EDITORIAL FOCUS
Cytokine-induced changes in chromatin structure and in vivo footprints in the inducible NOS promoter

Jane K. Mellott1, Harry S. Nick2, Michael F. Waters1, Timothy R. Billiar3, David A. Geller3, and Sarah E. Chesrown4

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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)-1beta , tumor necrosis factor (TNF)-alpha , and interferon (IFN)-gamma . 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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-1beta (National Cancer Institute), 500 U/ml of rhTNF-alpha (Genentech), and 250 U/ml of rhIFN-gamma (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 [alpha -32P]dATP and a random-primer DNA labeling kit (Life Technologies). Hybridization and washing were done as previously described (38) followed by autoradiography. The HS site analysis was reproduced in four independent experiments.

In vivo and in vitro DMS treatment. A549 and AKN-1 cells were incubated for 8 h with and without rhIL1-beta , rhTNF-alpha , and rhIFN-gamma 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).

Human genomic DNA previously isolated and purified of proteins was treated in vitro with DMS and served as a control to show all guanine residues in the area of interest. Purified DNA was digested with EcoRI at 37°C for 1 h and purified by organic extraction and ethanol precipitation. DNA was resuspended in 10 µl of sterile double-distilled H2O and 400 µl of 50 mM sodium cacodylate, pH 8.0, and 1 mM EDTA. To this solution, 0.25 µl of DMS was added and incubated for 20 s. The DMS reaction was stopped by the addition of 50 µl of ice-cold stop buffer (1.5 M sodium acetate, pH 7.0, and 100 µg/ml of tRNA) and 750 µl of ice-cold ethanol. Samples were frozen in a dry ice-ethanol bath and precipitated at 13,000 rpm for 30 min at 4°C. The DNA was resuspended in 250 µl of 0.3 M sodium acetate, precipitated, and washed in 95% ethanol. The vacuum-dried pellet was resuspended in double-distilled H2O before piperidine cleavage. DMS-treated DNA was heated to 95°C for 30 min in 200 µl of 1 M piperidine, precipitated as described by Kuo et al. (19a), and resuspended in 100 µl of 1× TE. Piperidine was completely removed by drying in a vacuum concentrator, and the pellets were rediluted to 1 µg/µl in 1× TE (17). All in vivo footprinting data were reproduced in three independent experiments.

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta , IFN-gamma , and TNF-alpha 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-1beta and IFN-gamma 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-gamma 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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Fig. 1.   Northern analysis of cells stimulated with all possible combinations of interleukin (IL)-1beta , tumor necrosis factor (TNF)-alpha , interferon (IFN)-gamma , and lipopolysaccharide (LPS). A549 (A) and AKN-1 (B) cells were grown to confluence and stimulated for 8 h with the inflammatory mediators as indicated. RNA was then harvested and subjected to Northern analysis. iNOS, inducible nitric oxide synthase. +, Presence of the mediator; -, absence of the mediator.

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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Fig. 2.   Chromatin structure analysis of human iNOS. A: restriction fragments and single-copy hybridization probes (regions 1-5) used to map DNase I-hypersensitive (HS) sites are illustrated below a restriction map of the iNOS promoter region. Arrow, transcription start site over the 1st exon. B: A549 cells were grown to confluence and either treated for 8 h with IL-1beta , TNF-alpha , and IFN-gamma or kept as control, unstimulated samples. After induction, the samples were permeabilized with lysophosphatidylcholine and digested with DNase I as described in EXPERIMENTAL PROCEDURES. After purification, the samples were digested with EcoRI and DNase I-HS sites were detected by Southern analysis. Top: analysis of region 1 in the promoter. The hybridization probe was a 500-bp fragment that abutted the EcoRI site at -3.8 kb. Control samples were not induced with cytokines. Bottom: samples that were digested in situ with increasing amounts of DNase I ([DNase I]). Lane 1 in each group received no DNase I digestion, and only the intact restriction fragment hybridized to the probe. *, Position of the inducible DNase I-HS site. C: AKN-1 cells treated as in B. Solid box, position of the constitutive HS site that was present in both control and cytokine-treated cells.

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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Fig. 3.   Chromatin structure analysis of region 3 in A549 (A) and AKN-1 (B) cells. Cell treatment and Southern analysis were performed as described in Fig. 2. The hybridization probe abutted the EcoRI site at -5.8 kb, detecting a 2.0-kb genomic restriction fragment. Two inducible (*) and two constitutive HS sites (solid boxes) mapped to this region in the A549 cells. A similar analysis in AKN-1 cells revealed only 3 constitutive HS sites.

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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Fig. 4.   Chromatin structure analysis of region 5. Cell treatment and Southern analysis were performed as described in Fig. 2. DNase I-HS sites were detected with a probe that hybridized to a sequence 5' to the EcoRI site at -5.8 kb, revealing the EcoRI fragment from -13 to -5.8 kb. A: this analysis revealed 4 cytokine-induced HS sites (*) in the A549 cells. B: in the AKN-1 cells, a similar analysis detected 2 constitutive (solid boxes) and 3 inducible (*) HS sites. C: summary of the chromatin structure analysis in the A549 and AKN-1 cells.

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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Fig. 5.   In vivo dimethyl sulfate (DMS) footprinting: analysis of DNA-protein interactions in region 3. A549 cells were treated with DMS as described in EXPERIMENTAL PROCEDURES. A: primers used for ligation-mediated (LM) PCR analysis of the top strand were 5'-CAAATGATAAAGGTGGG-3', 5'-GACGTGAAGGAAACACAGGGGCGTGG-3', and the common linker primer 5'- GCGGTGACCCGGGAGATCTGAATTC-3'. After LMPCR and denaturing gel electrophoresis, sequence was displayed by hybridization with a strand-specific radiolabeled oligonucleotide probe, 5'-TGCATGTCCCGTGGCGAGTCACG-3'. Lanes 1 and 2 were derived from protein-free DNA modified in vitro with DMS and served as controls to show each guanine residue. Lanes 3-6 were derived from cells modified in vivo with DMS. Control, unstimulated cells; induced, cytokine-treated cells. Right, sequence of the bracketed area. , Enhancements; open circle , protections. The basal footprints mapped within the constitutive DNase I-HS sites in region 3 of both the A549 and AKN-1 cells. Identical in vivo footprinting results were obtained in AKN-1 cells. B: identical in vivo footprint analysis of the bottom strand was performed as described in A by using the LMPCR primers 5'-ATCCTGGAGTGACCACCG-3' and 5'-TCCGTCATCTCCGAGTCAGGCAAGG-3'. The DNA sequence was displayed with the radiolabeled oligonucleotide probe 5'-AAGGTGGTGGAAACCGGGGAAGACC-3'. Right, constitutive in vivo contacts of the bracketed area.

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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Fig. 6.   In vivo DMS footprinting: identification of inducible DNA-protein interactions in region 3. Cells and DNA were prepared as described in Fig. 5. Top strand sequence 5' to the constitutive in vivo DMS footprints in Fig. 5 was analyzed with LMPCR primers 5'-ACAAATTCCAGCAGCTCC-3' and 5'-GTCTTCCCCGGTTTCCACCACCTTGC-3'. The sequence was displayed after hybridization with a radiolabeled oligonucleotide probe, 5'-GCCTGACTCGGAGATGACGG-3'. Right, sequence of the bracketed area. In this case, the enhanced hybridization signals () are seen only in the cytokine-treated samples. These cytokine-inducible footprints mapped near the region of the inducible DNase I-HS sites found in the A549 cells. Identical cytokine-induced enhancements were detected in AKN-1 cells, and interestingly, these inducible contacts were associated with the region of chromatin that was constitutively open in the AKN-1 cells, implying that a different mechanism was maintaining the open chromatin structure in these cells.



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Fig. 7.   Summary of DNase I-HS site (HSS) analysis and in vivo DMS footprint results in region 3. Shown is the sequence from -5,520 to -4,979 kb of the human iNOS promoter, summarizing the cytokine-inducible and constitutive in vivo footprints in A549 and AKN-1 cells relative to the DNase I-HS sites mapped in the A549 cells (HS sites II-IV). , Enhancements; open circle , protections. Arrows, approximate midpoints of the DNase I-HS sites.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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-gamma . Regulation of transcription in A549 cells is more involved, requiring both IFN-gamma and IL-1beta 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-kappa B binding sites within 7 kb of the transcription initiation site. Mutated nuclear factor-kappa 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.


    ACKNOWLEDGEMENTS

This work was supported by grants from the American Lung Association of Florida (to S. E. Chesrown).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Barnes, PJ. Nitric oxide and asthma. Res Immunol 146: 698-702, 1995[ISI][Medline].

2.   Basuyaux, JP, Ferreira E, Stehelin D, and Buttice GJ. The Ets transcription factors interact with each other and with the c-Fos/c-Jun complex via distinct domains in a DNA-dependent and -independent manner. J Biol Chem 272: 26188-26195, 1997[Abstract/Free Full Text].

3.   Blackford, JA, Jr, Antonini JM, Castranova V, and Dey RD. Intratracheal instillation of silica up-regulates inducible nitric oxide synthase gene expression and increases nitric oxide production in alveolar macrophages and neutrophils. Am J Respir Cell Mol Biol 11: 426-431, 1994[Abstract].

4.   Botelho, FM, Edwards DR, and Richards CD. Oncostatin M stimulates c-Fos to bind a transcriptionally responsive AP-1 element within the tissue inhibitor of metalloproteinase-1 promoter. J Biol Chem 273: 5211-5218, 1998[Abstract/Free Full Text].

5.   Charles, IG, Palmer RM, Hickery MS, Bayliss MT, Chubb AP, Hall VS, Moss DW, and Moncada S. Cloning, characterization and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte. Proc Natl Acad Sci USA 90: 11419-11423, 1993[Abstract].

6.   Del Pozo, V, de Arruda-Chaves E, de Andres B, Cardaba B, Lopez-Farre A, Gallardo S, Cortegano I, Vidarte L, Jurado A, Sastre J, Palomino P, and Lahoz CJ. Eosinophils transcribe and translate messenger RNA for inducible nitric oxide synthase. Immunology 158: 859-864, 1997.

7.   De Vera, ME, Shapiro RA, Nussler AK, Mudgett JS, Simmons RL, Morris SM, Jr, Billiar TR, and Geller DA. Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter. Proc Natl Acad Sci USA 93: 1054-1059, 1996[Abstract/Free Full Text].

8.   Evans, CH. Nitric oxide: what role does it play in inflammation and tissue destruction? Agents Actions Suppl 47: 107-116, 1995[Medline].

9.   Fritton, HP, Igo-Kemenes T, Nowock J, Strech-Jurk U, Theisen M, and Sippel AE. Alternative sets of DNase I-hypersensitive sites characterize the various functional states of the chicken lysozyme gene. Nature 311: 163-165, 1984[ISI][Medline].

10.   Fuchs, B, Wagner T, Rossel N, Antoine M, Beug H, and Niessing J. Structure and erythroid cell-restricted expression of a chicken cDNA encoding a novel zinc finger protein of the Cys + His class. Gene 195: 277-284, 1997[ISI][Medline].

11.   Geller, DA, Lowenstein CJ, Shapiro RA, Nussler AK, Di Silvio M, Wang SC, Nakayama DK, Simmons RL, Snyder SH, and Billiar TR. Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc Natl Acad Sci USA 90: 3491-3495, 1993[Abstract].

12.   Gross, DS, and Garrard WT. Nuclease hypersensitive sites in chromatin. Annu Rev Biochem 57: 159-197, 1988[ISI][Medline].

13.   Guo, FH, Comhair SAA, Zheng S, Dweik RA, Eissa NT, Thomassen MJ, Calhoun W, and Erzurum SC. Molecular mechanisms of increased nitric oxide (NO) in asthma: evidence for transcriptional and post-translational regulation of NO synthesis. J Immunol 164: 5970-5980, 2000[Abstract/Free Full Text].

14.   Hamid, Q, Springall DR, Riveros-Moreno V, Chanez P, Howarth P, Redington A, Bousquet J, Godard P, Holgate S, and Polak JM. Induction of nitric oxide synthase in asthma. Lancet 342: 1510-1513, 1993[ISI][Medline].

15.   Hangai, M, Yoshimura N, Hiroi K, Mandai M, and Honda Y. Inducible nitric oxide synthase in retinal ischemia-reperfusion injury. Exp Eye Res 63: 501-509, 1996[ISI][Medline].

16.   Hierholzer, C, Harbrecht B, Menezes JM, Kane J, MacMicking J, Nathan CF, Peitzman AB, Billiar TR, and Tweardy DJ. Essential role of induced nitric oxide in the initiation of the inflammatory response after hemorrhagic shock. J Exp Med 187: 917-928, 1998[Abstract/Free Full Text].

17.   Hornstra, IK, and Yang TP. In vivo footprinting and genomic sequencing by ligation-mediated PCR. Anal Biochem 213: 179-193, 1993[ISI][Medline].

18.   Koda, W, Harada K, Tsuneyama K, Kono N, Sasaki M, Matsui O, and Nakanuma Y. Evidence of the participation of peribiliary mast cells in regulation of the peribiliary vascular plexus along the intrahepatic biliary tree. Lab Invest 80: 1007-1017, 2000[ISI][Medline].

19.   Kroncke, KD, Fehsel K, and Kolb-Bachofen V. Inducible nitric oxide synthase in human diseases. Clin Exp Immunol 113: 147-156, 1998[ISI][Medline].

19a.   Kuo, S, Chesrown SE, Mellott JK, Rogers RJ, Hsu IL, and Nick H. In vivo architecture of the manganese superoxide dismutase promoter. J Biol Chem 274: 3345-3354, 1999[Abstract/Free Full Text].

20.   Laubach, VE, Zhang CX, Russell SW, Murphy WJ, and Sherman PA. Analysis of expression and promoter function of the human inducible nitric oxide synthase gene in DLD-1 cells and monkey hepatocytes. Biochim Biophys Acta 1351: 287-295, 1997[ISI][Medline].

21.   Li, G, Chandler SP, Wolffe AP, and Hall TC. Architectural specificity in chromatin structure at the TATA box in vivo: nucleosome displacement upon beta-phaseolin gene activation. Proc Natl Acad Sci USA 95: 4772-4777, 1998[Abstract/Free Full Text].

22.   Lieber, M, Smith B, Szakal A, Nelson-Rees W, and Todaro G. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer 17: 62-70, 1976[ISI][Medline].

23.   Linn, SC, Morelli PJ, Edry I, Cottongim SE, Szabo C, and Salzman AL. Transcriptional regulation of human inducible nitric oxide synthase gene in an intestinal epithelial cell line. Am J Physiol Gastrointest Liver Physiol 272: G1499-G1508, 1997[Abstract/Free Full Text].

24.   Majano, PL, Garcia-Monzon C, Lopez-Cabrera M, Lara-Pezzi E, Fernandez-Ruiz E, Garcia-Iglesiase C, Borque MJ, and Moreno-Otero R. Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a virus-induced gene upregulation. J Clin Invest 101: 1343-1352, 1998[Abstract/Free Full Text].

25.   Marks-Konczalik, J, Chu SC, and Moss J. Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappaB-binding sites. J Biol Chem 273: 22201-22208, 1998[Abstract/Free Full Text].

26.   Morris, SM, Jr, and Billiar TR. New insights into the regulation of inducible nitric oxide synthesis. Am J Physiol Endocrinol Metab 266: E829-E839, 1994[Abstract/Free Full Text].

27.   Nunokawa, Y, Oikawa S, and Tanaka S. Expression of human inducible nitric oxide synthase is regulated by both promoter and 3'-regions. Biochem Biophys Res Commun 233: 523-526, 1997[ISI][Medline].

28.   Nussler, AK, and Billiar TR. Inflammation, immunoregulation, and inducible nitric oxide synthase. J Leukoc Biol 54: 171-178, 1993[Abstract].

29.   Nussler, AK, Vergani G, Gollin SM, Dorko K, Gansauge S, Morris SM, Jr, Demetris AJ, Nomoto M, Beger HG, and Strom SC. Isolation and characterization of a human hepatic epithelial-like cell line (AKN-1) from a normal liver. In Vitro Cell Dev Biol Anim 35: 190-197, 1999[ISI][Medline].

30.   Nye, JA, Petersen JM, Gunther CV, Jonsen MD, and Graves BJ. Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif. Genes Dev 6: 975-990, 1992[Abstract].

31.   Pfeifer, GP, and Riggs AD. Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR. Genes Dev 5: 1102-1113, 1991[Abstract].

32.   Robbins, RA, Barnes PJ, Springall DR, Warren JB, Kwon OJ, Buttery LD, Wilson AJ, Geller DA, and Polak JM. Expression of inducible nitric oxide in human lung epithelial cells. Biochem Biophys Res Commun 203: 209-218, 1994[ISI][Medline].

33.   Salzman, AL. Nitric oxide in the gut. New Horiz 3: 352-364, 1995[Medline].

34.   Spitsin, SV, Koprowski H, and Michaels FH. Characterization and functional analysis of the human inducible nitric oxide synthase gene promoter. Mol Med 2: 226-235, 1996[ISI][Medline].

35.   Taylor, BS, Alarcon LH, and Billiar TR. Inducible nitric oxide synthase in the liver: regulation and function. Biochemistry (Mosc) 63: 766-781, 1998[ISI][Medline].

36.   Taylor, BS, de Vera ME, Ganster RW, Wang Q, Shapiro RA, Morris SM, Jr, Billiar TR, and Geller DA. Multiple NF-kappaB enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene. J Biol Chem 273: 15148-15156, 1998[Abstract/Free Full Text].

37.   Thomas, RS, Tymms MJ, McKinlay LH, Shannon MF, Seth A, and Kola I. ETS1, NFkappaB and AP1 synergistically transactivate the human GM-CSF promoter. Oncogene 14: 2845-2855, 1997[ISI][Medline].

38.   Visner, GA, Dougall WC, Wilson JM, Burr IA, and Nick HS. Regulation of manganese superoxide dismutase by lipopolysaccharide, interleukin-1, and tumor necrosis factor. Role in the acute inflammatory response. J Biol Chem 265: 2856-2864, 1990[Abstract/Free Full Text].

39.   Worrall, NK, Boasquevisque CH, Misko TP, Sullivan PM, Ferguson TB, Jr, and Patterson GA. Inducible nitric oxide synthase is expressed during experimental acute lung allograft rejection. J Heart Lung Transplant 16: 334-339, 1997[ISI][Medline].

40.   Yun, HY, Dawson VL, and Dawson TM. Nitric oxide in health and disease of the nervous system. Mol Psychiatry 2: 300-310, 1997[ISI][Medline].

41.   Zweidler-Mckay, PA, Grimes HL, Flubacher MM, and Tsichlis PN. Gfi-1 encodes a nuclear zinc finger protein that binds DNA and functions as a transcriptional repressor. Mol Cell Biol 16: 4024-4034, 1996[Abstract].


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