square bullets ( ). Primer mGHR1 used in primer extension and ribonuclease protection assay experiments to map the transcription start sites is indicated by an arrow. Putative TATA boxes are underlined. Intron sequences are in lowercase. Nucleotides +13 to +100 ( boldface type) are identical to the published cDNA (8).



Sequence analysis in the immediate upstream region from these transcription start sites revealed consensus sequences for TATA boxes located -23 and -52 from the proximal transcription start site (Fig. 1). In addition, this sequence analysis revealed the presence of consensus binding sequences for some common transcription factors such as AP-2 and NF-IL6 (Fig. 1).

Analysis of Promoter Activity of the 5`-Flanking Region of the GH Receptor Gene

The functional role of the 5`-flanking region in the regulation of transcription of the GH receptor gene was assessed by its ability to drive expression of the luciferase reporter gene. A 3.6-kb fragment of the 5`-flanking region of the GH receptor gene, devoid of the exon 1-intron 1 junction, was inserted immediately upstream of the luciferase reporter gene contained in the promoterless expression vector pGL2-Basic. This fusion construct (pGL2B-3.6[+53]) exhibited significant luciferase expression when transiently transfected into HepG2 hepatoma cells (Fig. 3). In multiple transfection experiments, the relative expression of luciferase using pGL2B-3.6[+53] was consistently about 10-fold greater than the background measured with the promoterless vector and 30% of that observed with the positive control that contains the SV40 promoter/enhancer. Ribonuclease protection assays with RNA isolated from the transfected cells indicated that the transcription initiation sites utilized by the fusion construct corresponded to the two major start sites identified in RNA extracted from whole liver tissue (Fig. 2 B). Hence, we conclude that expression of the reporter gene in the fusion construct was initiated at sites identical to that used by the GH receptor gene in liver tissue.

We observed that cotransfection of plasmid pCAT (Promega) as an internal reference significantly decreased the expression of the luciferase fusion construct. This decrease occurred with various preparations of pCAT and luciferase fusion construct DNA and over a range of molar ratios between the two DNAs. Transcriptional interference between a cotransfected reference plasmid and expression plasmids containing gene-specific promoters has also been observed by other investigators (18, 19) . Although this interference precluded the cotransfection of pCAT for purposes of normalization of transfection efficiency between experiments, we believe that the conclusions derived from our transient transfection experiments are valid for the following reasons. 1) The activity of pGL2B-3.6[+53] construct was tested at least six times in triplicate, and the same relative increase in transcriptional activity was observed in each experiment. 2) Triplicate samples in a single experiment varied by less than 20%. 3) Similar results were obtained when DNA prepared by different methods ( i.e. cesium chloride-ethidium bromide gradient (9) , polyethylene glycol precipitation (9) , or anion exchange resin (Qiagen, Chatsworth, CA)) was used for transient transfection. 4) Ribonuclease protection assays established that expression of the reporter gene in the fusion construct was initiated at sites identical to that used by the GH receptor gene in liver tissue. 5) The magnitude of promoter activity is similar to that reported for the recently described ovine GH receptor gene (19) .

To localize putative cis-acting elements regulating transcription of the GH receptor gene, we used exonuclease III to obtain progressively shorter fragments of GH receptor 5`-DNA (Fig. 3), and the expression of these deletion mutants was examined by transient transfection assays. Whereas deletion of the 552-bp of the 5`-upstream region (pGL2B-3.0[+53]) resulted in a significant decrease (60%) in promoter activity (Fig. 3), serial deletions of the next 2845 nucleotides (pGL2B-1.3[+53], pGL2B-422[+53], and pGL2B-385[+53]) did not result in a significant change in expression. These results indicate that the sequence between -3.6 and -3.0 kb is involved in regulation of expression, and the region between -3.0 and -385 bp is devoid of significant regulatory activity. The shortest fragment of DNA that exhibited activity was the 385-bp fragment; the luciferase-fusion construct containing the 155-bp fragment of the 5`-flanking DNA of the GH receptor DNA (pGL2B-155[+53]) did not show any significant activity (Fig. 3). Thus, the deletional analysis demonstrates that a fragment extending from +53 to between -155 and -385 is sufficient for basal promoter activity, and the 552-bp region between -3.6 and -3.0 kb is essential for enhanced expression of the reporter gene.


Figure 3: Transient expression analyses of the promoter-regulatory region of the GH receptor gene. Luciferase expression plasmids were generated by inserting the GH receptor transcription start sites and various portions of the 5`-flanking sequence of the GH receptor gene into the promoterless luciferase plasmid pGL2-Basic. These expression plasmids were transfected into HepG2 hepatoma cells, and luciferase specific activity was measured as described under ``Materials and Methods.'' Results represent the mean ± S.E. of three independent transfections performed in quadruplicate. The activity of promoterless vector (pGL2-Basic) transfected in the same experiments is indicated. Arrows indicate orientation of GH receptor DNA relative to direction of GH receptor gene transcription; exon 1 is indicated by the oblique bars.



To examine the potential cell-type specificity of the GH receptor promoter, the activities of the various reporter gene constructs were assayed in CHO and 3T3 fibroblasts. The expression profiles of these various constructs were essentially similar in HepG2, CHO, and 3T3 fibroblasts (data not shown). These results showed that the deletion of the 552-bp region between -3.6 and -3.0 kb resulted in a significant loss of activity in all of the cell lines tested (Fig. 4), suggesting that the enhancer element located within this 552-bp region may not be involved in tissue-specific regulation of GH receptor gene expression.

Protein Binding Activity of the Putative Enhancer Element

The reduced expression consequent on deletion of the 552-bp region located between 3.0 and 3.6 kb upstream of the transcription start site suggested the presence of an enhancer element(s) within this region. To localize and characterize the putative enhancer element, DNase I footprint assays were conducted to identify the protein binding regions within this DNA fragment. Based on the observation that expression of the GH receptor gene is maximum in liver tissue, crude nuclear extracts from female mouse liver were used in DNase I footprint assays. DNase I digestion of the 552-bp fragment labeled with P on one strand established that nuclear extracts from adult mouse liver protected a 30-bp region (designated FP1) within the 552 fragment (Fig. 5). Separate footprint assays performed with each strand of DNA uniquely labeled established that an identical region was protected on both strands.

EMSA was used to test the protein binding activity of the FP1 sequence with crude nuclear extracts from female mouse liver. Addition of nuclear extracts from female mouse liver to an aliquot of P-labeled FP1 resulted in the formation of a single protein-DNA complex (Fig. 6). To determine whether the DNA-protein complex was sequence specific, we performed competition experiments in which the binding reaction was carried out in the presence of increasing concentrations of either unlabeled FP1 or an unlabeled 30-bp oligonucleotide with random sequence (5`-CCCATGTTAGAATCCCAGCTTATACCCGCAGGCACAACA-3`). Whereas a 50-fold molar excess of unlabeled FP1 eliminated the formation of the DNA-protein complex, the random sequence oligonucleotide did not affect the binding even at a 400-fold molar excess (Fig. 6). Thus, these results demonstrate that these protein-DNA complexes are sequence specific.


Figure 6: Nuclear proteins from mouse liver bind to the GH receptor enhancer. P-Labeled fragment FP1 of the GH receptor enhancer was incubated with nuclear extracts prepared from liver of adult female mice and electrophoresed as described under ``Materials and Methods.'' Competition between labeled and unlabeled specific (FP1, lanes 2-4) or nonspecific (random oligonucleotide, lanes 5-7) DNA at molar excess ratios of 50, 100, and 200 is shown. The bands representing specific and nonspecific DNA-protein complexes are indicated as A1 and NS, respectively.



Experiments with nuclear protein extracts from liver tissue of adult male mice and adult male rats (Fig. 7) also revealed sequence-specific DNA-protein complexes of similar size. Since GH receptor expression in the kidney is second only to that in the liver (7) , we tested the ability of nuclear proteins from the kidney to bind to FP1. Nuclear extracts from adult mouse kidney formed a DNA-protein complex with FP1 that migrated with similar electrophoretic mobility as the complex formed with liver nuclear proteins (Fig. 7). Crude nuclear extracts from HepG2 human hepatoma cells also formed a sequence-specific DNA-protein complex with FP1 that migrated with an electrophoretic mobility similar to that formed with mouse liver and kidney nuclear proteins (data not shown), suggesting that there is a human homolog of the FP1 binding protein(s) present in rodent liver. In contrast to the experiments using FP1, no specific binding of nuclear proteins from liver was found with other regions of the 552-bp fragment (data not shown). Thus, these experiments along with the DNase I footprint assay demonstrate that the 30-bp FP1 sequence is the only element within the 552-bp fragment that exhibits protein binding activity.


Figure 7: Nuclear proteins from rat liver and mouse kidney bind to the GH receptor enhancer. P-Labeled GH receptor enhancer element FP1 was incubated with nuclear extracts prepared from livers of adult male mice ( lane 1), kidneys of male mice ( lane 2), and livers of male rats ( lane 3) and electrophoresed as described under ``Materials and Methods.'' The band representing a specific DNA-protein complex is indicated as A1.



Computer analysis of the nucleotide sequence of FP1 did not reveal any potential DNA binding motifs for known transcription factors. To exclude the possibility that this region contained binding sites for known transcription factors that did not closely match consensus sequences, competition experiments were also performed using readily available oligonucleotides with consensus sequences for some common transcription factors (AP-1, OCT-1, CTF/NF1, GRE, CREB, NF-kB, TFIID, SP-1) (Promega). None of these oligonucleotides competed for binding by liver nuclear proteins to FP1 (data not shown), suggesting that the protein(s) binding to FP1 are novel.

Analysis of Enhancer Element FP1 in a Heterologous Promoter

Deletion of 552 nucleotides from the 5`-end of the 3.6-kb GH receptor DNA (pGL2B-3.0[+53]) resulted in reduced expression, suggesting the presence of an enhancer element within this region (Fig. 3). To confirm the presence of enhancer element(s) within the (3.6-3.0) kb region, this 552-bp fragment was subcloned into the pTK81 luciferase plasmid, which contains a truncated Herpes Simplex I thymidine kinase promoter (12) . Since this promoter has low basal activity, it has been used to identify enhancers. Compared with the pTK81 vector alone, the pTK-552 fusion construct exhibited significantly increased luciferase activity, and this increased activity was similar in both forward and reversed orientation (Fig. 8). These results demonstrate that the enhancer element(s) present within this region can function independently of orientation and of its homologous promoter.

Enhancer function of the FP1 nucleotide sequence identified by DNase I footprinting was tested in transient transfection assays via the ability to enhance the activity of an heterologous promoter. When cloned into the vector pTK81, the FP1 fragment significantly increased the activity of the thymidine kinase promoter in an orientation-independent manner. The activity of this enhancer element is sequence specific, since insertion of a 30-bp oligonucleotide of random sequence into the pTK81 vector had no effect on activity of the thymidine kinase promoter (Fig. 8). In accordance with the previous results obtained with the homologous promoter, the enhancer activity of FP1 was similar in HepG2, CHO, and 3T3 fibroblasts and primary cultures of adult rat hepatocytes.


Figure 8: Analysis of enhancer element FP1 in a heterologous promoter. Expression plasmids were generated by inserting putative GH receptor enhancer fragments or an oligonucleotide with random sequence ( open bars) into the plasmid pTK81 containing the luciferase gene ( solid bars) driven by the thymidine kinase ( TK) promoter ( oblique bars). Luciferase fusion plasmids were transfected into HepG2 hepatoma cells, and luciferase activity was measured as described under ``Materials and Methods.'' Luciferase specific activity in cell homogenates is expressed as average -fold increase over activity obtained by transfecting equimolar amounts of plasmid pTK81. Results represent the mean ± S.E. of four to seven independent transfections performed in quadruplicate. Arrows indicate orientation of GH receptor DNA relative to direction of GH receptor gene transcription. Position of restriction site for XbaI ( X) is indicated.



Developmental Regulation of Functional and DNA Binding Activity of Enhancer Element FP1 in Liver

Previous studies have shown that in a number of species such as cow (20, 21) , lamb (6, 22, 23) , pig (3) , rat (24, 25) , and mouse,() GH receptor expression in the liver is very low in the fetus compared with the adult. To examine the potential involvement of the enhancer FP1 in the activation of GH receptor expression in postnatal life we compared the relative activities of pGL2B-3.6[+53] and pGL2B-3.0[+53] in transfected rat fetal and adult hepatocytes. These results demonstrate that -3.0 kb of the 5`-flanking sequence of the GH receptor gene was sufficient to promote the expression of the reporter luciferase gene in both fetal and adult hepatocytes (4-fold over the background) (Fig. 9). However, in contrast to the results obtained with adult hepatocytes, the activity of pGL2B-3.6[+53] was similar to that of pGL2B-3.0[+53] in fetal hepatocytes. Similarly, enhancer element FP1 had little effect on the activity of the heterologous thymidine kinase promoter in fetal hepatocytes compared to its robust enhancer activity in adult hepatocytes (Fig. 9). These results suggest that FP1 may play a role in the developmental expression of the GH receptor gene in the liver.


Figure 9: Activity of enhancer element in adult and fetal hepatocytes. Isolated adult and fetal hepatocytes were transfected with expression plasmids containing the 3.6- and 3.0-kb DNA fragment ( panel A) or FP1 enhancer element ( panel B) of the 5`-flanking region of the murine GH receptor DNA, and luciferase activity was measured as described under ``Materials and Methods.'' Activities are expressed relative to that of the respective enhancerless vector, pGL2-Basic ( panel A) or pTK81 ( panel B). Results represent the mean ± S.E. of three independent transfections performed in quadruplicate.



To test the hypothesis that the absence of activity of the enhancer element FP1 in fetal hepatocytes was due to differential interaction with the cognate DNA binding protein(s), the protein binding activity of FP1 with nuclear extracts from murine fetal liver was compared with that obtained with adult liver nuclear extracts. DNase I footprint assays were performed to compare the DNA-protein contact points in adult and fetal nuclear proteins. DNase I digestion of the P-labeled 552-bp region established that nuclear extracts from fetal and adult mouse liver protected the same 30-bp FP1 region (Fig. 10), indicating that the DNA-protein interaction with adult and fetal nuclear proteins occurred at the same DNA site. EMSA was used to further characterize the protein(s) interacting with the enhancer FP1 in fetal hepatocytes. These results demonstrate that FP1 formed a single sequence-specific protein-DNA complex with fetal (day 19 of gestation, term = 19-20 days) liver extracts that migrated with an electrophoretic mobility faster than that observed with the protein-DNA complex formed with adult nuclear proteins (Fig. 11). To ascertain the developmental profile of the protein binding activities of the enhancer region, we performed EMSA analysis with liver nuclear extracts from fetal, 1-week-old, and adult (4-week-old) mice. As shown in Fig. 11, the adult pattern of DNA-protein complex was present by 1 week of age. The different electrophoretic mobilities of the DNA-protein complexes formed with adult and fetal liver nuclear extracts could be explained by the expression of different DNA binding proteins, modified forms of a single binding protein, or differences in proteolytic degradation. Although conclusive resolution of this matter must await purification and comparison of these DNA binding proteins, proteolytic degradation is unlikely to be responsible for these results because these nuclear extracts were prepared in the presence of a mixture of protease inhibitors, and these findings were reproducible in three separate nuclear extracts prepared by two different protocols. Thus, the results from the EMSA and DNase I footprint assays indicate a differential interaction with nuclear proteins from fetal and adult hepatocytes and, in conjunction with the results of the transient transfection experiments, support a role for the enhancer element FP1 in the developmental expression of the GH receptor gene.


Figure 11: Upper panel, nuclear proteins from fetal mouse liver bind to the GH receptor enhancer. P-Labeled enhancer element (FP1) was incubated with nuclear extracts prepared from livers of fetal mice (day 19 of gestation) and subjected to EMSA. Competition between labeled and unlabeled specific (FP1, lanes 2-4) or nonspecific (random oligonucleotide, lanes 5-7) DNA at molar excess ratios of 0, 50, 100, and 200 is shown. The bands representing specific DNA-protein complexes are indicated as F1. Lower panel, ontogenic pattern of protein-DNA interaction between mouse liver nuclear proteins and enhancer element FP1. P-Labeled GH receptor enhancer (FP1) was incubated with nuclear extracts prepared from livers of fetal (day 19 of gestation, lane 1), 1-week-old ( lane 2), and 4-week-old mice ( lane 3) and subjected to EMSA. The bands representing specific DNA-protein complexes are indicated as A1 in the adult and F1 in the fetal nuclear extracts, respectively. The nonspecific DNA-protein complex is indicated as NS.




DISCUSSION

Current data suggest that the regulation of GH receptor expression is complex and possibly involves both transcriptional and post-transcriptional mechanisms. This study was undertaken to identify DNA sequences involved in the regulation of transcription of the murine growth hormone receptor gene. Utilizing RNA isolated from liver of male, female, and pregnant mice, two major transcription start sites were identified adjacent to a non-coding exon, which we have termed exon 1. These transcription start sites are appropriately located with respect to consensus TATA box sequences. It is noteworthy that in sheep liver, O'Mahoney et al. (19) identified a single transcription initiation site located in the context of consensus sequences for TATA and CCAAT boxes. Analysis of the genomic organization of the 5`-region of the gene established that the untranslated exon 1 is spliced into an acceptor site located 12 bp upstream of the initiator ATG in exon 2. A similar organization of the 5`-untranslated region adjacent to the first coding exon has been reported for the sheep, rabbit, bovine, and human GH receptor genes. Thus, all the currently available data indicate that the GH receptor gene exhibits a complex organization in its 5`-regulatory region. Whether this multiplicity of transcription start sites and 5`-untranslated exons plays a role in developmental or tissue-specific expression of the GH receptor gene remains to be elucidated.

This study has established that the 5`-flanking region of the murine GH receptor gene exhibits promoter activity when transfected into rat hepatocytes. Although the rate of transcription of the GH receptor gene is not known, the level of activity of the 3.6-kb fragment of 5`-flanking DNA is compatible with the relatively low concentration of GH receptor mRNA in the liver (24) and is comparable with that reported for the ovine GH receptor promoter (19) . Deletional analysis localized the minimal promoter activity to a region from +53 to between -155 and -385 bp. Sequence analysis of this region revealed consensus sequences for some common transcription factors such as NF-IL6, AP-2, and CTF/CBP. A similar array of consensus sequences was reported in the promoter region of the sheep GH receptor gene (19) . Whether these consensus sequences play any role in the regulation of transcription of the GH receptor remains to be determined.

In most species studied, GH receptor expression displays a distinct ontogenic pattern with the expression of receptor being absent or minimal before birth and exhibiting a marked increase postnatally. These experimental data correlate with the observation in humans that intrauterine growth is for the most part GH independent (27) . The factors regulating this ontogenic pattern and the molecular mechanism(s) involved in this development-specific expression of the GH receptor gene are not known. A previous study from this laboratory (25) had excluded thyroid hormone as one of the factors responsible for the initiation of postnatal expression of the GH receptor gene in the rat. In the current report, we have identified an enhancer element (termed FP1) in the 5`-flanking region of the murine GH receptor gene whose activity was significantly lower in fetal than in adult hepatocytes, correlating with the early ontogenic pattern of expression of the GH receptor gene in liver. Furthermore, the protein binding profile of FP1 was different with nuclear extracts from fetal and adult liver, suggesting that differences in expression of cognate trans-acting factors specific for FP1 may play a role in the postnatal expression of the GH receptor in liver. The presence of a DNA-protein complex with faster electrophoretic mobility in fetal nuclear extracts compared to that seen with adult nuclear extracts suggests either that the adult complex is formed by a protein that is simply larger than in the fetal DNA-protein complex or that the addition of a subunit(s) to the fetal complex results in the larger adult complex. The presence of identical footprints after DNase I digestion would favor the model where a subunit(s) is added to the fetal complex to form the adult complex. Since the subunit present in adult hepatocytes may not come in contact with DNA, this interaction would preserve the footprint obtained with fetal nuclear proteins. The enhancer element FP1 functions in both rat and HepG2 human hepatoma cells, indicating that the cognate DNA binding protein(s) is conserved across mammalian species and that its function is not species specific. The DNA sequence of FP1 did not reveal any homology to binding motifs for known transcription factors. In preliminary experiments (data not shown), the molecular mass of the protein binding to FP1 in adult hepatocytes was determined to be about 22 kDa. It is noteworthy that this size does not correspond to that of any of the well known transcription factors regulating liver-specific transcription, including HNF-1 (28) , C/EBP (29) , DBP (30) , HNF-3 (31) , HNF-4 (31) , and LF-A1 (26) . Our current efforts are directed toward the identification and characterization of the protein(s) binding to FP1.

In summary, we have identified major transcription start sites for the murine GH receptor gene, partially characterized the promoter region, and identified an enhancer element in the 5`-flanking region of the gene. Preliminary analysis of the enhancer region suggests that it defines a novel protein-DNA binding motif and is involved in the developmental expression of the murine GH receptor gene.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant K08-HD00986 (to R. K. M.), a New Investigator Award from the Child Health Research Center (NIH-HD28836), the Children's Hospital of Pittsburgh, and the Vira I. Heinz Foundation. 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/EMBL Data Bank with accession number(s) U06224.

§
To whom correspondence should be addressed: Division of Endocrinology, Dept. of Pediatrics, Children's Hospital of Pittsburgh, 3705 Fifth Ave., Pittsburgh, PA 15213. Tel.: 412-692-5806; Fax: 412-692-6449.

The abbreviations used are: GH, growth hormone; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); CHO, Chinese hamster ovary; EMSA, electromobility shift assay; nt, nucleotides.

R. K. Menon, M. Singh, and L. Zou, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to Dr. Mark A. Sperling for encouragement and support during the course of this project. The technical suggestions given by Drs. Trucco, Giorda, Franz, and Nararayanan are greatly appreciated.


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