©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Upstream Enhancer Activity in the Human Surfactant Protein B Gene Is Mediated by Thyroid Transcription Factor 1 (*)

(Received for publication, April 4, 1995; and in revised form, June 26, 1995)

Cong Yan Zvjezdana Sever Jeffrey A. Whitsett (§)

From the Children's Hospital Medical Center, Division of Pulmonary Biology, The Children's Hospital Research Foundation, Cincinnati, Ohio 45229-3039

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 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.


MATERIALS AND METHODS

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 pDelta5`-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).

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.


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^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 + 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-betagal 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 times 10^5 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 beta-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(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 µl 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 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).


RESULTS

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.


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-betagal, 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. (^2)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.


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-betagal, 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(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(-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(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(-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(1) fragment (Fig. 4B, lanes 2 and 3), confirming that TTF-1 interacts with multiple binding sites in the hSP-B(-439/-331) fragment.


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(1) fragment (f(1)) (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(1) 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(1) fragment (f(1)), or the F(2) fragment (f(2)) (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(1)) 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^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(-439/-331) 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 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(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(-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 TAF40(21) , as well as tumor suppressor protein p53 interacts with TAF40 and TAF60(22) , SP1, YY1, USF, CTF, and adenoviral E1A interacting with TAF55(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.


FOOTNOTES

*
This work was supported by Grant HL38859 from the National Institutes of Health and Grant HL51832 from the Center for Gene Therapy for Cystic Fibrosis and Other Lung Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Children's Hospital Medical Center, Division of Pulmonary Biology, TCHRF, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-559-4830; Fax: 513-559-7868.

(^1)
The abbreviations used are: SP-B, surfactant protein B; hSP-B, human SP-B; TTF-1, thyroid transcription factor 1; HNF-3, hepatocyte nuclear factor 3; PCR, polymerase chain reaction; bp, base pair; EMSA, electrophoresis mobility shift assay; HD, homeodomain; TAF, transcription-activating factor.

(^2)
J. A. Whitsett, C. Yan, and Z. Sever, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Robert Bohinski, Dr. Cindy Bachurski, and Ann Maher for support.


REFERENCES

  1. Avery, M. E., and Mead, J. (1959) Am. J. Dis. Child. 97,517-523
  2. Clark, J. C., Wert, S. E., Bachurski, C. J., Stahlman, M. T., Weaver, T. E., and Whitsett, J. A. (1995) Proc. Natl. Acad. Sci. U. S. A., 92,7794-7798 [Abstract]
  3. Whitsett, J. A., Nogee, L. M., Weaver, T. E., and Horowitz, A. D. (1995) Physiol. Rev. , in press
  4. Bohinski, R. J., Di Lauro, R., and Whitsett, J. A. (1994) Mol. Cell. Biol. 14,5671-5681 [Abstract]
  5. Clevidence, D. E., Overdier, D. G., Peterson, R. S., Porcella, A., Ye, H., Paulson, K. E., and Costa, R. H. (1994) Dev. Biol. 166,195-209 [CrossRef][Medline] [Order article via Infotrieve]
  6. Sawaya, P. L., and Luse, D. S. (1994) J. Biol. Chem. 269,22211-22216 [Abstract/Free Full Text]
  7. Guazzi, S., Price, M., Felice, M. D., Damante, G., Mattei, M. G., and Di Lauro, R. (1990) EMBO J. 9,3631-3639 [Abstract]
  8. Lazzaro, D., Price, M., Felice, M. D., and Di Lauro, R. (1991) Development 113,1093-1104 [Abstract]
  9. Ikeda, K., Clark, J. C., Shaw-White, J. R., Stahlman, M. T., Boutell, C. J., and Whitsett, J. A. (1995) J. Biol. Chem. 270,8108-8114 [Abstract/Free Full Text]
  10. Bohinski, R. J., Huffman, J. A., Whitsett, J. A., and Lattier, D. L. (1993) J. Biol. Chem. 268,11160-11166 [Abstract/Free Full Text]
  11. Yan, C., and Tamm, I. (1989) J. Biol. Chem. 265,20188-20194 [Abstract/Free Full Text]
  12. Damante, G., Fabbro, D., Pellizzari, L., Civitareale, D., Guazzi, S., Polycarpou-Schwartz, M., Cauci, S., Quadrifoglio, F., Formisano, S., and Di Lauro, R. (1994) Nucleic Acids Res. 22,3075-3083 [Abstract]
  13. Arnone, M. I., Zannini, M., and Di Lauro, R. (1995) J. Biol. Chem. 270,12048-12055 [Abstract/Free Full Text]
  14. Bruno, M. D., Bohinski, R. J., Huelsman, K. M., Whitsett, J. A., and Korfhagen, T. R. (1995) J. Biol. Chem. 270,6531-6536 [Abstract/Free Full Text]
  15. Beacley, P. A., Varkey, J., Young, K. E., Von Kessler, D. P., Sun, B. I., and Ekker, S. C. (1993) Mol. Cell. Biol. 13,6941-6956 [Abstract]
  16. LeBowitz, J., Clerc, R. G., Breiowitz, M., and Sharp, P. A. (1989) Genes & Dev. 3,1625-1638
  17. Galaup, C. K., and Hauser, C. A. (1992) New Biol. 4,558-568 [Medline] [Order article via Infotrieve]
  18. Pabo, C. O., and Sauer, R. T. (1992) Annu. Rev. Biochem. 61,1053-1095 [CrossRef][Medline] [Order article via Infotrieve]
  19. Zawel, L., and Reinberg, D. (1993) Prog. Nucleic Acid Res. Mol. Biol. 44,67-108 [Medline] [Order article via Infotrieve]
  20. Hoey, T., Weinzierle, R. O. J., Gill, G., Chen, J. L., Dynlacht, B. D., and Tjian, R. (1993) Cell 72,247-260 [Medline] [Order article via Infotrieve]
  21. Goodrich, J. A., Hoey, T., Thut, C. J., Admon, R., and Tjian, R. (1993) Cell 75,519-530 [Medline] [Order article via Infotrieve]
  22. Thut, C. J., Chen, J. L., Klemm, R., and Tjian, R. (1995) Science 267,100-104 [Medline] [Order article via Infotrieve]
  23. Chiang, C. M., and Roeder, R. G. (1995) Science 267,531-536 [Medline] [Order article via Infotrieve]
  24. Ptashne, M. (1992) A Genetic Switch Gene Control and Phage , pp. 114, Blackwell and Cell Press, Cambridge, UK
  25. Kelly, S. E., Burhans, M. S., Bachurski, C. J., and Glasser, S. W. (1995) Am. J. Respir. Crit. Care Med. 151,163 (abstr.)

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