Interactions between the Aryl Hydrocarbon Receptor and P-TEFb

The expression of the cytochrome P450 1A1 gene (cyp1a1) is regulated by the aryl hydrocarbon receptor (AhR), which is a ligand-activated transcription factor that mediates most toxic responses induced by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). In the nucleus, ligand-activated AhR binds to the xenobiotic response elements, initiating chromatin remodeling and recruitment of coregulators, leading to the formation of preinitiation complex followed by elongation. Here, we report that ligand-activated AhR recruits the positive transcription elongation factor (P-TEFb) and RNA polymerase II (RNA PII) to the cyp1a1 promoter with concomitant phosphorylation of the RNA PII carboxyl domain (CTD). Interestingly, the serine 2 and serine 5 of the heptapeptide repeats (YSPTSPS) were sequentially phosphorylated upon TCDD treatment. Inhibition of P-TEFb kinase activity by 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB) suppressed CTD phosphorylation (especially serine 2 phosphorylation) and abolished processive elongation without disrupting the assembly of the preinitiation complex at the cyp1a1 promoter. Remarkably, we found that activation of NF-κB by TNF-α selectively inhibited TCDD-induced serine 2 phosphorylation in mouse liver cells, suggesting that residue-specific phosphorylation of RNA PII CTD at the cyp1a1 promoter is an important regulatory point upon which signal “cross-talk” converges. Finally, we show that ligand-activated AhR associated with P-TEFb through the C terminus of cyclin T1, suggesting that AhR recruit the P-TEFb to the cyp1a1 promoter whereupon its kinase subunit phosphorylates the RNA PII CTD.

Per-ARNT-Sim (bHLH-PAS) family and mediates most TCDDinduced toxic responses. The AhR is at the high echelon of transcriptional regulatory circuitry regulating many aspects of physiological processes in addition to the xenobiotic metabolism (1,2).
It has been shown that, in mouse hepatoma cells, the AhR resides in the cytoplasm in association with heat shock protein 90 (hsp90) (3,4) and an immunophilin protein (5)(6)(7). Upon activation by ligand, the AhR translocates into the nucleus and binds to another bHLH-PAS protein called the AhR nuclear translocator (ARNT) (8). The heterodimeric protein complex then binds to the xenobiotic response elements (XREs) (9), which are enhancer sequences located in the regulatory regions of AhR-controlled genes such as the gene from cytochrome P450 1A1 (cyp1a1), and activates gene expression. Cyp1a1 is a member of the cytochromes P450 monooxygenase superfamily, which plays an important role in xenobiotic metabolism as well as in carcinogenesis. Historically, many important mechanistic aspects of AhR-regulated gene expression have been investigated utilizing transcriptional regulation of mouse cyp1a1 as a model system (1).
The ligand-dependent cyp1a1 transcriptional regulation is a dynamic process involving AhR binding to the XRE, controlled recruitment of coregulators as well as general transcription factors, chromatin remodeling and histone modifications (10 -14). These processes lead to the assembly of the preinitiation complex at the cyp1a1 promoter, which is followed by transcription elongation. Actinomycin D treatment, which blocks the nucleosomal changes downstream from the transcription start site has no effect on chromatin remodeling around the promoter region upstream from the transcription start site, suggesting that assembly of the preinitiation complex and elongation are distinct processes (15).
Transcription elongation by RNA PII is a highly regulated process and its complexity has only recently been appreciated. The largest subunit of mammalian RNA PII possesses 52 repeats of heptapeptide with consensus YSPTSPS motif (16). Hyperphosphorylated CTD is associated with active elongating RNA PII, while inactive RNA PII is hypophosphorylated. It is recognized that after formation of the preinitiation complex, RNA PII is subjected to negative regulation by the negative transcription elongation factor (N-TEF) (17,18). The N-TEF is composed of the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF). NELF is a transcription factor complex that cooperates with DSIF/hSpt4-hSpt5 to repress elongation by RNA PII (19). In order for RNA PII to overcome the negative regulation and engage in processive transcription, P-TEFb is required. P-TEFb consists of a regulatory subunit (either cyclin T1, T2, or K) and an enzymatic subunit (CDK9) (18). CDK9 phosphorylates the C-terminal * This research was funded in part by NIEHS, National Institutes of Health Grants ES 09859 and ES 09106 and American Heart Association Grant 0355131Y. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Phosphorylation status of specific serine residues, especially serine 2 and serine 5, are associated with different isoforms of RNA PII engaging at different stages of transcription cycle (20 -22). Serine 2 has been shown to be phosphorylated by P-TEFb (23) and is associated with RNA PII engaging in transcript elongation (22), while serine 5 phosphorylation is involved in recruitment and activation of the mammalian capping enzyme (24,25) and involved in the processing of primary RNA transcript.
In this study, we show for the first time, that activation of AhR leads to recruitment of P-TEFb to the cyp1a1 promoter followed by differential phosphorylation of the RNA PII CTD. NF-B activation antagonizes TCDD-induced serine 2 phosphorylation. Futhermore, we demonstrated that cyclinT1 directly interacts with AhR in vitro as well as in vivo. Our results suggest that elongation control is an important point of regulation of cyp1a1 expression.

MATERIALS AND METHODS
Plasmid Constructs and Vectors-pCyclin T1 and pCDK9 were kindly provided by D. Price (University of Iowa). For yeast two hybrid and glutathione S-transferase (GST) pull-down assays, Cyclin T1 expression plasmids were made by inserting the PCR-generated cyclin T1 fragments into the pGAD424 (Clontech) and pGEX-5X-3 (Amersham Biosciences). The PCR primers used are: fragment 1-726: OL1, GCG-GATCCCCACCATGGAGGGAGAGAGGAAG and OL726, ACGCGTC-GACTTACTTAGGAAGGGGTGGAAG; for fragment 1-250: OL 1 and OL250, ACGCGTCGACTTAGTTGGGAGTTTTCTCCAAAA; for fragment 233-726: OL233, GCGGATCCCCACCATGTTTAGATGAACT-GACACATG and OL726. The PCR products were modified with restriction enzymes BamH1 and SalI for insertion into the plasmid vectors. Plasmid pGL3-CYP1A1-Luc was created by inserting the PCR product of the mouse cyp1a1 upstream regulatory region (Ϫ1395 to ϩ 1) into the pGL3 basic vector (Promega). PCR primers used were: TTGAGTTAGA-CACGCCAAGTTCAG and AGTGAAGGAAGAGGGTTAGGGTGAAG-GCACCACCAC. In the PCR reaction, mouse genomic DNA was used as the template. The PCR product was cloned into the TA cloning vector (pCR2.1, Invitrogen) and restricted with HindIII and XhoI and then inserted into pGL3 basic vector. Plasmid DNAs used for transfection were purified using the Qiagen Maxi-Prep DNA Isolation system (Qiagen).
Cell Culture and Transient Transfection-Cells were maintained in ␣MEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 250 ng/ml amphotericin B (Invitrogen), 5% CO 2 and 37°C. For transient transfection, Hepa1c1c7 cells were seeded in 12-well plates on day Ϫ1 and transfection performed using LipofectAMINE (Invitrogen) when cell density reached 70% confluence. pSV-␤-galactosidase control plasmid (Promega) was used for normalization of transfection efficiency. Six hours after transfection, cells were treated with TCDD or Me 2 SO (solvent control) for 18 h before harvest for determination of luciferase activity.
RNA Isolation and Ribonuclease Protection Assay for Determination of cyp1a1 Transcripts-Hepa1c1c7 cells were seeded into 100-mm in diameter Petri dishes. When growth reached 80% confluence, cells were treated with TCDD (10 nM), DRB (30 M) ϩ TCDD (10 nM) for 2 h. Total cellular RNA was isolated using TRIzol reagent (Invitrogen) according to manufacturer's instructions.
The proximal promoter probe was generated by PCR amplification of mouse genomic DNA with PCR primers: TTGAGTTAGACACGCCAA-GTTCAG and GAAGTGAAGAGTGTTCTCTAGGAC. PCR products were cloned into a TA cloning vector (pCR2.1, Invitrogen). The DNA template was linearized with Msc1 and used as the template for generation of riboprobe corresponding to the proximal promoter region (Ϫ52 to ϩ65). The riboprobe for the cyp1a1 distal region was generated by PCR using plasmid DNA containing cloned distal region of mouse cyp1a1. The PCR primers used were: CAGAAACACAGATCCTGG and TAT-TTAGGTGACACTATAGAATCAAAGTAACCAGACACATCC, which contains the Sp6 promoter. Antisense probes were prepared using MAXIscript In Vitro Transcription Kit (Ambion) according to the manufacturer's instructions. The ribonuclease protection assays were performed on 20 g of total RNA and 20,000 cpm of antisense probes using RPA III Ribonuclease Protection Assay Kit (Ambion) according to manufacturer's recommendations.
Northern Blot-Total RNA from Hepa1c1c7 cells was isolated using TRIzol reagent. Twenty micrograms of total RNA from each sample were separated on a 1% agarose/formaldehyde gel and transferred overnight onto a nylon membrane. After UV-cross-linking, membrane was prehybridized for 4 h at 42°C in prehybridization buffer (6ϫ SSC, 5ϫ Denhardt's reagent, 0.5% SDS, 100 g/ml denatured salmon sperm DNA), and then probed overnight at 42°C with cyp1a1 cDNA probe labeled with [␣-32 P]dCTP using Radprime labeling systems (Invitrogen) at 1 ϫ 10 6 cpm/ml hybridization buffer (6ϫ SSC, 0.5% SDS, 100 g/ml denatured fragmented salmon sperm DNA, 50% formamide). After hybridization, the membrane was washed 3 ϫ 5 min in buffer I (2ϫ SSC, 0.5% SDS), 1 ϫ 15 min in buffer II (2ϫ SSC, 0.1% SDS), and then washed with buffer III (0.1% SSC, 0.1% SDS) at 65°C until the background is low. The wet membrane was exposed at Ϫ80°C overnight using Kodak film. As a control, the blot was stripped and re-probed with ␣-32 P-labeled cDNA for rat GAPDH. Plasmid that contained rat GAPDH cDNA (pBSSKII ϩ ) was obtained from Binas (Texas A&M University). Mouse cyp1a1 cDNA was obtained using RT-PCR using total RNA from Hepa1c1c7 cells. The PCR primers were: CCCACAGCACCA-CAAGAGATA and AAGTAGGAGGCAGGCACAATGTC. The PCR product was inserted to pGEM-T easy vector (Promega). The BamH1-HindIII fragment of GAPDH and PstI fragment of cyp1a1 were used as templates for labeling, respectively.
In Vivo Coimmunoprecipitation Assay-The coimmunoprecipitation assays were based on a published procedure with modifications (26,27). Hepa1c1c7 cells were maintained in 100-mm cell culture plates and when growth reached 80% confluence, the cells were treated with TCDD (10 nM) or Me 2 SO (vehicle control) for 60 min. Before harvest, the cells were washed twice with ice-cold phosphate-buffered saline, harvested by scraping, and collected by centrifugation at 600 ϫ g. Nuclei of the cells (two plates from each treatment) were isolated based on a published procedure (26). The isolated nuclei were lysed in buffer (20 mM Hepes, pH 7.4, 125 mM NaCl, 1% Triton X-100, 10 mM EDTA, 2 mM EGTA, 2 mM Na 3 VO 4 , 50 mM NaF, 20 mM ZnCl 2 , 10 mM sodium pyrophosphate, 1 mM PMSF, 1 mm DTT, 5 g/ml leupeptin) and centrifuged for 15 min at 12,000 ϫ g, and supernatant fractions were collected. For coimmunoprecipitation assays, the antisera were added to the lysate, and the binding reactions were performed at 4°C for 2 h on a rotary shaker. 30 l of GammaBind Plus Sepharose slurry (50% beads) (Amersham Biosciences) were added to precipitate the antibody-antigen complexes. The beads were washed three times in lysis buffer and then boiled in 2ϫ SDS sample buffer. The proteins were separated by 8% SDS-polyacrylamide gel. Proteins on the gel were transferred to nitrocellulose membranes (BioRad) and the membranes were blocked with 5% bovine serum albumin in TBST buffer (20 mM Tris-HCL, pH 7.6, 137 mM NaCl, 2.68 mM KCl, 0.05% Tween 20), and incubated with appropriate primary antibodies at 37°C for 60 min. Blots were washed three times with TBST, then incubated with a 1:2000 dilution of immunoaffinity-purified goat anti-rabbit IgG linked to alkaline phosphatase. Blots were washed three times with TBST and subsequently developed using NBT/BCIP (Sigma) as the substrate.
Yeast Two-hybrid Interaction Assays-Yeast two-hybrid assays were performed according to the Match Maker Gal4 two-hybrid user manual (Clontech). Human AhR cDNA was obtained by PCR amplification of pSport huAhR and the product was cloned into the trp ϩ yeast expression vector pGBT9 (Clontech) in-frame with the DNA binding domain of GAL4, resulting in the pGBT9AhR plasmid. Similarly, cyclin T1 cDNA fragments were cloned into the leu ϩ yeast vector pGAD424 (Clontech) in-frame with the GAL4 transactivation domain, resulting in plasmid pGAD424CycT1 (1-726), pGAD424CycT1 (1-250), and pGAD424CycT1 (233-726). As a positive control the human ARNT cDNA without the activation domain was also cloned into pGAD424 to yield pGAD424 ARNT. The primers used for generation of the cDNA fragment were: GGGCGGATCCCCATGGCGGCGACTACTGCCAAC and GGGCGTC-GACGGGAAATCTGGGCCAACATC. Assays for the interactions between AhR and cyclin T1 were performed as follow: Saccharomyces cerevisiae strain SFY526 was grown in YPD medium and transformed by electroporation with pGBT9AhR and pGAD424Cyc T1 plasmids. The transformation mixtures were plated on synthetic drop-out medium minus leucine and tryptophan. For ␤-galactosidase assays, minimal medium minus leucine and tryptophan was inoculated with single yeast colonies and grown overnight at 30°C to saturation. To activate the Ah receptor, ␤-naphthoflavone was added to fresh medium to a final concentration of 5 M, and the medium was inoculated with an aliquot of the saturated yeast cultures to an OD 600 around 0.2. Control cultures were treated with an equivalent amount of dimethyl sulfoxide. All cultures were grown 8 -12 h and harvested by centrifugation when OD 600 reached 1.0 -1.3. The cells were washed in Z buffer and after centrifugation, cells were resuspended in 300 l of Z buffer and lysed by freezing in liquid nitrogen and thawing at 37°C. Z buffer (0.7 ml) and ␤-mercaptoethanol (0.27 ml) were added to the cells. ␤-Galactosidase activity was determined by adding 0.16 ml o-nitrophenyl-␤-D-galactopyranoside (4 mg/ml in Z buffer), and reaction mixtures were incubated at 30°C. Cellular debris was pelleted by centrifugation, and the absorbance of supernatants was measured at 420 nm. Each determination was performed in triplicate.
GST Pull-down Analysis-[ 35 S]methionine-labeled full-length AhR protein was generated with a TNT-coupled Reticulocyte Lysate System (Promega) using the SP6 promoter-driven cDNA plasmid (phuAhR) as the template. PCR-generated cDNA fragments of cyclin T1 corresponding to amino acids 1-726, 1-250, and 233-726 were inserted in-frame into pGEX-5X-3 (Amersham Biosciences), yielding the expression plasmids for GST-cyclin T1 fusion proteins. The plasmids were expressed in E. coli (BL21), and fusion polypeptides were purified with the Bulk GST Purification Module (Amersham Biosciences) according to the manufacturer's instruction. Twenty micrograms of each fusion polypeptides (estimated by comparison with bovine serum albumin in an SDS-PAGE gel with Coomassie staining) was incubated with 10 l of radiolabeled AhR in a total of 100 l binding reaction buffer (20 mM Hepes (pH 7.9), 1% Triton X-100, 20 mM DTT, 0.5% bovine serum albumin, and 100 mM KCl) for 3 h at 4°C. After incubation, glutathione-Sepharose 4B beads were added and washed with the same buffer without bovine serum albumin three times. The bound proteins were eluted by boiling in the SDS-PAGE sample buffer, and resolved by 8% SDS-PAGE gel electrophoresis. The signals were detected by autoradiography.
Luciferase Reporter Gene Activity Assay-Luciferase assays were performed using the Luciferase Assay System (Promega). Briefly, the transfected cells were lysed in the culture plates with Reporter Lysis Buffer and the lysates centrifuged at maximum speed for 10 min in an Eppendorf microfuge. 10 l of the supernatant fraction were incubated with 50 l of luciferase substrate and relative luciferase activity determined with a luminometer (Turner Designs).
Chromatin Immunoprecipitation (ChIP) Assay-ChIP assay was performed based on published protocols (14,34) with modifications. Hepa1c1c7 cells were maintained in 10-cm plates under standard cell culture conditions. At 80% confluence, formaldehyde was added directed to the media to a final concentration of 1.0% for cross-linking, and the plates were incubated for 15 min at room temperature with gentle rocking. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 M. The plates were then rinsed twice with ice-cold phosphate-buffered saline. Cells were scraped off the plates and collected into 50-ml conical tubes by centrifugation (600 ϫ g for 10 min at 4°C). Pellets were washed once with 1ϫ phosphatebuffered saline containing 1 mM PMSF, resuspended in 2 ml of cell lysis buffer (5 mM PIPES, (pH 8), 1 mM EDTA, 0.5 mM EGTA, 85 mM KCl, 0.5% Nonidet P-40, 1 mM PMSF, 1 mM DTT, and 5 g/ml each of leupeptin and aprotinin), and incubated for on ice for 10 min. Cells were homogenized on ice using an A type Dounce homogenizer several times to aid the release of nuclei. The crude nuclei were collected by centrifugation (600 ϫ g for 10 min at 4°C) and then resuspended in nuclei lysis buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 0.5 mM EGTA, 1% SDS, 1 mM PMSF, 1 mM DTT, 5 g/ml each of leupeptin and aprotinin) and incubated again on ice for 10 min. The samples were then sonicated into DNA fragments of 0.5-1.5 kb and microcentrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was precleared by incubation with Staph A cells (2.5 g/per sample, Roche Applied Science) for 15 min at 4°C on a rotating platform and after centrifugation at 12,000 ϫ g for 5 min the supernatant was transferred to a clean tube. Appropriate antibodies (1 g) were added to the supernatant and then 25 l of precleared 50% protein A/G beads (Amersham Biosciences) were added to each tube. Final volume of each sample was adjusted to 400 l with IP dilution buffer (0.01% SDS, 1.1% Trition X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8.1, 167 mM NaCl, 100 g/ml sonicated salmon sperm DNA). The mixtures were incubated on the rotating platform at 4°C overnight. After incubation, the beads were collected by centrifugation at 5000 rpm for 1 min in a microcentrifuge, and pellets were washed three times with 1 ml of 1ϫ Dialysis buffer (2 mM EDTA; 50 mM Tris-Cl, pH 8.0; 100 g/ml sonicated salmon sperm DNA) and 5 times with 1 ml of IP Wash buffer (100 mM Tris-Cl, pH 9.0, 500 mM LiCl, 1% Nonidet P-40, 1% deoxycholic acid) for 10 min with rotation. After the wash, 200 l of digestion buffer (50 mM Tris, pH 8, 1 mM EDTA, 100 mM NaCl, 0.5% SDS, 100 g/ml proteinase K) was added to each tube, and tubes were incubated at 55°C for 3 h and followed by 6 h at 65°C to reverse the cross-linking. The samples were extracted once with phenol-chloroform-isoamyl alcohol and once with chloroform and then ethanol-precipitated in the presence of 20 g of glycogen overnight. The precipitated pellets were collected by centrifugation at top speed and the pellets were resuspended in 20 l of TE buffer. Aliquots from each tube were used for PCR amplification. The PCR products were either separated on 1.2% agarose gels and visualized by ethidium bromide staining or separated by 6% polyacrylamide gel electrophoresis. In later cases, one of the PCR primers was end-labeled by 32 P, and signals of the PCR products were visualized by autoradiography. The primer pairs for PCR of the cyp1a1 promoter region were: Ϫ1100 to Ϫ770, TTAAGAGCCT-CACCCACGG and GCGGGTGCAGAGCTATCTAAG; Ϫ285 to ϩ66, TT-TCCTCAAACCCCTCCCTC and GAAGTGAAGAGTGTTCTCTAGGAC.

Ligand-activated AhR Recruits Cyclin T1, CDK9, and RNA
Pol II to the cyp1a1 Promoter with Concomitant Phosphorylation of RNA PII CTD-To investigate the elongation control of cyp1a1 transcription, we used in vivo chromatin immunoprecipitation assay to analyze the recruitment of the cyclin T1, CDK9, and RNA PII to the cyp1a1 promoter and the residuespecific phosphorylation of the C-terminal domain (CTD) of the RNA PII in response to AhR activation. In Hepa1c1c7 cells, 30 min after TCDD treatment, AhR and ARNT began to associate with the cyp1a1 regulatory region followed by the recruitment of RNA PII at the promoter region (30 -60 min after TCDD treatment) (Fig. 1). P-TEFb complex was recruited to the promoter region at 60 min and coincided with strong phosphorylation of serine 2 of the CTD. Interestingly, although the strongest phosphorylation of serine 2 of RNA PII CTD was detectable at 60 min, strongest phosphorylation of serine 5 was detected after serine 2 phosphorylation. In addition, the increases of serine 5 phosphorylation correlated with decreases of serine 2 phosphorylation, suggesting that phosphorylations of serine 2 and serine 5 are controlled by separate mechanisms (Fig. 1).
Inhibition of Kinase Activity of P-TEFb Selectively Blocks cyp1a1 Transcript Elongation by RNA Pol II but Not the Assembly of the Preinitiation Complex-Phosphorylation of the CTD of RNA PII is associated with transition from the initiation to elongation stage of transcription (16). To further investigate the role of P-TEFb in cyp1a1 elongation control, we used the specific P-TEFb inhibitor DRB, to test the involvement of P-TEFb in cyp1a1 elongation. DRB treatment (30 M, 2 h) inhibited phosphorylation of serine 2 and reduced serine 5 phosphorylation of CTD and prevented the RNA Pol II from transcribing the cyp1a1 distal region as determined by ChIP assay (compare lanes 5 and 6 in Fig. 2A). It appeared that serine 2 was more sensitive to inhibition by DRB than serine 5. Interestingly, in contrast to the distal region, the association of the RNA PII with the cyp1a1 promoter region was not affected by DRB treatment (compare lanes 3 with 6 in Fig. 2A in Pol II rows). These results suggest that while elongation is sensitive to DRB treatment, assembly of the preinitiation complex is a distinct step and is resistant to CDK9 inhibition. As a confirmation of effects of inhibition of CTD phosphorylation on AhRregulated gene expression, we transiently transfected pGL3-CYP1A1-Luc in Hepa1c1c7 cells and treated the transfected cells with TCDD and DRB. The pGL3-CYP1A1-Luc is a luciferase reporter gene driven by the upstream sequences (Ϫ1395 to ϩ1) of mouse cyp1a1 and is activated by AhR ligands. Similar reporter gene plasmid has been constructed and used by other investigators (12). DRB treatment markedly suppressed the AhR-driven luciferase reporter gene activity (Fig. 2B).
To further analyze the effects of AhR activation on cyp1a1 transcription initiation and elongation, we performed RNase protection assays (RPA) with two different RNA probes to independently evaluate levels of initiated and elongated transcripts. Whereas the promoter-proximal probe measured total levels of transcription, the distal probe measured total levels of runoff transcription. When Hepa1c1c7 cells were treated with TCDD (10 nM, 2 h), robust transcription could be detected as the marked increases in the levels of transcripts at distal as well as the proximal promoter regions (Fig. 3A, compare lanes  1 and 2). Critically, DRB treatment reduced levels of total runoff transcripts to the basal level but did not affect the total level of transcription at the proximal promoter region (Fig. 3A,  lane 3), suggesting the RNA PII complex was still at the proximal promoter region engaging in abortive transcription, which generates only short transcripts.
The results of the RNase protection assays are in agreement with the promoter occupancy by RNA PII at the cyp1a1 promoter and association of RNA PII at distal regions in response to TCDD and DRB treatments (Fig. 2). As confirmation for the RNase protection results, we measured the mRNA levels of the treated samples by Northern blot analysis. Inhibition of P-TEFb by DRB completely suppressed the TCDD-induced mRNA of cyp1a1 in Hepa1c1c7 cells. Curiously, the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appeared to be much less sensitive to DRB treatment, although this could be due to the longer half-life of GAPDH mRNA (Fig. 3B).
Physical and Functional Interactions Between AhR and P-TEFb in Hepa1c1c7 Cells-Recruitment of P-TEFb complex to the cyp1a1 promoter in response to AhR activation suggests that there may be a direct interaction between AhR and P-TEFb. To test this possibility, we performed coimmunoprecipitation assays to detect potential complex formation between AhR and cyclin T1 in Hepa1c1c7 cells. Cyclin T1 is a regulatory subunit of P-TEFb and has been shown to directly associate with several transactivators (28 -31). The results of our experiment showed that AhR formed complex in vivo with cyclin T1 as determined by sequential immunoprecipitation and Western blot analysis, using antibody against cyclin T1 to immunoprecipitate the complex. The presence of AhR in the precipitated complex was detected by Western blot analysis with antibody against AhR (Fig. 4A). To analyze the functional consequences of the P-TEFb/AhR interaction, we transiently coexpressed pGL3-CYP1A1-Luc and cyclin T1 and/or CDK9 in Hepa1c1c7 cells. Transient expression of cyclin T1 markedly enhanced the dioxin-induced luciferase reporter gene activity (Fig. 4B). Without activation by agonist, expression of cyclin T1 or CDK9 alone was ineffective in enhancing the pGL3-CYP1A1-Luc reporter gene activity, which is consistent with the role of P-TEFb in the control of elongation downstream from the agonist-induced initiation. Transient expression of CDK9 in Hepa1c1c7 cells also enhanced the activation of pGL3-CYP1A1-Luc gene expression, although the enhancement by CDK9 was not as strong as that by cyclin T1. No further enhancement of the reporter gene activity was archived by coexpression of CDK9 FIG. 1. Activation of AhR by TCDD leads to sequential recruitment of AhR complex, RNA PII, and P-TEFb to the cyp1a1 promoter with differential phosphorylation of RNA PII CTD. ChIP assays were performed to determine the time-dependent promoter occupancy following TCDD treatment of Hepa1c1c7 cells. Antibodies (Ab) used for ChIP assay are indicated on the side of the panels. The associations of AhR complex with the cyp1a1 regulatory region was detected using antibodies against AhR and ARNT (lanes 1-6). The sequential recruitment of RNA PII, P-TEFb, and differential phosphorylations of RNA PII CTD were analyzed using antibodies against RNA PII, phosphoserine 2, 5 of CTD, cyclin T1, and CDK9 (lanes 7-12). The upstream regulatory and promoter regions are schematically illustrated (bottom panel).

FIG. 2. DRB inhibits the phosphorylation of RNA PII CTD and prevents the RNA PII from transcribing cyp1a1 sequences. A,
Hepa1c1c7 cells were treated with either TCDD (10 nM, 2 h) or cotreated with TCDD (10 nM, 2 h) and DRB (30 M, 2 h). ChIP assays were performed to determine the association of RNA PII with different regions of the cyp1a1 gene as well as the phosphorylation status of serines 2 and 5 of the RNA PII CTD after the treatments. B, DRB suppresses transcription by AhR as determined by luciferase reporter gene assay. Hepa1c1c7 cells were transfected with pGL3-CYP1A1-Luc. The transfected cells were treated with TCDD and/or DRB as indicted for 18 h. Luciferase reporter gene activity was determined using a luminometer after the treatment.
with cyclin T1. These results suggest that P-TEFb (particularly cyclin T1) plays a role in regulating the AhR-mediated gene expression through physical and functional interaction with AhR receptor complex.
Phosphorylation of RNA Pol II CTD at the cyp1a1 Promoter Is Co-regulated by AhR and NF-B-In earlier studies, we found mutual repression between AhR and NF-B signal pathways and this antagonism is reflected in the histone H4 acetylation and deacetylation at the cyp1a1 upstream regulatory region (14,27). Since the results of our current study suggest that transcription elongation is also a regulatory step for cyp1a1 gene expression, we are interested in analyzing if this step is subject to the positive and negative transcripitonal regulation imposed by AhR and NF-B. We analyzed the serine 2 and serine 5 phosphorylation of RNA PII CTD at the cyp1a1 promoter in response to TCDD and TNF-␣ treatments using ChIP assay. As expected, we found that both serine 2 and serine 5 were phosphorylated in response to TCDD treatment. However, TNF-␣ treatment selectively inhibited serine 2 phosphorylation while TCDD-induced serine 5 phosphorylation was not significantly suppressed (Fig. 5). These results are consistent with the reported glucocorticoid receptor (GR)-mediated suppression of NF-B where GR differentially inhibits RNA PII CTD serine 2 phosphorylation at the IL-8 and ICAM promoters, which are regulated by NF-B (34).
AhR and Cyclin T1 Interact Directly as Determined by Yeast Two-hybrid as Well as GST Pull-down Assays-The results from the coimmunoprecipitation assays (Fig. 4A) suggest that AhR associate with the cyclin T1 in vivo. To further characterize these interactions, we performed yeast two-hybrid assays as well as GST pull-down assays to analyze the interaction domains within cyclin T1 that mediate its interaction with AhR (Fig. 6). Human cyclin T1 is a polypeptide (726 amino acids) consisting of a N-terminal cyclin box (amino acids 1-250), which is conserved among cyclins T1, T2, and K, and the C-terminal domain, which contains a coiled-coil motif (amino acids 379 -430), a histidine-rich region (amino acids 506 -530), and a PEST sequence (amino acids 709 -726) (Fig. 6A) (32). The C-terminal domain is known to interact directly with RNA PII and is essential for elongation activity of P-TEFb (33). To perform yeast two hybrid assay, full-length human AhR cDNA was inserted in-frame into the yeast two hybrid bait vector (pASII, Clontech) and cyclin T1 cDNAs were inserted into pGAD 424 plasmid (Clontech). The chimeras were transfected into S. cerevisiae SFY56 and transfectants were grown in liquid culture and assayed for ␤-galactosidase activity after treatment with AhR ligand ␤-naphthoflavone (Fig. 6B). About 2-3fold induction by ␤-naphthoflavone was observed when AhR was co-expressed with pGAD424 (empty vector). However, coexpression of AhR with full-length cyclin T1 resulted in significant ligand-induced induction (about 8-fold). The interaction between cyclin T1 and AhR is mainly mediated by the C terminus of the cyclin T1 (CycT1 233-726) and the N terminus of cyclin T1 interacted marginally. As a positive control, coexpression of AhR with ARNT (a known interactive partner of AhR) resulted in high levels of ␤-galactosidase activity upon induction with BNF. These results suggest that cyclin T1 interacts directly with AhR in vivo in a ligand-dependent manner, and the association is mediated by the C-terminal domains of cyclin T1. Next we performed GST pull-down assays to further analyze the interaction between AhR and cyclin T1. In vitro translated and radiolabeled AhR protein was incubated with cyclin T1 deletion mutants fused with GST to determine the domains involved in the association. In agreement with the results of yeast two-hybrid assays, AhR was associated with full-length cyclin T1 and the C-terminal domain of the cyclin T1, while the interaction between AhR and the N terminus of the cyclin T1 was insignificant (Fig. 6C). DISCUSSION In 1992, Morgan and Whitlock (15) reported that TCDD induced changes in the nucleosomal positions in both the promoter and transcribed regions of mouse cyp1a1 gene with an interesting difference: the nucleosomal changes in the transcribed regions were sensitive to inhibition by actinomycin D, while the TCDD-induced nucleosomal changes in the promoter were insensitive to the same treatments. Actinomycin D binds to DNA and blocks the movement of RNA PII, thus inhibiting transcription. These results suggest that within a single cyp1a1 gene, the transcription is regulated by two interconnected but distinct mechanisms involving controls of initiation and elongation. In the last two decades, the transcriptional processes leading to initiation have been investigated extensively, but little is known about the process of transcription elongation of cyp1a1.
In this study, we have investigated transcription elongation control of cyp1a1 expression in response to AhR activation. Using the ChIP assay, we found that TCDD treatment in Hepa1c1c7 cells induced binding of the AhR complex to the regulatory region of cyp1a1 followed by promoter occupancy by RNA PII and recruitment of P-TEFb to the promoter region. The association of P-TEFb with the promoter was correlated with strong phosphorylation of serine 2 of the RNA PII CTD (Figs. 1 and 2). Intriguingly, although both serine 2 and serine 5 are phosphorylated in response to AhR activation, there is an interesting time lag between the serine 2 and serine 5 phosphorylation at the cyp1a1 promoter with serine 2 being phosphorylated in advance of serine 5 (Figs. 1 and 5). It is not known why strongest phosphorylation of serine 5 was detected after serine 2 phosphorylation at the cyp1a1 promoter. Using synthetic peptides containing 6 tandem repeats of YSPTSPS, Ho and Shuman (25) showed that phosphorylation of serine 5 induced capping of RNA transcript in vitro. Since capping is an early event during transcript elongation, it is somewhat surprising that our results with ChIP assay showed serine 5 was phosphorylated after serine 2 in vivo. Conceivably, because the time required for serine 5 phosphorylation associated with capping is much shorter than serine 2 phosphorylation associated with elongation, the serine 5-phosphorylated RNA PII moved away from the proximal promoter region and was not detected till the surge of serine 5 phosphorylation from second round of transcription. Interestingly, we observed that TCDDinduced serine 2 phosphorylation decreased over time and the decreases correlated with increases of the serine 5 phosphorylation ( Figs. 1 and 5), suggesting that phosphorylation of serine 2 and serine 5 is controlled by distinct mechanisms. It has been shown that CDK7 of TFIIH phosphorylates serine 5 while CDK9 of P-TEFb phosphorylates serine 2, the differential phosphorylation of serines 2 and 5 of CTD at the cyp1a1 promoter may be due to different kinases with different substrate specificity (20 -23). Thus, the CTD appears to function as a molecular switchboard where hyper-and hypo-phosphorylations sig- FIG. 5. Differential inhibition of TCDD-induced RNA PII CTD phosphorylation by TNF-␣. ChIP assays were performed on Hepa1c1c7 cells treated with TCDD (10 nM) and TCDD (10 nM) ϩ TNF-␣ (5 ng/ml). Phosphorylations of RNA PII CTD were analyzed by using either a phosphoserine 5 monoclonal antibody (H14, BAbCO) and a phosphoserine 2 monoclonal antibody (H5, BAbCo). PCR amplification was performed for 28 cycles using primer designed to amplify the promoter region of mouse cyp1a1. The PCR products were visualized by autoradiography.
FIG. 6. Cyclin T1 interacts directly with AhR in yeast twohybrid and GST pull-down assays. A, top panel, schematic illustration of the domains of cyclin T1 and deletion mutants used for yeast two-hybrid and GST pull-down assays. B, yeast two-hybrid assay. Expression plasmids of AhR and cyclin T1 were introduced into yeast cells, and positive transfectants were selected for assays of ␤-galactosidase activity after treatment with ␤-naphthoflavone for induction of the AhR as described under "Materials and Methods." C, GST pull-down analysis of the domains of cyclin T1 that interact with AhR. The radiolabeled AhR was incubated with various bacterial expressed GST-cyclin T1 fusion peptides as indicated in the upper part of the top panel. Lane i shows 1:10 of the input radiolabeled AhR. Arrow to the left points to the AhR band in the autoradiograph. The quantity of the fusion peptides was estimated by separation with SDS-PAGE and stained with Coomassie Blue (lower panel).
nify "on and off " states and differential phosphorylation of serine 2 and serine 5 may indicate the different stages of transcription at which RNA PII functions.
Interestingly, DRB treatment suppressed phosphorylation at the cyp1a1 promoter and blocked RNA PII from traveling downstream from the promoter and completely inhibited cyp1a1 mRNA synthesis (Figs. 2 and 3). It has been shown by in vitro experiments that DRB inhibits the kinase activity of CDK9 (18), which phosphorylates the serine 2 of RNA PII CTD. Our in vivo results with DRB-treated Hepa1c1c7 cells showed that phosphorylation of serine 2 and to a lesser extent serine 5 was inhibited (Fig. 2); therefore, supporting the role of CDK9 as a phosphorylating kinase for serine 2 of CTD (23). Treatment of Hepa1c1c7 cells with DRB inhibited the run-off transcript and blocked the RNA PII from transcribing the distal region of cyp1a1 without affecting the assembly of the preinitiation complex. Taken together, these results indicate that elongation is distinct regulatory step in cyp1a1 expression.
It has been shown that P-TEFb can directly associate with transactivators including NF-B (29), CIITA (28), c-Myc (30), androgen receptor (31), and nuclear receptor co-activator p160 (35), thereby directly bridging transactivators with the elongation controlling mechanism. The results of our study showed that AhR interacted with P-TEFb by forming complex in the nucleus. Transient expression of the P-TEFb significantly enhanced the AhR-directed luciferase reporter gene expression suggesting that elongation is an important regulatory step in cyp1a1 gene expression. We characterized the association between AhR and cyclin T1 using three complementary approaches: by coimmunoprecipitation assay, we found AhR/cyclin T1 complex formation in the nucleus of mouse liver cells and by yeast two-hybrid assays and GST pull-down assays we showed a direct binding of AhR with cyclin T1. Interestingly, AhR selectively associated with the C-terminal domain of cyclin T1, which contains coiled-coil, His, and PEST motifs. It has been shown that the C terminus of cyclin T1 directly interacts with CTD of RNA PII (33). Furthermore, when the C terminus of cyclin T1 is tethered to DNA through the Gal4 DNA binding domain, the Gal4-cyclin T1 can activate gene transcription by binding to the Gal4 enhancer sequences placed either upstream or downstream from a promoter (33), suggesting that one function of cyclin T1 is to connect the enhancer-bound transactivator with the RNA PII. Similarly, AhR may be capable of conveying the transactivating signal over the long distance to the promoter-bound RNA PII through its ability to interact with P-TEFb.
The results of this study suggest a model in which AhR regulates the transcription elongation by recruiting the P-TEFb complex to the promoter region of cyp1a1 gene through direct protein-protein interaction (Fig. 7). XRE-bound AhR recruits P-TEFb to the vicinity of cyp1a1 promoter where the CDK9 of P-TEFb phosphorylates the CTD of RNA PII, thereby releasing it from the arrested state for processive transcription. This model also suggests that transcription elongation is an important point of convergence for the interactions between the AhR and different transcription factors, since the activity of P-TEFb is regulated by many signaling mechanisms. Furthermore, as many therapeutic agents have now been developed targeting P-TEFb, their effects on AhR-regulated xenobiotic detoxification need to be analyzed (36).
The intricacy of transcriptional regulation of cyp1a1 expression continues to unravel. In addition to the transcriptional processes that lead to assembly of the preinitiation complex at the promoter region, AhR also regulates the transcription elongation by interaction with the general elongation factor P-TEFb, thereby regulating elongation through phosphorylation of the CTD of RNA PII.

FIG. 7. A working model for AhR-regulated transcription elongation.
Ligand-activated AhR binds to XRE sequences, which leads to the assembly of the preinitiation complex at the cyp1a1 promoter. Through its ability to associate with cyclin T1, the XRE-bound AhR also brings the P-TEFb complex to the vicinity of the cyp1a1 promoter. The CDK9 subunit of P-TEFb phosphorylates the CTD of RNA PII resulting in processive elongation. The C terminus of the cyclin T1 has been shown to interact directly with the RNA PII CTD (33) (not shown here).