Upstream enhancer activity in the human surfactant protein B gene is mediated by thyroid transcription factor 1.

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 pulmonary 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 bhe 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 than 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 1 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 5Ј 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 fam-ily of proteins, bind to cis-acting elements in the SP-B and Clara cell secretory protein genes (4 -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 Ϫ218 to ϩ41 bp in the SP-B gene (4). Point mutations in the TTF-1 and HNF-3 binding sites in this proximal SP-B promoter (Ϫ111 to Ϫ73 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 5Ј-flanking region of the human SP-B gene located Ϫ439 to Ϫ331 nucleotides upstream of the transcriptional initiation site. The hSP-B Ϫ439/Ϫ331 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 m , B-500 Bb m , and B-500 Bc m mutant luciferase constructs. The correctness of all the wild type and mutant plasmid constructs were confirmed by DNA sequencing.
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 ϫ 10 5 cells were seeded into 60-mm dishes. Each dish was transfected with 12.5 g 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 ϭ 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 2 , 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 l of nuclear buffer (0.5 M KCl, 20 mM Tris-Cl, pH 7.6, 0.2 mM EDTA, 1.5 mM MgCl 2 , 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 g) 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(Ϫ439/Ϫ331) fragment).

Expression of SP-B, SV40, and Thymidine Kinase Promoters in H441-4 Cells-The
Ϫ218 to ϩ41 bp (minimal promoter) and the Ϫ500 to ϩ41 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.
Transcriptional Activity and DNA Protein Binding of hSP-B (Ϫ439 to Ϫ331)-Nucleotide sequence in the 5Ј-flanking distal upstream regions of the human and mouse SP-B genes share 95% identity from Ϫ439 to Ϫ331 bp (human) and Ϫ382 to Ϫ282 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. 2 In order to determine the biological function of the stimulatory element in the human gene, the hSP-B(Ϫ439/Ϫ331) 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(Ϫ439 to Ϫ331) element.

TTF-1 Binds to the hSP-B(Ϫ439/Ϫ331) Fragment of the Human SP-B Gene-After carefully examining the hSP-B
Ϫ439 to Ϫ331 region, three distinct CAAG motifs (12) were found in the hSP-B(Ϫ439/Ϫ331) 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 Ϫ439 to Ϫ331 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 1 , 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(Ϫ439/Ϫ331) fragment was TTF-1. Fig. 4A demonstrates that the specific interaction between the H441-4 nuclear protein and the radiolabeled hSP-B(Ϫ439/Ϫ331) fragment was inhibited by adding 100-fold molar excess of F 1 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(Ϫ439/Ϫ331) 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 Ϫ439/Ϫ331. These TTF-1 complexes were inhibited by adding 50-fold molar excess of self-competitor and the F 1 fragment (Fig. 4B, lanes 2 and 3), confirming that TTF-1 interacts with multiple binding sites in the hSP-B(Ϫ439/Ϫ331) fragment.
Mutations in the hSP-B(Ϫ331/Ϫ439) Abolished or Reduced the TTF-1 Response-To further confirm that the putative TTF-1 binding to the sites in the hSP-B(Ϫ439/Ϫ331) 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  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. Ϫ, no competitor; ϩ, 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 m , Bb m , and Bc m 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 ϩ S.D. (n ϭ 6). TTF-1 HD were introduced into the B-500 luciferase expressing construct. Wild type and mutant B-500 constructs mutated at the positions Ba m , Bb m , and Bc m 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 m and Bb m 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 m only moderately impaired activity. Transcription from the hSP-B(Ϫ439/Ϫ331) fragment was therefore highly dependent on TTF-1 binding to the region. DISCUSSION 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 5Ј-flanking region of hSP-B(Ϫ439/Ϫ331). 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 Ϫ439 to Ϫ331 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 1 ) 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(Ϫ439/Ϫ331) 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 Ϫ438 to Ϫ331 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 Ϫ439 to Ϫ331 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 clus-tered 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 Ϫ111 and Ϫ73 bp in the hSP-B gene. Region II consists of three TTF-1 binding sites, located in the Ϫ439 to Ϫ331 bp region 5Ј 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 I summarizes 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.
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 Cterminal 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 II 110 (20), and the acidic activation domain of VP16 interacts with Drosophila TAF II 40 (21), as well as tumor suppressor protein p53 interacts with TAF II 40 and TAF II 60 (22), SP1, YY1, USF, CTF, and adenoviral E1A interacting with TAF II 55 (23), etc. The present findings demonstrate TTF-1-dependent enhancer activity in the distal upstream 5Ј 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.