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Endocrinology Vol. 141, No. 7 2361-2369
Copyright © 2000 by The Endocrine Society

ARTICLES

A Histone Deacetylase Inhibitor Potentiates Estrogen Receptor Activation of a Stably Integrated Vitellogenin Promoter in HepG2 Cells1

Chengjian Mao and David J. Shapiro

Department of Biochemistry, University of Illinois, Urbana, Illinois 61801

Address all correspondence and requests for reprints to: Dr. David J. Shapiro, Department of Biochemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801. E-mail: djshapir{at}uiuc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To compare the role of histone deactylation in estrogen activation of a transiently transfected vitellogenin (VIT) promoter and an integrated VIT promoter in the same cells, we produced three HepG2, human hepatoma, cell lines (HepG2ERV cells) stably expressing human estrogen receptor {alpha} (hER{alpha}) and containing an integrated VIT promoter-chloramphenicol acetyltransferase (VIT-CAT) reporter gene. The three ER-positive HepG2ERV cell lines and wild-type, ER-negative, HepG2 cells cotransfected with cytomegalovirus-hER{alpha} exhibited similar MOX-dependent inductions of 20- to 50-fold with a transiently transfected VIT-luciferase reporter and 15- to 50-fold with a transfected 4-estrogen response element-TATA-luciferase reporter gene. The histone deacetylase inhibitor, trichostatin A, did not enhance MOX induction of the transiently transfected VIT promoter in the HepG2ERV cells. In contrast, trichostatin A dramatically potentiated MOX induction of the stably integrated VIT-CAT reporter gene, resulting in MOX-ER-dependent increases in CAT activity of up to 600-fold. These data demonstrate that although liganded ER exhibits the capacity to fully activate a transiently transfected VIT promoter, under some circumstances the ability to reorganize a repressive chromatin structure may be limiting for steroid receptor action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACETYLATION and deactylation of the histones in nucleosomes have been linked to the formation of a transcriptionally active chromatin structure (reviewed in Refs. 1, 2, 3, 4). Acetylation of lysine residues in the N-terminal region of histones diminishes their positive charge, reducing the strength of their interaction with DNA and facilitating the chromatin remodeling critical for relief of chromatin repression of transcription. Histone acetylation can facilitate access to the DNA template by transcription factors and enhance the remodeling of chromatin required for transcription through long stretches of DNA. It is widely accepted that histone acetylation and deacetylation are dynamic processes that occur continuously and rapidly and thereby allow for rapid changes in histone acetylation/deacetylation and nucleosome organization at specific DNA sites. Histone deacetylase inhibitors, such as trichostatin A (TSA), trapoxin, and sodium butyrate, increase the level of histone acetylation in many types of cells (5, 6, 7). Unlike trapoxin, whose effects are irreversible, and sodium butyrate, which elicits a broad range of effects on cells, TSA is a highly specific, reversible inhibitor of histone deacetylase. Therefore, TSA has found wide application in studies of the effect of histone acetylation and deacetylation on transcription in vivo.

Histone acetylation and deacetylation play important roles in the regulation of gene expression by steroid/nuclear receptors (8, 9, 10). Several coactivators and integrators, including CBP/p300, SRC1, and ACTR, have intrinsic histone acetylase activity (11, 12, 13, 14). In addition, the complex formed by nuclear receptors, coactivators, and CBP/p300 contains the histone acetylase p300/CBP-associated factor. p300/CBP-associated factor also exhibits direct, ligand-dependent association with retinoid X receptor/retinoic acid receptor heterodimers (15). Unliganded nuclear receptors and antagonist-occupied steroid receptors recruit corepressors that inhibit target gene transcription through the recruitment of histone deacetylases (9, 10), such as Sin3 (16, 17, 18, 19, 20, 21).

Although the importance of chromatin reorganization in the regulation of gene expression is widely recognized, most in vivo studies of gene expression by steroid/nuclear receptors employ transient transfections. There have been a few in vivo studies of gene regulation by steroid/nuclear receptors that did not employ transiently transfected templates. Wolffe and co-workers exploited rapid DNA synthesis in microinjected Xenopus oocytes to demonstrate that replication-coupled chromatin assembly is required for repression of basal transcription (22). This group also used the Xenopus oocyte microinjection system to investigate the effects of a positioned nucleosome on thyroid hormone receptor regulation of transcription (23). Early studies suggested that plasmid DNA transiently transfected into cultured mammalian cells by several methods formed chromatin structures similar to those exhibited by cellular genes, and that butyrate, a nonspecific histone deacetylase inhibitor, in some cases stimulated the expression of transiently transfected genes (24, 25). Studies carried out with newer methodologies suggest that the chromatin structure on transiently transfected plasmid DNA is incompletely organized and is more open and assessable to transcription factors than cellular chromatin (26, 27). Using mouse mammary tumor virus (MMTV) as a model system (28), Hager and co-workers compared trans-activation by steroid hormones on the same MMTV promoter when it was transiently transfected and when it was in a stable replicating chromosomal template. They showed that transiently transfected and stable MMTV templates differ in the kinetics of glucocorticoid induction, in their responses to progestins, and in the effects of cAMP (29, 30, 31). Although ER action has been widely studied, to our knowledge there was no information comparing trans-activation of an ER-responsive promoter in the same cells when it was stably integrated and when it was transiently transfected.

A model system we use to study ER action is the estrogen induction of vitellogenin (VIT) gene transcription. In hepatocytes of Xenopus laevis estrogen induces a massive approximately 1000-fold increase in the rate of transcription of the genes encoding the egg yolk precursor protein, VIT (32, 33). Although this system has served as a useful model for estrogen-regulated gene expression (reviewed in Ref. 34), and the estrogen response element (ERE) was initially identified in studies of Xenopus VIT gene expression (35), more recent studies of VIT gene expression have been hampered by the limited estrogen induction of the VIT promoter in transiently transfected cell lines (36). As estrogen receptor (ER), ubiquitous transcription factors and liver-enriched transcription factors are all thought to play a role in VIT gene transcription (36, 37, 38, 39, 40), we reasoned that a hepatocyte-derived cell line might be superior for analysis of transcription from the VIT promoter. Although early passages of the widely used HepG2, human hepatoma cell line (41) contained ER (42), the cells subsequently lost receptor and became ER negative.

To develop a system allowing direct comparison of transiently transfected VIT promoters and stably integrated VIT promoters, we produced several HepG2 cell lines stably transfected to express human ER{alpha} (hER{alpha}), and containing an integrated VIT promoter-chloramphenicol acetyltransferase (VIT-CAT) reporter gene (termed HepG2ERV cells). In HepG2 cells cotransfected with an ER expression plasmid, and in the ER-positive HepG2ERV cell lines, the estrogen, MOX (MOX), trans-activated a transiently transfected VIT-luciferase (VIT-LUC) promoter far more effectively than previously reported for established vertebrate cell lines (36, 38). TSA did not enhance MOX-ER induction of the transiently transfected VIT-LUC reporter gene, indicating that the liganded ER was sufficient for full promoter activation. However, TSA dramatically potentiated MOX induction of the stably integrated VIT promoter. The ability of TSA to strongly potentiate transcription from the integrated VIT promoter indicates that the liganded ER was unable to elicit complete relief of chromatin repression, and that relief of chromatin repression was limiting for activation of the stably integrated VIT template.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
We recently described the construction of the bicistronic hER{alpha} expression plasmid (43), used for stable ER{alpha} expression and in the G418 selections. The VIT promoter driving expression of the CAT reporter gene (VIT-CAT) contains a 610-bp fragment (-596/+14) from the 5'-flanking region of a Xenopus VIT B1 genomic clone. The -596/+14 fragment contains the functional EREs, the upstream activator sequences, and the binding sites for liver-enriched transcription factors and is sufficient for full hormone regulation in transiently transfected primary Xenopus hepatocytes (39, 44, 45). To reduce background transcription, two copies of a polyadenylation signal were inserted upstream of the VIT promoter in pTZVIT-CAT3 (36). To produce the VIT-LUC gene used in the transient transfections, the HindIII/NcoI fragment, including the entire promoter region, from VIT-CAT was inserted into HindIII/NcoI-digested pGL3-basic (Promega Corp., Madison, WI).

The hER{alpha} expression plasmid [cytomegalovirus (CMV)-hER{alpha}] used in transient transfections has been described previously (46). The estrogen-responsive 4ERE-TATA-luciferase reporter gene was created by cloning the synthetic promoter in the 4ERE-TATA-CAT plasmid (46) into the pGL3-basic vector.

Cell culture, transfections, and reporter gene assays
HepG2 cells were maintained in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% charcoal dextran-treated FBS (Atlanta Biological, Norcross, GA). Chinese hamster ovary (CHO) cells were maintained in DMEM/Ham’s F-12 medium (Sigma, St. Louis, MO) supplemented with 5% charcoal dextran-treated newborn calf serum (CD-NBCS). The stable cell lines were maintained in HepG2 medium containing 800 µg/ml G418. Transfections were achieved by calcium-phosphate-DNA coprecipitation and were performed as previously described (46) with minor modifications. After glycerol shock, medium containing the desired concentrations of TSA and MOX was added, and 24 h later, the cells were harvested for assay. For TSA treatment, MOX or ICI 182,780 or 4-hydroxytamoxifen (OHT) induction in the stable cell lines, approximately 400,000 cells were plated in each well of a 6-well plate and maintained for 48 h in the base medium. The medium was then replaced with medium containing the indicated concentration of TSA, MOX, ICI 182,780, or OHT. The cells were maintained for 24 h in the test medium, harvested, and assayed for CAT activity (38) and for luciferase activity using the dual luciferase assay system (Promega Corp.).

Establishment of cell lines expressing transfected ER and VIT-CAT
Linearized human ER{alpha} expression plasmid and VIT-CAT reporter construct (10 µg each/100-mm plate of HepG2 cells) were used in calcium-phosphate transfections. After glycerol shock, the cells were maintained in HepG2 medium for 24 h, then switched to selection medium containing 50% HepG2-conditioned medium and 800 µg/ml G418. After 3 weeks of selection, isolated G418-resistant colonies were picked, subcultured under continued selection in 96-well plates, then transferred to larger plates. After 3 months, several G418-resistant colonies were obtained, and three of these cell lines showed the doubly transfected phenotype of MOX induction of the VIT-CAT reporter. The stably integrated VIT-CAT reporter in these cell lines exhibited similar levels of basal transcription and inducibility by the estrogen, MOX, over the several months needed to carry out these experiments. However, HepG2 cells tend to dedifferentiate over time in culture (42), and the HepG2ERV cell lines showed signs of dedifferentiation and reduced ER expression and inducibility of the VIT promoter after about 200 generations, requiring replacement with frozen stocks.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E2-ER effectively trans-activates the VIT promoter in HepG2 cells
As expression of a transiently transfected Xenopus VIT B1 promoter in established Xenopus cell lines resulted in only a modest 2- to 4-fold induction by estrogen (38), we compared expression of the VIT B1 promoter in transient transfections of HepG2, human hepatoma cells, and CHO cells. As HepG2 cells and CHO cells do not contain significant levels of ER, we cotransfected a CMV-hER{alpha} expression plasmid and the VIT-CAT reporter gene into the cells. 17ß-Estradiol (E2)-ER elicited a 30- to 40-fold induction of CAT activity in the HepG2 cells and a 3- to 4-fold induction of CAT activity in the CHO cells (Fig. 1Go). The minimal activity of the VIT promoter in the CHO cells is not due to either a lower transfection efficiency or a failure of E2-ER to function in CHO cells. CHO cells transfect with a higher efficiency than HepG2 cells and efficiently trans-activate simple reporter genes containing EREs (46). Liver-enriched transcription factors, such as hepatocyte nuclear factor-3 (HNF3), are thought to play a role in VIT expression (39). To determine whether the absence of HNF3{alpha} and HNF3ß in CHO cells was responsible for the low level of VIT-CAT expression, we cotransfected HNF3{alpha} or HNF3ß expression plasmids into the CHO cells. Optimal levels of transfected HNF3{alpha} and HNF3ß expression plasmids increased E2-ER-induced expression of the VIT-CAT promoter in CHO cells from 2- to 3-fold to 7- to 10-fold (Fig. 1Go), but did not elicit the approximately 30- to 40-fold induction of VIT-CAT seen in the HepG2 cells. Moreover, total activity of the VIT promoter was more than 5-fold higher in the HepG2 cells than in the CHO cells transfected with HNF3{alpha} or HNF3ß. Although these data demonstrate that HNF3 plays a significant role in liver-specific expression of the VIT promoter, it clearly represents only one of the factors important in liver-specific VIT gene expression. The 30- to 40-fold E2-ER-mediated induction of VIT gene expression in the HepG2 cells is similar to the fold induction observed in transient transfections of primary Xenopus liver cell cultures (44, 45). We therefore chose HepG2 cells to establish stable cell lines expressing the VIT promoter.



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  		Figure 1. The VIT promoter is efficiently induced by estrogen in HepG2 cells. HepG2 cells were transiently transfected with 2 µg VIT-CAT reporter plasmid, 300 ng pCMV-hER{alpha} expression plasmid, and 300 ng pCMV-luciferase as an internal standard. For CHO cells, 2 µg VIT-CAT reporter plasmid, 5 ng pCMV-hER plasmid (which is a saturating level) (46 ), and 200 ng internal standard plasmid were used. Five hundred micrograms of CMV-HNF3{alpha} or 100 µg CMV-HNF3ß were used when HNF3 was present. These levels of HNF3 expression plasmids were determined to be optimal for E2-ER induction of VIT-CAT expression in preliminary experiments (data not shown). After glycerol shock, the cells were maintained for 48 h in fresh medium containing 10 nM or 1 µM E2 (+E2, filled bars) for CHO or HepG2 cells, respectively. As HepG2 cells rapidly metabolize E2, a high (1 µM) concentration of E2 is required for effective estrogen induction (46 ). The cells were then harvested for CAT assay. An equivalent volume of the ethanol vehicle (-E2, open bars) was added to the control medium. The data present the average ± SEM for samples from three different transfections.

 
Isolation of HepG2 cells containing stably integrated VIT-CAT genes
Because expression of substantial levels of ER is quite toxic to most mammalian cells, we cotransfected the HepG2 cells with the bicistronic CMV-ER{alpha}/NeoR expression plasmid (43) and with the VIT-CAT plasmid. Several independent clones of G418-resistant cells were isolated and subcultured. As HepG2 cells rapidly metabolize E2 (46), we used the more slowly metabolized estrogen, MOX, in most of our studies. To determine whether these cell lines expressed functional ER, we transiently transfected an ER-responsive 4ERE-TATA-luciferase reporter or the VIT-LUC reporter into each cell line and determined whether expression of the reporter genes could be induced by MOX. We identified three cell lines (lines 10, 20, and 25), in which MOX induced expression of the transfected 4ERE-LUC (15- to 52-fold; Fig. 2AGo) and the VIT-LUC reporters (31-to 55-fold; Fig. 2BGo). In these three stably transfected HepG2 cell lines, the fold induction by MOX of the transfected synthetic 4ERE-LUC reporter and that of the native promoter in the VIT-LUC reporter were similar to those seen in the parental HepG2 cells cotransfected with these reporter genes and a saturating level of CMV-hER{alpha} (Fig. 2BGo and data not shown). In wild-type HepG2 cells and in other G418-resistant lines (lines 29, 32, and 47), reporter gene expression was not induced by MOX (Fig. 2AGo), indicating that these cell lines did not contain functional ER. As these cell lines appear to contain VIT-CAT reporter (see below), we designated the ER-positive cell lines HepG2ERV10, -20, -25, and the ER-negative cell lines HepG2V29, -32 and -47.



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  		Figure 2. Identification of ER-positive, stably transfected HepG2 cell lines. A, Wild-type HepG2 cells and the clonal lines of G418-resistant HepG2 cells were transfected with 1 µg 4ERE-TATA-luciferase and 30 ng pRL-SV40 luciferase internal control plasmid. After glycerol shock, the cells were maintained for 48 h in HepG2 medium containing 10 nM MOX (+Mox, filled bars) or an equivalent volume of ethanol vehicle (-Mox, open bars), and harvested for dual luciferase assays. The open bars are from single experiments, and the filled bars represent the average of two independent transfections. B, Comparison of the induction of VIT-luciferase activity in the stably transfected ER-positive cell lines and in HepG2 cells cotransfected with saturating levels of ER expression plasmid. For the stable cell lines, 50 ng VIT-luciferase and 10 ng pRL-SV40 luciferase internal control plasmid were transfected into each well of six-well plates. For HepG2 cells, 100 ng VIT-luciferase, 7.5 ng CMV-hER{alpha} expression plasmid, and 30 ng pGL-SV40 luciferase internal control plasmid were transfected into 60-mm dishes. The data for HepG2 cells and for the HepG2ERV10 cell line represent the average of three independent transfections. The other data are from single transfections.

 
The stably integrated VIT-CAT reporter gene is induced by MOX
To determine whether the ER-positive HepG2 cell lines contained functional VIT-CAT reporter gene, we performed dose-response curves for MOX induction of CAT activity in the three ER-positive cell lines (Fig. 3Go). A fourth ER-positive cell line contained the VIT-CAT gene (see below), but did not express it, and was not studied further (data not shown). For all three cell lines, CAT activity increased dramatically between 10-11 and 10-10 M MOX. The MOX dose-response curves are similar to those reported for MOX induction of cellular genes in stably transfected ER positive lines of HepG2 cells (47, 48). All three cell lines exhibited a very low, but reproducible, basal level of expression in the absence of MOX. Because the basal activity was so low, it proved difficult to use the pure antiestrogen ICI 182,780 to determine whether traces of estrogen in the medium were responsible for the basal activity of the integrated VIT-CAT genes (data not shown).



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  		Figure 3. Dose-response curves for MOX induction of the VIT-CAT gene in the stably transfected HepG2ERV cell lines. The three HepG2ERV cell lines were split and plated in 6-well plates 2 days before MOX treatment. After removal of the medium, fresh medium containing the indicated concentration of MOX was added to the plates. Twenty-four hours later, the cells were harvested for CAT assays. An equivalent volume of ethanol was used as a MOX-negative control (none).

 
To determine whether the cell lines in which there was no MOX induction of CAT activity contained the VIT-CAT promoter-reporter gene, we isolated genomic DNA from these cell lines and used it as a template in PCR reactions. Using PCR primers that covered either the whole promoter region or part of the promoter region and part of the CAT reporter gene-coding region, genomic DNA from all of these cell lines yielded PCR-amplified products, suggesting the presence of integrated VIT-CAT genes (data not shown). Of course, we cannot rule out the possibility that the VIT-CAT gene is truncated or mutated in these cell lines or integrated into a silent chromosomal site in the ER-positive cell line.

TSA strongly potentiates MOX induction of the VIT-CAT reporter gene
Moxestrol induction of the stably integrated VIT promoter is much less efficient than induction of the transiently transfected VIT promoter (compare Figs. 1Go and 2Go with Fig. 3Go). This suggested MOX-ER might be insufficient for full chromatin remodeling of the integrated VIT promoter. To test this possibility, we examined the effect of TSA on MOX induction of the integrated VIT-CAT gene. In preliminary studies we found that TSA enhanced the expression of the integrated VIT-CAT reporter, and that stimulation of VIT-CAT expression was maximal at 3 µM TSA (data not shown). We tested the effect of 3 µM TSA on induction of VIT-CAT activity by 50 pM MOX, a subsaturating concentration, and by 10 nM MOX, a concentration of MOX that should saturate the ER in HepG2ERV10 cells (Fig. 3Go). At both 50 pM and 10 nM MOX, TSA dramatically stimulated the expression of the VIT-CAT reporter gene. Induction of VIT-CAT activity increased from 3- to 4-fold in the presence of MOX alone to more than 500-fold in the presence of MOX plus TSA (Fig. 4Go). Similar results were obtained in the other two HepG2ERV cell lines, although the increase in fold expression with TSA was somewhat lower in HepG2ERV25 than in HepG2ERV10 and HepG2ERV20 cell lines.



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  		Figure 4. TSA potentiates the induction of the VIT-CAT gene in HepG2ERV10 cells. Approximately 400,000 cells/well were plated in 6-well plates 2 days before TSA and/or MOX treatment. The medium was removed, and fresh medium with or without TSA containing 50 pM MOX (gray bars) or 10 nM MOX (black bars), or the ethanol control (-Mox, open bars) was added. Twenty-four hours later, the cells were harvested for CAT assays. The data represent the average ± SEM for samples from three different wells. To represent the large, several hundred-fold induction in the presence of TSA, the scale of the y-axis is not continuous.

 
Antiestrogens interfere with TSA stimulation of VIT-CAT expression
TSA alone elicited a significant approximately 30-fold increase in VIT-CAT expression (Fig. 4Go). This might be due to synergism with traces of estrogen remaining in the culture medium or to activation of the promoter by TSA in the presence of unliganded ER. To test these possibilities we examined the effect of antiestrogens on the ability of TSA to stimulate VIT-CAT expression. To determine whether an estrogenic ligand bound to ER was required for synergy with TSA, we examined the ability of the antiestrogens OHT and ICI 182,780 to block induction of the integrated VIT-CAT gene by MOX-ER and TSA. A 200-fold molar excess of OHT over MOX blocked most of the synergistic TSA induction, and a 200-fold excess of ICI 182,780 almost completely abolished the synergistic TSA induction (Fig. 5Go). These data are in agreement with other observations suggesting that OHT can be a weak agonist in HepG2 cells (47, 48).



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  		Figure 5. Antiestrogens interfere with induction of the VIT promoter by MOX and TSA. Approximately 400,000 HepG2ERV10 cells/well were plated in 6-well plates and grown for 2 days. The cells were then transferred to medium containing or lacking TSA, MOX, ICI 182,780, or OHT as indicated. After 24 h the cells were harvested and assayed for CAT activity. The data represent the mean ± SEM for three separate sets of cells.

 
The pure antagonist, ICI 182,780 substantially reduced, but did not completely eliminate, TSA activation of the integrated VIT-CAT gene in the HepG2ERV10 cells (Fig. 5Go). As TSA plus a relatively low 50 pM concentration of MOX was sufficient for full activation of the VIT-CAT gene (Fig. 4Go), it is likely that a substantial part of the TSA activation results from synergy with very low levels of liganded ER in the HepG2ERV cells.

TSA activation of VIT-CAT expression requires ER
To determine whether ER was required for TSA activation of the integrated VIT-CAT gene in the absence of added MOX, we examined the ability of TSA to activate the VIT-CAT gene in two ER-negative HepG2V cell lines. Although PCR analysis indicates that the VIT-CAT gene is present in these two cell lines, the possibility that it has been mutated or inactivated could not be excluded. We therefore examined the ability of transiently transfected ER plus MOX to synergize with TSA in activating the VIT-CAT genes in the HepG2V29 and HepG2V32 cell lines. In the presence of transiently transfected ER, MOX plus TSA induced VIT-CAT expression 11-fold in the HepG2V29 cell line and 4.4-fold in the HepG2V32 cell line (Fig. 6Go). The fold induction by MOX plus TSA is low because only a small percentage (<5%) of HepG2 cells is transfected using calcium chloride (49), and the untransfected cells do not contain functional ER. These data demonstrate that a functional VIT-CAT gene is present in the HepG2V29 and HepG2V32 cell lines. When TSA is added to the medium of HepG2V29 and HepG2V32 cells transfected with carrier DNA, overall CAT activity and TSA activation are extremely low (Fig. 6Go). These data suggest that liganded ER is required for TSA activation of VIT-CAT expression.



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  		Figure 6. TSA does not activate the VIT promoter in stably transfected ER-negative HepG2 cell lines. ER-negative HepG2V29 and HepG2V32 cells were plated in six-well plates. Forty-eight hours later, the cells were either transfected with 200 ng pCMV-hER{alpha} expression plasmid (+ER) or mock transfected for TSA treatment (-ER). For the transfected cells, after glycerol shock the cells were maintained for 24 h in medium containing ethanol vehicle (hatched to the left), 3 µM TSA alone (hatched to the right), 50 pM MOX alone (horizontal hatching), or 3 µM TSA plus 50 pM MOX (black bars) and then harvested and assayed for CAT activity. For mock-transfected cells, the medium was replaced with fresh medium containing 3 µM TSA (cross-hatched) or an equivalent volume of ethanol vehicle (open bars). The cells were maintained for an additional 24 h, harvested, and assayed for CAT activity. The data represent the mean ± SEM for three separate sets of cells.

 
TSA does not potentiate MOX-ER activation of a transiently transfected VIT gene
When TSA was added to the medium of wild-type HepG2 cells transiently transfected with an ER{alpha} expression plasmid and a VIT promoter, it did not potentiate MOX induction of the transiently transfected VIT-reporter gene (data not shown). Cell density can affect the ability of TSA to stimulate gene expression (50), and it can sometimes be difficult to control when transfections are performed on separate plates. We therefore also carried out transfections using the HepG2ERV cell lines in which we could use the same cells to compare TSA effects on the transiently transfected VIT-LUC reporter and the stably integrated VIT-CAT reporter. When we compared reporter gene activity on the two VIT templates in the same cells, we found that TSA plus MOX resulted in high level expression of the stably integrated CAT reporter gene (Fig. 7AGo). Fully induced (+TSA +Mox) CAT activity from the integrated VIT promoter was similar (4000–5000 cpm/µg) in the transfected HepG2ERV cells and the untransfected HepG2ERV cells shown in Fig. 4Go. Although fully induced activity was unaffected, transient transfection elevated both basal and TSA-induced CAT activity in the stable cell lines. Addition of ICI 182,780 reduced the activation by approximately 60% (Fig. 7AGo), suggesting that traces of estrogens play a role in the higher basal activity and higher TSA activation in the transiently transfected cells.



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  		Figure 7. TSA does not potentiate the expression of a transiently transfected VIT promoter. A, TSA potentiates MOX induction of the stably integrated VIT-CAT gene, and ICI 182,780 significantly reduces the potentiation by TSA. HepG2ERV10 cells were plated into six-well plates 48 h before transfection. The cells were then transfected with 50 ng VIT-LUC plasmid and 10 ng pRL-SV40 plasmid as an internal standard. After glycerol shock, the cells were maintained for 24 h in medium containing 3 µM TSA and/or 50 pM MOX, 10 nM ICI 182,780, or ethanol vehicle. The cells were harvested, and samples were assayed for luciferase activity using the dual luciferase assay protocol (B) followed by assays for CAT activity (A). The data are presented as the relative CAT activity normalized to the -MOX +TSA sample and represent the mean ± SEM for three separate sets of cells. B, TSA does not potentiate induction of the transiently transfected VIT-LUC reporter gene.

 
Similar to the data for wild-type HepG2 cells, expression of the transiently transfected VIT-LUC reporter was not stimulated by TSA in the same HepG2ERV cells in which TSA stimulated expression of the integrated VIT-CAT template. Although TSA appears to significantly reduce MOX-ER induction of the transiently transfected VIT-LUC reporter (Fig. 7BGo), this apparent reduction in VIT-LUC activity by TSA is largely due to correcting for the TSA-mediated 2- to 5-fold increase in the activity of the simian virus 40 (SV40) promoter used as an internal control (51) (our unpublished data).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The massive estrogen induction of Xenopus VIT messenger RNA (mRNA) is a widely studied model used to establish many basic features of estrogen induction of gene expression (32, 33, 34, 35, 36, 37, 38, 39, 40). However, the substantial basal activity and poor estrogen inducibility of the VIT promoter in established vertebrate cell lines greatly hindered more recent studies. Only in primary Xenopus hepatocytes, which are difficult to isolate and work with, was a robust estrogen induction achieved. As promoter analysis suggested that ER, liver-specific, and ubiquitous transcription factors all play a role in expression of the VIT promoter, we tested CHO cells cotransfected with HNF3{alpha} or HNFß, which are enriched in hepatocytes, and HepG2 cells, which maintain a number of liver-specific functions (41, 42). Cotransfecting optimal amounts of HNF3{alpha} or HNF3ß expression plasmids into the CHO cells enhanced MOX-ER induction of transiently transfected VIT-CAT in the CHO cells from 2- to 3-fold to 7- to 10-fold. HepG2 cells may contain saturating levels of HNF3s, because cotransfecting HNF3{alpha} or HNF3ß did not increase the induction of VIT-CAT expression (data not shown). Moxestrol-ER induced a 30- to 40-fold increase in expression of the VIT-CAT reporter in HepG2 cells. The 30- to 40-fold induction in HepG2 cells is similar to the fold induction previously reported in primary Xenopus hepatocytes (44, 45). Human HepG2 cells, therefore, mimic Xenopus liver cells in the expression of a transiently transfected VIT promoter and represent a useful new system for analysis of those aspects of VIT gene expression that do not involve chromatin.

It would have been quite difficult to isolate HepG2 cell lines stably expressing three different cotransfected plasmids: the neomycin phosphotransferase gene for G418 selection, the toxic hER gene and the VIT-CAT gene. We recently described the use of a vector encoding a bicistronic mRNA encoding both the ER and the neomycin phosphotransferase genes (43). As the bicistronic system allowed us to use only two plasmids in the cotransfections, it substantially simplified isolation of the ER-positive, VIT-CAT-positive cell lines. The use of a bicistronic mRNA may prove generally useful in simplifying the production of cell lines in which stable integration of more than one gene is desired. Despite the fact that the VIT-CAT gene was on a different plasmid and was not ligated to the plasmid encoding the bicistronic mRNA before transfection, because we adjusted the ratios of the two plasmids used in the transfections, all of the ER-positive and ER-negative G418-resistant cell lines we isolated contained VIT-CAT. Despite translation of the ER and neomycin phosphotransferase genes from the same mRNA and transcription of the VIT-CAT mRNA from a different promoter, the toxicity of the ER resulted in our isolation of several G418-resistant cell lines that were ER negative and contained VIT-CAT DNA.

Although the most straightforward explanation for TSA potentiation of MOX-ER induction of the integrated VIT-CAT gene is based on the ability of TSA to inhibit histone deacetylase activity, other possibilities had to be considered. TSA stimulates expression from both stably integrated and transiently transfected CMV promoters, resulting in an increase of severalfold in ER levels in TSA-treated cells (52, 53) (our unpublished data). Several types of data indicate that TSA induction of additional ER is not responsible for its ability to potentiate expression of the VIT-CAT gene. TSA increases MOX-ER activation of the stably integrated VIT-CAT gene by up to 600-fold, a level far higher than would be expected from simply raising ER levels by severalfold. ER-positive HepG2 cell lines, including those used in this study, with levels of ER ranging from a few thousand molecules of ER per cell to about 30,000 molecules ER/cell, all show effective MOX-dependent induction of several mRNAs transcribed from chromosomal genes (47, 48). Without TSA treatment, the HepG2ERV cell lines used in this study all contain sufficient ER for strong MOX induction of the cellular proteinase inhibitor 9 gene (47). These data support the view that the level of ER in these HepG2 cell lines is not limiting for expression of chromosomal genes. Our observation that TSA has similar effects in potentiating activation by a subsaturating concentration of MOX (50 pM) when only a fraction of the ER is liganded and by a saturating concentration of MOX (10 nM) when all of the ER is liganded also supports the view that there is ample ER for activation of the chromosomal VIT-CAT genes in the HepG2ERV cells. In addition, three types of data indicate that the level of ER in the HepG2ERV cell lines not treated with TSA is sufficient for maximal activation of a transiently transfected VIT promoter. 1) The 30- to 50-fold induction of the VIT-luciferase and the 15- to 50-fold induction of the 4ERE-TATA-luciferase by estrogen in the stably transfected HepG2ERV cell lines are similar to the fold induction of these reporter genes in transiently transfected HepG2 cells cotransfected with high saturating levels of the ER expression plasmids (Fig. 2Go and data not shown). 2) TSA does not stimulate the expression of a transiently transfected VIT promoter in the ER positive cell lines. 3) Even in the absence of added estrogen, when only trace concentrations of estrogens are present in the medium and a very small proportion of the endogenous ER will be liganded, ICI 182,780 retains the ability to substantially reduce TSA stimulation of VIT-CAT expression. Taken together, these data indicate that changes in ER levels are not a major factor in TSA potentiation of MOX-ER induction of VIT-CAT expression. As the complexes involved in remodeling of chromatin at the VIT promoter are not known, we cannot formally exclude the possibility that some of the effects of TSA on VIT gene expression are indirect and are due to TSA inducing the expression of factors required for remodeling of VIT chromatin.

TSA significantly stimulates VIT-CAT expression in the absence of added MOX, raising the possibility that unliganded ER might be able to activate the integrated VIT gene in the presence of TSA. We previously reported that it is extremely difficult to eliminate trace concentrations of estrogens from cell culture medium, and that residual estrogens are present in both charcoal-stripped serum and serum-free culture medium (46). Although HepG2 cells rapidly metabolize E2, MOX is much more resistant to degradation. Some of the unidentified estrogens in the medium may be similarly resistant to degradation. As ICI 182,780 substantially reduces TSA stimulation of VIT-CAT expression, at least some of the ability of TSA to activate the VIT-CAT gene is therefore likely to be based on its ability to potentiate the effects of very low concentrations of estrogens in the culture medium. It is possible that TSA treatment results in the formation of a more open and accessible chromatin structure that facilitates binding to the VIT promoter by the very low concentration of liganded ER present in these cells. The inability of TSA to achieve significant activation of the VIT-CAT genes in ER-negative HepG2V cells suggests that although TSA dramatically potentiates ER activation of the VIT promoter, TSA may be unable to act as an independent activator of VIT expression.

Surprisingly, transient transfection results in increased levels of basal and TSA-mediated expression of the integrated VIT-CAT reporter. Determining whether transient transfection increases the expression level of other chromosomal genes will require further study. Increased VIT-CAT expression resulting from transient transfection could result from a combination of the glycerol shock used in transient transfections and the uptake of large amounts of DNA by the cells, resulting in redistribution of some chromatin proteins onto the transfected DNA. This redistribution of chromatin proteins might diminish chromatin repression of the VIT gene, and facilitate its expression. The idea that transient transfection and TSA treatment both decrease chromatin repression is consistent with our observation that the effects of transient transfection and TSA on VIT-CAT expression are not additive. Fully induced VIT-CAT activity (+TSA, +MOX) is similar in untransfected and transiently transfected cells. As ICI 182,780 reduced the expression of the VIT-CAT gene in transiently transfected HepG2ERV cells treated with TSA by about 60%, residual estrogens bound to ER are likely to also play a role in the expression of the integrated VIT-CAT gene in transiently transfected cells treated with TSA.

Although the fold induction of VIT-CAT expression by TSA differs substantially in untransfected and transiently transfected cells, the data presented in Figs. 4Go and 7AGo clearly establish that TSA treatment strongly potentiates MOX-ER-dependent expression of the stably integrated, chromatin-assembled, VIT-CAT gene. In contrast, in both ER-positive HepG2ERV cells and wild-type HepG2 cells cotransfected with an ER expression plasmid and a VIT reporter gene (data not shown), TSA does not stimulate, and may modestly decrease, the expression of the transiently transfected VIT-reporter gene.

In male Xenopus liver, very high levels of E2 induce an approximately 1000-fold increase in the absolute rate of VIT gene transcription (32, 33). In HepG2ERV cells, physiological concentrations of an estrogen (50 pM) and TSA induce VIT-CAT expression up to 600-fold, providing the first established cell lines that can mimic the induction of VIT transcription seen in liver. Although E2 is sufficient for full induction of VIT transcription in Xenopus liver, TSA is required in the HepG2ERV cells. The requirement for TSA may reflect imperfect homology between Xenopus and human chromatin remodeling systems, differences in the level of chromatin remodeling proteins in the Xenopus and human cells, or partial dedifferentiation of HepG2 cells over the many generations they have been in culture. An alternative possibility is that a round of DNA replication is required for full activation of the VIT genes. In Xenopus liver, pharmacological doses of estrogen stimulate a burst of DNA synthesis that parallels the activation of VIT transcription (54, 55). Because estrogen is toxic to the HepG2ERV cells, there appears to be limited cell division when the cells are maintained in MOX. It is therefore possible that TSA facilitates VIT activation by reproducing a loosening of chromatin structure that normally occurs during estrogen-stimulated DNA replication.

It is widely accepted that activation of gene transcription by liganded nuclear receptors occurs at two levels. First, there is relief of chromatin repression. This allows basal transcription at a rate intrinsic to the particular promoter. Steroid/nuclear receptors also act as de novo transcription activators, increasing transcription to rates beyond those exhibited by the basal promoter. It has been difficult to determine whether relief of chromatin repression or de novo activation is limiting for genes regulated by ER. The vast majority of regulatory studies either employ a promoter that is transiently transfected or in vitro transcribed, and therefore does not exhibit an organized chromatin structure, or study the expression of a cellular gene, in which effects due to relief of chromatin repression and de novo transcription activation are difficult to dissociate. This issue was addressed by the development of HepG2ERV cell lines, in which the activity of both a transiently transfected and an integrated VIT promoter can be studied in the same cell line. Our data indicate that acetylation-dependent chromatin remodeling can be limiting for induction of gene expression by liganded ER.


   Acknowledgments
 
We are grateful to Dr. W. Chen for the gifts of the HNF3 expression plasmids, to Dr. S. McMasters of the University of Illinois Cell/Media Facility for contributions to the production of the cell lines, and to Dr. R. Dodson for many helpful comments on the manuscript.


   Footnotes
 
1 This work was supported by NIH Grant HD-16720. Back

Received November 18, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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