A Role for A/T-Rich Sequences and Pit-1/GHF-1 in a Distal Enhancer Located in the Human Growth Hormone Locus Control Region with Preferential Pituitary Activity in Culture and Transgenic Mice
Yan Jin,
Rama Mohan Surabhi,
Agnes Fresnoza,
Aristides Lytras and
Peter A. Cattini
Department of Physiology University of Manitoba Winnipeg,
Manitoba, Canada, R3E 3J7
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ABSTRACT
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A region located remotely upstream of the human
pituitary GH (GH-N) gene and required for efficient GH-N gene
expression in the pituitary of transgenic mice was cloned as a 1.6-kb
BglII (1.6G) fragment. The 1.6G fragment in the forward or
reverse orientation increased -496GH-N promoter activity significantly
in pituitary GC and GH3 cells after gene transfer. The 1.6G fragment
was also able to stimulate activity from a minimal thymidine kinase
(TK) promoter which, unlike -496GH-N, lacked any Pit-1/GHF-1 element.
Enhancer activity was localized by deletion analysis to a 203-bp region
in the 3'-end of the 1.6G fragment and was characterized by the
presence of a diffuse 136-bp nuclease-protected site, observed with
pituitary (GC) but not nonpituitary (HeLa) cell nuclear protein. A
major low-mobility complex was observed by electrophoretic mobility
shift assay (EMSA) with GC cell nuclear protein, and the pattern was
distinct from that seen with a HeLa cell extract. The
nuclease-protected region contains three A/T-rich Pit-1/GHF-1-like
elements, and their disruption, in the context of the 203-bp region
fused to the TK promoter, reduced enhancer activity significantly in
pituitary cells in culture. A mutation in this region was also shown to
decrease enhancer activity in transgenic mice and correlated with a
decrease in the 203-bp enhancer region complex observed by EMSA. The
participation of Pit-1/GHF-1 in this complex is indicated by
competition studies with Pit-1/GHF-1 elements and antibodies, and
direct binding of Pit-1/GHF-1 to the A/T-rich sequences was shown by
EMSA using recombinant protein. These studies link the A/T-rich
sequences to the distal enhancer activity associated with the GH locus
control region in vitro and in vivo.
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INTRODUCTION
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The human pituitary GH (GH-N) gene is expressed efficiently in
pituitary somatotrophs and somatolactotrophs in vivo (1).
Sequences located in the first 140 bp of upstream GH-N flanking DNA
were implicated in pituitary-specific expression, based largely on the
use of rat pituitary cell lines, hybrid reporter genes, and gene
transfer (2, 3). Subsequently, a nuclear protein variously called Pit-1
or GHF-1 was shown to bind these sequences and direct efficient and
pituitary-specific GH-N promoter activity (4, 5). However, these
proximal promoter sequences were not sufficient to permit efficient or
tissue-specific expression of GH-N (in transgenic mice) in
vivo (6, 7), suggesting the absence and, thus, requirement for a
locus control region (LCR). LCRs allow position-independent and
efficient expression of their associated genes. The presence of
deoxyribonuclease I (DNase I)-hypersensitive sites is a characteristic
of LCRs (7, 8, 9). These sites often signal the presence of
cis-acting sequences associated with a variety of mechanisms
regulating gene expression. Two pituitary-specific hypersensitive sites
(HS I and HS II) were detected in a 1.6-kb region, contained in a
BglII (1.6G) fragment, and centred about 15 kb upstream of
the GH-N transcription initiation site (7), in the 5'-flanking
DNA of the adjacent B-lymphocyte-specific CD79b gene (10). GH-N
transgenes including the 1.6G fragment were expressed efficiently and
specifically in transgenic mice, suggesting that they constitute a
necessary component of the GH LCR (7, 11). Both HS I and HS II were
reconstructed in the transgenic mouse and were pituitary specific (7).
LCRs are expected to possess at least two important activities to
achieve position-independent high levels of expression. The first is
the ability to establish an open chromosomal domain containing the gene
or genes to be expressed, in this case, the GH-N gene in the pituitary,
and the second is to promote high levels of expression (8) and, thus,
possess enhancer activity.
In spite of the recent localization of enhancer activity to a 404-bp
region of the larger 1.6G fragment (11), there is scant information
about the sequences and factors involved in this component of the GH
LCR, largely because of a paucity of in vitro studies.
Specifically, the sequence of the 1.6-kb fragment containing HS I and
HS II was not reported initially (7). Due to the pituitary-specific
nature of HS I and HS II, it would be appropriate to question whether
sites for the pituitary-specific factor Pit-1/GHF-1 are present in the
1.6G fragment. Also, the available data were obtained using the
homologous human GH-N promoter to test the 1.6G fragment or
subfragments. Thus, it was still unclear whether the enhancer activity,
which is contained in the 5'-flanking DNA of the lymphocyte-specific
CD79b gene, required the Pit-1/GHF-1 sites in the proximal promoter
region for enhancer function in pituitary cells in vivo as
well as in vitro.
We have cloned the GH-N gene and all upstream sequences to exon 9 of
the SCN4A gene on chromosome 17 and characterized the 1605-bp 1.6G
fragment of this clone, which is reported to contain HS I and HS II, as
well as retain pituitary-specific activity in transgenic mice (7). We
report the localization of enhancer activity to a 203-bp subfragment of
the 1.6G fragment, which is characterized by the presence of a diffuse
136-bp nuclease-protected region using pituitary cell nuclear protein.
Analysis of this region revealed three A/T-rich Pit-1/GHF-1-like
elements. Mutation of these A/T-rich sites, in the context of the
203-bp subfragment, resulted in a decrease in enhancer activity in
transfected GC cells; however, this decrease was most significant with
the disruption of sequences at nucleotides 1426/1441. The effect of
this mutation was also tested in vivo. Both the 1.6G
fragment and the 203-bp subfragment were able to stimulate thymidine
kinase (TK) promoter activity efficiently in the pituitary, and to a
lesser extent in brain and testis, of founder transgenic mice. Mutation
of these A/T-rich sequences resulted in an overall loss (>99%) of
enhancer activity in the pituitary, and this correlated with a decrease
in the levels of a specific major low-mobility complex observed between
GC cell nuclear protein and the 203-bp subfragment, as seen by
electrophoretic mobility shift assay (EMSA). Pit-1/GHF-1 binding was
shown to contribute to complex formation. These data suggest the
participation of A/T-rich sequences in the enhancer activity associated
with a component of the GH LCR. Although our data implicate Pit-1/GHF-1
as a participant in the distal enhancer activity in the pituitary, it
occurs independently of the homologous GH-N promoter and the presence
of proximal Pit-1/GHF-1 sites.
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RESULTS
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Cloning and Sequencing of 1.6G Fragment
A clone, P13 containing about 45 kb of sequences
located upstream of the GH-N gene, was obtained by screening a P1
library of human genomic DNA with a 160-bp region of the GH-N
5'-flanking DNA (-3127/-2968) (12). The 1.6-kb BglII
(1.6G) fragment, identified by Jones et al. (7), containing
the pituitary-specific locus control activity and HS I and HS II, was
isolated from the clone and sequenced (Fig. 1
; EMBL/GenBank Data Library Accession
AF010280).

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Figure 1. Nucleotide Sequence (Upper Strand) of the 1.6-kb
BglII (1.6G) Fragment Located about 15 kb Upstream of
the GH-N Transcription Initiation Site
No sequence corresponding to the consensus Pit-1/GHF-1 binding site
(5'-WWTATNCAT-3') was identified. A 136-bp region protected by a
pituitary GC cell nuclear extract from nuclease digestion is indicated
by shading. Three A/T-rich regions containing
Pit-1/GHF-1-like elements in the footprint region are indicated by
double underlining. Details of the nucleotides mutated
in M1, M2, M3, and M4 are indicated in the text; however, their
positions are indicated by single overlining. A 72-bp
CA-rich region containing a 6-bp stretch of reverse complementary TG
residues is indicated by single underlining. The
EMBL/GenBank Data Library accession number for these sequences is
AF010280.
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To establish the presence and location of the 1.6G fragment in
the human genome, DNA from human choriocarcinoma JEG-3 cells was
digested with BglII, EcoRI, HindIII,
and HindIII/EcoRI and subjected to DNA blotting
using the 1.6G fragment as a probe. Based on the restriction map, bands
of predicted sizes (1.6 kb with BglII; 12 and 15 kb with
EcoRI and HindIII; and 11.5 kb with
HindIII/EcoRI) were detected in human genomic DNA
(not shown).
To determine the orientation of the 1.6G fragment within the
genome, the clone P13 was digested with BglII,
BglII/HindIII, and
BglII/XhoI, transferred to nitrocellulose, and
probed with a 27-bp oligonucleotide (5'-GCCTTCCAACCATGGCATGGGAGGTGG-3')
corresponding to nucleotides 1629/1656 (relative to the 11605
sequence of the 1.6G fragment). This oligonucleotide sequence was
determined by extending the sequence analysis outside of the 1.6G
fragment in the P13 clone and corresponds to the sequence reported
for the CD79b gene 5'-flanking DNA (10). Also, bands of 11.2 kb with
BglII, 4.7 kb with BglII/HindIII, and
3.3 kb with BglII/XhoI were detected (not shown).
Based on the reported restriction endonuclease map (7, 12, 13), these
sizes correspond to those predicted for sequences downstream of the
1.6G fragment.
No Pit-1/GHF-1 element was identified in the 1.6G fragment
after searching with a consensus sequence reported for Pit-1/GHF-1
(5'-WWTATNCAT-3')(14). However, A/T-rich domains, which might represent
potential binding sites for homeodomain-containing proteins such as
Pit-1/GHF-1, could be found throughout the fragment, including 21 bp
(18 of 21, A or T), 29 bp (28 of 29, A or T), 12 bp (11 of 12, A or T),
18 bp (13 of 18, A or T), and 16 bp (14 of 16, A or T) observed at
nucleotides 57/77, 894/922, 1368/1379, 1388/1405, and 1426/1441,
respectively (Fig. 1
). A 72-bp CA-rich region (58%) with 21
CA-dinucleotides, including a repeat of 14 bp, was identified at
nucleotides 1086/1153 and contains the consensus CACC-binding protein
site located at nucleotides 1141/1148. Interestingly, this CA-rich
region also contains a 6 bp stretch of reverse complementary TG
residues (nucleotides 1113/1118).
The 1.6G Fragment Confers Enhancer Activity on Minimal GH-N and TK
Promoters
A hybrid luciferase gene directed by the (-496/+1) GH-N promoter
(GHp.luc) was used to test the effect of the 1.6G fragment
on expression in rat pituitary GC and GH3 cells after gene transfer.
The 1.6G fragment was inserted upstream of GHp.luc in the
forward (1/1605G.GHp.luc) and reverse
(1605/1G.GHp.luc) orientation. A promoterless luciferase
reporter gene (pXP1) was used as a control for random transcription
initiation. All test plasmids were cotransfected with
RSVp.cat, and chloramphenicol acetyltransferase (CAT)
activity was used to control for variation in DNA uptake. Values were
obtained as luciferase activity per µg lysate protein divided by CAT
activity per µg lysate protein (luciferase/CAT activity). The results
are expressed as fold effect of the 1.6G region on GH-N promoter
activity (Fig. 2
). The GH-N promoter was
stimulated 5.1- and 2.6-fold (P < 0.005, n = 6)
in the presence of the 1.6G fragment in the GC and GH3 cells,
respectively. This enhancement of promoter activity was also seen when
the 1.6G fragment was present in the reverse orientation (Fig. 2
).

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Figure 2. The 1.6G Fragment Stimulates GH-N and TK Promoter
Activity in Pituitary Cells
The effect of the 1.6G fragment, in the forward (1/1605G) and reverse
(1605/1G) orientation, on -496/+1GH-N or -81/+52TK promoter activity
was tested in pituitary and nonpituitary cells after gene transfer. The
luciferase gene was used as the reporter, and cotransfection with
RSVp.cat was used to control for DNA uptake. Promoter
activity was determined as luciferase activity per µg lysate protein
divided by CAT activity per µg lysate protein and expressed as fold
effect of the 1.6G region on GH-N or TK promoter activity. The basal
GH-N promoter activity in pituitary GC and GH3 cells was 1083.9 ±
46.5 (n = 6) and 1181.8 ± 280.4 (n = 6), respectively.
The basal TK promoter activity in pituitary GC, cervical HeLa, and
glial C6 cells was 146.6 ± 6.1 (n = 12), 30.7 ± 1.3
(n = 12), and 17.2 ± 0.5 (n = 3), respectively. Error
bars represent SEM.
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To assess the 1.6G fragment for activity on a heterologous promoter
(lacking a Pit-1/GHF-1 element) as well as the possibility of a
tissue-specific effect, the 1.6G fragment was inserted in the forward
(1/1605G.TKp.luc) and reverse (1605/1G.TKp.luc)
orientation upstream of a hybrid luciferase gene directed by a minimal
(-81/+52) TK promoter and assessed in pituitary GC, cervical HeLa, or
glial C6 cells after gene transfer. The data, expressed as the fold
effect of the 1.6G region on TK promoter activity, are also shown in
Fig. 2
. The presence of the 1.6G fragment in GC cells resulted in about
a 5- and 3-fold increase in heterologous promoter activity in the
forward and reverse orientation, respectively (P <
0.0001, n = 12). In contrast, no significant effect was observed
in HeLa or C6 cells with either hybrid gene (n = 312).
Enhancer Activity Can Be Localized to a 260-bp Subfragment
(Nucleotides 1346/1605) at the 3'-End of the 1.6G Fragment
A series of deletions of the 1.6G fragment were made and inserted
upstream of the hybrid TK/luciferase gene. These included 1)
5'-deletions of nucleotides 1/745, 1/917, and 1/1298 to generate
746/1605G.TKp.luc, 918/1605G.TKp.luc, and
1299/1605G.TKp.luc; respectively; 2) an internal deletion of
nucleotides 601/1300 to generate
a601/1300G.TKp.luc; and
3) a 3'-deletion of nucleotides 1346/1605 to generate
1/1345G.TKp.luc (Fig. 3
).
These hybrid genes, together with the RSVp.cat gene, were
used to transiently transfect rat pituitary GC cells to localize the
enhancer activity. Values were generated as mean luciferase/CAT
activity plus or minus SEM and are expressed as the fold
effect of each of the truncated 1.6G fragments on TK promoter activity
(Fig. 3
). All 5'-deletions displayed significant (
5- to 6- fold)
stimulatory activity (P < 0.001; n = 1233),
which was indistinguishable from that observed in the presence of the
full-length 1.6G fragment (Fig. 3
). Similarly, the internal deletion
a601/1300G did not affect enhancer activity in pituitary GC cells
after gene transfer (4.1-fold, P < 0.01, n = 9).
However, a deletion of nucleotides 1346/1605 at the 3'-end of the 1.6G
fragment abolished all significant stimulatory activity (n = 9),
localizing 93% of the enhancer activity to this 260-bp region (Fig. 3
).

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Figure 3. Enhancer Activity Is Decreased Greater Than 90%
with Deletion of Nucleotides 1346/1605 from the 3'-End of the 1.6G
Fragment
Nucleotides 1/745, 1/917, 1/1297, 601/1300, and 1346/1605 were deleted
from the 1.6G fragment and tested upstream of a hybrid TK
promoter/luciferase gene in transiently transfected GC cells. Cells
were cotransfected with the RSVp.cat gene. Data
(luciferase/CAT activity) from at least nine experiments are expressed
as the fold effect (of these truncated 1.6G fragments) on TK promoter
activity ± SEM.
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Detection of DNA-Protein Interactions in the Truncated Enhancer
Region and Assessment of A/T-Rich Pit-1/GHF-1-Like Sequences Contained
in This Region for Enhancer Function in Culture
DNase I protection assays were done in an attempt to provide a
more focused basis for assessment of the 260-bp region. A fragment
corresponding to nucleotides 1298/1605 was radiolabeled (separate
reactions done to assess each strand), incubated without or with
nuclear protein from rat anterior pituitary GC or human cervical
carcinoma HeLa cells, treated with DNase I, and then subjected to
denaturing gel electrophoresis and autoradiography (Fig. 4
). A large region of 133136 bp showed
some protection with increasing amounts of GC but not HeLa nuclear
protein. This protection was observed on both strands and affected
nucleotides 1344/1476 and 1343/1478 on the upper and lower strands,
respectively (shaded, Fig. 1
).

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Figure 4. A Large 133- to 136-bp Domain of the Enhancer
Region Shows Some Protection with Increasing Amounts of GC but Not HeLa
Cell Nuclear Extract
A subfragment (1 ng) containing nucleotides (A) 1298/1605
(SmaI/BglII), upper strand, or (B)
1605/1298 (BglII/SmaI), lower strand, was
radiolabeled and incubated without or with nuclear extract and then
digested partially with DNase I. Samples were run in a denaturing 5%
or 8% acrylamide/8 M urea gel, dried, and assessed by
autoradiography. Lane a: pBR322/HpaII; lane b: G+A
sequencing reaction; lane c: G sequencing reaction; lane d: no nuclear
protein; lanes eg: 25, 12.5, and 6.3 µg of HeLa cell nuclear
protein; and lanes hj: 25, 12.5, and 6.3 µg of GC cell nuclear
protein. The boxes indicate the footprinted (FP) region
and the nucleotides protected.
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Given the tissue-specific nature of the protected region as well as the
activity of the 1.6G fragment in pituitary cells in vitro
(Fig. 2
) and in vivo (7), the possible participation of
Pit-1/GHF-1 in the complex(es) formed in this region was investigated.
Emphasis was given to the sequences 5'-ATGTTTATATTT-3' at nucleotides
1368/1379, 5'-TTTATTCCATGAACTGAA-3' at nucleotides 1388/1405, and
5'-AAATGTTTTTTCATTTg-3' at nucleotides 1426/1441 (double
underlined, Fig. 1
), because of the overrepresentation of A/T-rich
sequences in binding sites for homeodomain proteins (14, 15, 16, 17). Indeed, a
reexamination of these three sequences revealed that each contains a
site, 5'-TATAaACAT-3', 5'-TTTatTCCAT-3', and 5'-TTTtTTCAT-3',
respectively, that show some similarity with the consensus Pit-1/GHF-1
site, 5'-WWTATNCAT-3' (Fig. 5
). To assess
a possible role in enhancer function, site-directed mutagenesis was
used to disrupt these three sequences, in the context of a 203-bp PCR
product corresponding to nucleotides 1298/1500. This subfragment of the
1.6G fragment contains the entire nuclease-protected region at position
1343/1478. The element located at nucleotides 1368/1379 was converted
to 5'-ATGgcggccgcT-3' (M1), the element at nucleotide 1388/1405 was
converted to 5'-TTTATTCCgactctgtcA-3' (M2), and the element at
1426/1441 was converted to 5'-AAATGTTTTTTgtcgac-3' (M3). Sequences in
an adjacent region corresponding to nucleotides 1444/1450
(5'-AACATCT-3') at the 3'-end of the footprint were also mutated to
5'-AACgcgT-3' (M4) for comparison. The wild-type and mutated fragments
were then inserted upstream of TKp.luc to generate
1298/1500G.TKp.luc, M11298/1500G.TKp.luc,
M21298/1500G.TKp.luc, M31298/1500G.TKp.luc,
and M41298/1500G.TKp.luc, respectively, and tested for
activity in transfected GC cells. The expression for each of these
constructs was corrected using RSVp.cat activity, and the
results are presented relative to 1298/1605TKp.luc which,
based on the results presented in Fig. 3
, was set to 100% activity
(Fig. 6
). As expected, the truncated
region 1298/1500 containing the intact nuclease protected domain
retained (96%) enhancer activity. Although 58% and 56% reductions in
enhancer activity were observed with the disruption of sequences in M1
and M2 (P < 0.05, n = 18), the most significant
decrease (75%) was seen with the modification resulting in M3
sequences (P < 0.001, n = 30). In contrast, only
a 15% decrease in enhancer activity was seen with the disruption of
sequences in M4 DNA.

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Figure 5. Sequence of Pit-1/GHF-1-Like Sites Found in the
Region Protected by Pituitary GC Cell Nuclear Protein
A, Nucleotides identical to those in the consensus Pit-1/GHF-1 element
(14 ) are indicated by uppercase letters. The sequences
of previously characterized Pit-1/GHF-1 elements from the proximal
promoter region of the human GH-N (hGH-N) gene (18 ) and the proximal
promoter region from the rat PRL (rPRL) gene (19 ) are indicated. The
rPRL sequence was shown to represent a high-affinity binding site due
to the presence of the upstream A/T-rich sequences
(underlined) and is capable of supporting binding of
Pit-1/GHF-1 as a dimer (19 ). B, Sequence of the region protected by
pituitary GC cell nuclear protein corresponding to nucleotides
1343/1478. The three boxed domains indicate the
boundaries of the A/T-rich regions, A/T-1, A/T-2, and A/T-3, and the
Pit-1/GHF-1-like sequences are underlined. The region
described as A/T-1+2 corresponds to a fragment spanning nucleotides
1344/1425.
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Figure 6. Enhancer Activity Is Decreased Greater Than 50%
with Disruption of A/T-Rich Regions Containing Pit-1/GHF-1-Like
Elements
A fragment corresponding to nucleotides 1298/1500 and containing the
intact nuclease-protected region 1343/1478 was generated by PCR.
Site-directed mutagenesis was used to disrupt the A/T-rich
Pit-1/GHF-1-like elements located at nucleotides 1368/1379 (M1),
1388/1405 (M2), and 1426/1441 (M3), as well as an adjacent region
1464/1470 (M4) for comparison. The wild-type and mutated fragments were
then inserted upstream of TKp.luc to generate
1298/1500G.TKp.luc,
M11298/1500G.TKp.luc,
M21298/1500G.TKp.luc,
M31298/1500G.TKp.luc, and
M41298/1500G.TKp.luc, respectively, and tested for
activity in transfected GC cells. The results are expressed as a
percentage of the enhancer activity observed with
1298/1605TKp.luc, which was set to 100%. Error bars
represent SEM from at least six experiments.
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Disruption of 5'-AAATGTTTTTTCATTT-3' in M3 Interferes with Enhancer
Function in Transgenic Mice
Founder transgenics were used to assess the result of disrupted
sequences, referred to as M3, in vivo. Since previous
studies of enhancer function in vivo were restricted to
using the homologous human GH-N promoter, which contains Pit-1/GHF-1
DNA elements, the opportunity was also taken to examine the effect of
the 1.6G fragment on heterologous promoter activity in pituitary and
nonpituitary tissue. The hybrid genes 1/1605G.TKp.luc,
1298/1500G.TKp.luc, M31298/1500G.TKp.luc, and
TKp.luc were introduced into zygotes of CD1 mice by
pronuclear injection. Tissues were taken at embryonic day 19 or term,
and luciferase activity was determined. The results expressed as
luciferase activity per µg protein (per 30-sec period of measurement)
are presented in Table 1
. The
identification (or confirmation) of a transgenic mouse as well as an
estimate of copy number was done by DNA blotting using genomic tail
DNA. Even in the absence of consensus Pit-1/GHF-1 sites within the
context of the TK promoter, appreciable activity was detected in the
pituitary of transgenic mice in the presence of the 1.6G fragment.
However, significant activity was also seen in multiple tissues,
particularly the brain and testis. Although there was more variability,
a similar pattern of transgene expression was observed using the 203-bp
subfragment of the 1.6G fragment. However, enhancer activity was
disrupted by modification of sequences, M3, in the context of the
203-bp subfragment. Three of the four founders identified using
M31298/1500G.TKp.luc as the transgene had control levels
of activity in the pituitary (similar to that seen with the
TKp.luc gene). In contrast, one of the four transgenics
appeared to retain enhancer activity in the pituitary; however, levels
were almost 4 times lower than seen in the brain of this animal. The
mean results from 20 mice processed in an identical manner during the
course of this study but determined not to be transgenic are included
for comparison.
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Table 1. Luciferase activity (per µg protein per 30
sec) in Tissues Taken at the Time of Birth of Founder Transgenic
Mice
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Disruption of Sequences at Nucleotides 1426/1441 in M3 Interferes
with Complex Formation on the 203-bp Enhancer Fragment, Correlating
with a Loss of Function
The effect of the M3 mutation on protein/DNA complex formation
with the 203-bp fragment was assessed by EMSA (Fig. 7
). The patterns generated between the
wild-type 203-bp fragment, containing the intact nuclease protected
region 1343/1478, and GC vs. HeLa cell nuclear protein were
distinct, although some bands/complexes with similar mobilities were
observed. Most notably, a major high-mobility (smaller) and
low-mobility (larger) complex was detected with the pituitary GC cell
extract (Fig. 7
, lane b). Mutation of the A/T-rich region at the core
of the footprint region at nucleotides 1426/1441, referred to as M3,
resulted in a decrease in the levels of the low-mobility complex (see
arrow; Fig. 7
, compare lanes b and e). The result of the M4
modification of sequences (nucleotides 1447/1449) was also assessed for
comparison. In contrast to M3, this mutation appeared to have little
effect on the pattern of complexes observed (Fig. 7
, compare lanes b
and h).

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Figure 7. Complex Formation on the 203-bp Subfragment with
Pituitary Nuclear Protein Is Decreased after Mutation of Sequences
Associated with the A/T-Rich Pit-1/GHF-1-like Element (1426/1441) but
Not Adjacent Sequences (1464/1470)
EMSAs were done by incubating 32P-labeled wild-type 203-bp
fragment (wt) corresponding to nucleotides 1298/1500 (ac), as well as
the mutant form of this fragment M3 (eg) or M4 (hj), without (a, e,
and h) or with pituitary GC (b, f, and i) or cervical HeLa (c, g, and
j) cell nuclear extract (6 µg) in the presence of 2 µg of poly
(dI-dC). Complexes detected after gel electrophoresis and
autoradiography are shown. The major specific LMC formed between GC
nuclear protein and wild-type 203-bp fragment is indicated
(arrow).
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The 203-bp Enhancer Region Contains Pit-1/GHF-1 Binding Sites
EMSA of the 203-bp enhancer region with pituitary GC nuclear
protein was done in combination with DNA competitors for low- and
high-affinity Pit-1/GHF-1 binding. Pit-1/GHF-1 elements were used from
the human GH-N gene (18) and rat PRL (19) proximal promoter regions
(Fig. 5
). The PRL sequence was shown to be a high-affinity site capable
of binding Pit-1/GHF-1 as a dimer (19). An unrelated RF-1 element (20)
was used as a negative control. The low-mobility complex was competed
efficiently with the PRL (high affinity) but not GH-N Pit-1/GHF-1 or
RF-1 sites (Fig. 8
). Three overlapping
fragments containing one or more of the A/T-rich regions (Fig. 5B
)
corresponding to nucleotides 1344/1425, which contain two putative
elements at 1368/1379 and 1388/1400 (A/T-1+2), 1378/1412 (A/T-2, not
shown) and 1416/1455 (A/T-3), containing the Pit-1/GHF-1-like elements
in the 203-bp enhancer region, were generated and also used as
competitors. Efficient competition was seen only with the A/T-1+2
region (Fig. 8
).

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Figure 8. A Major Complex Formed on the 203-bp Subfragment
with Pituitary Nuclear Protein Is Competed Using a Pit-1/GHF-1 DNA
Element
EMSA of the 203-bp enhancer region (a) with pituitary GC nuclear
protein (b,r) was done in combination with a 50, 100, and 200
pM excess of the following DNA competitors: rat PRL
Pit-1/GHF-1 element (ce); human GH-N Pit-1/GHF-1 element (fh);
nucleotide region 1416/1455 (ik); nucleotide region 1344/1425 (ln);
and RF-1 element (oq). The major specific LMC formed between GC
nuclear protein and wild-type 203-bp fragment, as seen in Fig. 7 , is
indicated (arrow).
|
|
The overlapping fragments corresponding to A/T-1+2 (nucleotides
1344/1425), A/T-1 (nucleotides 1365/1379), A/T-2 (nucleotides
1378/1412), and A/T-3 (nucleotides 1416/1455) were also used as probes
and competed with the GH-N and PRL Pit-1/GHF-1 elements (Fig. 9
). For the
fragment A/T-1+2 multiple specific complexes were observed based on
their competition by excess unlabeled fragment (Fig. 9A
). Both low- and
high-mobility bands, reflecting larger, low-mobility (LMC) and smaller
(SMC) complexes, respectively, were competed with the PRL Pit-1/GHF-1
element (Fig. 9A
). In contrast, the GH-N Pit-1/GHF-1 element was only
able to compete the smaller high-mobility complexes. A specific
intermediate complex (IMC) with a mobility between those indicated (LMC
and SMC) was not competed with either Pit-1/GHF-1 elements (Fig. 9A
).
For the A/T-1 region, a specific intermediate mobility complex was
observed (Fig. 9B
). This complex was competed by the GH-N Pit-1/GHF-1
site to the same extent as observed with the wild-type unlabeled probe
fragment, but more efficiently by the PRL Pit-1/GHF-1 element. A minor
low- (LMC) and two major high-mobility complexes (SMC) were observed
with A/T-2 (Fig. 9C
), which contains the second putative A/T-rich
Pit-1/GHF-1-like element at nucleotides 1388/1400 in the A/T-1+2
fragment (Fig. 5
). These complexes all showed some degree of
competition, and thus specificity, with excess unlabeled fragment.
Evidence for competition of the high-mobility complexes (SMC) was
observed with the GH-N Pit-1/GHF-1 element, and to a greater extent
with the PRL Pit-1/GHF-1 element (Fig. 9C
). For the A/T-3 region, which
contains a putative Pit-1/GHF-1-like element at nucleotides 1426/1441
(Fig. 5
), multiple specific complexes ranging in mobility/size were
detected (LMC, IMC1, IMC2, and SMC; Fig. 9D
). Complexes represented by
IMC2 and SMC were competed by the PRL Pit-1/GHF-1 element. Competition
of these complexes by the GH-N Pit-1/GHF-1 site was also observed,
although competition of SMC was to a lesser extent (Fig. 9D
).

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Figure 9. Multiple Complexes Are Associated with A/T-Rich
Regions in the 136-bp Nuclease-Protected Domain
Pituitary GC cell nuclear extract (5 µg) was incubated with 2 µg of
poly (dI-dC) and 32P-labeled DNA probes (1 ng)
corresponding to nucleotides 1344/1425 (A/T-1+2), 1365/1379 (A/T-1),
1378/1412 (A/T-2), and 1416/1455 (A/T-3). For competition assays, EMSA
of each fragment with pituitary GC nuclear protein was done in
combination with DNA competitors for Pit-1/GHF-1 binding. Pit-1/GHF-1
elements were used from the human GH-N (18 ) and rat PRL (19 ) gene
proximal promoter regions (Fig. 5 ). Large, intermediate, and small
complexes were detected after gel electrophoresis and autoradiography
and are indicated by LMC, IMC, and SMC, respectively. A, 1344/1425
probe (a); probe/pituitary protein extract (b) in the presence of 25
(c), 50 (d), and 100 (e) pM excess 1344/1425 fragment, or
25 (f), 50 (g), and 100 (h) pM excess PRL Pit-1/GHF-1
oligonucleotide, or 25 (i), 50 (j), and 100 (k) pM excess
GH-N Pit-1/GHF-1 oligonucleotide. B, 1365/1379 probe (a);
probe/pituitary protein extract (b) in the presence of 50 (c), 100 (d),
250 (e), and 500 (f) pM excess 1365/1379 fragment, or 100
(g), 250 (h), and 500 (i) pM excess PRL Pit-1/GHF-1 oligonucleotide, or 100 (j), 250 (k),
and 500 (l) pM excess GH-N Pit-1/GHF-1 oligonucleotide. C,
1378/1412 probe (a); probe/pituitary protein extract (b) in the
presence of 25 (c), 50 (d), and 100 (e) pM excess 1378/1412
fragment, or 25 (f), 50 (g), and 100 (h) pM excess PRL
Pit-1/GHF-1 oligonucleotide, or 25 (i), 50 (j), and 100 (k)
pM excess GH-N Pit-1/GHF-1 oligonucleotide. D, 1416/1455
probe (a); probe/pituitary protein extract (b) in the presence of 25
(c), 50 (d), and 100 (e) pM excess 1416/1455 fragment, or
25 (f), 50 (g), and 100 (h) pM excess PRL Pit-1/GHF-1 oligonucleotide,
or 25 (i), 50 (j), and 100 (k) pM excess GH-N Pit-1/GHF-1
oligonucleotide.
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Pit-1/GHF-1 Participates in Complexes Formed on Subfragments of the
203-bp Enhancer Region
Antibodies to GHF-1 were used in an attempt to obtain a more
direct assessment of the participation of GHF-1/Pit-1 in complexes
formed on the A/T-1+2 (nucleotides 1344/1425) and A/T-3 (nucleotides
1416/1455) subfragments, which contain the A/T-rich Pit-1/GHF-1-like
elements identified in the 1.6G fragment (Figs. 1
and 5
). More
specifically, antibodies to GHF-1 were used to compete complexes formed
with these regions and GC cell nuclear extract in a gel mobility shift
assay. Evidence for competition and the appearance of low- mobility
complexes was detected with these fragments in the presence of GHF-1
antibodies but not normal rabbit serum (Fig. 10
). Most notably, complexes identified
as IMC2 in the A/T-1+2 region and SMC in the A/T-3 region that were
competed with a Pit-1/GHF-1 DNA element (Fig. 9
) were also competed by
the GHF-1 antibodies (Fig. 10
).

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Figure 10. EMSA and Competition with Specific Antibodies
Indicates that Pit-1/GHF-1 Participates in Complexes Formed on
Nucleotide Regions 1344/1425 and 1416/1455
Radiolabeled DNA probes (1 ng) corresponding to nucleotide regions (A)
1344/1425 (A/T-1+2) and (B) 1416/1455 (A/T-3) were incubated with 2
µg poly (dI-dC) and pituitary GC cell nuclear extract in the absence
(a) or presence (b) of GHF-1 antiserum or (c) normal rabbit serum
(NRS). Some competition of bands and the appearance of LMCs are
detected in the presence of GHF-1 antibodies but not NRS with both
fragments.
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|
The A/T-1+2, A/T-1, A/T-2, and A/T-3 fragments were used with purified
Pit-1/GHF-1 in EMSAs to assess any interaction in the absence of
additional nuclear proteins. Specific binding of a commercially
available rat Pit-1 preparation to well characterized Pit-1/GHF-1
elements was established initially. Pit-1/GHF-1-specific bands were
observed when PRL and GH-N Pit-1/GHF-1 DNA elements were used as
probes, but not with the unrelated RF-1 site (Fig. 11
). Assessment of the A/T-rich regions
revealed evidence of Pit-1 binding to A/T-1+2 and A/T-3 sequences (Fig. 11
). A band corresponding to the higher mobility Pit-1/GHF-1-specific
complex was also seen with the A/T-2 fragment on prolonged
autoradiographic exposure.

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Figure 11. Pit-1 Binds to A/T-Rich Regions of the 136-bp
Nuclease-Protected Domain in the Absence of Other Nuclear Proteins
Radiolabeled DNA probes (1 ng) corresponding to Pit-1/GHF-1 elements
from the rat PRL and human GH-N gene proximal promoter regions, the
RF-1 element (RF-1E, negative control), as well as the A/T-1+2, A/T-1,
A/T-2, and A/T-3 regions of the distal enhancer were incubated in the
absence (-) or presence (+) of 714 ng Pit-1. In each panel, the
open arrowheads indicate Pit-1/GHF-1-specific complexes
observed with the rPRL Pit-1/GHF-1 element. The solid
arrowhead indicates a, presumably, DNA-dependent complex seen
with the A/T-3 region with a mobility that would, unfortunately,
obscure one of the Pit-1/GHF-1-specific complexes. The lower-mobility
Pit-1/GHF-1-specific complex is, however, observed with the A/T-3
region.
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Pit-1/GHF-1 Alone Is Not Sufficient to Stimulate 1.6G Enhancer
Activity
To further assess a possible contribution of Pit-1/GHF-1 to 1.6G
enhancer activity, possibly through protein-protein interactions,
expression of GHp.luc and 1/1605G.GHp.luc was
compared in HeLa cells cotransfected with increasing amounts of a
Pit-1/GHF-1 cDNA expression vector (RSVp.GHF-1). Results for promoter
activity, corrected using RSVp.cat activity, were expressed
as mean luciferase/CAT activity ± SEM (Fig. 12
). Comparison of GHp.luc
and 1/1605G.GHp.luc expression in the absence of Pit-1/GHF-1
reveals only a 1.2-fold increase (not significant) in (albeit low
levels of) basal expression in the presence of the 1.6G fragment.
Overexpression of Pit-1/GHF-1 using 5 and 10 µg of expression vector
resulted in 3.2- and 15.5-fold increases in GHp.luc
activity, respectively (Fig. 12
). Similar increases (3.6- and
13.6-fold) in 1/1605G.GHp.luc expression were also observed
with Pit-1/GHF-1 overexpression (Fig. 12
). The increase in GH-N
promoter activity observed as a consequence of Pit-1/GHF-1
overexpression is Pit-1/GHF-1 DNA element dependent, since the
corresponding effect of Pit-1/GHF-1 overexpression using 5 and 10
µg of expression vector on Rous sarcoma virus (RSV) promoter activity
(expressed as cpm/mg protein) was a 2-fold decrease and only a 1.5-fold
increase (not significant), respectively (n = 6).

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Figure 12. Pit-1/GHF-1 Coexpression in HeLa Cells Results in
a Similar Level of TK Promoter Activity in the Presence or Absence of
the 1.6G Fragment
Expression of GHp.luc and 1.6G/GHp.luc
were compared in HeLa cells cotransfected with 0, 5, or 10 µg of
RSVp.GHF-1 to increase Pit-1/GHF-1 levels. Cells were also
cotransfected with RSVp.cat. Promoter activity from at
least six experiments is expressed as mean luciferase/CAT activity
± SEM.
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|
 |
DISCUSSION
|
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In the present study we have done sequencing, gene transfer, and
protein/DNA interaction experiments to investigate the enhancer
activity associated with a component (1.6G) of the GH LCR, located
remotely (15 kb) upstream of the GH-N transcription initiation site (7, 10). The 1.6G region was reported to contain two pituitary-specific
hypersensitive sites (HS I and HS II) (7), and enhancer activity was
localized to a 404-bp region (11). However, a more detailed
investigation of the sequences and protein/DNA interactions involved in
enhancer activity was lacking. In this study we have characterized
functional and structural properties of a 203-bp distal GH-N enhancer
region. We report that the 1.6G fragment, in both the forward and
reverse orientation, was able to significantly enhance the GH-N
promoter in two rat pituitary cell lines (GC and GH3) after gene
transfer (Fig. 2
). Thus, 1.6G enhancer activity can be observed in both
transient transfection as well as transgenic mouse experiments.
Similarly, enhancer sequences within the context of the globin genes
LCRs, function efficiently both in vitro and in
vivo (7, 21, 22, 23). The ability of the 1.6G fragment to act as an
enhancer in a promoter-independent manner and without a requirement for
a Pit-1/GHF-1 element in the proximal promoter region, was confirmed
when the 1.6G fragment was shown to stimulate a heterologous (minimal
TK) promoter in pituitary cells in culture (Fig. 2
) and transgenic mice
(Table 1
). In the case of the latter, enhancer activity was detected in
tissues other than pituitary, most notably the brain and testis.
However, on a per µg protein basis, levels were significantly less
than measured in the pituitary of all three founder transgenic mice
(Table 1
).
Deletion analysis with subsequent testing of truncated fragments
upstream of the minimal TK promoter in GC cells localized the enhancer
activity, at least in part, to a 203-bp region corresponding to
nucleotides 1298/1500 in the 3'-end of the 1.6G fragment (Figs. 3
and 6
). These sequences were also capable of stimulating TK promoter
activity in founder transgenic mice (Table 1
). A similar pattern to
that seen with the 1.6G fragment was observed, as three of the four
founders displayed more efficient enhancer activity in the pituitary
vs. brain, testis, and other tissues, including kidney,
liver, placenta, and spleen. The 1.6G fragment was shown previously to
contain insufficient information to permit copy number-dependent
transgene expression (7). Consistent with this result, we did not
observe copy number-dependent luciferase gene expression and, thus,
integration independence with the 203-bp subfragment. Considerable
variation in expression was seen with three founders determined to have
a single copy of the transgene inserted, and a fourth with five copies
was determined to display the lowest level of stimulation (Table 1
).
Given this observation, the fifth founder (), which showed
efficient enhancer activity in the pituitary but also a comparable
level in the brain, likely reflects a consequence of site of
integration.
Nuclease protection assay of the 203-bp region using pituitary GC cell
nuclear protein resulted in the detection of a large 136-bp footprint,
which appears diffuse because of difficulty in detecting any clear
internal boundaries. However, this level of protection was evident on
both strands, corresponding to nucleotides 1344/1476 and 1343/1478 on
the upper and lower strands, respectively (Fig. 4
). This footprint was
not seen with nonpituitary HeLa cell nuclear extract (Fig. 4
). Although
some of these results were obtained using human DNA sequences with
proteins isolated from rat pituitary cells, both human
pituitary-specific hypersensitive sites (HS I and HS II) were
reconstructed in the transgenic mouse pituitary when the
1.6G-containing fragment, including the GH-N promoter, was used as a
transgene (7). Thus, protein(s) responsible for generating the
chromosomal organization at HS I and HS II that is recognized by
nuclease in the human pituitary must be conserved in the mouse
pituitary. By extension, it is likely then that equivalent proteins are
present in the rat anterior pituitary GC cells used for our
studies.
The size and nature of the nuclease-protected region suggested that
more than one protein, a complex, likely involving DNA-protein as well
as protein-protein interactions, was responsible for this event and,
presumably efficient enhancer function. However, this also excluded any
simple rational approach for mutagenesis to investigate protein/DNA
interactions. The idea of a multicomponent complex was supported,
however, by the detection of a major low-mobility (larger) band with
pituitary (GC) but not nonpituitary (HeLa) cell nuclear protein by EMSA
(Fig. 7
). The failure to see a similar level of nuclease protection or
pattern of shifted bands with cervical HeLa cell nuclear protein
correlates with the lack of any stimulatory activity in these cells
after gene transfer (Fig. 2
). Given the broad range of enhancer
activity seen in various tissues (Table 1
), it is unclear why no
stimulatory activity was seen in HeLa or C6 cells. However, although
cervical tissue and glial cells specifically (as opposed to brain) were
not assessed for luciferase activity in transgenic mice, the lack of
activity observed is likely due to the enormous difference in
sensitivity between the in culture vs. in vivo assay.
This difference is reflected in the levels of enhancer activity seen in
pituitary GC cells vs. pituitary tissue (Fig. 2
and Table 1
). A difference between transient transfection in culture and stable
integration in the transgenic mice could also contribute to this
effect.
The apparent tissue-specific nature of the 136-bp footprint and the
efficient enhancer activity observed in pituitary cells suggested the
possible involvement of the POU-homeodomain protein Pit-1/GHF-1, which
plays an essential role in the normal development and function of the
pituitary. No consensus Pit-1/GHF-1 DNA element of the form
5'-WWTATNCAT-3' is present in the 136-bp nuclease-protected region
(Fig. 1
). However, examination of the sequences in this region did
reveal three relatively A/T-rich sequences that contained
Pit-1/GHF-1-like DNA elements (Fig. 5
). Mutation of each of these three
elements resulted in some, but not total, loss of enhancer function in
the context of the 203-bp subfragment (Fig. 6
). The greatest and most
significant loss was observed with disruption of the Pit-1/GHF-1-like
element at nucleotides 1426/1441 within A/T-3. This element can be
distinguished from the other two by virtue of a sequence
(5'-AAATGTTTTTTCATTT-3') with the capacity to
form a stem-loop structure, which was also disrupted by the mutation,
M3. In the context of the ß-globin locus, palindromes with the
potential to form cruciform structures under torsional stress were
found preferentially near clusters of possible homeodomain
protein-binding sites and or matrix attachment sites (MARs)(16).
The involvement of A/T-3 in enhancer activity was confirmed in
transgenic mice. As a result of the A/T-3 mutation (M3), three of the
four founders displayed only background levels of activity, comparable
to that seen using the minimal TK promoter alone, in the pituitary as
well as other tissues (Table 1
). A fourth founder () retained
stimulatory activity in the pituitary, but the level was relatively
lower than seen in all but one of the nine founders expressing the wild
type 1.6G or 203-bp enhancer transgenes. Also, the pattern of enhancer
activity was different from the majority of founders, as more than 3
times the activity was seen in the brain and, thus, it is assumed that
this reflects a consequence of site of integration. Although not shown,
three founders () were also generated with
the double mutation corresponding to M1 and M3, and none displayed
pituitary-enhancer activity (14.08.4 luciferase units/µg
protein).
The loss of enhancer activity in culture, as well as transgenic mice
with the M3 mutation, correlates with a decrease in the levels of a
major low-mobility specific pituitary complex seen with a 203-bp
subfragment bearing the M3 mutation (Fig. 7
). This decrease was not
observed with mutation (M4) of adjacent sequences (nucleotides
1444/1450). The Pit-1/GHF-1-like site in A/T-3 lies at the core of the
footprint domain detected with pituitary nuclear protein at position
1343/1478 (Figs. 4
and 5
). The efficient enhancer activity observed in
pituitary cells and knowledge that A/T-rich sequences are a common
feature of homeodomain protein-binding sites raised the possibility
that Pit-1/GHF-1 or a Pit-1/GHF-1-like protein interacts with this DNA.
Data from EMSAs done using the 203-bp fragment and pituitary nuclear
extract, in combination with competitors of Pit-1/GHF-1 binding,
indicated that Pit-1/GHF-1 binds with high affinity to the major LMC
(Fig. 8
). Given the competition using the rat PRL, but not the human
GH-N Pit-1/GHF-1 element, the binding likely reflects formation of a
dimer (19). When the three A/T-rich regions were used as competitors,
only the A/T-1+2 region (nucleotides 1344/1425) competed the same
complex effectively (Fig. 8
). This suggested that these sequences might
contain a high-affinity Pit-1/GHF-1 binding site. This was confirmed by
EMSA and competition with the rat PRL Pit-1/GHF-1 element (Figs. 8
and 9
). Pit-1/GHF-1 binding to A/T-3 (nucleotides 1416/1455), the region
containing the putative stem-loop structure, as well as A/T-1
(nucleotides 1365/1379) and A/T-2 (nucleotides 1378/1412), were also
indicated by competition with Pit-1/GHF-1 elements (Fig. 9
). Indeed,
these appeared to contain low-affinity Pit-1/GHF-1 binding sites when
assessed individually, as homologous competition was relatively weak.
Furthermore, competition was not only possible by the high-affinity PRL
Pit-1/GHF-1 site but also by the GH-N Pit-1/GHF-1 element. The
participation of Pit-1/GHF-1 in complexes formed on fragments
containing these sequences was confirmed using antibodies to GHF-1
(Fig. 10
) and through their ability to directly interact with
recombinant rat Pit-1 in the absence of additional nuclear protein
(Fig. 11
). Interestingly, when the A/T-1+2 region, which contains two
of the Pit-1/GHF-1-like elements, was used as a probe or competitor,
evidence for high-affinity Pit-1/GHF-1 binding was obtained. The
presence of Pit-1/GHF-1 binding sites spanning a significant portion of
nuclease-protected region raises the possibility that there is
interaction between these sites and is consistent with the formation of
a large complex on the 203-bp enhancer fragment. Of course this does
not rule out the participation of proteins other than Pit-1/GHF-1 in
this complex. Indeed, the detection of bands formed on the A/T-1+2
(IMC, Fig. 9A
), A/T-2 (LMC, Fig. 9C
), and A/T-3 (LMC and IMC1, Fig. 9D
)
regions, which are not competed by a Pit-1/GHF-1 element (under the
EMSA conditions employed), indicates the participation of other
protein(s) (Fig. 9
). The importance of a factor(s) in addition to
Pit-1/GHF-1 for distal enhancer activity was suggested by transfection
studies using nonpituitary HeLa cells overexpressing Pit-1/GHF-1.
Although there was sufficient overexpression of Pit-1/GHF-1 to see a
stimulation of basal GH-N promoter activity, there was no further
increase resulting from the inclusion of the 1.6G fragment (Fig. 12
).
This additional participant might be tissue- or cell-specific, but the
possibility that it is present at low levels or modified in the HeLa
cells cannot be ruled out. A review of the properties of POU domain
transcription factors like Pit-1/GHF-1 supported the possibility that
Pit-1/GHF-1 might act as a coactivator but that the specificity of its
function would be determined in part by the unique configuration it
assumes on binding its own DNA element (24). With regard to the 203-bp
enhancer fragment, the need for multiple binding events to produce a
stable and functional complex allowing pituitary enhancer activity is
indicated not only by the 136-bp nuclease-protected region (nucleotides
1343/1478; Fig. 5
) but also by the functional assessment of mutant
enhancer regions (Fig. 6
and Table 1
). Also, although not shown, two
transgenic founders () were generated using the
A/T-1+2 region (nucleotides 1344/1425) alone to stimulate TK/luciferase
gene expression. No pituitary enhancer activity was detected with
either 3853 (3.8 luciferase units/µg protein) or 3983 (0.03
luciferase units/µg protein). The case for multiple binding events
with the GH-N distal enhancer region may be analogous to the situation
observed with both human and rat PRL genes. Their distal enhancers are
both Pit-1/GHF-1-dependent, and multiple Pit-1/GHF-1 elements are
required for efficient enhancer activity (25, 26). However, both
contain additional protein-binding sites within these regions and, in
the case of the rat gene, these are necessary to confer regulatory
potential (25, 26).
The 1.6G fragment is located in the 5'-flanking DNA of the
B-lymphocyte-specific CD79b gene (10). The ability of these sequences
to stimulate not only the GH-N promoter, but also the minimal TK
promoter (Fig. 2
), raises the possibility that they might also be able
to influence the expression of the CD79b gene in lymphocytes, where the
B lymphocyte-specific CD79b gene locus is open. Furthermore, we and
others have detected GH-N in lymphocytes (27, 28), which raises the
question of whether the 1.6G fragment participates in the activation of
the GH-N gene in these cells. Interestingly, the presence of
Pit-1/GHF-1 transcripts in lymphocytes has been reported (29). Thus, it
is possible that sequences in the 1.6G region might serve two
functions. The first relates to a role in the GH LCR where it
contributes to the establishment of an open chromatin conformation in
pituitary. This is betrayed by the generation of pituitary-specific
hypersensitive sites, which presumably involves chromatin
reorganization, perhaps through association with the nuclear matrix.
The second could involve stimulation of promoter activity outside as
well as inside the pituitary. These functions would be consistent with
multiple regulatory roles associated with A/T-rich sequences, which
include binding sites for homeobox-containing transcription factors, as
well as MARs. A striking similarity has been suggested between the
A/T-rich sequence motifs present in MARs and homeobox-containing
transcription factors (15). Indeed, the POU-specific domain in
Pit-1/GHF-1 has been shown recently to contain a necessary and
sufficient signal for targeting to the nuclear matrix (30). Among
several functions ascribed to MARs is their ability to mediate
cell-specific expression and define the borders of chromatin domains
(Ref. 15 and references therein and Ref. 16). We have not examined
whether the A/T-rich Pit-1/GHF-1-like sequences that we identified
within the distal GH enhancer located in the GH LCR participate in
changing chromatin configuration and the establishment of an open
locus. However, the A/T-rich Pit-1/GHF-1-like sequences identified in
the 136-bp protected region are excellent candidates as mediators of
the enhancer activity associated with the GH LCR, since we have
demonstrated that their disruption diminishes enhancer function in
pituitary in vivo.
 |
MATERIALS AND METHODS
|
---|
Cloning the 1.6-kb BglII Fragment of the GH Locus
and Sequencing
A P1 library containing human genomic DNA was screened by PCR
using an amplicon corresponding to 160 bp located in a region 3 kb
upstream of the GH-N transcription initiation site (Genome Systems Inc., St. Louis, MO). The amplicon was generated using
the primers: 5'-CAGCCTCTGATCTCAAGGAAG-3' and
5'-GTGGGGTTGAGGACGATCAC-3', and a clone was obtained (P13; clone
address: DMPC-HFF#11434-A). The boundaries of P13 were confirmed by
sequence analysis, since both the 5'- and 3'-ends reside in reported
sequences (exon 9 of SCN4A and downstream of the GH-N gene,
respectively)(12, 31). P13 was digested with BglII and
electrophoresed, and the desired 1.6-kb BglII (1.6G)
fragment size was eluted and subcloned into the BglII site
of pSP73. Nucleotide sequence was determined by the dideoxy method
using the f-mol sequencing kit (Promega Corp., Madison,
WI). Analysis of the sequence for consensus elements was done using
GeneWorks v2.4 (Intelligenetics, Inc., Mountain View, CA)
including a consensus sequences for Pit-1/GHF-1 (14).
DNA (Southern) Blotting
Human genomic DNA was isolated from choriocarcinoma JEG-3 cells
(32), digested with various restriction endonucleases, electrophoresed
in 0.7% (wt/vol) agarose gels, and blotted to nitrocellulose.
Fragments or oligonucleotides used as probes were radiolabeled
routinely to a specific activity of approximately 1 x
109 cpm/µg or 1.6 x 107 cpm/µg using
[32P]dATP or [32P]ATP, respectively. DNA
blots were hybridized to radiolabeled probes at 42 C (DNA fragments) or
48 C (oligonucleotides) in the presence of 50% formamide for 2024 h.
For DNA fragment probes, blots were washed three times for 15 min each
time at 65 C in 0.1x SSC (20x SSC: 3 M sodium chloride,
0.3 M sodium citrate) with 0.1% SDS, and for
oligonucleotides, washed for 10 min each with 6x SSC/0.1% SDS and 2x
SSC/0.1% SDS. Blots were visualized by autoradiography.
Plasmid Construction and Site-Directed Mutagenesis
The 1.6G fragment was blunted into the HindIII site
of pBR322 in the forward and reverse orientation, with the 2.6 kb GH-N
gene located downstream in the EcoRI site (33). The 1.6G
fragments (forward and reverse) with the GH-N promoter region
(-496/+1) were released by BamHI digestion and introduced
in the BamHI site of pXP1 (34) to generate
1/1605G.GHp.luc and 1605/1G.GHp.luc,
respectively. To generate GHp.luc, the 1.6G fragment was
removed from 1/1605G.GHp.luc by
EcoRV/ClaI digestion. The 1.6G fragment was
blunted and inserted into the SmaI site of
pT81luc (34) in the forward (1/1605G.TKp.luc) and
reverse (1605/1G.TKp.luc) orientation. To generate 5'-deletions, the
1.6G fragment was first blunted into the SmaI site of pUC19.
A PstI digestion was done to remove an internal 745-bp
sequence containing a SacI site and then reclosed resulting
in a subfragment of the 1.6G fragment corresponding to nucleotides
741/1605. This vector was then digested with PstI
(blunted)/SacI, SspI/SacI or
SmaI/SacI. The resulting subfragments of the 1.6G
fragment were introduced into the SmaI/SacI sites
of pT81luc to generate 746/1605G.TKp.luc,
918/1605G.TKp.luc, and 1298/1605G.TKp.luc,
respectively. The plasmid containing 1/1605G.TKp.luc was
digested with SmaI or NcoI/XhoI and
religated to generate the internal deletion
a601/1300GTKp.luc, or the 3'-deletion
1/1345G.TKp.luc, respectively. A 203-bp subfragment of the
1.6G fragment corresponding to nucleotides 1298/1500 was synthesized by
PCR with HindIII and SacI ends and inserted into
pT81luc cut with HindIII/SacI to
generate 1298/1500G.TKp.luc. Site-directed mutagenesis (35, 36) was done to disrupt sequences in the 203-bp region (1298/1500) of
the 1.6G fragment by PCR, using 5 ng of template and the primers: M1,
5'-CGGGCCCATGGGCCTCAAGCTGACCTCAGGTGATGgcg-gccgcTCTGAGCTGTTTATTCC-3';
M2,
5'-CGGGCCCATGGGCCTCAAGCTGACCTCAGGTGATGTTTATATTTCTGA-GCT
GTTTATTCCgactctgtcACATCTGACAGCTTTTC-3'; M3, 5'-TAAGGTGAGCTCCGAGGAAC
AGCCCGTTCCGGGCAGCCCCAGATGTTCTTTCTTGTTTCCAGATGTTCgtcgac
AA-AAAACATTTCTCT-3'; and M4 5'-TAAGGTGAGCTCCGAGGAACAGCCCGTTCCGGGC
AGCCCCAGATGTTCTTTCTTGTTTCCAcgcGTTCCAAATGAAAAAAC-3'. The M1 and M2
primers were paired with the reverse primer
5'-TAAGGTGAGCTCCGAGGAACAGCCCGTTCC G-3', and M3 and M4 were paired with
the forward primer 5'-GATATCAAGCTTCC CGGGTCAGTCTCTCTCCAG-3'. After an
initial step at 94 C for 4 min, amplification was done (in 10
mM Tris-HCl pH 8.3, 2.5 mM MgCl2,
50 mM KCl, 200 µg/ml gelatin, 50 µM of each
deoxynucleotide triphosphate, 1 µM of each primer,
and 2 U Taq polymerase in a final volume of 50 µl) with
2939 cycles of denaturation at 95 C for 1 min, annealing at 5558 C
for 45 sec, and extension at 72 C for 90 sec. In the final cycle the
extension time was increased to 10 min. The products were digested with
either NcoI/SacI (M1 and M2) or
HindIII/SacI (M3 and M4), resolved by 4% agarose
gel electrophoresis, isolated, and inserted upstream of
1298/1605G.TKp.luc cut with NcoI/SacI
(M1 and M2) or pT81luc cut with
HindIII/SacI (M3 and M4). The chloramphenicol
acetyltransferase (CAT) gene directed by the RSV promoter
(RSVp.cat) was described elsewhere (37). The expression
vector containing the rat GHF-1 cDNA directed by the RSV promoter
(RSVp.GHF-1) was a generous gift from Dr. M. Karin
(University of California, San Diego, CA).
Cell Culture and Gene Transfer
Monolayer cultures of rat anterior pituitary GC and GH3 cells,
as well as glioma C6 cells, and human cervical carcinoma HeLa cells
were grown on 100-mm dishes and maintained at 37 C in 8% FBS-DMEM
medium at a density of 1 x 106 cells per plate.
Cells, in triplicate, were transfected with 10 µg of test
(luciferase) plasmid DNA and 2 µg of RSVp.cat 1824 h
after plating, by the calcium phosphate/DNA precipitation method as
previously described (38). Cells were harvested 48 h after
transfection. Luciferase activity per µg of lysate protein was
determined using the Luciferase Assay System (Promega Corp.) and a luminometer (ILA911 Luminometer, Tropix Inc.,
Bedford, MA) according to manufacturers instructions. CAT activity
was measured using a modification of the two-phase fluor diffusion
assay (39). Quantitative values for CAT activity were determined by
regression analysis to give counts per min/µg of cell lysate protein.
Values for promoter activity are expressed as the mean
(luciferase/CAT) ± SEM.
Nuclease Protection Assay
Nuclear extracts were made from GC and HeLa cell lines according
to published protocols (40). A subfragment of the 1.6G fragment
containing nucleotides 1298/1605 (SmaI/BglII) was
radiolabeled at convenient restriction enzyme sites in adjacent vector
sequences using Klenow and [32P]dATP. For the protection
assay, 0.51 ng of radiolabeled DNA was incubated without or with
nuclear extract (6.25, 12.5, and 25 µg) on ice for 30 min, and then
at room temperature for 5 min. Each sample was treated with 0.05 U of
deoxyribonuclease I (DNase I) (Promega Corp.) for 90 sec.
The DNase I digestion was stopped with 1% (wt/vol) SDS, 0.1
M NaCl, 0.02 mM EDTA, 10 µg proteinase
K, and 4 µg tRNA and incubated at 37 C for 30 min. Samples were
extracted once with phenol-chloroform-isoamyl alcohol and precipitated
with 2 volumes of ethanol. Pellets were resuspended in 80% formamide,
1 mM EDTA, 0.1% (wt/vol) xylene cyanol and 0.1% (wt/vol)
bromophenol blue, and run in a denaturing 5% acrylamide/8
M urea gel, and assessed by autoradiography.
Gel Mobility Shift Assay
For the gel mobility shift assay (41), pituitary GC or cervical
HeLa cell nuclear protein (2 or 6 µg) was incubated with 2 µg of
poly (dI-dC) and 32P-labeled DNA fragments (250 pg; 1
x 104 cpm). Reactions were done in binding buffer (10
mM HEPES-NaOH, pH 7.9, 50 mM KCl, 2.5
mM EDTA, 10% glycerol, and 1 mM
dithiothreitol) for 30 min at room temperature. For competition assays,
competitor double-stranded oligonucleotides were added with nuclear
extract for 10 min at room temperature and then radiolabeled fragment
for a further 20 min. The specific Pit-1/GHF-1 competitors,
corresponding to the proximal promoter site in the human GH-N gene, as
described previously (18), and the proximal promoter site in the rat
PRL gene, a high-affinity DNA element capable of supporting Pit-1/GHF-1
dimerization (19), were synthesized (Fig. 5
). A nonspecific RF-1
competitor corresponding to a region in the 3'-flanking region of the
CS-B gene, described previously (20), was also synthesized as a
control. For supershift assays, rabbit GHF-1 antiserum or normal rabbit
serum (1 µl) was added to the binding reaction (20 µl final volume)
after 20 min preincubation of the other components at room temperature
and incubated for a further 10 min. Antibodies to the carboxy-terminal
region (amino acids 274285) of GHF-1 (lots 50322 and 50333) were
kindly provided by Drs. M. Karin and C. Caelles (University of
California, San Diego, La Jolla, CA). Full-length rat Pit-1 produced in
Escherichia coli as a 40-kDa polyhistidine-tagged fusion
protein was obtained from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). The Pit-1 binding assay was done essentially as
described (19) except that reactions were done with 714 ng of Pit-1
protein at room temperature for 20 min in 20 µl of 20 mM
HEPES, pH 7.9, 1 mM EDTA, 0.1% NP-40, 15% glycerol, and 2
µg of poly-(dIdC). The DNA-protein complexes were resolved in
nondenaturing 5% polyacrylamide gels.
Transgenic Mice
All animal experiments were done in accordance with the
standards of the Canadian Council for Animal Care. The plasmids
1/1605G.TKp.luc, 1298/1500G.TKp.luc,
M31298/1500G.TKp.luc, and pT81luc were
linearized with BamHI and PvuI and introduced
into the pronuclei of single-cell zygotes from CD1 mice. Injected
embryos were subsequently transferred to the oviduct of surrogate
mothers and brought to embryonic day 19 or term. Genomic DNA was
extracted from tail tissue using Proteinase K digestion followed by
phenol-chloroform extraction and ethanol precipitation. The DNA was
blotted to nitrocellulose membrane as well as an amount of each of the
transgenes estimated to reflect one or five copies in the genome. The
presence of the transgene was determined by probing with a 834-bp
PvuI/PstI fragment of pTK81. The fragment was
labeled with 32P using the random priming method
(Promega Corp.) and Prime-A-Gene Kit Fisher Scientific, Pittsburgh, PA).
Statistics
Statistical analysis of the data was done using the Mann-Whitney
(nonparametric) test. Alternatively, the Kruskal-Wallis (nonparametric)
ANOVA with Dunns multiple comparisons post hoc test was employed. In
all cases, a value was considered statistically significant if
P was determined to be <0.01.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Peter A. Cattini, Gene Technology and Department of Physiology, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E.
This work was supported by a Medical Research Council of Canada grant
(MT-10853). P.A.C. is the recipient of a Medical Research Council of
Canada Scientist award.
Received for publication March 15, 1999.
Revision received May 3, 1999.
Accepted for publication May 14, 1999.
 |
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