Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
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ABSTRACT |
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We used
transgenic mice to identify cis-active regions of the human
pulmonary surfactant protein C (SP-C) gene that impart tissue-
and cell-specific expression in vivo in the lung.
Approximately 3.7 kb of genomic SP-C DNA upstream of the transcription
start site was sufficient to direct chloramphenicol acetyltransferase (CAT) reporter gene expression specifically in bronchiolar and alveolar epithelial cells of the lung. To further define
cis-active regulatory elements that mediate cell-specific
expression, we tested deletions of the parental 3.7-kb human SP-C
sequence in transgenic mice. Tissue CAT assays of mice generated with
truncations or overlapping internal deletions of the 3.7-kb construct
functionally map alveolar cell-specific regulatory elements to within
215 bp of the SP-C promoter. Analysis of SP-C promoter deletions
demonstrate that sequences between
3.7 kb and
1.9 kb
contain enhancer sequences that stimulate SP-C transgene
expression. In situ hybridization studies demonstrate that deletion of
the
1,910- to
215-bp region abolishes the ectopic
bronchiolar expression seen with the original 3.7-kb SP-C promoter
construct. Comparison of sequences from
215 to +1 bp identified
consensus binding sites for the homeodomain transcription factor
thyroid transcription factor-1 (TTF-1). Cotransfection assays of the
human 3.7-kb SP-C or
1,910- to
215-bp SP-C deletion construct with a TTF-1 expression plasmid demonstrates that TTF-1 transactivates the human SP-C gene. These results suggest that the TTF-1 cis-active sites are important in directing
cell-specific expression of the SP-C gene in vivo.
thyroid transcription factor-1; chloramphenicol acetyltransferase
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INTRODUCTION |
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PULMONARY SURFACTANT is a protein and phospholipid complex that is essential for normal respiration and functions by reducing surface tension at the alveolar surface (6). Four surfactant-associated proteins (SP-A, SP-B, SP-C, and SP-D) have been identified (16). Of these proteins, the two small hydrophobic proteins (SP-B and SP-C) are lung specific. They enhance the surface active properties of synthetic surfactant phospholipids in vitro and are a component of replacement surfactant preparations used to treat neonatal respiratory distress syndrome (15, 16, 30). SP-C gene transcription is initiated early in embryonic lung development with a temporal-spatial pattern of expression distinct from other surfactant protein genes (18, 31, 34). Murine SP-C mRNA is first detected on fetal day 11 (fetal day 20 is birth) and is maintained in epithelial cells at the distal migrating edge of the developing primitive airways (31). During prenatal development, SP-C mRNA expression is progressively extinguished in conducting airway cells along the proximal-to-distal axis of airway tubule growth. In the adult mouse lung, SP-C is expressed exclusively in mature type II cells, whereas SP-A and SP-B expression is detected in both bronchiolar epithelial and alveolar type II cells (7, 20). Thus SP-C is the only surfactant protein expressed exclusively in type II cells.
Several growth factors and humoral mediators have been identified that are required to sustain the type II phenotype and SP-C gene expression (24, 27). Such studies do not reveal the molecular basis for type II cell-specific SP-C transcription. The first insights into this complex process have come from limited in vitro gene activation studies of the SP-C promoter and candidate transcription factors. The murine SP-C gene is activated in vitro by coexpression of the homeodomain transcription factor thyroid transcription factor-1 (TTF-1) (3). Activation requires protein interactions with two adjacent cis-active sites, TTF-1 binding elements (TBE) of the murine SP-C promoter (17), implicating TTF-1 as a critical regulator of SP-C gene expression. Biochemical analysis of the murine SP-C promoter demonstrates that several other protein to DNA interactions occur near the TBE. The nature of these molecules and their role in specifying type II cell-specific transcription are unknown.
As a complement to in vitro studies, transgenic mouse models have been
used to detect cis-active DNA that is required to drive the
correct temporal and cell-specific pattern of gene expression. The
transgenic analysis of promoter function has often identified relevant
cis-active regions that are not identified in vitro. We have
generated transgenic mice with segments of human SP-C DNA to identify
cis-active sequences that direct lung-specific transcription in
vivo. Analysis of transgenic mice demonstrated that 3.7 kb of DNA
upstream from the transcription start site were sufficient to cause
reporter gene expression in lung epithelial cells of the developing
mouse (9, 10). That study identified the first genomic sequence that
could be used to selectively drive lung-specific expression in vivo.
The 3.7-kb SP-C promoter has been used subsequently to express a
variety of biologically active molecules in vivo to alter lung
development and to model lung diseases. Examples include 3.7-kb
SP-C-driven transforming growth factor- expression, which has
produced a model of progressive pulmonary fibrosis (21).
The 3.7-kb SP-C- promoted expression of the SV40 T antigen has
generated a model of bronchoalveolar lung cancer (32). Further
characterization of multiple 3.7-kb SP-C-driven chloramphenicol
acetyltransferase (CAT) transgenic lines would be useful
then to define the range of cell specificity that can be obtained when
using this promoter to develop new transgenic models. The high levels
of expression from 3.7-kb SP-C-CAT mice indicated that analysis of
3.7-kb SP-C deletions in vivo could be successful in mapping regions
essential for type II-specific expression. In the present study, we
have identified transcriptional control elements of the SP-C
gene that activate and sustain transgene expression in subpopulations
of distal respiratory epithelial cells.
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MATERIALS AND METHODS |
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Generation of SP-C-CAT constructs and transgenic mice. A 3.7-kb
fragment of human SP-C genomic DNA containing basal promoter elements
and additional 5' sequences (extending from +21 of exon 1 to
3686) was subcloned into the unique Hind III restriction site of the expression plasmid pSVO-CAT. This plasmid and the SP-C
genomic clones have been described previously (9, 10). For this study,
the
1,225- and
590-bp regions of the promoter were cloned
into pSVO-CAT with Hind III linkers and oriented by Xba
I and Ava I restriction digests. Internal deletion constructs were generated by restriction digest of the original 3.7-kb SP-C-CAT plasmid at paired restriction sites unique to the genomic SP-C sequence, followed by plasmid self-ligation with T4 DNA ligase. Deletion clones were mapped relative to restriction sites 5' and 3' of the deleted SP-C DNA. Nde I-BamH I
double-digested fragments were purified by CsCl gradient
ultracentrifugation, and transgenic mice were generated at the
University of Cincinnati transgenic core facility in FVB/N mice.
Founder (F0) transgenic mice were identified by genomic
Southern blot of tail-clip DNA using the bacterial CAT gene as
probe, as previously described (10). F0 mice were bred to
wild-type FVB/N mice to establish heterozygotes, which were bred to
generate mice homozygous for the SP-C-CAT transgene. For in
vitro expression analyses, the 3.7-kb SP-C segment of DNA and deletions
of the SP-C DNA were subcloned into the promoterless CAT expression
plasmid pBLCAT6.
Cell culture and DNA transfection. HeLa cells were maintained
in DMEM supplemented with 5% fetal bovine serum at 37°C, 5% CO2. Cells were plated into 60-mm dishes and
grown to ~30% confluency for transfection using FuGene 6 transfection reagent according to the manufacturer's instructions
(Boehringer Mannheim). Cotransfection experiments were performed with 1 pmol of SP-C-CAT test construct, 0.5 pmol of pRC-TTF-1 as constitutive
transactivator or 0.5 pmol of pRC without TTF-1 insert and 0.05 pmol of
the pCMV--gal plasmid (TTF-1 and pRC kindly provided by Dr. R. Di
Lauro). The medium was changed on the following day, cells were
maintained 24-48 h until confluent, and then the cells were
harvested as described previously (17).
CAT assays of tissue and cell culture extracts. CAT assays of
cell transfection experiments in vitro were performed on cell extracts
that were normalized to equivalent amounts of -galactosidase activity to control for transfection efficiency. CAT assays of tissue
from nontransgenic and transgenic mice were normalized to uniform
amounts of protein from the homogenized tissue extracts. Tissue extract
preparation and conditions for CAT assay have been described previously
(10). Chromatograms were visualized by autoradiography, and reactions
were quantitated in a Molecular Dynamic PhosphorImager using the
ImageQuant program (Molecular Dynamics; Sunnyvale, CA).
In situ hybridization. Sense and antisense 35S-labeled riboprobes were synthesized from pGEM plasmids containing 550 bp of the bacterial CAT gene or 758 bp of the murine SP-C cDNA. Tissue preparation, probe synthesis, hybridization, and wash conditions have been described previously (31). The specific activity of CAT and SP-C antisense probes, varied by only 7% with equivalent total counts of probe used in individual hybridizations (106 counts / min in 15 µl of hybridization overlay). Sense strand probe hybridization was used to establish specificity of hybridization. Equivalent hybridization of SP-C antisense probe was detected over either transgenic or nontransgenic lungs. CAT antisense probe only hybridized to sections of transgene-positive lung.
Quantitation of SP-C and transgene expression. Dark-field images of the in situ hybridization sections were visualized and transferred via an ImageQuant video system for morphometric analysis using the Metamorph program. The diameter of individual sites of SP-C probe hybridization were used to establish minimal and maximal thresholds to be counted. A minimal threshold level was selected to size eliminate individual silver grain (nonspecific) signals. Fields were randomly selected, and bronchiolar and vascular tissues were excluded. Four separate fields were counted from the left lobe and averaged. The cellular sites of CAT transgene expression were quantitated with the identical size parameters. Results are expressed as percentage of cells expressing CAT compared with cells expressing endogenous SP-C.
Northern blot analysis. Total RNA was prepared from lungs by previously described methods (9). Five micrograms of total lung RNA was size fractionated in formaldehyde-agarose gels, transferred to nylon membranes, and subject to hybridization analysis as previously described (9). The probes are described in In situ hybridization.
Sequence analysis of human and murine SP-C promoter regions. Segments of DNA that extended 5' of the human and murine SP-C promoters were restriction mapped and subcloned into pUC plasmid vectors. Restriction fragments of the genomic DNA was either subcloned into M13 vectors for conventional dideoxynucleotide sequencing or directly sequenced in the plasmid vectors using a model 377 Applied Biosystems automated sequencer at the University of Cincinnati DNA-sequencing core facility. Human and murine SP-C DNA sequences were compared using the alignment and matrix programs of MacVector 6.5 (Symantec).
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RESULTS |
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Tissue-specific expression of the transgene was assessed in adult
transgenic mice generated with the human 3.7-kb SP-C-CAT construct.
Transgenic lines were subsequently generated with deletions of the
3.7-kb human SP-C DNA to identify regions of DNA that determine lung-specific expression. The deletion constructs tested and number of
transgenic mouse lines expressing each construct are summarized in Fig.
1.
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The 3.7-kb SP-C-CAT transgene expression does not alter levels of
endogenous SP-C expression. To determine whether the level of
transgene expression altered the overall level of endogenous SP-C
expression by competing for essential transcription factors, we
compared CAT mRNA and SP-C mRNA levels in lungs from the various founder lines. Total lung RNA was prepared from lungs of nontransgenic mice and four 3.7-kb SP-C-CAT founder lines and used for Northern blot
analysis. Transgene mRNA expression was detected using a 550-bp portion
of the bacterial CAT gene as probe (Fig.
2A). CAT mRNA was not detected in
the nontransgenic control (Fig. 2A, lane 1), whereas high
levels of CAT mRNA were detected in the lungs from transgenic lines
5.5, 5.7 and 3.8.8. Relatively low levels of CAT mRNA were
detected from line 3.8.1 (Fig. 2A, lane 4). To determine the level of endogenous SP-C expression, the blot was stripped of CAT probe and rehybridized with an SP-C cDNA probe (Fig.
2B). Similar levels of SP-C expression were detected in lungs
from all transgenic lines and were similar to the nontransgenic wild-type mice, indicating that transgene expression did not alter the
level of endogenous SP-C mRNA. Differences in SP-C signal levels
reflected slight differences in loading as determined by methylene blue
staining of the membrane before hybridization analysis.
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Lung-specific expression of SP-C-CAT transgenes. To determine sequences required to direct lung-specific gene expression in vivo, we generated transgenic mice with progressively smaller deletions of the 3.7-kb sequence directing the CAT reporter gene. Constructs were generated that were either 5' directional, eliminating segments of DNA distal to the transcription start site, or overlapping internal deletions of proximal SP-C DNA (Fig. 1).
Transgenic lines were established that retained either 1,225 or
590 bp of promoter proximal genomic SP-C DNA. CAT activity was
detected in 4 of the 10 independent lines, two
1,225-bp lines and two
590-bp lines and was lung specific. CAT activity was not
detected in other examined tissues. Representative tissue CAT activity
of a
590-bp SP-C-CAT transgenic line is shown (Fig. 3). The level of lung CAT activity from the
1,225-bp transgenic lines was nearly identical to the
590-bp SP-C-CAT transgenic lines and is not shown. The levels of
lung-specific CAT activity in lung tissue from transgenic mice with the
1,225- and
590-bp SP-C, promoters were much less than the
activity detected in the 3.7-kb SP-C-CAT lines (Fig. 3). Therefore,
loss of enhancer elements located in the deleted distal sequence may
account for the diminished level of in vivo expression. Nevertheless,
lung-selective CAT expression from the two independent
590-bp
SP-C-CAT transgenic lines demonstrated that cis-active
determinants of lung-specific transcription were located within this
small region of the promoter.
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To more precisely identify sequences conferring lung-specific
expression, we generated transgenic mice with small internal deletions
that would overlap into the 590-bp region retaining only 365 bp
(
860 to
365) or 215 bp (
1,910 to
215) of
the promoter proximal DNA. CAT activity was detected in lung tissue
from two independent founder lines bearing each construct (Fig.
4). The 3' boundaries of these
constructs functionally map cis-active elements that
mediate lung-specific expression to within 215 bp of the human SP-C
promoter.
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Cell-specific expression of SP-C-CAT transgenes. The cellular
sites of SP-C-CAT transgene expression were localized by in situ hybridization with a CAT mRNA-specific probe and compared with the
pattern of endogenous murine SP-C mRNA expression. Three independent
lines of the 3.7-kb SP-C-CAT construct and the four internal deletion
transgenic lines were evaluated. CAT mRNA was detected along the distal
bronchiolar epithelium and focally throughout the lung parenchyma of
the 3.7-kb SP-C-CAT lines. The focal CAT expression in the parenchyma
is a pattern consistent with the distribution of type II cells (Fig.
5). A uniform pattern of focal sites of
endogenous SP-C mRNA expression was detected throughout the lung
parenchyma, consistent with the distribution of alveolar type II cells
(Fig. 6). To determine if all SP-C
mRNA-expressing cells also expressed the CAT transgene mRNA,
the number of alveolar cellular sites of CAT expression and endogenous
SP-C expression were determined. In 3.7-kb SP-C-CAT transgenic
lines 5.5 and 5.7, 36 and 56% of alveolar cells
expressing SP-C also expressed the CAT transgene. CAT
expression in line 3.8.8 most closely approximated the
endogenous pattern of SP-C expression, with 72% of SP-C-expressing cells also expressing the transgene (compare Fig. 5B and Fig. 6B). The number of sites of SP-C expression in nontransgenic
mice and transgenic mice varied by ±12%, indicating that endogenous SP-C expression is unaffected by expression of SP-C-CAT
transgene constructs in vivo.
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In a similar manner, the expression patterns of the internal deletion
constructs were determined by in situ hybridization. The 860- to
365-bp SP-C-CAT transgene was detected in bronchiolar and alveolar cells similar to the pattern of CAT expression produced by
the parental 3.7-kb SP-C-CAT transgene (Fig.
7). The pattern of expression found in the
1,910- to
215-bp SP-C-CAT mice lacked the bronchiolar
expression but maintained focal alveolar-specific expression (Fig.
8). As with the other transgenes, the
number of sites of alveolar expression were reduced compared with the endogenous SP-C gene. The loss of bronchiolar expression in the
1,910- to
215-bp line, but not in
860- to
365-bp lines, suggests that deletion of the more distal DNA
(from
860 out to
1,960 bp) removed sequences that
mediated the incorrect bronchiolar expression. Collectively, the in
vivo CAT assays, which map lung specificity to
215 bp, and the
in situ data, which show sustained alveolar expression, suggest that
key cis-active elements that guide type II cell-specific
expression are located within
215 bp of the human SP-C promoter.
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Sequence comparison of murine and human SP-C promoters. The
pattern of bronchiolar expression obtained with the 3.7-kb human SP-C
promoter suggested that there may be sequence differences between the
promoters of the human and murine genes. We have sequenced the human
3.7-kb DNA and DNA upstream from the murine SP-C promoter to determine
the extent of sequence similarity between both SP-C promoter regions.
Sequence homology alignment of the two SP-C sequences demonstrated that
there is considerable sequence divergence with portions of the human
3.7-kb fragment common to both species (Fig.
9). Segments of the human promoter
sequences are arranged as three blocks of conserved DNA dispersed over
~4.8 kb of murine genomic DNA (Fig. 9A). The highest degree
of homology was found in the promoter proximal sequences (81%). The
three regions of sequence conservation account for ~2.8 kb of DNA
common to the two SP-C promoters. The position and size of the human-
or murine-specific sequence differences are diagrammed as insertions
relative to the 2.8 kb of sequence common to both human and mouse SP-C
DNA (Fig. 9B). In the human 3.7-kb SP-C sequence, four small
regions were identified that were absent in the murine SP-C sequence. One of these inserts (250 bp in Fig. 9B) has identity with
the human Alu family of repetitive elements. The largest of the human inserts was a sequence of ~550 bp in length. Both the distal Alu and
proximal 550-bp inserts begin with 32-bp direct repeats. These unique
direct repeats suggest sites for specific insertional or recombination
events. The largest region of nonhomology between human and murine
sequences can be accounted for by three adjacent but distinct
insertions in the murine DNA relative to the human sequence in the
promoter proximal region (~110 bp, 1,120 bp, and 650 bp, Fig.
9B).
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Activation of human SP-C promoter requires transcription factor
TTF-1. TTF-1 is expressed in airway epithelial cells and stimulates the murine surfactant protein gene promoters (3, 4, 17). The consensus
site for TTF binding to DNA has been identified as the conserved
nucleotide motif CAAG. Two TTF-1 elements essential for in vitro
activation of the murine SP-C gene lie within the 215-bp
region, which is highly conserved between the human and mouse
SP-C genes (17). To determine whether the human gene is also
regulated by TTF-1, we cotransfected the human 3.7-kb SP-C-CAT construct into HeLa cells with a TTF-1-expressing plasmid. Constitutive expression of TTF-1 with the 3.7-kb SP-C-CAT construct stimulated a
sevenfold increase in CAT expression (Fig.
10). The increased stimulation of the
human SP-C promoter by TTF-1 was similar to the increased induction
reported for the murine SP-C promoter (17). However, the
relative level of human 3.7-kb SP-C-CAT expression in these transient
transfection assays was significantly less than expression levels
obtained with the murine 4.8-kb SP-C-CAT construct. TTF-1 did not
transactivate the empty CAT reporter plasmid, pBLCAT6 (Fig.
10A) nor the plasmid pBLCAT5 where the CAT reporter
gene is driven by the herpes thymidine kinase gene promoter (data not
shown). The
1,910- to
215-bp human SP-C construct was
also transactivated by TTF-1 in vitro (Fig. 10B,
Ava
SP-C). This construct retained the two proximal CAAG elements that were shown by site-specific mutation to mediate the TTF-1 response of the
murine SP-C promoter (17). Thus the human SP-C sequences that mediate
the TTF-1 response in vitro map to the region necessary for in vivo
SP-C transgene expression.
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DISCUSSION |
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We have analyzed the in vivo expression of SP-C-CAT transgenic constructs to identify the cis-active regulatory regions that mediate lung cell-specific expression of SP-C. We previously determined that 3.7 kb of human DNA extending upstream from the SP-C gene transcription start site directed lung-specific expression of reporter genes in transgenic mice (11). In the current study, we have determined that an essential cis-active region for in vivo SP-C expression in alveolar epithelial cells maps to within 215 bp of the basal promoter. In transgenic lines generated with extensive deletions of distal 5' SP-C sequence, the level of lung-specific expression was dramatically reduced in comparison to expression levels obtained with the parental 3.7-kb SP-C promoter fragment. These results indicate that a significant enhancer region(s) lies upstream from the tissue-specific sequences.
Transgene expression overlaps but is not identical to endogenous
SP-C expression. The 3.7-kb SP-C-CAT transgene expression was detected in both bronchiolar and alveolar epithelial cells of each
3.7-kb SP-C-CAT founder line, whereas native SP-C gene transcription was detected only in the surfactant-producing type II
cell. To determine whether position effects or cis-active
sequences of the human SP-C transgene controlled bronchiolar
expression, levels and cellular sites of transgene expression were
compared. Eight independent SP-C-CAT lines were generated by
independent microinjection and subsequent transgene integration events.
Four of these transgenic lines were bred to homozygosity and subject to
further analysis. Additionally, the 3.7-kb SP-C-CAT transgenic lines resulted from low and high copy number transgene integrations, yet all the lines sustained bronchiolar CAT expression with no alteration of endogenous SP-C expression. Therefore, it is unlikely that the bronchiolar transgene expression is due to position effects or
copy number. The possibility that multiple copies of transgene SP-C
cis-active sequences sequester an essential SP-C
transcriptional repressor is unlikely to account for the observed
bronchiolar expression because the endogenous SP-C expression is not
activated in bronchiolar epithelial cells by the loss of such a
hypothetical repressor. A plausible interpretation is suggested by
sequence homology between the human segment of SP-C DNA and the murine equivalent of upstream SP-C sequence. Homology between the 3.7 kb of
human DNA is not sequence identical but is arranged as three large
regions of homology distributed over 4.8 kb of murine genomic DNA (Fig.
9A). Near identity between the human and murine DNA is
preserved only in the promoter proximal 320 bp. When the two SP-C
sequences are compared, multiple distinct inserts are identified that
are specific to either the human or murine gene. Such species differences in sequence may partially disrupt precise binding of
transcription factors to their intended cognate site or may displace
the alignment of bound factors necessary to silence airway epithelial
cell expression of the human transgene and sustain only alveolar type
II-specific expression. Moreover, the type II cell only expression of
the 1,910- to
215-bp deletion construct suggests that
alveolar cell specificity resides within the conserved proximal region.
When transgenic mice were generated with 2.4 kb of rat Clara cell
secretory protein (CCSP) promoter, the 2.4-kb CCSP-CAT pattern of
expression was similar but not identical to the pattern of murine CCSP
expression. The 2.4-kb CCSP-CAT mRNA was detected in epithelial cells
along the trachea, bronchi, and large bronchioles. The 2.4-kb CCSP-CAT
was diminished in distal bronchioles where murine CCSP
expression was still maintained (29). Such species differences may
account for variations in patterns of transgene expression.
A second difference between the sites and levels of activity of the
3.7-kb SP-C-CAT transgene and endogenous SP-C mRNA was a
reduced number of alveolar cellular sites of transgene expression. Each
3.7-kb SP-C-CAT and 3.7-kb SP-C-CAT deletion transgenic
line had significantly less transgene-expressing alveolar cells than endogenous SP-C-expressing cells. Transgene expression levels were high
enough that loss of hybridization signal could not account for reduced
numbers of transgene-expressing cells. Variation in the number of
target cells, which express a transgene, has been widely reported for
tissue-specific genes analyzed in transgenic mice. Extensive transgene
analysis of -globin gene regulation represents the most complete
studies to date, which define complex elements that determine uniform
cell appropriate expression in vivo and minimize insertion
site effects. Minimal
-globin transgenes demonstrated cell type- and
developmental stage-specific expression but with a dramatic
variation in the percentage of cells expressing the transgene between
different lines made with the same construct (12). Transgene-expressing
cells ranged from 0.3 to 88% in different lines, whereas
little variation was seen in the relative levels of
expression per cell. The variation in number of sites of globin transgene expression is similar to the variation in alveolar sites of
human SP-C transgene expression described in this study.
Further analysis of the globin locus control regulatory sites in
transgenic mice defined multiple elements that cooperate to open
chromatin structure and mediate uniform transgene transcription (5). To
date, such equivalent locus control regions of the surfactant protein
genes have not been identified. The
-globin transgene studies
provide the conceptual basis for future experiments to characterize
elements from the SP-C locus that mediate uniform alveolar cell
expression. Other studies have indicated heterogeneity in type II
cells. Wikenheiser and co-workers (33) noted that a subpopulation of
type II cells maintained SP-B expression in the lungs of mice exposed
to high levels of oxygen. In a separate study, Piedboeuf et al. (25)
determined that in mice a subset of SP-C-positive type II cells
expressed intercellular adhesion molecule-1 in response to oxygen
exposure. Therefore, levels of gene expression can vary among type II
cells in vivo. The present study suggests that additional
cis-active sequences in the transgenes may be required to
reduce the variation in number of type II cells expressing a
SP-C transgene.
Localization of a regulatory region that is essential for lung
epithelial cell transcription. An in vivo deletion analysis of the
3.7-kb promoter region was used to map sequences that mediate lung-specific gene transcription. CAT activity was detected only in the
lungs of transgenic mice when the SP-C promoter was reduced to
1,225 or
590 bp in length (Fig. 3). In a separate study, lung-specific expression of a single transgenic line generated with
1277 bp of human SP-C promoter DNA was reported. Expression was
localized to type II cells, but bronchiolar expression was not
evaluated (8). This report is consistent with our transgenic analysis.
The deletion SP-C-CAT transgenic lines were similar in high
copy number to the 3.7-kb SP-C lines, yet the lung CAT expression was
at the lower limits of detection. These results suggested that the
distal sequences eliminated from the constructs included an enhancer(s)
and that essential lung-specific elements were located in the
590-bp region. The presumptive enhancer may be associated with
the two distal segments of conserved homology eliminated in the
deletion constructs (Fig. 9).
Because of the very low level lung-specific CAT activity detected in
the 590-bp SP-C-CAT lines, it was unlikely that we would be able
to detect expression from shorter deletions that might further reduce
the CAT reporter signal. We therefore generated two sets of internal
deletions that removed regions of DNA closer to the transcription start
site than
590 bp but with intact distal sequences. By including
distal regions of the original construct, we hoped to maintain
transgene expression levels that were high enough to detect. The
3' boundary of the constructs retained either 365 or 215 bp of
DNA adjacent to the basal TATAA box of the SP-C gene. CAT
activity was present only in lung extracts from transgenic animals
generated with these transgenes. The results of this analysis precisely
map a cis-active region for lung-specific expression to the 215 bp directly adjacent to the human SP-C promoter.
Deletion analysis of the rabbit SP-A gene promoter in
transgenic mice demonstrated that a region between 378 and
47 bp is critical for bronchiolar and alveolar cell expression
of SP-A-hGH (human growth hormone) transgenes. Low-level
SP-A-hGH expression was also detected in heart, thymus, and spleen of
some
991-bp SP-A-hGH or
378-bp SP-A-hGH
transgenic lines. Extended
4,000-bp SP-A promoter constructs
were expressed exclusively in the lung, indicating that distal
sequences may be required to silence nonlung SP-A expression (1). In a
separate study, regions necessary for cell-specific expression of the
murine CCSP gene were identified in transgenic mice. A
166-bp region was shown to confer bronchiolar-specific expression, whereas a second region between
803 and
166
bp was required for high-level CCSP expression (26). Consistent with our findings, both of these studies indicate that multiple
cis-active regions contribute to pulmonary cell-specific expression.
Transcription from human SP-C promoter is stimulated in vitro by
TTF-1. TTF-1 is a member of the dispersed family of Nkx homeodomain transcription factors and is expressed in developing thyroid, brain,
and lung (22). Targeted mutation of the TTF-1 gene disrupted development of the distal lung, demonstrating that TTF-1 is a critical
determinant of lung maturation (19). The onset of TTF-1 expression
precedes SP-C expression and is maintained along the entire airway
epithelium during development (22, 37). These observations implicated
TTF-1 as a potential regulator of surfactant protein gene expression.
Cotransfection of TTF-1 expression plasmids with test promoter
constructs demonstrated that TTF-1 activates expression of murine SP-A,
human and murine SP-B, murine SP-C, and rat CCSP promoters (3, 4, 17,
36). TTF-1 transactivation of the murine SP-C promoter was mediated by
binding to two adjacent sites at 186 to
163 bp of the
murine SP-C promoter (17). These two TTF-1 responsive sites are
conserved between the murine and human SP-C promoters and are located
within the
215-bp human SP-C region shown in the current study
to be essential for lung-specific transgene expression. We demonstrate
that the human 3.7-kb SP-C-CAT and
1,910- to
215-bp
deletion SP-C-CAT constructs are transactivated by coexpression of
TTF-1. These results indicate that TTF regulation of SP-C is conserved
across species and that the deletion clone, which mapped
alveolar-specific expression at
215 bp in vivo, also harbors the
TTF-1 responsive region of the human SP-C promoter.
Each TTF-1 responsive gene has been shown to have a unique arrangement and number of TTF-1 binding sites with distinct TTF-1 binding affinities. In addition to distinct configurations of TTF-1 sites, the DNA binding activity of TTF-1 has been shown to be dependent on phosphorylation state and responsive to redox regulation (2, 35). Analysis of the TTF-1 gene identifies multiple promoter and transcription initiation sites that might produce alternate isoforms of TTF-1 as well (13, 23). Collectively these reports suggest that numerous subtle modifications of TTF-1 may alter selective expression of a gene in type II cells relative to Clara or other airway epithelial cells.
It is unlikely that TTF-1 alone is sufficient to dictate the precise
expression of SP-C and other surfactant protein genes in
differentiated airway and alveolar cells because each surfactant protein gene is expressed in a unique cellular and developmental pattern. The transcription factors hepatocyte nuclear factor (HNF)-3 and forkhead homolog (HFH) family members have been shown to be expressed in the bronchiolar epithelium where the SP-A,
SP-B, and CCSP genes are expressed (37).
HNF-3 stimulates human SP-B and human TTF-1 gene
expression, whereas HNF-3
suppresses HNF-3
stimulation of the
TTF-1 gene (3, 14). The transcription factor GATA-6 is
expressed along the developing airway epithelium and transactivates the
TTF-1 promoter in transient transfection experiments (28). These
experiments are the first evidence for complex networks regulating gene
expression in developing lung cells. Footprint analysis of the active
promoter regions of SP-A, SP-B, and SP-C indicate that numerous other
protein-DNA interactions have occurred (4, 3, 17). The combination of
this current biochemical data with HNF-TTF-1 transactivation data
suggests that combinatorial interactions of TTF-1 and other
transcription factors orchestrate the abundance and distribution of
SP-A, SP-B, SP-C, and CCSP in a cell-specific manner along the
respiratory epithelium.
The present study demonstrates that the 590-bp segment of human
SP-C DNA is sufficient to initiate and sustain lung-specific transgene
expression. Transgenic mice with internal deletions of the SP-C DNA map
cis-active elements, which mediate transcription in alveolar
cells to
215 bp and a second distal segment of DNA that sustains
a high level of SP-C expression in vivo. An extensive deletion analysis
of the 4.8-kb murine SP-C promoter in vitro did not identify any distal
enhancer activity, suggesting that elevated in vivo expression
requiring the distal human SP-C region is chromatin dependent (17). The
3.7-kb human SP-C promoter has been used to generate transgenic mice
expressing growth factors, cytokines, and other reporters in vivo. The
present findings indicate that in such models the extent of type II
cell expression may vary for each founder line and should be considered
in the interpretation of phenotypes. The human SP-C promoter is also
transactivated by TTF-1 at cis-active sites in close proximity
to the SP-C promoter, supporting the concept that TTF-1 is critical in
regulating cell-specific SP-C gene expression.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jeffrey A. Whitsett for a critical reading of the manuscript. We also appreciate the assistance of Dr. Susan Wert in morphological analysis and Ann Maher in manuscript preparation.
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FOOTNOTES |
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Portions of this work were supported by the National Institutes of Health Grant HL-50046 (S. W. Glasser) and American Lung Association Grant RG-182-N (S. W. Glasser).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. W. Glasser, Children's Hospital Medical Center, Div. of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: glass0{at}chmcc.org).
Received 13 September 1999; accepted in final form 13 December 1999.
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