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(The FASEB Journal. 2007;21:535-542.)
© 2007 FASEB

Identification of a classic cytokine-induced enhancer upstream in the human iNOS promoter

Zhong Guo, Lifang Shao, Qiang Du, Kyung Soo Park and David A. Geller1

Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

1 Correspondence: University of Pittsburgh, Kaufmann Medical Bldg., Ste. 300, 3471 Fifth Ave., Pittsburgh, PA 15213-3442, USA. E-mail gellerda{at}upmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human inducible NOS (iNOS) promoter transcriptionally regulated by 5' flanking region extending 16 kb upstream that contains cytokine-responsive DNA motifs. In this study, we further identified a classic inducible enhancer located between –5 and –6 kb in the hiNOS upstream promoter. This 1 kb promoter sequence functions as a cytokine-inducible enhancer in an orientation- and position-independent manner in human lung A549 and liver AKN1 cells. This DNA enhancer also confers cytokine inducibility to the heterologous thymidine kinase (TK) promoter. Chromatin immunoprecipitation (ChIP) analysis was applied, and confirmed cytokine-inducible in vivo DNA-protein interactions within this enhancer region. In vivo functional binding of both NF-{kappa}B (p65/p50) and Stat-1{alpha} at the –5.8 kb human iNOS promoter site was significantly increased in A549 cells after cytokine stimulation, while only Stat-1{alpha} bound at the –5.2 kb site. These results identify the –5 to –6 kb promoter region as a classic transcriptional enhancer for the human iNOS gene and provide definitive in vivo evidence of specific NF-{kappa}B and Stat-1 nuclear protein binding that mediates transcription of the hiNOS gene under cytokine stimulation.—Guo, Z., Shao, L., Du, Q., Park, K. S., Geller, D. A. Identification of a classic cytokine-induced enhancer upstream in the human iNOS promoter.


Key Words: NO • ChIP


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NO IS PRODUCED AT HIGH LEVELS by iNOS during hemorrhagic, septic, and inflammatory conditions. It can result in either protective or damaging effects in clinical diseases depending on the physiological or pathophysiological state (1 , 2) . The iNOS gene is typically not expressed in normal resting cells but activated by various infectious agents, microbial products, or cytokines. The molecular mechanism of iNOS gene is complex and is believed to be mainly regulated at the transcriptional level (3 , 4) .

Expression of iNOS was initially described in murine macrophages (5) , and the murine iNOS cDNA was subsequently cloned from lipopolysaccharide (LPS) and IFN-{gamma}-stimulated macrophages (6 7 8) . Human iNOS expression in primary human hepatocytes was originally identified by stimulating with a cytokine mixture (CM) of TNF-{alpha}, IL-1ß, IFN-{gamma}, and LPS (9) . Subsequently, the human iNOS (hiNOS) cDNA was cloned from LPS and cytokine-stimulated primary human hepatocytes (10) . This cloned gene reveals a 4145 bp cDNA, containing a 3459 bp open reading frame that encodes 1153 amino acids with a 131 kDa molecular mass. Overall, human iNOS shares 80% homology to murine iNOS at the amino acid level. The genomic cloning of human iNOS clarified that the completed human iNOS gene consists of 26 exons and 25 introns spanning 37 kb of genomic region and specifically mapped to chromosome 17 at position 17 cen-q11.2 (11) .

The induction of iNOS gene may vary in different species or different cells. iNOS expression in murine cells is induced by bacterial LPS (lipopolysaccharide) and inflammatory cytokines such as IFN-{gamma}, IL-1ß, IL-6, TNF-{alpha}, or other compounds. In contrast, the majority of human cells require a complex cytokine combination that includes IFN-{gamma}, IL-1ß, and TNF-{alpha} for iNOS induction (4 , 12) . Interleukin (IL)-1ß alone at high doses can induce iNOS mRNA in cultures of primary human hepatocytes (13) and chondrocytes (14) . IFN-{gamma} alone can induce only a low level of iNOS mRNA expression. However, either TNF-{alpha} or IL-1ß can synergize with IFN-{gamma} for the increased iNOS expression, whereas CM is the strongest inducing combination (15) .

The predominant mode of regulation for iNOS expression had been shown at the transcriptional stage (15 16 17) , although post-transcriptional mechanisms are also active (18) . Previously, we isolated and sequenced the 5'-flanking promoter region of the hiNOS gene and demonstrated that regions as far as –16 kb upstream were required for maximal cytokine-inducibility (15 , 19) . In human AKN-1, A549, or DLD1 cells, only human iNOS promoter fragments larger than –3.8 kb showed any significant induction with cytokines (15 , 19 20 21 22) . We have further shown that TNF-{alpha} or IL-1ß signal through NF-{kappa}B binding to –5.5, –5.8, and –6.1 kb cis-acting DNA elements, while IFN-{gamma} signals through Stat-1 by binding to motifs at –5.2 and –5.8 kb in the hiNOS promoter (19 , 23 , 24) . Another group showed that cytokine-responsiveness required 5'-flanking DNA regions extending to –8 kb and demonstrated CM-inducible activating protein (AP)-1 binding sites at –5.1 and –5.3 as well as a functional role for a NF-{kappa}B element located at –8.2 kb in the human iNOS promoter (25) . Interestingly, NF-{kappa}B repressing factor (NRF) protein binding to a novel negative response element (NRE) at –6.7 kb in the hiNOS promoter was recognized to mediate constitutive silencing of hiNOS transcription (26) .

In addition to gel shift assays, promoter transfections, and mutagenesis experiments, several studies have also shown functional regulators of the upstream hiNOS promoter in vivo. By analyzing cytokine-induced DNaseI hypersensitive sites (DHS) in human iNOS 5'-flanking region in A549 lung and AKN 1 liver cells, constitutive and inducible DHS were identified in an overlapping but cell type-specific pattern (27) . One major cluster of DHS was found around –5 kb in the hiNOS promoter region. Another DHS corresponded to a downstream AABS response element that exhibited a dual role in regulating hiNOS gene expression (28) . The AABS site mediated basal hiNOS transcription in a tissue-specific pattern in liver cells, and it also functioned as a "switch point" for cytokine inducibility where C/EBPß (LAP) binding to an intact AABS was necessary, but not sufficient, for inducible promoter activity (28) . ChIP assay showed that both NF-{kappa}B and STAT-1 bound to the hiNOS promoter region spanning from –5.2 to –5.8 kb (24) . Additional chromatin-based mechanisms in the control of hiNOS gene transcription include DNA methylation of CpG dinucleotides (29) . Recent work has also identified ß-catenin/TCF4 binding in vivo to two TBE motifs at –3.8 and –6.1 kb in the hiNOS promoter (30) .

While the studies cited above have identified several cytokine-inducible cis-acting DNA motifs that regulate hiNOS transcription, none of the studies have addressed whether these promoter elements function as an inducible "classical enhancer", nor have they shown in vivo protein-DNA binding to a classical enhancer region. Herein, we identified an important cytokine-inducible region located at –5 to –6 kb in the hiNOS promoter that acts as a classic enhancer for hiNOS transcription in a manner independent of its position or orientation. Furthermore, we show specific in vivo binding of NF-{kappa}B and Stat1{alpha} proteins at –5.8 and –5.2 kb sites in this enhancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Human recombinant TNF-{alpha} and IFN-{gamma} were obtained from R&D Systems (Minneapolis, MN, USA), and IL-1ß was kindly provided by Craig Reynolds of the National Cancer Institute (Bethesda, MD, USA). LipofectAMINE was purchased from GIBCO/Life Technologies, Inc. (Gaithersburg, MD, USA) Antibodies used in ChIP assay were obtained from Santa Cruz (Santa Cruz, CA, USA). All other reagents were obtained from Sigma (St. Louis, MO, USA).

Plasmids
The human iNOS promoter reporter plasmid vector piNOS (7.2)luc contains –7.2 kb of upstream 5'-flanking DNA linked to the luciferase reporter gene and has been described previously (15 , 31) . The fragment of –6 to –5 kb hiNOS promoter was excised by using Eco47III and Pmel enzymes and subcloned back in the construct in different location and/or in different orientation. The regions between –7.2 to –5.8, –5.8 to –3.8, and –6 to –5 kb or three copies of either NF-{kappa}B/ Stat 1 motif at –5.8 kb or Stat 1 motif at –5.2 kb were inserted into pTK-luc, a plasmid carrying a minimal promoter fragment of the herpes thymidine kinase gene with luciferase reporter gene to create different heterologous constructs in either orientations. Confirmation of the recombinant constructs was accomplished with DNA sequencing analysis by the University of Pittsburgh Sequencing Facility.

Cell culture
The A549 human lung cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in medium as recommended. AKN-1 human liver cell line had been described (32) . AKN-1 liver cells were grown in Dulbecco’s modified Eagle medium (DMEM) with 10% low endotoxin CS (GIBCO/Life Technologies, Inc.), Penicillin (100 U/ml), streptomycin (100 µg/ml), and HEPES (15 mM, pH 7.4). Cells were plated onto 6-well cell culture plates (Corning Co., Corning, NY, USA) and stimulated with a cytokine mixture (CM) of TNF-{alpha} (1000 U/ml) + IL-1ß(100 U/ml) + IFN-{gamma}(250 U/ml) for human cells.

Transfections and reporter gene assays
DNA transfections of cells were performed in 6-well plates (Corning) using lipofectamine for human cells. Briefly, cells were exposed to serum-free medium containing 1 µg DNA of different iNOS promoter constructs with 10 µg of liposomes for 4 h, washed, and replenished with medium supplemented with 5% calf serum. To control for transfection efficiency between groups, 0.5 µg of a plasmid containing a cytomegalovirus (CMV) promoter-driven ß-galactosidase gene (pIEP-Lacz) was added to each well. Cells were lysed with reporter lysis buffer (Promega, Madison, WI, USA) or buffer containing 1% Triton X-100, 5 mM dithiotreitol, 50% glycerol, 10 mM EDTA, and 125 mM Tris-phosphate (pH 7.8). Luciferase activity was assayed with 20 µl of lysate in a Berthold (Nashua, NH, USA) AutoLumat LB 953 luminometer using a commercially available kit (Promega). ß-galactosidase activity was determined as recommended (Promega), using a 96-well multiplate reader with SOFTMAX software (Molecular Devices, Sunnyvale, CA, USA). Luciferase activity was normalized to ß-galactosidase activity.

Chromatin immunoprecipitation (ChIP) assay
A549 cells or AKN1 cells (10x106) are divided into two groups. One group is untreated cytokines as control, and the other was treated with CM for 2 h. The ChIP assay is performed following the recommendations of Upstate Biotechnology, Inc. (Lake Placid, NY, USA). Briefly, formaldehyde is added the culture medium at a final concentration of 1% to freeze the DNA-protein and protein-protein interactions. Cells are washed twice with ice-cold PBS, resuspended in cell lysis buffer (5 mM Pipes, pH 8.0; 85 mM KCl; and 0.5% Nonidet P-40) containing 0.5 mM PMSF and kept on ice for 15 min. Then cell lysates are sonicated on ice until the cross-linked chromatins are sheared to yield DNA fragments between 200 bp and 1 kb. The supernatants are diluted 10 times with ChIP dilution buffer (0.01% SDS; 1.1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris–HCl, pH 8.0; and 167 mM NaCl). Salmon sperm DNA/protein agarose-50% slurry is added to the diluted cell supernatant to reduce nonspecific background. Half supernatants are incubated overnight at 4°C with anti- NF-{kappa}B or Stat 1 antibody (Ab), while the other half is served as negative control without Ab incubation. These supernatants are added 5M NaCl and heated at 65°C to reverse histone-DNA crosslinks. The immunocomplexes are further treated with DNase- and RNase-free proteinase K, and DNA is purified using a DNA purification kit (Qiagen Inc., Santa Clara, CA, USA). Polymerase chain reaction (PCR) is performed with specific primers for 28 cycles. Amplified DNA fragments are analyzed on a 1% agarose gel by electrophoresis. The sequences of primers are listed as followed: p1 primer: 5'-GAGGGCTTTCCCAGAACCAAG-3';p2 primer: 5'-GCTGGGCTACTGACCCAGCAGTTCCAG-3'; p3 primer: 5'-TTGAGTCACACTCCAGG-3'; p4 primer: 5'-TTACTGGAAACGCCCGG-3'.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chromatin structure analysis of the human iNOS (hiNOS) promoter with major functional binding motifs
Computational analysis of the –16 kb human iNOS promoter for relevant transcription factor binding sites reveals numerous potential regulatory motifs, however only a handful of these elements have been shown to exert a functional role in regulating hiNOS transcription (Fig. 1 ). Gene transcription is associated with changes (or unfolding) of chromatin structure, which allows transcription factors to bind to specific cis DNA elements to regulate gene expression (33) . By using DNaseI digestion, certain regions of chromatin involving DNA-protein interactions can be cleaved and exposed their in vivo footprints (34) . These DNA sequences are defined as the DNaseI hypersensitive sites, which are highly correlated with promoter enhancers, silencers, insulators, and local control regions (34 , 35) . Mapping of HS serves as a powerful guide to identify the functional transcriptional motifs in gene regulation (36) . Mellott et al. performed a chromatin structure analysis utilizing both in vivo footprinting and DNaseI hypersensitive site (DHS) mapping for the hiNOS gene (27) . A DHS cluster around –5 Kb was observed in the hiNOS promoter and also correlated well with several critical NF-{kappa}B, Stat1{alpha}, and AP-1 binding sites in that region (Fig. 1) (19 , 23 , 25) . Notably, most of the inducible hypersensitive sites were beyond –5 kb upstream in the hiNOS promoter. Therefore, we focused on the region between –5 and –6 kb to determine if it functioned as a "classical enhancer" mediating cytokine-inducibility.


Figure 1
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  		Figure 1. Structure of 16 kb human iNOS promoter. Unique restriction enzymes and DNaseI footprints({diamondsuit}) were indicated at the positions in the promoter. Functional motifs of major transcriptional factors were mapped in the detailed location.

The –5 to –6 kb region of the hiNOS promoter functions as a classical enhancer in an orientation- and position-independent manner
To determine whether the fragment located at –5 to –6 kb for hiNOS promoter could function as a classic enhancer, ~1 kb Eco47III-Pmel DNA fragment (–6006 to –4956 bp), was deleted from the –7.2 kb wild-type (WT) hiNOS promoter luciferase construct [piNOS (6–5)-D] and reinserted to establish position- and orientation-independence (Fig. 2 A). Specifically, the ~1 kb fragment (–5 to –6 kb region) was subcloned back into the –7.2 kb deletional hiNOS promoter in reverse orientation to create the piNOS (5–6)-O construct to test for orientation-independence, or inserted in the far upstream position at the BamHI cloning site in either direction [piNOS (6–5)-P or piNOS (5–6)-OP] to test for position-independence (Fig. 2A ).


Figure 2
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  		Figure 2. Deletion, orientation, and position effect of –6 to –5 enhancer on the endogenous hiNOS promoter activity and its cytokine inducibility. A) A series of constructs containing the hiNOS promoter fragments with different size, orientation, or position in the context of endogenous hiNOS promoter. The arrow sign indicates the orientation and position of the –5 to –6 kb enhancer as well as direction for the WT human iNOS promoter. The –7.2 kb WT human iNOS promoter construct and its constructs with the deleted, inverted, or positioned enhancer were transfected into A549 cells (B) and AKN 1 cells (C). Basal and stimulated luciferase activity was determined 6 h after cytokine stimulation. Relative luciferase activity (RLA) were normalized to the cotransfected ß-galactosidase as an internal standard. Values shown are the means ± SD of at least three separate experiments performed in triplicate. The cytokine inducibility is referred as the relative ratio of the CM stimulated and basal luciferase activities. White bars represent the expression of luciferase reporter gene in untreated cells as control; gray bars represent the expression of luciferase reporter gene with cytokine mixture treatment. *Indicates P < 0.05 vs. control.

AKN-1 liver cells and A549 lung cells were transiently transfected with these hiNOS promoter constructs. Promoter activities were measured as relative luciferase activities (RLA) in the lysed cells and normalized with ß-gal cotransfection. As we have previously shown, the cytokine mixture of TNF-{alpha} + IL-1ß + IFN-{gamma} induced a 4.3-fold induction in luciferase activity with the –7.2 kb WT hiNOS promoter construct [piNOS (6–5)] in A549 cells (Fig. 2B ). Deletion of the ~1 kb (–5 to –6 kb) fragment abrogated all cytokine-inducibility when the piNOS (6–5)-D construct was transfected in the A549 cells. Reversing the direction (orientation) of the ~1 kb fragment [piNOS (5–6)-O] still maintained a >4-fold cytokine inducibility when transfected in the A549 cells. Moving the ~1 kb fragment to a far upstream position in either orientation [piNOS (6–5)-P or piNOS (5–6)-OP] also maintained significant cytokine-inducibility (Fig. 2B ). Relatively similar results were also observed in the AKN1 cells (Fig. 2C ). Although the reverse orientation of the ~1 kb enhancer exhibited slightly higher inducible luciferase activity, the differences were not significant. These results indicate that –6 to –5 kb fragment functions as a classical inducible enhancer for hiNOS gene transcription in an orientation- and position-independent manner.

The ~1 kb enhancer confers cytokine-inducibility to a heterologous minimal TK promoter
To validate that the ~1 kb hiNOS enhancer contains active cytokine-stimulated DNA elements, we examined for the ability of this enhancer to confer cytokine-inducibility to a minimal HERPES thymidine kinase (TK) luciferase promoter construct. The ~1 kb enhancer fragment was subcloned in both directions in front of the TK promoter and compared to overlapping WT hiNOS promoter regions (–3.8 to –5.8 kb) and (–5.8 to –7.2 kb), which overlapped with the ~1 kb enhancer and were also ligated in front of the TK promoter in either orientation (Fig. 3 A).


Figure 3
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  		Figure 3. Functional analysis of a serial of hiNOS promoter fragments ligated into a heterologous thymidine kinase (TK) promoter. A) A series of constructs containing the hiNOS promoter fragments with different size, orientation, or position in the context of minimal heterologous tk promoter. Various promoter constructs of different sized promoter fragments in either orientation were transfected into A549 cells (B) and AKN 1cells (C). Basal and stimulated luciferase activity was determined 6 h after cytokine stimulation. White bars represent the expression of luciferase reporter gene in untreated cells as control; gray bars represent the expression of luciferase reporter gene with cytokine mixture treatment. Relative luciferase activity (RLA) was normalized to cotransfected ß-galactosidase as an internal standard. Values shown are the means ± SD of at least three separate experiments performed in triplicate. The cytokine inducibility is referred as the relative ratio of the CM stimulated and basal luciferase activities. * Indicates P < 0.05 vs. control.

As shown in Fig. 3B , the ~1 kb hiNOS enhancer could confer a 10.8-fold cytokine inducibility on the minimal heterologous TK promoter in A549 cells [pTK (6–5)]. Reversing the direction of this enhancer still conferred 9.1-fold cytokine inducibility [pTK (5–6)]. Using the longer fragment of –5.8 to –3.8 kb, which included most of –5 to –6 kb enhancer, still gave 3- to 4-fold in either orientation. But, the –7.2 to –5.8 kb fragment of hiNOS promoter did not elicit any cytokine inducibility in A549 cells. Similar results were also observed in AKN1 cells, but with relatively lower inducibilities (Fig. 3C ). The fragments in –6 to –5 kb orientation yielded 2.6-fold cytokine inducibility, whereas the same fragment in reverse order still had 2.5-fold cytokine inducibility. The larger fragment of –5.8 to –3.8 kb still retained the cytokine induction in either direction. Interestingly, the fragments of –7.2 to –5.8 kb in either orientation could produce low level cytokine induction only in AKN 1 cells, suggesting this region might have cell type-specific cytokine-responsive elements. Thus, these results further proved that the –6 to –5 enhancer can mediate the cytokine inducibility in the heterologous promoter but in a relatively cell type-dependent manner.

NF-{kappa}B and Stat 1 interact in vivo with human iNOS chromatin at –5.8 kb and –5.2 kb
Previously, we have characterized the NF-{kappa}B binding motif at –5.8 kb and two Stat-1 sites at –5.2 kb and –5.8 kb, which are essential for cytokine-induced hiNOS transcriptional activation by using site-directed mutagenesis and in vitro gel shift assay. In this study, ChIP assay was further applied to detect in vivo functional DNA-protein interactions for NF-{kappa}B and Stat-1 in the A549 cells. We designed human iNOS-specific PCR primers spanning the –5.8 and –5.2 kb sites (Fig. 4 A). Using ChIP assay, in vivo NF-{kappa}B and Stat 1 binding at the –5.8 kb site were significantly increased under cytokine stimulation, compared with the control (unstimulated) group (Fig. 4B ). No observable DNA binding was found in IgG immunoprecipitation as background control. In comparison, in vivo Stat 1 binding was slightly increased while NF-{kappa}B binding was not detectable at –5.2 kb site in A549 cells. These results provide definitive evidence for in vivo NF-{kappa}B DNA and Stat 1 protein-DNA binding in the hiNOS promoter enhancer region. To further clarify which NF-{kappa}B proteins were binding in vivo at the –5.8 kb promoter site, specific antibodies against p50 or p65 were used in the ChIP experiments. Both p50 and p65 protein binding were detected at rest and were enhanced by cytokine stimulation. (Fig. 4C ). These results corroborate our previous in vitro findings and further support a role for cytokine-induced transcriptional regulation of the hiNOS promoter at this enhancer region.


Figure 4
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  		Figure 4. A) Schematic diagram of detailed positions and sequences for two sets of primers used in ChIP analysis. B) NF-{kappa}B and Stat 1 in vivo interacting with human iNOS chromatin structure. Chromatin immunoprecipitation (ChIP) analysis for NF-{kappa}B and Stat-1 in vivo bindings at –5.8 and –5.2 kb sites of the human iNOS promoter in A549 cells. C) ChIP analysis for specific NF-{kappa}B dimer (p50 or p65) bindings at –5.8 kb site of the human iNOS promoter.

NF-{kappa}B/Stat 1 motifs at –5.8 and –5.2 kb enhanced cytokine-inducible promoter activity on a minimal heterologous promoter
The results in Fig. 3 show that the –6 to –5 kb enhancer region can confer cytokine inducibility to a minimal heterologous promoter. Next, within this 1 kb enhancer, we sought to confirm the binding localization of the specific DNA motifs at –5.8 and –5.2 kb. Therefore, to test the functionality of the NF-{kappa}B/Stat 1 motifs at –5.8 and –5.2 kb in the hiNOS promoter as independent transcriptional regulators, we developed a reporter gene construct that contained three copies of either the NF-{kappa}B/Stat 1 motif at –5.8 kb or the Stat 1 motif at –5.2 kb. A triplet copy of the motifs was ligated in front of the minimal herpes Thymidine kinase (TK) promoter driving the luciferase reporter gene (Fig. 5 ). The minimal TK-luc construct served as control. Transfection experiments in the A549 cells showed that three copies of NF-{kappa}B/Stat 1 motif at –5.8 kb dramatically increased CM-induced reporter gene activity 6.9-fold. Three copies the Stat 1 motif at –5.2 kb also produced 4.3-fold inducibility under CM stimulation (Fig. 5) . Experiments were repeated, and similar results were obtained in the AKN 1 cells (data not shown). These results indicate that NF-{kappa}B and Stat 1 motifs at –5.8/–5.2 kb in the hiNOS promoter can contribute to cytokine-inducible transcriptional activity that is not limited to the endogenous hiNOS promoter.


Figure 5
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  		Figure 5. NF-{kappa}B and Stat 1 motifs at –5.8 and –5.2 kb confer cytokine-inducible transcription activation to a heterologous thymidine kinase (TK) promoter. A549 cells were transiently transfected with 1 µg of a luciferase reporter gene construct driven by three copies of NF-{kappa}B/Stat 1 motifs at –5.8 or –5.2 kb hiNOS promoter.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We previously reported that the promoter regions required for cytokine-induced hiNOS transcription were located far upstream from –4.7 kb in the human iNOS promoter. Several functionally important NF{kappa}B, Stat-1, and AP-1 elements have been identified by gel shift assay and site-directed mutagenesis. However, essentially nothing is known about cytokine-inducible functional enhancers governing hiNOS transcription. Furthermore, functional DNA-protein interaction localized to a cytokine-responsive enhancer has not been investigated. The major and novel findings of this study are the following: i) the region from –6 to –5 kb in the hiNOS promoter functions as a classic enhancer that confers cytokine inducibility for hiNOS transcription in a position- and orientation-independent manner; ii) the enhancer exhibits the cytokine-induced transcriptional activity in a relatively cell type-specific pattern; iii) in vivo functional bindings for NF-{kappa}B and Stat-1 within this enhancer region are shown by ChIP assay at –5.8 and –5.2 kb in hiNOS promoter; iv) the cis-acting DNA motifs at –5.8 and –5.2 kb bind cognate NF-{kappa}B and Stat-1 nuclear proteins that mediate cytokine-stimulated transcription in the hiNOS gene as well as a heterologous gene promoter.

Transcriptional regulation is controlled by multiple regulatory DNA motifs located in the core promoter and distant enhancer. A typical enhancer is usually a control region at several kilobases upstream or downstream of the transcriptional initial site that displays position- and orientation-independence for conferring transcriptional activity on its cognate gene (37) . However, its modulated transcriptional efficiency may also depend on the tissue/cell type and developmental stage. The enhancer identified in this study fulfills the criteria for a "classic" enhancer, i.e., its cytokine inducibility is independent of its position and orientation in relation to the promoter.

In the past, we have identified the functional cytokine-responsive elements far upstream in the hiNOS promoter by deletion analysis and site mutagenesis. A significant increase in cytokine inducibility required promoter regions upstream from –4.7 kb in human A549 lung cells or human AKN 1 liver cells. Other groups (21 , 25) obtained comparable results with the human –8.3 kb iNOS promoter. The deletion of –2.1 to –4.7 kb in the context of the 7.2 hiNOS promoter construct did not affect any cytokine-induction in either human A549 or AKN 1 cells. Mellott et al successfully mapped the DHS in the full-length human iNOS promoter (27) . Interestingly, DHS clusters from –5.2 to –5.8 kb in the hiNOS promoter correlated well with several functional NF{kappa}B and Stat-1 binding sites mapped in this region, which have proved to play a vital role in regulating cytokine-stimulated hiNOS expression. We hypothesize that this region might be the most critical one governing hiNOS cytokine-induced transcription. We used the –7.2 kb hiNOS promoter as native context and designed a series of plasmid constructs to address whether this region functions as a classic enhancer.

Our present study demonstrates that the –5 to –6 kb region functions for cytokine-induced activities in a position- and orientation-independent manner not only in A549 lung cells but also in AKN-1 liver cells. Almost identical patterns were found for the cytokine induction as well as their inducibilities for both cell types. However, some differences were found in the values for their relative luciferase activities. AKN1 lung cells seemed to display a relatively higher promoter activity with higher enhancer activity than A549 cells did. Conversely, A549 cells showed a relatively higher cytokine-induced activity and higher enhancer inducibility in the context of heterologous TK promoter. Notably, this hiNOS enhancer was more effective in activating cytokine-induced transcription from its endogenous promoter than from a heterologous promoter in AKN 1 cells, whereas it functioned well both in endogenous and heterologous promoter in A549 cells. Thus, this enhancer demonstrated that it is not only position- and orientation-independent but also relatively cell type- and promoter type-dependent in cytokine-induced transcription activation.

We previously mapped and mutated multiple NF-{kappa}B response elements from –6.0 to –5.0 kb. Subsequently, we identified that TNF-{alpha} or IL-1ß signal is through NF-{kappa}B binding at –5.8 kb motif, while IFN-{gamma} signals through Stat-1 for binding to cis-acting iNOS promoter elements at –5.2 and –5.8 kb. Furthermore, we have identified that the NF-{kappa}B repressing factor (NRF) protein binds to a novel negative response element (NRE) at –6.7 kb in the hiNOS promoter to mediate constitutive silencing of hiNOS transcription. Here, we applied ChIP assay to successfully detect in vivo binding of NF-{kappa}B/Stat-1 protein at –5.2 to –5.8 kb, respectively, in the hiNOS promoter. These results provide definitive evidence for in vivo NF-{kappa}B DNA binding in this hiNOS promoter region and corroborate our previous in vitro findings (19 , 23) . In conclusion, the promoter region from –6 to –5 kb plays a vital role in regulating CM-stimulated hiNOS expression with functional bindings of NF-{kappa}B and Stat 1. This classic enhancer also displays cell type-specific in the context of endogenous and heterologous promoter.


   ACKNOWLEDGMENTS
 
This study was supported by NIH grant R01-GM52021 and R01-DK62313.

Received for publication August 28, 2006. Accepted for publication August 29, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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