(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 ()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.
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, 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.
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, 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).
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-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.
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-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).
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-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).
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 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.
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) 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.
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 TAF110(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.