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(Received for publication, April 4, 1995; and in revised form, June 26, 1995) From the
Surfactant protein B (SP-B) is selectively expressed in
bronchiolar and alveolar epithelial cells of the lung. We identified an
upstream enhancer located in the 5`-flanking region of the human SP-B
gene (-439 to -331 base pair, hSP-B(-439/-331))
by deletion analysis of SP-B-luciferase constructs assessed in
transfection assays in vitro. The element cis-activated the
expression of an SV40 promoter-luciferase reporter gene in a human
pulmunary adenocarcinoma cell line (H441-4). Three distinct binding
sites for the nuclear transcription protein, thyroid transcription
factor 1 (TTF-1), were identified, and the purified TTF-1 homeodomain
was bound to the region of hSP-B(-439/-331).
Co-transfection of H441-4 cells with the expression vector pCMV-TTF-1
trans-activated the native human SP-B promoter and the SV40 promoter
fused with the SP-B enhancer. Mutations of the TTF-1 binding sites in
the upstream enhancer blocked TTF-1 binding and transactivation
activity. In summary, TTF-1 interacts with distinct proximal (-80
to -110) and distal (-439 to -331) cis-acting
elements that regulate lung epithelial cell-specific transcription of
the human SP-B gene.
Surfactant protein B is a small, hydrophobic protein that
interacts with phospholipids to reduce surface tension at the
air-liquid interface of the alveoli in the lung. Deficiency of SP-B ( Analysis of the
SP-B gene promoter demonstrated that both TTF-1 and HNF-3 were
activators of SP-B gene transcription mediated by cis-acting elements
located between -218 to +41 bp in the SP-B gene(4) .
Point mutations in the TTF-1 and HNF-3 binding sites in this proximal
SP-B promoter (-111 to -73 bp) eliminated binding of both
transcription factors and decreased transcriptional activity of the
SP-B promoter construct(4) . In the present report, we
identified a distinct enhancer activity in the 5`-flanking region of
the human SP-B gene located -439 to -331 nucleotides
upstream of the transcriptional initiation site. The hSP-B
-439/-331 element activated SV40 promoter activity in
forward and reverse orientations in H441-4 cells. Mobility shift assay,
point mutation, and transfection assays showed that TTF-1 is the
critical nuclear transcription protein activating this SP-B enhancer
element in the human gene.
To generate the site-specific mutants of
B-500 construct at the TTF-1 binding sites, two steps of PCR were
conducted. For the first PCR, proper mutant PCR oligonucleotides were
synthesized with mutations at the position indicated in Fig. 6A. The mutant primers were mixed with the PGL2-B
vector primer GLprimer 1 and GLprimer 2 to make two sets of PCR
products that were subsequently purified by low melting point agarose
gel electrophoresis and the QIAquick gel extraction kit. The purified
PCR products were then mixed together along with GLprimer 1 and
GLprimer 2 primers for the second PCR. The second PCR products were
digested with MluI/XhoI restriction enzymes for 3 h
at the 37 °C. The DNA fragments (553 bp) with MluI- and XhoI-flanking sites at each end were purified by low melting
point gel electrophoresis as described above and ligated into the MluI/XhoI-digested pGL2-B plasmid to generate B-500
Ba
Figure 6:
A, site-specific mutagenesis of the TTF-1
binding sites in the hSP-B(-439/-331). Wild type and mutant
oligonucleotides were used for EMSA analysis. The core nucleotide
(CAAG) of the TTF-1 binding sites were changed to atcc in the mutants
as underlined. The locations of the Ba, Bb, and Bc
oligonucleotides in the hSP-B(-439/-331) enhancer fragment
are indicated in Fig. 1A. B, EMSA of the wild
type and mutant Ba, Bb, and Bc with the TTF-1 HD. Oligonucleotides were
end-labeled by T4 kinase. Probes (100,000 dpm) were incubated with 2 ng
of purified recombinant TTF-1 homeodomain, separated on a 4%
polyacrylamide gel, and subjected to autoradiography. -, no
competitor; +, self-competitor. C, transfection analysis
of the mutant B-500 in H441-4 cells. TTF-1 site mutations described in A were introduced into B-500 by PCR as described under
``Materials and Methods.'' The promoterless construct B, wild
type B-218, B-500, and mutant B-500 at Ba
Figure 1:
SP-B
promoter activity in H441-4 cells. Plasmid DNA (12.5 µg/60-mm dish)
was used to transfect H441-4 cells. Cells were transfected with 5
µg of pCMV-
Figure 2:
A, nucleotide sequence of
hSP-B(-439/-331) of the human SP-B gene. The underlined nucleotide consensus sequences (CAAG) are the putative TTF-1
binding sites. Bars (Ba, Bb, and Bc) represent the regions used to design the oligonucleotides
for mutagenesis study (see details in Fig. 5). B,
plasmid constructs used in transfection assays. a,
promoterless pGL2-B luciferase reporter vector (B); b, pGL2-B vector containing the human SP-B promoter region
from -218 to +41 bp (B-218); c, pGL2-B
vector containing the human SP-B promoter region from -500 to
+41 bp (B-500); d, pGL2-B vector containing the
SV40 promoter (SV40-P); e, SV40-P vector fused with
hSP-B(-439 to -331), the enhancer is forward orientated (SV40-P F); f, SV40-P vector fused with
hSP-B(-439 to -331), the enhancer is in reverse orientation (SV40-P R); g, PCP II-C vector containing the
hSP-B(-439 to -331) fragment from -218 to +41 bp
at the EcoRI site (PCR
II-C).
Figure 5:
A,
TTF-1-dependent enhancer activity of the hSP-B(-439/-331)
element on the human SP-B promoter. TTF-1 enhances hSP-B transcription.
H441-4 cells were transfected with plasmid DNA (12.5 µg/60-mm dish)
containing 2.5 µg pCMV-
Figure 3:
hSP-B(-439/-331) inhibits
hSP-B (-500 to +41 bp) promoter activity in H441-4 cells.
Total plasmid DNA of 12.5 µg/60-mm dish was used in transfection,
which contains 2.5 µg pCMV-
Figure 4:
A, TTF-1 binds to the
hSP-B(-439/-331) enhancer fragment. Radiolabeled
hSP-B(-439/-331) enhancer probe (35,000 dpm) was incubated
with 2 µg of H441-4 cytoplasmic (C) or nuclear (N) extracts in the presence of no competitor(-),
self-competitor (S), or F
Surfactant deficiency in premature infants causes respiratory
distress syndrome(1) . SP-B plays an important role in
maintaining the alveolar stability by enhancing the rate of spreading
and the stability of phospholipid at the air-water interface. SP-B
exerts important effects on phospholipid structures, contributing to
tubular myelin formation, and enhances phospholipid uptake by Type II
epithelial cells (3) . Genetic ablation of the SPB gene in
transgenic mice caused perinatal respiratory failure associated with
atelectasis and the lack of both lamellar bodies and tubular myelin in
the lungs of newborn SP-B deficient mice(2) . Precise
regulation of SP-B expression is therefore likely critical to
surfactant homeostasis and is mediated, at least in part, by
transcriptional mechanisms. In the present work, an upstream
enhancer sequence was identified in the 5`-flanking region of
hSP-B(-439/-331). This distal element is active in the
context of the proximal SP-B promoter-enhancer region and also
stimulates transcription from a minimal SV40 promoter construct
regardless of the orientation. TTF-1 binds to and activates the
enhancer at three distinct sites located within the region -439
to -331 of the human SP-B gene. This conclusion is based on
several observations: 1) TTF-1 HD binds to the enhancer sequence and
forms multiple distinct complexes; 2) nuclear proteins bind to the
upstream SP-B enhancer sequence and were competed by a known TTF-1
binding sequence (F There is increasing evidence supporting the role of TTF-1 in lung
development and lung-specific gene expression. The amino acid sequence
of TTF-1 has been strongly conserved among mammalian species, canine,
rat, and human TTF-1 sharing up to 98% identity(9) . In the
lung, the distribution of TTF-1 expression is consistent with its role
in the modulation of surfactant protein expression.
Immunohistochemistry and in situ hybridization analysis showed
that TTF-1 protein and mRNA were present in a subset of nonciliated
bronchiolar epithelial cells in the conducting airways and in the Type
II epithelial cells in alveoli. TTF-1 was excluded from the ciliated
respiratory epithelial cell and from terminally differentiated Type I
epithelial cells in human and rat(9) , cells that do not
express the surfactant proteins. The present work demonstrates that
homeodomain-containing TTF-1 transcription factor binds complex
cis-acting elements in an enhancer located -439 to -331 bp
from the start of transcription of the human SP-B gene. These findings,
as well as those derived from the analysis of SP-A gene(14) ,
demonstrate that several TTF-1 proteins bind to closely clustered TTF-1
binding sites. Disruption of individual TTF-1 binding sites in each
``unit'' of the SP-B promoter either abolished or severely
impaired the regulatory activity of the element. As shown in Fig. 7, there are at least two such units in the human SP-B
gene. Region I consists of two TTF-1 and one HNF-3 binding site and is
located between -111 and -73 bp in the hSP-B gene. Region
II consists of three TTF-1 binding sites, located in the -439 to
-331 bp region 5` to the transcriptional start site. The TTF-1
cluster sites were also identified in the SP-A, Clara cell secretory
protein, and SP-C promoter and enhancer regions. Table 1summarizes the TTF-1 binding sites in the promoter and
enhancer regions of the lung specific genes. These sites have been
confirmed to be essential for TTF-1 function. Mutations at these sites
either abolished or reduced TTF-1 DNA binding or transactivation
activity.
Figure 7:
Schematic illustration of TTF-1
interactions with the SP-B promoter-enhancer. TTF-1 binds in clustered
sites in two distinct regions in the 5`-flanking sequence of the human
SP-B gene. The proximal element (-to -80) contains two
TTF-1 sites and activates transcription in concert with HNF-3 member by
interacting with basal transcriptional apparatus of the SP-B gene.
TTF-1 also binds to three distinct, clustered sites located from
-439 to -331 that act as an enhancer influencing gene
transcription from both the SP-B and SV40
promoters.
Many naturally occurring homeodomain DNA binding sites are
found in tandem clusters. Cooperativity among the binding sites of
homeodomain proteins may serve to increase occupancy of the cis-acting
site. This cooperativity may also be influenced by oligomerization of
TTF-1 proteins through the Cys residues(13) . Cooperativity of
clustered protein-DNA binding sites was observed in ultrabithorax
gene(15) . POU family proteins (16) and homeobox
containing human HOX 2.1 proteins (17) also bind to their
cognate cis-active elements in a cooperative manner. The functional
significance of these binding site clusters may lie in a fine tuning of
a target gene regulation by limiting concentrations of the
transcription factors. It is tempting to speculate that precise
temporal-spatial regulation of TTF-1 concentration in the developing
foregut endoderm may determine organ-specific gene expression and
development of the lung and thyroid. On the other hand, clusters of
homeodomain regulatory proteins may provide potential interacting
surfaces and facilitate contact with other nuclear proteins modulating
gene expression. A region rich in glutamine and alanine is located
C-terminal to the homeodomain region of the TTF-1 peptide(7) .
This sequence strongly resembles activator regions of other
transcription factors. In general, upstream transcription activators
are thought to interact with the basal transcription factors in the
promoter (e.g. TAFs in TFIID) to increase gene
transcription(18, 19) . For example, the
glutamine-rich activation domain of human SP1 interacts with Drosophila TAF
Volume 270,
Number 42,
Issue of October 20, 1995 pp. 24852-24857
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)is associated with lethal neonatal respiratory failure in
humans (1) and in transgenic mice in which the SP-B gene was
deleted by homologous recombination(2) . Immunohistochemical, in situ hybridization and the promoter analysis indicated that
surfactant protein B is expressed in a lung epithelial cell-specific
manner(3) . The lung epithelial cell specificity of surfactant
protein gene expression is mediated at the level of gene
transcription(4) . Analysis of the 5` regions of several genes
expressed in a lung-specific manner (SP-A, -B, -C, and Clara cell
secretory protein) supports an important role for thyroid transcription
factor 1 (TTF-1) in the control of surfactant protein gene expression.
The homeodomain proteins, TTF-1, and hepatocyte nuclear factor 3
(HNF-3)/forkhead family of proteins, bind to cis-acting elements in the
SP-B and Clara cell secretory protein
genes(4, 5, 6) . TTF-1 was originally
identified as a thyroid transcription factor controlling the expression
of thyroid-specific genes, such as thyroperoxidase and thyroglobulin
genes(7) . However, the temporal and spatial distribution of
TTF-1 expression in the lung supports the concept that may play a role
in lung development and gene expression. In the lung, TTF-1 mRNA and
protein are present at the earliest stages of differentiation and are
later confined to the bronchial and alveolar epithelium(8) .
TTF-1 protein is present in human fetal lung at 11 weeks of gestation,
where it is found in the nuclei of epithelial cells of the developing
airways(9) , consistent with its potential important role in
lung epithelial cell differentiation and function.
Plasmid Constructions and PCR-mediated
Site-directed Mutagenesis
The human SP-B promoters with
various lengths and regions were generated by polymerase chain reaction
(PCR) using Taq DNA polymerase (Life Technologies, Inc.),
synthetic oligonucleotide primers, and the p
5`-650 SP-B CAT
construct as a template(10) . The upstream primer with the MluI site for the B-281 construct is
5`-CGCACGCGTGAACATGGGAGTCTGGGCAGG. The upstream primer with the MluI site for the B-500 construct is
5`-CGCACGCGTCAGAAGATTTTTCCAGGGGAA. The downstream primer with the XhoI site for the B-281 and the B-500 construct is
5`-GCGCTCGAGCCACTGCAGCAGGTGTGACTC. The upstream primer with the MluI site for the SV40-P F construct is
5`-CGCACGCGTCAGGGCTTGCCCTGGGTTAAG. The downstream primer with the XhoI site for the SV40-P F construct is
5`-GCGCTCGAGGCCTGGGTGTTCCCCTCCCAT. The upstream primer with the MluI site for the SV40-P R is
5`-CGCACGCGTGCCTGGGTGTTCCCCTCCCAT. The downstream primer with the XhoI site for SV40-R construct is
5`-GCGCTCGAGCAGGGCTTGCCCTGGGT TAAG. The PCR products were digested with MluI and XhoI restriction enzymes (Life Technologies,
Inc.) and ligated with MluI/XhoI-digested pGL2-B or
pGL2-P luciferase reporter plasmids (Promega). The oligonucleotide
sequences for the PCR II-C are upstream primer 5`-CAGGGCTTGCCCTGGGTTAAG
and downstream primer 5`-GCCTGGGTGTTCCCCTCCCAT. The PCR product was
directly subcloned into the PCR II vector as described by the
manufacturer (Invitrogen).
, B-500 Bb, and B-500 Bc mutant
luciferase constructs. The correctness of all the wild type and mutant
plasmid constructs were confirmed by DNA sequencing.
, Bb,
and Bc were transfected into H441-4 cells, and activity was
assessed by luciferase assays. pCMV-Rc (Invitrogen) is the parent
plasmid for pCMV-TTF-1 and used as a control, which contains no TTF-1
cDNA insert. Mutations in the TTF-1 binding sites decreased
transcriptional activity of all three B-500 mutants. Values are mean
+ S.D. (n = 6).
gal and 7.5 µg of construct B, SV40-P,
thymidine kinase (a pGL2-B luciferase reporter construct containing the
minimal thymidine kinase promoter), B-218, and B-500. Luciferase assays
were carried out in duplicate 2 days after
transfection.
Cell Culture, Transfection, and Reporter Gene
Assays
H441-4 cells were maintained in RPMI medium
supplemented with 2 mM glutamine and 10% fetal calf serum
(Life Technologies, Inc.). One day before transfection, 5 
10
cells were seeded into 60-mm dishes. Each dish was
transfected with 12.5 µg of total plasmid DNA using the calcium
phosphate precipitation method and incubated in Dulbecco's
modified Eagle's medium overnight. The next day, the media were
changed to RPMI, and the cells were incubated for 2 days prior to
assay. Cell lysis and luciferase assays were performed using the
luciferase assay system purchased from Promega. The light units were
assayed by luminometry (monolight 2010, Analytical Luminescence
Laboratory, San Diego, California). Transfection efficiency was
normalized to -galactosidase activity. Multiple transfections (n = 2-8) were carried out for each experiment,
and the mean values were used for data presentation. Standard
deviations were generally less than 20%. Plasmids pCMV-Rc (Invitrogen)
and pCMV-TTF-1 were kind gifts from Dr. R. Di Lauro, Stazione Biologic,
Naples, Italy.Nuclear Extracts and EMSA
H441-4 cells
were grown on 75-mm flasks. Before harvesting, cells were washed twice
in Hanks' solution. The cell pellet was then resuspended in 5
volumes of lysis buffer (50 mM Tris-Cl, 100 mM NaCl,
5 mM MgCl
, and 0.5% (v/v) Nonidet P-40) for 5 min
on ice. After centrifugation, the supernatant was saved as cytoplasmic
protein extract. The nuclear pellet was resuspended in a 100 µl of
nuclear buffer (0.5 M KCl, 20 mM Tris-Cl, pH 7.6, 0.2
mM EDTA, 1.5 mM MgCl, 25% glycerol, and 1
mM dithiothreitol) and incubated on ice for 30 min. The
resulting DNA pellet was spun down, and the supernatant was used as
nuclear extract. Protein extract (5 µg) was used for EMSA as
described previously(11) . Recombinant rat TTF-1 homeodomain
(HD) was a kind gift from Dr. Di Lauro. The probes for EMSA were made
from either the synthetic oligonucleotides or the PCR product
(hSP-B(-439/-331) fragment).
Expression of SP-B, SV40, and Thymidine Kinase
Promoters in H441-4 Cells
The -218 to +41 bp
(minimal promoter) and the -500 to +41 bp regions of the
human SP-B gene were subcloned into the pGL2-B luciferase reporter gene
producing constructs B-218 and B-500 (Fig. 2B). When
the B-218 and B-500 promoters were compared with the SV40 and thymidine
kinase promoters in H441-4 cells using transient transfection assays,
both B-218 and B-500 constructs were more active than the SV40 and
thymidine kinase promoters (Fig. 1). Activity of B-500 was
3-4-fold greater than B-218, indicating a potential enhancer
element located in the distal upstream region.
gal, 5 µg of construct B, B-218,
B-500, and 5 µg of pCMV-Rc(-) or pCMV-TTF-1 (+). B-218
activity is set as 1. pCMV-Rc (Invitrogen) is the parent plasmid for
pCMV-TTF-1 and used as a control, which contains no TTF-1 cDNA insert.
TTF-1 transactivated both B-218 and B-500. Values are mean ±
S.D. (n = 8). B, TTF-1-dependent enhancer
activity of the hSP-B(-439/-331) element on the SP-B SV40
promoter. SV40 promoter stimulation by TTF-1. Assay conditions were the
same as in A, except construct B, SV40-P, SV40-P F, and SV40-P
R were co-transfected with pCMV-Rc or pCMV-TTF-1. SV40-P activity is
set as 1. TTF-1 trans-activated both SV40-P F and SV40-P R. Activity of
the constructs after transfection with pCMV-Rc is consistant with
activation by endogenous TTF-1 in H441-4 cells. Values are mean
± S.D. (n = 4).
Transcriptional Activity and DNA Protein Binding of
hSP-B (-439 to -331)
Nucleotide sequence in the
5`-flanking distal upstream regions of the human and mouse SP-B genes
share 95% identity from -439 to -331 bp (human) and
-382 to -282 bp (mouse). Deletion of this region in mouse
SP-B gene dramatically reduced the transcriptional activity (50-fold
reduction) as assayed by transient transfection of the mouse lung
epithelial (MLE-15) cell line using the chloramphenicol acetyl
transferase reporter gene. ()In order to determine the
biological function of the stimulatory element in the human gene, the
hSP-B(-439/-331) sequence was subcloned into the PCR II
vector. The final construct, PCR II-C (Fig. 2B, g), was generated using the standard PCR procedure. Transient
transfection of the B-500 construct with an excess amount of PCR II-C
competitor plasmid reduced transcriptional activity from B-500 to the
level of B-218 activity (Fig. 3, lane 4), compared with
the 4-fold activity without the PCR II-C competitor. The competition
experiments suggested the presence of trans-acting factors that
interact with the hSP-B(-439 to -331) element.
gal, 1.5 µg of construct B, and
8.5 µg of PCR II (lane B); 1.5 µg of B-218 and 8.5
µg of PCR II (lane B-218); 1.5 µg of B-500 and 8.5
µg of PCR II (lane B-500); or 1.5 µg of B-500 and 8.5
µg of PCR II-C (lane B-500 + PCR II-C). PCR II
(Invitrogen) is the parent plasmid of PCR II-C, which contains no
hSP-B(-439/-331) insert. Values are mean ± S.D. (n = 4).
TTF-1 Binds to the hSP-B(-439/-331)
Fragment of the Human SP-B Gene
After carefully examining
the hSP-B -439 to -331 region, three distinct CAAG motifs (12) were found in the hSP-B(-439/-331) fragment,
supporting the likelihood that the element contains TTF-1 binding sites (Fig. 2A). EMSA was used to examine the nuclear
proteins binding to the hSP-B -439 to -331 region. Smeared
DNA-protein complexes with slow mobility were identified using H441-4
cell nuclear extracts (Fig. 4A). No shift in mobility
was observed with the cytoplasmic fraction from H441-4 cells (Fig. 4A). DNA oligonucleotide F, a TTF-1
binding site previously identified in the proximal element of the human
SP-B gene (4) , was used as a competitor in EMSA to test
whether the nuclear protein binding to the hSP-B(-439/-331)
fragment was TTF-1. Fig. 4A demonstrates that the
specific interaction between the H441-4 nuclear protein and the
radiolabeled hSP-B(-439/-331) fragment was inhibited by
adding 100-fold molar excess of F fragment or
self-competitor. The protein-DNA complexes were retarded with TTF-1
antibody in the supershift analysis (data not shown). When the
radiolabeled hSP-B(-439/-331) fragment was incubated with
the purified TTF-1 HD protein, three protein-DNA complexes were
observed (Fig. 4B, lane 1), consistent with
the presence of multiple TTF-1 binding sites in the DNA fragment
-439/-331. These TTF-1 complexes were inhibited by adding
50-fold molar excess of self-competitor and the F fragment (Fig. 4B, lanes 2 and 3), confirming
that TTF-1 interacts with multiple binding sites in the
hSP-B(-439/-331) fragment.
fragment (f) (containing known TTF-1 binding sites
of the human SP-B gene) and run on a 4% polyacrylamide gel. The
DNA-binding protein (BP) complex was inhibited by
self-competitor or F DNA competitors. B, DNA
binding study of TTF-1 HD to the hSP-B(-439/-331) enhancer
fragment. Radiolabeled hSP-B(-439/-331) enhancer probe
(40,000 dpm) was incubated with 3 ng of purified recombinant TTF-1
homeodomain protein in the presence of no competitor (-),
self-competitor (S), F fragment (f), or the F fragment (f) (containing an HNF-3 binding site) of
the human SP-B gene and separated on 4% polyacrylamide
gel.
hSP-B(-439/-331) Activates Transcription
from SV40 and SP-B Promoters
pCMV-TTF-1 was co-transfected
with B-218 and B-500 into H441-4 cells. pCMV-TTF-1 activated
transcription of B-218 approximately 4-fold. pCMV-TTF-1 further
activated B-500 transcription (11-fold) (Fig. 5A).
Because there are two active TTF-1 sites in B-218, it was not possible
to discern the distinct contributions of the activity from the three
putative TTF-1 sites in the hSP-B(-439/-331) fragment from
those in the proximal (F) element located -111 to
-73 bp. The hSP-B(-439/-331) fragment was therefore
isolated and ligated to an SV40 promoter-luciferase construct in the
forward and reverse orientation producing SV40-P F and SV40-P R (Fig. 2B). The hSP-B(-439/-331) fragment
stimulated the SV40 promoter transcriptional activity in both
orientations. SV40-P R was more active than SV40-P F (Fig. 5B). Co-transfection of H441-4 cells with
pCMV-TTF-1 increased SV40-P F activity 9-fold and SV40-P R activity
19-fold (Fig. 5B).Mutations in the hSP-B(-331/-439)
Abolished or Reduced the TTF-1 Response
To further confirm
that the putative TTF-1 binding to the sites in the
hSP-B(-439/-331) fragment mediated transactivation, three
wild type TTF-1 sites and three mutant oligonucleotides were
synthesized (Fig. 6A), radiolabeled, and incubated with
recombinant TTF-1 HD protein and separated by EMSA. Although all three
wild type oligonucleotides were shifted by TTF-1 HD, the mobility of
mutant oligonucleotides was not altered (Fig. 6B).
TTF-1 HD binding to the wild type oligonucleotides were inhibited by
100-fold molar excess of self-competitor. The mutants lacking binding
to TTF-1 HD were introduced into the B-500 luciferase expressing
construct. Wild type and mutant B-500 constructs mutated at the
positions Ba, Bb, and Bc were
transfected into H441-4 cells. As illustrated in Fig. 6C, site-specific mutations in the B-500
constructs decreased transcriptional activity. Mutations at positions
Ba and Bb reduced transcription to the level of
the minimal promoter (B-218) and completely abolished the stimulatory
response produced by co-transfection with pCMV-TTF-1. Mutation at the
position Bc only moderately impaired activity.
Transcription from the hSP-B(-439/-331) fragment was
therefore highly dependent on TTF-1 binding to the region.
) and supershifted by the TTF-1
antibody; 3) pCMV-TTF-1 expression vector stimulated the SP-B and the
SV40 promoters linked to the upstream SP-B enhancer sequence; 4)
mutations at the three putative TTF-1 binding sites on the
hSP-B(-439/-331) fragment reduced or abolished TTF-1 HD
binding transcriptional activity. Dr. Di Lauro and co-workers recently
demonstrated that TTF-1 forms intermolecular protein oligomers through
its cysteine residues(13) , likely accounting for the
heterogeneity of the -438 to -331 region of the SP-B gene.
110(20) , and the acidic
activation domain of VP16 interacts with Drosophila TAF
40(21) , as well as tumor suppressor
protein p53 interacts with TAF
40 and
TAF
60(22) , SP1, YY1, USF, CTF, and adenoviral E1A
interacting with TAF
55(23) , etc. The present
findings demonstrate TTF-1-dependent enhancer activity in the distal
upstream 5` region of the SP-B promoter. From previous studies in this
laboratory, the binding of TTF-1 proteins in region I of the SP-B gene
was dependent upon interactions with general transcriptional factors in
the SP-B promoter(4) , functioning in a manner distinct from
that of region II. Region I is indispensable for basal transcription of
the SP-B promoter and does not act as an enhancer when linked to other
basal promoters(4) . In contrast, mutations in the TTF-1
binding sites in the distal element (region II) block TTF-1-dependent
enhancer activity but do not block the activity of region I of the
human SP-B promoter. It follows that the ``extrinsic
cooperativity'' model described by Ptashne (24) may
provide a mechanism explaining the distinct behavior of the distal and
proximal hSP-B elements. Clusters of TTF-1 proteins in each region
would increase the stability of the complex forming a higher order
complex with the basal transcription factor machinery.
))
We thank Dr. Robert Bohinski, Dr. Cindy Bachurski, and
Ann Maher for support.
, pp. 114, Blackwell and Cell Press, Cambridge, UK
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 C. M. Moya, G. Perez de Nanclares, L. Castano, N. Potau, J. R. Bilbao, A. Carrascosa, M. Bargada, R. Coya, P. Martul, E. Vicens-Calvet, et al. Functional Study of a Novel Single Deletion in the TITF1/NKX2.1 Homeobox Gene That Produces Congenital Hypothyroidism and Benign Chorea But Not Pulmonary Distress J. Clin. Endocrinol. Metab., May 1, 2006; 91(5): 1832 - 1841. [Abstract] [Full Text] [PDF]
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 M. Dentice, C. Luongo, A. Elefante, R. Ambrosio, S. Salzano, M. Zannini, R. Nitsch, R. Di Lauro, G. Rossi, G. Fenzi, et al. Pendrin Is a Novel In Vivo Downstream Target Gene of the TTF-1/Nkx-2.1 Homeodomain Transcription Factor in Differentiated Thyroid Cells Mol. Cell. Biol., November 15, 2005; 25(22): 10171 - 10182. [Abstract] [Full Text] [PDF]
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 C. Yan and H. Du Alveolus formation: what have we learned from genetic studies? J Appl Physiol, October 1, 2004; 97(4): 1543 - 1548. [Abstract] [Full Text] [PDF]
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 L. Yang, X. Lian, A. Cowen, H. Xu, H. Du, and C. Yan Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-{alpha} in Regulation of the Surfactant Protein B Gene in the Lung Mol. Endocrinol., June 1, 2004; 18(6): 1520 - 1532. [Abstract] [Full Text] [PDF]
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 K.-S. Park, J. A. Whitsett, T. Di Palma, J.-H. Hong, M. B. Yaffe, and M. Zannini TAZ Interacts with TTF-1 and Regulates Expression of Surfactant Protein-C J. Biol. Chem., April 23, 2004; 279(17): 17384 - 17390. [Abstract] [Full Text] [PDF]
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L. Yang, D. Yan, C. Yan, and H. Du Peroxisome Proliferator-activated Receptor {gamma} and Ligands Inhibit Surfactant Protein B Gene Expression in the Lung J. Biol. Chem., September 19, 2003; 278(38): 36841 - 36847. [Abstract] [Full Text] [PDF]
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 L. Yang, A. Naltner, and C. Yan Overexpression of Dominant Negative Retinoic Acid Receptor {alpha} Causes Alveolar Abnormality in Transgenic Neonatal Lungs Endocrinology, July 1, 2003; 144(7): 3004 - 3011. [Abstract] [Full Text] [PDF]
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L. Yang, A. Naltner, A. Kreiner, D. Yan, A. Cowen, H. Du, and C. Yan An enhancer region determines hSP-B gene expression in bronchiolar and ATII epithelial cells in transgenic mice Am J Physiol Lung Cell Mol Physiol, March 1, 2003; 284(3): L481 - L488. [Abstract] [Full Text] [PDF]
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T. Di Palma, R. Nitsch, A. Mascia, L. Nitsch, R. Di Lauro, and M. Zannini The Paired Domain-containing Factor Pax8 and the Homeodomain-containing Factor TTF-1 Directly Interact and Synergistically Activate Transcription J. Biol. Chem., January 24, 2003; 278(5): 3395 - 3402. [Abstract] [Full Text] [PDF]
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J. Weidenfeld, W. Shu, L. Zhang, S. E. Millar, and E. E. Morrisey The WNT7b Promoter Is Regulated by TTF-1, GATA6, and Foxa2 in Lung Epithelium J. Biol. Chem., May 31, 2002; 277(23): 21061 - 21070. [Abstract] [Full Text] [PDF]
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C. Yan, A. Naltner, M. Martin, M. Naltner, J. M. Fangman, and O. Gurel Transcriptional Stimulation of the Surfactant Protein B Gene by STAT3 in Respiratory Epithelial Cells J. Biol. Chem., March 22, 2002; 277(13): 10967 - 10972. [Abstract] [Full Text] [PDF]
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C. Liu, S. W. Glasser, H. Wan, and J. A. Whitsett GATA-6 and Thyroid Transcription Factor-1 Directly Interact and Regulate Surfactant Protein-C Gene Expression J. Biol. Chem., February 1, 2002; 277(6): 4519 - 4525. [Abstract] [Full Text] [PDF]
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R. Lonigro, D. Donnini, E. Zappia, G. Damante, M. E. Bianchi, and S. Guazzi Nestin Is a Neuroepithelial Target Gene of Thyroid Transcription Factor-1, a Homeoprotein Required for Forebrain Organogenesis J. Biol. Chem., December 14, 2001; 276(51): 47807 - 47813. [Abstract] [Full Text] [PDF]
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 Z. Luo and R. N. Hines Regulation of Flavin-Containing Monooxygenase 1 Expression by Ying Yang 1 and Hepatic Nuclear Factors 1 and 4 Mol. Pharmacol., December 1, 2001; 60(6): 1421 - 1430. [Abstract] [Full Text] [PDF]
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R. H. Costa, V. V. Kalinichenko, and L. Lim Transcription factors in mouse lung development and function Am J Physiol Lung Cell Mol Physiol, May 1, 2001; 280(5): L823 - L838. [Abstract] [Full Text] [PDF]
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 R. N. Hines, Z. Luo, T. Cresteil, X. Ding, R. A. Prough, J. L. Fitzpatrick, S. L. Ripp, K. C. Falkner, N.-L. Ge, A. Levine, et al. Molecular Regulation of Genes Encoding Xenobiotic-Metabolizing Enzymes: Mechanisms Involving Endogenous Factors Drug Metab. Dispos., April 13, 2001; 29(5): 623 - 633. [Abstract] [Full Text]
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 K. Doronin, M. Kuppuswamy, K. Toth, A. E. Tollefson, P. Krajcsi, V. Krougliak, and W. S. M. Wold Tissue-Specific, Tumor-Selective, Replication-Competent Adenovirus Vector for Cancer Gene Therapy J. Virol., April 1, 2001; 75(7): 3314 - 3324. [Abstract] [Full Text]
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V. V. Kalinichenko, L. Lim, B. Shin, and R. H. Costa Differential expression of forkhead box transcription factors following butylated hydroxytoluene lung injury Am J Physiol Lung Cell Mol Physiol, April 1, 2001; 280(4): L695 - L704. [Abstract] [Full Text] [PDF]
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 Y.-S. Yang, M.-C. W. Yang, B. Wang, and J. C. Weissler BR22, a Novel Protein, Interacts with Thyroid Transcription Factor-1 and Activates the Human Surfactant Protein B Promoter Am. J. Respir. Cell Mol. Biol., January 1, 2001; 24(1): 30 - 37. [Abstract] [Full Text]
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R. A. Pierce, G. L. Griffin, J. H. Miner, and R. M. Senior Expression Patterns of Laminin alpha 1 and alpha 5 in Human Lung during Development Am. J. Respir. Cell Mol. Biol., December 1, 2000; 23(6): 742 - 747. [Abstract] [Full Text]
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A. Naltner, S. Wert, J. A. Whitsett, and C. Yan Temporal/spatial expression of nuclear receptor coactivators in the mouse lung Am J Physiol Lung Cell Mol Physiol, December 1, 2000; 279(6): L1066 - L1074. [Abstract] [Full Text] [PDF]
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T. Yano, R. J. Mason, T. Pan, R. R. Deterding, L. D. Nielsen, and J. M. Shannon KGF regulates pulmonary epithelial proliferation and surfactant protein gene expression in adult rat lung Am J Physiol Lung Cell Mol Physiol, December 1, 2000; 279(6): L1146 - L1158. [Abstract] [Full Text] [PDF]
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A. Losada, J. A. Tovar, H. M. Xia, J. A. Diez-Pardo, and P. Santisteban Down-Regulation of Thyroid Transcription Factor-1 Gene Expression in Fetal Lung Hypoplasia Is Restored by Glucocorticoids Endocrinology, June 1, 2000; 141(6): 2166 - 2173. [Abstract] [Full Text] [PDF]
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M. D. Bruno, T. R. Korfhagen, C. Liu, E. E. Morrisey, and J. A. Whitsett GATA-6 Activates Transcription of Surfactant Protein A J. Biol. Chem., January 14, 2000; 275(2): 1043 - 1049. [Abstract] [Full Text] [PDF]
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A. Naltner, M. Ghaffari, J. A. Whitsett, and C. Yan Retinoic Acid Stimulation of the Human Surfactant Protein B Promoter Is Thyroid Transcription Factor 1 Site-dependent J. Biol. Chem., January 7, 2000; 275(1): 56 - 62. [Abstract] [Full Text] [PDF]
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M. D. Bruno, J. A. Whitsett, G. F. Ross, and T. R. Korfhagen Transcriptional Regulation of the Murine Surfactant Protein-A Gene by B-Myb J. Biol. Chem., September 24, 1999; 274(39): 27523 - 27528. [Abstract] [Full Text] [PDF]
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