©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
cis-Acting Elements Involved in the Regulation of Mouse Clara Cell-specific 10-kDa Protein Gene
IN VITRO AND IN VIVO ANALYSIS (*)

(Received for publication, October 18, 1994; and in revised form, November 10, 1994)

Manas K. Ray Susan W. Magdaleno Milton J. Finegold (1) Francesco J. DeMayo (§)

From the Department of Cell Biology and Pathology, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(2)(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 (^1)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.


EXPERIMENTAL PROCEDURES

Construction of CC10-CAT and CC10-hGH Plasmids for Transfection and Transgenic Analysis

Fig. 1shows the overall strategy for the identification of the elements regulating mCC10 gene expression. The CAT gene was used as a reporter gene for transient transfection analysis. The plasmid pBLCAT3 (24) served as the parent CAT vector into which various lengths of the mCC10 5`-flanking DNA was placed. The human growth hormone gene (hGH) was used as the reporter gene for transgenic mouse analysis. The BamHI-EcoRI fragment containing the human growth hormone (hGH) gene was cloned into Bluescript, pKS (Stratagene), generating the plasmid pKShGH. As with the CAT constructions, various deletion fragments of mCC10 were placed 5` to the hGH gene.


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(2), 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.

Generation and Identification of Transgenic Mice

The mCC10-hGH transgenes were isolated from plasmid sequences by digestion with the appropriate restriction enzymes and separated by electrophoresis through low melting point agarose (Seaplaque, FMC Bioproducts, Rockland, ME). The mCC10-hGH transgenes were excised, and the DNA was isolated using QIAEX gel extraction kit (Qiagen Inc., Chatsworth, CA). The isolated fragment was diluted to 2 µg/ml in a modified TE buffer (10 mM Tris (pH 7.5) and 0.25 mM EDTA). Transgenic mice were produced as described by Hogan et al.(25) . DNA fragments were microinjected into ICR times B6C3F1 embryos. The transgenic founder mice were identified by using Southern blot analysis (26) of PvuII-digested mouse tail DNA. The Southern blots were probed with a random primed [alpha-P]dCTP-labeled hGH probe(27) .

RNA Isolation and RNA Blot Analysis

To facilitate the speed of this analysis, the founder mice were sacrificed for expression analysis. The tissues were removed from euthanized transgenic mice and were homogenized in RNAzol B (Cinna/Biotex Laboratories Inc., Houston, TX). The RNA was isolated following the procedure described by Chomczynski et al.(28) . The mRNA for the hGH transgene and the endogenous mCC10 gene were identified by Northern blot analysis(29) . The isolated RNA was denatured in a formaldehyde-containing buffer and separated by 1.5% agarose gel electrophoresis in a buffer containing 2.2 M formaldehyde. RNA was then transferred to nylon membrane (Hybond-N, Arlington Heights, IL) by capillary blotting in 20 times SSC overnight. The filters were hybridized with 20 million counts of either the [P]dCTP-labeled mCC10 cDNA or the [P]dCTP-labeled hGH cDNA probe. The probes were labeled to equivalent specific activities of 2 times 10^9 cpm/µg of DNA. Hybridization, washing of the membranes, and autoradiography were performed as previously described(29) . The mRNA levels for both mCC10 and hGH in Northern blots were quantitated using a Betascope 603 blot analyzer (Betagen, Waltham, MA).

Immunohistochemical Analysis

Tissues were removed from mice at autopsy and placed in 10% buffered formalin for 4-6 h and processed using a Miles VIP automatic tissue processor set for 4 cycles at 13.4 °C. Immunohistochemical staining for hGH or mCC10 was performed using immunoperoxidase staining as described by Sepulveda et al.(30) . A rabbit anti-mCC10 antibody (^2)and a commercially available hGH antibody (DAKO, Carpinteria, CA) were used.

Cell Cultures, Transient Transfection, and Assay of CAT Expression

Human lung adenocarcinoma cells, H441, were grown in RPMI medium containing 10% fetal calf serum. All medium contained 100 units of penicillin and 0.1 mg/ml streptomycin. The transient transfection analysis was conducted using 0.4 pmol of the above mCC10CAT constructs along with 0.2 pmol of pCMV-betaGAL. The DNAs were mixed with 25 µl of lipofectamine (Life Technologies, Inc.) in 1 ml of Dulbecco's modified Eagle's medium (without serum). Cells growing in logarithmic phase (50-70% confluent) on 100 mM dishes were washed twice with phosphate-buffered saline and fed with 5 ml of Dulbecco's modified Eagle's medium (without serum). After washing, 1 ml of plasmid-lipofectamine mixture was added to the cells. The cells were then incubated for 24 h at 37 °C. Cells were then washed with phosphate-buffered saline and fed with RPMI plus 10% fetal calf serum and incubated at 37 °C for 48 h.

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 ^14C-acetylated derivatives using a Beckman LS-8000 scintillation counter. The assay for beta-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 beta-galactosidase activity. The relative strength of the promoters are expressed as CAT activity (cpm)/beta-galactosidase activity (units).

Reverse Transcriptase PCR

H441 cells were harvested from T175 flasks, and total RNA was isolated using RNAzol B as previously described. Human lung total RNA was purchased from Clontech. H441 and human lung RNA were treated with RNase-free DNase, RQ1 (Promega), for 15 min and extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1). The RNA was then precipitated with ethanol. As an internal control for the RNA samples, oligonucleotides were added to amplify the human glyceraldehyde-3-phosphate dehydrogenase mRNA. The primers were designed from the human CC10 and glyceraldehyde-3-phosphate dehydrogenase cDNA sequence to amplify a 348- and 891-bp fragment, respectively. Forward and reverse primers for hCC10 are 5`-GGAATTCACCAGACTCAGAGACGGAAC-3` and 5`-GGAATTCTACACAGTGAGCTTTGGG-3`, respectively. Forward and reverse primers for human glyceraldehyde-3-phosphate dehydrogenase are 5`-ATGGGGAAGGTGAAGGTCGGA-3` and 5`-AGCGTCAAAGGTGGAGAGTG-3`, respectively. For each reaction, 1 µg of RNA template, 0.1 µg poly(dT), 9 units of RNasin (Promega), 1 mM of each dNTP, 10 times PCR buffer (500 mM KCl, 100 mM Tris (8.4), 30 mM MgCl(2)), and 14 units of Moloney murine leukemia virus reverse transcriptase (Boehringer Mannheim) were added and incubated at 37 °C for 30 min. Reaction mixture was incubated at 90 °C for 5 min to inactivate the enzyme.


RESULTS

Transgene Expression of hGH under the Control of the mCC10 Promoter

Transgenic mice were generated for the hGH transgene fused to 5.1, 2.1, 0.803, 0.166, 0.123, and 0.087 kb of mCC10 5`-flanking DNA. Several independent founder animals, 12, 7, 3, 6, and 2, respectively, were generated for each hGH construction. Fig. 2shows the results of the expression of the hGH transgenes under the control of various lengths of the mCC10 5`-flanking DNA. Pulmonary expression of hGH could be detected with as little as 0.166 kb of mCC10 5`-flanking DNA driving the transgene. Expressed as a percentage of the endogenous mCC10 mRNA signal, the hGH transgene was expressed at a level comparable with the endogenous gene for the constructions with as little as 0.8 kb of mCC10 5`-flanking DNA. Although the average level of expression was comparable with the expression of the endogenous gene, there was a high degree of variability between the transgenic founders. The level of expression in these mice was not correlated with the copy number of the transgenes (data not shown). However, when only 0.166 kb of 5`-flanking DNA was used, there was a significant decrease in the level of transgene expression. The level fell to 10% that of the endogenous gene.


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 200times). 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 200times).




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.



Transient Transfection Assay

To further identify the precise cis-acting elements regulating the tissue-specific expression of the mCC10 promoter, transient transfection into H441 cells was employed. H441 cells, which were derived from a human adenocarcinoma and displayed a Clara cell-like phenotype, have previously been used as a source of Clara cells for identifying cis-acting elements and trans-acting factors involved in the regulation of the rat and rabbit CC10 gene(33, 34) . Initially, the ability of the mCC10 promoter was compared with the ability of the rabbit promoter to drive CAT expression in H441 cells. Fig. 5shows the results of this experiment. Although previous reports have shown the mouse promoter to be poorly expressed in H441 cells, the use of lipofectamine was sufficient to give detectable expression in H441 cells. However, expression of CAT under the control of the rabbit uteroglobin promoter was undetectable. Although the 3.3-kb fragment of the rabbit promoter was sufficient to direct the expression of several transgenes appropriately in mice, it was incapable of driving expression in H441 cells. Also, a 0.4-kb fragment of the rabbit promoter was also ineffective in driving CAT expression in H441 cells (data not shown). The rabbit promoter has been used by other investigators to direct CAT expression in Ishikawa cells and H441 cells (34) . However, these experiments used DNA constructs that contained the SV40 early gene enhancers. Thus, the rabbit uteroglobin promoter may be incapable of driving detectable gene expression in these cells without the aid of additional enhancer elements.


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 beta-galactosidase activity expressed from cytomegalovirus promoter in CMV-beta 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.



Expression of hCC10in H441 Cells

Data obtained from the transfection of H441 cells and the transgenic mouse data are consistent and indicate that 0.166-kb of the promoter elements were capable of directing the expression of the mCC10-driven gene constructions. However, the transfection analysis in H441 cells was not capable of identifying elements between 0.803 and 0.166 kb, which is necessary for the full level of expression of the mCC10 gene in vivo. Although the expression of hCC10 was not observed in H441 cells, it has been speculated that the level of hCC10 expression may be below the level of detection(33) . Since reverse transcriptase PCR represents a very sensitive method of detection of mRNA, reverse transcriptase PCR was used to identify hCC10 mRNA in H441 cells. Fig. 7shows that hCC10 mRNA could not be detected in these cells.


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.




DISCUSSION

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 alpha-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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL47620. 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: Tel.: 713-798-6241; Fax: 713-790-1275.

(^1)
The abbreviations used are: kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; hGH, human growth hormone.

(^2)
M. K. Ray, S. W. Magdaleno, M. J. Finegold, and F. J. DeMayo, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. Bert W. O'Malley and Ming-Jer Tsai for critically reviewing this paper and for valuable suggestions. We also thank the following people for their contribution: Lou Ann Stanley, Joyce L. Pike, John Stockton for microinjection, Osvaldo Trujillo for screening the mice, and Irene A. Harrison for help in preparation of this manuscript.


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