(Received for publication, October 18, 1994; and in revised form, November 10, 1994)
From the
Transient transfection and murine germ line gene transfer analysis was used to determine the regions of DNA necessary to confer the appropriate level and cell specificity of the expression of the gene coding for the murine Clara cell 10-kDa protein, mCC10. To identify the cis-acting elements involved in the regulation of mCC10 gene, different lengths of the 5`-flanking sequence were ligated to the bacterial chloramphenicol acetyltransferase gene for transient transfection to H441 cells (human lung adenocarcinoma cell line). The corresponding sequences were also fused to the human growth hormone gene and transferred to the murine genome for an in vivo analysis of mCC10 promoter activity. The results of the transient transfection analysis identified the region from -166 to -124 of the 5`-flanking region of the mCC10 gene as necessary for the expression of this gene in H441 cells. The transgenic mouse analysis confirmed that the 166 base pairs of 5`-flanking DNA was sufficient to confer cell-specific expression. However, the transgenic mouse analysis also showed that, to achieve the full quantitative level of transgene (human growth hormone) expression, regions between -803 and -166 base pairs of the 5`-flanking sequences are required for maximum expression of mCC10 gene promoter activity.
The lung is composed of several distinct cell populations that
function to facilitate the exchange of gases between the tissues of the
body and the external environment(1) . This cellular
heterogeneity makes the lung an intriguing model to investigate the
molecular determinants of pulmonary cell differentiation. One distinct
cell type, the nonciliated secretory cells lining the airways of the
lung, is the Clara cell(2) . The major secretory product of
Clara cells is a homodimeric 10-kDa protein known as the Clara cell
10-kDa protein (CC10), Clara cell secretory protein, polychlorobiphenyl
binding protein, or
uteroglobin(3, 4, 5, 6, 7, 8, 9, 10, 11) .
This protein binds polychlorinated biphenyl compounds (8) and
can inhibit phospholipase A(12) . An
anti-inflammatory role for this protein has been hypothesized, but the
physiological role of this protein is yet to be defined. The tissue
distribution of the expression of CC10 is similar among the
different species investigated. The primary site of expression of CC10 is the nonciliated secretory cells of the respiratory
tract(13) . However, under certain endocrine conditions, CC10 mRNA can also be detected at very low levels in the male
and female reproductive tracts(5) . In lagomorphs, CC10, also
called uteroglobin, is expressed at highest levels in the female
reproductive tract during implantation of the embryo. Despite the lack
of a defined physiological role of this protein, CC10 serves as a
marker for the analysis of the genetic control of the cellular and
tissue specificity of pulmonary gene expression.
Transgenic and
transient transfection analysis has been conducted on the rabbit (14) and rat (15) CC10 gene to determine the
location of the DNA elements necessary for the control of the tissue
and cell-specific expression. Analysis of the rabbit gene has shown
that 3.3 kb ()of the 5`-flanking DNA was sufficient to
confer the full tissue-specific expression of this gene in transgenic
mice(14) . Similarly, analysis of rat CC10 promoter
sequence has demonstrated that the 2.4-kb upstream promoter sequence
was sufficient for tissue-specific expression of reporter
transgenes(15) . Using the human lung adenocarcinoma cell line,
which displays a Clara cell phenotype, the H441 cell transient
transfection analysis of the 5`-flanking region of the rat and rabbit
gene fused to the bacterial chloramphenicol acetyltransferase (CAT)
gene has shown that elements more proximal to the start of
transcription were sufficient to direct the cell-specific expression (14, 15) . Electrophoretic mobility shift assay and
DNase I footprint analysis with H441 nuclear extract identified
transcription factors in the octamer, hepatic nuclear factor, and AP1
families as being the potential regulators for the expression of this
gene(15, 16) . Analysis of other lung-specific
promoters including surfactant proteins SP-A, SP-B, and SP-C have been
reported(15, 16, 17, 18, 19, 20) .
Even though the cellular specificity of these promoters is not as
restricted as the CC10 promoter, several of the factors
involved in CC10 gene expression are also important for the
expression of the surfactant proteins. Specifically, the transcription
factors HNF3 (21, 22) and thyroid transcription factor
1 (23) seem to be important for the expression of all pulmonary
epithelial genes analyzed to date. Therefore, pulmonary cell
specificity may be the result of a combination of common and
cell-specific transcription factors regulating pulmonary gene
transcription.
In this study, we report the in vitro and in vivo analysis of the cis-acting elements responsible for tissue-specific expression of the mouse CC10 gene, mCC10. Using H441 cells for transient transfection analysis, the proximal promoter regions necessary for the transcriptional regulation of mCC10 was determined. Using transgenic mouse analysis, the elements involved in cell-specific regulation were determined and compared with the expression of the endogenous gene. The in vivo transgenic analysis verified the findings of the in vitro analysis but also identified additional regions in the 5`-flanking region of mCC10 needed for the full quantitative level of expression of the reporter gene in transgenic mice. Since the mouse has become the model system for investigating genetic regulation of mammalian gene expression, these data will serve as the foundation for future investigations into the elements regulating cell-specific expression of the mCC10 gene in a homologous system. The identification of these elements will be useful for somatic gene therapy of human pulmonary diseases.
Figure 1: Strategy for the constructions used in transfection and isolation of microinjection fragments. Successive deletions of mCC10 5`-flanking sequences were ligated to either pBLCAT3 for transfection of H441 and CV1 cells or human growth hormone gene and isolated CC10 growth hormone fragment for microinjection.
The initial 5`-flanking DNA
constructions of the mCC10 gene were generated by placing the
2.1-kb HindIII-HphI (-2.1 to +0.007) mCC10 fragment into HindIII- and EcoRV-digested
Bluescript (KS, Stratagene) yielding the plasmid
pKS-mCC10(2.1). To place this region 5` to the reporter genes, the
2.1-kb mCC10 insert was liberated by digestion with HindIII and BamHI and subcloned into the appropriate
restriction sites 5` to the CAT and hGH reporter genes of the plasmids,
pBLCAT3 and pKShGH. The mCC10 fragment containing 5.1 kb of
5`-flanking sequences was generated by subcloning the HindIII
fragment of the mCC10 5`-flanking DNA, spanning -5.1 to
-2.1 kb, 5` to the 2.1-mCC10-CAT and hGH constructs. This yielded
CAT and hGH constructions with 5.1 kb of contiguous mCC10 5`-flanking DNA. Subsequent deletion of the mCC10 5`-flanking DNA constructions were made by digestion of
pKS-mCC10(2.1) with StuI/BamHI, SacI/BamHI to generate fragments of 0.803 and 0.166
kb of 5`-flanking DNA. These fragments were subcloned 5` to CAT and hGH
in their respective plasmids.
Constructions with 0.123, 0.087, and
0.023 kb of 5`-flanking DNA were generated by PCR amplification of
pKS-mCC10(2.1) using oligonucleotides corresponding to the appropriate
junctions. The 5`-upstream oligonucleotide primer sequences for 0.123-,
0.087-, and 0.023-kb fragments were 5`-ATTATTTGCTTATTCCACGGAG-3`,
5`-ATAATGCAATCTCCTAAGTG-3`, and 5`-TATAAAAAGCCACACACCCAC-3`,
respectively. The 5`-XbaI restriction site was added to the
5`-end of each oligonucleotide sequence to aid in future subcloning of
these fragments. The reverse primer, 5`-CACTATAGGGCGAATTGG-3`, was
selected from the Bluescript multiple cloning cassette. This sequence
amplifies the BamHI restriction site present in the polylinker
site of the plasmid, which was used in the subsequent subclonings. The
PCR amplification of the DNA fragments was accomplished by adding 0.01
µg of linearized template and 1 µM of each
oligonucleotide primer to a 100-µl reaction mixture containing 50
mM KCl, 10 mM Tris (pH 8.4), 2.5 mM MgCl, 200 µM of all four dNTPs, and 2.5
units of Taq polymerase. The polymerase chain reaction was
performed for 30 cycles using the following program: 1 min at 94 °C
for denaturation, 2 min at 65 °C for annealing, and 3 min at 72
°C for amplification. PCR-amplified products were pooled together
and further purified. The fragments were digested with XbaI
and BamHI and subcloned into the appropriate sites of pBLCAT3
and pKShGH. All clones generated from PCR amplification were sequenced
to ensure faithful amplification of the mCC10 flanking DNA.
Finally, the cells were
harvested and lysed in 0.2 M Tris-HCl (pH 7.5) using 3 cycles
of freezing and thawing. Protein concentration of the cell lysate was
measured using the Bradford assay (Bio-Rad). The CAT activity of the
lysate was assayed as described by Seed and Sheen(31) . The CAT
assay was conducted using 50 µg of protein for each reaction. The
reaction was incubated at 37 °C for 5 h. The reaction mixture was
stopped by extracting with 200 µl of
2,6,10,14-tetramethyl-pentadecane/Xylene (2:1). The CAT activity was
measured by counting C-acetylated derivatives using a
Beckman LS-8000 scintillation counter. The assay for
-galactosidase was performed according to the protocol described
by Sambrook and co-workers(32) . The CAT enzymatic activity was
corrected for variability due to transfection efficiency by normalizing
the CAT activity to
-galactosidase activity. The relative strength
of the promoters are expressed as CAT activity
(cpm)/
-galactosidase activity (units).
Figure 2: The pulmonary expression of the hGH transgene under the control of various lengths of the mCC10 5`-flanking DNA. The bars on the graph represent the mean level of expression of the transgene. The opencircles represent the data point for individual mice.
In the mice where hGH mRNA was detected in the lungs, hGH protein was also detected by radio-immuno assay (Nichols Institute, San Juan Capistrano, CA) in both lung protein extract and serum (data not shown). Although hGH protein was detected, no fetal or neonatal growth retardation was observed. The phenotype of fetal growth retardation caused by the expression of hGH has been reported by Hackett et al. (20) when hGH was placed under the control of the rat CC10 promoter. This phenotype must be unique to that transgene since it has not been observed for either the mouse or rabbit CC10 promoter(6) .
Immunohistochemical localization of the hGH transgene protein showed that for all transgene constructions in which mRNA was detected, the cellular specificity of the transgene expression was maintained. An example of the immunohistochemical analysis is shown in Fig. 3. Thus, although the 0.166-kb fragment was not able to confer the full level of transgene expression, it was sufficient to maintain the cellular specificity of the mCC10 gene expression. Although the cell-specific pulmonary expression was the focus of this investigation, the tissue distribution of the transgene was investigated. The tissue distribution of the transgene followed the distribution of the endogenous gene. Fig. 4shows that of the tissues examined, only the lung and uterus showed expression of the transgene. In the cases when the uterus showed expression of the transgene, the expression of the endogenous gene could also be detected. Since the uteri were taken from randomly cycling female mice, the expression could not be detected in every mouse. This is due to the hormonal regulation of the transgene. However, the uterine expression of the transgene was at a level comparable with the expression of the endogenous gene.
Figure 3:
The
cellular localization of endogenous CC10 and hGH in CC10-hGH
mice. A, anti-hGH antibody was used in immunohistochemical
analysis of mouse lung bearing the mCC10-hGH transgene with 0.166 kb of
5`-flanking DNA (magnification 200). B, anti-mCC10
antibody was used on a similar section of the same mouse lung to
determine the extent of expression of the endogenous mCC10 gene (magnification 200
).
Figure 4: Northern blot analysis of the tissues from transgenic mice for the hGH gene under the control of varying lengths of the mCC10 5`-flanking DNA. PanelA was probed with the hGH cDNA, and panelB was probed with the mCC10 cDNA. The lanes are labeled by the lengths of 5`-flanking DNA used for each mCC10-hGH transgene.
Figure 5: Transient transfection of H441 cells with the CAT gene under the control of the mCC10 promoter and the rabbit uteroglobin gene. The CAT activity is expressed as counts per minute of CAT activity per unit of galactosidase activity. The data presented here is the mean of several independent transfections, and the errorbars represent the standard error for each construction.
To
identify precisely the cis-acting elements as mentioned
before, we used a series of CC10-CAT constructs having progressive
deletions of the 5`-flanking sequences. Expression of CAT from each
construct was normalized to -galactosidase activity expressed from
cytomegalovirus promoter in CMV-
plasmid. Fig. 6shows that
deletion of DNA sequences between -5.1 and -2.1 kb lowers
the CAT activity by 58%, which indicates some positive transcriptional
modulators within that region. Deletion of DNA sequences between
-2.1 and -0.166 kb increases the CAT activity by 242%,
indicating the presence of some negative regulatory elements. Deletion
of another 43 nucleotides to 123 bp of 5`-flanking DNA lowers the CAT
activity by 300%. Transfection of H441 cells with CAT constructs having
123 and 87 bp showed no difference in their ability to express CAT in
H441 cells. Deletion to -23 bp, a fragment containing only the
TATA sequences, does not show any appreciable CAT expression, which is
comparable with the negative control, pBLCAT3. From this transient
transfection data it could be concluded that cell-specific
transcriptional regulatory elements reside within 166 nucleotides
upstream of the transcription start site of mCC10 gene, and
presence of the above mentioned positive and negative regulatory
elements are consistent with previous reports in the
literature(15, 35) .
Figure 6: Analysis of CAT expression from mCC10-CAT constructs with successive deletion of the 5`-flanking sequence. The CAT activity is expressed as counts per minute of CAT activity per unit of galactosidase activity. The data presented here are the mean of several independent transfections, and the errorbars represent the standard error for each construction.
Figure 7:
Reverse transcriptase (RT) PCR of
total RNA from H441 cells. PCR products were analyzed in 1.5% agarose
gel. Lane1 is -DNA digested with EcoRI/HindIII. Lanes2 and 3, total RNA from H441 cells in absence and presence of
reverse transcriptase. Lanes4 and 5, human
lung total RNA in absence and presence of reverse transcriptase. The upperband indicates the amplification of the
internal control human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) mRNA. The lowerband indicates the
amplification of the hCC10 mRNA.
This analysis demonstrates that 803 bp of the 5`-flanking DNA of the mCC10 gene is capable of driving the expression of a heterologous gene in transgenic mice at the appropriate level and cellular specificity. Although 166 bp of the mCC10 5`-flanking DNA is capable of supporting the appropriate Clara cell-specific expression of transgenes in mice, this fragment is not capable of driving the level of expression to the same magnitude as that of the endogenous gene. Therefore, there must exist enhancer(s) elements between -803 and -166 needed to support full expression. A similar phenomenon was observed when deletion analysis was conducted on the rabbit uteroglobin transgene. Although 0.6 kb of 5`-flanking DNA of the rabbit promoter was capable of directing pulmonary-specific gene transcription, the level of transgene expression was lower than when 3.3 kb of 5`-flanking DNA was used(14) .
Although the -803-bp and larger fragments of the mCC10 promoter are capable of supporting transgene expression to the appropriate cell type and, on average, to the appropriate level, the expression of transgenes is highly variable and could not be correlated with the number of copies of the transgene. If such integration site and copy number independence in transgene expression can be achieved with mCC10 regulatory regions, then there must exist domains located outside of the regions used in this study that will confer these properties.
Analysis of the deletion mutants of the mCC10 gene demonstrated a similar pattern of expression in the H441 cells as previously reported for the rCC10 promoter. In the distal 5`-flanking region, there could be identified positive and negative elements that modulate the basal expression of this gene. This was not observed in the transgenic mouse analysis. The inability to identify these distal elements using the transgenic mouse analysis could have been due to the high degree of variability of transgene expression in the mice. This high degree of variability of transgene expression, due to the influences of the site of transgene integration, made the detection of subtle differences impossible. As observed in the rCC10 gene, elements proximal to the promoter were necessary for expression in H441 cells. The importance of these elements was observed in transgenic mice where 166 bp of the 5`-flanking DNA was capable of directing the appropriate cellular expression of the transgene in mice. However, the transfection analysis in H441 cells was not capable of detecting elements between -803 and -166, which are needed for the full quantitative expression of the gene. The lack of detection of enhancer(s) in this region by the H441 cells may be due to the fact that the CC10 gene is not expressed in this cell line and the trans-acting factors that interact with the DNA sequences in -803 to -166 fragment may not be present in these cells. The lack of expression of hCC10 in H441 cells may be due to the lack of the ability of the in vitro culture conditions to support CC10 expression. Attempts to generate murine-transformed Clara cell lines have resulted in the initial expression of CC10. However there is loss of expression in subsequent cell passages(36) .
An alternative explanation for the inability of transient transfection analysis of mCC10 promoter to detect regulatory elements upstream of the -166 bp of 5`-flanking DNA may be due to species differences between human and mouse Clara cells. This species heterogeneity in Clara cells can be observed by the level of expression of the reporter gene driven by various CC10 promoters in these cells. The rat promoter drives the expression of the reporter gene to a higher level than the mouse promoter in these cells(33) , and the expression of CAT under the control of the rabbit promoter cannot be detected unless heterologous enhancers are added(34) . Therefore, the elements between -803 and -166, which regulate the mouse gene expression, may not be present in this human cell line. Despite this species variability, at present the H441 cells serve as the only tool to precisely identify the cis-acting elements involved in the regulation of CC10 gene expression. Therefore, the distal elements must be identified by further transgenic analysis.
Stripp et al. (15) identified a nonamer sequence at -90, and a perfect copy of the nonamer sequence in reverse orientation is present and centered at -118 in rat CC10 promoter. Interestingly, the same nonamer sequence is present in the rabbit uteroglobin promoter sequence at positions -98 and -127. We identified the nonamer sequence, AAGTAAATA, present at position -93, and the reverse orientation of the same sequence is present at -117 of the mCC10 promoter. These sequences are partially similar to octamer motif (ATGCAAAT) of transcription factors Oct-1 and Oct-2. The cis-acting elements regulating the expression of the rat gene have been identified. Sawaya and co-workers (16) have dissected the elements required for expression of genes under the control of the rCC10 promoter. DNase 1 footprinting analysis identified two regions in which H441 nuclear proteins interacted with the rCC10 5`-flanking region, region 1 and region 2. Region 1 of the rat gene spanned from -123 to -86. Using linker scanner mutations, an AP1 site and several octamer and HNF3 sites were identified in this region. As previously reported(7) , several potential factor binding sites in mCC10 promoter were identified within this region by computer analysis. Alignment of the mouse and rat region 1 shows a high degree of sequence homology. In designing the deletion construct for this investigation, 123 bp of 5`-flanking DNA was selected because this fragment contained region 1, having the potential transcription factor binding sites. The next construct having 87 nucleotides upstream was designed to delete this region. There were no significant differences between the -123- and -87-bp fragment to confer the expression of CAT in H441 cells. However, addition of the fragment with sequences from -166 to -124 was sufficient to give maximum expression in H441 cells. When DNase 1 footprinting was conducted on the mCC10 promoter with H441 cell nuclear extract, two protected regions were identified(33) . These regions were identified at -137 to -117 and -101 to -84. Thus, the protected regions in the mouse gene extends outside the defined region 1 in the rat gene. Analysis of the sequences in this region revealed the sequence of TATGAAAGA as a potential octamer binding site. This sequence is also found at the identical location in the rat gene, yet linker scanner mutations of this region did not alter rCC10 promoter activity(16) . Since the mouse gene lacks region 2, this additional octamer site may be important for mCC10 gene expression. The rat gene may not require this region, and transcription factor activity in region 2 may compensate for the mutation of this octamer region.
The Clara cell 10-kDa
protein represents an interesting model system to identify the elements
regulating both tissue- and cell- specific expression. In this report,
transgenic mouse technology and transient transfection analysis was
used to investigate the elements required for the expression of the
murine CC10 gene. The transient transfection experiments
represent a functional identification of the boundaries of the core
elements regulating cell-specific expression. The transgenic analysis
verified the importance of the proximal promoter region in Clara
cell-specific expression of this gene and also identified distal
regions necessary for the appropriate level of expression of this gene.
The analysis of the mouse promoter also showed subtle species
differences in the elements required for expression in H441 cells.
Transgenic data presented here will be able to dissect the mechanism of mCC10 gene expression in bronchiolar epithelium by identifying
the cis-acting elements. The advantage of executing this
analysis in the murine gene is that future analysis on the interactions
of these factors in the developmental biology of the lung will be
accomplished in vivo as well as in vitro using the
mouse as a genetically manipulated system. Besides this, identification
of cis-acting elements and trans-acting factors has a
much more clinical importance in the treatment of genetic diseases like
cystic fibrosis and -antitrypsin using somatic gene
therapy(37) . Thus, these manipulations can be accomplished in
a system where the elements regulating the expression of the homologous
gene has been identified.