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Previous Article | Next Article 
Molecular and Cellular Biology, December 2003, p. 9014-9024, Vol. 23, No. 24
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.24.9014-9024.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Division of Pulmonary Biology, Cincinnati Children's Research Foundation, Cincinnati, Ohio 45229,1 Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 142142
Received 19 May 2003/ Returned for modification 21 July 2003/ Accepted 18 September 2003
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ABSTRACT |
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INTRODUCTION |
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Isoforms of NFI arise from differential splicing of the gene products from the four NFI genes, Nfia, Nfib, Nfic, and Nfix. Although NFI is sometimes thought to be a ubiquitous transcription factor, these genes are expressed in specific overlapping patterns during fetal development and at lower levels in specific tissues in the adult (10). The relative expression levels of the four NFI genes vary with cell type, cell cycle, and differentiation status (16). Mice homozygous for deletion of Nfib die at birth from respiratory distress due to a block in lung maturation, suggesting that this NFI family member is required for late-fetal or perinatal lung development (18).
NFI family members are highly homologous in the amino-terminal DNA binding and dimerization domain but are divergent in the carboxyl-terminal transactivation-repression domain (1, 24; reviewed in reference 17). Further diversity of the transactivation domain is accomplished by alternative splicing that creates regions of variable proline richness. The significance of the proline-rich areas is not understood but they may form sites of protein-protein interaction. To test the hypothesis that changes in the repertoire of NFI family members directly modulate transcription of the SP-C gene, we measured promoter activation by selected isoforms of each of the NFI genes, alone and in combination with one another and TTF-1.
TTF-1 is a homeodomain-containing transcription factor
that regulates morphogenesis and differential gene expression in the
lung, thyroid, and ventral forebrain. Mice lacking TTF-1 protein do not
undergo proper lung or thyroid differentiation and die at birth from
respiratory distress
(21). TTF-1 is expressed
in the pulmonary epithelium during development and regulates the
expression of the surfactant protein genes (reviewed in reference
42). TTF-1 interacts with
retinoic acid receptor alpha (RAR
) and TIF2 by mammalian
two-hybrid analysis and synergistically interacts with RAR,
SRC-1, TIF2, ACTR, CBP, and STAT3 to stimulate SP-B
(31,
46,
44) and with CBP/p300 and
SRC-1 to stimulate SP-A
(47) promoter activity.
TTF-1 was recently shown to directly interact with GATA-6 in the
activation of SP-C transcription
(28). This study was
designed to test whether NFI family members interact with TTF-1 to
regulate mouse SP-C gene transcription in vitro and in vivo. We now
show that cotransfection of all NFI family members with TTF-1 causes
synergistic activation of SP-C promoter activity, but to different
extents. Mammalian two-hybrid and coimmunoprecipitation analysis
demonstrated that TTF-1 interacts with all NFI family members by
binding to the conserved DNA binding and dimerization domain.
Additionally, doxycycline (Dox)-induced expression of a
dominant-negative NFI-engrailed chimeric protein inhibited SP-C
expression in double-transgenic mice, suggesting that SP-C is an
NFI-regulated gene in vivo.
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MATERIALS AND METHODS |
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To facilitate subcloning, a polylinker site was inserted into pBET, a chicken beta actin promoter-driven expression vector (19), replacing the BglII-to-SalI sequence with a BglII-NotI-HindIII-SalI polylinker to create pBETpl. Mouse NFI isoform cDNAs containing an amino-terminal hemagglutinin (HA) epitope were subcloned from pCHM-NFI-A1, -B2, -C2, and -X2 (11) as NotI-HindIII fragments into pBETpl and as HpaI-HpaI fragments into EcoRV-digested pPAC5Cpl, an expression vector containing the Drosophila beta actin promoter (23). To generate an HA-tagged carboxyl-terminal truncation mutant of NFI-A, pCHM-NFIA1 was digested with NotI and BstXI and the 978-bp fragment encoding amino acids 1 to 308 was subcloned into pPAC5Cpl to create pPAC-HANFI-A-5'. The cytomegalovirus promoter-driven rat TTF-1 expression plasmid was previously described (5, 20).
For mammalian two-hybrid analysis, NFI and TTF-1 cDNAs were subcloned into the pM and pVP16 vectors (Clontech, Palo Alto, Calif.). These constructs direct the expression of NFI or TTF-1 fusion proteins containing an amino-terminal GAL4 DNA binding domain (dbd) from the pM vector or a VP16 transactivation domain (tad) from the pVP16 vector. The NFI constructs were designated either pM or pVP16 followed by NFI-A1, for the full-length coding sequence of mouse NFI-A1 from pBETNFIb1f (19); NFI-A-5'dbd for the DNA binding and dimerization domain of NFI-A (amino acids 1 to 308); and NFI-A1-3'tad for the NFI-A1 transactivation domain (amino acids 296 to 509). Construction of pMTTF-1 was previously described (31). The TTF-1 coding sequence was subcloned into pVP16 as an EcoRI fragment.
The dominant-negative NFI chimeric protein (NFIen) was constructed by fusing the amino-terminal HA-tagged DNA binding and dimerization domain (amino acids 1 to 296) of mouse NFI-A (11), via an eight-amino-acid linker, to the Drosophila engrailed transcriptional repression domain (amino acids 1 to 298) (8). This fusion protein was placed under control of the (TetO)7CMV minimal promoter (15, 39), and the 3' untranslated sequence and polyadenylation signal from the bovine growth hormone gene were added to generate a stable mRNA. All constructs were verified by sequencing. An expression vector containing the reverse tetracycline transactivator, pUHG17-1, was obtained from Hermann Bujard (University of Heidelberg).
Cell culture, transfection, and reporter gene assays. Human JEG-3 choriocarcinoma cells were maintained in minimum essential medium (MEM; Gibco) with 10% fetal bovine serum (FBS). JEG-3 cells, which do not express endogenous TTF-1 (data not shown) and have very low levels of endogenous NFI (7, 11), were used for the functional analysis of SP-C promoter reporter constructs by transient transfection. Cells were transfected by the calcium phosphate coprecipitation method, with modifications (3). Briefly, 6-well plates of JEG-3 cells at 50 to 60% confluence were transfected with 2 µg of SP-C luciferase plasmid, the indicated amounts of NFI or TTF-1 expression constructs, and 0.25 µg of pCMV-ßgal per well in 2 ml of Dulbecco's modified Eagle's medium with 10% FBS. After 18 h, the precipitate was removed and the cells were fed MEM with 10% FBS. Two days after transfection, the cells were washed with phosphate-buffered saline, lysed in 150 µl of 1x reporter lysis buffer (Promega) per well, and frozen at -20°C. Luciferase and beta-galactosidase (ß-Gal) assays were performed with 10 µl of the cleared lysates as described previously (3). Luciferase activity was normalized for ß-Gal activity, and the relative activity of the p0.32SP-C promoter plus empty vector(s) was set to 1. All data for cotransfection reactions were reported as activities relative to the control. Transfections were performed in duplicate and the data were plotted as means ± standard errors (SE) for at least three independent experiments (n = 6) unless stated otherwise in figure legends.
Drosophila melanogaster Schneider line 2 (SL2) cells were maintained in Schneider's Drosophila medium (Gibco) with 10% FBS at 27°C in ambient air. SL2 cells were transiently transfected by the calcium phosphate coprecipitation method in 100-ml tissue culture dishes, with 20 µg of a Drosophila expression vector, pPAC5cpl, per dish driving the expression of the indicated HA-tagged mouse NFI family member or rat TTF-1. Two days after transfection, cells were washed and collected for preparation of nuclear extracts.
Coimmunoprecipitation and Western blot analysis. Drosophila SL2 cell nuclear extracts containing individual HA-tagged NFI family members or TTF-1 were prepared by a miniextract procedure essentially as previously described (6). The indicated nuclear extracts were incubated with polyclonal anti-HA antibody and protein A/G-agarose beads in IP buffer (20 mM TRIS [pH 7.6], 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg of pepstatin A per ml, 10 µg of leupeptin per ml, 30 µg of bacitracin per ml, 1 mM o-phenanthroline) with rotation at 4°C overnight. The beads were washed extensively with IP buffer without Triton X-100 and were boiled in sodium dodecyl sulfate (SDS) loading dye. The coimmunoprecipitated proteins were separated by electrophoresis under reducing conditions on SDS-10 to 20% polyacrylamide gradient gels (Novex, San Diego, Calif.) in Tris-glycine buffer and were then transferred to nylon membranes. Western blots were blocked with 5% nonfat dry milk in TBST (10 mM Tris [pH 8], 150 mM NaCl, 0.1% Tween 20) and incubated with anti-TTF-1 monoclonal antibody (a kind gift from J. A. Whitsett) followed by horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin G (Calbiochem, La Jolla, Calif.). Western blots were developed with the enhanced chemiluminescence system (Amersham Biosciences, Arlington Heights, Ill.) and exposed to X-OMAT film (Kodak). Western blots were stripped and the procedure was repeated with a mouse monoclonal anti-HA antibody (12CA5; Roche Molecular Biochemicals) to detect the HA-tagged NFI isoforms.
Mammalian two-hybrid analysis. The reporter construct pG5LUC, containing five GAL4 DNA binding sites and the adenovirus E1b minimal promoter, was previously described (31). For two-hybrid analysis, JEG-3 cells were cotransfected with the indicated combinations of GAL4 DNA binding domain and VP16 transactivation domain chimeric plasmids, 2 µg of pG5LUC reporter construct, and 1 µg of pRSVßgal per well. Data are presented as relative luciferase activities, with the activity of pG5LUC cotransfected with the indicated pM chimeric plasmid and pVP16 empty vector set to 1. Since TTF-1 has been previously reported to self-associate (2), cotransfection of pMTTF-1 with pVP16TTF-1 was used as a positive control.
EMSA.
Annealed oligonucleotides were gel
purified by use of 4% Biogel and the Mermaid kit (Bio 101,
Vista, Calif.) for use as electrophoretic mobility shift assay (EMSA)
probes. Purified probes were end labeled with
[
-32P]ATP and T4 polynucleotide kinase.
EMSAs were performed as previously described, with slight modifications
(3). Briefly, nuclear
extracts from transiently transfected cells expressing similar amounts
of HA-tagged NFI family members were incubated in EMSA binding buffer
[20 mM Tris (pH 7.6), 50 mM KCl, 2 mM MgCl2, 40 ng of
poly(dI-dC) (Boehringer Mannheim) per µl, 10% glycerol,
1 mM dithiothreitol, 0.1 mM fresh phenylmethylsulfonyl fluoride]
for 5 to 10 min at room temperature. End-labeled probe was added
(100,000 cpm) and the mixture was incubated for an additional 10 min at
room temperature. Bound and free probes were separated by nondenaturing
polyacrylamide gel electrophoresis using 5%
acrylamide-bisacrylamide (29:1)-2.5% glycerol gels in
0.5x Tris-borate-EDTA
buffer.
Generation of double-transgenic mice. Homozygous CCSP-rtTA activator transgenic mice (39), containing the reverse tetracycline transactivator under the control of the 2.3-kb rat Clara cell secretory protein (CCSP) promoter, were kindly provided by Jeffrey Whitsett (Cincinnati Children's Hospital, Cincinnati, Ohio). Animals were housed under pathogen-free conditions according to federal and institutional guidelines. The (TetO)7CMV promoter-driven dominant-negative NFI-engrailed (NFIen) construct was microinjected into F/VBN mouse oocytes by the transgenic core at CCHMC, and NFIen transgene-positive mice were bred with CCSP-rtTA transgenic activator mice. Double-transgenic mice were identified by PCR using primers specific for each transgene. Primers used for the (TetO)7CMV-NFIen transgene were a 5' primer for the CMV minimal promoter (5'-GCCATCCACGCTGTTTTG) and a 3' primer for the Drosophila engrailed sequence (5'-CCAGTTGCTGTTGGTGCAAC). Primers used to detect CCSP-rtTA were a 5' primer for the rat CCSP promoter sequence (5'-ACTGCCCATTGCCCAAACAC) and a 3' primer in the reverse tetracycline transactivator sequence (5'-AAAATCTTGCCAGCTTTCCCC).
Double- and single-transgenic mice at 6 weeks of age were fed Dox-containing mouse chow for 3 or 8 days as previously described (34) to induce expression of the NFIen transgene in a subset of TII cells and Clara cells. Total lung RNA (10 µg/lane) was separated in 1% agarose-formaldehyde gels and Northern blot analysis was performed as described previously (4), using cDNA probes for mouse SP-C, TTF-1, ribosomal protein L32, and the Drosophila engrailed domain to detect NFIen transgene expression.
ISH and IHC.
Lungs were inflation fixed with
4% paraformaldehyde at 25-cm water pressure and were
processed for paraffin embedding. Serial 5-µm sections were
prepared for in situ hybridization (ISH) and immunohistochemistry
(IHC). ISH was performed by using
[-35S]UTP-labeled riboprobes for sense
and antisense mouse SP-C cDNA under high-stringency conditions
essentially as previously described
(41). Slides were dipped
in Kodak NTB2 emulsion, exposed for 3 to 4 days, and developed with
Kodak D19 developer. Slides were imaged by dark field illumination to
detect the ISH signal and by phase-contrast microscopy to detect the
underlying structures.
To detect NFIen transgene expression, antigen retrieval was performed by heating of slides in the microwave oven in citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with hydrogen peroxide, and slides were incubated with polyclonal anti-HA antiserum (1:200) (HAprobe1; Santa Cruz). For immunodetection, sections were incubated with biotinylated goat anti-rabbit immunoglobulin G and visualized with the Vectastain Elite kit. The diaminobenzamidine reaction products were enhanced with nickel cobalt and the slides were counterstained with nuclear fast red essentially as previously described (48).
Statistical analysis. Relative luciferase activity from transient transfections was analyzed by the two-tailed t test statistic with Statview 4.5 software (Abacus Concepts, Berkeley, Calif.). Significance was accepted when P < 0.05.
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RESULTS |
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47 kDa for NFI-B2 to
56 kDa for NFI-A1. Consistent with previously published data
(16), all NFI family
members interacted with the palindromic NFI binding site with similar
affinities (Fig. 2B,
compare lanes 1, 4, 7, 10, and 13). However, NFI family members bound
NFI half-site probes from the C1 and C3 footprinted regions of the SP-C
promoter with variable lesser affinities (Fig.
2B, lanes 2, 5, 8, 11, and
14 [probe C1] and lanes 3, 6, 9, 12, and 15 [probe
C3]). NFI-C2 had the strongest relative binding to the NFI
half-site probes in titration experiments (data not shown), suggesting
that binding affinity does not correlate precisely with transcriptional
activation (see Discussion).
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NFI-TTF-1 protein-protein interactions were confirmed by use of the mammalian two-hybrid system. Fusion protein expression vectors were constructed containing the putative interaction domains fused to the GAL4 DNA binding domain (dbd) or the VP16 transactivation domain (tad). Protein-protein interactions between fusion partners induce promoter activity from a cotransfected luciferase expression vector containing multiple copies of the GAL4 DNA binding site adjacent to a minimal promoter (diagrammed in Fig. 5). Relative luciferase activity was compared to control transfections with the respective GALdbd fusion protein and VP16tad alone. The full-length TTF-1 and NFI-A1 coding sequences were fused to the GAL4dbd and VP16tad domains and the NFIA-dbd and -tad domains were fused to VP16tad. Since TTF-1 has been shown to self-associate in vitro (2, 38), we used cotransfection with GAL4TTF-1 and VP16TTF-1 as a positive control for protein-protein interactions (Fig. 5). NFI-A-TTF-1 cotransfection reactions induced luciferase activity to levels similar to those of the positive controls for both fusion partner combinations tested. Consistent with the coimmunoprecipitation assays, the conserved NFIA-dbd showed binding activity to TTF-1, whereas the NFIA-tad did not.
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The SP-C gene is an NFI-regulated gene in vivo. To examine the functional consequences of NFIen expression in the pulmonary epithelium, we generated double-transgenic mice using the lung epithelial cell-specific Dox-inducible system (34) (Fig. 8A). Single-transgenic "operator" mice were generated that contained the (TetO)7-CMV HA-Nfi-a-engrailed transgene. These single-transgenic mice do not express NFIen and have no detectable phenotype. When crossed with transgenic activator mice expressing rtTA protein in the pulmonary epithelium, double-transgenic progeny express NFIen in a tissue-specific, Dox-regulated manner. We used CCSP-rtTA activator mice from J. A. Whitsett (Cincinnati Children's Hospital) in which the rat CCSP (mouse genome designation Scgb1a1) promoter drives rtTA expression in a subset of bronchiolar Clara cells and alveolar TII cells. Double-transgenic mice harboring CCSP-rtTA and (TetO)7CMV-NFIen were fed Dox for 3 or 8 days to induce NFIen expression in the pulmonary epithelium. NFIen expression was not detected in untreated double-transgenic mice by Northern blot analysis of 10 µg of total lung RNA (Fig. 8B). Dox treatment induced expression of NFIen at both time points tested and decreased SP-C mRNA by approximately 55%. TTF-1 mRNA expression was not affected by Dox treatment.
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All NFI family members are expressed in the lung (10). In the developing mouse lung, NFI family members are expressed in both epithelium and mesenchyme (10; C. Bachurski, unpublished observations), whereas TTF-1 is restricted to the epithelium (48). We previously showed by transient transfection analysis that NFI and TTF-1 independently regulate the minimal SP-C promoter (3, 20). This study suggests that all NFI family members can directly interact with the homeodomain transcription factor TTF-1 in the pulmonary epithelium to regulate SP-C promoter activity. Our data support the hypothesis that TTF-1 interactions with different NFI family members modulate SP-C promoter activity. TTF-1 interaction with NFI-A1 or -B2 induced SP-C promoter activity to high levels, whereas NFI-C2 was least effective (Fig. 2).
NFI-B2, the strongest transactivator of SP-C transcription, had the lowest apparent binding affinity and consistently produced a slower mobility complex in EMSA, even though it is the smallest protein as assessed by SDS gel electrophoresis. The discrepancy between apparent protein size and relative mobility in EMSA could be caused either by intrinsic surface charge differences between isoforms or by covalent modifications such as phosphorylation or glycosylation. NFI family members are both glycosylated and phosphorylated (reviewed in reference 17), but the sites of these modifications have not been mapped. Domain swap experiments are being considered to map the region(s) in NFI that is involved in differential SP-C promoter activation.
The NFI proteins have a modular structure with a highly conserved amino-terminal DNA binding and dimerization domain followed by a central region and a more divergent C-terminal transactivation-repression domain. NFI family members form stable hetero- and homodimers through interactions between the conserved amino-terminal 250 to 300 amino acids (25). This region is sufficient to drive specific protein-DNA interactions of fusion proteins. We took advantage of the heterodimerization ability of the NFI DNA binding domain to create a dominant-negative NFI-engrailed domain chimeric gene. Previously, Schuur et al. reported the creation of a dominant-negative NFI truncation mutant that partially inhibited the activity of some NFI family members (36). To improve the transcriptional repression of NFI-regulated promoters, we fused the transcriptional repressor domain from the Drosophila engrailed gene to the NFIA-dbd. This strategy was previously used to create a dominant-negative GATA-6 transcriptional repressor expressed in the pulmonary epithelium (28, 45). These studies also showed that expression of the engrailed domain alone had no effect on lung development or epithelial maturation.
Dox-induced expression of NFIen inhibited SP-C promoter activity in transient transfection analysis and SP-C gene expression in TII cells in the adult mouse lung. Whether specific NFI family members regulate SP-C promoter activity in vivo is unknown. Three of the four NFI genes, Nfia (12), Nfib (18), and Nfic (37), have been targeted in mice. Nfia and Nfic gene-targeted mice have no known lung defects, whereas lung development fails to progress through the canilicular phase in Nfib gene-targeted mice, causing respiratory failure and perinatal death (18). Whether the pulmonary defects are caused by epithelial or mesenchymal deficiencies remains to be determined.
TTF-1 regulates lung morphogenesis and SP gene expression in TII cells of the adult lung (reviewed in reference 42). Protein-protein interactions with TTF-1 regulate lung epithelial, thyroid follicular, and brain-specific gene expression. Calreticulin, a Ca2+ binding chaperone, binds to the homeodomain of TTF-1 and stimulates transcriptional activity (35). In the lung, TTF-1 interacts with GATA-6 to activate SP-C (28) and Wnt7b transcription (40). RAR/RXR, CBP, SRC-1, and BR22 have been shown to interact with TTF-1 to regulate SP-B transcription (43, 46). TTF-1 also interacts with two coactivator proteins, CBP and SRC-1, to activate SP-A transcription (47). We now add NFI to the list of transcription factors that interact with TTF-1 to activate lung epithelial cell-specific gene expression.
In thyroid follicular cells, TTF-1 interacts with TTF-2 and Pax8, both of which are tissue-restricted transcription factors, to activate thyroglobulin and thyroid peroxidase expression (13, 29, 33). NFI also directly interacts with TTF-2 (a forkhead domain transcription factor; also termed Foxe1) to modulate hormone-induced expression of thyroid peroxidase in thyroid cells (32). In the FRTL-5 rat thyroid follicular cell line, NFI was shown to regulate constitutive expression of TTF-1 (30). Inhibition of NFI binding to the TTF-1 promoter was suggested to be a mechanism by which thyroglobulin mediates inhibition of thyroid-specific gene expression. However, Dox-induced expression of dominant-negative NFIen in the pulmonary epithelium had no effect on TTF-1 expression in TII cells (Fig. 8B and 9F). These apparently contradictory findings suggest that either TTF-1 expression is regulated differently in the lung and thyroid or TTF-1 regulation in FRTL-5 cells is distinct from that in lung cells. Conditional expression of NFIen could be used to clarify the role of NFI in other target systems, including the thyroid, mammary gland, brain, and liver, where NFI family members have been implicated in gene regulation (reviewed in reference 17).
Our data also suggest that the coactivator CBP/p300 can interact with the NFI-TTF-1 transcription complex to further enhance SP-C transcription. p300 synergistically enhanced the cooperative NFI-TTF-1 transactivation of SP-C promoter activity by all NFI family members (Fig. 6). NFI-C interacts with the CREB binding domain of CBP in the two-hybrid assay (26). Whether all NFI family members interact directly with CBP or p300 is unknown. Leahy et al. showed that different NFI family members have opposite effects on transcription of the PEPCK gene. They proposed a model in which CBP coordinates diverse signals to regulate gene transcription by acting as a scaffold for bringing together specific transcription factors with the basal transcriptional machinery in response to insulin, steroid hormones, and cyclic AMP (cAMP) (26). Treatment of fetal human lung explants with cAMP induces interaction of CBP with TTF-1, acetylation of TTF-1, and increased SP-A transcription (47). In the present experiments, p300 had no effect on cotransfected NFI or TTF-1 alone, and synergistic activation only occurred in the presence of both transcription factors and when both NFI and TTF-1 binding sites were intact in the SP-C promoter sequence. CBP/p300 may act as a scaffold protein to bring together the cotransfected factors with the basal transcription complex or may act to open chromatin and allow access of transcription factors to their binding sites through its histone acetyltransferase activity (reviewed in reference 9). The present experiments do not distinguish between the two possible roles for p300 in SP-C promoter activation by NFI and TTF-1.
Our studies indicate that TTF-1 and NFI family members cooperate to regulate the transcription of SP-C in vitro. In addition, the ability of the dominant-negative NFIen protein to repress SP-C expression both in vitro and in vivo indicates that NFI family members likely play an important role in SP-C expression in pulmonary TII cells. Finally, the strong enhancement of TTF-1 and NFI-dependent SP-C reporter expression by p300 suggests that p300 or related coactivators may play essential roles in SP-C expression. Together with recent findings that lung development is delayed in Nfib-deficient mice (18), these data strongly support the hypothesis that NFI family members have multiple important roles in lung development and SP-C gene expression.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grants HL60907 (C.J.B.), HD34908, and DK58401 (R.M.G.) and the Charlotte R. Schmidlapp Women Scholars Award (C.J.B.).
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FOOTNOTES |
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