Departments of 1 Biochemistry and Molecular Biology, 2 Neuroscience, and 4 Pediatrics, University of Florida, Gainesville, Florida 32610; and 3 Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Transcription of the human
inducible nitric oxide synthase (iNOS) gene is regulated by
inflammatory cytokines in a tissue-specific manner. To determine
whether differences in cytokine-induced mRNA levels between
pulmonary epithelial cells (A549) and hepatic biliary epithelial
cells (AKN-1) result from different protein or DNA regulatory
mechanisms, we identified cytokine-induced changes in DNase
I-hypersensitive (HS) sites in 13 kb of the iNOS 5'-flanking region.
Data showed both constitutive and inducible HS sites in an overlapping
yet cell type-specific pattern. Using in vivo footprinting and
ligation-mediated PCR to detect potential DNA or protein interactions, we examined one promoter region near 5 kb containing both
constitutive and cytokine-induced HS sites. In both cell types, three
in vivo footprints were present in both control and cytokine-treated
cells, and each mapped within a constitutive HS site. The remaining
footprint appeared only in response to cytokine treatment and mapped to an inducible HS site. These studies, performed on chromatin in situ,
identify a portion of the molecular mechanisms regulating transcription
of the human iNOS gene in both lung- and liver-derived epithelial cells.
nitric oxide synthase; deoxyribonuclease I-hypersensitive sites; inflammatory cytokines; lung epithelial cells; liver epithelial cells
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INTRODUCTION |
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EXPRESSION OF THE HUMAN inducible nitric oxide (NO·) synthase (iNOS) gene is dramatically induced by inflammatory cytokines in many tissues including lung and liver epithelia (11, 13, 32). Production of NO· by iNOS is thereby increased during the inflammatory response, adding to the local defense against invading microbes (8, 28) or potentially contributing to free radical-mediated tissue injury in inflammatory disorders affecting these and other organs (3, 15, 16, 33, 39, 40).
Kroncke et al. (19) recently reviewed evidence of iNOS involvement in human diseases and concluded that the functional role of epithelial iNOS activity is not well understood. In lung epithelium, iNOS expression is dramatically increased in patients with asthma, and nitration of proteins in these cells provides evidence of the functional consequences of increased NO· (13). Because corticosteroid treatment of asthmatic patients leads to clinical improvement and decreased levels of exhaled NO·, it is often speculated that epithelial iNOS activity contributes to the pathogenesis of asthma (1, 13). Within the liver, both hepatocytes and biliary lining epithelium show iNOS expression in response to proinflammatory Th1 cytokines. Although iNOS levels are increased in chronic viral hepatitis (24) and cirrhosis (18), the functional significance is unknown. Hepatic regulation and function of iNOS in the liver was recently reviewed by Taylor et al. (35). Based on pharmacological blockade of iNOS, these authors cite the interesting hepatic cytoprotective effect of iNOS expression in sepsis and ischemia-reperfusion.
Cytokines induce iNOS expression at the level of
transcription, but the specific combination of cytokines leading to a
maximal increase in mRNA levels is different among tissues (7,
13). Tissue-specific regulation of human iNOS may be
important, therefore, in determining its local physiological role and
pathophysiological potential. To begin to define those regions of
iNOS that confer cytokine responsiveness, deletion analyses
with the 5'-flanking sequence have been done in liver, lung, and
colonic epithelial cell lines (7, 20, 23, 27, 34). Results
have shown that compared with the murine iNOS promoter, a
much larger region of the 5'-flanking sequence of human iNOS
is required for maximal cytokine induction. More than 3.8 kb of the
human iNOS 5'-flanking sequence is required for cytokine
induction of reporter genes in both liver and colonic cell lines
(7, 23). In DLD-1 colonic adenoma-derived cells, Linn et
al. (23) showed that sequences between 8.7 and 10.7 kb
upstream from the transcription initiation site were necessary for
cytokine responsiveness and functioned as a classic enhancer. In
contrast, Spitsin et al. (34) reported that sequences
between 0.4 and
1.6 kb supported modest twofold cytokine
responsiveness in the A549 lung epithelial adenocarcinoma cell line,
whereas Nunokawa et al. (27) showed that 3.2 kb of the
iNOS 5'-flanking sequence supported a threefold increase in reporter gene expression in these same cells. These data suggest interesting and important tissue-specific differences in human iNOS regulation at the transcriptional level.
In addition to deletion analysis, large regions of chromatin can be
screened for potential regulatory regions with DNase I-hypersensitive (HS) site analysis. Local changes in chromatin structure accompany the
activation of gene transcription through the binding of
trans-acting protein factors to DNA response elements
(12). To localize regulatory sites within 13 kb of the
5'-flanking region of human iNOS, permeabilized control and
cytokine-treated cells were exposed to increasing concentrations of
DNase I to probe the chromatin structure. In both the A549 human
pulmonary adenocarcinoma cell line and the AKN-1 biliary epithelial
cell line, we compared DNase I-digested chromatin isolated from control
cells and cells treated with a combination of interleukin (IL)-1,
tumor necrosis factor (TNF)-
, and interferon (IFN)-
. The
results identified both unique and overlapping constitutive and
cytokine-induced HS sites in these cell types extending from the
transcriptional start site to
12 kb.
Using the locations of DNase I HS sites as a guide, we performed
dimethyl sulfate (DMS) ligation-mediated (LM) PCR in vivo footprint analysis to identify potential DNA binding sites of regulatory proteins at single-nucleotide resolution. The data identified three constitutive and two cytokine-induced binding sites on
guanine residues between 5 and
5.5 kb in the human iNOS
5'-flanking sequence from both cell types.
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EXPERIMENTAL PROCEDURES |
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Cell culture.
A549 cells, a human pulmonary epithelial-like cell line originally
isolated from a lung adenocarcinoma (22), were cultured in
90% Ham's F-12K medium (Sigma, St. Louis, MO) and 10% fetal bovine
serum supplemented with 10 mM L-glutamine and an
antibiotic-antimycotic solution (Sigma). AKN-1 cells, a hepatic biliary
epithelial cell line isolated by serial dilution from a normal human
liver (7, 29), were cultured in serum-free, chemically
defined medium as previously described (7). To
induce iNOS, eight 10-cm tissue culture dishes of 90%
confluent A549 or AKN-1 cells were incubated in serum-free Ham's F-12K
medium or serum- and dexamethasone-free medium for 8 h in the
presence of 100 U/ml of recombinant human (rh) IL-1 (National Cancer
Institute), 500 U/ml of rhTNF-
(Genentech), and 250 U/ml of
rhIFN-
(Genentech). Control cells were incubated in serum- and
dexamethasone-free medium without the cytokines. RNA isolation and
Northern analysis were performed as previously described
(38).
Cell permeabilization. Control and treated cells were trypsinized and pooled in separate centrifuge tubes and then washed in 30 ml of medium with serum. The cells were pelleted at 1,500 rpm for 5 min at 4°C and then gently resuspended in 10 ml of ice-cold solution A (150 mM sucrose, 80 mM KCl, 35 mM HEPES, pH 7.4, 5 mM K2HPO4, 5 mM MgCl2, and 0.5 mM CaCl2) (31). After recentrifugation, the pellets were resuspended in 4.0 ml of ice-cold solution A. The cells were permeabilized with 4.0 ml of room temperature 0.1% lysophosphatidylcholine (Calbiochem) in solution A, triturated, incubated on ice for ~2 min, and diluted to 40 ml with ice-cold solution A. Permeabilized cells were centrifuged at 1,800 rpm for 5 min at 4°C. The pellets were resuspended in 3 ml of ice-cold solution A and placed on ice before immediate DNase I digestion.
DNase I digestion and genomic DNA isolation. Duplicate sets of tubes containing increasing amounts (range 0-36 U/ml) of DNase I (Worthington Biochemical) were prepared on ice. Three hundred microliters of a permeabilized cell suspension from control or treated samples were added, gently mixed, and incubated for 4 min in a 37°C water bath. The cells were lysed by the addition of 300 µl of freshly prepared DNA lysis buffer (4% SDS, 0.2 M EDTA, and 800 µg/ml of proteinase K). DNase I-digested genomic DNA was purified by sequential organic extraction followed by RNase digestion and ethanol precipitation. Genomic DNA was resuspended to a final concentration of ~1 µg/µl in 1× Tris-EDTA (TE). To ensure that DNase I digestion had occurred, all samples were size fractionated and stained with ethidium bromide. Loss of high molecular weight DNA and the appearance of a continuous gradient of smaller molecular weight DNA was evidence of DNase I cleavage.
Southern analysis. Fifteen micrograms of DNase I-cleaved DNA from control and cytokine-induced cells were digested with an appropriate restriction enzyme and size fractionated on either 0.8% high gelling temperature agarose (FMC) or 2.5% NuSieve 3:1 (FMC) gels overnight at 40 V. The gels were washed, and DNA was electrotransferred to Hybond N+ nylon membranes (Amersham).
Double-strand fragments of ~400 bp were obtained by either restriction digestion of the human iNOS promoter and cDNA constructs or by PCR amplification with gene-specific primers. Probes were verified to be single copy and to hybridize to restriction fragments of the predicted sizes by Southern hybridization to both human genomic DNA and cosmid iNOS constructs. Single-copy probe fragments were radiolabeled by random-hexamer primer extension with [In vivo and in vitro DMS treatment.
A549 and AKN-1 cells were incubated for 8 h with and without
rhIL1-, rhTNF-
, and rhIFN-
as described in Cell
culture. After a wash with room temperature PBS, the cells
were incubated with 20 ml of room temperature PBS containing 0.5% DMS
for 1 min. The DMS reaction was terminated, and the cells were lysed
with 10 ml of DNA lysis buffer (50.0 mM Tris · HCl, pH 8.5, 50 mM NaCl, 25 mM EDTA, pH 8.0, 0.5% SDS, and 300 µg/ml of proteinase
K) and incubated overnight at room temperature. The DMS-treated genomic DNA was purified by organic extraction, RNase digestion, and ethanol precipitation. After purification, DMS-treated DNA was dissolved in
double-distilled water and cleaved with piperidine as described below (17).
Ligation-mediated PCR. Piperidine-cleaved DMS-treated DNA samples and primers were amplified for 1 cycle at 95°C for 3.5 min, 65°C for 30 s, and 76°C for 3 min, then for 19 cycles at 95°C for 30 s, 65°C for 30 s, and 76°C for 3.0 min + 5 s/cycle for each of the 19 cycles, and finally for 1 cycle at 95°C for 30 s, 65°C for 30 s, and 76°C for 10 min. See Figs. 5 and 6 for a description of the respective primers.
To resolve the sequencing ladder, the samples were size fractionated in 5% LongRanger acrylamide (FMC) in 50 mM Tris-borate-EDTA at 90 W. The gel was electrotransferred to Hybond N+ nylon membrane (Amersham), prehybridized at 45°C for 15 min in hybridization buffer (0.5 M sodium phosphate, pH 7.2, 7% SDS, 1% BSA, and 1 mM EDTA) followed by overnight hybridization with a 32P end-labeled gene-specific oligonucleotide at 45°C. A computer analysis of the sequence surrounding the inducible and constitutive footprints was carried out with TFSEARCH (http://pdap1.trc.rwcp.or.jp/research/db/TFSEARCH.html) with all matrices and an 85.0 threshold score and with MatInspector version 2.2 (http://transfac. gbf.de/cgi-bin/matSearch/matsearch.pl) with all matrices, a core similarity of 0.80, and a matrix similarity equal to 0.85. ![]() |
RESULTS |
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Human iNOS is regulated primarily at the level of
transcription, and its mRNA is often undetectable in cells that have
not been stimulated with proinflammatory mediators (26).
In contrast to murine iNOS that is induced by a single cytokine or
lipopolysaccharide (LPS), various combinations of cytokines are
necessary to maximally induce human iNOS mRNA (5, 6,
11). We therefore determined the precise combinations of
cytokines necessary for the induction of human iNOS
steady-state mRNA levels in A549 and AKN-1 cells. Figure
1 shows a Northern analysis of both A549
and AKN-1 cells stimulated with all possible combinations of the
cytokines IL1-, IFN-
, and TNF-
and bacterial LPS. Maximal
induction in A549 cells (Fig. 1A) required treatment with a
combination of all three cytokines, whereas the addition of LPS, a
potent inducing agent of murine iNOS, had no detectable
effect. Both IL-1
and IFN-
were necessary to minimally induce
message levels in the A549 cells. In AKN-1 cells (Fig. 1B),
maximal induction also required the addition of all three cytokines.
However, IFN-
alone was sufficient to induce iNOS
expression in the AKN-1 cells. Because these two epithelial cell lines
have different requirements for cytokines to minimally induce
iNOS steady-state message levels, we hypothesized that
transcriptional regulation of human iNOS may be regulated by
distinct molecular mechanisms in a tissue-specific manner.
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The cytokine-responsive cis-acting elements of the human
iNOS promoter are contained within 16 kb of the start of
transcription (7). To identify potential regulatory
regions within this large 5'-flanking sequence, we analyzed this region
for changes in chromatin structure in control and cytokine-stimulated
cells. DNase I-HS sites may be used to map areas of chromatin
functionally associated with trans-acting factors involved
in the transcriptional regulation of a gene. To digest intact
chromatin, we made cells permeable to DNase I with
lysophosphatidylcholine. Pfeifer and Riggs (31) showed
that cells permeabilized with lysophosphatidylcholine display in vivo
DNase I footprints that are lost in samples from isolated nuclei; thus
this method may preserve DNA-protein interactions that are
disrupted by nuclear isolation methods. Shown in Fig. 2A are the
restriction fragments and hybridization probes used in the chromatin
structure analysis. The region analyzed was ~16 kb and included the
first exon and intron and 13 kb of the 5'-flanking region.
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Figure 2, B and C, shows a representative
chromatin structure analysis of region 1 in A549 and AKN-1
cells, respectively. We defined region 1 by an
EcoRI fragment from 3.8 to +2.3 kb that mediated only
basal transcriptional activity based on promoter deletion analysis in
AKN-1 cells (7). In control A549 samples (Fig.
2B), the probe hybridized only to the large intact
restriction fragment; however, in cytokine-treated samples, an
additional DNase I-cleaved fragment appeard. In contrast, AKN-1 cells
(Fig. 2C) demonstrated one constitutive DNase I-cleaved
fragment in both control and treated cells. In both A549 and AKN-1
cells, this HS site mapped near the transcription initiation site.
Although there was essentially no DNase I digestion of the control
samples (see Fig. 2B), DNase I digestion was documented in
these and all samples by ethidium bromide staining of size-fractionated
DNA before restriction enzyme digestion.
An analysis of region 2, which mapped from 5.8 kb to +110
bp, shows a single DNase I-cleaved fragment in both control and cytokine-treated A549 and AKN-1 cells (data not shown). Because this
DNase I-cleaved fragment migrated just slightly below the intact
restriction fragment on a 0.8% high gelling temperature agarose gel,
we employed a high-resolution analysis on a smaller restriction
fragment contained within region 2 to more clearly observe
the DNase I cleavage pattern in this area. Figure
3 shows the analysis of region
3, defined by an EcoRI fragment that maps from
5.8 to
3.8 kb. These samples were size fractionated on NuSieve 3:1 agarose,
which finely resolves DNA fragments < 2 kb in size. In both
control and cytokine-treated A549 cells (Fig. 3A) digested
with DNase I, two constitutive HS sites as well as two additional
cytokine-specific HS sites were revealed. The chromatin structure of
this region in the AKN-1 cells (Fig. 3B) revealed only three
constitutive HS sites. An analysis of the chromatin structure in
region 4 (defined by a BamHI-EcoRI
fragment that maps from
7.1 to
5.8 kb) revealed no detectable DNase
I-HS sites in either cell type (data not shown).
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Figure 4 is a
chromatin structure analysis of region 5 evaluated with an
EcoRI fragment extending from 13 to
5.8 kb. This fragment contained all but the upstream 3 kb of the 5'-flanking region
shown to have a 10-fold transcriptional induction with cytokines in
AKN-1 cells (7). In control A549 cells digested with DNase
I (Fig. 4A), no HS sites were found; however, on stimulation with cytokines, four inducible DNase I HS sites appeared. In contrast, control AKN-1 cells had two constitutive HS sites (Fig. 4B),
and cytokine-treated AKN-1 cells showed an additional three
cytokine-inducible HS sites.
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Figure 4C is a summary of the chromatin structure changes found in the 5'-flanking region of human iNOS in both control and cytokine-treated A549 and AKN-1 cells. The promoter had constitutive HS sites in both cells lines; however, the induction of iNOS with cytokines was accompanied by dramatic changes in chromatin structure as revealed by the appearance of several inducible HS sites. Although most sites were shared between the two cell lines, each also had unique, tissue-specific sites.
To begin to localize specific DNA binding sites of
trans-acting factors, we used DMS in vivo footprinting with
LMPCR to analyze region 3 in the vicinity of the HS sites.
We chose this region because it contained both constitutive, inducible,
and tissue-specific HS sites and because a genomic DNA sequence was
available. Figure 5A shows in
vivo footprinting on the top strand of constitutive HS sites
II and III (Fig. 3) in A549 cells. Identical results were obtained in AKN-1 cells (data not shown). Enhanced DMS reactivity is seen at two guanine residues at 5,106 and
5,081 bp relative to
the start of transcription. Additionally, two protected guanine residues are located at
5,113 and
5,098 bp. The results of in vivo
footprinting on the bottom strand of this region are seen in Fig.
5B. A cluster of protections is seen at
5,111,
5,109,
5,107,
5,105,
5,104,
5,103,
5,101, and
5,100 bp.
Additionally, an isolated protected guanine at
5,079 bp also shows
decreased DMS reactivity. All of these constitutive in vivo footprints
map to the HS sites II and III depicted in Fig.
3.
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We also analyzed the inducible HS sites IV and V
in region 3 (Fig. 3A) by in vivo footprinting. In
the A549 cells, the chromatin structure in this region contains
cytokine-inducible HS sites IV and V; however,
this region of chromatin is constitutively accessible in the AKN-1
cells (HS site IV). The results of in vivo footprinting on
the top strand in A549 cells revealed enhanced DMS reactivity at
5,502 and
5,490 bp only in the samples prepared from cells treated
with cytokines (Fig. 6). Interestingly,
this cytokine-inducible footprint is seen in a constitutively
accessible region of the chromatin in the AKN-1 cells. DMS footprint
analysis of the bottom strand of this region showed no differential DMS reactivity (data not shown). Figure 7
summarizes the results of in vivo footprinting and the location of the
DNase I-HS sites in region 3.
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DISCUSSION |
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The physiological and pathophysiological roles of NO· can be placed into the three broad categories of neurotransmission, vasodilation, and inflammation, although its specific function in human diseases is incompletely understood (19). NO· produced by iNOS is generated predominantly in settings of inflammation and infection and is elevated in many disease states such as asthma (1, 13, 14) and acute hepatic injury (15, 18, 35). iNOS expression is therefore coordinately upregulated with many other cytokine-induced genes such as manganese superoxide dismutase (38). Human iNOS is tightly regulated at the level of transcription, and multiple cytokines are required for maximal induction of steady-state mRNA levels, potentially implicating a complex array of trans-acting factors that mediate cytokine responsiveness. Adding to the complexity of the regulatory machinery, cells originating from different tissues have dissimilar requirements for cytokines to generate maximal induction, and the same cells from different species respond to dissimilar stimuli. It is perhaps not surprising, therefore, that sequences of human iNOS dispersed over 16 kb, as defined by promoter deletion analysis, confer 10-fold cytokine inducibility to a reporter gene. There is also a lack of cytokine-inducible activity in the proximal promoter as reported by de Vera et al. (7) and Linn et al. (23) in deletion analysis in liver and colonic epithelial cells, respectively. Our chromatin structure analysis of the first 3.8 kb of the 5'-flanking region, which identified a single constitutive HS site located at the transcription start site, supports the lack of cytokine-inducible regulatory elements in the proximal promoter in AKN-1 cells. This HS site is cytokine inducible in the A549 cells, however, and binding of factors to this region may account for the threefold cytokine inducibility reported by Nunokawa et al. (27) in these cells.
Overall, our chromatin structure analysis corroborates the promoter
deletion data reported by de Vera et al. (7). In their analysis, however, the reporter gene construct that contained 7.1 kb of
promoter sequence conferred additional cytokine inducibility compared
with the construct that contained only 5.8 kb. When we examined the sequence between 5.8 and
7.1 kb for
DNase I-HS sites, none were found. To address whether any HS sites
located very close to the ends of the restriction fragment may not have been resolved, we repeated the analysis of this region with a BamHI-HindIII restriction fragment that maps from
7.1 to
5.3 kb and an EcoRI fragment that maps from
5.8
to
13 kb, with the same result. The transcriptional activity found
with sequences out to
7.1 kb may be a consequence of the inability of
a plasmid-based construct to attain a chromatin structure resembling
that of the endogenous gene. Thus the role that chromatin structure
normally plays in transcriptional repression would not have been seen, and this region could be functionally active in this in vitro promoter
deletion assay. An analogous situation was uncovered in the case of the
-phaseolin (phas) gene (21). In
these studies, tobacco plants stably transformed with constructs of the
phas promoter and the lethal diphtheria toxin A-chain
reporter gene developed normally until phas activation in
the heart stage of embryogenesis that resulted in death. In contrast,
similar constructs containing the nonlethal uidA reporter
transiently transfected by bombardment or electroporation showed
abundant transcription. Thus the same promoter sequence when ligated
into the chromatin was regulated in a radically different manner than
when transiently transfected. A similar scenario could explain the
additional cytokine-inducible promoter activity displayed by the region
of the human iNOS promoter that does not demonstrate open
chromatin regions defined by DNase I-HS sites.
When de Vera et al. (7) tested 16 kb of the human
iNOS promoter in a reporter gene construct, they attained a
10-fold induction of reporter gene activity on cytokine treatment of
the AKN-1 cells. When we examined sequences between 5.8 and
13 kb,
we found dramatic changes in the chromatin structure in both the A549
and the AKN-1 cell lines, changes that were reminiscent of those seen
in the chicken lysozyme gene (9). This promoter region in
the AKN-1 cells contained constitutive, cytokine-inducible, and unique
tissue-specific HS sites. This same region of the promoter in A549
cells contained only cytokine-inducible HS sites.
When the results of the chromatin structure data are examined as a
whole, interesting details emerge. Although many of the DNase I-HS
sites map to the same promoter regions in both cell lines, the HS sites
are more likely to be constitutive in the AKN-1 cells and inducible in
the A549 cells. These chromatin structure differences may reflect the
different requirements that the cytokines required to minimally induce
the iNOS steady-state message levels. Many of the HS sites
in the AKN-1 cells are constitutively accessible and thus presumably
already bound by transcription factors, and these cells require only
the single cytokine IFN-. Regulation of transcription in A549 cells
is more involved, requiring both IFN-
and IL-1
to minimally
induce the message. Cytokine induction in these cells is therefore
accompanied by additional changes in the chromatin structure. In
addition, the two cell lines also have unique, tissue-specific HS sites
reflecting the tissue-specific regulation of iNOS.
Promoter deletion studies implicate sequences up to 16 kb upstream from the transcription start site in the cytokine-inducible transcriptional regulation of iNOS (7), whereas our chromatin structure analysis successfully narrowed the promoter regions in which to search for functional elements. Using DNase I-HS sites as a guide, we used in vivo footprinting with LMPCR to examine one functionally relevant area at single-nucleotide resolution for sequence-specific DNA-protein interactions.
Based on the availability of sequence data and the presence of
constitutive, cytokine-inducible, and tissue-specific HS sites, we
chose to analyze the HS sites in region 3 from approximately 5.45 to
4.95 kb. We found a good correlation between the functional role of the promoter region analyzed, the conformation of its chromatin, and the nature of the in vivo footprints. The promoter deletion analysis identified region 3 (downstream from
5.8
kb) as conferring a modest threefold increase in reporter gene activity on stimulation with cytokines in the AKN-1 cells, and the inducible footprints map to the location of the inducible HS sites IV
and V in the A549 cells (Fig. 3). Thus the binding of a
cytokine-inducible trans-acting factor(s) likely causes the
cytokine-inducible DMS footprints. Additionally, the constitutive
footprints map within an area of chromatin that is constitutively
accessible in both cell lines. Interestingly, the inducible footprints
bind within an area of constitutively accessible chromatin in the AKN-1
cells. This may be related to the transcriptional mechanism that allows a single cytokine to minimally stimulate the iNOS
steady-state message levels, whereas the A549 cells require two
cytokines for minimal induction.
A computer analysis of the sequence surrounding the inducible and
constitutive footprints identified numerous putative consensus sequences. Only the growth factor independence (Gfi-1)
transcription factor consensus sequence significantly overlapped the
inducible DMS footprints. However, we have ruled out involvement of
Gfi-1 in the transcriptional regulation of human
iNOS based on its limited tissue distribution in adult
animals (10, 41). Independently, Taylor et al.
(36) have used computer sequence analysis to identify putative nuclear factor-B binding sites within 7 kb of the
transcription initiation site. Mutated nuclear factor-
B sites
evaluated by transient transfection studies implied that specific
elements located within the region we analyzed by in vivo footprinting affected reporter gene activity. Our in vivo footprinting data do not
show any evidence of differential DMS reactivity at these sites in this
region. Therefore, our inducible DMS footprints are likely caused by
the binding of a cytokine-inducible, previously undescribed
sequence-specific DNA binding protein.
Based on the number of nucleotides that separate them, the footprints
located from 5,113 to
5,098 bp define constitutive binding
site I, and the enhancement at
5,081 bp and the protection at
5,079 bp define binding site II. Computer analysis
identified activator protein (AP)-1 and Ets-1 consensus sequences
within two palindromes that span binding site I. The Ets-1
consensus sequence in binding site I contains the invariant GGA, shown
to be critical for DNA binding activity by methylation interference analysis (30). As seen in constitutive binding site
I, not only the invariant GGA but also every G residue in the
putative Ets-1 consensus sequence is protected in vivo.
In addition to the footprints in the Ets-1 consensus sequence, top and bottom strand guanine residues in the AP-1 consensus sequence are also protected, possibly indicating that these residues are also involved in DNA binding of an AP-1-Ets-1 heterotrimer. Another study (2) has shown that the Ets-1 and AP-1 transcription factors can interact to form a heterotrimer. For example, this heterotrimeric complex forms on the granulocyte-macrophage colony-stimulating factor promoter as shown by Thomas et al. (37). The arrangement of the AP-1 and Ets-1 consensus sequences on this promoter is very similar to that seen in binding site I. Specifically, the AP-1 and Ets-1 consensus sequences are in very close proximity with the Ets-1 sequence positioned on the bottom strand. Binding site II also overlaps a palindrome that contains an AP-1 consensus sequence. The footprint pattern in this AP-1 consensus sequence is different from that seen in binding site I. Formation of an AP-1-Ets-1 heterotrimer may affect the interaction of AP-1 with its consensus sequence, thus affecting DMS reactivity of the guanine residues. The only caveat to the potential involvement of AP-1 is its almost exclusive distinction as an inducible factor. Based on recent evidence, however, constitutive AP-1 binding has been shown in some unstimulated cells (4).
Based on computer analysis, Marks-Konczalik et al. (25)
mutated, among others, putative AP-1 sites at 5,115 and
5,301 bp
and found a decrease in reporter gene activity. The AP-1 consensus sequence at
5,115 bp is identical to the AP-1 sequence found in our
footprinted binding site I. However, we found no evidence for binding
of the consensus sequence at
5,301 bp. A potential explanation for
the differences between our in vivo footprinting data and results from
transient transfection studies is the inability of plasmid constructs
to reduplicate the in vivo chromatin structure of the endogenous gene.
In conclusion, we have shown direct evidence that human iNOS is regulated by cytokines in a tissue-specific manner. Furthermore, we have identified DNA-protein interactions in AP-1 and Ets-1 binding sites within a region of the promoter known to play a functional role in the transcriptional regulation of this gene. We have also identified a novel, previously undescribed, cytokine-inducible in vivo DNA footprint.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the American Lung Association of Florida (to S. E. Chesrown).
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. E. Chesrown, Box 100296 HSC, Dept. of Pediatrics, Univ. of Florida, Gainesville, FL 32610 (E-mail: chesrse{at}peds.ufl.edu).
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.
Received 17 February 2000; accepted in final form 29 September 2000.
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