Angiotensinogen Gene Expression Is Dependent on Signal Transducer and Activator of Transcription 3-Mediated p300/cAMP Response Element Binding Protein-Binding Protein Coactivator Recruitment and Histone Acetyltransferase Activity
Sutapa Ray,
Christopher T. Sherman,
Muping Lu and
Allan R. Brasier
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.
 |
ABSTRACT
|
---|
Angiotensin II, a potent vasoactive peptide produced by proteolysis of the angiotensinogen (AGT) prohormone, plays a critical role in cardiovascular homeostasis. Recently we showed that IL-6 induces human (h)AGT transcription by activating the signal transducers and activators of transcription (STATs). Here we investigated the role of the coactivator p300/cAMP response element binding protein-binding protein (CBP) in STAT3-mediated hAGT gene expression. Overexpression of adenovirus 12S E1A, which binds and inactivates p300/CBP, strongly inhibited basal and stimulated hAGT transcription, whereas a mutant E1A defective in binding p300/CBP did not. Conversely, ectopic expression of p300 and CBP potentiated inducible hAGT promoter activity. Coimmunoprecipitation assays revealed STAT3-p300 interaction upon IL-6 stimulation. The STAT3-p300 association requires the STAT3 C-terminal transactivation domain, as STAT3 deleted of transactivation functions as a dominant-negative inhibitor and does not associate with p300/CBP. The observation that IL-6 stimulation increases histone H4 acetylation of the endogenous hAGT promoter, and expression of p300 deficient in histone acetyltransferase activity down-regulates hAGT promoter activity both suggest that p300 histone acetyltransferase activity is required for hAGT expression. Finally, treatment of HepG2 cells with a histone deacetylase inhibitor increased the hAGT mRNA abundance by 2- to 3-fold. Taken together, our results indicate that IL-6-inducible expression of the hAGT promoter is mediated by physical association of the COOH terminus of STAT3 with p300/CBP, the recruitment of which targets histone acetylation and results in chromatin remodeling.
 |
INTRODUCTION
|
---|
THE INTRAVASCULAR renin-angiotensin system (RAS) is a central regulator of blood pressure and intravascular volume homeostasis in humans by its effects on vascular tone, aldosterone secretion, catecholamine release and cardiac growth (1). Angiotensin II (Ang II), the effector octapeptide of the renin-angiotensin system, is produced by sequential proteolysis initiated through a rate-limiting processing of angiotensinogen (AGT) into Ang I mediated by the aspartylprotease, renin (1). Because AGT circulates in the plasma at concentrations close to the MichaelisMenten constant (Km) of renin (1), changes in circulating human (h)AGT concentrations lead to a parallel increase in the velocity of Ang II formation. Consistent with this relationship, genetic or pharmacological manipulations that induce increases in circulating AGT produce Ang II-dependent hypertension (2). In fact, recent genetic linkage studies have found that the hAGT locus appears to be linked to human autosomal dominant hypertension in which the hyperexpressing M235T AGT allele is associated with approximately 2-fold increases in circulating AGT concentrations and Ang II-dependent hypertension (3). An important determinant of circulating hAGT concentration is its transcription in the liver, an organ responsible for the majority of circulating plasma proteins (4).
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 13 [hAPRE13 (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.
 |
RESULTS
|
---|
Adenovirus 12S E1A Interferes with Endogenous hAGT Transcription in HepG2 Cells
To analyze the effect of E1A on endogenous hAGT transcription and to evaluate the role of p300/CBP, HepG2 cells were transiently transfected with construct expressing the 12S splice variant of adenovirus E1A (12S E1A), which binds to the third cysteine-histidine-rich (CH3) domain of p300 and inhibits its coactivator function (17, 18). hAGT transcript abundance was assessed by Northern blot analysis of RNA isolated from control or 72-h-stimulated cells transfected with empty or 12S E1A vectors. Relative to empty vector, cells transfected with 12S E1A showed a significant decrease in control and stimulated hAGT mRNA abundance (Fig. 1A
). 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.

View larger version (21K):
[in this window]
[in a new window]
|
Figure 1. Northern Blot Analysis of hAGT mRNA in HepG2 Cells Transfected with Adenovirus E1A Expression Vector
A, HepG2 cells were transiently transfected with an empty vector (lane 1 and 2) or with adenovirus 12S E1A expression vector (lanes 3 and 4) were serum starved [media supplemented with 0.5% BSA (wt/vol) 24 h before and during IL-6 (8 ng/ml) +Dex (100 nM) stimulation for indicated times]. Serum starvation was to remove the confounding effect of growth factor stimulation. Top, Autoradiogram after hybridization with 32P-labeled hAGT cDNA probe. Bottom, Autoradiographic exposure of the same blot with 32P-labeled 18S probe as an internal control. B, Hybridization signals of the hAGT and 18S mRNAs were quantitated by a PhosphorImager, and the ratio of hAGT/18S band intensity was calculated and and plotted. 12S E1A decreased the absolute value of basal and stimulated hAGT transcript abundance relative to 18S. Experiment was repeated two times with similar results.
|
|
Adenoviral 12S E1A Inhibits Basal and Stimulated hAGT Promoter Activity
Our previous studies showed that 1 kb of the hAGT promoter, spanning -991/+22 nucleotides (nt) relative to the cap site of the M235T allele, was sufficient to drive inducible luciferase reporter activity similar to expression of the endogenous gene (8). We first examined the possibility that p300/CBP is required for basal hAGT promoter expression. Groups of HepG2 cells were cotransfected with the hAGT/LUC reporter, an internal control plasmid pSV2-PAP, and with increasing amounts of 12S E1A expression vector. 12S E1A expression inhibited unstimulated (basal) hAGT promoter activity in a dose-dependent manner (Fig. 2A
), 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.

View larger version (16K):
[in this window]
[in a new window]
|
Figure 2. E1A Inhibits Basal and Stimulated hAGT Promoter Activity
A, Adenovirus 12S E1A down-regulates basal hAGT promoter activity. HepG2 cells were transiently transfected with 5 µg of hAGT/LUC, internal control pSV2-PAP, and the indicated amounts of adenovirus 12S E1A expression plasmids. Luciferase activity was measured 48 h after transfection and normalized to alkaline phosphatase activity. The figure represents the mean ± SD of fold changes (relative to untreated, n = 3 independent transfections). *, P < 0.05 relative to empty vector, t test. B, IL-6-mediated induction of hAGT promoter is inhibited by adenovirus 12S E1A. HepG2 cells were transiently transfected with 5 µg of hAGT/LUC reporter and 0.4 µg of 12S E1A or 0.4 µg of mutant E1A 236 expression plasmid. After 24 h, cells were stimulated with either IL-6 (8 ng/ml) or with IL-6 and Dex (100 nM). Luciferase activity was measured 24 h later. The relative luciferase activity was normalized to that of untreated cells and plotted as in Fig. 2 A . *, P < 0.05 relative to empty vector, t test. C, Effect of E1A on multimerized hAGT promoter. HepG2 cells were transiently transfected with (hAPRE1)5-hAGT/LUC and with 0.4 µg of 12S E1A or with 0.4 µg of mutant 12S 236 expression plasmids. After 24 h of transfection, cells were stimulated with either IL-6 (8 ng/ml) or with IL-6 + Dex (100 nM). Luciferase activity was measured after 24 h and plotted in Fig. 2C . *, P < 0.05 relative to empty vector, t test.
|
|
IL-6 alone, and in combination with the potent synthetic glucocorticoid, dexamethasone (Dex), induced hAGT-dependent luciferase reporter activity that peaked at 24 h after stimulation (8). To determine whether the effects of 12S E1A were mediated through p300/CBP sequestration, HepG2 cells were cotransfected with an expression plasmid encoding E1A
236, 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
236 had a neutral effect on basal or IL-6-stimulated activity (Fig. 2B
, right lanes). In the case of IL-6 + Dex, E1A
236 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
236 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
236 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.

View larger version (17K):
[in this window]
[in a new window]
|
Figure 3. p300 and CBP Enhance IL-6-Mediated Induction of hAGT Promoter
HepG2 cells were cotransfected with 5 µg of hAGT/LUC and either with 100 ng of human p300 expression plasmid (pCMVß-p300; panel A) or with (pRC/RSV mCBP; panel B). Amount of transfected DNA was kept constant using an empty DNA expression plasmid. cells were stimulated 24 h later with either IL-6 (8 ng/ml) or with IL-6 +Dex (100 nM). Fold induction of luciferase activity was determined after 24 h of stimulation. *, P < 0.05, t test.
|
|
Endogenous STAT3 Complexes with p300/CBP in Cellulo
Coactivators are non-DNA-binding bridges that stabilize complexes between sequence-specific DNA-binding proteins and general transcription factors through direct protein-protein interactions (23, 24, 25). Because 12S E1A is an inhibitor of hAPRE1-driven transcription (hAPRE 1 being a combined STAT1 and -3 DNA-binding site), the interaction of STAT1 and STAT3 with p300/CBP was investigated by a nondenaturing coimmunoprecipitation/immunoblot assay.
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 13) and IL-6-stimulated (lanes 46) 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.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 4. STAT3 Complexes with p300/CBP
A, Relative amount of STAT-1, -3 p300 association upon IL-6 induction. HepG2 cells were transfected with pCMVßp300, pCMV IL2R, and either pEF6/V5-His STAT1, (lane 1) or pEF6/V5-His STAT3 (lane 2). Cells were stimulated (IL-6, 15 min) and isolated with magnetic beads (Materials and Methods), and WCE were analyzed for STAT expression by Western immunoblot with anti-V5 antibody (top). Extracts were then immunoprecipitated with anti-p300 antibody and relative amounts of p300-associated STAT proteins were analyzed by Western with anti-V5 antibody (bottom). B, Inducible STAT3-p300 association upon IL-6 stimulation. Unstimulated and IL-6-stimulated NEs (1 mg) were immunoprecipitated with rabbit preimmune serum (lanes 1 and 6), anti-STAT3 antibody (lanes 2 and 5), and anti-p300 antibody (lanes 3 and 4). The immunoprecipitated proteins were probed with rabbit polyclonal STAT3 antibody. C, STAT3 coimmunoprecipitates with p300 and CBP. IL-6-stimulated HepG2 NEs (2 mg) were immunoprecipitated with rabbit preimmune sera (lane 1), anti-STAT3 (lane 2), anti-p300 (lane 3), and anti-CBP (lane 4). p300/CBP-bound STAT3 was analyzed by immunoblot using anti-STAT3 polyclonal antibodies (top) or STAT3-bound p300 was analyzed with anti-p300 antibody (lane 2) in a separate 6% SDS-PAGE gel, (bottom). Only one third of the immunoprecipitated STAT3 sample was loaded onto lane 2 (top).
|
|
p300/CBP-STAT3 Association Requires the STAT3 COOH Terminus
Although the primary amino acid sequences of the COOH-terminal domain of STAT proteins are poorly conserved among the STAT family members [in contrast to the high degree of conservation for the DNA-binding and dimerization domains (9)], its function is highly conserved, where it is required for STAT-dependent transactivation (16). We therefore investigated whether the COOH terminus of STAT3 was a site of p300 interaction in HepG2 cells. HepG2 cells were cotransfected with expression vectors encoding FLAG epitope-tagged full-length STAT3 (1770) or STAT3
TA (1715) 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
).

View larger version (17K):
[in this window]
[in a new window]
|
Figure 5. STAT3 COOH Terminus Is Required for p300/CBP Transactivation
A, TA domain of STAT3 is required for binding p300/CBP. HepG2 cells were cotransfected with pCMV IL2R and p300 expression plasmid and either with FLAG-tagged wild-type human STAT3 (lanes 1 and 2) or with FLAG-tagged STAT3 TA (lanes 3 and 4). IL-6-stimulated cells were affinity isolated and immunoprecipitated either with anti-FLAG (lanes 1 and 3) or with anti-p300 (lanes 2 and 4) antibodies. Coimmunoprecipitated proteins were detected in Western immunoblot with anti-FLAG antibody. B, Transactivation of UAS/LUC with STAT3 (1770). HepG2 cells were transfected with 6 µg of UAS/LUC and either with 1.6 µg of GAL4 (1147), GAL4 STAT3 (1770), or with GAL4-STAT TA (1715) expression vectors. Cells were harvested after 24 h of transfection and luciferase activity was measured. GAL4-STAT3 (1770) transactivates UAS/LUC more efficiently than GAL4STAT3 TA (1715). Expression level of the approximately 110-kDa GAL4 STAT3 and the approximately 104-kDa GAL4-STAT3 TA fusion proteins in transient transfectants is shown by Western immunoblot (inset). C, Functional association of p300 with STAT3 (1770) but not with STAT3 TA (1715). HepG2 cells were transfected with 6 µg of UAS LUC and either with 1.6 µg of GAL4 (1147), GAL4 STAT3 (1770), or with GAL4 STAT TA (1715) expression vectors in the presence of pcDNA3 empty vector or pcDNA encoding p300/VP16. Cells were harvested after 24 h of transfection and luciferase activity was measured. For each GAL4 expression plasmid, the degree of induction by p300/VP16 was calculated by subtracting luciferase reporter activity of p300/VP16 from that of empty vector and is plotted. p300/VP16 transactivated UAS/LUC in the presence of GAL4 STAT3 but not GAL4 STAT TA (1715). *, P < 0.05, t test.
|
|
To confirm and further extend the coimmunoprecipitation experiments, we tested whether STAT3
TA was capable of transactivating target genes or functionally interacting with p300. For this we first assayed whether GAL4-STAT3 (1770) or GAL4-STAT3
TA (1715) could transactivate GAL4 binding sites (UAS/LUC) in HepG2 cells. Although GAL4-STAT3 (1770) transactivated UAS/LUC, GAL4-STAT3
TA (1715) 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 (1147), GAL4-STAT3 (1770), or GAL4-STAT3
TA (1715). p300/VP16 was unable to transactivate UAS/LUC alone, but strongly transactivated UAS/LUC in the presence of GAL4-STAT3 (1770) (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 (1715). Together, these data indicate that STAT3 associates with p300/CBP in a mechanism requiring an intact COOH terminus (amino acids 715770).
STAT3
TA, 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 715770), 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 STAT3
TA is a dominant negative inhibitor of hAPRE-dependent transcription, confirming our earlier studies with the STAT3 (Tyr705Phe) mutant (8).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 6. Effect of STAT3 TA on IL-6-Induced Activation of hAGT Promoter
HepG2 cells were transiently transfected with either 5 µg of hAGT/LUC reporter (panel A) or (hAPRE1)5 LUC construct (panel B), internal control pSV2-PAP, and with indicated amounts of STAT3 TA. After 24 h of transfection, cells were stimulated with either IL-6 (8 ng/ml) or with IL-6 and Dex (100 nM). Luciferase activity was measured after 24 h of stimulation. Normalized luciferase activity is shown. *, P < 0.05, t test.
|
|
p300 HAT Activity Is Involved in hAGT Gene Expression
HAT activity is recognized to be an important chromatin-remodeling enzyme associated with inducible expression of some genes (23). To determine whether STAT3 inducibly associated with HAT activity, we performed immunoprecipitation-HAT assay on control or IL-6-stimulated HepG2 NE. We consistently observed a 1.93 ± 0.32 (n = 3)-fold increase in HAT enzymatic activity in STAT3 immunoprecipitates from stimulated vs. control NE (Fig. 7A
). 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.

View larger version (18K):
[in this window]
[in a new window]
|
Figure 7. p300 HAT Domain is Required for STAT3-Mediated hAGT Promoter Induction
A, STAT3 inducibly associates with HAT activity. Unstimulated (lane 1) and IL-6-stimulated (lane 2) NEs were immunoprecipitated with STAT3 antibody. Immune complexes were then subjected to HAT enzymatic assay with core histone as a substrate and [3H]acetyl-coenzyme A as a cofactor (Materials and Methods). [3H] Incorporation of histone was measured by liquid scintillation counting. B, HAT activity of p300 is required for IL-6-mediated induction of hAGT promoter. HepG2 cells were transiently transfected with 5 µg of hAGT/LUC and the indicated amounts of p300Hm. After 24 h of transfection, cells were stimulated with either IL-6 (8 ng/ml) or with IL-6 and Dex (100 nM). Luciferase activity was measured after 24 h of stimulation. The amount of transfected DNA was kept equivalent using empty vector pcDNA3. *, P < 0.05, t test.
|
|
IL-6 Stimulation Increases Abundance of Hyperacetylated Histone H4 Bound to the Endogenous hAGT Promoter
The observation that p300 HAT activity is required for hormone-stimulated hAGT promoter activity led us to determine whether IL-6 stimulation changed the extent of histone acetylation of the endogenous hAGT promoter. For this, a chromatin immunoprecipitation (ChIP) assay (29) was performed. Briefly, control and IL-6-treated HepG2 cells were treated with formaldehyde, and the cross-linked chromatin was fragmented and immunoprecipitated with antibody to hyperacetylated histone H4. The amount of hAGT promoter associated with hyperacetylated H4 was determined by semiquantitative PCR using gene-specific primers [PCR conditions were optimized to reflect changes in product formed as a function of input hAGT promoter DNA (data not shown)]. PCR amplifications were carried out on a fixed amount of immunoprecipitated DNA using hAGT promoter-specific primer pairs; controls for initial loading included determining the amount of hAGT promoter present in the chromatin extracts before immunoprecipitation (input sample), and for similar total chromatin immunoprecipitated by quantitating ß-actin in the immunoprecipitate (ß-actin is a non-IL-6-inducible gene). Product abundance was quantitated both by ethidium bromide staining (Fig. 8A
) 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.

View larger version (52K):
[in this window]
[in a new window]
|
Figure 8. IL-6 Induces Acetylated Histone H4 Association with the Endogenous hAGT Promoter
HepG2 cells were treated with IL-6 for 12 h, and ChIP assays were performed as described in Materials and Methods. The sequences of the promoter regions in the antiacetylated histone H4 immunoprecipitate were amplified by PCR primer pairs, -493 HATS (sense primer) and Hagt L1 (antisense primer), and the products were run either in 1% agarose gel and stained with EtBr (panel A), or 32P-labeled PCR products were separated in 6% polyacylamide gel followed by autoradiography (panel B).
|
|
Activation of hAGT Transcription with the Histone Deacetylase (HDAC) Inhibitor Trichostatin A (TSA)
The relationship between HAT activity and hAGT promoter activation was further explored with the potent HDAC inhibitor TSA. To determine whether HAT activity was sufficient to induce hAGT expression, HepG2 cells were treated for various times with TSA (100 ng/ml), and changes in hAGT transcripts were quantitated by Northern blot. The abundance of 1.9-kb transcript normalized to 18S rRNA was approximately 2.5-fold higher after 24 h of TSA treatment (Fig. 9
). 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.

View larger version (25K):
[in this window]
[in a new window]
|
Figure 9. Treatment of Deacetylase Inhibitor TSA Increases the Abundance of hAGT mRNA in HepG2 Cells
HepG2 cells were serum starved 24 h before and during stimulation with 100 ng/ml TSA for the indicated times and total RNA was extracted. Serum starvation was performed to remove confounding effects of serum growth factors. Top, Autoradiogram after hybridization with hAGT cDNA probe. Bottom, Exposure after hybridization with 18S internal control.
|
|
 |
DISCUSSION
|
---|
The RAS contributes to the regulation of blood pressure by controlling peripheral vascular resistance and electrolyte homeostasis and plays a significant role in the etiology of renovascular and essential hypertension (1). The effector peptide of the RAS, Ang II, is produced by sequential proteolysis of angiotensinogen (AGT), a prohormone the synthesis of which is dynamically regulated in the liver (4). The circulating level of AGT is an important determinant of RAS activity because it circulates at concentrations that are rate limiting for the formation of Ang II (1). The hAGT gene probably arose as a gene duplication event in the serine protease inhibitor gene family (5), and interestingly retains regulatory features of a hepatic APR. The hepatic acute-phase response is a switch in secretory protein transcription in the vertebrate liver in response to systemic inflammation (30).
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,
236 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 715770 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 STAT3
TA, 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.
 |
MATERIALS AND METHODS
|
---|
Cell Culture, Reagents, and Transfection
The human hepatoblastoma cell-line HepG2 was obtained from ATCC (Manassas, VA) and grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% (vol/vol) heat-inactivated FBS, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and antibiotics (penicillin/streptomycin) in a humidified atmosphere of 5% CO2. Transient transfections in exponentially growing HepG2 cells were performed using Lipofectamine reagent (Life Technologies, Inc.). Five micrograms of hAGT (-991/+22) promoter-luciferase construct (hAGT/LUC) were cotransfected into a 60-mm plate (106 cells) with expression vectors for the human
-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-STAT3
TA/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 (1147) under control of the SV40 early region promoter/enhancer. STAT3 (1770) and STAT3
TA (1715) coding sequences were cloned as BamHIXbaI 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,
236 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 manufacturers 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 212 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.
 |
ACKNOWLEDGMENTS
|
---|
We thank P. K. Roychoudhury (University of Illinois, Chicago, IL), R. H. Goodman (Oregon Health Sciences University, Portland OR), S. Grossman (Dana Farber Cancer Research Institute, Boston, MA), and J. Wong (Baylor College of Medicine, Houston, TX) for gifts of plasmids and reagents.
 |
FOOTNOTES
|
---|
This work was supported in part by National Heart, Lung, and Blood Institute Grant 55630 (to A.R.B.), American Heart Association Grant-in-Aid 9950345N (to A.R.B.), and National Institute on Environmental Health Sciences Grant ES06676 for core laboratory support. (R. S. Lloyd, University of Texas Medical Branch).
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.
 |
REFERENCES
|
---|
-
Reid IA, Morris BJ, Ganong WG 1978 The renin-angiotensin system. Annu Rev Physiol 40:377410[CrossRef][Medline]
-
Brasier AR, Li J 1996 Mechanisms for inducible control of angiotensinogen gene transcription. J Hypertens 27:465475
-
Jeunemaitre X, Soubrier F, Kotelevtsev YV, Lifton RP, Williams CS, Charru A, Hunt SC, Hopkins PN, Williams RR, Lalouel JM 1992 Molecular basis of human hypertension: role of angiotensinogen. Cell 71:169180[Medline]
-
Brasier AR, Youqi H, Sherman C 1999 Transcriptional regulation of angiotensinogen gene expression. Vitam Horm 57:217247[Medline]
-
Doolittle RF 1983 Angiotensinogen is related to the antitrypsin-antithrombin-ovalbumin family. Science 222:417419[Medline]
-
Baumann H, Gauldie J 1990 Regulation of hepatic acute phase plasma protein genes by hepatocyte stimulating factors and other mediators of inflammation. Mol Biol Med 7:147159[Medline]
-
Fischer CP, Bode BP, Takahashi K, Tanabe KK, Souba WW 1996 Glucocorticoid-dependent induction of interleukin-6 receptor expression in human hepatocytes facilitates interleukin-6 stimulation of amino acid transport. Ann Surg 223:610618[CrossRef][Medline]
-
Sherman CT, Brasier AR 2001 Role of signal transducers and activators of transcription1 and 3 in inducible regulation of the human angiotensinogen gene by interleukin-6. Mol Endocrinol 15:441457[Abstract/Free Full Text]
-
Darnell Jr JE 1997 STATs and gene regulation. Science 277:16301635[Abstract/Free Full Text]
-
Darnell Jr JE, Kerr IM, Stark GR 1994 Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:14151421[Medline]
-
Eckner R, Ewen ME, Newsome D, Gerds M, Decaprio JA, Lawrence JB, Livingston DM 1994 Molecular cloning and functional analysis of the adenovirus E1A-associated 300 Kd protein (P300) reveals a protein with properties of a transcriptional adaptor. Genes Dev 8:869884[Abstract]
-
Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MN, Goodman RH 1993 Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855859[CrossRef][Medline]
-
Dallas PB, Cheney IW, Liao DW, Bowrin V, Byam W, Pacchione S, Kobayashi R, Yaciuk P, Moran E 1998 p300/CREB binding protein-related protein P270 is a component of mammalian SWI/SNF complex. Mol Cell Biol 18:35963603[Abstract/Free Full Text]
-
Nakajima T, Uchida C, Anderson SF, Lee C-G, Hurwitz J, Parvin JD, Montminy M 1997 RNA helicase A mediates association of CBP with RNA polymerase II. Cell 90:11071112[Medline]
-
Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87:953959[Medline]
-
Paulson M, Pisharody S, Pan L, Guadagno S, Mui AL, Levy DE 1999 STAT protein transactivation domains recruit p300/CBP through widely divergent sequences. J Biol Chem 274:2534325349[Abstract/Free Full Text]
-
Arany Z, Newsome D, Oldread E, Livingston DM, Eckner R 1995 A family of transcriptional adapter proteins targeted by the E1A oncoprotein. Nature 374:8184[CrossRef][Medline]
-
Bannister AJ, Kouzarides T 1996 The CBP co-activator is a histone acetyltransferase. Nature 384:641643[CrossRef][Medline]
-
Owen GI, Richer JK, Tung L, Takinoto G, Hortwitz KB 1998 Progesterone regulates transcription of the p21 WAF1 cyclin-dependent kinase inhibitor gene through SP1 and CBP/p300. J Biol Chem 273:1069610701[Abstract/Free Full Text]
-
Bhakat KK, Mitra S 2000 Regulation of the human O6-methylguanine-DNA methyltransferase gene by transcriptional coactivators cAMP response element-binding protein-binding protein and p300. J Biol Chem 275:3419734204[Abstract/Free Full Text]
-
Horvai AE, Xu L, Korzus E, Brad G, Kalafus D, Mullen T, Rose DW, Rosenfeld MG, Glass CK 1997 Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP And p300. Proc Natl Acad Sci USA 94:10741079[Abstract/Free Full Text]
-
Kamei Y, Xu Y, Heinzel T 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403414[Medline]
-
Blobel GA 2000 CREB-binding protein and p300: molecular integrators of hematopoietic transcription. Blood 95:745755[Free Full Text]
-
Shikama N, Chan HM, Krstic-Demonacos M, Smith L, Lee C-W, Cairns W, La Thangue NB 2000 Functional interaction between nucleosome assembly proteins and p300/CREB-binding protein family coactivators. Mol Cell Biol 20:89338943[Abstract/Free Full Text]
-
Korzus E, Torchia J, Rose DW, Xu L, Kurkawa R, McInerney EM, Mullen TM, Glass CK, Rosenfeld MG 1998 Transcription factor-specific requirements for coactivators and their acetyltransferase functions. Science 279:703707[Abstract/Free Full Text]
-
Gingras S, Simard J, Groner B, Pfitzner E 1999 p300/CBP is required for transcriptional induction by interleukin-4 and interacts with STAT6. Nucleic Acids Res 27:27222729[Abstract/Free Full Text]
-
Caldenhoven E, Van Dijk TB, Solari R, Armstrong J, Raaijmaker JAM, Lammers J-WJ, Koenderman L, de Groot RP 1996 STAT3ß, a splice variant of transcription factor STAT3, is a dominant negative regulator of transcription. J Biol Chem 271:1322113227[Abstract/Free Full Text]
-
Li J, OMalley B, Wong J 2000 p300 Requires its histone acetyltransferase activity and SRC-1 interaction domain to facilitate thyroid hormone receptor activation in chromatin. Mol Cell Biol 20:20312042[Abstract/Free Full Text]
-
Braunstein M, Rose AB, Holmes SG, Allis CD, Broach JR 1993 Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev 7:592604[Abstract]
-
Gabay C, Kushner I 1999 Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 340:448454[Free Full Text]
-
Bhattacharya S, Eckner R, Grossman S, Oldread E, Zoltan A, DAndrea A, Livingston DM 1996 Cooperation Of STAT2 and p300/CBP in signalling induced by interferon-
. Nature 383:344347[CrossRef][Medline]
-
Pfitzner E, Jahne R, Wissler M, Stoecklin E, Groner B 1998 p300/CREB-binding protein enhances the prolactin-mediated transcriptional induction through direct interaction with the transactivation domain of STAT5, but does not participate in the STAT5-mediated suppression of the glucocorticoid response. Mol Endocrinol 12:15821593[Abstract/Free Full Text]
-
Vinkemeier U, Cohen SJ, Moarefi I, Chait BT, Kuriyan J, Darnell Jr JE 1996 DNA binding of in vitro activated STAT1
, STAT1ß and truncated STAT1: interaction between NH2-terminal domains stabilizes binding of two dimers to tandem DNA Sites. EMBO J 15:56165626[Abstract]
-
Zhu M, John S, Berg M, Leonard WJ 1999 Functional association of Nmi with STAT5 and STAT1 in IL-2 And IFN
-mediated signaling. Cell 96:121130[Medline]
-
Kim H, Baumann H 1997 The carboxyl-terminal region of STAT3 controls gene induction by the mouse haptoglobin promoter. J Biol Chem 272:1457114579[Abstract/Free Full Text]
-
Wen Z, Zhong Z, Darnell Jr JE 1995 Maximal activation of transcription by STAT1 and STAT3 requires both tyrosine and serine phosphorylation. Cell 82:241250[Medline]
-
Yuan J, Wegenka UM, Lutticken C, Buschmann J, Decker T, Schindler C, Heinrich PC, Horn F 1994 The signalling pathways of interleukin-6 and
interferon converge by the activation of different transcription factors which bind to common responsive DNA elements. Mol Cell Biol 14:16571668[Abstract]
-
Stark GR, Kerr I, Williams BRG, Silverman RH, Schreiber RD 1998 How cells respond to interferons. Annu Rev Biochem 67:227264[CrossRef][Medline]
-
Chakravarti D, Ogryzko V, Kao H, Nash A, Chen H, Nakatani Y, Evans RM 1999 A viral mechanism for inhibition of p300 and PCAF acetyltransferase activity. Cell 96:393403[Medline]
-
Giguere V, Hollenberg SM, Rosenfeld MG, Evans RM 1986 Functional domains of the human glucocorticoid receptor. Cell 46:645652[Medline]
-
Brasier AR 1990 Reporter system using firefly luciferase. In: Ausubel FM, ed. Current protocols in molecular biology. New York: John Wiley & Sons; 9.6.109.6.13
-
Sadowski I, Ptashne M 1989 A vector for expressing GAL4 (1147) in mammalian cells. Nucleic Acids Res 17:75397539[Medline]
-
Li J, Brasier AR 1996 Angiotensinogen gene activation by angiotensin II is mediated by the Rel A (nuclear factor-
B p65) transcription factor: one mechanism for the renin angiotensin system positive feedback loop in hepatocytes. Mol Endocrinol 10:252264[Abstract]
-
Bannister AJ, Kouzarides T 1995 CBP induced stimulation of c-Fos activity is abrogated by E1A. EMBO J 14:47584762[Abstract]
-
Jamaluddin M, Meng T, Sun J, Bolodogh I, Han Y, Brasier AR 2000 Angiotensin II induces nuclear factor (NF)-
B1 isoforms to bind the angiotensinogen gene acute-phase response element: a stimulus-specific pathway for NF-
B activation. Mol Endocrinol 14:99113[Abstract/Free Full Text]
-
Han Y, Brasier AR 1997 Mechanism for biphasic Rel A:NF-
B1 nuclear translocation in tumor necrosis factor
-stimulated hepatocytes. J Biol Chem 272:98239830