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INTRODUCTION |
Growth hormone-releasing hormone
(GHRH)1 plays a major role in
stimulating both synthesis and release of growth hormone (GH) in the
anterior pituitary through its specific receptor. So far, the GHRH
receptor (GHRH-R) has been cloned in human (1, 2), mouse (3), rat (1),
and pig (4). The GHRH-R is a member of G protein-coupled receptor
family and transduces GHRH-dependent increase in intracellular cAMP via
Gs activation for stimulating somatotroph proliferation and
GH gene expression (1-4). Lin et al. (5) demonstrated that
one amino acid substitution of the GHRH-R in the little
mouse, showing genetically transmitted dwarfism, caused GH deficiency
and somatotroph hypoplasia. In addition, an amber-type mutation
(Glu72Stop) of the GHRH-R in humans was demonstrated to cause profound
GH deficiency (6). These genetic disorders suggest the physiological
significance of GHRH-R in hypothalamo-pituitary GH axis.
The relationship between the dynamics of GHRH-R expression and GH
secretion remains to be clarified. Not only the functional defects of
GHRH-R but also the amount of GHRH-R should affect GH synthesis and
secretion in the pituitary. Hypothalamic hormone, neurotransmitters,
various hormonal states, and nutrition all could modulate the activity
of the GHRH-GH axis. For instance, glucocorticoids potentiate GHRH
action and enhance GH secretion in rats (7-9), whereas they have a
biphasic effect in humans (10, 11). A recent study showed that GHRH-R
gene expression was increased by glucocorticoids in rats (12). The
result suggests that glucocorticoids stimulate GH synthesis and
secretion, at least in part, via up-regulation of GHRH-R gene
expression. Interestingly, Horikawa et al. (13) observed a
marked reduction of GHRH-R mRNA in GHRH-deprived neonatal rats.
This observation suggests that GHRH up-regulates GHRH-R expression in
addition to direct action to stimulate GH synthesis and secretion.
Thus, we need to take the alteration of GHRH-R expression into account
to understand the regulatory mechanism of GH secretion.
The GHRH-R transcripts have a highly specific distribution in the
anterior pituitary, even though the GHRH-R gene is also expressed in
the rat hypothalamus (14). During the development of the anterior
pituitary gland, the GHRH-R transcripts were detected at embryonic day
16 in somatotrophs following the gene activation of Pit-1, a
pituitary-specific POU domain transcription factor (15). The GHRH-R are
not expressed in the pituitary of Pit-1-defective Snell/Jackson dwarf
mice (16, 17). Thus, it appears that Pit-1 is required for the GHRH-R
gene expression. However, it remains unknown whether the Pit-1-binding
site is present in the GHRH-R gene and whether it functions for the
GHRH-R expression.
In the present study, we have cloned and characterized the 5'-flanking
region of the human GHRH-R gene to elucidate the regulatory molecular
mechanism of the human GHRH-R gene expression. Furthermore, we
demonstrated that the 5'-flanking region contained a functional promoter and that its promoter activity was dependent on Pit-1.
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EXPERIMENTAL PROCEDURES |
Cloning of the 5'-Flanking Region of the Human GHRH-R Gene by PCR
Amplification Method--
We cloned 5'-flanking region of the human
GHRH-R gene using a PCR-based method for DNA walking in uncloned
genomic DNA. (PromoterFinder DNA walking kit
(CLONTECH)) (18). Adaptor-ligated genomic DNA was
amplified with two consecutive PCRs using two oligonucleotide primer
pairs. One set of primers used in the primary PCR was the adaptor-specific primer ASP1 (5'-CCATCCTAATACGACTCACTATAGGGC-3') corresponding to the synthetic adaptor and the gene-specific antisense primer GSP2 (5'-TCCATGGTGAGCCCAGCAGTGGCT-3') corresponding to the
5'-end of the human GHRH-R cDNA (nucleotides position +5 to
19
according to Mayo (1)). Another set of primers for the secondary PCR
was the adaptor specific primer ASP2 (5'-CTATAGGGCACGCGCGTGGT-3'), nested to ASP1, and the nested gene-specific antisense primer GSP1
(5'-GGGAAGCTTCCCTCCACCAGCCTCAGTAAGCCTT-3',
nucleotides position
19 to
44). The underlined
HindIII site was added to the 5'-end of GSP1 to facilitate
subcloning. PCR was performed according to the manufacturer's
instructions. The PCR conditions were 32 cycles of 94 °C for 25 s and 67 °C for 4 min following 7 cycles of 94 °C for 25 s
and 72 °C for 4 min. PCR products were analyzed by electrophoresis
on a 1% agarose gel.
DNA Sequencing--
PCR products were subcloned into pT7Blue
vector (Invitrogen) or pBluescript SK(+) (Stratagene) and sequenced
with a DNA sequencer (model 373A or 310, Perkin-Elmer, Applied
Biosystems.).
Rapid Amplification of 5'-cDNA Ends--
5'-RACE was
performed using Marathon-Ready cDNA library
(CLONTECH) derived from human pituitary glands
according to the manufacturer's instructions. In the first experiment,
PCR was performed using ASP1 (5'-CCATCCTAATACGACTCACTATAGGGC-3')
specific to the adaptor ligated to cDNA ends and GSP5
(5'-TTGTACAGGCACTCTCATCCTCTCTCA-3') corresponding to the coding region
from +101 to +127 of the human GHRH-R cDNA (1). Cycling parameters
used in this 5'-RACE protocol were 35 cycles of 94 °C for 30 s,
64 °C for 30 s and 68 °C for 2 min 30 s. PCR products
were analyzed by electrophoresis on an ethidium bromide-stained 1.2%
agarose gel. The PCR products were subcloned into pT7Blue vector and
sequenced with a DNA sequencer.
RNase Protection Analysis--
RNase protection assays were
performed, based on the method of Gilman (19). A DNA fragment
corresponding to nucleotides
2207 to
19 was inserted into
pBluescript SK(+). The plasmid containing the DNA fragment from
2207
to
19 was linearized with BalI and used as a template for
antisense cRNA probe. The antisense cRNA probe was synthesized by
T3 RNA polymerase and [
-32P]CTP. The
labeled probe (105 cpm) was hybridized to 4 µg of normal
human pituitary gland poly(A)+ RNA
(CLONTECH) in 30 µl of hybridization buffer
containing 80% formamide, 40 mM PIPES, 400 mM
NaCl, and 1 mM EDTA overnight at 50 °C. Following the
hybridization step, RNases A and T1 were added to the hybridization
mixture, and the mix was incubated for 1 h at 37 °C. The
protected fragments were analyzed by electrophoresis on a 6%
polyacrylamide/50% urea sequencing gel. A bacteriophage M13 mp18
single strand DNA sequencing ladder was prepared using primer (
40) to
size the protected fragment.
Cell Culture--
Cells from the rat pituitary-derived cell
line, GH3, were cultured in Ham's F-10 medium supplemented with 15%
(v/v) horse serum and 2.5% (v/v) fetal bovine serum. HeLa and BeWo
cells were maintained in Dulbecco's modified Eagle's medium with 10%
(v/v) calf serum. All culture media contained penicillin (100 units/ml) and kanamycin (100 µg/ml).
Reporter Gene Construction and Transient Expression
Assays--
A 2189-nucleotide HindIII fragment of the
5'-flanking genomic sequence (from
2207 to
19, when the translation
start site was numbered as +1) of GHRH-R gene was subcloned into a
promoterless luciferase reporter vector, pGL3 basic vector (Promega),
and the resulting reporter gene was named as hGHRHR-Luc(
2207,
19).
This hGHRHR-Luc(
2207,
19) was digested with SacI, and
after removing SacI/SacI fragment, the remaining
vector was self-ligated with T4 DNA Ligase to form
hGHRHR-Luc(
1377,
19). A DraI/HindIII fragment was prepared from hGHRHR-Luc(
1377,
19) by digesting with
DraI and HindIII. This fragment was inserted into
the multicloning site of pGL3 basic vector digested with
SmaI and HindIII to form hGHRHR-Luc(
941,
19).
hGHRHR-Luc(
399,
19) was prepared by self-ligating after the
digestion of hGHRHR-Luc(
2207,
19) with BalI and
SmaI. hGHRHR-Luc(
310,
19) was prepared by self-ligating
after the digestion of hGHRHR-Luc(
2207,
19) with ApaI and
SmaI. hGHRHR-Luc(
130,
19), hGHRHR-Luc(
120,
19),
hGHRHR-Luc(
110,
19), and hGHRHR-Luc(
100,
19) were prepared
by inserting PCR-amplified fragments from
130 to
19, from
120 to
19, from
110 to
19, and from
100 to
19 into pGL3 basic
vector, respectively. The regions from
130 to
120 and
310 to
130 have putative Pit-1-binding sites P1 and P2, respectively. P1
contains one Pit-1-binding element from
129 to
123, and P2 contains
two Pit-1-binding elements from
166 to
160 in upper strand (P2
upper) and from
165 to
171 in lower strand (P2 lower). To clarify
their role in stimulating GHRH-R gene expression, reporter plasmid
containing mutation of P1 (P1m; TGTACGA, mutated
sequences are underlined), P2 upper (P2m;
TAGTAAG), or both P2 upper and lower (dP2m;
GTCAGTCGTAAG), was made.
These plasmids were introduced into GH3, HeLa, or BeWo cells using
LipofectAce (Life Technologies, Inc.). 2 µg of GHRH-R-luciferase reporter construct, pGL3 basic vector alone, or rat PRL-luciferase reporter construct, with or without RSV-Pit-1 expression vector, was
transfected to the cells in 60-mm dishes. Cells were harvested 48 h after transfection, and luciferase activity was measured using
Luciferase Assay System and Luminometer TD-20/20 (Promega). Protein
concentration was measured by Bio-Rad protein assay based on the method
of Bradford (29).
DNase-I Footprint Analysis--
For footprint studies,
recombinant His-tagged Pit-1 was produced in Escherichia
coli and partially purified with nickel-nitrilotriacetic acid
Resin (Qiagen) according to the manufacturer's instructions. His-tagged Pit-1 appeared to have same function compared with recombinant Pit-1 previously described (20). The DNA fragment used for
DNase-I footprint analysis of the promoter and enhancer regions of
GHRH-R gene was prepared by digestion of GHRH-R 5'-flanking region with
Bsu36I and HindIII and uniquely radiolabeled at
the upstream end by filling recessed 3'-termini with
[
-32P]deoxy-ATP using DNA polymerase-I. The
radiolabeled DNA fragment was first incubated with purified His-tagged
Pit-1 in a 25-µl reaction containing 10 mM Tris-HCl (pH
7.5), 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and 2 µg of salmon sperm DNA.
After incubation for 30 min at room temperature, 10 µl of DNase-I
(0.05 unit; RQ-1 DNase-I, Promega) in 100 mM Tris-HCl (pH
7.5) and 35 mM MgCl2 were added, and the sample
was incubated for 8 min at room temperature. The reaction was
terminated by the addition of 85 µl of 30 mM EDTA, and
then the sample was extracted with and equal volume of 1:1 (v/v)
phenol-chloroform mixture. After precipitation of the aqueous fraction
with ethanol, the samples were analyzed by denaturing polyacrylamide
gel electrophoresis and autoradiography.
Mobility Shift Assays for Analysis of Pit-1 DNA
Binding--
Binding reactions contained 5000 cpm
32P-labeled oligonucleotide probe, varying amounts of
Pit-1, 5 µg of poly(dI-dC), 10 mM Tris-HCl (pH 7.5), 5%
glycerol, 50 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol in a total volume of 25 µl. The
oligonucleotide probes used were as follows: P1 probe,
5'-TTGGCTAGCTCCTGCCTATGCAAACAGCCACCTG-3' (
145 to
112); P2 probe,
5'-CCTGGTGAATATTCAGCGGTCTGTGTCCCCTTGG-3' (
175 to
142); P1m probe,
5'-TTGGCTAGCTCCTGCCTGTACGAACAGCCACCTG-3' (mutated sequences are underlined); and P2m probe,
5'-CCTGGTGAATAGTAAGCGGTCTGTGTCCCCTTGG-3'. The mutated oligonucleotides had the same mutations as the
reporter gene used in the transfection analysis. Reactions were
incubated for 30 min at room temperature and then analyzed on
nondenaturing, 4% polyacrylamide gels.
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RESULTS |
Cloning of 5'-Flanking Region of the Human GHRH-R Gene by PCR-based
Method--
First, we cloned 5'-flanking region of the human GHRH-R
gene as described under "Experimental Procedures." We obtained
three genomic fragments, which were about 6, 2.7, and 0.9 kb in size, respectively. The nucleotide sequence of the 0.9-kb fragment was identical to that of 3'-side of the 2.7-kb fragment. In addition, restriction mapping of those fragments indicated that the 6-kb fragment
contained the 2.7- and 0.9-kb fragments. The complete sequence of the
2.7-kb genomic fragment is shown in Fig.
1.

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Fig. 1.
Nucleotide sequence of the 5'-flanking region
of the human GHRH-R gene. The 5'-flanking region is shown in
uppercase letters. The coding region is shown in
lowercase letters. Numbers are relative to the
adenosine of the initiation codon. The putative Pit-1-binding elements
are indicated with open boxes, and other putative regulatory
elements are underlined. The locations of primers used for
PCR-DNA walking are indicated by dotted lines.
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According to the analysis of the 5'-flanking sequence of the human
GHRH-R gene by Mac DNASIS Pro software (Hitachi), no typical mammalian
TATA boxes were present, but a CAAT box was present at nucleotides
399 to
393. In addition, there were several putative regulatory
elements similar to consensus elements, AP-1 (
1558 to
1552), AP-2
(
241 to
230), SP1 (
1179 to
1171), CRE (
524 to
517), GREs
(
983 to
969,
1506 to
1492, and
2474 to
2460), two EREs
(
662 to
648 and
1887 to
1873), and eleven putative Pit-1-binding elements. The locations of one nucleotide mismatched Pit-1-binding site consensus are as follows: nucleotides
129 to
123,
166 to
160,
171 to
165,
434 to
428,
839 to
833,
1049 to
1043,
1138 to
1132,
1336 to
1330,
1631 to
1625,
1816 to
1810, and
1881 to
1875.
Analysis of the 5'-Flanking Region of the Human GHRH-R
Gene--
To analyze the 5'-end of the human GHRH-R cDNA, we
performed 5'-RACE experiments. The 5'-RACE analysis using ASP1 and GSP5 as primers showed that about 300-nucleotide band was amplified (Fig.
2A). From the sequence
analysis of these products, the 5'-end of the longest clone was found
to extend to 137 nucleotides upstream from the translation start site.
The 5'-ends of all the RACE products are indicated with solid
circles in Fig. 2B. To confirm the results of 5'-RACE
experiments, RNase protection assays were performed. RNase mapping with
a 381-nucleotide labeled cRNA probe complementary to
399 to
19 gave
a major protected band of 104 nucleotides with several faint protected
bands (Fig. 3). The result indicated that
the major transcription start site was 122 nucleotides upstream from
the translation start site. In addition, the several faint bands
suggested the existence of possible minor start sites.

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Fig. 2.
Analysis of the 5'-untranslated region of
human GHRH-R mRNA. A, analysis of the 5'-RACE products.
The 5'-RACE experiment was performed using a pair of primers, AP1 and
GSP5, as described under "Experimental Procedures." Marathon-Ready
cDNA library (CLONTECH) derived from human
pituitary gland was used as a template. 5'-RACE products were analyzed
on an ethidium bromide-stained 1.2% agarose gel. Lanes 1 and 2, molecular weight standards. Lane 3, RACE
products. bp, base pairs. B, nucleotide sequence
of the 5'-noncoding region of the human GHRH-R gene. The 5'-flanking
region is shown in uppercase letters. The coding region is
shown in lowercase letters. Numbers are relative
to the adenosine of the initiation codon. The 5'-ends of all the RACE
products are indicated with solid circles. A solid
triangle indicates the major transcription start site determined
by RNase protection assay.
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Fig. 3.
RNase mapping of the transcription start
sites of the human GHRH-R gene. A, autoradiogram of RNase
protection assay. RNase protection assay was performed as described
under "Experimental Procedures." A uniformly labeled probe
corresponding to bases 399 to 19 was hybridized to 4 µg of human
pituitary poly(A)+ RNA or 20 µg of yeast tRNA and
digested with RNases A and T1. The protected fragments were analyzed by
electrophoresis on a 6% polyacrylamide/50% urea sequencing gel.
Lane 1, human pituitary poly (A)+RNA. Lane
2, tRNA. Lanes M, bacteriophage M13 mp18 single strand DNA
sequence when primer ( 40) was used. The major protected band is
indicated by an arrow. bp, base pairs.
B, schematic representation of the probe used for RNase
protection assay and protected fragment. The major transcription start
site is indicated by a bent arrow. Numbers are
relative to the adenosine of the initiation codon.
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Promoter Activity of the Human GHRH-R Gene--
We performed
transient transfection studies to determine whether our cloned
5'-flanking region is functional. Plasmids containing cloned
5'-flanking sequences of the human GHRH-R gene fused to the luciferase
gene were transfected into rat pituitary tumor cell line GH3 and
heterologous cell lines HeLa and BeWo. As illustrated in Fig.
4, a significant increase of luciferase
activity was observed in GH3 cells transfected with
hGHRHR-Luc(
2207,
19), but not in HeLa cells and BeWo cells. The
result indicated that the 5'-flanking region has pituitary-specific
promoter activity.

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Fig. 4.
Promoter activity of the 5'-flanking region
of the human GHRH-R gene in heterologous cells. The human GHRH-R
5'-flanking 2.2-kb fragment ( 2207 to 19) was subcloned into the
promoterless luciferase plasmids, pGL3 basic vector. Two µg of these
expression plasmids were transfected into GH3, HeLa, or BeWo cells, and
luciferase activity was measured as described under "Experimental
Procedures." rPRL-Luc, a reporter plasmid containing rPRL gene, was
used as a positive control. Each value was normalized for total
protein. Results represent the means ± S.E. for three
transfections from a representative experiment.
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Next, to define DNA regions important for gene expression, deletion
mutants of the 5'-flanking sequences were constructed and used to
transfect GH3 cells. Deletion of upstream between
2207 and
310
resulted in a moderate increase in luciferase activity. Further
deletion to
130 decreased activity remarkably. Deletion to
120 or
100 resulted in a level of activity that was comparable with that of
the pGL3 basic vector (Fig.
5A). This finding suggested that the regions between
310 and
130 and between
130 to
120 were important for the expression in GH3 cells. The regions from
130
to
120 and
310 to
130 have putative Pit-1-binding sites P1 and
P2, respectively. P1 contains one Pit-1-binding element (
129 to
123), and P2 contains two Pit-1-binding elements, one from
166 to
160 (P2 upper) and one from
171 to
165 in the opposite
orientation (P2 lower). hGHRHR-Luc(
310,
19) containing mutations in
either P2 upper or P1 showed significantly lower luciferase activity
than wild hGHRHR-Luc(
310,
19), and hGHRHR-Luc(
310,
19) containing mutations in both P2 upper and P2 lower showed a
marked reduction in activity, which was almost the same as that of the pGL3 basic vector (Fig. 5B). These findings, consistent with
the results from the deletion study, suggested that P1, P2 upper, and
P2 lower contribute to stimulation of GHRH-R expression.

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Fig. 5.
Expression analysis of the 5'-flanking region
of the human GHRH-R gene in GH3 cells. A, 2 µg of reporter
plasmids were transfected into GH3 cells by lipofection method and
harvested 2 days after transfection. rPRL-Luc, a reporter plasmid
containing rPRL gene, was used as a positive control. Luciferase
activity was measured as described under "Experimental Procedures."
Each value was normalized for total protein. Results represent the
means ± S.E. for three transfections from a representative
experiment. Deletion of the 5'-flanking sequences from the 5'-end
showed a moderate increase in luciferase activity as the deletion
progressed from 2207 to 310. The deletion mutant, progressed to
130, showed a remarkable decrease in activity. The deletion mutant to
120 further decreased its activity. B, the regions from
130 to 120 and 310 to 130 have putative Pit-1-binding sites P1
and P2, respectively. P1 contains one Pit-1-binding element from 129
to 123, and P2 contains two Pit-1-binding elements from 166 to
160 in upper strand (P2 upper) and from 171 to 165 in lower
strand (P2 lower). To clarify their role in stimulating GHRH-R gene
expression, reporter plasmid containing mutation of P1 (P1m;
TGTACGA, mutated sequences are
underlined), P2 upper (P2m; TAGTAAG), or both
P2 upper and lower (dP2m;
GTCAGTCGTAAG) was made
and transfected to GH3 cells. Also, pGL3 basic vector (negative
control) or rPRL-Luc (positive control) was transfected into GH3 cells,
and luciferase activity was measured. hGHRHR-Luc( 310, 19) containing
P2 upper mutation (P2 m) or P1 mutation (P1m) showed significantly
lower luciferase activity than wild hGHRHR-Luc( 310, 19), and
hGHRHR-Luc( 310, 19) containing both P2 upper and lower mutations
(dP2 m) showed marked reduction in the activity, which was almost same
as background activity of pGL3 basic vector. On the other hand, when
hGHRHR-Luc( 130, 19) was used as the reporter plasmid, P1 mutation
did not change the luciferase activity, suggesting that P1 may work in
the reporter plasmid with a longer 5'-region.
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On the other hand, when hGHRHR-Luc(
130,
19) was used as the reporter
plasmid, a P1 mutation did not change the luciferase activity,
suggesting that P1 may work in the reporter plasmid with a longer
5'-region (Fig. 5B). The drop in luciferase activity due to
deletion of the region between
130 to
120, which was observed in
the deletion study, could not be due to the loss of Pit-1 binding
because inactivation of the Pit-1 site by mutation has no effect in the
context of a short upstream region. In BeWo cells, co-transfection of
the deletion mutants down to
310 and RSV-Pit-1 expression vector
increased luciferase activity compared with control. However, deletion
to
130 resulted in a decrease in the activity in BeWo cells even when
Pit-1 expression vector was co-transfected (Fig.
6). Results of these transfection studies indicated that the region from
310 to
130 was important for Pit-1-dependent GHRH-R gene expression in BeWo cells.

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Fig. 6.
Expression analysis of the 5'-flanking region
of the human GHRH-R gene in BeWo cells co-transfected with RSV-Pit-1
expression vector. Two µg of reporter plasmids containing
various deletion mutants of the human GHRH-R 5'-flanking sequences were
transfected respectively into BeWo cells by lipofection method with or
without 1 µg of Pit-1 expression vector, and luciferase activity was
measured as described under "Experimental Procedures." Each value
was normalized for total protein. Results represent the means ± S.E. for three transfections from a representative experiment. The
deletion mutant, progressed to 130, showed a decrease in the activity
in BeWo cells when Pit-1 expression vector was co-transfected.
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DNase-I Footprint Analysis--
The region from
310 to
130,
which was important for the Pit-1-dependent expression of
GHRH-R gene, contains DNA sequence P2 (
171 to
160), similar to the
Pit-1-binding element. To confirm that Pit-1 binds to the sequence,
DNase-I footprint studies were used. Recombinant Pit-1 protected the
region from
176 to
155, in which P2 was included, in a
dose-dependent fashion. However, Pit-1 did not protect P1
(
129 to
123), which is also similar to the Pit-1-binding element
(Fig. 7).

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Fig. 7.
DNase-I footprint analysis. The DNA
fragment used for DNase-I footprint analysis of the promoter and
enhancer region of GHRH-R gene was prepared by digestion of GHRH-R
5'-flanking region with Bsu36I and HindIII and
uniquely radiolabeled at the upstream end by filling 3'-termini with
[ -32P]deoxy-ATP using DNA polymerase. The DNA fragment
contains two putative Pit-1-binding sites located from 129 to 123
(P1) and from 171 to 160 (P2). Recombinant His-tagged Pit-1
protected the region from 176 to 155, in which P2 is included
(indicated by arrow-ended bracket). However, recombinant
Pit-1 did not protect P1.
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Mobility Shift Assays for Analysis of Pit-1 DNA Binding--
To
further examine Pit-1 binding to P1 and P2, a mobility shift assay was
used. A radiolabeled oligonucleotide probe spanning
175 to
142 (P2
probe) formed two specific DNA-protein complexes when incubated with
Pit-1, because P2 has two putative Pit-1-binding elements. The two
complexes were specifically competed by excess unlabeled
oligonucleotide. When the consensus sequence of Pit-1 was mutated in
the P2 probe, the upper DNA protein complex disappeared, confirming
that the element was important for binding of Pit-1. Another
radiolabeled oligonucleotide probe spanning
145 to
112 (P1 probe)
formed only one DNA-protein complex when incubated with the same amount
of Pit-1. When the consensus sequence of Pit-1 was mutated in the P1
probe, the binding of Pit-1 to P1 was reduced, confirming that the site
was a Pit-1-binding element. However, the amount of DNA-protein complex
was smaller than that when P2 probe was used, suggesting that the
binding affinity of P1 to Pit-1 was weaker than that of P2 (Fig. 8).
The finding appeared to be consistent with the result observed in the
DNase-I footprint analysis that Pit-1 did not protect P1 (Fig.
7).

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Fig. 8.
Mobility shift assays for analysis of Pit-1
DNA binding. A radiolabeled oligonucleotide probe spanning 175
to 142 (P2 probe) formed two specific DNA-protein complexes when
incubated with Pit-1, because P2 have two putative Pit-1-binding
elements. The two complexes were specifically competed by the excess of
unlabeled oligonucleotide. When a consensus sequence of Pit-1 was
mutated in P2 probe, upper DNA-protein complex disappeared, confirming
that the site was important for binding of Pit-1. Another radiolabeled
oligonucleotide probe spanning 145 to 122 (P1 probe) formed only
one DNA-protein complex when incubated with the same amount of Pit-1.
When the consensus sequence of Pit-1 was mutated in P1 probe, the
binding of Pit-1 was reduced, confirming that the site was a
Pit-1-binding element. However, the amount of DNA-protein complex was
smaller than that when P2 probe was used, suggesting that the binding
affinity of P1 to Pit-1 was weaker than that of P2.
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 |
DISCUSSION |
In the present study, we cloned the 5'-flanking region of the
human GHRH-R gene by the PCR-based DNA walking method and determined transcription start site with RNase protection assay and 5'-RACE. Furthermore, we analyzed its function and Pit-1 dependence using transient transfection assay and determined Pit-1-binding sites with
DNase-I footprint analysis and mobility shift assay. PCR-based methods
are available for walking from a known region to an unknown region of
the genome. Recent improvement of PCR technique allowed us to obtain
longer and more accurate sequences of genomic DNA of interest. We
applied the method reported by Siebert et al. (18) to clone
the 5'-flanking region of the human GHRH-R gene.
Recently Petersenn et al. (21) reported that 5'-flanking
region of human GHRH-R. They (22) and we (23, 24) have been independently trying to clone the 5'-upstream sequence of GHRH-R to
analysis the mechanism of GHRH-R gene expression. The sequence reported
here is almost identical to that reported by Petersenn et
al., although minor differences are present. The reason why minor
difference exists between theirs and ours is unknown. This difference
may be explained by polymorphism.
The result of our RNase protection assay indicated that the major
transcripts appeared to start at
122 nucleotides upstream from the
translation start site in the normal pituitary gland (Fig. 3). Also,
the 5'-RACE products were close to that of the major band obtained in
RNase protection assay, supporting the results in RNase protection
assay (Fig. 3). However, some shorter bands were detected in 5'-RACE
experiments and in RNase protection assay. This finding suggests that
some transcripts start from the downstream sites of the major
transcript start site, which is often observed in TATA-less promoter.
Petersenn et al. (21) reported that the transcription start
site of GHRH-R gene was 40 nucleotides upstream from the translation start site. However, the site reported by Petersenn et al.
is located in the 5'-untranslated region of GHRH-R mRNA already
reported by Mayo (1). In addition, our results obtained from RNase
protection assay and 5'-RACE showed that the site was located in the
sequence to be transcribed to mRNA. Thus, it is unlikely that the
site reported by Petersenn et al. is a major transcription
start site.
No intron was found in the 5'-flanking region of the human GHRH-R gene,
because the sequence of the 5'-RACE products was completely identical
to that obtained from the analysis of genomic DNA flanking to GHRH-R
coding region. In addition, these results suggest the accuracy of the
PCR-based method. The structure of the GHRH-R gene resembles that of
recently characterized human vasoactive intestinal peptide receptor
gene (25), which belongs to the same subfamily of G protein-coupled
receptors, in respect to lack of intron in its 5'-untranslated region.
Typical TATA homologies were not present in the 5'-flanking sequence
upstream from the transcription start site. A number of genes are known
to lack obvious TATA boxes. Such TATA-less promoters can be divided
into two classes. One class consists of GC-rich promoters (26), found
in housekeeping genes, which usually contain several transcription
start sites spread over a fairly large region and several Sp1-binding
sites (27, 28). Another class has no apparent TATA boxes and is not
GC-rich (27). Many of these promoters are not constitutively active but
rather are regulated during differentiation and development (27). In the human GHRH-R gene, GC content appeared not so rich, and neither a
CAAT box nor a typical Sp1-binding site was at the site appropriate to
the start site. Thus, the human GHRH-R promoter may belong to the
latter class.
Despite the absence of typical TATA homologies, analysis of the 2.7-kb
nucleotide sequence of the 5'-flanking region revealed that it
contained a number of putative regulatory cis-acting
elements. Of particular interest, we identified several sequences
corresponding to Pit-1-binding consensus (TATNCAT) (Fig. 1). As
described previously, the GHRH-R gene expression is considered to be
controlled by Pit-1 (15-17). In fact, we demonstrated that our cloned
5'-flanking region of the gene increased luciferase activity in
Pit-1-expressing GH3 cells and that the 5'-flanking region increased
luciferase activity in non-Pit-1-expressing BeWo cells exclusively when
RSV-Pit-1 expression vector was co-transfected. The region
310 to
130 was required for Pit-1-dependent promoter activity in
BeWo cells according to the deletion mutant analysis. In the region,
there are two Pit-1-binding elements, P2 upper and P2 lower. The
mutation of P2 upper caused 50% reduction in the luciferase activity,
and the mutation of both P2 upper and P2 lower suppressed the activity to basal levels in GH3 cells. These findings demonstrated that P2 is a
functional site for GHRH-R gene expression. Another important region for GHRH-R gene expression was located from
130 to
120. Although the Pit-1-binding site (P1) was present in the region, mutation of P1 did not affect the luciferase activity when
hGHRHR-Luc(
130,
19) was used as a reporter plasmid. This
finding was consistent with the results that P1 is a low affinity
binding site for Pit-1 compared with P2, which was obtained from
mobility shift assay and DNase-I footprint analysis. In addition, this
appears to explain why co-transfected RSV-Pit-1 expression vector did
not stimulate GHRH-R reporter gene deleted to
130 in BeWo cells.
However, this finding does not exclude the possibility that P1 did not
play a role in stimulating GHRH-R gene expression, because P1 mutation
decreased luciferase activity by 40% when hGHRHR-Luc(
310,
19) was
used as a reporter gene. P1 may function in combination with P2 or
other cis-elements, located from
310 to
130.
On the other hand, the hGHRHR-Luc(
2207,
19) exhibited two times
lower luciferase activity than serial deletion mutant constructs. The
luciferase activity appeared to drop between
2207 and
1377 (Fig.
5A). The results might imply that the region
2207 to
1377 serves as a significant repressor in GH3 cells.
In summary, we have cloned and characterized the 5'-flanking region of
the human GHRH-R gene. The 5'-flanking region contained a functional
promoter, and its promoter activity was dependent on Pit-1. The binding
site of Pit-1 was located from
176 to
155 judging from DNase-I
footprint analysis, and the region containing the Pit-1-binding sites
was important for Pit-1-dependent expression of GHRH-R
gene. Characterization of this 5'-flanking region will further clarify
the regulatory mechanism of human GHRH-R gene expression.