From the Department of Pharmacology and Toxicology,
University of Utah, Salt Lake City, Utah 84112 and
§ Department of Pediatrics, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226-4801
Received for publication, January 10, 2003, and in revised form, February 13, 2003
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ABSTRACT |
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The CYP2F1 gene encodes a cytochrome
P450 enzyme capable of bioactivating a number of pulmonary-selective
toxicants. The expression of CYP2F1 is highly
tissue-selective; the highest expression is observed in the lung with
little or no hepatic expression. The objective of these studies was to
elucidate the mechanisms that govern the unique tissue-specific
regulation of CYP2F1. Cosmid and bacterial artificial
chromosome clones were screened and sequenced to identify a gene that
spanned 14 kbp containing 10 exons, including an untranslated exon 1. Primer extension analysis and 5'-rapid amplification of cDNA ends
were used to identify the transcription start site. Several sequences
homologous to known cis-elements were identified in the
5'-upstream region of the CYP2F1 promoter. Transient
transfection studies with luciferase reporter constructs demonstrated a
significant functional lung cell-specific CYP2F1 promoter
region (from position Cytochrome P450 proteins
(P450s)1 are a superfamily of
heme-containing enzymes that generally catalyze the metabolism of
endogenous and foreign compounds to metabolites that can easily be
eliminated from the body. However, for many foreign compounds
cytochrome P450 metabolism produces "bioactivated" metabolites,
which are highly reactive with endogenous proteins and DNA, causing
cell death and gene mutations (1). Cytochrome P450 expression, which can be important for organ-specific functions, can also lead to tissue-selective bioactivation and toxicity of drugs and other xenobiotic compounds. Cytochrome P450-mediated bioactivation of toxicants is a particularly relevant process to lung diseases because
the lungs are exposed directly to environmental pollutants, such as
cigarette smoke. Characterizing the mechanisms that regulate tissue-selective P450 expression is vital to understand organ-specific toxicity and individual differences in susceptibility to environmental pollutants and drugs (2).
Due to the propensity of human lung to bioactivate procarcinogens and
other xenobiotics, several screening processes have been performed to
identify new P450s that are potentially involved in population
susceptibility to cancers caused by cigarette smoke and other
environmental pollutants. A cDNA library screen from human lung
tissue identified a P450 gene, designated CYP2F1, that was
sequenced and mapped to chromosome 19 (3). Expression of recombinant CYP2F1 showed that this enzyme bioactivates two prototypical
pneumotoxicants, naphthalene and 3-methylindole. CYP2F1 metabolizes
naphthalene to its highly pneumotoxic intermediate,
naphthalene-1,2-oxide, and 3-methylindole to its dehydrogenated
pneumotoxic product, 3-methyleneindolenine (4-7). CYP2F1 can also
bioactivate styrene to its carcinogenic epoxide (8).
The expression of P450 enzymes is controlled by diverse regulatory
mechanisms such as inducible transcriptional activation by
ligand-activated receptors and constitutive expression by
tissue-enriched transcription factors, each of which generally bind to
specific regulatory elements in the 5'-upstream regions of genes. It is not known which regulatory factors are responsible for expression of
P450 genes in pulmonary tissues or whether these factors might be
involved in population susceptibility to lung cancers. However, it has
been shown that many P450 genes, CYP1A1, CYP1B1, CYP2B6, CYP2E1,
CYP2F1, CYP2S1, CYP3A5, and CYP4B1, are transcribed in lung tissues (9-13), and lung tissues activate carcinogens to produce
organ-selective damage (14-17). Despite the extensive knowledge of
chemically induced changes in P450 expression, little is known about
the transcription factors responsible for constitutive or tissue-selective induction of P450 enzymes. Hepatocyte-enriched transcription factors have been the most extensively studied mechanisms of tissue-selective P450 gene expression, whereas mechanisms of pulmonary-selective gene expression have only received minimal attention (2). One elegant example used transgenic mice with the rat
CYP2B1 promoter to drive reporter gene expression in a pulmonary specific manner (18). Additional studies (19) showed that
C/EBP proteins in pulmonary epithelium controlled CYP2B1 gene expression. Other superb studies (20, 21) have demonstrated that
NF1-like factors control nasal-selective expression of
CYP1A2 and CYP2A3.
Although there are no examples of endogenous substrates of CYP2F1, its
expression appears to be under tight transcriptional control that
confines expression predominantly to lung tissues. Therefore, CYP2F1 is
an ideal model to elucidate the mechanism of tissue-selective
transcription of cytochrome P450 enzymes. Insight into the mechanisms
of transcriptional regulation of drug-metabolizing enzymes in lung
tissues should provide relevant information regarding tissue-selective
toxicity, individual susceptibility to lung cancers, and basic
knowledge of constitutive transcriptional mechanisms in lung tissues,
where little is known.
Materials--
Human lung tissue was obtained from a 35-year-old
male Caucasian donor (Intermountain Donor Services, Salt Lake City,
UT). Qiagen plasmid/RNA isolation kits were purchased from Qiagen
(Valencia, CA). Cosmid clones were kindly provided by Dr. Harvey
Morhenweizer, Lawrence Livermore National Laboratories (Livermore, CA).
Human CYP2F1 cDNA in pUC9 (pUC9-2F1) was generously provided by Dr. Frank Gonzalez, NCI, National Institutes of Health (Bethesda). Dr.
Michael Lehmann, Institut fur Genetik der Freien Universitat Berlin
(Berlin, Germany), provided anti-AP4 antibody. The AP4 expression
plasmid was obtained from Dr. Laura Bridgewater, Brigham Young
University (Provo, UT) with permission of Dr. Robert Tjian (University
of California, Berkeley, CA). The expression plasmid for Cell Culture--
BEAS-2B, A549, and HepG2 cells were obtained
from American Type Culture Collection (ATCC, Manassas, VA). BEAS-2B
cells were cultured in the serum-free medium, LHC-9 (Biofluids,
Rockville, MD). For sub-culturing, cells were trypsin-dissociated
and plated onto fibronectin/collagen-coated culture plates (22). A549
cells were maintained with Dulbecco's modified Eagle's
medium/nutrient mixture F12 containing 10% fetal bovine serum. HepG2
were cultured on Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum and 10 mM sodium pyruvate.
Cloning and Sequencing of the CYP2F1 Gene--
Cosmid clones
F15749, R20003, F16767, R28543, and ED7850 (average insert size of 40 kbp) that hybridized to CYP2F1 (23) were cleaved with
HindIII for Southern blot verification of CYP2F1
using a 298-bp EcoRI/KpnI pUC9-2F1 cDNA
fragment as a probe. Positive clones were subsequently sequenced (see
below). In addition, BAC clones were identified by screening the CITB human BAC DNA library (Research Genetics, Huntsville, AL) using "whole cell" PCR (24) with primers designed from the cDNA to amplify exon regions of CYP2F1. The CITB BAC library
represents a 13-17× coverage of the human genome, and BAC clones
contain an average insert size of 100-150 kbp. Positive BAC clones
identified during the library screening and others purchased based on
sequence comparisons with the human genome had plate addresses that
corresponded to the following clones: CITB-HSP-D 2356P16
(GenBankTM accession number AC008962), CITB-HSP-C
490E21 (GenBankTM accession number AC008537), and
CITB-HSP-D 2415I10. All positive clones were sequenced using primers
designed based on putative CYP2F1 exonic sequences. All
sequencing was performed at the University of Utah core sequencing
facility using fluorescent DNA sequencing methods and automated ABI 377 (Applied Biosystems) sequencers.
Sequencing of CYP2F1 cDNA--
The vector pUC9-2F1 (3) was
sequenced to confirm the nucleotide sequence of the CYP2F1 cDNA.
First strand cDNA synthesis for reverse transcriptase-coupled PCR
(RT-PCR) was performed using Superscript II reverse transcriptase
(Invitrogen) and 5 µg of total cellular RNA isolated from confluent
BEAS-2B cells using RNeasy kit (Qiagen) and a QIAshredder microspin
homogenizer according to the manufacturer's recommendations. PCR
amplification utilized primers designed to amplify 1726 bp, which spans
the entire coding region. The primers were 5'-GCA TCC CAG CCA GTG CTC
C-3' (sense) and 5'-GAA AAG GGC GTG CCA TAG AAC AAG-3' (antisense),
designed for selective binding to the CYP2F1 cDNA sequence
(GenBankTM accession number J02906) using the OLIGO
5.0 program (Molecular Biology Insights, Cascade, CO). The PCRs were
performed using 2 µl of cDNA reaction, 2.5 units of
Pfx DNA polymerase (Invitrogen), 5 µl of 10× PCR buffer,
1.5 µl of 50 mM MgCl2, 1 µl of 10 mM dNTP mix, 0.2 µM each primer, and water to
a final volume of 50 µl. PCR conditions were to denature at 94 °C
for 3 min, followed by 30 cycles of melting at 94 °C for 1 min,
annealing at 55 °C for 1 min, extending at 72 °C for 2 min, and a
10-min final extension. The PCR product was cloned using Zero Blunt
TOPO PCR Cloning Kit (Invitrogen) for sequencing. Clones were sequenced
on both strands to verify the sequence of the cDNAs.
Primer Extension Analysis and 5'-RACE--
To identify the
transcription start site (TSS) of CYP2F1, primer extension
analysis and rapid amplification of cDNA ends (5'-RACE) were
performed. Donor human lung tissue was used to isolate total RNA using
an RNeasy miniprep kit (Qiagen). Total RNA (5 µg) was hybridized to a
32P-end-labeled primer (5'-GGC TGT GCT TAT GCT GTC CAT-3')
and extended using Superscript II reverse transcriptase (as above). The
cDNA product was denatured and analyzed by electrophoretic
fractionation on an 8% polyacrylamide gel along with products from a
sequencing reaction generated using the same primer and a 10-bp DNA
standard ladder. The TSS was determined by comparing the size and
sequence of the product with the sequence of the CYP2F1
gene. Human lung Marathon-Ready cDNA (Clontech)
was used for 5'-RACE analysis. Briefly, PCR amplification was performed
using the gene-specific primer (GSP1) 5'-CAG GAG ACA CTG GCT GGG
ATG-3', the nested gene-specific primer (GSP2) 5'-GGC TGT GCT TAT GCT
GTC CAT-3', and an adapter primer (AP1) according to the
manufacturer's recommendations. The 5'-RACE products were cloned into
the pCR2.1 TOPO-TA vector (Invitrogen) for sequencing using the m13
forward and m13 reverse primers. The resulting sequence was compared
with the sequence of the CYP2F1 gene obtained from the BAC
clone (CITB-HSP-D 2356P16), which was the only BAC clone to contain the
full-length CYP2F1 gene. In addition, analysis of the
5'-upstream region was performed using Matinspector version 2.2 and the
TRANSFAC 4.0 matrices to identify putative transcription
regulatory-binding motifs (25).
Transient Transfection Studies--
Luciferase reporter assays
were performed to identify functional promoter regions.
CYP2F1 reporter constructs were produced using PCR
amplification with multiple primers that introduced a
5'-SacI at positions Preparation of Nuclear Extracts--
Human lung nuclear extracts
were prepared using a combination of the protocols described by Ueno
and Gonzalez (26) and Dignam (27). All solutions were at 4 °C
throughout the procedure and contained a 1:1000 dilution of a protease
inhibitor mixture solution (1 mg/ml 4-(2-aminoethyl)benzenesulfonyl
fluoride, 0.5 mg/ml aprotinin, 1 mg/ml leupeptin, 1.2 mg/ml bestatin, 1 mg/ml pepstatin A, 0.5 mg/ml E-64) (Sigma). Ten grams of minced tissue
was suspended in 100 ml of homogenization buffer (10 mM
HEPES, pH 7.6, 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 2 M
sucrose, 10% glycerol) and homogenized using a motor-driven
Teflon-glass homogenizer. Tissue homogenate was layered over four 10-ml
cushions of the same buffer and centrifuged for 30 min at 24,000 rpm
and 4 °C in an SW28 rotor (Beckman Instruments, Fullerton, CA).
After discarding the supernatant fraction, the nuclear pellets were combined, washed with a low salt buffer (20 mM HEPES, pH
7.9, 25% glycerol, 1.5 mM MgCl2, 0.02 M KCl, 0.2 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol),
and collected by centrifugation at 3,300 × g for 10 min. After determining the volume of the nuclear pellet, it was
resuspended in a 1:1 (v/v) ratio with the same low salt buffer. The
nuclear proteins were extracted by addition of an equal volume of the
same buffer, adjusted to 1.2 M KCl. The resulting
suspension was shaken gently on ice for 30 min and centrifuged in a Ti
50 rotor (Beckman Instruments) at 18,000 rpm for 60 min at 4 °C. The
supernatant fraction was dialyzed against 500 volumes of 20 mM HEPES, pH 7.9, 20% glycerol, 0.2 mM EDTA, 100 mM KCl, 0.2 mM phenylmethylsulfonyl
fluoride, and 0.5 mM dithiothreitol for 4 h on ice to
reduce the KCl concentration to 100 mM. After dialysis, the
extract was centrifuged for 20 min at 18,000 rpm to remove precipitated
material. The supernatant fraction was aliquoted into working volumes,
flash-frozen, and stored at DNase Footprinting Assay--
Interactions of 5'
CYP2F1 upstream sequence (position Electrophoretic Mobility Shift Assays (EMSA)--
EMSA was
performed using the gel shift assay system from Promega, essentially as
described by the manufacturer. Binding reaction mixtures were
preincubated at room temperature for 10 min. The mixtures contained 4 µl of nuclear extract (4 µg for lung tissue and 6 µg for cells)
and 2 µl of 5× binding buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 20% glycerol,
0.25 mg/ml poly(dI-dC)·poly(dI-dC)) in a total volume of 10 µl. For
competition experiments, a 100-fold molar excess of unlabeled
double-stranded oligonucleotide was incubated for 15 min with nuclear
extract prior to the addition of 1 µl of 32P-labeled
double-stranded probe (0.005-0.01 pmol). The mixtures were incubated
for another 20 min at room temperature. Before electrophoresis, gel
loading dye (25 mM Tris-HCl, pH 7.5, 0.02% bromphenol
blue, 4% glycerol) was added to all binding reaction mixtures. The
DNA-protein complexes and unbound probes were separated by
electrophoresis using 4% polyacrylamide gels and detected by autoradiography. OCT1, AP1, Sp1, and NF Cloning and Nucleotide Sequence of the Human CYP2F1
Gene--
Library screening and sequencing was used to identify one
full-length CYP2F1 gene and one pseudogene. Sequencing was
performed by walking upstream and downstream from within each exon,
using primers based on the published cDNA sequence
(GenBankTM accession number J02906). Three cosmid
clones, F16767, F15749, R20003, and one BAC clone, CITB-HSP-D 2356P16,
were used to sequence the full-length CYP2F1 gene. Two other
cosmid clones (ED7850 and R28543) and the two BAC clones (CITB-HSP-C 490E21 and CITB-HSP-D 2415I10) all appeared to contain a pseudo CYP2F1 gene, missing exons 1-4, with mutated exons 5-10,
and were not used for further sequencing. Sequencing of
CYP2F1 revealed a disparity from what was originally
identified as the genomic localization of CYP2F1 (23). Two
functional and one pseudo CYP2F1 gene loci were proposed,
but our sequencing identified two genes with identity to the CYP2F1
cDNA, one with an incomplete sequence at the centromeric end of the
CYP2 family gene cluster (middle of intron 4 through exon
10, CYP2F1P), and the full-length sequence at the telomeric
end of the cluster (CYP2F1) in opposite orientations. This
information has subsequently been confirmed by the completion of the
human genome and reconstruction of the CYP2 family gene cluster (30).
The full-length CYP2F1 gene was deduced and found to span
~14 kbp and contain 10 exons (Fig. 1).
The CYP2F1 gene structure, which contains 10 exons and 9 introns, is unique from all genes of the CYP2 family, which
have been shown to contain 9 exons and 8 introns (31). The structure
described here recognizes an additional 5'-untranslated (UTR), exon 1, separated from exon 2 by 1685 bp, which was missing from the
CYP2F1 gene structures reported earlier (3, 32). Earlier
reports concluded that CYP2F1 contained nine exons, but the
results presented here demonstrate that the previously reported exon 1 is actually composed of exons 1 and 2, separated by a 1685-bp intron.
The cDNA sequence contained 56 bp of 5'-UTR sequence spanning exon
1 and the first 11 bp of exon 2. Similar gene structures, containing a
5'-UTR exon 1, were also observed for CYP2F genes in mouse
and rat (NCBI, National Center for Biotechnology Information, genome
resources).
Sequence of the Human CYP2F1 cDNA--
Sequencing of the
CYP2F1 gene revealed several differences from the published
cDNA sequence. Therefore, we sequenced clone pUC9-2F1 (used for
submission of the sequence, GenBankTM accession
number J02906) along with an RT-PCR product that was obtained from
BEAS-2B lung epithelial cells. Comparison of pUC9-2F1 and the sequence
obtained from the RT-PCR product confirmed the sequence obtained from
the genomic clones, indicating that there were several errors in the
original published cDNA sequence. These differences were confirmed
by the "curated RefSeq project" at NCBI with submission of CYP2F1,
accession number NM_0007744 (33). The correct cDNA sequence is
illustrated in Fig. 2A, with the differences in the predicted amino acid sequence illustrated in
Fig. 2B. The variant CYP2F1 sequence identified by Nhamburo et al. (3) was not observed in RNA isolated from either
BEAS-2B cells or human lung tissue. It was originally proposed that the variant was a product of a pseudogene in the CYP2 cluster.
However, the "variant" would appear to be a product of alternate
CYP2F1 splicing, because the incomplete gene at the
centromeric region of the gene cluster does not contain exons 1-4, and
other CYP2F subfamily genes have not been identified.
Identification of the Transcriptional Start Site of
CYP2F1--
Primer extension analysis and 5'-RACE were used to
identify TSSs with mRNA isolated from human lung tissue (Fig.
3) and the BEAS-2B cell line (not shown).
The positions of the primer extension-determined TSSs were revealed by
comparison of the reverse transcriptase reaction product (primer
extension) to a sequencing reaction performed using the same primer and
a radiolabeled 10-bp DNA ladder (L). This analysis identified two
putative TSSs at positions Nucleotide Sequence Analysis of the 5'-Region of CYP2F1--
To
identify possible transcription regulators, the sequence upstream of
the major TSS was analyzed using Matinspector version 2.2 and the
TRANSFAC 4.0 data base (Fig. 4). The
analysis revealed no TATA box element but did identify a
pyrimidine-rich region within the vicinity of the TSS that contained a
putative Sp1 site and a transcription initiator element. Several other
putative cis-acting elements that have been implicated in
regulating pulmonary selective gene expression were identified. These
elements included recognition sequences for the forkhead family of
transcription factors and the hepatonuclear factor family of proteins.
These transcription factors have been shown to control the pulmonary selective expression of surfactant proteins in lung epithelial cells
and other genes during lung development (34). Other sites in this
region included binding sites for the C/EBP proteins, which are known
to regulate differentiation in several tissues and are implicated in
CYP2B1 pulmonary specific expression (19). Overlapping a C/EBP site, a
basic transcription element (BTE) was identified. A BTE site in the
promoter of the carcinogen-metabolizing CYP1A1 gene has been
demonstrated to control the pulmonary specific regulation of
CYP1A1 (35, 36). In addition, several elements that have
been implicated in regulating constitutive gene expression were also
identified (Sp1 and AP2).
Transcriptional Activity of the 5'-Region of CYP2F1 in Lung
Epithelial Cells--
To localize functional cis-acting
element(s) responsible for tissue-specific CYP2F1 promoter
activity, a series of luciferase reporter constructs were designed and
generated for use in transient transfection assays. Two human lung
epithelial cell lines were chosen for analysis: BEAS-2B, an
immortalized bronchial epithelial cell line with low levels and
variable CYP2F1 transcript expression, and A549, an alveolar epithelial
cell line with no detectable CYP2F1 transcripts, as well as HepG2, a
hepatocellular carcinoma cell line which also contained no detectable
CYP2F1 mRNA (data not shown). The CYP2F1 reporter
constructs demonstrated reasonable functional transcriptional activity
in both lung epithelial cell lines but essentially no activity in the
human liver HepG2 cell line, despite relatively high HepG2 transfection
efficiency (>50%) compared with the BEAS-2B cell line (Fig.
5A). Surprisingly, the CYP2F1-directed reporter activity in the BEAS-2B cells that
contain CYP2F1 was slightly lower and more variable than the activity observed in the A549 cells. The cellular variability may have been due
to the low transfection efficiency (<15%) observed with BEAS-2B cells
compared with the relatively higher efficiency (>40%) observed with
the A549 cells. Maximal reporter activity in A549 cells was observed
with the construct containing CYP2F1 position
To demonstrate the functionality of the promoter, without potential
upstream elements, the Identification of Lung-specific Binding Factor(s) by DNase I
Footprinting--
To investigate potential DNA-protein-binding sites
involved in CYP2F1 promoter activity, DNase I footprint
analysis was performed using nuclear extracts from human lung tissues.
Sequential overlapping DNA fragments of 200-400 bp from position
Investigation of the Binding Specificity of Lung Nuclear Extracts
for LSF-binding Motif Using EMSA--
The binding specificity of
protein(s) to a radiolabeled LSF probe (Table
I) was investigated by EMSA analysis
using nuclear extracts isolated from lung tissues. Two DNA-protein
complexes were observed using 4 µg of lung nuclear extract that were
abolished by coincubation with a 100-fold molar excess of unlabeled LSF probe (Fig. 7A). Further
confirmation of the specificity of these DNA-protein complexes was
demonstrated by the inability of a 100-fold molar excess of unlabeled
OCT1 probe to abolish binding. Tissue-specific binding of the 31-bp LSF
radiolabeled probe was demonstrated using 10-12 µg of commercially
available nuclear extracts prepared from human lung, liver, and heart
tissues (Fig. 7B). Specific DNA-protein binding complexes
were only observed in the nuclear extracts prepared from lung tissues,
with essentially no binding to nuclear extracts from human liver or
heart tissues. We also examined the binding affinity of nuclear
extracts from BEAS-2B, A549, and HepG2 (data not shown) cells using 6 µg of nuclear extract. Of the cell lines investigated, only BEAS-2B
nuclear extracts contained proteins that bound to the 31-bp LSF
radiolabeled probe (Fig. 7C). Specific binding was
determined in BEAS-2B cells by including a 100-fold molar excess of
non-radiolabeled specific and nonspecific competitor probes AP1 and
NF Characterization of the LSF-binding Site of CYP2F1--
A series
of mutated oligonucleotide probes (Table I) was used as competitors to
deduce the core sequence of the LSF-binding site by EMSA competition
analysis using human lung and BEAS-2B nuclear extracts. This analysis
allowed us to identify the core LSF-binding site as 5'-CTC CCA
CGG CAC CTT TCC
AGC TGG CTG TGA G-3' (underlines represent nucleotides that
are critical for maximum competition). Similar results were obtained
with both lung and BEAS-2B nuclear extracts, suggesting that the same
protein(s) in human lung tissue and BEAS-2B cells bind to LSF. It is
difficult to infer the number of proteins that bind to this sequence.
However, because the protein/DNA-binding pattern remained unchanged
regardless of the competitor, the LSF-binding motif may recruit a
single protein or protein complex. Interestingly, the CA site of each of the E box element was important for maximum binding, which suggests
that a member of the E box family of transcription factors may bind to
this core sequence. Strong competition was observed with a 20-bp
double-stranded oligonucleotide containing the 12-bp core element and a
minimum 5 bp of 5'- and 3 bp of 3'-flanking sequence (20 bp, Table
I).
The potential of known trans-acting factors to bind to the
LSF motif was investigated with competitive oligonucleotides
representing known AP4, NFAT, and E box-binding elements (Table I). The
mouse MCK E box consensus sequence (37), which has two E box sites separated by 11 bp versus 2 bp for LSF, strongly competed
for protein(s) that bound radiolabeled LSF. Likewise the immunoglobulin
A luciferase reporter construct containing the pGLP-LSF ( CYP2F1 is expressed primarily in lung tissues with little or no
expression in hepatic or other extrahepatic tissues (3, 13, 43). The
expression of CYP2F1 has been implicated in the tissue-specific
toxicity associated with many pulmonary toxicants including styrene,
3-methylindole, naphthalene, and benzene (6, 8, 44). All of these
compounds are environmental and occupational toxicants found in sources
such as cigarette smoke, gasoline, and industrial by-products.
Understanding the transcriptional regulation of cytochromes P450, which
are expressed in tissues that are directly exposed to environmental
toxicants, such as the lung, may help predict the susceptibility of an
individual to acute toxicities or chemically induced cancer. Despite
the importance of pulmonary transcriptional regulation of cytochromes P450, few mechanisms of tissue-specific regulation have been identified (2). Understanding the transcriptional regulation of CYP2F1, which is uniquely expressed in pulmonary tissues, should provide vital
information about organ-selective transcription regulatory mechanisms
and pulmonary specific bioactivation. Therefore, the primary objectives
of the studies described herein were to characterize the
CYP2F1 gene and identify specific regulatory motifs and
trans-activating factors that may be involved in its expression.
To identify the factors that regulate CYP2F1 transcription
in lung cells, it was necessary to clone the gene and sequence its
regulatory region. Sequencing of an amplified cDNA product from
CYP2F1 mRNA revealed several sequence variations from the published
cDNA. It was concluded from sequence analysis that the original
published cDNA had several sequencing errors, which were confirmed
by sequencing the pUC9-2F1 clone obtained from the original authors
(3). The corrected sequence has because been updated in
GenBankTM as accession number NM_000774. This information
may be important for protein modeling studies that depend on correct sequences.
Genomic library screening identified a single BAC clone that contained
the entire CYP2F1 gene, which spans 14 kbp and contains 10 exons, exon 1 encoding a 5'-UTR. Primer extension analysis was used to
identify two major TSSs at positions CYP2F1 tissue-specific expression was demonstrated using
luciferase reporter constructs containing up to To identify protein-binding sites present within the first 1.6 kb of
5'-flanking region of the CYP2F1 gene, we performed DNase I
footprinting assays using nuclear extracts from human lung tissues. By
scanning the 5'-flanking region, we discovered only one strong DNA-binding site at positions Two DNA-protein binding complexes were observed with the LSF motif
using lung nuclear extracts and EMSA analysis in the presence or
absence of specific or nonspecific oligonucleotides. Tissue specificity
was demonstrated by complex formation with lung nuclear extracts but
not liver or heart nuclear extracts. This specificity is consistent
with CYP2F1 lung-specific expression. Nuclear extracts from
BEAS-2B cells also produced two bands. Surprisingly, no complexes were
observed with nuclear extracts from A549 cells despite the observation
that reporter activity was driven by the CYP2F1 promoter. This difference may explain the lack of CYP2F1 mRNA detected in A549 cells in contrast to the low but detectable expression of CYP2F1
mRNA in BEAS-2B cells. No complexes were observed with HepG2 cell
nuclear extract, which is consistent with the lung-specific binding
observed with nuclear extracts from human tissues. We defined the core
consensus sequence of the LSF-binding site as 5'-CCCACGGCACCTTTCCAGCT-3'
(underlined) using mutant oligonucleotide competitors and
EMSA with lung nuclear extracts. Analysis of the protected region
revealed two putative E box sites, separated by 2 bp.
The consensus sequence of the E box sites showed a high degree of
similarity to the binding sites for ATPase 1 Due to the high similarity of the consensus AP4 factor-binding site
within the 5'-upstream region, and despite the lack of competition of
AP4 oligonucleotides with the LSF complex, supershift assays with
anti-AP4 were performed. The anti-AP4 antibody was unable to supershift
the LSF-binding complexes with lung nuclear extracts. However, nuclear
extracts enriched by overexpression of AP4 in the cells produced
DNA/protein bands with different mobilities than those observed from
normal cell extracts, and the new AP4-LSF complexes were supershifted
by anti-AP4. Therefore, AP4 is capable of binding to the LSF-binding
sequence when enriched in nuclear extracts, but this result was
expected, given the high degree of similarity of the AP4 consensus site
within LSF. In fact, recent evidence has shown that the AP4 interacts
with the immunoglobulin These studies provide specific structural characterization of the
unique CYP2F1 gene and identification of a tissue-specific promoter. A novel nuclear factor-binding site, which may regulate the
selective expression of CYP2F1 in human lung tissue, was
also identified. The nuclear protein(s) (LSF) that binds to this domain appears to be a protein uncharacterized previously but may belong to
the E box family of transcription factors. The functional consequence of LSF binding to the CYP2F1 promoter was investigated but not elucidated in these studies. Ongoing studies to characterize the LSF
binding activity include the following: finding an improved cell
culture model, in vitro transcription studies using human lung extracts, and isolation of LSF for identification using mass spectrometry. Characterization of additional regulatory elements of the
CYP2F1 gene should yield insight into the biochemical
mechanisms of P450 gene expression and the tissue-selective expression
of CYP2F1 in human lung.
129 to +115). DNase footprinting analysis of
1.6 kbp of the upstream sequence with nuclear extracts from human lung
tissues revealed one strong DNA-protein complex at
152 to
182. This
nuclear protein (called lung-specific factor, LSF) was present only in
lung but not liver or heart tissues. Competitive electrophoretic
mobility shift assays characterized a DNA consensus site, within the
LSF-binding domain, that was highly similar to two E box motifs, but no
known "E box" trans-factors were identified. These
studies identified a novel LSF and its consensus sequence that may
control tissue-specific expression of CYP2F1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EF1 and the
anti-
EF1 antibody were provided by Dr. Hisato Kondoh, Osaka
University (Osaka, Japan). Expression plasmids for E47 and E12 were
provided by Dr. Cornelis Murre, University of California, San Diego
(La Jolla, CA). Anti-E47 and anti-E12 antibodies were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). The pGL3 luciferase reporter
vectors and dual-luciferase reporter assay system were purchased from
Promega (Madison, WI). Marathon-Ready cDNA and Advantage cDNA
PCR kits were purchased from Clontech (Palo Alto,
CA). Superscript II reverse transcriptase, the TOPO cloning kits, the
double-stranded DNA cycling system, Taq polymerase, cell
culture media, restriction enzymes, and all other molecular biology
reagents were purchased from Invitrogen.
1681,
1468,
1299,
1168,
992,
893,
748,
493, and
129 paired with a single 3'-antisense primer that generated a 3'-BglII restriction site at position +115.
After digestion with SacI/BglII, the fragments
were cloned into the pGL3 basic vector. Reporter constructs, the
pGL3SV40 positive control, and the pGL3 basic negative control vectors
were used for transient transfection studies. To verify the basic
promoter region, the pGLE vector that contains a strong SV40 enhancer
element was used to construct pGLE-129 (
129 to +115). To investigate the functionality of the putative lung-specific transcription factor
(LSF), the pGLP vector that contains the SV40 promoter without any
enhancers was used to construct pGLP-LSF (
182 to
152). Two human
lung epithelial cell lines, BEAS-2B and A549, and one human liver cell
line, HepG2, were transfected with 0.1 µg of the reporter constructs
and 0.001 µg of pRL-SV40 using a 3:1 ratio of FuGENE 6 reagent (Roche
Molecular Biochemical) in 96-well plates. Cells were lysed 36 h
post-transfection, and luciferase activities were assayed using the
dual luciferase assay (Promega). Firefly luciferase activity was
normalized for transfection efficiency using Renilla
luciferase activity (pRL-SV40) and expressed as fold luminescence over
the activity observed with the promoter-less pGL3 basic vector. The
data are presented as mean fold luminescence ± S.E. for three
independent experiments performed in triplicate.
80 °C. Additional human lung, liver,
and heart nuclear extracts were purchased from Geneka Biotechnology
(Montreal, Canada). Nuclear extracts from cultured cells were prepared
as described by Dignam et al. (27) with addition of the
protease mixture solution. Protein concentrations of all nuclear
extracts were calculated using the Bio-Rad Protein Assay Kit I.
1,681 to +115) with
human lung nuclear proteins were identified using the Core Footprinting
System (Promega) with slight modification (20). Binding reaction
mixtures (50 µl), which were preincubated on ice for 10 min,
contained 20-30 µg of human lung nuclear extract in 10 mM Tris-HCl buffer, pH 7.9, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 8% glycerol, 20 mM KCl, 3.5 mM MgCl2, 7 µM ZnSO4, and 32P-end-labeled DNA
fragments (~2 ng, 40,000-50,000 cpm). An equal volume of DNase
reaction buffer (10 mM MgCl2 and 5 mM CaCl2) was added and incubated at room
temperature for 1 min. DNase I was added (0.3 units to probe without
protein and 3 units to probe with protein), and the DNase digestion was
allowed to proceed for 90-120 s at room temperature. The reactions
were terminated by the addition of 100 µl of DNase stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, 100 µg/ml yeast
RNA) provided in the Core Footprinting System. DNA was recovered from
the reaction mixture by phenol/chloroform extraction and ethanol
precipitation. Precipitated DNA was collected by centrifugation, washed
with cold 70% ethanol, air-dried, and resuspended in 10 µl of gel
dye loading buffer (1:2 NaOH/formamide (v/v), 0.1% xylene cyanol,
0.1% bromphenol blue). The DNA samples were denatured at 90 °C for
4 min, quickly placed on ice, and analyzed by electrophoretic
fractionation on a 6% polyacrylamide, 7 M urea DNA
sequencing gel. DNA sequence ladders of the same DNA were prepared by
the method of Maxam and Gilbert (28) and fractionated simultaneously
for identification of the DNA-protein-binding site.
B double-stranded
oligonucleotides were included in the gel shift assay system (Promega).
All other oligonucleotide probes were synthesized by Integrated DNA
Technologies (Coralville, IA). Sequences of the synthesized DNA probes
are listed in Table I. EMSA supershift assays were performed with antibodies to AP4, and the E box factors E47, E12, and
EF1 as described previously (29).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The gene structure of
CYP2F1. Sequencing of BAC clone CITB-HSP-D 2356P16 revealed
a cytochrome P450 gene that spans 14 kbp comprising 10 exons.
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Fig. 2.
The nucleotide sequence of the CYP2F1
cDNA and comparison of the corresponding amino acid sequence to the
published sequence. A, the cDNA sequence of
CYP2F1, determined by sequencing the RT-PCR product obtained from
BEAS-2B cells (see "Experimental Procedures"), is shown with the
translational start and stop sites boldface and
underlined. B, the cDNA sequence was
translated (boldface) and compared with the published amino
acid sequence (GenBankTM accession number J02906) with the
differences between the sequences highlighted in
black.
1781 and
1741 relative to the A of the
ATG start codon. Sequence analysis of five clones containing 5'-RACE
fragments revealed a single TSS corresponding to the
1741 site. Based
on both of these results, we chose to assign the CYP2F1 +1
position to the
1741 site that was identified by both approaches and
also agrees with the reported cDNA sequence
(GenBankTM accession number NM_0007744).
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Fig. 3.
The transcriptional start sites of
CYP2F1. Primer extension methods were used to
identify the TSS of CYP2F1. Human lung total RNA (5 µg)
was hybridized to a 32P-end-labeled primer and extended
using Superscript reverse transcriptase II (see "Experimental
Procedures"). The product (PE) was denatured and
electrophoresed next to a sequencing reaction (T,
G, C, and A) using the same
radiolabeled primer and a 10-bp ladder (L) on an 8%
polyacrylamide gel. The TSS was determined by comparing the size and
sequence of the product with the genomic sequence. Major and minor TSSs
determined from primer extension and 5'-RACE analyses are depicted
(arrows) next to the corresponding sequence from the
CYP2F1 gene.
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Fig. 4.
Possible known transcription
factor-binding elements of CYP2F1. The sequence from 1775
to +125 relative to the major TSS was analyzed by Matinspector version
2.2 using TRANSFAC matrices 4.0. The major TSSs are marked
with right-angled arrows. Exon 1 of the CYP2F1
gene is italicized and underlined. All putative
transcription factor-binding sites are highlighted in
gray and labeled above the sequence. The 31-bp
LSF-binding site is in boldface and underlined.
The
indicates the 5'-most nucleotide sequence positions of the DNA
fragments used for construction of the luciferase reporter constructs.
All constructs had the same 3'-most nucleotide sequence (
).
1681 to +115
sequence directing luciferase expression. However, maximal reporter
activity in BEAS-2B cells was observed with the construct containing
1168 to +115. Removing the sequence +1 to +115 in several constructs
had little to no effect on directing luciferase activity in BEAS-2B
cells (data not shown).
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Fig. 5.
The minimal promoter region of
CYP2F1, identified with progressive deletions of the
5'-flanking region in luciferase reporter constructs.
A, series of heterologous luciferase reporter constructs
were generated by PCR amplification and contained progressive deletions
of the 5'-flanking regions of CYP2F1 as illustrated
(X = position of 5'-most sequence relative to the TSS).
These constructs were transfected into BEAS-2B, A549, and HepG2 cells
using a ratio of 3:1 FuGENE 6 transfection reagent to 0.1 µg of
pGLB(X to +115) reporter construct and 0.001 µg
of pRL-SV40 to normalize for transfection efficiency. Cells were lysed
36 h post-transfection, and luciferase activities were assayed
using the dual luciferase assay from Promega. Firefly luciferase
activity was normalized for transfection efficiency using the control
Renilla luciferase activity and calculated as fold
luminescence over the promoter-less pGLB activity. The data are
presented as fold luminescence ± S.E. for three independent
experiments, each performed in triplicate. Analysis of variance and
post hoc tests (Fisher's protected least significant
difference, p 0.05) for individual experiments
revealed that all reporter construct activities in A549 and BEAS-2B
cells were significantly different from the promoter-less pGLB
activity, and none of the reporter activities in HepG2 cells were
significantly different. B, the promoter region position
129 to +115 was cloned into pGLE, which contains an SV40 enhancer
element separated by vector DNA (saw-tooth line). The
construct, pGLE(
129 to +115), was transfected into BEAS-2B, A549, and
HepG2 cells and assayed for luciferase activity as described above.
Analysis of variance and post hoc tests (Fisher's protected
least significant difference, p
0.05) revealed that
pGLE(
129 to +115) activities in BEAS-2B, A549, and HepG2 cells were
significantly different from the pGLE empty vector activities.
129 to +115 CYP2F1 fragment was cloned into the pGLE vector that contains a strong SV40 enhancer element but no functional promoter (pGLE-129). The transcriptional activity of this construct was assayed in BEAS-2B, A549, and HepG2 cells (Fig. 5B). Significant activity was observed in all
three cell lines, suggesting the necessary promoter elements for basal activity were present within the first 129 bp upstream of the TSS.
Interestingly, the HepG2 cells showed a 2-fold increase in activity,
which suggested that, in the presence of a strong transcriptional signal, the elements present in this minor regulatory region are sufficient to recruit basal transcriptional machinery, even in liver cells.
1681 to +115 were amplified or digested from the largest reporter
construct, and 32P-end-labeled on either the sense or
antisense strands and utilized for DNase I footprinting. Interestingly,
only one strong DNA-protein complex (position
182 to
152) was
observed (Fig. 6A) over the entire region. This result was confirmed when the opposite strand was
similarly analyzed for DNase I protection (Fig. 6B). Only limited amounts (30 µg/reaction) of nuclear extracts were used, due
to the low yields of nuclear protein from human lung tissue preparations. This may explain why only one strongly protected site was
observed. The protected site, which is named LSF for lung-specific factor-binding site,
is 31 bp long and located 152 bp from the TSS. Analysis of the region
using the TRANSFAC data base revealed that the binding site partially
included a potential Sp1 site (89.8% similar) and contained two E
box-binding elements separated by 2 bp (Fig. 6C). The E box
sites scored 94.3% similarity to the human ATPase 1
1 regulatory
element-binding protein 6 DNA-binding site, 93.6% to
EF1, 97.3% to
myogenic differentiation factor, and 95.3% similarity to AP4. Another
site identified, unrelated to E box domains, was NFAT (93.1%).
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Fig. 6.
DNase I footprint analysis identified a 31-bp
region in the proximal promoter of CYP2F1 that bound
LSF. A, 32P-end-labeled DNA probe (position
223 to +117) was incubated with 30 µg of human lung nuclear extract
(P + NE) before treatment with DNase I (see "Experimental
Procedures"). A negative control incubation (P) was
performed in the absence of nuclear extract, and a DNA sequencing
ladder (A + G) was included for identification of the protected
location. The bar shows the region protected by the unknown
protein(s), and numbers indicate nucleotide positions
relative to the CYP2F1 TSS. Arrows indicate DNase
I-hypersensitive sites. B, the opposite strand (position
413 to
106) was end-labeled, incubated with 30 µg of human lung
nuclear extract, then treated with DNase I, and analyzed as above.
C, the sequence surrounding the DNase I-protected region was
analyzed with the TRANSFAC data base. The results of the analysis are
shown, with their relative positions above (+, sense
direction) and below (
, antisense direction) the
LSF-binding motif. The consensus sequence of the known factor is
reported using IUPAC nomenclature (similarity score in
parentheses).
B to the reactions (Fig. 7D).
Oligonucleotides used to identify the core nucleotide sequence of the
LSF-binding site of CYP2F1 utilizing EMSA competition analysis
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Fig. 7.
Specific binding of lung nuclear protein(s)
to the LSF motif. A, EMSA was performed with the 31-bp
LSF (Table I) radiolabeled oligonucleotide probe (P) and
nuclear extract prepared from human lung (NE) tissue as
described under "Experimental Procedures." Competition reactions
were performed with a 100-fold molar excess of unlabeled LSF and OCT1.
The positions of DNA-protein complexes throughout are indicated by
arrows. B, the LSF motif binds specifically to
lung nuclear protein(s). EMSA was performed with the LSF radiolabeled
oligonucleotide probe (P) and commercial nuclear extracts
prepared from human lung (Lu), liver (Li), and
heart (H) tissues. C, EMSA analysis demonstrated
that specific binding was observed with BEAS-2B (BS) but not
A549 (A) lung cell nuclear extracts using the LSF probe.
D, specific binding to the LSF probe (P) was
determined with BEAS-2B cell nuclear extracts (NE) by
including a 100-fold molar excess of unlabeled LSF, AP1, and NF B
probes in the incubations. All competitor probes other than LSF were
obtained using the gel shift system from Promega.
enhancer element (
E2) containing an E box consensus sequence demonstrated strong competition with LSF. Interestingly, competitive oligonucleotides designed from the human MLC1/3 enhancer (MLC) (38) and
the brachyury gene, which both contain E box sites, did not compete
with LSF. The oligonucleotides for the MCK,
E2, and brachyury E box
promoter elements were designed analogously to competition studies that
were used to characterize
-crystallin enhancer-binding protein
(
EF1) (39). In addition, coincubation with the oligonucleotides
containing the NFAT consensus sequence from human aldose reductase
promoter (40) or AP4 consensus sequence from human proenkephalin
promoter (41) did not compete, suggesting that these factors may not be
important. EMSA supershift assays were performed using antibodies to
AP4 and the E box specific factors E47/E12, SIP1, a member of
EF1
family that binds to CACCT motifs (42), and
EF1 (data not shown).
None of the antibodies were able to supershift the LSF complexes with
lung nuclear proteins. However, when nuclear extracts were enriched by
overexpression (cotransfection) of AP4, E47, E12, or
EF1 proteins,
only the AP4-enriched extracts produced a new protein complex, which
was shifted by anti-AP4 (data not shown).
182 to
152) was generated to investigate the function of the LSF-binding site in BEAS-2B and HepG2 cells (data not shown). The reporter activity
of pGLP, in both cell lines, was not affected by introduction of the
LSF-binding motif. This finding was surprising because BEAS-2B cell
extracts appeared to contain the LSF. In addition, cotransfection of
AP4, E47, E12, and
EF1, with pGLP-LSF, had no effect on reporter
activity compared with cotransfection with pGLP alone.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1781 and
1741 relative to the
ATG start codon in exon 2 and several minor sites upstream of the first
exon. However, 5'-RACE only identified the site at position
1741,
which is in agreement with the full-length sequence reported in
NM_000774. As such, this site was designated as +1. The promoter region
of CYP2F1 does not contain a TATA box, but the sequence
proximal to the TSS (+1) contains a putative Sp1 site and
pyrimidine-rich (transcription initiator element) element, possibly
facilitating initiation (45). The promoter also contains potential
binding sites for several other well characterized transcription
factors, such as C/EBP. There is substantial evidence that the CYP2B
enzyme in pulmonary cells is regulated by C/EBP
and C/EBP
(46). A
1.3-kbp sequence from the CYP2B promoter was shown to drive
lung-specific reporter gene expression in a transgenic mice, and it was
shown that C/EBP
and C/EBP
regulate CYP2B pulmonary expression
during differentiation (19). In addition, the promoter contains a BTE
site that has been demonstrated in numerous studies (35, 36, 47) to
regulate CYP1A1 transcription. The BTE site of
CYP1A1 has been recognized to bind a number of transcription
factors from the Sp/XKLF family of factors, which are
involved in both constitutive and tissue-specific regulation (48). The
functional significance of these binding sites was consistent with the
reporter assays that demonstrated promoter activity with the
CYP2F1 fragment, position
129 to +115.
1.6 kbp of
5'-flanking region in two lung epithelial cell lines but not a liver
cell line. When the sequence up to
129 bp was inserted into a vector containing a strong SV40 enhancer element, transcription occurred in
both lung and liver cells indicating that the region indeed contained
the minimal promoter, with the elements necessary for transcriptional
initiation. The minimal promoter also showed tissue specificity for
lung cells. Although the
129 to +115 fragment appears to contain the
minimal promoter, additional experiments are required to identify
the cis-elements responsible for the promoter activity. Thus
the minimal promoter may be shorter than this fragment. The transient
transfection studies demonstrated tissue-specific expression of
CYP2F1 in BEAS-2B cells; however, they were not useful in
demonstrating functionality of the LSF-binding motif, despite that
BEAS-2B cell extracts contained the LSF. An explanation for this result
is that LSF in BEAS-2B cells requires another transcription
factor-binding site or an additional cofactor protein for activity. We
are currently investigating additional basal and tissue-specific
binding motifs within this region.
182 to
152, which we termed LSF. Interestingly, the location of this fragment was upstream of the minimal promoter that drove promoter activity in both lung cell lines.
Additional DNase protection regions undoubtedly exist, which could have
been identified with higher amounts of nuclear proteins than we could
reasonably obtain. DNase I-hypersensitive sites were also observed,
which are strong indicators of protein binding-induced conformation
changes in DNA structure.
1 regulatory element-binding protein 6 (49), SIP1 (Smad-interacting protein 1) (42),
myogenic differentiation factor (37), and
EF1 (39). However, there
was also considerable similarity to NFAT (40) and AP4 (41). Additional
competitive EMSA analyses using oligonucleotides that bind NFAT, AP4,
and E box factors were investigated. Only the oligonucleotides that
contained E box-binding sites from MCK and
E2 (39) competed with LSF
binding, suggesting that LSF may be an E box binding transcription
factor. Although the lack of competitive binding with the other E
box-binding sites may suggest that additional nucleotide interactions,
other than the E box core CA(C/G)CT(T/G), or additional cofactors are
required. It has been shown that two adjacent E box sites are required
for the binding of E box factors, SIP1 and
EF1 (50), with weaker binding observed with 3 versus 24 bp spacing between the E
boxes. Both LSF and MCK have adjacent E box sites, but the spacing is quite different, 2 bp in LSF and 11 bp in MCK, yet the MCK element was
a strong competitor. However, the
E2 oligonucleotide, which contains
only one E box site, also efficiently abolished LSF binding. Thus,
these studies were not informative with respect to the potential need
for adjacent E box sites for LSF binding or the possible spacing
between these sites if they are required. By using competition studies,
we could not conclude whether the established E box factors bind to the
LSF sequence. Therefore, EMSA supershift analysis with antibodies
directed against two major E box binding factors (E47 and E12) were
performed. Neither anti-E47 nor anti-E12 bound to the LSF complex using
lung nuclear extracts or extracts from cells that were enriched with
E47 or E12 proteins. Similarly, supershift assays that were designed to
identify SIP1 and
EF1 showed no reactivity, even with
EF1-enriched extracts.
promoter E box of the E47/E12 type with
higher affinity than E47, suggesting that AP4 is the major ligand for Ig-
promoter E boxes (29). Although AP4 is thought to be a ubiquitous transcription factor, we are conducting additional studies
to compare the specificity of AP4 and other E box-binding factors for
LSF, and to relate multiple factor complexes in the tissue-specific
expression of CYP2F1.
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FOOTNOTES |
---|
* This work was supported by NHLBI Grant HL60143 from the National Institutes of Health.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. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) NM_000774.
¶ To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, 30 S. 2000 E., Rm. 201, University of Utah, Salt Lake City, UT 84112. Tel.: 801-581-7956; Fax: 801-585-3945; E-mail: gyost@pharm.utah.edu.
Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M300319200
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ABBREVIATIONS |
---|
The abbreviations used are:
P450s, cytochrome
P450 proteins;
BAC, bacterial artificial chromosome;
LSF, lung-specific
factor;
C/EBP, CCAAT enhancer-binding protein;
TSS, transcription start
site;
RACE, rapid amplification of cDNA ends;
EMSA, electrophoretic
mobility shift assay;
AP4, activator protein 4;
NFAT, nuclear factor of
activated T-cells;
EF1,
-crystallin enhancer-binding protein 1;
UTR, untranslated region;
BTE, basic transcription element;
SIP1, Smad-interacting protein 1;
MLC, myosin light chain;
MCK, muscle
creatine kinase;
RT-PCR, reverse transcriptase-coupled PCR.
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