CBP Recruitment and Histone Acetylation in Differential Gene Induction by Glucocorticoids and Progestins
James R. Lambert and
Steven K. Nordeen
Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado 80262
Address all correspondence and requests for reprints to: Steven K. Nordeen, Department of Pathology and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262. E-mail: steve.nordeen{at}uchsc.edu.
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ABSTRACT
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We have analyzed histone acetylation at the steroid-responsive mouse mammary tumor virus (MMTV) promoter in five separate cell lines that express functional glucocorticoid and/or progesterone receptors. Chromatin immunoprecipitation assays reveal that glucocorticoid and progesterone receptors bind the MMTV promoter after hormone addition but that receptor binding is not associated with an increase in acetylation of histone H3 or H4. We have, however, found one exception to this rule. Previously we described a cell line [T47D(C&L)] that displayed a remarkable differential induction of MMTV by glucocorticoids and progestins. At one chromosomal locus (MMTV-luciferase), MMTV is preferentially induced by glucocorticoids, whereas at another locus within the same cell (MMTV-CAT), MMTV is activated by both glucocorticoids and progestins. Here we show that the glucocorticoid-mediated induction of MMTV-luciferase is accompanied by increased recruitment of CBP to the promoter and increased histone H3 and H4 acetylation, whereas the hormonal induction of MMTV-CAT in the same cell exhibits a more modest CBP recruitment without any increase in histone acetylation. These studies suggest that increased histone acetylation may serve a potentiating function for MMTV promoter activation at certain loci. However, increased histone acetylation is not requisite for steroid-mediated induction of transcription at all genes.
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INTRODUCTION
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TRANSCRIPTIONAL REGULATION in eukaryotic genomes occurs within highly compacted chromatin. The basic structural unit of chromatin is the nucleosome, which consists of DNA wound around the core histone octamer composed of histones H2A, H2B, H3, and H4 (1). The N terminus of each of the core histones contains highly conserved residues that are targets for covalent modification by a variety of cellular factors. Studies in Saccharomyces cerevisiae have shown that the tails of histones H3 and H4 are important for transcriptional regulation. Strains carrying mutations in the tails of histones H3 and H4 that render them unable to be modified exhibit increased derepression and reduced activation of numerous genes (2, 3). The modifications of histone tails observed include acetylation, methylation, and phosphorylation. These modifications have been implicated in processes such as transcriptional regulation, chromosomal condensation, mitosis and apoptosis (4). It has been proposed that histone modification facilitates the accession of target sites by DNA-interacting factors by reducing the electrostatic attraction between the negatively charged phosphate backbone of DNA and the positively charged N-terminal tails of histones (5). Strahl and Allis (4) have offered an alternative, termed the histone code hypothesis, that suggests the modifications serve as recognition targets for proteins involved in the assembly and activity of regulatory complexes.
The correlation between histone acetylation and gene activation was first described by Allfrey (6) and recently, the role of histone acetylation in transcriptional regulation has become an intense area of investigation. Increases in histone acetylation have generally been correlated with increases in transcriptional activity. For example, the human B-globin locus has been shown to be hyperacetylated when being actively transcribed in chicken erythrocytes (7). Conversely, decreased histone acetylation often accompanies gene silencing or repression (8, 9). Inactive X chromosomes in female mammals have been shown to lack histone H4 acetylation, presumably leading to repression of gene activation (10). However, the correlation between histone acetylation levels with transcriptional activity at target promoters is not universal. An analysis of 40 yeast genes that respond to specific activators and repressors demonstrated that gene activation is not always accompanied by an increase in histone acetylation nor is repression of transcription always associated with a decrease in histone acetylation (11).
Direct links between steroid hormone activation of gene expression and histone acetylation have recently been described (12, 13). Steroid hormone receptors are members of the nuclear receptor family of ligand-activated transcriptional regulatory proteins. Recent work in the field of nuclear receptor action has demonstrated an association of receptors with coregulatory proteins termed coactivators and corepressors. When bound by hormonal antagonists, or in some cases, in the absence of ligand, nuclear receptors repress transcription through their association with corepressors. Upon binding hormonal agonists, receptors recruit transcriptional coactivators to target promoters. In turn, coactivators functionally, and perhaps physically, bridge DNA-bound receptors and the general transcription machinery forming a complex capable of promoting the activation of gene expression (12, 14, 15). A developing picture of the molecular mechanism of nuclear receptor coregulatory protein function suggests that their roles in receptor-mediated gene expression are dependent on histone-modifying activities that are either intrinsic or associated. For example, steroid receptor coactivator 1 has been shown to possess intrinsic histone acetyltransferase (HAT) activity (16) as has CBP/p300 (17), general coactivators of steroid receptors and other transcription factors. Conversely, the corepressors nuclear receptor corepressor (N-CoR) and silencing mediator of retinoic acid receptor and thyroid receptor (SMRT) have been shown to be part of a large multisubunit complex containing histone deacetylase activity (18, 19, 20). The ability of coregulatory proteins to affect the levels of histone acetylation at target promoters thus appears to be a key step in the activation and/or repression of nuclear receptor target genes.
Studies on estrogen actions have observed increased histone acetylation at several estrogen-responsive promoters in response to hormone (13, 21, 22, 23) as well as a global increase in histone acetylation (24). Published studies on glucocorticoid and progestin action have been largely confined to the study of the MMTV promoter thus far. The mouse mammary tumor virus (MMTV) promoter has been a model system for studying the molecular mechanisms of steroid hormone receptor action, especially the role of chromatin in steroid mediated transcriptional regulation. MMTV can be activated by several steroid hormones including androgens, glucocorticoids, mineralocorticoids and progestins (25, 26, 27, 28, 29, 30, 31, 32). Previous studies using histone deacetylase inhibitors gave conflicting data suggesting that increased acetylation was associated with either a decreased or increased hormone induction (33, 34). Examination of histone acetylation at a stably integrated MMTV promoter in cell lines, whose hormone induction is inhibited by trichostatin A, suggests that glucocorticoids and progestins inhibit histone acetylation at the MMTV promoter (35, 36). However, the MMTV promoter responds differently in other cell lines and even within the same cell two MMTV promoters can behave differently (37). Based on these observations, we have suggested that the regulation of the MMTV promoter is locus dependent and that, therefore, it can be regulated very differently in different chromatin contexts (37).
In these studies, we have used chromatin immunoprecipitation (ChIP) assays to assess the role of histone acetylation in steroid-mediated induction of the MMTV promoter in a variety of cell and chromatin contexts. The MMTV-luciferase transgene in T47D(C&L) cells is induced by glucocorticoids to a much greater degree than progestins. The differential responsiveness of this MMTV promoter to the two hormones may be the result of differential induction of histone acetylation at the promoter. The observation of differential recruitment of the coactivator and histone acetyltransferase, CBP, may provide a mechanistic underpinning accounting for the differential acetylation evoked by the two hormones. No change in histone acetylation at the MMTV promoter is seen at the MMTV-CAT transgene in the same cell or at MMTV promoters in several other cell lines. Based on these data, we propose that increased histone acetylation is not required for response to an activated steroid receptor but, in certain chromatin environments, acetylation may accompany induction and potentiate the transcriptional response.
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RESULTS
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Increased Histone Acetylation Is Not Required for Induction of the MMTV Promoter But May Potentiate Activation
Previously, we described the generation of human breast cancer cell lines that maintain stably replicating MMTV-luciferase and MMTV-CAT transgenes at separate chromosomal loci. We showed that remodeling of MMTV chromatin and the concomitant activation of the MMTV promoter was induced equally by glucocorticoids and progestins in one chromosomal context (MMTV-CAT) but was preferential for glucocorticoids in another (MMTV-luciferase). Furthermore, treatment of these cells with the histone deacetylase inhibitors butyrate and trichostatin A modulated MMTV promoter regulation disparately at the two chromosomal locations (37).
The preferential induction of MMTV-luciferase is illustrated in Fig. 1A
. Although the progestin induction is dwarfed by the robust glucocorticoid induction, there is a significant luciferase induction over background by progestins. To test the hypothesis that differential induction of MMTV-luciferase in T47D(C&L) cells is a consequence of the ability of glucocorticoid receptor (GR) and progesterone receptor (PR) to promote histone acetylation at the MMTV-luciferase promoter, we analyzed acetylation of MMTV promoter histones H3 and H4 by ChIP assay. Because the MMTV-CAT transgene present in T47D(C&L) cells is activated to a similar degree by both glucocorticoids and progestins, this gene provides a control for steroid-mediated influences on histone acetylation at the MMTV promoter using the same immunoprecipitated chromatin samples. As anticipated, glucocorticoid treatment resulted in a robust increase in acetylation at the MMTV-luciferase gene (Fig. 1B
). Increased acetylation of both histone H3 and H4 was observed. A lesser increase in acetylation in response to progestins was observed consistent with the lesser induction by progestins. Surprisingly, no change in acetylation of H4 or H3 was seen at the MMTV-CAT locus in the same immunoprecipitations (Fig. 1B
). To confirm that GR and PR were in fact bound to the MMTV-CAT and MMTV-luciferase promoters at 1 h after hormone, we performed ChIPs using antibodies specific for either GR or PR. Both GR and PR were shown to be bound to the MMTV promoters in T47D(C&L) cells in response to the appropriate hormone (Fig. 1B
).

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Figure 1. ChIP Analysis of Histone H3 and H4 Acetylation and Steroid Receptor Loading in Response to Dexamethasone and R5020 at Two MMTV Promoters in T47D(C&L) Cells
A, Hormonal induction of luciferase and CAT activity directed by pHHLuc and pHHCAT in T47D(C&L) cells. Cells were seeded at a density of 0.6 x 106 cells/60-mm dish and 18 h later were treated with ethanol vehicle (-), the synthetic progestin, R5020 (10 nM) (P) or the synthetic glucocorticoid, dexamethasone (10 nM) (G) for 24 h. Luciferase and CAT activities were determined on aliquots of the same extract and the data normalized to protein content of the extract. Results represent the average of three experiments where each condition was done in duplicate. LU, Light units. B, Chromatin was prepared from T47D(C&L) cells treated with ethanol, dexamethasone or R5020 for 1 h and immunoprecipitated with antibodies against acetylated histone H3 ( AcH3), acetylated histone H4 ( AcH4), GR, or PR. The purified DNA was amplified with primers to either the MMTV-luciferase or MMTV-CAT promoters (see Materials and Methods). Input DNA is shown as a control for equal amounts of DNA in the samples.
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Kinetics of Histone Acetylation and PR Binding at the MMTV Promoter
The above observations on the MMTV-CAT transgene indicate that an increase in histone acetylation does not accompany steroid-mediated chromatin remodeling and transcriptional induction of the promoter. However, it is possible that increases in histone acetylation could have been missed if the induction was transient or cyclical in nature. Shang et al. (13) have shown that activated estrogen receptors promote histone acetylation at the cathepsin D promoter in a time-dependent manner. They showed that receptor binds within 15 min following addition of estradiol and promotes maximal induction within 30 min following hormone addition and that increases in histone acetylation followed these same kinetics on the cathepsin D promoter. We, therefore, performed a time course of progestin-mediated induction of MMTV-CAT in T47D(C&L) cells. Cultures were treated with hormone and harvested at 20-min intervals (up to 1 h) and subjected to ChIP assays using anti-acetyl histone H4 and anti-PR antibodies. As shown in Fig. 2
, we did not observe an increase in histone H4 acetylation at any time point. We have also analyzed 15-min intervals and see no change (data not shown). Significantly, we were able to show that PR is bound at all time points at which we assayed for histone acetylation (Fig. 2
). Analysis of the MMTV-luciferase promoter from the same immunoprecipitates confirmed the finding that, in contrast to MMTV-CAT, increased acetylation does accompany induction of MMTV-luciferase by progestins, even though this increase and the subsequent biological response is less than with glucocorticoids (Fig. 2
). Our previous studies have also shown that progesterone and glucocorticoid-dependent chromatin remodeling in T47D(C&L) cells occurs within 30 min at the MMTV promoter (37). Together, these data suggest that increased histone acetylation may accompany hormone-mediated induction but is not required for chromatin remodeling or transcriptional induction to occur at the MMTV promoter.

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Figure 2. Kinetics of PR Loading to the MMTV-CAT Promoter in T47D(C&L) Cells
Soluble chromatin was prepared from T47D(C&L) cells that had been treated for 0, 20, 40, and 60 min with R5020 and immunoprecipitated with antibodies against acetylated histone H4 ( AcH4) and against PR. Primers to the MMTV-CAT and MMTV-luciferase promoters were used to amplify the DNA from the same immunoprecipitates.
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Induction of the MMTV Promoter by Steroid Hormones Does Not Require Histone H4 Acetylation in Breast Cancer Cells or Fibroblasts
To test the generality of the observation that induction of histone acetylation is not required for hormone-induced transcription at the MMTV promoter, we performed ChIP assays on another subline of T47D cells that maintains a stably replicating MMTV-CAT transgene. The T47D(pAHCAT) cell line expresses functional PR. Treatment of these cells with the progestin, R5020, results in a dose-dependent induction of CAT activity of the integrated MMTV-CAT reporter gene (38). As shown in Fig. 3A
, the MMTV-CAT gene is induced after addition of R5020, indicating the presence of a functional PR and MMTV promoter in these cells. However, no change is observed in histone H4 acetylation after hormone treatment (Fig. 3B
). These findings suggest that, in T47D-based cell lines, stably replicating MMTV promoters do not require histone acetylation to elicit transcriptional induction by steroid hormones.

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Figure 3. ChIP Analysis of MMTV Promoter Histone H4 Acetylation after Progestin Treatment of T47D(pAHCAT) Cells
A, CAT reporter gene activity of MMTV-CAT after 24 h induction with R5020. B, ChIP analysis of acetylated histone H4 at the MMTV promoter. Cells were treated with either ethanol (-) or the synthetic progestin R5020 (P) and soluble chromatin subjected to immunoprecipitation with antibodies against acetylated histone H4 ( AcH4). Primers to the MMTV-CAT promoter were used to amplify the final DNA.
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We next sought to determine whether the lack of requirement for MMTV histone acetylation is peculiar to influences of the CAT reporter or T47D- derived cell lines. Using the mouse fibroblast cell line, 4F, that expresses GR and PR, we generated stable transfectants of 4F cells using the same MMTV-luciferase reporter present in the T47D(C&L) cells. Several, independent clones were recovered and three isolates were subjected to treatment with the glucocorticoid, dexamethasone, or the progestin, R5020. As shown in Fig. 4A
, all three clones analyzed exhibited hormone-mediated inductions of MMTV-luciferase activity. However, neither hormone treatment resulted in an increase in histone H4 acetylation (Fig. 4B
). ChIP assay of the same chromatin samples indicate that GR and PR bind at the MMTV promoter after hormone addition (data not shown). These experiments confirm our initial findings in T47D-based cell lines that transcriptional activation of the MMTV promoter by steroid hormones does not require an increase in histone acetylation and suggest that, in general, a mechanism not involving increased histone acetylation is used in the activation of MMTV by steroid hormones.

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Figure 4. ChIP Analysis of MMTV Promoter Histone H4 Acetylation in Three Mouse Fibroblast Clones
A, Three 4F clones (# 4, 7, 10) stably maintaining MMTV-luciferase transgenes were treated with either ethanol (-), dexamethasone (G) or R5020 (P) and assayed for luciferase activity. B, ChIP assays were performed on the three clones using antibodies against acetylated histone H4. The final DNA was amplified with primers to the MMTV-luciferase promoter.
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Differential Recruitment of CBP at MMTV-Luciferase
To explore the basis of the differential ability of glucocorticoids and progestins to induce transcription of MMTV-luciferase and histone acetylation at the MMTV-luciferase promoter, we performed further chromatin immunoprecipitation analyses. As shown in Fig. 5
, the differential acetylation of the MMTV promoter is associated with a differential recruitment of the coactivator and potent histone acetyltransferase, CBP. In contrast to progestins, glucocorticoid treatment resulted in a robust increase in CBP recruitment. Loading of CBP at the MMTV promoter was observed within 15 min of hormone exposure (data not shown) consistent with the kinetics of the increase in histone acetylation. Chromatin immunoprecipitation assays for the related coactivator p300 did not yield consistent changes, and the interaction of the p160 family of coactivators (Src-1, -2, -3) was not detected. Likewise, neither Brg nor Brm was detected by this assay.

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Figure 5. ChIP Analysis of CBP Loading on the MMTV Promoter after Glucocorticoid and Progestin Treatment of T47D(C&L) and 4F#10 Cells
Chromatin was prepared from T47D(C&L) (A) and 4F#10 (B) cells treated with ethanol, dexamethasone, or R5020 for 1 h and immunoprecipitated with antibodies against CBP. The purified DNA was amplified with primers to either the MMTV-luciferase of MMTV-CAT promoters. Input DNA is shown as a control for equal amounts of DNA in the samples.
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At the MMTV-CAT promoter in the same cell, glucocorticoids and progestins exhibited an ability to recruit CBP even though no change in histone acetylation is seen. However, consistent with the transcriptional induction, no difference in CBP recruitment by the two hormones was seen. In 4F fibroblasts, neither glucocorticoids nor progestins resulted in an increased recruitment of CBP to the promoter, possibly reflecting the different set of coactivators in this cell compared with the mammary carcinoma cell line or instead the influence of the chromatin context of this particular transgene. These results suggest there are cell- and/or context-specific assemblies of transcription complexes initiated by ligand-activated steroid receptors.
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DISCUSSION
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Recent reports have directly demonstrated histone acetylation at target promoters after hormone activation of several nuclear receptors including the estrogen, retinoic acid, and vitamin D receptors (13, 21, 22, 23, 39). Furthermore, these increases in histone acetylation were shown to be dependent on receptor association with coactivators that contain HAT activity. Thus, the favored model for transcriptional activation by nuclear receptors envisions a receptor-mediated recruitment of nuclear receptor-coactivator complexes to target promoters resulting in increased histone acetylation and consequent induction of transcription. However, the studies presented here demonstrate that this model cannot be strictly applied to the MMTV promoter.
Our studies were initiated in an effort to determine the mechanism of differential transcriptional regulation of the MMTV-luciferase promoter in T47D(C&L) cells by glucocorticoids and progestins. These cells also contain an MMTV-CAT transgene that, unlike MMTV-luciferase, is equally induced by activation of GR or PR (37). We also have previously shown that hormonal induction of the MMTV promoters in T47D(C&L) cells may be potentiated or inhibited by modulation of global levels of histone acetylation with histone deacetylase inhibitors depending on the integration site of the transgene (37). The idea that chromatin context may determine the response of the MMTV promoter to the inhibition of histone deacetylases reconciles disparate findings of earlier work (33, 34). We reasoned that the selective ability of glucocorticoids to remodel MMTV-luciferase chromatin and activate transcription, compared with progestins, might be a consequence of a superior ability of GR to promote histone acetylation at the MMTV-luciferase promoter. In an effort to experimentally address this hypothesis, we performed ChIP assays using antibodies directed against acetylated forms of histones H3 and H4. Because T47D(C&L) cells also contain a stably replicating MMTV-CAT transgene that is equally induced by both GR and PR, we could analyze the effect of hormone treatment on histone acetylation on two MMTV transgenes within the same cell. Whereas the MMTV-luciferase promoter exhibited preferential histone acetylation directed by GR, we observed no change in histone acetylation at MMTV-CAT in response to either hormone under conditions where we observe both chromatin remodeling and gene induction. We then analyzed stably integrated MMTV-CAT and MMTV-luciferase promoter/reporters in breast cancer and fibroblast cell lines. In no case was an increase in histone acetylation observed after hormone induction. To reconcile these findings and current understanding of receptor-coactivator action, we propose that hormone-dependent induction of the MMTV promoter does not require increased acetylation of histones at the promoter. Nonetheless, in a permissive chromatin environment increased acetylation can accompany receptor activation and potentiate the hormone response as seen with the glucocorticoid induction of MMTV-luciferase locus in T47D(C&L) cells. At this particular locus, we see a large increase in histone H3 and H4 acetylation after treatment with glucocorticoids but a lesser increase in acetylation in response to progestins. This result suggests the intriguing possibility that the site of MMTV integration can govern the responsiveness to related activators, in this case distinguishing between GRs and PRs. This discrimination is not observed at another MMTV-transgene in the same cell. The ability of the GR to provoke a robust increase in histone acetylation at MMTV-luciferase in T47D(C&L) cells may provide a mechanistic explanation for the extraordinary induction of this transgene and the differential induction compared with progestins that is not observed at other transgenes we have examined.
The mechanistic basis of the differential acetylation induced by glucocorticoids is still uncertain. However, we did observe that activation of the GR was able to differentially recruit CBP to the promoter (Fig. 5
), suggesting that in this chromatin context, GRs and PRs have differential ability to assemble an active transcription complex. The finding that transcriptional activation of the MMTV-CAT gene in T47D(C&L) cells could occur without an accompanying increase in histone acetylation was confirmed with both MMTV-CAT and MMTV-luciferase transgenes in breast cancer and fibroblast cell lines. In stable chromatin, the MMTV long terminal repeat directs the assembly of an ordered nucleosome array that represses loading of basal transcription factors on the promoter (40). Activation of steroid hormone receptors by agonists promotes the remodeling of the MMTV chromatin and the loading of basal transcription factors (41). Our findings indicate that the remodeling of MMTV chromatin directed by steroid receptors does not require increases in histone acetylation. This does not rule out the possibility that some basal level of histone acetylation is needed to remodel chromatin and activate the MMTV promoter or is needed for activation once remodeling has occurred. Indeed, in all ChIP assays where we examined histone acetylation at the MMTV promoters, we observed a significant signal in the samples from untreated cells. Basal levels of histone acetylation in uninduced cells was observed in other studies as well. In these other chromatin contexts, hormone induction of the MMTV promoter is associated with a decrease in histone acetylation at the promoter (35, 36). Together with the present studies, these findings suggest that the binding of an activated steroid receptor to the MMTV promoter can be interpreted differentially in different chromatin environments. Although changes in histone acetylation are not required for induction, at least in some promoter contexts, in one example we find a receptor-specific increase in acetylation that is associated with a greatly potentiated hormone response. The idea that chromatin context can greatly influence the response of a promoter to a signal has implications for understanding the basis of tissue-specific gene expression as well as developmentally regulated gene expression.
An alternative explanation for induction of MMTV transcription without an increase in histone acetylation is that the histones may be undergoing another covalent modification after hormone addition. Studies utilizing growth factor treatment of mouse fibroblasts have shown that histone H3 is rapidly phosphorylated upon stimulation and that this phosphorylation event corresponds to transcriptional activation of immediate-early genes (42, 43, 44). Experiments performed in Drosophila have shown that acetylation of histone H3 and H4 in polytene chromosomes does not change during the heat shock response but that phosphorylation of histone H3 at the heat shock loci increased dramatically after heat shock (45). Furthermore, mutations in the histone H3 kinase Rsk-2 have been shown to be associated with Coffin-Lowry syndrome in humans (46). Rsk-2 mutations result in the loss of epidermal growth factor-stimulated histone H3 phosphorylation that corresponds to the altered transcriptional response to mitogens in Coffin-Lowry cells (47, 48). These findings suggest that histone H3 phosphorylation has an important role in the induction of transcription. Our preliminary results from ChIP assays employing antibodies that recognize phosphorylated histone H3 or phosphorylated and acetylated H3 give no evidence for a change in the levels of these modifications at the MMTV promoter after hormone (our unpublished data).
Another histone modification that plays a role in transcription of the MMTV promoter is methylation. Coactivator-associated arginine methyltransferase 1 has been shown to methylate histone H3 at arginine 17 and arginine 26 in vitro and to synergize with the p160 family of coactivators in the induction of transcription by steroid hormones (49, 50, 51, 52, 53). Histone H4 is also a target for methylation. Methylation of arginine 3 by protein arginine methyltransferase I facilitates transcriptional activation by nuclear hormone receptors perhaps by promoting subsequent H4 acetylation (54). Clearly, there are several examples of histone modifications that effect transcription other than acetylation. Future experiments in our laboratory will address the roles, if any, of histone acetylation, phosphorylation and methylation or the combination of these modifications in the regulation of gene expression mediated by steroid hormones. The induction of the MMTV promoter without any apparent increase in histone acetylation is not inconsistent with the observation that a number of nuclear receptor coactivator proteins exhibit HAT activity. Clearly acetylation of other critical proteins involved in the transcriptional complex could play a central role in receptor-mediated induction. Indeed, although there is no induction of histone acetylation of the MMTV-CAT promoter in T47D(C&L) cells, both glucocorticoids and progestins increase recruitment of CBP. Unlike the case of MMTV-luciferase however, no differential in CBP recruitment was observed. Finally, in vitro transcription studies indicate that acetyl coenzyme A is absolutely required for activator-dependent transcription on chromatin templates (55, 56, 57). Studies are underway to identify acetylated substrates required for receptor-dependent transcription on chromatin templates.
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MATERIALS AND METHODS
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Cell Culture
All cells were grown in MEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum, penicillin/streptomycin and glutamine. T47D(C&L) cell medium contained 200 µg/ml G418 and 200 µg/ml hygromycin B. T47D(pAHCAT) cell medium contained 200 µg/ml G418. 4FpHHLUC cell medium contained 200 µg/ml G418 and 1x hypoxanthine-aminopterin-thymidine supplement (Life Technologies, Inc.).
Stable Transfections
The plasmid pHHLUC was used in stable transfections of 4F cells and has been described previously (58). The MMTV sequence in pHHLUC comprises a HaeIII (-224)-HpaII (+100) fragment containing the hormone response elements. For stable transfections, 1 x 106 4F cells/10-cm dish were plated and allowed to grow for 24 h before transfection in minimal essential medium plus 1x hypoxanthine-aminopterin-thymidine supplement. One hour before transfection, the culture medium was changed to allow proper pH equilibrium. A calcium phosphate/DNA precipitate was prepared essentially according to Wigler et al. (59). One milliliter of this mixture containing a total of 21 µg/ml DNA (20 µg/ml pHHLUC plus 1 µg/ml pSV2neo) was added to the culture dishes containing 10 ml of growth medium. After 24 h, the medium was removed and the cells returned to fresh medium containing 1x hypoxanthine-aminopterin-thymidine supplement for 24 h. At that time, the medium was replaced with medium containing 1x hypoxanthine-aminopterin-thymidine supplement plus 200 µg/ml G418. Individual colonies were harvested with cloning rings. Three independent clones demonstrating luciferase activity after induction with the glucocorticoid dexamethasone and the progestin R5020 were expanded and designated 4FpHHLUC-4, 4FpHHLUC-7, and 4FpHHLUC-10.
Reporter Gene Assays
For reporter gene assays, cell monolayers were rinsed twice with wash buffer (40 mM Tris-Cl, pH 7.4; 150 mM NaCl; 1 mM EDTA). Cells were lysed by the addition of 1 ml lysis buffer (20 mM K2PO4, pH 7.8; 5 mM MgCl2; 0.5% Triton X-100). The lysate was transferred to a microfuge tube and centrifuged for 2 min to pellet cell debris.
Luciferase and CAT assays were performed exactly as described previously (60). The protein concentration of each extract was determined by dye binding using a commercial kit (Bio-Rad Laboratories, Inc., Hercules, CA).
ChIP Assays
Hormone treatments (dexamethasone 10 nM, R5020 10 nM) were carried out for 1 h or as indicated. Approximately 2 x 107 cells were fixed with formaldehyde (1% final concentration) at room temperature for 15 min. Cells were washed once with ice-cold PBS (140 mM NaCl; 2.5 mM KCl; 8.1 mM Na2HPO4; 1.5 mM KH2PO4, pH 7.5) containing protease inhibitors (1 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin; Sigma, St. Louis, MO). Cells were scraped and collected in PBS containing protease inhibitors and pelleted for 5 min at 1000 x g at 4 C. Soluble chromatin was prepared by resuspension of the cell pellet in 0.5 ml of lysis buffer [1% sodium dodecyl sulfate (SDS); 10 mM EDTA; 50 mM Tris-HCl, pH 8.0], followed by sonication of the lysates, and centrifugation at 14,000 rpm for 10 min at 4 C. Supernatants were diluted 6-fold in immunoprecipitation dilution buffer containing protease inhibitors (0.01% SDS; 1.1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris-HCl, pH 8.0; 167 mM NaCl; 1 µg/ml phenylmethylsulfonyl fluoride; 1 µg/ml aprotinin; 1 µg/ml pepstatin) followed by immunoclearing with 80 µl protein A+G agarose (50% slurry in 10 mM Tris-HCl, pH 8.0; 1 mM EDTA containing 200 µg/ml salmon sperm DNA; 500 µg/ml BSA; 0.05% sodium azide) for 1 h at 4 C. One-milliliter aliquots were subjected to immunoprecipitation with 5 µl of antiacetylated histone H3, antiacetylated histone H4, anti-GR, anti-PR or anti-CBP antibodies with rotation at 4 C for 16 h. Anti-acetylated histone antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY) antiacetyl histone H4 antibodies (catalog no. 06-866) were generated against the peptide AGG[K*]GG[K*]GMG[K*]VGA[K*]RHSC and anti-acetyl histone H3 antibodies (catalog no. 06599) were generated against the peptide ARTKQTAR[K*]STGG[K*]APRKQLC where [K*] denotes acetylated lysine residues. Anti-GR antibodies (PA1510A, MA1510) were from Affinity BioReagents, Inc. (Golden, CO) and anti-PR antibodies (MAb 1294) were a gift from Dr. Dean Edwards. Anti-CBP antibodies (sc-369) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). After immunoprecipitation, 50 µl of the same protein A + G agarose slurry used for preclearing was added and the incubation continued for another 1 h. Precipitates were washed sequentially for 5 min each in low salt wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.0; 150 mM NaCl), high salt buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.0; 500 mM NaCl), LiCl wash buffer (250 mM LiCl; 1% Nonidet P-40; 1% sodium deoxycholate; 1 mM EDTA; 10 mM Tris-HCl, pH 8.0) and twice with TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). All traces of the final TE wash were removed and the beads extracted twice with 250 µl of 1% SDS, 0.1 M NaHCO3. A total of 20 µl of 5 M NaCl was added to the pooled eluates and the samples were heated to 65 C for 4 h to reverse the formaldehyde cross-links. The samples were extracted with phenol/chloroform/isoamyl alcohol (25:24:1, vol/vol) and the DNA recovered by ethanol precipitation of the aqueous phase. MMTV-luciferase sequences were detected using 2 µl DNA in PCRs containing an MMTV-specific primer (5'-GCGGTTCCCAGGGCTTAAGT-3') and a luciferase-specific primer (5'-CCATTTTACCAACAGTACCG-3') for 26 cycles. MMTV-CAT sequences were detected using 5 µl DNA in PCRs containing the same MMTV-specific primer as above and a CAT-specific primer (5'-TCAACGGTGGTATATCCAGTG-3') for 28 cycles. In addition to using modest cycle numbers, care was taken to ensure that no signals saturated the digital camera image (Bio-Rad Gel Doc 1000). Control experiments with serial dilutions of input DNA were done to affirm that the PCRs were performed within the linear range of amplification cycles. All experiments shown are representative of experiments performed three to six times.
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ACKNOWLEDGMENTS
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We thank Drs. James T. Kadonaga and Brian D. Strahl for critically reading the manuscript.
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FOOTNOTES
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This work was supported by NIH Grants DK-09554 (to J.R.L.) and DK-37061 (to S.K.N.).
Abbreviations: CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; CREB, cAMP response element binding protein; GR, glucocorticoid receptor; HAT, histone acetyltransferase; MMTV, mouse mammary tumor virus; PR, progesterone receptor; SDS, sodium dodecyl sulfate.
Received for publication August 10, 2001.
Accepted for publication March 5, 2003.
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