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Departments of Pediatrics (C.G.G., G.Z., H.Z., G.N.H.), Medicine (C.G.G., G.Z., H.Z., G.N.H.), Human Genetics (G.N.H.), and Physiology (G.N.H.), McGill University, Montréal, Québec, Canada H3Z 2Z3; and Department of Pediatrics, University of Pittsburgh School of Medicine, Children’s Hospital of Pittsburgh (G.S., R.K.M.), Pittsburgh, Pennsylvania 15213-2583
Address all correspondence and requests for reprints to: Dr. Cynthia G. Goodyer, McGill University-Montreal Children’s Hospital Research Institute, 4th Floor, Place Toulon, Room 415/1, 4060 St. Catherine Street West, Westmount, Québec, Canada H3Z 2Z3. E-mail: cindy.goodyer{at}muhc.mcgill.ca
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we have identified a novel 5'UTR variant, V9, cloned approximately 40 kb of the 5'-flanking region of the hGHR gene and have precisely mapped all of the known variant sequences within this region. We demonstrate that seven of the 5'UTR exons are clustered within two modules. Transcriptional start sites have been established for one variant exon (V1 and V9) within each module, and promoter analyses were carried out for V9. In addition, we analyzed expression patterns of all variants in a panel of human fetal and postnatal tissues, which confirmed the complex regulation of the hGHR gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genomic cloning
A human lymphocyte genomic 
DASH library
(Stratagene, La Jolla, CA) was screened with V1- and
V3-screening oligonucleotide probes (Table 1
), and specific clones containing
approximately 16-kb inserts were isolated, as previously described
(9). Specific primers (V7[S]-V1[AS] and V3[S]-V3
intronic[AS]; Table 1) and PCR protocols (10) were used
to isolate an approximately 120-kb BAC clone positive for both
V1 and V3 and an approximately 110-kb PAC clone positive for V3
from libraries at the Whitehead Institute Center for Genome Research
(Cambridge, MA). Additional clones were obtained after Down-to-the-Well
PCR (Genome Systems, Inc., St. Louis, MO) screening of a
human PAC library. Three overlapping clones (V267, V268, and V269)
contained V2, V9, and V3 sequences, but not V1, were found. One (V268)
was used for subcloning and mapping.
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) and pooled RNA from eight male and female
human hearts (aged 25–59 yr; Marathon-Ready cDNA, CLONTECH Laboratories, Inc., Palo Alto, CA) according to the
manufacturer’s instructions. PCR products were cloned into pCRIITOPO
(Invitrogen, Carlsbad, CA) and sequenced using either SP6
or T7 primers and the Sequenase 2.0 kit (U.S. Biochemical Corp., Cleveland, OH).
Restriction enzyme mapping
Southern blots of phage, bacterial artificial chromosome
(BAC), P1 artificial chromosome (PAC), and lymphocyte
genomic DNA, digested with SalI, XbaI,
HindIII, EcoRI, and BamHI, were probed
with 32P end-labeled V1, V2, and V3
oligonucleotides (Table 1), variant-specific
32P-labeled probes (for V1, 0.58-kb
EcoRI-BamHI genomic fragment), and riboprobes
(for V3, 0.39-kb V3[S]-V3 intronic[AS] PCR fragment) to generate
the structural map of the 5'-flanking region. A 4.4-kb
XbaI/XbaI V1-containing genomic DNA fragment, a
4.3-kb HindIII/XbaI V3-containing phage DNA
fragment, and an 8-kb BamHI-BamHI V9-containing
PAC DNA fragment were subcloned into Bluescript II (SK)
(Stratagene, La Jolla, CA) and sequenced.
RT-PCR and long range PCR assays
One to 5 µg total RNA were reverse transcribed using AMV-RT
(Life Technologies, Inc., Gaithersburg, MD). Reverse
transcriptase products or genomic DNA were amplified for 23–30 cycles
with Taq polymerase (Life Technologies, Inc.)
and primer pairs for V3 (V3[S]-Exon 2[AS]) and V4 mRNA
(V4[S]-exon 5B[AS]; Table 1), as previously described
(11). The internal standard for V4 mRNA assays was
generated using PCR, as previously described (5, 12),
using a hybrid V4/exon2 sense primer and an exon 7 antisense primer
(Table 1). CLONTECH Laboratories, Inc., Multiple Tissue
cDNA (MTC) Panels were amplified for 32–36 cycles using AdvanTaq DNA
polymerase (CLONTECH Laboratories, Inc.) and
V1–V9-specific primer pairs (Table 1) according to the manufacturer’s
instructions. Fragments of DNA more than 2.0 kb were amplified using
the Expand Long Template PCR System (Roche, Laval, Canada)
and variant-specific primer pairs (Table 1), according to the
manufacturer’s instructions. PCR products were resolved on 0.7–2%
agarose gels and transferred to either Nytran+
(Xymotech, Schleicher & Schuell, Inc., Montréal,
Canada) or GeneScreen Plus (NEN Life Science Products,
Boston, MA) membranes. Blots were hybridized with the appropriate
nested 
-32P end-labeled oligonucleotide
probes, and hGHR bands were revealed by autoradiography, as previously
described (11, 13, 14).
Transcription start site assays
Primer extension (PE) and ribonuclease protection assays (RPA)
were performed as described previously (13, 14, 15), using
human adult liver RNA, yeast transfer RNA as control, and specific PE
primers and RPA probes (see Figs. 3, 4, and 6–8 for details). RPA
probe templates were generated from liver DNA and cloned into
Bluescript II (SK); antisense RNA probes were generated with T7 or T3
RNA polymerase and gel-purified.
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). PCR
fragments were digested with XhoI and HindIII and
ligated into pGL3-Basic (Promega Corp., Madison, WI).
Direction and nucleotide fidelity of the inserts were verified by
sequencing.
Transient transfections
H9C2.2 cells (rat muscle; American Type Culture Collection, Manassas, VA) were maintained in DMEM with 10% FBS,
penicillin (100 U/ml) and streptomycin (100 µg/ml; Life Technologies, Inc.). Cells (5 x 105)
were seeded into six-well plates 24 h before transfection. Cells
were exposed to 3 µg reporter plasmid DNA/well complexed with
Lipofectamine (Life Technologies, Inc.) in serum-free
OptiMEM for 5 h, washed, and then grown for 40 h in
FBS-supplemented medium. Transfections were performed in duplicate, and
efficiency was monitored by cotransfection of 100 ng pRL-CMV
(Promega Corp.) expressing Renilla luciferase.
For the luciferase assays, cells were rinsed twice in PBS, and 200 µl
lysis buffer were added (Dual Luciferase Assay System, Promega Corp.) followed by a freeze-thaw cycle and centrifugation at 4 C
for 2–3 min at 12,000 x g. Supernatants were
sequentially assayed for firefly and Renilla luciferase
activity, and results are expressed as normalized light units relative
to pGL3-Basic activity.
In vivo V9 promoter activity
Female adult CD-1 mice (>30 g) were anesthetized with chloral
hydrate (0.4 g/kg). After left lateral thoracotomy, the heart was
partially removed from the thorax, and 10 µl of a 500 ng/ml DNA
solution in normal saline were injected into the left ventricle; the
heart was replaced, and the wound was sutured (16). Mice
were killed 6 days later by CO2 asphyxiation, and
the hearts were removed and washed in ice-cold PBS. The lower thirds of
both ventricles were weighed, homogenized, lysed in 4 vol/wet wt in 1x
lysis buffer, and assayed for firefly and Renilla luciferase
activity as described above. Activities are reported as relative light
units, with firefly luciferase reporter activity normalized to the
Renilla luciferase activity (100 ng coinjected internal
control plasmid pRL-CMV).
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from Hep3B cells (American Type Culture Collection) as previously described
(13). Double stranded oligomer DNA probes were end-labeled
on the sense strand with [-32P]ATP and T4
polynucleotide kinase before annealing of the complementary strand. Ten
femtomoles of labeled probe were incubated with 2 µg nuclear extract
in 20 µl containing 1 mg poly(dA-dT), 20 mM Tris (pH
8.0), 50 mM NaCl, 50 µg/ml BSA, 10% glycerol, 1%
Nonidet P-40, 1 mM EDTA, and 1 mM
dithiothreitol at room temperature for 30 min. DNA-protein complexes
were resolved by electrophoresis through a 6% polyacrylamide gel with
90 mM Tris borate (pH 8.5) and 2 mM EDTA buffer
at room temperature. Gels were dried and subjected to autoradiography
with intensifying screens (NEN Life Science Products) at
-80 C. Competition experiments included the addition of a 200-fold
excess of unlabeled, double stranded oligonucleotide. For the
supershift assay, nuclear extracts were incubated with 2 µg
polyclonal antibodies against NFY-A or NFY-B subunits (gift from Dr. D.
Mathis) for 1 h at 4 C before EMSA.
Statistical analyses
Data were analyzed by Student’s t test or ANOVA,
followed by the Tukey-Kramer multiple comparisons test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cloning and structure of the hGHR 5'- flanking region
Screening of BAC, PAC, and phage libraries using V1-, V2-, and
V3-specific primers and probes resulted in isolation of several
positive clones (Fig. 1A). Restriction
enzyme mapping and Southern blotting, followed by subcloning and
sequencing of smaller fragments as well as long range PCR, localized
the nine hGHR 5'UTR variant sequences within a 40-kb region
upstream of exon 2 (Fig. 1, B–D). Three of the sequences (V2-V9-V3)
are located in a 1.6-kb module (module A) 36 kb upstream of exon 2
(Fig. 1, C and D). The placental 5'UTR reported by Urbanek et
al. (19) was also placed on the map, beginning 306 bp
upstream of the 5'-end of the V2 sequence reported by Pekhletsky
et al. (4) and overlapping the 3'-end of V2.
Another four of the variants (V7-V1-V4-V8) are clustered in a second
module of 2 kb (module B), 18 kb upstream of exon 2 (Fig. 1, C and D).
V6 is located midway between modules A and B, whereas V5 is contiguous
with exon 2 (Fig. 1, C and D).
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and data not shown). V4 RNA was
not expressed in fetal liver (n = 6; 11–20 weeks fetal age),
hepatoblastomas (n = 12; 6–36 months), or hepatocarcinomas
(n = 4; 50–75 yr; Fig. 2A and data not shown). In addition, V4
mRNA was not detected in a large series of normal fetal and postnatal
tissues (lung, kidney, small intestine, skeletal muscle, adrenal, and
related tumors), although the same RT products were positive for V3
(data not shown). Assays of the MTC Panels confirmed the results with
V1 and V4 and showed that V7 and V8 are also expressed only in
postnatal liver (data not shown). Thus, the four hGHR
variant exons clustered in module B appear to be under similar control,
being induced only after birth.
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and data not shown for fetal spleen,
thymus, placenta, and adult pancreas); the one exception was fetal
brain, which did not have detectable levels of V9. We also noted two
variants of V5. One corresponds to an intronic 5'-extension of exon 2,
described previously (4), and the second is a novel
smaller variant, splicing out at position 152 of the previously
described sequence (GenBank Z11850; Fig. 1D). The larger V5 mRNA is
ubiquitously expressed and always at a higher level than the smaller
transcript (data not shown). V6 was detectable in individual adult
liver RNA samples, but at low levels relative to the other variants
(data not shown). There was a consistent increase in all variant mRNAs
in postnatal liver, whereas the other tissues showed variable changes
during development (Fig. 2B and data not shown).
Start site of transcription and promoter region of V9
Primer extension assays revealed a predominant start site of
transcription 302 bp upstream of the 3'-end of exon V9 (Figs. 3 and 4).
The region 5' to this start site has several potential transcription
factor-binding sites, including those for Sp1 family members (-62 bp
relative to the start site), a CCAAT core (-132), NKX2 (-212), and
GATA factors (-214), but no canonical TATA motif (Fig. 4).
V9 promoter activity was shown in vivo. After intracardiac
injection of promoter/reporter constructs, the V9 promoter construct
demonstrated 7-fold greater luciferase activity than the control (Fig. 5A). Information
on potential regulatory elements was obtained by transiently
transfecting H9C2 cells with luciferase reporter constructs with
progressive 5'-deletions of the V9 promoter. Sequences between
-585 and -306 bp from the start site of transcription contribute
approximately 40% of the activity (Fig. 5B). This region contains the
V2 5'UTR, a GC-rich exon with several putative Sp1-binding sites.
However, no significant drop in promoter activity was observed until
removal of the sequence from -165 to -39 bp from the start site (Fig. 5B). Although this region contains a potential Sp1-binding motif, no
consistent binding to this site could be detected by EMSA (data not
shown). In contrast, specific binding of NF-Y to the CCAAT box at -132
was demonstrated by antibody supershift of the bound protein complex in
another EMSA, and no binding was observed when the CCAAT core was
mutated to CACAT (Fig. 5C). As shown previously, the NFY-B antibody
resulted in a more marked supershift than the NFY-A antibody
(13).
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). The
start sites of transcription for V1 associated with both of these
elements were determined by RPA and primer extension assay. A riboprobe
antisense to the genomic sequence between EcoRI and
BamHI (see Figs. 6 and 8) was
hybridized with human postnatal liver RNA; two major [325 and 172
nucleotides (nt)] and several minor protected fragments were
identified (Fig. 6). In a parallel primer extension assay (PE#1; Figs. 7 and 8),
three major extended products resulted, one of which (155 nt) matched
the start site of transcription corresponding to the 172-nt
ribonuclease-protected fragment. These data suggest that the major
start site of transcription for the first (-308) V1 TATA-like element
lies 19 bp downstream of it (numbering relative to the last A; Fig. 8).
A second riboprobe, antisense for the StyI-DraI
genomic sequence (see Figs. 6 and 8), protected a fragment of 140 nt
and several minor fragments. This start site was confirmed by the
169-nt product of a second primer extension assay (PE#2; Figs. 7 and 8)
as being 25 bp downstream of the second (-248) TATA box.
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) (24, 25); the comparable promoter regions are all TATA-less and
highly GC rich, with a putative CCAAT box and numerous Sp1
elements.
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There is more than 99% agreement between the V7-V8 module B sequence
of the present study and human genomic sequences in GenBank (4, 28, 29) (AJ131868). In addition, there is high homology
(64–83%) between the V1 sequence after the downstream consensus TATA
box and the ovine/bovine 1A exons (30, 31), a rabbit GHR
mRNA 5'UTR sequence (1), the mouse L1 exon (23, 32), and the rat GHR1 exon (33) (Fig. 9B). All
known postnatal liver-dominant 5'UTR mRNA sequences in these subprimate
species match V1. However, the BLAST analysis of the ovine and bovine
GHR genomic sequences both upstream and downstream of the 1A
exon revealed regions that were 74–83% homologous with V7, V4, and
V8, suggesting that equivalent exons may be present in subprimate
GHR genes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The most striking aspect of the hGHR 5'-flanking region is the arrangement of seven of the exons in two modules, each comprising about 2 kb of genomic sequence. We have designated the cluster of V2, V9, and V3 exons as module A, and that of V7, V1, V4, and V8 as module B. These exons are not only physically clustered, but their expression patterns are quite distinct. Although module A-derived mRNAs are widely expressed at almost every stage in development, those derived from module B are detectable only in normal postnatal liver.
Emerging knowledge of the GHR regulatory region in several subprimates points to a high level of complexity in other mammalian species as well. V2-like exons, regulating ubiquitous expression of GHR mRNAs, have been demonstrated for the ovine (o1B) (21, 34), the bovine (b1B) (22, 35), the mouse (mL2) (13, 23), and the rat (GHR2) (25, 33). More recently, V9-like exons have been reported for the mouse (mL3–5) (26) and the bovine (b1C) (27) GHR, and a V5-like exon has been found in the mouse (mL5) (36). All of these exons are located in homologous positions within the GHR 5'-flanking regions (Fig. 9, A and B). In addition, although only a single liver-specific (V1-like) 5'UTR exon has been described for the ovine (o1A), bovine (b1A), mouse (mL1), and rat (GHR1), comparative sequence analyses revealed that in the ovine and bovine GHR, there are regions highly homologous to V7, V4, and V8 (Fig. 9B). We have also shown that V3-like mRNAs can be detected in bovine, rabbit, mouse, and rat tissues (unpublished data), although no equivalent genomic sequences have been reported to date. Thus, all of the human 5'UTR exons, except V6 and V3, have highly homologous sequences in the GHR gene of several subprimate species, suggesting that the complex arrangement of these exons may be a common feature in mammals.
The newly discovered V9 exon overlaps with the GHR genomic sequences reported for bovine and mouse; however, there are significant structural and expression differences. V9 consists of a single exon that yields one mRNA transcript that is widely expressed (present study). The bovine has been reported to also have a single exon (b1C), but three transcripts with the same 3'-GT splice site (b1C1–3). Although the shortest transcript, b1C1, is undetectable by RT-PCR, the other two are ubiquitously expressed, representing 10–40% of the total GHR mRNAs in the tissues examined (27). The mouse GHR, in contrast, has three separate exons, each transcribing its own unique mRNA, but all at very low levels relative to total GHR transcripts (26). The alignment analysis of the present study revealed a significant amount of variance among species, especially between the end of V2 and the start site of transcription for V9. This degree of variance is not surprising given that the mRNA transcription mechanisms within this genomic region are so different among the three species.
Promoter analysis of transcription of V9-containing
sequences was carried out as an example of the widely expressed module
A mRNAs. Deletion analysis of the 5'-sequence flanking V9 suggests
coordinated as well as separate regulation of V2 and V9 expression.
Although a 585-bp promoter fragment, including part of the V2 exon as
well as the V9 promoter, had the highest in vitro activity
in H9C2 cells, a smaller region spanning -165 to -39bp upstream of
the V9 transcription start site also displayed promoter activity.
Similar regulatory mechanisms may operate in the bGHR gene;
a 2.3-kb fragment that includes both b1B and b1C promoters has high
activity, while a 1-kb fragment that includes only the b1C promoter has
significant activity in several cell lines (27). Within
the proximal promoter sequence for V9, we have identified a binding
site for the nuclear transcription factor NF-Y. NF-Y can act on
tissue-restricted promoters such as phospholamban (37) and
the Na,K-adenosine triphosphatase 
3-subunit
(38), even though binding sites for NF-Y (CCAAT-boxes)
exist in many mammalian promoters, and NF-Y itself may be ubiquitously
expressed (39). Clearly, promoter context is important in
determining the specificity of gene expression (40).
Numerous binding sites for Sp1 are present in the proximal V9 promoter
region, and members of this family are responsible for regulation of
the murine V2-like transcript, L2 (14) and the homologous
ovine as well as bovine 1B transcripts (22, 34).
Interestingly, NF-Y has been shown to act synergistically with Sp1 to
direct gene expression (38, 41). Although the V9
transcript is widely expressed in human fetal and postnatal tissues, it
is most abundant in skeletal muscle. Future studies will need to
address the factor(s) responsible for directing high level muscle
expression of the V9 transcript.
We studied the V1 exon as an example of liver-specific expression of module B exons, A transcriptional start site for the upstream nonconsensus TATA box was defined by RPA and primer extension assay close to the site suggested previously by 5'RACE (29). In addition, we determined a second start site, for the downstream consensus TATA box. The presence of two functional TATA boxes for this exon is similar in the human and mouse GHR genes, whereas the ovine and bovine GHR V1-like exons have a single functional consensus TATA box in a position similar to that of the downstream V1 TATA element (Fig. 9B). The question of why, in addition to having four different exons that code for expression of hGHR mRNAs only in postnatal liver, one of the exons produces two mRNAs remains to be answered.
Preliminary V1 promoter analyses have been carried out by two groups. Zou et al. (28) reported that a 2-kb region upstream of the consensus V1 TATA box had promoter activity in HepG2 human hepatoma cells. Rivers and Norman (29) used the same cells to define a minimal (-0.16 kb) promoter as well as negative elements more than 0.65 kb upstream of the nonconsensus TATA box. Nuclear extracts from the hepatoma cells were used in a footprint assay to identify four putative regulatory sites, -0.58 to -0.27 kb upstream of the same TATA element. Because there are low or undetectable levels of V1 mRNA in liver tumor cells (5, 42), these sites may be primarily for repressive elements. In addition, all of the V1 promoter constructs used to date also contain V7 exon and putative promoter sequences. Thus, the question of whether clustered hGHR regulatory exons in each module are under separate or coordinated control mechanisms remains to be explored.
In summary, we have significantly advanced our understanding of the hGHR gene, cloning a new 5'UTR exon and mapping the relative locations of nine 5'UTR exons. Seven of these exons are clustered within two modules that exhibit marked structural as well as functional differences.
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Acknowledgments |
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Footnotes |
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2 C.G.G., G.N.H., and R.K.M. contributed equally to this study.
3 G.Z. and G.S. contributed equally to this study.
Received November 7, 2000.
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X174 HinfI
DNA markers (M) and sequencing reactions (not shown).
, primers),
restriction enzyme mapping [XbaI (X),
HindIII (H), EcoRI (E), and
BamHI (B); 
, arrowheads], Southern
blotting (
, genomic probes), and sequencing (C). D, Long range
(i–iii) and standard (iv) PCR Southern blots, using BAC and genomic
(Gen1–4; 25–57 yr) DNA, showing that module B is located
about 18 kb downstream from module A and about 18 kb upstream from exon
2 (D, i–iii). Approximately halfway between the two modules lies V6
(D, i and ii). V5 lies just upstream from and is contiguous with exon 2
(D, iv). The DNA size markers (in kilobases or base pairs) are
indicated.
