From the
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.
The essential role of growth hormone (GH)
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.
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.
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
To demonstrate
that the full 5`-untranslated region was contained within the putative
first exon (exon 1), ribonuclease protection assays were performed.
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.
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
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.
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.
The
nucleotide sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) U06224.
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
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) .
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.
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
10
cells 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.
10
in 2 ml of adhesion
medium per plate. After incubation in 95% O
, 5% CO
at 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(dI
dC), 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.
Cloning of 5`-Flanking Region of the GH Receptor
Gene
Six clones were isolated after screening
approximately 2
10
plaque-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.
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.
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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.