Thyroid Transcription Factor-1 Activates the Promoter Activity of Rat Thyroid Na+/I- Symporter Gene
Toyoshi Endo,
Masahiro Kaneshige,
Minoru Nakazato,
Masayuki Ohmori,
Norikazu Harii and
Toshimasa Onaya
Third Department of Internal Medicine, Yamanashi Medical
University, Tamaho, Yamanashi 40938, Japan
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ABSTRACT
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We have cloned 15 kbp of rat thyroid
Na+/I- symporter gene
from liver genomic library, which contains 6 kbp upstream sequence from
the translation initiation site. Southern blot analysis of the genomic
DNA from the liver has revealed that thyroid
Na+/I- symporter gene
is the single gene in the rat. To study the tissue-selective expression
mechanism of the gene, we at first determined the transcriptional start
site of the gene. Results of a rapid amplification of cDNA end
procedure as well as that of primer extension analysis indicated that
the transcriptional start sites clustered between -96, -95, and -93
bp of the gene (A in ATG is designated as +1). Chimeras containing 1.9
kbp (-1967 to -46 bp) of the 5'-flanking sequence of the
Na+/I- symporter gene
and luciferase gene expressed significant enzyme activity when
transfected into a rat thyroid cell line, FRTL-5, but little activity
was observed in BRL-3A rat liver cells. Deletion analysis of the
constructs indicated that a minimal region, exhibiting promoter
activity and cell specificity, is located between -291 and -134 bp of
the gene. Deoxyribonuclease I footprinting shows that nuclear extracts
from FRTL-5, but not BRL-3A, cells protect a region between -245 and
-230 bp. Electrophoretic mobility shift assays have demonstrated that
nuclear extracts from FRTL-5 cells formed a specific DNA-protein
complex with an oligonucleotide probe corresponding to -250 to -211
bp of the gene, but that from BRL-3A cells did not, suggesting that
thyrocyte-selective nuclear factors bind to the region. When the
nuclear extracts from FRTL-5 cells were preincubated with antibody
against thyroid transcription factor-1 (TTF-1), homeodomain containing
nuclear protein, formation of the complex was abolished and the band
was supershifted. We also found that the probe formed a DNA-protein
complex with the recombinant TTF-1 homeodomain, and mutations of the
binding site eliminated factor binding. When pRc/CMV-TTF-1 was
cotransfected with the minimal promoter fragment of thyroid
Na+/I- symporter gene
into FRT cells, which express no TTF-1, it caused a significant
increase in the transcriptional activity of the reporter construct, but
not of the construct having mutated TTF-1-binding element. These
results suggest that TTF-1 confers the cell-selective expression of
Na+/I- symporter gene
in thyrocytes.
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INTRODUCTION
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Thyroid gland is unique in its ability to accumulate iodide, and
the process is the first step of the hormonogenesis. Iodide excess or
deficiency largely affects thyroid function (1, 2) and, in thyroid
tumor, the process is important for its diagnosis and radioiodine
therapy (3). Therefore, iodide uptake mechanism by thyrocytes has been
studied extensively (4, 5, 6, 7, 8), and recent studies have revealed that the
transport of iodide is catalyzed by Na+/I-
symporter (NIS) (9).
Very recently, Dai et al. (10) succeeded in cloning of rat
NIS cDNA and have revealed that it encodes an intrinsic membrane
protein with 12 putative transmembrane domains (10). By Northern
analysis, they identified NIS mRNA in the thyroid, but not in the
liver, kidney, intestine, brain, or heart, suggesting that the gene is
primarily expressed in thyrocytes.
Recent studies have revealed that three genes encoding thyroid-specific
proteins, thyroglobulin (Tg), thyroid peroxidase (TPO), and TSH
receptor (TSH-R), are regulated by thyroid transcription factor-1
(TTF-1) and/or Pax-8 (11). TTF-1, which belongs to a family of
homeobox-containing genes in Drosophila NK2 protein (12), is
expressed in adult rat thyroid, lung, and restricted regions of the
forebrain (13). Pax-8, a member of the murine family of paired
box-containing genes (Pax genes), is present in adult rat thyroid and
kidney (14). Therefore, simultaneous presence of TTF-1 and Pax-8 is a
specific feature of thyroid follicular cells (11).
The structures of Tg and TPO promoters are very similar to each other.
TTF-1 and Pax-8 bind to some specific region, site C, of both
promoters, and DNA sequences recognized by both factors largely overlap
(11). It has been believed that TTF-1 and Pax-8 could be used as
alternatives to each other depending on functional requirements of Tg
and TPO promoters (15). On the other hand, the structure of TSH-R
promoter is different from that of Tg and TPO (16). TSH-R promoter
lacks the Pax-8-binding site, and the gene expression depends on TTF-1
and other factors that interact with cAMP response element (CRE) (17, 18).
To investigate the structure and function of NIS gene, we have isolated
rat NIS gene, defined its transcription start sites, and determined its
promoter region. We also studied cell-selective expression mechanism of
NIS gene in thyrocytes.
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RESULTS
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Isolation and Sequencing of the Genomic Fragment Containing the
5'-Franking Sequence of the Rat NIS Gene
Four positive plaques were isolated by screening rat liver genomic
library (1.5 x 106 plaques) with
32P-labeled rat NIS cDNA (-29 to 1975 bp; in the remainder
of the report, the A in the ATG initiation codon is designated as +1).
A restriction map of one of the positive clones (Clone-KT3, 15 kbp
long) is shown in Fig. 1a
. Since KT3 as
well as rat NIS cDNA contain only one cutting site for
SacII, the genomic fragment possesses 6 kbp of upstream
sequence from the translation initiation site. After ligating Clone-KT3
into pBluescript SK (pBS-NIS·KT3), we determined its nucleotide
sequence from -2264 bp to +114 bp of the gene (DDBJ, EMBL, and NCBI
accession No. D89570).

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Figure 1. Cloning of Rat NIS Gene
a, Schematic representation of one of the clones (KT-3) of rat NIS gene
fragment. Restriction sites are shown as follows: N,
NotI; Sp, SpeI; X, XbaI;
SII, SacII; Sm, SmaI; SI,
SacI; M, MluI. The translational
initiation site (ATG) is indicated by arrows. b, Total
Southern blot analysis of rat liver genomic DNA. Rat liver DNA (20
µg) (lanes 2 and 4) and pBS-KT3 (10 ng) (lanes 1 and 3) were digested
with SpeI (lanes 1 and 2) and SmaI (lanes
3 and 4). After being electrophoresed on agarose gel and transferred to
the cellulose acetate membrane, they were hybridized with
32P-labeled SmaI-SmaI
fragment from pBS-KT3.
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We then performed total Southern blot analysis of the rat liver genomic
DNA. When the genomic DNA as well as pBS-NIS·KT3 were digested by
SpeI or SmaI and then hybridized with 3 kbp of
32P-labeled SmaI-SmaI fragment from
KT3, only one band, the size of which was identical to that from
pBS-NIS ·KT3, was detected for each digestion (Fig. 1b
). The results
indicate that there is a single NIS gene in the rat genome.
Determination of the Transcriptional Start Sites
To identify the transcriptional start sites of NIS gene, we at
first performed a rapid amplification of cDNA ends (RACE) procedure
using mRNA from functional rat thyroid cells, FRTL-5 (19) (see
Materials and Methods). Using anchor primer and NIS
gene-specific primer (GSP1), we obtained the major PCR products, the
size of which was about 150 bp. After isolation and subcloning, 10
positive clones were identified and sequenced. 5'-Ends of these clones
were mapped as follows; -96 bp (five clones), -95 bp (three clones),
and -93 bp (two clones). In these clones, we could not find any
intronic sequence between their 5'-end and the ATG initiation
codon.
Primer extension analysis was performed to validate the results from
the RACE procedure. As shown in Fig. 2a
, extension products were observed only with the FRTL-5 cell poly
(A)+ RNA, but not with that of BRL-3A cells. The major
transcriptional initiation sites were also mapped at -96, -95, and
-93 bp.

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Figure 2. The Transcriptional Start Sites and 5'-Flanking
Sequence of Rat NIS Gene
a, Primer extension analysis for determining the transcriptional start
sites of the NIS gene. The end-labeled GSP-2 was hybridized to 10 µg
poly(A)+ RNAs from BRL-3A (lane 1) and FRTL-5 (lane 2)
cells. The primer was extended by AMV RT, and the products were
analyzed on polyacrylamide gel. A sequence ladder, using the same
primer, was run in parallel. The major transcription initiation sites
are indicated by arrows. b, Nucleotide sequence of the
5'-flanking region of the rat NIS gene. The transcription start sites
are indicated by arrows. TATA-like motif and GC box
motif are marked by solid circles and dashed
line, respectively.
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Figure 2b
shows the nucleotide sequence of 5'-flanking sequence (-400
to -1) of the NIS gene. The region is GC rich (62%), and a TATA-like
sequence, AATAAAT, is located in -125 to -119 bp. Thirty base pairs
further upstream, there exists a GC box motif (from -152 to -149 bp),
but we could not find a consensus sequence for CRE or thyroid hormone
response element in this portion.
These results indicate that the major start sites were -96, -95, and
-93 bp. This conclusion is supported below by demonstrating that they
are encompassed in a region with promoter activity.
Identification of Cell-Selective Promoter Activity in the
5'-Flanking Region of Rat NIS Gene
Chimeric constructs were made in which 1.9 kbp of the 5'-flanking
region, or deletions thereof, were ligated to the luciferase reporter
gene. By electroporation, each was transiently transfected into FRTL-5
and BRL-3A cells. The pNIS-Luc
-1968 expressed significant
luciferase activity when transfected into FRTL-5 cells, compared with
the promoterless control, pGL2 Basic (Fig. 3
). The level of transient expression of
pNIS-Luc
-1968 in BRL-3A cells was below 5% of that in FRTL-5
cells.

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Figure 3. Promoter Activity of Rat NIS-Luciferase Chimeric
Plasmids
Luciferase (Luc) activity in cell lysates from FRTL-5 ( ) and
BRL-3A( ) cells 72 or 48 h, respectively, after transfection
with the NIS-Luc deletion mutants as indicated. All cells were
cotransfected with plasmid pCH110-ß Gal, and the transfection
efficiency was normalized to ß-Gal. The activity is expressed as
relative units of luciferase per unit of ß-galactosidase, and the
values are the mean ± SE for three separate
experiments.
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5'-Deletion mutants, pNIS-Luc
-1550, which remove the sequence
from -1968 to -1551 bp, showed similar activity to the pNIS-Luc
-1968 construct, but 5'-deletion mutants, pNIS-Luc
-1129,
-621,
-476,
-370, and
-291 expressed higher levels of
luciferase activity, 1.8-, 3.5-, 4.2-, 3.5-, and 1.9-fold,
respectively, than that of pNIS-Luc
-1968. However, further deletion
of 5'-end pNIS-Luc
-134 showed little promoter activity. Therefore,
the smallest region necessary for cell-selective promoter activity of
rat NIS gene is -291 to -135 bp, and the region is encompassed by
pNIS-Luc
-291.
TTF-1 Binds to the 5'-Flanking Region of NIS Gene and
Stimulates Its Minimal Promoter Activity
Nuclear extracts from FRTL-5 cells as well as those from
FRT or BRL-3A cells were incubated with a probe spanning nucleotides
-290 to -185 bp, and the resultant complexes were evaluated in DNase
I protection assay (Fig. 4a
). Nuclear
extracts from FRTL-5 cells protected the region between nucleotides
-245 to -230 bp. In contrast, this region was not similarly protected
by nuclear extracts from FRT cells, also derived from rat thyroid
epithelial cells, which contain a trace amount of Pax-8 but not TTF-1
(15, 18, 20, 21, 22, 23), or BRL-3A cells.

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Figure 4. DNase I Protection Assay of the Promoter Region of
the Rat NIS Gene from -290 to -185 bp by Nuclear Extracts from
FRTL-5, FRT, BRL-3A Cells, and TTF-1 HD Protein
a, Lane 1 contains the G + A ladder determined by Maxam and Gilbert
sequence reaction. Lane 2 is unprotected probe digested with DNase I
(Free). Other lanes contain the probe preincubated with nuclear
extracts from FRTL-5 cells, FRT or BRL-3A cells, or BSA. b, Lane 1: G +
A; lane 2, free; lane 3, nuclear extracts from FRTL-5 cells; lane 4,
probe was preincubated with 50 ng bacterial expressed TTF-1 HD
protein.
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We then performed electrophoretic mobility shift assays (EMSAs) using
oligonucleotides that contain the sequence protected by nuclear
extracts from FRTL-5 cells. Oligo-W spanning -250 to -211 bp, which
includes the protected area, formed a specific protein-DNA complex with
extracts from FRTL-5 cells, but not with extracts from FRT or BRL-3A
cells (Fig. 5a
). Formation of the complex
was inhibited by the homologous unlabeled oligonucleotide
(self-competition) and also by Oligo-DS, which contains the TTF-1
binding site on a TSH-R promoter (18, 24) (Fig. 5b
). Since the extracts
from FRT cells do not contain TTF-1 and Oligo-DS interacts with TTF-1
but not with Pax-8 (18, 24), the results suggest that TTF-1 might be
involved in the formation of the complex. Indeed, when the nuclear
extracts from FRTL-5 cells were preincubated with antiserum to TTF-1,
the complex was supershifted (Fig. 5c
). Next, we prepared prokaryotic
expressed and purified TTF-1 homeodomain (HD) to examine its binding
ability to Oligo-W. TTF-1 HD protein formed a protein-DNA complex with
the probe, and mutation of the putative TTF-1 binding sequence, GTTC,
to GTGA eliminated TTF-1 HD binding (Fig. 5d
). To confirm the TTF-1
binding ability to the region between nucleotides -245 to -230 bp, we
repeated the DNase I protection assay using TTF-1 HD protein. TTF-1 HD
protein also protected the region between -248 to -230 bp of the gene
(Fig. 4b
).

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Figure 5. EMSAs with Oligo-W, Oligo-M, and Oligo-DS
a, Nuclear extracts from FRTL-5, FRT, and BRL-3A (BRL) were incubated
with radiolabeled Oligo-W (wild type), which corresponds to -250 to
-211 bp of the NIS gene. b, Nuclear extracts from FRTL-5 cells were
incubated in the presence of unlabeled self-competitor (250-fold, lane
2), unlabeled Oligo-DS, which contains downstream TTF-1 binding site of
TSH-R promoter (250-fold, lane 3), or absence of the competitor (lane
1). c, Radiolabeled Oligo-W was incubated in the absence (lane 1) or
presence of anti-TTF-1 serum (1:250, lane 2) or preimmune serum (PIS)
(1:250, lane 3). d, Ability of recombinant TTF-1 HD protein to form a
protein-DNA complex with radiolabeled synthetic oligonucleotides,
Oligo-W and Oligo-M. The recombinant TTF-1 HD protein (50 ng) was
incubated with radiolabeled Oligo-W (lane 1) or with radiolabeled
Oligo-M (mutant type). EMSAs were performed as described in
Materials and Methods. Arrows on the left
of each gel set denote the specific protein-DNA complexes and the
arrow on the right is the up-shifted complex.
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Next, to investigate whether TTF-1 is truly involved in the
transcriptional expression mechanism of NIS gene, we cotransfected
pRc/CMV-TTF-1 with pNIS-Luc
-291 into FRT cells. TTF-1 significantly
increased the luciferase activity expressed by pNIS-Luc
-291 (Fig. 6a
). However, when we also mutagenized
the putative TTF-1-binding sequence, GTTC, to GTGA of pNIS-Luc
-291,
TTF-1 failed to transactivate the mutated promoter (pNIS-Luc
-291
M) activity in FRT cells (Fig. 6b
).
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DISCUSSION
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In the present report, the 5'-flanking region of the rat NIS gene
has been isolated and characterized. Results of RACE as well as that of
primer extension analysis revealed that the major transcription sites
were mapped at -96, -95, and -93 bp relative to the ATG initiation
codon. In the first report of rat NIS cDNA cloning, Dai et
al. (10) stated that their cDNA had a 5'-untranslated region of
109 bp nucleotides. However, comparison of their published sequence
with our genomic sequence of NIS suggests that the most 5'-end of their
cDNA corresponds to -84 bp of the gene and that the further upstream
sequence of the insert they cloned is probably some multiple cloning
sequence. Therefore, the existence of transcription initiation sites in
this locus is consistent with their report.
Sequential deletion mutants of rat NIS-luciferase chimeras have
revealed that minimal promoter activity is encompassed within the
sequence between -291 to -135 bp relative to the ATG codon. In
addition, comparison of the minimal promoter activity in thyroid cells
with that in the liver cells suggests that this region is important for
cell-selective expression of NIS gene.
It has been reported that thyroid iodide transport activity is markedly
stimulated by TSH and (Bu)2cAMP (4, 5, 6, 7). So, we searched for
the existence of CRE in this portion. However, we could not find the
CRE consensus or CRE-like sequence in this minimal promoter region and
also in more upstream portion (from -2264 to -380 bp of the gene).
Therefore, if TSH or (Bu)2cAMP stimulates the gene
expression of NIS, it is likely that they enhance it via
non-CRE-mediated mechanism.
Our additional concern is the thyroid-selective expression mechanism of
rat NIS gene, because Dai et al. (10) identified NIS mRNA in
the thyroid, but not in other tissues. In the thyroid, it has been
revealed that genes of thyroid-specific proteins such as Tg, TPO, and
TSH-R are regulated by TTF-1 and/or Pax-8 (11). In Tg and TPO
promoters, overlapping binding sites for TTF-1 and Pax-8, dominated as
site C, are similarly arranged, and both factors compete for their
target promoters (14, 15), which leads to modulation of the ratio of Tg
and TPO mRNAs depending on physiological requirement. Adjacent to the
site C region is TTF-2, another thyroid-specific transcription factor
binding sites in Tg and TPO promoters (21, 22, 23). TTF-2 is involved in
hormonal regulation of the expression of these genes (25, 26). On the
other hand, TSH-R gene transcribed not only in the thyroid but also in
nonthyroidal tissues (27), and structure of TSH-R promoter is different
from those of Tg and TPO promoters (16). The presence of only one
binding site for TTF-1 and lack of binding sites for Pax-8 and TTF-2
suggest that molecular events responsible for the thyroid-specific
expression of Tg and TPO do not operate in TSH-R gene in the thyroid.
Recently, it has been reported that TTF-1 and other factors that
interact with CRE are involved in the expression of TSH-R gene in the
thyroid (28, 29).
These lines of evidence, as well as the previous report that not only
thyroid glands, but also salivary glands and gastric mucosa possess
iodide uptake activity (30), prompted us to study the structure of the
NIS gene and also the role of TTF-1 in the tissue-selective expression
mechanism of rat NIS gene in the thyroid. The results of EMSA and DNase
I footprinting analysis, as well as that of cotransfection assay, have
suggested that TTF-1 is also involved in the thyrocyte-selective
expression mechanism of the NIS gene.
The DNA sequence of the NIS gene protected by DNase I and the results
of EMSAs with Oligo-M (Fig. 5
) suggest that the sequence 5'-GTTC-3' in
the sense strand, accordingly, 5'-CAAG-3' in the antisense strand,
spanning from -240 to -237 bp, is important for its TTF-1 binding.
Damante et al. (31) identified the TTF-1 binding site as
minimally CAAG (31) in the sense strand, but in no case 5'-GTTC-3'. Our
data suggest, however, that 5'-CAAG-3' in the antisense strand has a
binding ability to TTF-1.
However, TTF-1 exerts only a modest effect on NIS transcriptional
activity, like that on TSH-R promoter (17, 18). In addition, the
minimal promoter activity of NIS gene, pNIS-Luc
-291, in FRT cells
is about 4-fold higher than in BRL-3A cells even in the absence of
TTF-1 (data not shown). The results suggest the possibility that
factor(s) other than TTF-1, as in the case of the TSH-R gene, might
also be involved in the expression of NIS gene in the thyroid.
Identification and characterization of these factors remain to be
clarified.
It is well known that iodide deficiency or excess largely influences
the thyroid function and induces variable pathological changes in the
thyroid (1, 2). Furthermore, if differentiated thyroid cancer tissues
retained iodide uptake activity, administration of 131I is
very useful for their therapy (3). Very recently, we have demonstrated
that autoantibody against NIS frequently exists in the sera from
patients with autoimmune thyroid disease (32, 33). In these contexts,
analysis of the expression mechanism of NIS might contribute to our
further understanding of the role of iodide and its transporter in the
pathophysiology of the various thyroid diseases.
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MATERIALS AND METHODS
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Cloning of Rat NIS Gene
EMBL3-liver genomic library (CLONTECH Lab. Inc. RL1022j, Palo
Alto, CA) from adult male Sprague-Dawley rats was screened for NIS gene
using 32P-labeled rat NIS cDNA that contains full coding
sequence (10, 32). Positive clones were subcloned into pBluescript
(Stratagene Cloning Systems, La Jolla, CA). Selected restriction
fragments were further subcloned into pBluescript or M13 phage and
sequenced by the dideoxynucleotide method.
RACE and Primer Extension Analysis
RACE was performed using 5'-RACE System [GIBCO BRL (Life
Technologies, Inc., Gaithersburg, MD)] to determine the 5'-end of the
NIS mRNA. Poly (A)+ RNA (2 µg) from FRTL-5 cells (16)
cultured in the presence of TSH was used to synthesize the first strand
cDNA with the first primer (GSP1) (5'-AAGTCGTCGGCACTGCGTTG). After TdT
tailing of the cDNA, PCR amplification of dC-tailed cDNA was performed
using anchor primer (5'-CUACUACUACUAGGCCACGCGTCGACTAGTACG
GGIIGGGIIGGG IIG) and NIS-specific second primer, GSP2
(5'-TCGGATCCCTCCATGGAG ACAGGTGACT). PCR reaction was carried out at 94
C (1 min), 55 C (2 min), and 74 C (2 min) for 30 cycles using a
Perkin-Elmer Cetus Thermal Cycler. PCR products were then ligated into
pCR II vector (Invitrogen, San Diego, CA) and further subcloned into
M13 phage for sequencing.
Primer extension analysis was performed as described previously (34)
using GSP1 as a primer. The primer was labeled with
[
-32P]ATP using T4 nucleotide kinase (Takara Shuzo
Co., Tokyo, Japan). The primer was hybridized with Poly
(A)+ RNA from FRTL-5 cells or BRL-3A rat liver cells (18)
at 42 C overnight and extended with avian myeloblastosis virus (AMV)
reverse transcriptase (Takara Shuzo Co.) for 2 h at 37 C. The
resulting products were analyzed on 8% polyacrylamide-8.3
M urea gel in parallel with a sequencing reaction generated
with the extension primer.
Reporter Plasmids and cDNA Expression Vectors
A 1932 bp SacI-AatII fragment (from -1968
to -36 bp) and MluI-AatII fragment (-624 to
-36 bp) of rat NIS genomic upstream sequence were cloned to
SacI-BglII and MluI-BglII
sites of pGL2-Basic vector (Promega Co., Madison, WI) (these constructs
are designated as pNIS-Luc
-1968 and pNIS-Luc
-621). pNIS-Luc
-1550 and
-1128 were obtained by internal deletion of pNIS-Luc
-1968, and pNIS-Luc
-476, -370, -291, and -134 were from pNIS-Luc
-621. The TTF-1 binding site on NIS promoter was mutagenized by PCR
with the mismatched primers
MP1(5'-GGGGTACCTATACGGAACAAGCCCTAGATGTGGGAGAAAGGGTCAGGA
GACACGAGTGTGACCCCACCCCGAC), and MP2 (5'-GTACAGATCTGACGTCGGGGA
CTCTCG GTC), to obtain the pNIS-Luc
-291 M
construct. Rat TTF-1 cDNA ligated into pRc/CMV was kindly donated by
Professor R. Di Lauro, Naples, Italy.
Recombinant TTF-1 HD, DNase I Protection Analysis and EMSA
The cDNA corresponding to the homeobox of TTF-1 (12) was
amplified by PCR using oligos 5'-TATCTGCAGCACGCCGGAAGCGTCGGG-3' and
5'AGACAA GCTTCTGCTGCGCCGCC-3'. The amplified cDNA (222 bp) was cut with
PstI and HindIII and cloned into pTrcHis B
(Invitrogen). The cDNA encodes 68 amino acids of TTF-1 HD, which is the
same as the recombinant TTF-1 HD reported by Guazzi et al.
(12). The plasmid was then used to transform the BL21 pLysS strain.
Recombinations were grown at 37 C, inducted by 1 mM
isopropyl-ß-D-thiogalactopyranoside, and the protein was
purified using ProBond column (Invitrogen).
DNase I protection analysis was performed as previously described (15).
In brief, NIS genomic fragment from -290 to -185 bp was synthesized
by PCR and was subcloned into pBluescript SK. After end-labeling with
[a-32P]dCTP and Klenow fragment, the plasmid was cut with
HindIII and was purified on 5% native polyacrylamide gel.
For DNase I footprinting, 30 µg of nuclear extracts or 50 ng of TTF-1
HD were incubated for 15 min at room temperature in 25 mM
HEPES/KOH at pH 7.6 containing 5 mM MgCl2, 34
mM KCl, and 1 µg poly(deoxyinosinic-deoxycytidylic)acid.
Extracts were then incubated 20 min in the presence of the probe
(50,000 cpm) in a 20 µl reaction volume. The DNA probe was digested
with 1 U of DNase I (Promega) for 1 min at room temperature before the
addition of 80 µl of stop solution (20 mM Tris-HCl, pH
8.0, 250 mM NaCl, 20 mM EDTA, 0.5% SDS, 10
µg proteinase K, and 4 µg sonicated calf thymus DNA). After
incubation at 37 C for 15 min, the digested products were phenol
extracted, ethanol precipitated, and separated on 8% sequencing
gel.
Oligos used for EMSAs were as follows. Oligo-W (wild type):
5'-AGACAC GAGTGTTCCCCCACCCCGACTGCCCGCACCCCTG-3', which corresponds to
-250 to -211 bp of the NIS gene; Oligo-M:
5'-AGACACGAGTGTGACCCCACCCCGACTG CCCGCACCCCTG-3', which is the mutant
type of Oligo-W; Oligo-DS: 5'-GTTCG CCTCGTGAACTCTCGGAGAGG, which
contains the sequence of the downstream TTF-1 binding site of the TSH-R
promoter (19, 20). EMSAs were performed as described previously (18) as
follows: 1 µg nuclear extract from FRTL-5, FRT, and BRL-3A cells or
50 ng recombinant TTF-1 HD were incubated in a 30 µl reaction volume
for 20 min at room temperature, with or without unlabeled competitor
oligonucleotides in the following buffer: 10 mM Tris-HCl,
pH 7.6, 50 mM KCl, 5 mM MgCl2, 1
mM dithiothreitol, 1 mM EDTA, 12.5% glycerol,
0.1% Triton X-100, and 1 µg poly(deoxyinosinic-deoxycytidylic)acid.
End-labeled probe, 50,000 cpm (0.5 ng DNA), was added and incubated for
an additional 20 min at room temperature. DNA-protein complexes
were separated on 5% native polyacrylamide gel.
Nuclear extracts from FRTL-5, FRT, and BRL-3A cells were prepared as
described (18). Antibody to TTF-1 was produced in rabbits by using
purified recombinant partial rat TTF-1 residue, corresponding to 1 to
126 amino acids, expressed in bacteria with pGEX-2T (Pharmacia Biotech,
Uppsala, Sweden) as an antigen. The ability and specificity of the
antibody were reported previously (35).
Transfection and Luciferase Reporter Assay
FRTL-5 cells (ATCC CRL 8305) cultured in the presence or absence
of TSH, FRT cells, and BRL-3A cells (ATCC CRL 14429) were grown to 80%
confluency. FRT cells, which are known to contain Pax-8 but not TTF-1
(15, 18, 20, 21, 22, 23), were kindly donated from Dr. L. D. Kohn (NIH,
Bethesda). These cells were transfected by an electroporation technique
(Gene Pulser, Bio-Rad Laboratories, Hercules, CA). Ten milligrams of
pNIS-Luc
-1968 or equivalent molar amount of the deletion mutant, or
pGL2-Basic, were introduced into the cells together with pCH110 ß-Gal
to correct for variability in transfection efficiency. Cells were
pulsed (300 V for FRTL-5 and FRT cells; 270 V for BRL-3A cells, 960
µFarads), plated (6 x 106 cells per dish) and
cultured for 72 h in the case of FRTL-5 cells or 48 h for FRT
and BRL-3A cells. All transfections were carried out in triplicate
batches using at least two different DNA preparations. Cells were lysed
by three to four freez-thaw cycles and centrifuged at 4 C in a
microfuge for 5 min. Luciferase assay was performed as described
previously (36). ß-Galactosidase assay was carried out according to
Sambrook et al. (34). Statistical analysis was performed by
paired t test.
 |
FOOTNOTES
|
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Address requests for reprints to: Toshimasa Onaya, Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Yamanashi 40938, Japan.
Received for publication May 19, 1997.
Accepted for publication July 28, 1997.
 |
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