Department of Animal Sciences University of Missouri Columbia, Missouri 65211
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ABSTRACT |
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INTRODUCTION |
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In the bovine, the liver-specific GHR 1A mRNA appears to be the most important GHR mRNA variant because it is the most abundant GHR mRNA variant in liver, and its levels are highly correlated with the blood levels of IGF-I under various physiological conditions (15, 16). The objective of the present study was to understand how the liver-specific GHR 1A mRNA is generated in the bovine and to identify transcription factors that are involved in the liver-specific expression of this GHR mRNA variant. Our study indicates that, like its homologs in other species, the bovine GHR 1A mRNA is transcribed from an alternative leader exon (exon 1A) by a promoter (GHR P1) in the 5'-flanking region of exon 1A. In addition, our study indicates that the liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4) plays a role in the expression of the GHR 1A mRNA in bovine liver.
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RESULTS |
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Identification of the Transcription Start Site for the GHR 1A
mRNA
The transcription start site for the bovine GHR 1A mRNA was first
determined by using a ribonuclease protection assay (RPA). Using an
antisense riboprobe containing a 130-bp GHR DNA region that was
composed of 105 bp of exon 1A and 25 bp of its 5'-flanking region, the
RPA generated an abundant protected fragment and several less abundant
fragments from bovine liver RNA (Fig. 2A). The size of the most abundant
fragment was 105 nucleotides (nt) (Fig. 2A
), and it placed a
transcription start site at the nucleotide 19 bp downstream from a
putative TATA box (Fig. 1B
). This site was designated as +1 in Fig. 1B
.
The less abundant RPA bands may correspond to minor transcription start
sites or may be artifacts of the RPA.
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Although the 5'-RACE identified a second transcription start site at
nucleotide -115, the 5'-RACE did not indicate whether this start site
was a major start site or a minor start site. For this reason, a second
RPA using a probe corresponding to the long 5'-RACE product was
performed to determine the relative abundance of the GHR 1A mRNA
transcribed from this upstream transcription start site. This RPA
generated one abundant and several less abundant protected fragments
(Fig. 2B). The length of the most abundant RPA band was 262 nt, and it
placed a transcription start site at nucleotide +1 (Fig. 1B
), the same
transcription start site that was mapped by both the short 5'-RACE
product (262 bp) and the 105-nt product of the first RPA. A 377-nt RPA
fragment corresponding to the long 5'-RACE product was not observed on
the same autoradiograph, suggesting that the transcription start site
corresponding to the long 5'-RACE product is a minor transcription
start site. Like the first RPA, the second RPA also generated several
less abundant bands (Fig. 2B
), but none of these less abundant bands
matched with those generated by the first RPA, further suggesting that
the less abundant bands on both RPA autoradiographs were artifacts.
Promoter Analysis of the 5'-Flanking Region of Exon 1A in Cell
Lines
To determine whether the 5'-flanking region of exon 1A serves as a
liver-specific promoter (designated GHR P1) and to identify potential
regulatory regions that control promoter activity, serially truncated
5'-flanking regions of exon 1A were tested in luciferase reporter gene
constructs using human hepatoma-derived cell lines Hep G2 and
PLC/PRF-5, baby hamster kidney-derived cell line BHK-21, and human
cervix adenocarcinoma-derived cell line HeLa. The transcriptional
activities of the various 5'-flanking regions of exon 1A were low and
similar in both liver cell lines (Hep G2 and PLC/PRF-5) and nonliver
cell lines (BHK-21 and HeLa) (Fig. 3). A
relatively high transcriptional activity was observed in the
approximately 500 bp 5'-proximal region (4 to 6 times the promoterless
pGL2B activity) across all of the four cell lines (Fig. 3
). The
transfection analyses did not indicate a positively regulatory region
for greater GHR P1 activity in liver cells than nonliver cells (Fig. 3
). An RPA of the total RNA isolated from cultured Hep G2 and PLC/PRF-5
cells did not detect mRNA for the human liver-specific GHR mRNA variant
(named V1 in Ref. 3) in either cell line (data not shown). Thus, Hep G2
and PLC/PRF-5 cells may not be optimal models for direct
transcriptional analysis of the liver-specific bovine GHR P1.
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Identification of Protein-Binding Sites within the Proximal GHR P1
by Deoxyribonuclease I (DNase I) Footprinting Analysis
The liver cell lines were not ideal models for transient
transfection analysis of the bovine GHR P1. Therefore, DNase I
footprinting analyses were used to analyze the bovine GHR P1 for
transcription factor-binding sties using nuclear extracts from bovine
liver and other tissues. Two DNA fragments corresponding to the
-299/+21 and -469/-250 region of the proximal GHR P1 were incubated
with nuclear extracts from bovine liver, kidney, and spleen tissues and
then digested with DNase I. Incubation of the -299/+21 DNA fragment
with liver nuclear extracts generated an extended footprint within the
-218/-151 region on both strands of the DNA fragment (Fig. 4, A and B). Incubation of the -299/+21
DNA fragment with kidney nuclear extracts generated a weak footprint in
the same region on the antisense strand but not on the sense strand
(Fig. 4
, A and B). Incubation of the same DNA fragment with spleen
nuclear extracts did not generate a footprint on either strand (Fig. 4
, A and B). These results together suggest that the -218/-151 region of
the proximal GHR P1 contains binding sites for liver nuclear proteins.
The liver nuclear proteins that bound to the -218/-151 region may not
be expressed in spleen because the same region was not footprinted by
the spleen nuclear extracts. The same proteins may be expressed in
kidney because the same DNA region was footprinted by kidney nuclear
extracts. The proteins in kidney are probably expressed at a lower
level compared with liver because the footprint generated by kidney
nuclear extracts was weaker and was only observed on one strand of the
DNA fragment (Fig. 4
, A and B). The footprinting analysis of the
-469/-250 DNA fragment revealed a weak footprint in the -464/-446
region for the liver nuclear extracts and a weak footprint in the
-423/-403 region for all of the three nuclear extracts (data not
shown). The specificity of these two weak footprints was not further
evaluated.
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Identification of HNF-4 as the Bovine Liver Nuclear Protein Binding
to the -218/-151 Region
The DNase I footprinting analysis and EMSA demonstrated that the
-218/-151 DNA region contained binding sites for liver nuclear
proteins. An analysis of the -218/-151 DNA sequence with the computer
program MatInspector (17) revealed the presence of putative binding
sites for Nkx-1, Sp1, C/EBPß, E4BP4, HNF-4, COUP-TF, VBP, and
C/EBP within this region. Among these putative binding proteins,
HNF-4, C/EBP
, and C/EBPß were the most likely candidates because
they are liver-enriched transcription factors that play a critical role
in the regulation of many liver-specific gene promoters (18, 19). EMSAs
using specific antibodies for C/EBP
, C/EBPß, and HNF-4 showed that
the DNA-protein complex formed between the -218/-151 DNA fragment and
the liver nuclear extracts could be supershifted by the anti-HNF-4
antibody (Fig. 5D
). This result indicated that HNF-4 was present in the
DNA-protein complex formed between the -218/-151 region and the liver
nuclear extracts. The DNA-protein complex formed between the
-218/-151 DNA fragment and the liver nuclear extracts was not
supershifted by the anti-C/EBP
or anti-C/EBPß antiserum (Fig. 5D
).
Confirmation of Binding of Liver HNF-4 Protein to the Putative
HNF-4 Binding Site in the GHR P1
The putative HNF-4 binding site in the -218/-151 DNA fragment is
located between nt -196 and -178 (Fig. 1B), as revealed by the
computer program (17). To confirm the binding of this site by HNF-4
protein from bovine liver nuclear extracts, EMSAs were performed on a
double-stranded oligonucleotide confined to the putative HNF-4 binding
site (-196/-178). Incubation of the -196/-178 oligonucleotide with
the bovine liver nuclear extracts resulted in the formation of two
DNA-protein complexes (Fig. 6
, A and B).
Both complexes were efficiently competed away by an excess of an
unlabeled oligonucleotide containing the consensus HNF-4 binding site
(see Fig. 1B
for the sequence of the consensus HNF-4 binding site)
(Fig. 6A
). Both complexes were supershifted to near completion by the
anti-HNF-4 antibody (Fig. 6B
). The observation of two DNA-protein
complexes between the HNF-4 binding site and liver nuclear extracts is
consistent with previous studies (20, 21). The formation of two
DNA-protein complexes indicates that in addition to HNF-4, the bovine
liver may express a second protein that contains an identical or
similar DNA-binding region to the HNF-4 protein and an identical or
similar region to the epitope against which the anti-HNF-4 antibody was
raised (21). The possibility that this second protein is a splice
variant of HNF-4 was not studied. Nevertheless, the results of the EMSA
confirm that the -196/-178 region of the bovine GHR P1 is a HNF-4
binding site that can interact with the HNF-4 protein in bovine
liver.
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The HNF-4 Transactivates the GHR P1 by Binding to the HNF-4 Binding
Site
To determine whether the HNF-4 binding site (-196/-178) within
the GHR P1 was required for HNF-4 transactivation, HNF-4 expression
vector was cotransfected into cells with the HNF-4 site-deleted GHR P1
construct (-469/+21/delHNF-4/pGL2). Deletion of the HNF-4 binding site
completely blocked the effects of cotransfected HNF-4 on the GHR P1 in
Hep G2, BHK-21, and PLC/PRF-5 cells (Fig. 7). Deletion of the HNF-4
binding site blocked approximately 90% of the effect of cotransfected
HNF-4 on the GHR P1 in HeLa cells (Fig. 7
). These results confirm that
the cotransfected HNF-4 increased the activity of GHR P1 by binding to
the HNF-4 binding site between nt -196 and -178.
When the HNF-4 expression vector was not cotransfected, the HNF-4
site-deleted GHR P1 and the HNF-4 site-intact GHR P1 had similar
activities in each of the four cell lines (Fig. 7). This result
suggests that either the cell lines do not contain endogenous HNF-4 or
that the endogenous HNF-4 in these cell lines does not activate the
bovine GHR P1. The Hep G2 cells do contain endogenous HNF-4, but the
levels are 5 to 10 times less than in liver (Dr. F. M. Sladek,
personal communication). Perhaps the levels of HNF-4 in Hep G2 cells
are too low to fully or partially activate the transfected GHR P1.
Tissue Distribution of the HNF-4 mRNA and the GHR 1A mRNA in the
Bovine
The above experiments demonstrated that the GHR P1 contained a
HNF-4 binding site, and binding of HNF-4 to this site increased the
activity of the GHR P1 in cell lines. These results suggest that HNF-4
may be involved in the expression of the GHR 1A mRNA in
vivo. In rats, the HNF-4 mRNA was primarily expressed in liver
(20). To determine whether HNF-4 was also primarily expressed in bovine
liver, a 311-bp bovine HNF-4 cDNA fragment was cloned. The bovine HNF-4
cDNA sequence (deposited in GenBank under accession no. AF250028) was
90% identical to the human HNF-4 mRNA (Genbank accession no.
NM_000457) and 87% identical to the rat HNF-4 mRNA (GenBank accession
no. X57133). An RPA revealed that the HNF-4 mRNA in cattle was
primarily expressed in liver. The HNF-4 mRNA was also detected in
kidney, but the level in kidney was approximately 15% of that in liver
(Fig. 8). The HNF-4 mRNA was not detected
in other bovine tissues that were examined (Fig. 8
). An RPA was also
performed to determine the expression of the GHR 1A mRNA in the same
samples in which the HNF-4 mRNA was measured. As shown in Fig. 8
, the
GHR 1A mRNA was detected only in liver (Fig. 8
). The enrichment of
HNF-4 in liver and absence of HNF-4 in most other bovine tissues
support a role of HNF-4 in the liver-specific expression of the GHR 1A
mRNA.
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DISCUSSION |
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The mechanism for liver-specific expression of the GHR mRNA variants is
unknown in any species (5, 13, 14). This mechanism, therefore, was the
focus of the present study. An attractive hypothesis for this mechanism
is that liver-specific transcription factors interact with respective
DNA elements in the GHR P1 and thereby cause the transcription from
exon 1A in liver. Such DNA elements are traditionally defined by
transient transfection analysis in liver-derived cell lines
vs. nonliver cell lines. However, the transient transfection
analyses of the 4.5-kb ovine (5) or the 3.6-kb mouse (13) GHR
5'-flanking sequence in liver-derived cell lines vs.
nonliver cell lines did not identify a positively regulatory region
that defines the liver-specific activity of the GHR P1. The
liver-specific regulatory regions may reside in more distal 5'-flanking
regions. For this reason, the transfection analyses in the present
study were extended to the 8-kb 5'-flanking DNA region of exon 1A in
the bovine GHR gene. The analyses did not reveal a region within the
8-kb 5'-flanking region that conferred a significantly greater
transcriptional activity in liver cell line Hep G2 or PLC/PRF-5 than in
nonliver cell line BHK-21 or HeLa (Fig. 3). Subsequently, we found that
Hep G2 and PLC/PRF-5 cells did not express the human liver-specific GHR
mRNA variant V1, even though they were derived from human liver. The
liver-specific GHR mRNA variant V1 was also not expressed in two other
frequently used human liver-derived cell lines Hep3B and Huh7 (29, 30).
Therefore, liver cell lines may not be optimal models for studying the
transcriptional control of the liver-specific GHR mRNA variant. This
may explain why the transient transfection analysis of the bovine GHR
P1 in Hep G2 and PLC/PRF-5 cells in the present study, the transient
transfection analysis of the ovine GHR P1 in Huh7 cells (5), and the
transient transfection analysis of the mouse GHR P1 in Hep G2 cells
(13) failed to identify a liver-specific regulatory region within the
GHR P1.
Most, if not all, of the liver cell lines are derived from
hepatomas. Because hepatomas lack the liver-specific GHR mRNA
variant (30), we suspected that a liver cell line suitable for the
transient transfection analysis of the liver-specific GHR promoter was
probably not available. We therefore used DNase I footprinting analysis
and EMSA to directly analyze the 500-bp proximal region of the GHR P1
for protein binding sites with nuclear extracts isolated from bovine
liver. The 500-bp proximal GHR P1 was chosen to study because this
region of the GHR gene shared a high degree of sequence similarity
among species (Fig. 1B), and similarities in the 5'-flanking DNA
sequence among different species may represent important regulatory
regions. We found that the -218/-151 proximal GHR P1 region bound
nuclear proteins from liver and that the protein binding to the
putative HNF-4 binding site (-196/-178) was the liver-enriched
transcription factor HNF-4. Overexpression of HNF-4 increased the
activity of the cotransfected GHR P1 in various cell lines. In
addition, we found that in the bovine, HNF-4 mRNA was enriched in liver
and absent in many other tissues (Fig. 8
). These observations together
suggest that HNF-4 may play a role in the liver-specific expression of
the GHR 1A mRNA in the bovine.
HNF-4 is a highly conserved member of the nuclear receptor/HNF-4 family
(19). The HNF-4 binding site has been found in many genes (18, 19, 22, 31, 32, 33), and the reported HNF-4 binding sequences are heterogeneous
(34). The consensus sequence binding to HNF-4 with high affinity is
NG/AGGNCAAAGG/TTCA/GN, which consists of direct
repeats of the hexamer AGGTCA (underlined) intervened by one
or two nucleotides (34). The HNF-4 binding site in the bovine GHR P1,
CTGGGCAAAGGTCGG, is only one nucleotide
different from the consensus HNF-4 binding site, suggesting that the
HNF-4 binding site in the bovine GHR P1 is a high-affinity binding site
for HNF-4. A sequence comparison reveals that a similar HNF-4 binding
site is also present in the ovine GHR P1 and human GHR P1 (Fig. 1B).
Conservation of the HNF-4 binding site in the GHR P1 across species
further supports a role for HNF-4 in the expression of the
liver-specific GHR mRNA variant. Interestingly, the HNF-4 binding site
is less conserved in the mouse GHR gene (Fig. 1B
). Perhaps, the lack of
a HNF-4 binding site is one of the reasons why the mouse GHR P1 is
inactive except during pregnancy (8). A unique mechanism seems to be
responsible for the activation of the mouse GHR P1 during pregnancy
(35).
Given the fact that HNF-4 plays a critical role in the expression of
several liver-specific genes (18, 19), we suspect that HNF-4 plays an
important role in regulating the expression of the GHR 1A mRNA in
bovine liver. However, in addition to liver, HNF-4 is also expressed in
tissues such as kidney where the GHR 1A mRNA is not expressed (Fig. 8).
Thus, HNF-4 alone is probably not sufficient for the expression of GHR
1A mRNA in liver. Because a strictly liver-specific transcription
factor has never been identified for any liver-specific genes, we
postulate that the liver-specific expression of GHR 1A mRNA is probably
not regulated by one or two master liver-specific transcription factors
but may be regulated by many transcription factors that are not
necessarily liver specific. Thus, additional proteins remain to be
identified in future studies to completely understand the mechanism
underlying the liver-specific expression of the GHR 1A mRNA in the
bovine.
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MATERIALS AND METHODS |
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PCR and RT-PCR
A standard PCR was used to amplify the GHR DNA fragment from nt
-25 to +105 (see Fig. 1 for locations) with specific primers. The PCR
product was cloned into the PCR 2.1 vector. This plasmid was then used
to generate an antisense riboprobe for the RPA to map the transcription
start site for the GHR 1A mRNA.
A standard RT-PCR was used to clone a bovine HNF-4 cDNA fragment. Briefly, the bovine liver total RNA was oligo-d(T) primed and reverse transcribed into first-strand cDNA. The first-strand cDNA was then amplified with a forward primer 5'-ATGAAGGAGCAGCTGCTGGT-3' and a reverse primer 5'-AAGAAGATGATGGCTTTGAGG-3' that were designed based on the human HNF-4 cDNA sequence (GenBank accession no. NM 000457). The PCR products were cloned into the pGEM-T vector (Promega Corp., Madison, WI). This HNF-4 cDNA plasmid was used to generate an antisense riboprobe for the RPA to analyze the expression of the HNF-4 mRNA in bovine tissues.
5'-RACE
The 5'-RACE was used to map the transcription start site in the
GHR gene for the GHR 1A mRNA. The 5'-RACE was performed by using a
commercially available 5'-RACE kit according to the manufacturers
instructions (Life Technologies, Inc.). The starting RNA
was total RNA isolated from bovine liver. The three GHR-specific
reverse primers used in the reverse transcription and in the two
sequential rounds of PCR reactions were 5'-ATTTAGGATTCCCAGA-3',
5'-CTGCAGACTCTGAGATGCTC-3', and 5'-CCTGCCACTGCCAAGGTCAA-3' that
were each specific to the GHR exon 4, 3, and 2, respectively. The
products from the second PCR were electrophoresed on agarose gels, and
DNA bands of interest were isolated and cloned into the PCR2.1
vector.
Cloning and Sequencing
Cloning and subcloning were done according to standard
procedures. The nucleotide sequence of cloned DNA/cDNA was determined
by automated fluorescent sequencing (University of Missouri DNA Core
Facility, Columbia, MO).
RNase Protection Assay
The RPA was used to map the transcription start site for the GHR
1A mRNA and to measure the expression of the GHR 1A mRNA and the HNF-4
mRNA in bovine tissues. Two different antisense probes were used in the
RPAs for mapping the transcription start site for the GHR 1A mRNA. One
probe corresponded to a GHR cDNA region that consisted of 52 bp exon 2
and 325 bp exon 1A. The other probe corresponded to the GHR DNA region
nt -25 to +105 (Fig. 1). The former probe was also used in the RPA of
the GHR 1A mRNA expression in bovine tissues. For the RPA that was used
to determine the tissue distribution of the HNF-4 mRNA, the probe was
generated from the HNF-4 cDNA clone that was cloned by the RT-PCR. The
GHR 1A or HNF-4 mRNA- specific probe was generated by standard
in vitro transcription and each probe had a specific
activity greater than 5 x 108 cpm/µg. In
each RPA, approximately 4 x 104 cpm of
riboprobe were hybridized with 20 µg of total RNA. When used to
analyze the tissue distribution of the GHR 1A or HNF-4 mRNA, the RPA
also contained 4 x 104 cpm of bovine
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe. The bovine
GAPDH probe was generated as described (15) with a specific activity of
approximately 5 x 107 cpm/µg. The RPAs
were performed essentially as described previously (10).
Construction of Promoter-Reporter Plasmids
The promoter-reporter plasmid constructs were made by inserting
the GHR genomic DNA fragment into the polycloning sites upstream from
the luciferase reporter gene in the promoterless vector pGL2B
(Promega Corp.). The GHR DNA regions from nt -2,730 to
+21, -1,719 to +21, and -469 to +21 (numbering relative to the
transcription start site +1, Fig. 1) were each amplified by using
standard PCR with specific primers linked with a SacI site
and a NheI site. The PCR fragments were cloned in the
SacI/NheI sites of pGL2B to form
promoter-reporter plasmids -2,730/+21/pGL2, -1,719/+21/pGL2, and
-469/+21/pGL2, respectively. A DNA fragment corresponding to the GHR
5'-flanking region nt -8,133 to -2,730 was amplified by using
long-PCR with specific primers containing SacI linkers and
cloned in the SacI site of plasmid 2,730/+21/pGL2 to form
plasmid -8133/+21/pGL2.
Construct -469/+21/delHNF-4/pGL2 contained a deletion in the HNF-4
binding site (-196/-178) compared with construct -469/+21/pGL2. The
deletion was made by using PCR-based site-directed mutagenesis as
described previously (37). In all of the PCR reactions, proofreading
polymerase Tli (Promega Corp.) was added to minimize PCR
errors. The direction of the insert, insert-vector junction, and
deletion were confirmed by DNA sequencing. The HNF-4 expression vector
pMT7-rHNF41 that encodes rat HNF-4 was provided by Dr. Frances
Sladek (University of California at Riverside, Riverside, CA) (20).
Transient Transfection Analysis
Hep G2, PLC/PRF-5, HeLa, and BHK-21 cells were each plated on
12-well plates and cultured according to the manufacturers
instructions (ATCC, Manassas, VA). In the transient
transfection analyses that were used to determine the transcriptional
activity of the GHR 5'-flanking region of exon 1A, cells in each well
were transfected with 0.5 pmol of GHR promoter-reporter plasmid, 0.002
µg of pRLSV40 plasmid, and an appropriate amount of pCR 2.1 plasmid
(without insert) to bring the total amount of DNA to 5 µg/well. In
the transient transfection analyses that were used to determine the
effect of overexpressed HNF-4 on GHR P1, cells in each well were
transfected with 2 µg of plasmid -469/+21/pGL2 or 2 µg of plasmid
-469/+21/delHNF-4/pGL2, 0.002 µg of pRLSV40, the indicated amount of
pMT7-rHNF-41, and an appropriate amount of pMT7 (without HNF-4 cDNA
insert) to bring the total amount of DNA to 3 µg/well. The
transfection was done by using the calcium phosphate method as
described (10). Cotransfection of pRLSV40 plasmid was used as a control
for transfection efficiency. The firefly luciferase activity encoded by
the promoter-reporter plasmid and the Renilla luciferase activity
encoded by the pRLSV40 plasmid were measured by using the Dual
Luciferase Reporter Assay System (Promega Corp.),
essentially as described (10). To correct for variance in transfection
efficiency, the firefly luciferase activity of each GHR 1A
promoter-reporter construct was divided by the Renilla luciferase
activity of the cotransfected pRLSV40 plasmid. The transcriptional
activity of a GHR promoter-reporter construct was expressed as relative
to the activity of the promoterless vector pGL2B, which was arbitrarily
designated as 1. Data from independent transfections were subjected to
statistical analysis by using ANOVA and the Duncans multiple range
test.
Isolation of Nuclear Extracts from Bovine Tissues
The nuclear protein extracts from bovine liver, kidney, and
spleen were prepared as described (38) except that a protease
inhibitor cocktail (1 ml/20 g tissue) (Sigma, St. Louis,
MO) was included in the homogenization buffer (37). The bovine tissues
were collected from nonlactating cows at slaughter.
DNase I Footprinting Analysis
The DNase I footprinting analysis was used to identify the
5'-proximal region of GHR exon 1A that bound to liver nuclear proteins.
The footprinting analysis was done by using the Core Footprinting
System (Promega Corp.) as described previously (37).
Briefly, two DNA fragments corresponding to the 5'-flanking region of
exon 1A nt -299 to +21 (-299/+21) and nt -469 to -250 (-469/-250)
were released from their respective plasmids. The DNA fragments were
labeled at the 5'-end of either the sense or the antisense strand with
32P by using 32P--ATP
and T4 polynucleotide kinase (Promega Corp.).
Approximately 2 x 104 cpm of the labeled
DNA fragment were incubated with 40 µg of nuclear extracts in 50 µl
of total volume on ice for 45 min in 1x binding buffer supplemented
with 1 µg of poly d(A-T) and 1 µg of poly d(I-C)
(Sigma). The probe-protein mixture was then digested with
0.01 U/µl DNase I in the presence of
Ca2+/Mg2+ at room
temperature for 1 min. The DNase I digestion was analyzed by
electrophoresis on a 6% polyacrylamide sequencing gel. The (G+A)
Maxam-Gilbert sequencing ladders of the same DNA fragment were
generated essentially as described previously (39) and served as size
markers.
EMSA
Complementary oligonucleotides were annealed in 10
mM Tris-HCl (pH 8.0), 1 mM EDTA, and 5
mM MgCl2 by heating to 90 C for 10
min and slowly cooling to 25 C over 1 h. Both 5'-ends of the
double-stranded oligonucleotide were end-labeled with
32P as described above. The EMSA was performed as
described previously (37). Briefly, 2 x 104
cpm (0.02 ng) of probe were mixed with 5 µg or indicated amounts
of nuclear extracts in a total volume of 20 µl that contained 1x
binding buffer (Promega Corp.), 1 µg of poly d(A-T), 1
µg of poly d(I-C), and 2% Ficoll 400. For the competition shift
assays, the nuclear extracts were incubated with a molar excess of the
unlabeled oligonucleotide on ice for 45 min before the addition of the
labeled oligonucleotide. For the supershift assay, the nuclear extracts
were incubated with 1 µl or 2 µl of 1:5 diluted anti-HNF-4
antiserum (provided by Dr. Frances Sladek, University of California at
Riverside) (20) or 1 µl of anti-C/EBP
or anti-C/EBPß antiserum
(provided by Dr. Simon Williams, Texas Tech University) (40) before the
addition of the labeled oligonucleotide. The binding reactions were
further incubated on ice for 45 min and analyzed by electrophoresis on
a native 5% polyacrylamide gel.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported in part by National Research Initiative Competitive Grants USDA CSREES 9537205-2312 (M.C.L.) and 200135205-09963 (H.J.). This is contribution 13,127 from the Missouri Agricultural Experiment Station Journal Series.
Received for publication January 19, 2001. Revision received February 27, 2001. Accepted for publication March 7, 2001.
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REFERENCES |
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