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Department of Internal Medicine and Sealy Center for Molecular Sciences, The University of Texas Medical Branch, Galveston, Texas 77555-1060
Address all correspondence and requests for reprints to: Dr. Allan R. Brasier, Division of Endocrinology, MRB 8.138, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1060. E-mail: arbrasie{at}utmb.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Although AGT expression is regulated by factors governing cardiovascular homeostasis, the AGT gene also retains features of an hepatic acute phase reactant (APR). AGT arose as a distant gene duplication event that gave rise to the serine protease inhibitor family including 
1-antitrypsin and 1 antichymotrypsin (5). In this regard, AGT expression is controlled by inflammatory cytokines and glucocorticoids at the transcriptional level (reviewed in Ref. 2), features characteristic of type II APRs (6). Recently we have investigated the mechanism for up-regulation of hAGT gene expression by IL-6 alone and in combination with glucocorticoids. Although glucocorticoids do not directly activate the hAGT gene, they enhance IL-6 signaling by up-regulating IL-6 receptor abundance (7). IL-6 stimulates hAGT through an approximately 230-nucleotide domain containing three functionally distinct cis-regulatory elements, termed acute-phase response elements 1–3 [hAPRE1–3 (Ref. 8)]. The upstream hAPRE1 is necessary and sufficient to confer IL-6 responsiveness by binding members of the signal transducers and activators of transcription [STATs, (9)]. STATs are latent cytoplasmic transcription factors activated via a tyrosine phosphorylation cascade growth factor or cytokine receptor stimulation (reviewed in Ref. 9). Activated STATs form homo- or heterodimers through intermolecular SH2 phosphotyrosine interactions and are subsequently translocated into the nucleus where they bind to specific DNA sites in responsive genes (10). Although IL-6 induces tyrosine phosphorylation, nuclear translocation, and DNA binding of both STAT1 and -3 subunits, only STAT3 is predominantly responsible for mediating the IL-6-dependent hAGT activation (8). Expression of a dominant negative STAT3 isoform, mutated in the COOH-terminal tyrosine phosphoacceptor site (Tyr705Phe) critical in STAT3 translocation and DNA binding (9), completely inhibits IL-6-inducible hAGT reporter activity. In contrast, expression of a dominant negative STAT1, even to levels greater than that of the dominant negative STAT3, has no effect on hAGT-dependent transcription; moreover, a downstream hAPRE, hAPRE3, which binds primarily STAT1, is not IL-6 inducible (8). Recent studies have shown that the activities of STAT 1, -2, -5, and -6 isoforms can be modulated by their interactions with non-DNA-binding proteins such as coactivators p300 and CREB (cAMP response element binding protein)-binding protein (CBP). p300, cloned through its interaction with adenovirus E1A protein (11), and CBP, identified as a specific protein binding the kinase-inducible domain of CREB (12), are functional homologs referred to as p300/CBP. p300/CBP regulates gene expression as a molecular integrator, bridging sequence specific DNA-bound transcription factors and basal transcription machinery (13, 14) with associated chromatin-modifying activities including intrinsic histone acetyltransferase (HAT) activity (15) thought to open chromatin structure, allowing additional proteins to bind to DNA and activate transcription.
The ability of STAT3 to weakly bind p300/CBP relative to other STAT members (16) has raised the question of whether STAT3-mediated transcription truly requires p300/CBP. In this report, we examine the role of p300/CBP coactivator and HAT activity in STAT3mediated transactivation of the hAGT gene in HepG2 cells. Surprisingly, we find that STAT3 interacts with p300/CBP in the nuclei of IL-6-stimulated HepG2 cells. STAT3 interaction with p300/CBP requires an intact STAT3 COOH terminus, as a STAT3 isoform lacking the COOH terminus functions as a dominant negative inhibitor of hAGT expression and fails to associate with p300/CBP. p300/CBP HAT activity is required for promoter activation as expression of p300 defective in its HAT activity functions as a dominant-negative inhibitor of hAGT expression. Finally, in chromatin immunoprecipitation assays, IL-6 stimulation increased histone acetylation of the endogenous hAGT gene. We conclude that STAT3 associates with p300/CBP in HepG2 cells through its COOH terminus. p300/CBP association and HAT activity are both required for efficient hAGT promoter activation, demonstrating p300/CBP is an important coactivator for this type II APR.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Although 12S E1A slightly reduced the 18S signal as well, the effect was greater on hAGT mRNA abundance seen after normalizing transcript signal to 18S (Fig. 1B). This observation indicated a possible role of the p300/CBP coactivators in constitutive and inducible hAGT expression.
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), where normalized luciferase activity was reduced to 40% with the lowest amount (0.05 µg) of cotransfected pCMVE1A expression plasmid and plateaued at 20% of control activity with higher amounts (>0.2 µg). Although normalized values are shown, 12S E1A expression had no effect on the internal control alkaline phosphatase activity.
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2–36, an NH2-terminal deletion mutant defective in p300/CBP binding but still capable of binding Rb (11). Separate plates were left unstimulated or stimulated with IL-6 (8 ng/ml, 24 h) or IL-6 (8 ng/ml, 24 h) + Dex (100 nM, 24 h) before harvest and assay of reporter gene activity. In the presence of empty expression vector (pcDNA3) and relative to unstimulated transfectants, IL-6 stimulation induced a approximately 5-fold increase in normalized luciferase activity; Dex treatment further induces reporter activity to approximately 10-fold over unstimulated levels (Fig. 2B
). Overexpression of wild-type 12S E1A (0.4 µg pCMV12SE1A) not only inhibited basal, but also completely inhibited both IL-6- and IL-6 + Dex-stimulated promoter activity (Fig. 2 B, middle lanes). By contrast, overexpression of the p300/CBP-binding defective E1A 2–36 had a neutral effect on basal or IL-6-stimulated activity (Fig. 2B, right lanes). In the case of IL-6 + Dex, E1A 2–36 expression produced a further stimulation; this phenomenon has been previously described with other p300-dependent promoters (19, 20). Together, these data indicate a requirement for the p300/CBP coactivator in the constitutive and inducible activity of the hAGT promoter.
The human angiotensinogen promoter contains three tandemly repeated motifs, termed human acute phase response elements (hAPREs), which are found to be necessary and sufficient for IL-6 induction and glucocorticoid synergism. Among these three elements, hAPRE1 (which binds to the latent STAT1 and -3 transcription factors) is the only bona fide IL-6-inducible enhancer (8). To determine whether E1A could inhibit hAPRE1-driven transcription, basal and stimulated reporter gene activity of a promoter driven by five copies of hAPRE1 was determined in the presence of wild-type 12S E1A and E1A 2–36 coexpression. As previously reported, IL-6 stimulation of (hAPRE1)5-hAGT/LUC reporter causes 45-fold induction of luciferase activity and a 90-fold induction in the presence of Dex (Fig. 2C). Expression of 12S E1A inhibits IL-6-inducible activity by about 65% and IL-6 + Dex-inducible activity by approximately 85%. Conversely, expression of E1A 2–36 had an approximately 30% inhibition of IL-6-stimulated activity and a negligible effect on the IL-6 + Dex-stimulated activity (Fig. 2C). These observations strongly suggested that the STAT3 binding site was an E1A-sensitive element in the hAGT promoter and required p300/CBP association for its inducible activity.
p300/CBP Potentiates Basal and Inducible hAGT Promoter Activity
p300/CBP is thought to be found in a limiting concentration in eukaryotic cells (21, 22). To show more directly that the E1A-mediated inhibition of hAGT promoter was due to inactivation of p300 or related factors, we examined whether overexpression of p300 or CBP could further activate the hAGT promoter activity. Ectopic expression of full-length human p300 increased the stimulated IL-6 and IL-6 + Dex-induced wild-type promoter activity by about 2-fold in each case (Fig. 3A). Ectopic expression of CBP produced a similar effect (Fig. 3B). Together, these results indicate that p300/CBP is a candidate coactivator controlling hAGT promoter activity.
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HepG2 cells were transiently transfected with V5 epitope-tagged STAT1 and STAT3 expression vectors. Transfected cells were isolated after IL-6 stimulation and whole-cell extracts (WCEs) prepared. Equivalent STAT expression was confirmed by Western blot for the common V5 epitope (Fig. 4A, top, compare lane 1 vs. 2). Nondenaturing coimmunoprecipitation with p300 antibody was done on V5-STAT1- and V5-STAT3-expressing extracts and analyzed for the abundance of each STAT isoform using antibody recognizing the common V5 epitope (Fig. 4A, bottom). This result shows that both STAT3 (lane 2) and STAT1 (lane 1) qualitatively associate with p300 in HepG2 cells. A similar coimmunoprecipitation study was performed on endogenous protein (Fig. 4B) with unstimulated and IL-6-stimulated HepG2 cell nuclear extract (NE) to determine whether the association of STAT3-p300 is stimulus dependent. Unstimulated (lanes 1–3) and IL-6-stimulated (lanes 4–6) NEs were immunoprecipitated with rabbit preimmune serum, anti-STAT3, or anti-p300 antibodies, and immune complexes were analyzed for the presence of STAT3 (Fig. 4B). IL-6 induces strong STAT3 nuclear translocation (compare lane 5 with lane 2, Fig. 4B). Moreover, STAT3 interacts with p300 in IL-6-stimulated but not in unstimulated NE (compare lane 4 with lane 3, Fig. 4B), indicating that the interaction is primarily stimulus dependent. To determine whether STAT3 interacts with CBP (a p300 homolog), IL-6-stimulated NE of HepG2 cells was immunoprecipitated with anti-CBP antibody, and the association of STAT3 was detected by Western immunoblot. STAT3, not detectable in the preimmune immunoprecipitation, was readily detectable in the STAT3- as well as the p300- and CBP-immune complexes (Fig. 4C). As additional confirmation of STAT3-p300 association in cellulo, the presence of p300 protein was detected in the STAT3-immune complex (Fig. 4C, lower panel). These data indicate that endogenous STAT3 interacts with p300 and CBP upon IL-6 stimulation.
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TA (1–715) with p300. After IL-6 stimulation, WCEs from a homogeneous population of transfected cells (Materials and Methods) were separately immunoprecipitated with anti-FLAG or anti-p300 antibodies, and presence of STAT3 was detected by Western immunoblot (Fig. 5A). Although full-length STAT3 and STAT3 TA were expressed at approximately similar levels, STAT3 TA does not detectably interact with p300 (compare lanes 2 and 4, Fig. 5A).
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TA was capable of transactivating target genes or functionally interacting with p300. For this we first assayed whether GAL4-STAT3 (1–770) or GAL4-STAT3 TA (1–715) could transactivate GAL4 binding sites (UAS/LUC) in HepG2 cells. Although GAL4-STAT3 (1–770) transactivated UAS/LUC, GAL4-STAT3 TA (1–715) could do so only weakly (Fig. 5B). To confirm that both GAL4 chimeras were expressed at similar levels, Western immunoblots were performed on a homogeneous population of transfected cells where similar levels of expressed proteins were seen (Fig. 5B, inset). Next, to measure specifically p300/CBP-dependent transcription, we employed an established GAL4 two-hybrid assay (16, 26). In this assay, the ability of a transactivation-competent p300 fusion protein (created by fusing p300 with the potent transactivating domain of HSV VP16) to activate UAS/LUC was tested with GAL4 (1–147), GAL4-STAT3 (1–770), or GAL4-STAT3 TA (1–715). p300/VP16 was unable to transactivate UAS/LUC alone, but strongly transactivated UAS/LUC in the presence of GAL4-STAT3 (1–770) (Fig. 5C). Consistent with the lack of physical interaction between STAT3 TA and p300, p300/VP16 was unable to transactivate UAS/LUC in the presence of GAL4-STAT3 TA (1–715). Together, these data indicate that STAT3 associates with p300/CBP in a mechanism requiring an intact COOH terminus (amino acids 715–770).
STAT3TA, Deficient in p300/CBP Binding, Is a Dominant Negative Inhibitor of hAGT Expression
To determine whether p300/CBP binding was required for inducible transcription, we determined the effect of expressing a 55-amino acid COOH-terminal STAT3 truncation (lacking amino acids 715–770), STAT3 TA, on hAGT-driven reporter activity. STAT3 TA corresponds to the naturally occurring splice variant STAT3ß and functions as a dominant-negative inhibitor by competing for DNA binding with the endogenous full-length STAT3 [containing the transactivating domain, (8, 27)]. In this experiment, HepG2 cells were transfected with either native -991/+22 hAGT/LUC or the multimeric (hAPRE1)5-hAGT/LUC reporter with increasing amounts of STAT3 TA and stimulated with IL-6 or IL-6+Dex (Fig. 6). For the native -991/+22 hAGT/LUC reporter, STAT3 TA expression vector inhibited both basal and IL-6-induced reporter activity to approximately 20% of that seen with empty expression vector in a dose-dependent manner. A virtually superimposable dose-dependent inhibition was observed for the (hAPRE1)5 hAGT/LUC reporter gene (Fig. 6B). These data indicate that STAT3TA is a dominant negative inhibitor of hAPRE-dependent transcription, confirming our earlier studies with the STAT3 (Tyr705Phe) mutant (8).
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). Together, these data indicated that STAT3 inducibly associated with p300/CBP (Fig. 4) and HAT activity (Fig. 7A). We next determined the functional role of p300/CBP HAT activity in basal and stimulated induction of the hAGT promoter by cotransfection of a p300 expression vector encoding a mutant defective in acetyl coenzyme A binding and HAT activity (p300Hm) (28). As shown in Fig. 7B, expression of increasing concentrations of p300Hm greatly reduced the normalized hAGT/LUC reporter activity in a dose-dependent manner. At 1.5 µg of p300Hm expression vector, basal, IL-6-, and IL-6 + Dex-stimulated activity was 76% and 79% that produced by empty expression vector. The effect on basal and stimulated reporter activity was superimposable with that produced by 12S E1A (cf. Fig. 2). Thus we conclude that p300 HAT activity is essential for hAGT promoter activation.
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) and by PCR incorporation of radionucleotide (Fig. 8B); both are shown. Comparison of Fig. 8A (lanes 1 and 2) and 8B (lanes 1 and 2) shows that the specific 538-bp hAGT promoter sequence was significantly increased (3-fold) compared with that of IL-6untreated cells. Importantly, there was no starting differences in hAGT abundance in the IL-6-treated and control cellular extracts before immunopreciptation (Fig. 8A, lanes 3 and 4); moreover, immunoprecipitates in the absence of antiacetylated H4 antibody or in the presence of nonimmune IgG did not amplify any PCR products (data not shown). Similar experiments showed that the addition of Dex to IL-6 did not detectably increase the amount of chromatin-associated H4, compared with IL-6 alone (data not shown). Together these data indicate that IL-6 induces a relative increase in histone H4 acetylation on the hAGT promoter.
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). hAGT expression remained elevated until 72 h, after which it declined to control values. Together, these indicates that inhibition of histone deacetylation is necessary and sufficient for hAGT gene activation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have recently demonstrated that hAGT is a type II APR regulated by IL-6 and glucocorticoids (8). This type II APR is activated through the STAT family of transactivators, latent transcription factors that are activated by tyrosine phosphorylation in response to cytokine or growth factor stimulation of cells. Recent studies have indicated that most members of STAT proteins (STAT1, -2, -5, and -6) recruit coactivators as a mechanism to couple binding to changes in RNA polymerase II activity on target genes (21, 26, 31, 32). Here we show that basal and inducible hAGT expression is dependent on STAT3-p300/CBP association and recruitment of HAT activity. Our observations indicate that p300/CBP interaction is essential for transcription of the hAGT promoter in HepG2 cells because: 1) expression of a form of adenovirus E1A protein that strongly binds to the CH3 domain of p300/CBP strongly reduces the basal and STAT3-mediated transactivation of hAGT promoter in transient transfection assay, whereas a p300/CBP-binding mutant, 2–36 E1A, does not; 2) ectopic expression of p300/CBP activated hAGT gene expression, an observation consistent with p300/CBP limiting abundance in eukaryotic cells (21, 22); 3) STAT3-p300/CBP and HAT association can be detected by coimmunoprecipitation assay; 4) STAT3 mutants unable to bind p300/CBP are dominant negative inhibitors; and 5) inhibition of HDAC activity is sufficient for hAGT expression.
STATs are modular proteins composed of an NH2-terminal domain important in DNA binding through dimer stabilization (33), a coiled-coil protein binding domain required for protein-protein interactions (34), a central immunoglobulin-fold containing DNA-binding motif, a COOH-terminal SH2 domain, a highly conserved tyrosine residue, and a COOH-terminal transactivation (TA) domain (9). Several studies have shown the importance of the STAT3 TA in target gene activation (35), an observation reproduced herein. The STAT3 TA domain contains a critical phosphoacceptor site, Ser (727), the phosphorylation of which is apparently required for transcriptional activation (36). Our observations are the first to show that amino acids 715–770 of the STAT TA domain are required for stable STAT3-p300/CBP association in HepG2 cells. Moreover, this STAT3-p300/CBP association is required for hAGT transactivation, as STAT3TA, defective in p300/CBP binding, is a dominant-negative inhibitor of hAGT expression (our work and Ref. 27). In future studies, it will be of interest to determine whether the p300/CBP association with the STAT3 TA is phosphorylation dependent; we note that p300/CBP binding to the CREB transcription factor is phosphorylation selective (12).
Despite apparent superficial similarities, the STAT family of transcription factors retains highly specific function and stimulus responsiveness. Although IL-6 activates both STAT1 and -3 in HepG2 cells, as measured similarly by tyrosine phosphorylation, nuclear translocation, and DNA binding (8, 37), STAT1 appears not to be transcriptionally active. We earlier showed that a dominant negative STAT3 isoform, mutated in the COOH-terminal tyrosine phosphoacceptor site Tyr705Phe, completely inhibits IL-6-inducible hAGT reporter activity and induction of the endogenous hAGT gene (8). In contrast, expression of a dominant negative STAT1, even to levels greater than that of the dominant negative STAT3, had no effect on hAGT-dependent transcription. These observations, in conjunction with the observation that high-affinity STAT1 binding sites are not IL-6 inducible (8), argues that STAT1 does not play a role in hAGT transcriptional activation. Potential explanations for this surprising result may be dependence on promoter context in which STAT1 acts, the presence of STAT1-selective inhibitors, such as protein inhibitors of activated STATS (PIAS), or quantitative or qualitative differences in the ability of STAT1 to complex with p300 or other coactivators in HepG2 cells. This point will require additional experimentation.
p300/CBP are highly homologous and functionally interchangeable proteins that function as molecular integrators of nuclear signaling. p300/CBP binds to sequence-specific transcription factors, members of the core polymerase complex (13) including RNA polymerase II itself (14), other histone acetylases [such as P/CAF, a protein that complexes with STAT3 in HepG2 cells (Ray, S., unpublished)], and nucleosomal assembly proteins (Refs. 25 and 38 ; reviewed in Ref. 23). HAT activity may be important in chromatin remodeling by weakening restraints imposed by histone assembly, thereby allowing other components of the transcription machinery to access the hAGT promoter. In this way, one of the functions of the p300/CBP binding is to produce relief from chromatin-mediated repression. Our work indicates an important role for histone acetylation of the hAGT promoter in its activation, as IL-6 stimulation induces increase in the amount of hAGT associated with hyperacetylated histone H4, and overexpression of the HAT-deficient p300 mutant, p300Hm, causes pronounced down-regulation of both basal and IL-6-mediated hAGT promoter activity and HDAC inhibition activates hAGT expression. We note the recent finding of Chakravarti et al. (39), who showed that 12S E1A is a potent inhibitor of the p300/CBP HAT activity, as well as that of free and p300/CBP-associated P/CAF. Although we recognize this complicates the interpretation of the specific target of 12S E1A somewhat, the potent inhibitory activity of the p300Hm strongly argues that p300/CBP HAT activity participates in hAGT promoter activation.
In summary, hAGT is a precursor of the potent octapeptide Ang II, a vasopressor that plays an important role in cardiovascular homeostasis and hypertrophy. Our results suggest that STAT3 is a transcriptional mediator of IL-6-inducible transcription through a mechanism requiring binding and recruitment of the p300/CBP coactivator. These further suggest that chromatin remodeling is p300/CBP dependent through histone acetylation and is required for inducible hAGT expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-GR [pRShGR (40)], and the transfection efficiency control plasmid pSV2PAP (41), and indicated expression plasmids. Twelve hours later, cells were stimulated with 8 ng/ml of recombinant IL-6 (Calbiochem, San Diego, CA) and 100 nM dexamethasone (Calbiochem) for 24 h and before harvest and assay of luciferase and alkaline phosphatase activity. All transfections were carried out (in triplicate plates) in three independent experiments. Deacetylase inhibitor (TSA, Sigma, St. Louis, MO) was used to treat with serum-starved HepG2 cells at a concentration of 100 ng/ml.
Plasmids
The 1-kb human AGT promoter driving a luciferase reporter -991/+22 hAGT/LUC was constructed by PCR as previously described (8). The multimeric hAPRE1 construct was produced by annealing oligonucleotides 5'-GATCCTCCCGTTTCTGGGAACCTTGGA-3' (sense) and 5'-GAGGGCAAAGACCCTTGGAACCTCTAG-3' (antisense), phosphorylated with T4 polynucleotide kinase, ligated, and agarose gel purified. The eluted fragments were ligated into the BamHI linearized hAGT minimal promoter (-46/+22 hAGT/LUC). The dominant negative STAT3 expression plasmid encoding a FLAG epitope-tagged COOH-terminal-truncated STAT3 (by inserting a stop codon at residue 715) was constructed using PCR. The upstream gene-specific primer, 5'-GGATCCGCCCAA TGGAATCAGCTAC-3' and downstream primer 5'-AAGCTTTCATGGTGTCA CACAGATAAACTTGGTC-3' (BamHI and HindIII sites underlined; stop codon double underlined) was used in PCR with cloned human STAT3 as the template (8). The PCR product was digested with BamHI/HindIII, gel purified, and ligated into a modified pcDNA3 expression vector (pcDNA3-FLAG) digested with BamHI and HindIII producing the plasmid pcDNA3-STAT3TA/FLAG. V5-epitope-tagged wild-type STAT1 and STAT3 were also made by cloning into pEF6/V5-His TOPO vector (Invitrogen, San Diego, CA). GAL4-STAT3 expression vector was constructed using expression vector pSG424 (42) that produces GAL4 (1–147) under control of the SV40 early region promoter/enhancer. STAT3 (1–770) and STAT3TA (1–715) coding sequences were cloned as BamHI–XbaI fragments into the GAL4 pSG424 plasmid. The UAS LUC reporter plasmid was constructed using tandem GAL4-binding sites ligated upstream of the -59 nt rat AGT minimal promoter (43). Plasmids encoding the wild-type adenovirus 12S E1A protein (pCMVE1A) and p300/CBP binding mutant, 2–36 E1A (44), pRC/RSV mCBP encoding the full-length mouse CBP (12), human pCMVßp300 (11), p300VP16 (31), and p300Hm, a p300 site mutant that lacks HAT activity (28), have been previously described.
Immunoaffinity Isolation of Transfected HepG2 Cells
To isolate a homogenous population of transiently transfected cells, HepG2 cells (2 x 106) were transfected in a 10-cm2 dish using Lipofectamine PLUS reagent (Life Technologies, Inc.) and with 1.5 µg plasmid pCMV IL2R (a plasmid encoding the IL-2 receptor Tac subunit) and 3 µg of indicated expression plasmids. After 48 h, cells were left untreated or stimulated for 15 min. The IL-2 Tac subunit is an inert cell surface marker (without effect on STAT signaling) that is used to selectively capture transiently transfected cells (45).
Transfected cells were then washed twice with cold PBS and incubated for 30 min at 4 C with 1 µg of antihuman CD25 monoclonal antibody (Caltag Laboratories, Inc., Burlingame, CA) in 8 ml of PBS/0.01% BSA. After this incubation, cells were washed twice with PBS, and 25 µl of a slurry of goat antimouse IgG-conjugated magnetic beads (Dynabeads, M-450, Dynal Inc., Great Neck, NY) were added to 8 ml of PBS/0.01% BSA. After incubation for 30 min at 4 C, cells were washed, trypsinized and captured on a magnetic stand in Eppendorf tubes. In control experiments, transfection efficiency was determined by fluorescence microscopy to detect expression of the IL-2 receptor. In this assay, transfected cells were directly stained with fluorescein isothiocyanate-conjugated CD25 monoclonal antibody (Caltag Laboratories, Inc.) and scored for IL-2 receptor expression. Transfection efficiency averages approximately 19% (n = 3 of 456 individual cells counted). Capture efficiency of this method was determined by assay of the bound and flow-through fractions for specific activity of luciferase in comparison with the luciferase-specific activity made in the whole-cell lysates before affinity isolation. Under these conditions, 99% of reporter gene activity is bound to the magnetic beads, representing a 19- to 30-fold enrichment in specific activity (8, 45). Bound cells in magnetic beads were either dissolved in RNA ZOL B (Tel-Test, Friendswood, TX; for RNA extraction) or RIPA (for whole-cell lysate preparation).
Northern Blots
Ten micrograms of total HepG2 RNA were fractionated by electrophoresis on 1% agarose-formaldehyde gel, capillary transferred to a nylon membrane (Hybond, Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK), and then hybridized with random labeled 32P hAGT cDNA probe. Both prehybridization and hybridization were carried out at 62 C with QuickHyb hybridization solution (Stratagene, La Jolla, CA), and the membrane was subsequently washed according to the manufacturer’s protocol. For internal control, the same membrane was reprobed with 32P-labeled human 18S rRNA cDNA and exposed briefly. The signal intensity was analyzed using a Storm PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) with ImageQuant software.
Sucrose Density-Purified NEs
Nuclear proteins were purified from unstimulated or IL-6-stimulated (8 ng/ml, 15 min unless otherwise indicated) cells as follows: HepG2 cells were resuspended in Buffer A [50 mM HEPES (pH 7.4), 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 0.1 µg/ml phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml aprotinin, 20 mM NaF, 1 mM NaP207, 1 mM Na3VO3, and 0.5% (vol/vol) Nonidet P-40]. After 10 min on ice, the lysates were centrifuged at 4,000 x g for 4 min at 4 C. The supernatant was saved as the cytoplasmic fraction. The nuclear pellet was then resuspended in Buffer B (Buffer A with 1.7 M sucrose) and centrifuged at 15,000 x g for 30 min at 4 C (46). The purified nuclear pellet was then incubated in Buffer C [10% glycerol, 50 mM HEPES (pH 7.4), 400 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 µg/ml PMSF, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 20 mM NaF, 1 mM NaP207, 1 mM Na3VO3, and 10 µg/ml aprotinin] with frequent vortexing for 30 min at 4 C. After centrifugation at 15,000 x g for 5 min at 4 C, the supernatant was saved as NE. Both the cytoplasmic and nuclear fractions were normalized for protein amounts determined by protein assay (Protein Reagent; Bio-Rad Laboratories, Inc., Hercules, CA).
Coimmunoprecipitation
WCEs of HepG2 cells were prepared using modified RIPA buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, 1 mM PMSF, 1 mM NaF, 1 mM Na3VO4, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin). For immunoprecipitation, either WCE or NE extracts were precleared with protein A-Sepharose 4B (Sigma) for 10 min at 4 C and then immunoprecipitation was carried out for 2–12 h at 4 C with primary antibody. Sources of primary antibody included anti-STAT3 (K15, Santa Cruz Biotechnology, Inc.), anti-p300 (N15, Santa Cruz Biotechnology, Inc.), anti-CBP (A22, Santa Cruz Biotechnology, Inc.) or anti-FLAG M2 (Sigma) antibodies. Immune complexes were then captured by adding 30 µl of protein A-Sepharose beads (50% slurry) and rotated for 1 h at 4 C. Beads were washed three times with 5 min each time with cold PBS, and immune complexes were separated by SDS-PAGE using 6% (in the case of p300 immunoblot) or 10% gels. Proteins were transferred onto polyvinylidine difluoride membranes and probed with indicated secondary antibodies. The band was visualized by enhanced chemiluminescence (ECL) as directed by the manufacturer (Amersham Pharmacia Biotech).
ChIP Assay
Chromatin was immunoprecipitated with antiacetylated histone H4 antibody (Upstate Biotechnology, Inc., Lake Placid, NY). Approximately 2 x 107 HepG2 cells were treated with or without IL-6 (8 ng/ml, 12 h) and fixed with 1% formaldehyde at 37 C for 10 min. The cells were washed in PBS, lysed with SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1 mM PMSF, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin protease inhibitors), and sonicated 10 times for 10 sec at 0 C to shear DNA to lengths between 200 and 1,000 bp. After clarification of the lysate by centrifugation, 0.1 ml of supernatant containing solubilized chromatin was diluted 10-fold with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). To reduce nonspecific background, the diluted chromatin was precleared with salmon sperm DNA/Protein A slurry for 30 min at 4 C. For immunoprecipitation, the treated chromatin solution was incubated overnight at 4 C with 5 µg of antiacetylated H4 antibody. The immunocomplex was then purified by binding to 60 µl of protein A agarose slurry for 1 h at 4 C. Beads were then collected by centrifugation and washed sequentially with low-salt buffer [0.1% (wt/vol) SDS, 1% (vol/vol) Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl], high-salt buffer [0.1% (wt/vol) SDS, 1% (vol/vol) Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl], LiCl immune complex wash buffer [0.25 M LiCl, 1% Nonidet P-40, 1% (vol/vol) deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1], and finally in 1x TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The immune complex was then eluted with buffer containing 1% SDS and 0.1 M NaHCO3 with 15 min rotation at room temperature. The eluates were heated to 65 C for 4 h to reverse the formaldehyde cross-links and then treated with proteinase K for 1 h at 45 C. The DNA was subsequently extracted with phenol/chloroform, precipitated with ethanol, and dissolved in TE. The hAGT gene sequence was detected by PCR under conditions in which product yield was dependent on input DNA concentration, with the promoter-specific primer pair, -493 HATS (5'-AGGGTAGGATCCTTGGAGGGGGGCCACCTGAAGGTC-3') as a sense primer and HagtL1 (5'-CCGGCTTACCTTCTGCTGTAGTA-3') as an antisense primer amplifying the nt -493 to nt + 45 region of the hAGT promoter. The 538-bp PCR products were run in either 1% agarose gel and stained with ethidium bromide or 32P-labeled PCR products were separated in 6% nondenaturing polyacrylamide gel and detected by autoradiography.
Immunoprecipitation (IP) HAT Assay
HAT assay (15) was performed in nondenaturing immunoprecipitates. For this, unstimulated or IL-6-stimulated NE extracts were immunoprecipitated with anti-STAT3. The immune complex was captured by protein A agarose beads and washed three times with ice-cold PBS and once with HAT assay buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol, 0.1 mM EDTA, and 1 mM DTT). HAT activity was assayed by incubation at 30 C in a shaking incubator for 30 min in 50 µl HAT assay buffer containing 10 µg of reconstituted histone as a substrate and 6 pmol [3H] acetyl coenzyme A (4.3 mCi/mmol, Amersham Pharmacia Biotech). After incubation, the reaction mixture was spotted onto P-81 phosphocellulose filter paper (Whatman, Clifton, NJ) and washed six times for 15 min each in 50 ml of 50 mM NaHPO4, pH 9.0, at room temperature. The dried filters were resuspended in scintillation cocktail and the cpm in the IgG control was subtracted from experimental samples.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AGT, Angiotensinogen; Ang II, angiotensin II; APR, acute phase reactant; CBP, p300/cAMP response element binding protein-binding protein; ChIP, chromatin immunoprecipitation; Dex, dexamethasone; DTT, dithiothreitol; HAT, histone acetyltransferase; HDAC, histone deacetylase; NE, nuclear extract; nt, nucleotide; PMSF, phenylmethylsulfonyl fluoride; RAS, renin-angiotensin system; STAT, signal transducer and activator of transcription; TA, transactivation; TSA, trichostatin A; WCE, whole-cell extract.
Received for publication August 23, 2001. Accepted for publication December 18, 2001.
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