From the Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211
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ABSTRACT |
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The use of alternative promoters represents an
important mechanism for the regulation of growth hormone receptor (GHR)
gene expression. Two promoters have been isolated previously for the GHR gene: the P1 promoter that drives liver-specific expression, and
the P2 promoter that drives ubiquitous expression. In the present
study, we isolated a third GHR promoter termed P3. The P3 promoter was
GC-rich and TATA-less. The P3 promoter was able to drive the expression
of a luciferase reporter gene in cell lines Hep G2, PLC/PRF/5, and
BHK-21. In vivo, the P3 promoter initiated transcription
from two major sites in exon 1C of the GHR gene in many tissues. In the
adult bovine liver, the P3-transcribed GHR mRNA represented only
10% of the total GHR mRNA pool. In non-hepatic tissues such as
kidney, skeletal muscle, mammary gland, and uterus, P3-transcribed GHR
mRNA represented 30-40% of the total GHR mRNA pool. Within
the bovine GHR gene, the P3 promoter was located immediately downstream
from the P2 promoter. In transfected cells, the P2 promoter served as
an enhancer for the P3 promoter. Existence and co-regulation of two
ubiquitous promoters may be a mechanism for achieving a high level of
expression of the GHR gene in multiple tissues.
The growth hormone (GH)1
receptor (GHR) is a member of the cytokine/hematopoietin receptor
superfamily that includes receptors for prolactin, hematopoietin,
erythropoietin, thrombopoietin, granulocyte colony-stimulating factor,
interferons, and many interleukins (1). The highest levels of GHR
expression are found in liver. Expression of GHR is also readily
detectable in many other tissues such as muscle, fat, kidney, and heart
(2-5). In liver, GHR mediates the action of GH on the synthesis and
systemic secretion of insulin-like growth factor-I (IGF-I), which is
essential for growth as well as numerous metabolic processes (6, 7). In
non-hepatic tissues, GHR may mediate the local effect of GH on both
proliferation and differentiation at the cellular level, through
IGF-I-dependent or -independent mechanisms (8-11).
Although the expression of GHR increases dramatically during postnatal
life (5), functional GHR is also found in the early embryo (12) and
fetus (13, 14), suggesting a role of GH/GHR in early embryogenesis and fetal development.
Little is known about the mechanism regulating the ontogeny of the GHR
gene. GHR mRNA is transcribed from a single-copy gene (15, 16),
from which GH-binding protein, a shortened GHR lacking transmembrane
and intracellular domains, is also generated by alternative splicing
(2, 4, 17). The cDNA sequence for GHR has been determined in
various species (2-5, 17-20). The structural organization of the GHR
gene, however, is only known for the human. The human GHR gene is
composed of 10 exons and spans 87 kb from exon 2 to exon 10. The
translation start codon (AUG) is located in exon 2, and transcription
is initiated from exon 1 (15). Cloning of GHR cDNAs in various
species has revealed that the GHR cDNA are heterogeneous in their
5'-untranslated regions (5'-UTRs) (18, 21, 23, 24) and that alternative
5'-UTRs are spliced onto a common splice site 11 bp upstream from the
translation initiating codon AUG in exon 2. This has led to the
hypothesis that the transcription of the GHR gene is controlled by
multiple promoters in exon 1. Consistent with this hypothesis, a
liver-specific GHR promoter (P1) has been isolated in various species
including sheep (25), mouse (26), and human (27). The P1 promoter drives the expression of GHR in the liver of postnatal animals. A
second GHR promoter (P2) has also been isolated in sheep and appears to
drive the expression of GHR in various tissues including liver (13).
Thus, transcription from multiple promoters probably represents an
important mechanism controlling the amount and tissue specificity of
GHR gene expression.
In this paper, we report the isolation and characterization of a novel
promoter for the bovine GHR gene. Initially, we identified 5'-end
sequences for the bovine GHR mRNA in the uterus in an attempt to
isolate GHR with an NH2-terminal extended extracellular
domain that acts as a unique receptor for bovine placental lactogen. This strategy was implemented because the bovine uterus has high affinity binding sites for bovine placental lactogen, and these binding
sites are antigenically similar to the GHR (28). Using rapid
amplification of cDNA ends (RACE), we isolated three new 5'-UTRs
for the bovine GHR cDNA from uterus. However, RNase protection analysis (RPA) showed that the new 5'-UTRs were expressed in many other
tissues including uterus. Cloning and transient transfection analysis
of the genomic region corresponding to these 5'-UTRs have defined a
third promoter (designated P3) for the bovine GHR gene.
RNA Isolation--
Various bovine tissues were collected at
slaughter. Tissues were immediately frozen in liquid nitrogen and
stored at 5'-RACE--
Amplification of the 5'-end sequences of the bovine
GHR cDNA in uterus was performed using 5'-RACE (Life Technologies,
Inc.). 2 µg of total uterine RNA was reverse transcribed into
first-strand cDNA using SuperScript II reverse transcriptase and
the reverse primer E4R (Table I), which
was specific for the exon 4 region of the bovine GHR cDNA. After
degrading the original RNA template with RNase H, the first-strand
cDNA was extended from its 3'-end using terminal transferase to
form a poly(dCTP) tail. The poly(dCTP)-tailed cDNA was then used as
a template in 30 cycles of polymerase chain reaction (PCR) of 94 °C
for 30 s, 55 °C for 45 s, and 72 °C for 1 min with the
reverse primer E3R (Table I) and the forward 5'-RACE abridged anchor
primer 1 (AAP) (Table I). The product of this PCR amplification was
subjected to a second PCR amplification with abridged universal
amplification primer (AUAP) and primer E2R (Table I) for 30 cycles with
an annealing temperature at 58 °C. The PCR components other than
primers and template were included in Ready To Go PCR beads (Amersham
Pharmacia Biotech). The PCR product of the second amplification was
separated by gel electrophoresis in a 2% agarose gel, and bands of
interest were gel purified by using Qiaquick (Qiagen, Valencia, CA).
Purified PCR fragments were cloned into the PCR 2.1 vector (Invitrogen, Carlsbad, CA) and sequenced on an Applied Biosynthesis 377A DNA Sequencer (University of Missouri DNA Core, Columbia, MO). Sequence comparison was made by using the GCG program. Primers except AAP and
AUAP were synthesized by the University of Missouri DNA Core. All
enzymes were purchased from Promega (Madison, WI) unless otherwise indicated.
RPA--
The RPA was used to determine the expression of 5'-UTR
for GHR cDNA in bovine tissues and to map the transcription start
site. The PCR 2.1 plasmid containing the longest 5'-UTR, named 1C3, was
lineralized with SpeI. An antisense probe was generated by in vitro transcription using [32P]CTP (800 mCi/mmol, NEN Life Science Products) and T7 RNA polymerase (Stratagene,
La Jolla, CA). The specific activity of the probe was approximately
5 × 108 cpm/µg. The RPA was performed using the RPA
IITM kit according to the manufacturer's instructions
(Ambion, Austin, TX). Briefly, 5 × 104 cpm of probe
was hybridized with 20 µg of total RNA in 80% formamide at 42 °C.
Following hybridization overnight, the reactions were digested in 200 µl of a 1:100 diluted mixture of RNase A and RNase T1 at 37 °C for
45 min. The protected fragments were analyzed by electrophoresis on a
6% polyacrylamide gel containing 8 M urea (Promega). The
gel was dried and exposed to X-Omat AR film (Kodak) with intensifying
screens at
The density of each protected fragment on the RPA autoradiogram was
measured using GPTools (BioPhotonics Corp., Ann Arbor, MI). The
relative amount of mRNA represented by each fragment was calculated
by normalizing the density with the number of [32P]CTP
incorporated into each fragment (197, 87, and 18 CTP for 1C3, 1C2, and
52 bp of E2, respectively). The amount of total GHR mRNA was
obtained by summing the amounts of 1C2-, 1C3-, and E2-containing GHR mRNA.
Identification of GHR Genomic Clones--
We previously isolated
genomic clones containing the bovine GHR 5'-flanking DNA by screening a
bovine genomic library in vector EMBL3 SP6/T7
(CLONTECH) with probes generated from the cDNA
for the bovine GHR 1A or 1B 5'-UTR (21, 22). The GHR 1A and 1B 5'-UTRs
are transcribed by the P1 and P2 promoter, respectively. We therefore
used PCR to screen the GHR genomic clones for the presence of newly
isolated 5'-UTRs. The PCR amplification was carried out on 2 µl of
Promoter-Reporter Plasmid Constructs--
The respective
GHR genomic region was tested for its ability to drive the
expression of the firefly luciferase reporter gene in the promoter-less
plasmid pGL2-Basic (pGL2B) (Promega). A 2.3-kb EcoRI-SacI fragment digested from pSK 41-1-6 was
inserted into the polycloning sites SmaI and SacI
upstream from the firefly luciferase reporter gene in pGL2B. This
construct was designated 2300C-GL2. The second construct, designated
1000C-GL2, was generated by fusing a 1-kb
NotI-SacI fragment, released from the pSK 41-1-6 plasmid and blunted at the NotI end, into pGL2B at the
polycloning sites SmaI and SacI. The orientation
and sequence of the inserts were verified by sequencing through the
insert-vector junctions. Large scale plasmid DNA purification was done
using the Qiagen Maxiprep kit (Qiagen).
Cell Culture and Transient Transfection Assay--
Cell lines
were purchased from the American Type Culture Collection (ATCC)
(Rockville, MD). Media and reagents for cell culture were purchased
from Sigma unless otherwise indicated. Human liver cell line Hep G2 and
hamster kidney cell line BHK-21 were maintained in minimal essential
medium containing L-glutamine, 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C under 5%
CO2. A second human liver cell line PLC/PRF/5 was
maintained in the same medium but without L-glutamine.
Cells were plated on 12-well plates, each well containing 3 × 105 Hep G2 cells or 1 × 105 PLC/PRF/5
cells or 1 × 105 BHK-21 cells. Following a 24-h
incubation, cells in each well were transfected with 2 µg of
promoter-reporter construct and 0.002 µg of pRLSV-40 plasmid
(Promega) using the calcium phosphate method. The pRLSV-40 plasmid was
co-transfected to normalize the variation in transfection
efficiency. pRLSV-40 encodes the Renilla luciferase, and its
activity can be distinguished from that of the firefly luciferase
encoded in pGL2B in the Dual-Luciferase Assay System (Promega). After
12 h of transfection, the cells were washed with
phosphate-buffered saline and then maintained in fresh medium for
36 h prior to luciferase assay. In each experiment, the pGL2B
plasmid was also transfected in separate wells to compare the specific
activity of promoter-reporter constructs with the basic activity of the
promoter-less plasmid.
Dual-Luciferase Activity Assay--
Activities of the firefly
luciferase and Renilla luciferase in a single sample were
measured sequentially using the Dual-Luciferase Reporter Assay
(DLRTM) system (Promega) according to the manufacturer's
instructions. Briefly, cells were rinsed twice with phosphate-buffered
saline and then lysed in 200 µl of Passive Lysis Buffer at room
temperature for 15 min. 20 µl of cell lysate was quickly mixed with
100 µl of Luciferase Assay Reagent II in a luminometer tube. The
light emission for the firefly luciferase was recorded immediately for 15 s after a 3-s premeasurement delay using a TD-20e
Luminometer (Tuner Designs, Sunnyvale, CA). Subsequently, 100 µl of Stop&GloTM reagent was added to the same tube to
inactivate the firefly luciferase while activating the Renilla
luciferase. The light output from the Renilla
luciferase was integrated under the same conditions.
Variation in transfection efficiency was normalized by dividing the
measurement for the firefly luciferase activity with that for the
Renilla luciferase activity. The luciferase activity of a
construct plasmid was expressed as relative to that of pGL2B.
Novel 5'-UTRs for the Bovine GHR mRNA--
Three distinct PCR
products (designated 1C1, 1C2, and 1C3) were obtained after amplifying
the 5'-end sequence of the bovine GHR mRNA in the uterus by using
the RACE procedure (Fig. 1A). Cloning of these three PCR products and subsequent DNA sequencing showed that 1C1, 1C2, and 1C3 were each flanked by the reverse primer
E2R (used in the last PCR amplification) at the 3'-end (Fig.
1B) and the tailed poly(dCTP) sequence at the 5'-end. This indicated that they were specific RACE products. Comparison of sequences of 1C1, 1C2, and 1C3 with the previously isolated bovine GHR
1A and 1B 5'-UTRs (21) revealed that they all shared an identical 52-bp
region of exon 2 in the bovine GHR cDNA from their 3'-termini (Fig.
2A). The 5'-UTRs, however,
diverged in the remaining upstream sequence, 11 bp upstream from the
translation start codon ATG (Fig. 2A). Thus, the newly
isolated RACE products were new 5'-UTRs for the bovine GHR cDNA.
Sequence comparison for 1C1, 1C2, and 1C3 also revealed that they were
identical sequences with different extensions at the 5'-end (Fig.
1B). Whether 1C3, 1C2, and 1C1 are contiguous in the genomic
sequence will be examined below.
Alignment of 1C3 with the eight 5'-UTRs for the human GHR mRNA (23)
did not reveal significant similarity (between 30 and 50% identity)
for DNA sequence except for the homologous 52-bp fragment of exon 2 (>95% identity). Sequence alignment of 1C3 with the five 5'-UTRs for
the rat GHR mRNA (24) showed that rat V4 may be homologous to the
bovine 1C (Fig. 2B). Approximately 60% of the nucleotides
within the 144-bp rat V4 sequence (24, GenBank accession S77487) were
identical to the bovine 1C; higher identity (75%) was found within an
80-bp exon 1 region proximal to exon 2 (Fig. 2B).
Besides the translation start codon (ATG) in exon 2, a second ATG codon
was found 68 bp upstream of the former one within 1C3, 1C2, and 1C1
(Fig. 1B). However, the upstream ATG codon was not in-frame
with the major open reading frame for the bovine GHR (3). Assumed
translation from the upstream ATG codon would lead to an early
termination (codon TGA) within 30 amino acids. Therefore, expression of
an NH2-terminal extended extracellular domain of the bovine
GHR from 1C3, 1C2, or 1C1 is unlikely.
Expression of 1C3, 1C2, and 1C1 5'-UTRs in Bovine Tissues--
An
RPA was done to determine whether 1C3, 1C2, and 1C1 were expressed in
non-uterine tissues. The RPA yielded two major protected fragments
(Fig. 3). The long fragment was generated
from protecting the 585-bp 1C3; the short fragment was likely generated
from protecting the 247-bp 1C2. Besides uterus, expression of 1C3 and
1C2 was detected in most adult bovine tissues, with greater levels in liver, muscle, uterus, mammary gland, kidney, and adrenal gland than in
lung, heart, pituitary gland brain, placenta, and spleen (Fig. 3).
Expression of 1C3 and 1C2 was also detected in fetal liver and kidney
(Fig. 3). Across all tissues examined, the fetal lung was the only
location that did not appear to express 1C3 or 1C2. Expression of 1C3
and 1C2 was also expressed poorly in lung of adult cattle (Fig. 3).
Although the RPA was not designed as a strict quantitative assay, the
observed pattern of tissue distribution of 1C3 and 1C2 was comparable
with that of the total GHR mRNA (2-5).
The 1C1 5'-UTR, as isolated by RACE, was not detected by RPA, perhaps
because the RPA was less sensitive than the RACE procedure. Alternatively, 1C1 may have been amplified from a shortened
first-strand cDNA caused by incomplete reverse transcription and
therefore was a RACE artifact. The 52-bp exon 2 fragment generated from GHR mRNA with other 5'-UTRs (i.e. GHR 1A and GHR 1B) was
not shown (Fig. 3) because small fragments were electrophoresed out of
the gel.
Proportions of 1C2- and 1C3-derived GHR Transcripts within the
Total GHR mRNA Pool--
Subsequent RPAs were done to determine
the ratios of 1C2- and 1C3-derived GHR transcripts to the total GHR
mRNA pool. Tissues that had a relatively high level of GHR mRNA
were examined. The gel was electrophoresed for a shorter time so that
the 52-bp fragment of exon 2 could be visualized (Fig.
4). The ratios of 1C2- and 1C3-derived
GHR transcripts to the total GHR mRNA pool are shown in Table
II. In the adult bovine liver, where most
of the GHR mRNA is transcribed from exon 1A by the liver-specific
promoter P1 (25-27), 1C-derived GHR transcripts (1C3 + 1C2)
represented only 11% of the total GHR mRNA (Table II). In
non-hepatic tissues such as kidney, mammary gland, uterus, and muscle,
28-35% of the total GHR mRNA was represented by 1C-derived
transcripts. The percentages of 1C-derived GHR mRNA in non-hepatic
tissues were significantly higher than that in liver (p < 0.05). Within fetal muscle and kidney, percentages of 1C-derived
transcripts to the total GHR mRNA pool were similar to those
observed in the adult tissues (Table II). Much less GHR mRNA was
detected in the fetal liver than the adult liver because the primary
GHR promoter in liver is P1, and the P1 promoter is not activated until
birth (22). For the same reason, 1C-derived GHR transcripts represented a much higher proportion (30%) of the total GHR mRNA in the fetal liver than in the adult liver (Table II).
Within all tissues examined, expression of 1C2 was greater than 1C3
(p < 0.05) (Table II). Ratios of 1C2 to 1C3 varied
between 1.6 and 3.3 among various tissues (Table II). Higher ratios of 1C2 to 1C3 were found in liver and kidney than in other tissues (p < 0.05). Although the absolute amount of 1C2- and
1C3-derived GHR mRNA as well as the total GHR mRNA appeared to
be decreased in the uterus and mammary gland of pregnant cattle (Fig.
4), the ratios of 1C2 and 1C3 to the total GHR mRNA pool within
various tissues were not affected by pregnancy. The present study was designed to measure the relative levels of 1C2- and 1C3-derived GHR
mRNA to the total GHR mRNA pool. Absolute levels of 1C2- and 1C3-derived GHR mRNA across tissues as well as changes in these transcripts during pregnancy should be examined in controlled studies
with additional animals.
Genomic Sequence of 1C3, 1C2, and 1C1 5'-UTRs--
The DNA
fragment corresponding to 1C3 was amplified by PCR from the bovine
genomic clones that contained GHR 1B 5'-UTR. Sequencing of the subclone
pSK 41-1-6 that contained a 2.3-kb XhoI-XhoI
fragment of the GHR 1B genomic clone demonstrated that 1C3, 1C2, and
1C1 were contiguous in the genomic sequence (Fig.
5; the DNA sequence has been deposited in
GenBank under accession number AF046861) as they were in the cDNA
sequence (Fig. 1). This indicated that 1C3, 1C2, and 1C1 are generated
by initiating transcription from different sites in the same exon. The
exon was named 1C in agreement with the nomenclature used for the
bovine exon 1A and 1B, from which the 1A and 1B 5'-UTR are generated.
Restriction enzyme mapping and DNA sequencing also revealed that the
3'-border of exon 1C is 770 bp downstream from exon 1B (Fig. 5). The
exact distance of exon 1C to exon 2 remains to be determined. However,
based on the preliminary results of PCR mapping and partial sequencing of several bovine GHR genomic clones, at least 20 kb separate exon 1C
and exon 2 (data not shown). Preliminary results also indicated that
exon 1A is at least 10 kb downstream from exon 1B and at least 6 kb
upstream from exon 2 (Fig. 5).
Transcription Start Sites in Exon 1C--
The 5'-terminal
nucleotide of the 5'-end sequence is typically regarded as the
transcription start site when the 5'-end of a mRNA transcript is
isolated by the 5'-RACE procedure (30-32). The 5'-terminal nucleotide
A of 1C3 and T of 1C2 (Fig. 1B) were therefore assigned to
be the transcription start sites in exon 1C. The assigned transcription
start site for 1C2 was close to the transcription start site (8 bp
apart) mapped by using RPA in which a DNA sequencing ladder was run
alongside (Fig. 6). The 1C2 and 1C3 start
sites are two major start sites in exon 1C because levels of 1C2- and
1C3-derived GHR mRNA are relatively high. The start site for 1C1
may be a minor start site or amplification of 1C1 was due to RACE
artifact.
Promoter Activity--
The GHR promoter activity for 5'-UTR 1C3
and 1C2 in vivo was suggested by the relatively high
expression of 1C3 and 1C2 5'-UTRs in hepatic and many non-hepatic
tissues. The promoter activity of the 5'-flanking region of exon 1C was
assessed in its ability to drive the expression of the luciferase
reporter gene in the promoter-less pGL2B plasmid in liver-derived cell
lines Hep G2, PLC/PRF/5, and kidney-derived cell line BHK-21. After
transient transfection in these cells, construct 2300C-GL2, which
contained a 2.3-kb 5'-flanking region of 1C2 (Fig. 5), showed 20-, 18-, and 15-fold greater activity than the promoter-less pGL2B plasmid alone
(p < 0.05) in Hep G2, PLC/PRF/5, and BHK-21 cells,
respectively (Fig. 7). It was possible
that the promoter activity of this 2.3-kb region was associated with
the P2 promoter because the P2 promoter and exon 1B were included in
the upstream region of the 2.3-kb fragment. To examine this
possibility, a second construct, 1000C-GL2, was made by including only
the 1,000-bp 5'-flanking region of 1C2 fused to the luciferase reporter
gene. The 1,000-bp 5'-flanking fragment excluded the 5'-flanking region
of exon 1B (Fig. 5). As shown in Fig. 7, the second construct
maintained significant promoter activity, being 11-, 15-, and 5-fold
over the pGL2B plasmid alone (p < 0.05) in Hep G2,
PLC/PRF/5, and BHK-21 cells, respectively. However, removal of the P2
promoter region also significantly decreased (p < 0.05) the promoter activity of 1000C-GL2 when transfected into BHK-21
and Hep G2 cells, compared with that of 2300C-GL2. This result
indicated that transcription of 5'-UTR 1C3/1C2 is initiated by a
separate functional promoter from the P2 promoter. The new promoter was
named P3. The activity of promoter P3 can be enhanced by the upstream
P2 promoter.
In this study, we initially isolated three new 5'-UTRs for the
bovine GHR mRNA from bovine uterine total RNA by using RACE (Fig.
1, A and B). Expression of two of the 5'-UTRs,
1C2 and 1C3, was detected by RPA in various tissues including uterus
(Figs. 3 and 4). Cloning of the corresponding GHR genomic region (Fig. 5) and subsequent transfection analysis (Fig. 7) defined a new promoter
(P3) that initiated the transcription of these 5'-UTRs from different
sites in exon 1C (Fig. 5). Isolation of a third promoter for the GHR
gene adds to the complexity of the structural organization of the GHR
regulatory region and the molecular mechanisms for the regulation of
GHR gene expression.
Use of alternative promoters to initiate transcription has been
demonstrated as a strategy to achieve developmental and tissue-specific expression of many genes including IGF-II (33), the aldolase A gene
(34), Although liver is the primary tissue for the expression of GHR, the
activity of the P3 promoter seems to be more important in non-hepatic
tissues. In the liver of adult animals, the P3-derived GHR transcripts
represented only a small portion (11%) of the total GHR mRNA
(Table II), whereas the majority of the GHR mRNA was transcribed
from the liver-specific P1 promoter. In non-hepatic tissues, GHR
transcripts generated from the P3 promoter represented about 30-40%
of the total GHR mRNA pool (Table II). Most, if not all, of the
remaining GHR transcripts are probably transcribed from a second
ubiquitous promoter, P2. Exon 1C-derived GHR 5'-UTRs (Fig. 3) and exon
1B-derived 5'-UTR (22)2 are
expressed in a similar pattern of tissue distribution: ubiquitous but
with higher amounts in uterus, liver, muscle, kidney, and mammary gland
than in other tissues. The consistency of the relative expression of
1C- and 1B-derived GHR mRNA across tissues was also demonstrated by
the similar ratios of 1C-derived GHR mRNA in the total GHR mRNA
pool across tissues in pregnant and non-pregnant animals, and in adult
and fetal animals (Table II and Fig. 4). This suggests that the
activities of P3 and P2 promoters might be co-regulated. The
co-regulation mechanism of P3 and P2 promoters, which together generate
most of the GHR transcripts in non-hepatic tissues, is consistent with
the wide ranging actions of the GH-IGF-I axis in stimulating cellular
growth, proliferation, and differentiation in multiple tissues
(8-11).
The P3 promoter was located 700 bp downstream from exon 1B in the
bovine GHR gene (Fig. 5). The physical proximity of promoter P3 to P2
allows for an interaction between the two promoters as well as the
co-regulation of their activities. The transient transfection analyses
demonstrated that inclusion of the upstream P2 promoter region in the
P3 promoter-reporter construct increased the activity of the P3
promoter (Fig. 7). This result indicated that the P2 promoter may serve
as an enhancer for the P3 promoter, or that enhancer(s) for the P2
promoter may be shared by the P3 promoter. A binding site (CCAAT) for
ubiquitous factor CTF/NF-1 and several SP1 sites (GGGCGG) are located
in the proximal region of the P2 promoter. Perhaps the CTF/NF-1 and SP1
binding sites constitute a ubiquitous enhancer for both promoters.
Similar mechanisms for co-regulation of two ubiquitous promoters were
reported for the human aldolase gene (38). Whether this mechanism
applies to the bovine GHR gene remains to be verified.
Although P3-derived GHR transcripts were ubiquitously expressed (Fig.
3), differential regulation of P3 activity between tissues cannot be
excluded. Greater expression of 1C-derived 5'-UTRs in tissues such as
liver, muscle, uterus, and kidney than in other tissues such as lung,
heart, ovary, and brain (Fig. 3) indicates that the P3 promoter may be
controlled by constitutive as well as tissue-specific transcription
factors. Previous studies have reported that the GHR mRNA in kidney
(39) and in brain (40) (predominately P2- and P3-transcribed 5'-UTRs)
was regulated differentially by GH and steroids from that in liver
(predominately P1-transcribed 5'-UTR). In the present study, the levels
of 1C-derived GHR mRNA and the total GHR mRNA in uterus and
mammary gland appeared to be decreased in pregnant animals compared
with non-pregnant animals (Fig. 4), further suggesting that P3 and P2
promoters may be subject to regulation by hormones. However, unlike the
P1 promoter, whose activity increases dramatically after birth (22),
developmental up-regulation of the activity of the P3 promoter in
tissues such as liver and kidney was not suggested because similar
levels of 1C3 and 1C2 5'-UTRs were detected in both adult and fetal
tissues (Figs. 3 and 4).
Examination of the proximal region upstream of 1C2 or 1C3 within the
bovine GHR gene failed to identify a consensus TATA box. Absence of a
consensus TATA box supports the presence of at least two different
1C-derived 5'-UTRs because TATA-less promoters usually initiate
transcription from multiple start sites (41, 42). The proximal region
of the P3 promoter had a high content of GC (83% in the 700-bp
proximal region) and lacked a CCAAT box. These features were thought to
be unique to many constitutive gene promoters (43) but now are also
found in regulated genes (44-46). A search for potential transcription
factor binding sites using the computer program MatInspector (47)
identified several sites for constitutively expressed transcription
factor SP1 (48) and also a number of sites for regulated factors such
as STAT, AP1, AP2, and AP4 in the proximal region as well as in the
more upstream region of the P3 promoter. Thus, the molecular mechanism
for the hormonal regulation of the P3 and P2 promoters remains an
important area of study.
Mapping of the P3 promoter relative to exon 2 was not completed partly
because of the large size of the exon 1 region in the GHR gene.
Preliminary results generated from sequencing genomic clones indicate
that there are at least 20 kb between exons 1C and 2. Although a
definite map or DNA sequence for this region has not been established,
our estimate is comparable to an estimate of 34 kb between 1B and exon
2 in the ovine GHR gene (13).
In most species, one or two 5'-UTRs homologous to the bovine 1A or 1B
or both have been isolated for the GHR mRNA (49). In human, eight
GHR mRNA 5'-UTRs (namely V1 to V8) were reported (23). Among them,
the DNA sequences of V1 and V2 are about 80% identical to the bovine
1A and 1B, respectively. Comparison of the bovine 1C with the human V3
to V8 only revealed a 30-50% similarity, suggesting that the human
homolog of the bovine 1C was not included among the remaining six human
5'-UTRs. In the bovine GHR gene, exon 1C was located 700 bp from exon
1B (Fig. 5). The DNA sequence of the bovine GHR exon 1B and its
proximal 5'- flanking region was more than 90% identical to the
corresponding region in the human GHR gene (GenBank accession AJ002175)
and also to the corresponding region in the ovine GHR gene (13). Thus,
the human and ovine homologs of the bovine exon 1C might exist in the
human and ovine GHR mRNA. Five 5'-UTRs (V1 to V5; nomenclature for
the rat and for the human 5'-UTRs are not equivalent) were also
reported for the rat GHR mRNA (24). The rat 5'-UTR V2 and V1 are
similar to the bovine 5'-UTR 1A (therefore ovine 1A, human V1) and the bovine 5'-UTR 1B (ovine 1B, human V2) in both DNA sequence
(approximately 70% identity) and expression pattern (liver-specific
and ubiquitous, respectively). Our alignment of cDNA sequences
suggests that the rat V4 might be the homolog of bovine 1C because rat
V4 and bovine 1C share a region with 75% identity (Fig.
2B). Furthermore, the rat V4 and the bovine 1C 5'-UTRs are
expressed in similar locations, i.e. liver and many
non-hepatic tissues (24), suggesting the ubiquitous activity of the P3
promoter in the rat and perhaps in other species. However, compared
with 30-40% of representation of 1C-derived GHR transcripts in the
total GHR mRNA within non-hepatic tissues, V4 is the predominant
form of GHR mRNA transcripts in rats (24). The relative activity of
P3 and P2 promoters, therefore, may be different for different species.
Although three GHR promoters (P1, P2, and P3) appear to exist in
various species, isolation of other 5'-UTRs for the human GHR mRNA
(23), the rat GHR mRNA (24), and the bovine GHR
mRNA2 suggests that the GHR promoters may not be
limited to P1, P2, and P3. Other promoters remain to be identified and
characterized for the GHR gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
80 °C until used. Extraction of total RNA was carried
out by using TrizolTM reagent (Life Technologies, Inc.)
according to the manufacturer's instructions. The RNA samples were
checked for integrity by gel electrophoresis. Concentrations of RNA
samples were determined from absorbance of duplicate samples at 260 nm.
Quantitation was confirmed by gel electrophoresis.
Oligonucleotides used for RACE, PCR, and Sequencing
"
are forward and reverse, respectively. AAP, abridged anchor primer;
AUAP, abridged universal amplification primer.
80 °C for overnight or longer.
phage stocks (1 × 109 plaque-forming units in
50% glycerol) with two primers (1C3F1 and 1C3R1) that were specific
for the 1C3 5'-UTR (Table I). The PCR was for 30 cycles of 96 °C for
10 s, 55 °C for 30 s, and 72 °C for 2 min. The 1C3 was
amplified from several GHR 1B genomic clones that were isolated using
the 1B 5'-UTR as a probe. A 2.3-kb XhoI-XhoI
fragment was released from one genomic clone and subcloned into the
pBluescript SK plasmid (Stratagene) to form pSK 41-1-6. This plasmid
was sequenced as described before. Restriction sites for
NotI, SmaI, SacI, XhoI, and
HindIII were identified within the insert by the GCG program
and confirmed by restriction endonuclease digestion.
RESULTS
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Fig. 1.
Amplification of three 5'-end fragments of
the bovine GHR cDNA in uterus. Panel A, photograph
of an ethidium bromide-stained agarose gel showing three RACE products,
1C3, 1C2, and 1C1. Panel B, nucleotide sequences of three
RACE products, 1C3, 1C2, and 1C1. Sequences of 1C3, 1C2, and 1C1 are
co-linear. Numbering is relative to the start site for 1C2 (+1). Start
sites for each RACE product are denoted with arrows and
bold letters. The sequence complementary to the reverse
primer E2R is underlined. The GHR exon 2 is
italicized.
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Fig. 2.
Sequence alignment of the bovine 1C 5'-UTR
with the bovine 1A and 1B 5'-UTRs (panel A) and with
the rat 5'-UTR V4 (panel B). In panel
A, only the 100-bp sequence upstream of the 3'-end of 1C is
aligned with the corresponding regions within the previously isolated
bovine 1A and 1B 5'-UTR variants (21, 29); the three bovine 5'-UTRs
share an identical 52-bp exon 2 region and diverge in the upstream
region. In panel B, the 144-bp rat V4 sequence (24) is
aligned with the corresponding region within the bovine 1C 5'-UTR (+71
to +215, Fig. 1B). In each 5'-UTR, exon 2 is
italicized, and the translation initiation codon (ATG) is
underlined.
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Fig. 3.
RPA of expression of 5'-UTRs in bovine
tissues. The autoradiograph is representative of three
repeated RPAs, in which tissues from two adult cows and one 8-month-old
fetus were used. SK muscle, skeletal muscle. 20 µg of
total RNA was hybridized with 5 × 104 cpm of
32P-labeled riboprobe that contained a 585-bp antisense
sequence of 1C3 5'-UTR. Two major protected fragments, each
corresponding to the expression of 1C3 and 1C2 5'-UTR of GHR mRNA,
are denoted with arrows. M indicates a
32P end-labeled 100-bp DNA ladder (Promega).
tRNA denotes the negative control containing 20 µg of
yeast tRNA.
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Fig. 4.
RPA of proportions of 1C 5'-UTRs to the total
GHR mRNA pool in bovine tissues. The autoradiograph is
representative of two replicated RPAs. The RPA included liver, kidney,
muscle, mammary gland, and uterine samples from nonpregnant or pregnant
(p) cows, as well as a liver, muscle, and kidney sample from
an 8-month-old fetus. Mam, mammary gland; Muscle,
skeletal muscle. The RPA was carried out as described in Fig. 3. Three
major protected fragments, each corresponding to 1C3, 1C2, and 52 bp of
exon 2 (E2), are denoted with arrows.
Ratios of 1C3- and 1C2-derived GHR mRNA to the total GHR mRNA
pool and ratio of 1C2-derived GHR mNRA to 1C3-derived GHR mRNA
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Fig. 5.
Mapping exon 1C in the bovine GHR gene.
The middle diagram is a schematic representation of the
5'-region of the bovine GHR gene, showing the relationships of exon 1A,
exon 1B, and exon 1C to exon 2 and to each other. The structure of exon
2 to exon 10 is deduced from that of the human GHR gene (15). Exons are
represented as boxes, introns represented as
lines between exons. The 5'-UTRs generated from each leader
exon (exons 1A, 1B, and 1C) are indicated with arrows. The
GHR mRNA transcribed from each exon is also shown at the
bottom. The relative distances between exons are shown at
the top (not drawn to scale). Restriction sites for
XhoI (X), HindIII (H),
NotI (N), SacI (S), and
SmaI (Sm) are indicated within the 2.3-kb
5'-flanking region of exon 1C.
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Fig. 6.
Mapping of the transcription start site for
5'-UTR 1C2. The RPA was done as described in Fig. 3. An antisense
probe to 1C3 was hybridized with 20 µg of bovine liver and kidney
RNA. The sequencing ladder (lanes G, A,
C, and T), corresponding to the complementary
strand to 1C3 5'-UTR in Fig. 1, was generated with
32P-labeled primer E2R (Table I) by using the
fmol DNA Sequencing System (Promega). The 5'-end nucleotide
T of the protected 1C2 fragment is denoted by an
asterisk.
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Fig. 7.
Transient transfection analysis of the
promoter-reporter plasmid constructs in Hep G2, PLC/PRF/5, and BHK-21
cell cultures. Constructs 2300C-GL2 and 1000C-GL2 each contain
2,300-bp and 1,000-bp portions of the 5'-flanking region of the bovine
GHR gene inserted immediately upstream from the firefly luciferase
reporter gene in the promoter/enhancerless plasmid pGL2B. 2 µg of
promoter-reporter construct and 0.002 µg of pRL-SV40 (encoding the
Renilla luciferase) as transfection efficiency control were
co-transfected into cells using the calcium phosphate method. The
transfection of each construct was repeated five, five, and three times
in BHK-21, Hep G2, and PLC/PRF/5 cells, respectively. Activity of the
firefly luciferase and Renilla luciferase was assayed using
the Dual-Luciferase Assay System (Promega). The activity of
promoter-reporter plasmids, relative to that of the pGL2B plasmid, is
expressed as mean ± S.E. of repeated transfections. The
difference between the relative luciferase activities of the two
constructs within each cell line was compared by using Student's
t test. a and b denote
p < 0.05. For details on plasmid construction,
transient transfection, and luciferase assay, see "Experimental
Procedures."
DISCUSSION
-glutamyl transpeptidase (35), mineralocorticoid receptor gene
(36), and Na+-Ca+ exchanger gene
(ncx1) (37). In the case of the GHR gene, two promoters had
been isolated and characterized before this study. The first promoter,
termed P1, was originally isolated in sheep (25) and later in mouse
(26), cattle (29), and most recently in humans (27). The P1 promoter is
liver-specific. The second promoter, termed P2, was only isolated in
sheep (13), but its transcripts were also present in cattle (21, 22)
and humans (23). The P2 promoter appears to be a ubiquitous promoter
that governs the expression of the GHR gene in various tissues
including liver. The new GHR promoter, named P3, isolated in the
present study, appears to be a second ubiquitous promoter for the
bovine GHR gene. The evidence for this conclusion included that
P3-derived 5'-UTRs were detected in all bovine tissues examined (Fig.
3) and that P3 was able to drive the expression of a reporter gene in
both hepatic and non-hepatic cell lines (Fig. 7).
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FOOTNOTES |
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* This work was supported in part by National Research Initiative Competitive Grant USDA CSREES 95-37205-2312 (to M. C. L.). This is contribution 12837 from the Missouri Agricultural Experiment Station Journal Series.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) AF085281 (1C3 5'-UTR).
Supported by a Food for the 21st Century postdoctoral fellowship
from the University of Missouri-Columbia.
§ To whom correspondence should be addressed: 164 ASRC, Dept. of Animal Sciences, University of Missouri, Columbia, MO 65211. Tel.: 573-882-9897; Fax: 573-882-6827; E-mail: lucym{at}missouri.edu.
2 H. Jiang and M. C. Lucy, unpublished result.
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ABBREVIATIONS |
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The abbreviations used are: GH, growth hormone; GHR, growth hormone receptor; IGF, insulin-like growth factor; kb, kilobase(s); UTR, untranslated region; bp, base pair(s); RACE, rapid amplification of cDNA ends; RPA, RNase protection analysis; PCR, polymerase chain reaction; BHK, baby hamster kidney.
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