Edited by Fujio Kawamura. Hitoshi Ashida: Corresponding author. E-mail: ashida@kobe-u.ac.jp
Index
INTRODUCTION
MATERIALS AND METHODS
Cell lines and culture conditions
ChIP
Construction of ChIP DNA library
SW-ELISA
Genomic mapping
Quantitative real-time RT-PCR analysis
RESULTS
Screening of AhR-binding ChIP fragments
Validation of the binding of AhR to the ChIP fragments
Transcription of AhR-target candidates
DISCUSSION
References

INTRODUCTION

Dioxins, such as halogenated aromatic hydrocarbon and polycyclic aromatic hydrocarbon compounds, are widespread environmental contaminants (Safe, 1994). TCDD is one of the prototypical and most potent dioxins. Exposure to TCDD is known to result in a variety of toxic and biological effects including carcinogenicity, teratogenicity, immunosuppression, and changes in cell proliferation and differentiation (Brown et al., 1998; Gibbons, 1993; Mimura et al., 1997; Poland and Knutson, 1982; Shimizu et al., 2000). These effects were thought to be mediated by AhR, a ligand-dependent transcription factor, which is activated upon binding to TCDD. In the absence of TCDD, a group of Hsp90-related proteins in the cytoplasm captures inactive AhR to form a multimeric complex and thereby abolishes the DNA-binding ability of AhR (Hankinson, 1995; Henry and Gasiewicz, 1993). In contrast, after binding to TCDD, AhR is released from the complex and translocated into the nucleus (Hankinson, 1995; Henry and Gasiewicz, 1993), and then forms a heterodimer with Arnt to bind to DNA stretches containing a DRE sequence (GCGTG) within the regulatory region of target genes, resulting in xenobiotic responses (Hankinson, 1995; Lusska et al., 1993; Probst et al., 1993; Wu and Whitlock, 1993). The toxic effects of TCDD are probably due to superfluous activation of AhR provoked by an excess of TCDD (DeVito and Birnbaum, 1995; Hankinson, 1995).

To our knowledge, only a limited number of functional AhR-binding sites have been identified, mostly within the promoter regions of AhR-regulated genes (Sun et al., 2004; Tijet et al., 2006), including Cyp1A1 (Cytochrome P450, 1A1) (Denison et al., 1988; Lusska et al., 1993), Cyp1A2 (Cytochrome P450, 1A2) (Quattrochi et al., 1998; Sogawa et al., 2004), Cyp1B1 (Cytochrome P450, 1B1) (Eltom et al., 1999), Nqo1 (NAD(P)H: quinone oxidoreductase 1) (Favreau and Pickett, 1991), Ugt1A1 (UDP-glucuronosyltransferase 1A1) (Emi et al., 1996), Gsta1 (Glutathione S-transferase Ya) (Pimental et al., 1993), Aldh3A1 (Aldehyde dehydrogenase 3A1) (Boesch et al., 1999), Nrf2 (NF-E2 p45-related factor) (Miao et al., 2005), Pon1 (Paraoxonase 1) (Gouedard et al., 2004), Cyp19A1 (P450 aromatase) (Baba et al., 2005), c-myc (Yang et al., 2005), Cyp2S1 (Cytochrome P450, 2S1) (Rivera et al., 2007), and Cyp2A5 (Cytochrome P450, 2A5) (Arpiainen et al., 2005); Cyp1A1 and Cyp1B1 being studied most extensively as model AhR-induced genes. Previous gene expression profiling by the DNA microarray technique in mouse hepatoma Hepa-1c1c7 cells stimulated with TCDD revealed that at least 285 genes exhibited obvious changes in their expression (Dere et al., 2006), but experimental genome-wide research for direct AhR-target genes has not been carried out so far.

ChIP is a powerful tool with which to analyze DNA-binding factors and obtain chromosomal DNA fragments to which the factors bind in vivo, which can suggest possible targets. ChIP-chip is a technique used to isolate DNA fragments interacting with a DNA-binding factor, which are labeled and hybridized on to a DNA microarray to map binding sites of the factor within the entire genome (Carroll et al., 2006). However, an inevitable amount of non-specific DNA is contained in the ChIP DNA mostly due to weaker interactions between proteins and DNA, and additional validation is often required to distinguish specific from non-specific interactions. To validate the specificity of ChIP DNA, ChIP fragments can be cloned and analyzed further in biochemical ways to examine the interaction between the transcription factor and DNA (Weinmann et al., 2001, 2002). We previously set up a southwestern chemistry-based enzyme-linked immunosorbent assay (SW-ELISA) technique to determine the affinity between the AhR-Arnt heterodimer and fluorescein isothiocyanate (FITC)-labeled DNA probes (Fukuda et al., 2004; Nishiumi et al., 2007, 2008). In this study, our SW-ELISA technique was used to evaluate DNA fragments containing possible AhR-binding sites selected from mouse hepatoma Hepa-1c1c7 cells stimulated with TCDD through ChIP-based screening.


MATERIALS AND METHODS

Cell lines and culture conditions

The mouse hepatoma cell lines, Hepa-1c1c7 and c12 obtained from the American Type Culture Collection, were maintained in Eagle’s medium (Nissui Pharmaceutical) supplemented with 5% (v/v) fetal bovine serum, 4 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in at atmosphere containing 5% CO2.

ChIP

Hepa-1c1c7 cells were stimulated with 10 nM TCDD for 2 h, and then fixed by adding formaldehyde at a final concentration of 1% for 10 min at 37°C to cross-link protein and DNA. The cross-link reaction was terminated by addition of glycine at 125 mM at room temperature to quench the excess formaldehyde. Next, the cells were washed three times with ice-cold phosphate-buffered saline, suspended in 1 ml of SDS lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-HCl, pH 8.0), and sonicated to shear DNA into 0.1-1 kb fragments. After spinning down, the supernatant was diluted 10-fold with ChIP dilution buffer (0.01% SDS, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl, and 1.1% Triton X-100), and precleared with the addition of Protein G-sepharose 4 fast flow beads (GE Healthcare) to reduce non-specific DNA binding before antibody addition. Precleared chromatin was incubated overnight at 4°C in the presence of a specific polyclonal antibody raised against the C-terminal part of AhR, Ah receptor (M-20) (Santa Cruz Biotechnology), together with Protein G-sepharose 4 fast flow beads, that had been treated previously with 1.5% bovine serum albumin, to form precipitating materials containing the immune complex. The precipitating materials were washed and spun down for 5 min at 4°C successively with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, and 150 mM NaCl), with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, and 500 mM NaCl), with LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.0), and finally twice with TE buffer. The cross-links between DNA and proteins were released by heating the complex at 65°C, and proteins were digested by proteinase K, to extract the ChIP DNA fragments with ChIP elution buffer (1% SDS, and 0.1 M NaHCO3).

Construction of ChIP DNA library

The ChIP DNA fragments were blunt-ended by treatment with T4 DNA polymerase and recovered by ethanol precipitation. The precipitated ChIP DNA fragments were subjected to linker-mediated (LM)-PCR as amplification (Ren et al., 2000) as follows. Two unidirectional linkers (JW102 and JW103 linkers, Table 1) were annealed, phosphorylated, and ligated to both termini of the ChIP DNA fragments. The linker-ligated DNA fragments served as the templates in PCR using the primer JW102 (5 min at 95°C, then 20 cycles of 30 s at 95°C and 1 min at 72°C). The PCR products were purified with a PCR clean-up system (Promega), cloned into pT7blue2-T (Novagen) and introduced into E. coli DH5α resistant to ampicillin (50 μg/ml), to yield a ChIP DNA library.


View Details		Table 1
Oligonucleotides used in this study


SW-ELISA

SW-ELISA was performed essentially as described previously (Fukuda et al., 2004). DNA probes were the individual insert fragments of the ChIP DNA library amplified by PCR with a 5’ FITC-labeled JW102. Each of the FITC-labeled probes (25 fmoles) was put into the respective wells of a microtiter plate, and captured by a rabbit anti-FITC antibody (DakoCytomation) previously fixed on the bottom. Nuclear extract prepared from Hepa-1c1c7 or c12 cells, stimulated with 1 nM TCDD for 2 h, served as the source of the AhR-Arnt heterodimer. The nuclear extract (15 μg) was applied to the wells containing the probe, and incubated at 20°C for 2 h. After washing, a goat anti-Arnt antibody (Santa Cruz Biotechnology) was added to form the immune complex containing the AhR-Arnt heterodimer bound to the probe. After another washing step, the immune complex was further reacted with a biotinylated rabbit anti-goat IgG antibody (Jackson Immuno Research Lab), which was visualized by the labeled-streptavidin biotin method (Giorno, 1984) with peroxidase-conjugated streptavidin and tetramethylbenzidine. After sulfuric acid was added to terminate the visualizing reaction, absorbance at 450 nm in the wells was measured by using a Wallac ARVO sx multilabel counter (Perkin-Elmer Life Sciences). The AhR-binding score of a probe, representing its specific affinity for AhR, was calculated by dividing the absorbance obtained in the presence of the probe by that in its absence.

Genomic mapping

Nucleotide sequencing of the ChIP DNA fragments was performed using the BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems) and a capillary automatic sequencer (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems). Sequences thus obtained were mapped on the mouse genome using the UCSC genome browser (version mm9 at the UCSC Genome Browser website, http://genome.ucsc.edu) (Karolchik et al., 2003).

Quantitative real-time RT-PCR analysis

Quantitative real-time RT-PCR analyses were performed based on the SYBR green gene-expression technology (Morrison et al., 1998; Wittwer et al., 1997). Total RNA extracted from Hepa-1c1c7 cells stimulated with 1 nM TCDD or a vehicle control (0.1% DMSO, dimethyl sulfoxide) for 24 h was reverse transcribed into cDNA using the Primescript RT reagent kit (Takara Bio) and subjected subsequently to quantitative real-time PCR (45 cycles of 95°C for 10 s and 60°C for 20 s) in a LightCycler detection system (Roche Molecular Biochemicals) using a SYBR Premix EX-Taq perfect real time kit (Takara Bio). The relative gene expression values were calculated by the comparative CT method (Livak and Schmittgen, 2001), using expression of the β-actin gene (Actb) as the internal control. Specific primers used are listed in Table 1.


RESULTS

Screening of AhR-binding ChIP fragments

ChIP fragments prepared from mouse hepatoma Hepa-1c1c7 cells using the antibody specific for AhR were subjected to PCR to test whether the AhR binding to the Cyp1A1 promoter region was dependent on TCDD treatment. The very faint band amplified from the TCDD-untreated cells might suggest some weak interaction between AhR and the Cyp1a1 promoter in the absence of TCDD (Fig. 1A, lane 3). However, TCDD-treatment obviously enhanced the corresponding band to indicate that the tight AhR binding to the Cyp1A1 promoter region depends on TCDD (Fig. 1A, lane 1). When the ChIP DNA fragments were amplified by LM-PCR, PCR products 0.1–1 kb long were obtained (Fig. 1B, lane 1). Similar products were also obtained from the template prepared without the anti-AhR antibody (Fig. 1B, lane 2). However, the ChIP DNA prepared with the anti-AhR antibody exclusively gave the right product after another PCR using the primer set specific for the Cyp1A1 promoter region containing a well-characterized AhR-binding site (Fig. 1C). These results indicated that the ChIP DNA prepared with the anti-AhR antibody contains the specific AhR-binding fragments but also a certain amount of non-specific fragments. The products amplified by LM-PCR from the ChIP DNA were cloned into a plasmid, and introduced into E. coli to confer ampicillin resistance, yielding a ChIP DNA library. Clones from the ChIP DNA library were picked randomly, and their inserts were converted into FITC-labeled probes, which were subjected to SW-ELISA experiments to examine their AhR-binding.


View Details		Fig. 1
PCR-based analyses of ChIP DNA fragments. (A) TCDD-dependency of the AhR binding to the Cyp1A1 promoter region. Hepa-1c1c7 cells were treated with 10 nM TCDD (lanes 1 and 2) or the vehicle control (lanes 3 and 4) 2 h prior to ChIP. ChIP was performed in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of AhR-specific antibody. The ChIP fragments were subjected to PCR to amplify the Cyp1A1 promoter region with the specific primer pair (Table 1). (B) LM-PCR. Hepa-1c1c7 cells were stimulated with 10 nM TCDD 2 h prior to ChIP. ChIP DNA fragments were prepared in the presence (lane 1) or absence (lane 2) of AhR-specific antibody, and used as templates in LM-PCR performed as described in the text. (C) Specific-PCR of the Cyp1A1 promoter region. Lane assignment is the same as in panel B.


To certify the suitability of our SW-ELISA system, a series of control experiments was performed as follows. Nuclear extract prepared from Hepa-1c1c7 cells stimulated with TCDD was used as the material containing activated AhR for DNA binding. First to be used were FITC-labeled probes designed for the promoter region of well-characterized AhR target genes, including Cyp1A1, Cyp1B1, Cyp2S1, Cyp2A5, and Cyp19A1 (Arpiainen et al., 2005; Baba et al., 2005; Denison et al., 1988; Eltom et al., 1999; Rivera et al., 2007). These probes gave AhR-binding scores ranging from 2.2 to 3.2 (Fig. 2A), suggesting that the system could detect specific interaction between AhR and the probes. Second, another set of probes used randomly picked out 94 DNA fragments derived from the LM-PCR products prepared without the anti-AhR antibody (Fig. 1B, lane 2). AhR-binding scores given by these probes were mostly between 1.0 and 1.5 and never higher than 2.1 (Fig. 2B). Our SW-ELISA system might be sensitive enough to detect weaker interactions between AhR and DNA, and we decided to set a threshold to discard the probes giving AhR-binding scores of less than 2.1 which were possibly due to non-specific interactions. Finally as a pilot case, 92 probes prepared randomly from the ChIP DNA library were examined. As shown in Fig. 2C, some probes appeared to give AhR-binding scores as high as the positive control of the Cyp1A1 probe, suggesting that our strategy allowed for the screening of additional DNA fragments with greater affinity for the AhR.


View Details		Fig. 2
SW-ELISA. (A) AhR-binding scores of known targets. SW-ELISA was performed with probes designed for the promoter region of target genes as indicated, each of which contains well-characterized AhR-binding sites. The AhR-binding scores were calculated from three independent measurements as described in the text and shown as the means ± SE (n = 3). (B) AhR-binding scores of non-specific fragments. Probes used were specific for the Cyp1A1 promoter (left end) and 94 randomly picked DNA fragments derived from LM-PCR products prepared without anti-AhR antibody. Values are means for three independent measurements. Dotted line indicates the threshold level (AhR-binding score at 2.1). (C) AhR-binding scores of ChIP DNA fragments. Probes were 92 randomly picked fragments from the ChIP DNA library. Arrowheads indicate the fragments giving scores over the threshold.


So far we have screened approximately 1,700 probes prepared from the library, and selected 87 ChIP fragments exhibiting higher AhR-binding scores. Nucleotide sequences of the 87 ChIP fragments were determined and mapped on to the mouse genome. It was found that the same ChIP fragment was selected three times and another 8 fragments, twice (Table 2). Therefore, the ChIP-based SW-ELISA revealed 77 different AhR-binding fragments (Table 2). Sequencing revealed that 39 of the 77 fragments contained at least one typical DRE while the others did not, suggesting that in some cases, a DRE might not be required for the binding of AhR. In addition, 75 of the 77 fragments were not located within promoter-proximal regions but in downstream introns/exons or intergenic regions, implying that AhR could bind to promoter-distal regions regularly.


View Details		Table 2
AhR-binding ChIP fragments isolated in this study


Validation of the binding of AhR to the ChIP fragments

Nuclear extracts were prepared from Hepa-1c1c7 and c12 cells (an AhR-deficient counterpart to Hepa-1c1c7) (Eltom et al., 1999; Hankinson, 1979; Zhang et al., 1996) stimulated with TCDD to be compared in SW-ELISA experiments using the 77 ChIP fragments selected above. The extract from Hepa-1c1c7 gave AhR-binding scores of more than 2.2 with all 77 probes (Fig. 3A), while that from c12 mostly gave scores of around 1.5, which was clearly lower than the threshold and regarded as non-specific (Fig. 2B). Therefore, the higher scores obtained from the SW-ELISA experiments were dependent on the AhR, and the assay system was reliable enough to guarantee the specific interactions between AhR and the DNA fragments. Next, another nuclear extract was prepared from Hepa-1c1c7 cells without TCDD-stimulation and employed in the assay. With this extract, most of the ChIP fragments gave AhR-binding scores of no more than 2 (Fig. 3B), suggesting the binding of AhR to the selected ChIP fragments to be dependent on TCDD. However, there were several exceptions that showed higher scores without TCDD stimulation (Fig. 3B). The most remarkable exceptions were two fragments giving AhR-binding of more than 3; one corresponded to the intergenic region between Tspyl5 and Mtdh, and the other to the intragenic region of Dchs2. It was likely that for unknown reasons, these fragments possess higher affinity for AhR independent of TCDD.


View Details		Fig. 3
Validation of the AhR-binding to the ChIP DNA fragments. (A) AhR-dependency. Nuclear extracts were prepared from Hepa-1c1c7 cells (black bars) and c12 cells (gray bars) stimulated by TCDD. Values are means + SE (error bar) from three independent measurements. (B) TCDD-dependency. Nuclear extracts were prepared from Hepa-1c1c7 cells cultured with TCDD (black bars) or the vehicle control (gray bars). Dotted line indicates the threshold level (AhR-binding score at 2.1).


Transcription of AhR-target candidates

As candidates for AhR-target genes, we chose genes containing or located nearest to the genomic positions where the 77 AhR-binding fragments were assigned; when a fragment was mapped within an intergenic region, the two genes flanking it were chosen regardless of their orientation. As listed in Table 2, our ChIP-based screening allowed us to select 121 potential AhR-targets. These included two well-characterized AhR targets, Cyp1A1 and Cyp1B1, as well as others previously suggested to be AhR-responsive under certain conditions, including Abcb1b (MDR1b) (Mathieu et al., 2001), Igf1r (Tanaka et al., 2007), Pax9 (Sahlberg et al., 2007), and Slc2a3 (GLUT3) (Ishimura et al., 2002), (Table 2). However, 11 of 13 known AhR targets were not included, such as Aldh3A1, Cyp1A2, Cyp19A1, Cyp2A5, Cyp2S1, Gsta1, c-myc, Nqo1, Nrf2, Pon1, and Ugt1A1. Nevertheless, we have already isolated the same ChIP fragment three-times (Igf1r) and the other 8 twice (Alcam < S < Zpld1, Dlx5 < S > Acn9, F13a1 < S > Ly86, Nras, Uba2, Spata16, Flt3 < S > Pan3, and Zfp64 < S > Tshz2) in the total of 87 isolates after screening almost 1,700 individual clones (Table 2), implying that the number of different fragments isolatable from our library might be limited under the current conditions. In any case, these results might suggest that our screening was still insufficient.

Previously, the transcriptome in Hepa-1c1c7 cells stimulated with TCDD was analyzed using a DNA microarray technique to reveal that at least 285 of 8284 genes exhibited ±1.5-fold altered expression, suggesting these 285 genes might include AhR targets (Fig. 4) (Dere et al., 2006). Indeed, two of the 121 AhR-target candidates selected in this study, Cyp1A1 and Pla2g4a, were included in the 285 genes (Fig. 4 and Table 2). In addition, the induction of Cyp1A1 and Pla2g4a expression in the presence of TCDD (29.13- and 1.84-fold, respectively) was confirmed by quantitative real-time RT-PCR (Table 3). Therefore, in these two cases, our strategy was successful in finding the AhR-binding sites associated with the AhR targets. However, the 121 candidates were found to include 36 genes (Table 2) judged not to be affected by TCDD in a previous transcriptome-based analysis (Dere et al., 2006), including Abcb1b, Acadm, Cyp1B1, Cyp7B1, Nras, Slc2a3, and Slc25a12 (Fig. 4). We reinvestigated the expression of these 7 genes by quantitative RT-PCR only to find the induction of Cyp1B1 expression (2.41-fold) but no change in the expression of the other 6 genes (Table 3). The DNA microarray used in the previous study was not equipped with probes for 83 of the 121 candidates (Fig. 4). Among the 83 genes, Igf1r in breast carcinoma MCF-7 cells (Tanaka et al., 2007), Slc2a3 (GLUT3) in the placenta (Ishimura et al., 2002) and Pax9 involved in tooth development (Sahlberg et al., 2007) were previously induced by TCDD, and Spata16 required for spermatogenesis (Dam et al., 2007) was associated with two different AhR-binding fragments (Table 2). Their expression was examined by quantitative RT-PCR, revealing the induction of Pax9 expression (3.39-fold). However, the others did not exhibit meaningful change in expression dependent on TCDD at all (Table 3). It is conceivable that Igf1r (Tanaka et al., 2007), Slc2a3 (GLUT3) (Ishimura et al., 2002) and Spata16 (Dam et al., 2007) were not induced in hepatoma Hepa-1c1c7 cells stimulated with TCDD, because induction of these three genes was not evidenced in the liver. On the other hand, Abcb1b is induced in the liver depending on AhR in the presence of 3-methylcholanthrene-activated but not TCDD (Mathieu et al., 2001). Pax9 functions in tooth development, however, its induction in Hepa-1c1c7 was unexpected and might be worthwhile for further studies. All these results together suggested that the majority of the AhR-binding sites selected by our ChIP-based approach would not always be involved in transcriptional regulation. TCDD-activated AhR can bind to promoter-distal regions often without a DRE and its DNA-binding alone could not be enough to establish its regulatory function.


View Details		Fig. 4
Venn diagram of gene sets from the previous transcriptome and our ChIP SW-ELISA screening data.





View Details		Table 3
TCDD-induced fold-change in the expression of AhR-target candidates determined by quantitative real-time RT-PCR



DISCUSSION

We isolated 77 different AhR-binding fragments from a ChIP DNA library made from Hepa-1c1c7 cells stimulated with TCDD (Table 2). The interaction between these fragments and AhR (AhR-Arnt heterodimer) was confirmed to be specific and dependent on the receptor’s activation by TCDD (Fig. 3). Previous searches for AhR-binding sites were designed to investigate regions containing transcription start sites of AhR-target genes, and thus all known AhR-binding sites were mapped within promoter-proximal regions. However, in this study we revealed that the vast majority of the AhR-binding sites were contained in downstream introns/exons or intergenic regions, implying that the AhR regularly binds to promoter-distal regions.

Sequence-specific DNA-binding factors generally recognize degenerate motifs of cis-elements comprising 5–10 base pairs. Consequently, potential recognition sequences for many transcription factors may frequently throughout the genome and participate in actual protein-DNA interactions. According to this hypothesis, most of the AhR-binding site found in promoter-distal regions might be due to the distribution of DREs throughout the genome. Similarly to our findings, for estrogen receptor α (ERα), a well-studied model of ligand-activated eukaryotic transcriptional regulators, a very low frequency of promoter-proximal binding sites was reported, and almost all in vivo ERα binding events seemed to occur in regions previously unannotated as cis-regulatory elements within the genome (Carroll et al., 2005, 2006; Lin et al., 2007).

However, the possible genome-wide distribution of DRE was not enough to explain everything, since 38 of the 77 AhR-binding ChIP fragments did not contain a typical DRE (Table 2). In fact, it is known that in some cases, AhR can bind to DNA without a typical DRE motif. For instance, a DRE-like sequence (GCGGG) in the Pon1 promoter was involved in the recognition of AhR (Gouedard et al., 2004), and the AhR bound to the Cyp1A2 enhancer without a DRE only in the presence of unknown additional cellular factors coordinating specific AhR-DNA interaction (Sogawa et al., 2004). These findings suggest that the AhR was able to bind the 38 DRE-negative fragments because of degeneracy of the DRE sequence or possible coordinators. On the other hand, AhR-binding scores of the 39 DRE-positive AhR-binding ChIP fragments were ranged from 2.1 to 4.3 (mean ± SD, 2.9 ± 0.5), while those of the 38 DRE-negative fragments were from 2.1 to 3.4 (2.6 ± 0.3). In addition, the histogram in Fig. 5 indicates that fragments with lower affinity predominated more among the DRE-negative than DRE-positive fragments. Therefore, a DRE might not always be required for the binding to AhR, but it still must be one of the important determinants enabling tighter interaction, although in any case it is likely that the binding participates in the transcriptional regulation of target genes only when it occurs in the right place at the right time.


View Details		Fig. 5
Histogram of the AhR-binding scores of 39 ChIP fragments with a DRE (Black bars) and 38 without a DRE (white bars).


In this study, Pla2g4a was found as a possible new target of the AhR, which associated with at least one AhR-binding site (Table 2) and exhibited +1.84-fold increase in expression in the presence of TCDD (Table 3). This gene encodes cytosolic phospholipase A2-α (cPLA2-α) playing a central role in the release of arachidonic acid (AA) from sn2 position of membrane phospholipids, and AA metabolism is involved in a number of biological events such as inflammatory and carcinogenic processes. Previous studies revealed that TCDD promoted the release of AA from membranes as a result of its ability to stimulate membrane lipid oxidation and phospholipase A2 (PLA2) activity (al-Bayati and Stohs, 1991; Forsell et al., 2005; Tithof et al., 1996, 2002). Therefore, our finding might fill-in the missing link between TCDD stimulation and phospholipase A2 induction. On the other hand, TCDD can induce the expression of P450s metabolizing AA, such as Cyp1A1, which convert AA to prostaglandins (Lee et al., 1998; Schwarz et al., 2004). Recently, it was hypothesized that some AA metabolites, such as lipoxin A4 and several prostaglandins, serve as endogenous AhR ligands (Denison and Nagy, 2003). If this hypothesis is correct, once TCDD activated AhR, P450s would produce AA metabolites activating AhR again, and this positive feedback loop would provoke superfluous activation of AhR (Schaldach et al., 1999; Seidel et al., 2001). Further investigation might provide insight into how AhR may be involved in the regulation of AA metabolism.

This work was supported in part by the Japan Science and Technology Agency [research for promoting technological seeds 11–112 (2007) to H. A.].

References
al-Bayati, Z. A., and Stohs, S. J. (1991) The possible role of phospholipase A2 in hepatic microsomal lipid peroxidation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Arch. Environ. Contam. Toxicol. 20, 361–365.
Arpiainen, S., Raffalli-Mathieu, F., Lang, M. A., Pelkonen, O., and Hakkola, J. (2005) Regulation of the Cyp2a5 gene involves an aryl hydrocarbon receptor-dependent pathway. Mol. Pharmacol. 67, 1325–1333.
Baba, T., Mimura, J., Nakamura, N., Harada, N., Yamamoto, M., Morohashi, K., and Fujii-Kuriyama, Y. (2005) Intrinsic function of the aryl hydrocarbon (dioxin) receptor as a key factor in female reproduction. Mol. Cell. Biol. 25, 10040–10051.
Boesch, J. S., Miskimins, R., Miskimins, W. K., and Lindahl, R. (1999) The same xenobiotic response element is required for constitutive and inducible expression of the mammalian aldehyde dehydrogenase-3 gene. Arch. Biochem. Biophys. 361, 223–230.
Brown, N. M., Manzolillo, P. A., Zhang, J. X., Wang, J., and Lamartiniere, C. A. (1998) Prenatal TCDD and predisposition to mammary cancer in the rat. Carcinogenesis 19, 1623–1629.
Carroll, J. S., Liu, X. S., Brodsky, A. S., Li, W., Meyer, C. A., Szary, A. J., Eeckhoute, J., Shao, W., Hestermann, E. V., Geistlinger, T. R., et al. (2005) Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1. Cell 122, 33–43.
Carroll, J. S., Meyer, C. A., Song, J., Li, W., Geistlinger, T. R., Eeckhoute, J., Brodsky, A. S., Keeton, E. K., Fertuck, K. C., Hall, G. F., et al. (2006) Genome-wide analysis of estrogen receptor binding sites. Nat. Genet. 38, 1289–1297.
Dam, A. H., Koscinski, I., Kremer, J. A., Moutou, C., Jaeger, A. S., Oudakker, A. R., Tournaye, H., Charlet, N., Lagier-Tourenne, C., van Bokhoven, H., et al. (2007) Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia. Am. J. Hum. Genet. 81, 813–820.
Denison, M. S., and Nagy, S. R. (2003) Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu. Rev. Pharmacol. Toxicol. 43, 309–334.
Denison, M. S., Fisher, J. M., and Whitlock, J. P., Jr. (1988) The DNA recognition site for the dioxin-Ah receptor complex. Nucleotide sequence and functional analysis. J. Biol. Chem. 263, 17221–17224.
Dere, E., Boverhof, D. R., Burgoon, L. D., and Zacharewski, T. R. (2006) In vivo-in vitro toxicogenomic comparison of TCDD-elicited gene expression in Hepa1c1c7 mouse hepatoma cells and C57BL/6 hepatic tissue. BMC Genomics 7, 80.
DeVito, M. J., and Birnbaum, L. S. (1995) Dioxins: model chemicals for assessing receptor-mediated toxicity. Toxicology 102, 115–123.
Eltom, S. E., Zhang, L., and Jefcoate, C. R. (1999) Regulation of cytochrome P-450 (CYP) 1B1 in mouse Hepa-1 variant cell lines: A possible role for aryl hydrocarbon receptor nuclear translocator (ARNT) as a suppressor of CYP1B1 gene expression. Mol. Pharmacol. 55, 594–604.
Emi, Y., Ikushiro, S., and Iyanagi, T. (1996) Xenobiotic responsive element-mediated transcriptional activation in the UDP-glucuronosyltransferase family 1 gene complex. J. Biol. Chem. 271, 3952–3958.
Favreau, L. V., and Pickett, C. B. (1991) Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Identification of regulatory elements controlling basal level expression and inducible expression by planar aromatic compounds and phenolic antioxidants. J. Biol. Chem. 266, 4556–4561.
Forsell, P. K., Olsson, A. O., Andersson, E., Nallan, L., and Gelb, M. H. (2005) Polychlorinated biphenyls induce arachidonic acid release in human platelets in a tamoxifen sensitive manner via activation of group IVA cytosolic phospholipase A2-α. Biochem. Pharmacol. 71, 144–155.
Fukuda, I., Nishiumi, S., Yabushita, Y., Mukai, R., Kodoi, R., Hashizume, K., Mizuno, M., Hatanaka, Y., and Ashida, H. (2004) A new southwestern chemistry-based ELISA for detection of aryl hydrocarbon receptor transformation: application to the screening of its receptor agonists and antagonists. J. Immunol. Methods 287, 187–201.
Gibbons, A. (1993) Dioxin tied to endometriosis. Science 262, 1373.
Giorno, R. (1984) A comparison of two immunoperoxidase staining methods based on the avidin-biotin interaction. Diagn. Immunol. 2, 161–166.
Gouedard, C., Barouki, R., and Morel, Y. (2004) Dietary polyphenols increase paraoxonase 1 gene expression by an aryl hydrocarbon receptor-dependent mechanism. Mol. Cell. Biol. 24, 5209–5222.
Hankinson, O. (1979) Single-step selection of clones of a mouse hepatoma line deficient in aryl hydrocarbon hydroxylase. Proc. Natl. Acad. Sci. USA 76, 373–376.
Hankinson, O. (1995) The aryl hydrocarbon receptor complex. Annu. Rev. Pharmacol. Toxicol. 35, 307–340.
Henry, E. C., and Gasiewicz, T. A. (1993) Transformation of the aryl hydrocarbon receptor to a DNA-binding form is accompanied by release of the 90 kDa heat-shock protein and increased affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem. J. 294, 95–101.
Ishimura, R., Ohsako, S., Miyabara, Y., Sakaue, M., Kawakami, T., Aoki, Y., Yonemoto, J., and Tohyama, C. (2002) Increased glycogen content and glucose transporter 3 mRNA level in the placenta of Holtzman rats after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 178, 161–171.
Karolchik, D., Baertsch, R., Diekhans, M., Furey, T. S., Hinrichs, A., Lu, Y. T., Roskin, K. M., Schwartz, M., Sugnet, C. W., Thomas, D. J., et al. (2003) The UCSC Genome Browser Database. Nucleic Acids Res. 31, 51–54.
Lee, C. A., Lawrence, B. P., Kerkvliet, N. I., and Rifkind, A. B. (1998) 2,3,7,8-Tetrachlorodibenzo-p-dioxin induction of cytochrome P450-dependent arachidonic acid metabolism in mouse liver microsomes: evidence for species-specific differences in responses. Toxicol. Appl. Pharmacol. 153, 1–11.
Lin, C. Y., Vega, V. B., Thomsen, J. S., Zhang, T., Kong, S. L., Xie, M., Chiu, K. P., Lipovich, L., Barnett, D. H., Stossi, F., et al. (2007) Whole-genome cartography of estrogen receptor α binding sites. PLoS Genet. 3, e87.
Livak, K. J., and Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT Method. Methods 25, 402–408.
Lusska, A., Shen, E., and Whitlock, J. P., Jr. (1993) Protein-DNA interactions at a dioxin-responsive enhancer. Analysis of six bona fide DNA-binding sites for the liganded Ah receptor. J. Biol. Chem. 268, 6575–6580.
Mathieu, M. C., Lapierre, I., Brault, K., and Raymond, M. (2001) Aromatic hydrocarbon receptor (AhR)·AhR nuclear translocator- and p53-mediated induction of the murine multidrug resistance mdr1 gene by 3-methylcholanthrene and benzo(a)pyrene in hepatoma cells. J. Biol. Chem. 276, 4819–4827.
Miao, W., Hu, L., Scrivens, P. J., and Batist, G. (2005) Transcriptional regulation of NF-E2 p45-related factor (NRF2) expression by the aryl hydrocarbon receptor-xenobiotic response element signaling pathway: direct cross-talk between phase I and II drug-metabolizing enzymes. J. Biol. Chem. 280, 20340–20348.
Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., et al. (1997) Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes Cells 2, 645–654.
Morrison, T. B., Weis, J. J., and Wittwer, C. T. (1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. BioTechniques 24, 954–962.
Nishiumi, S., Yamamoto, N., Kodoi, R., Fukuda, I., Yoshida, K., and Ashida, H. (2008) Antagonistic and agonistic effects of indigoids on the transformation of an aryl hydrocarbon receptor. Arch. Biochem. Biophys. 470, 187–199.
Nishiumi, S., Yoshida, K., and Ashida, H. (2007) Curcumin suppresses the transformation of an aryl hydrocarbon receptor through its phosphorylation. Arch. Biochem. Biophys. 466, 267–273.
Pimental, R. A., Liang, B., Yee, G. K., Wilhelmsson, A., Poellinger, L., and Paulson, K. E. (1993) Dioxin receptor and C/EBP regulate the function of the glutathione S-transferase Ya gene xenobiotic response element. Mol. Cell. Biol. 13, 4365–4373.
Poland, A., and Knutson, J. C. (1982) 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22, 517–554.
Probst, M. R., Reisz-Porszasz, S., Agbunag, R. V., Ong, M. S., and Hankinson, O. (1993) Role of the aryl hydrocarbon receptor nuclear translocator protein in aryl hydrocarbon (dioxin) receptor action. Mol. Pharmacol. 44, 511–518.
Quattrochi, L. C., Shih, H., and Pickwell, G. V. (1998) Induction of the human CYP1A2 enhancer by phorbol ester. Arch. Biochem. Biophys. 350, 41–48.
Ren, B., Robert, F., Wyrick, J. J., Aparicio, O., Jennings, E. G., Simon, I., Zeitlinger, J., Schreiber, J., Hannett, N., Kanin, E., et al. (2000) Genome-wide location and function of DNA binding proteins. Science 290, 2306–2309.
Rivera, S. P., Wang, F., Saarikoski, S. T., Taylor, R. T., Chapman, B., Zhang, R., and Hankinson, O. (2007) A novel promoter element containing multiple overlapping xenobiotic and hypoxia response elements mediates induction of cytochrome P4502S1 by both dioxin and hypoxia. J. Biol. Chem. 282, 10881–10893.
Safe, S. H. (1994) Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment. Crit. Rev. Toxicol. 24, 87–149.
Sahlberg, C., Peltonen, E., Lukinmaa, P. L., and Alaluusua, S. (2007) Dioxin alters gene expression in mouse embryonic tooth explants. J. Dent. Res. 86, 600–605.
Schaldach, C. M., Riby, J., and Bjeldanes, L. F. (1999) Lipoxin A4: a new class of ligand for the Ah receptor. Biochemistry. 38, 7594–7600.
Schwarz, D., Kisselev, P., Ericksen, S. S., Szklarz, G. D., Chernogolov, A., Honeck, H., Schunck, W. H., and Roots, I. (2004) Arachidonic and eicosapentaenoic acid metabolism by human CYP1A1: highly stereoselective formation of 17(R), 18(S)-epoxyeicosatetraenoic acid. Biochem. Pharmacol. 67, 1445–1457.
Seidel, S. D., Winters, G. M., Rogers, W. J., Ziccardi, M. H., Li, V., Keser, B., and Denison, M. S. (2001) Activation of the Ah receptor signaling pathway by prostaglandins. J. Biochem. Mol. Toxicol. 15, 187–196.
Shimizu, Y., Nakatsuru, Y., Ichinose, M., Takahashi, Y., Kume, H., Mimura, J., Fujii-Kuriyama, Y., and Ishikawa, T. (2000) Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. USA 97, 779–782.
Sogawa, K., Numayama-Tsuruta, K., Takahashi, T., Matsushita, N., Miura, C., Nikawa, J., Gotoh, O., Kikuchi, Y., and Fujii-Kuriyama, Y. (2004) A novel induction mechanism of the rat CYP1A2 gene mediated by Ah receptor-Arnt heterodimer. Biochem. Biophys. Res. Commun. 318, 746–755.
Sun, Y. V., Boverhof, D. R., Burgoon, L. D., Fielden, M. R., and Zacharewski, T. R. (2004) Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences. Nucleic Acids Res. 32, 4512–4523.
Tanaka, J., Yonemoto, J., Zaha, H., Kiyama, R., and Sone, H. (2007) Estrogen-responsive genes newly found to be modified by TCDD exposure in human cell lines and mouse systems. Mol. Cell. Endocrinol. 272, 38–49.
Tijet, N., Boutros, P. C., Moffat, I. D., Okey, A. B., Tuomisto, J., and Pohjanvirta, R. (2006) Aryl hydrocarbon receptor regulates distinct dioxin-dependent and dioxin-independent gene batteries. Mol. Pharmacol. 69, 140–153.
Tithof, P. K., Elgayyar, M., Cho, Y., Guan, W., Fisher, A. B., and Peters-Golden, M. (2002) Polycyclic aromatic hydrocarbons present in cigarette smoke cause endothelial cell apoptosis by a phospholipase A2-dependent mechanism. FASEB J. 16, 1463–1464.
Tithof, P. K., Schiamberg, E., Peters-Golden, M., and Ganey, P. E. (1996) Phospholipase A2 is involved in the mechanism of activation of neutrophils by polychlorinated biphenyls. Environ. Health Perspect. 104, 52–58.
Weinmann, A. S., and Farnham, P. J. (2002) Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods 26, 37–47.
Weinmann, A. S., Bartley, S. M., Zhang, T., Zhang, M. Q., and Farnham, P. J. (2001) Use of chromatin immunoprecipitation to clone novel E2F target promoters. Mol. Cell. Biol. 21, 6820–6832.
Wittwer, C. T., Herrmann, M. G., Moss, A. A., and Rasmussen, R. P. (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques 22, 130–138.
Wu, L., and Whitlock, J. P., Jr. (1993) Mechanism of dioxin action: receptor-enhancer interactions in intact cells. Nucleic Acids Res. 21, 119–125.
Yang, X., Liu, D., Murray, T. J., Mitchell, G. C., Hesterman, E. V., Karchner, S. I., Merson, R. R., Hahn, M. E., and Sherr, D. H. (2005) The aryl hydrocarbon receptor constitutively represses c-myc transcription in human mammary tumor cells. Oncogene 24, 7869–7881.
Zhang, J., Watson, A. J., Probst, M. R., Minehart, E., and Hankinson, O. (1996) Basis for the loss of aryl hydrocarbon receptor gene expression in clones of a mouse hepatoma cell line. Mol. Pharmacol. 50, 1454–1462.