Epigenetic Mechanisms in the Dopamine D2 Receptor-Dependent Inhibition of the Prolactin Gene
Jeffrey C. Liu,
Ross E. Baker,
Winsion Chow,
Christopher K. Sun and
Harry P. Elsholtz
Department of Laboratory Medicine and Pathobiology, Banting and Best Diabetes Centre, University of Toronto and the Toronto General Hospital Research Institute, Toronto, Ontario M5G 1L5 Canada
Address all correspondence and requests for reprints to: Harry P. Elsholtz, PhD, Department of Laboratory Medicine and Pathobiology, University of Toronto, 100 College Street, Toronto, Ontario, Canada M5G 1L5. E-mail: h.elsholtz{at}utoronto.ca.
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ABSTRACT
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Transcription of the prolactin gene is dynamically controlled by positive and negative hormone signals that target the regulatory promoter region. Based on the inducibility of prolactin gene expression by inhibitors of histone deacetylases (HDACs), we examined the role of histone acetylation at the genomic prolactin promoter as a late step in transcriptional regulation. Chromatin immunoprecipitation analysis of GH4 cells revealed elevated levels of acetylated histones in the promoter and enhancer regions of the gene, compared with downstream intron sequences. 17ß-Estradiol stimulated histone H4 acetylation in the promoter region by 2- to 3-fold within 30 min. Dopamine inhibited histone H4 acetylation by 2-fold in 30 min, an effect mimicked by the MAPK kinase (MEK1) inhibitor U0126. In contrast, the synthetic glucocorticoid dexamethasone, which inhibits prolactin transcription, failed to alter histone acetylation over the same time frame. Association of transcription activator Pit-1 with the prolactin promoter was unchanged by hormone treatment. However, in response to dopamine, histone deacetylase HDAC2 and corepressor mSin3A were rapidly recruited to the prolactin promoter, and association was sustained above basal levels over a 1-h period. Consistent with this corepressor function, depletion of endogenous mSin3A by small interfering RNA was sufficient to enhance prolactin gene expression by 70%, comparable to the induction by the HDAC inhibitor, trichostatin A. These studies demonstrate that dopamine D2 receptor activation and inhibition of MAPK (ERK1/2) signaling lead to rapid deacetylation of histones at the genomic prolactin promoter. Recruitment of specific HDAC/ corepressor complexes may be an important mechanism for repression of target gene transcription by Gi/o-coupled receptors.
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INTRODUCTION
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THE ANTERIOR PITUITARY is a key endocrine tissue that secretes polypeptide hormones required for regulation of growth, metabolism, reproduction, and lactation. Prolactin, which controls diverse reproductive functions, is under tonic suppression primarily from dopamine released by the tubero-infundibular neurons of the hypothalamus (1). Dopamine binds to Gi/o-coupled D2-type receptors (D2Rs) on lactotrophs and effectively inhibits both prolactin secretion and synthesis. This model of negative control of pituitary prolactin is unique, as other pituitary hormones are normally expressed at low levels and are stimulated in response to endocrine-releasing signals from the hypothalamus. In addition to dopaminergic inhibition, a subset of mammalian pituitary lactotrophs expresses glucocorticoid receptors, which, in response to corticosteroid hormones, can contribute to negative regulation of prolactin synthesis by a direct mechanism (2).
Inhibition of prolactin synthesis by activated D2Rs and glucocorticoid receptors has been demonstrated at the transcriptional level (3, 4, 5). Regulation by both receptor types maps to sequences in the proximal promoter of the prolactin gene (6, 7, 8). Lactotroph D2Rs coupled to Gi proteins cause a reduction in intracellular cAMP (9) and protein kinase A activity, which can suppress transactivation by factors associated with the prolactin gene promoter (10). In addition, D2Rs couple to Go proteins, which appear not to inhibit adenylate cyclase in lactotroph cells; we have shown that gain-of-function Go mutants effectively inhibit prolactin promoter function without an accompanying reduction in cAMP (11). Therefore, D2Rs may employ several pathways to suppress prolactin gene transcription.
We and others demonstrated that in rat GH4 cells (12, 13) and primary pituitary cells (12, 14), dopamine causes a marked reduction in MAPK (ERK1/2) phosphorylation and activity, and that a Go-dependent signaling pathway is involved. ERK1/2 is known to be a key activator of prolactin gene transcription, possibly through direct or indirect phosphorylation of DNA-binding factors. Several hormones, like TRH, fibroblast growth factors, IGF-I, and even estrogen, that activate prolactin transcription in GH3 or GH4 cells are strong activators of the ERK signaling pathway (15, 16, 17, 18). Moreover, by signaling cross-talk, increased cAMP (e.g. by forskolin treatment) can activate ERK1/2 in these pituitary cells (19, 20, 21). Using a number of structurally unique MAPK kinase (MEK1) inhibitors with a range of potencies, we observed a close correlation between ERK suppression and a decrease in prolactin promoter activity (12). These experiments suggested that dopaminergic inhibition of ERK1/2 signaling could constitute an effective mechanism for suppression of prolactin gene transcription.
Hormone regulation of the prolactin gene is conferred by upstream elements located proximal and distal to the transcription start site (22, 23), whereas sequences in the proximal region contain binding elements of transcription factors and are sufficient for responding to both negative and positive hormone signals. These include the POU-domain factor Pit-1 (24, 25, 26), and the ETS domain factors ETS-1 (27, 28), GA-binding protein (29, 30), and ETS-2 repressor factor (12). In vitro DNA-binding studies using nuclear extracts from lactotroph cell lines have not revealed significant hormone-dependent changes in DNA interactions of these factors with the prolactin promoter. This could suggest that hormone regulation of the prolactin gene involves epigenetic changes to nucleosomal histones (e.g. acetylation, phosphorylation) brought about by the assembly or dissociation of cofactors from transcription preinitiation complexes (31, 32).
In this study, we show in GH4 pituitary cells that prolactin gene expression is strongly induced by histone deacetylase (HDAC) inhibitors, indicating a potential role for protein acetylation in regulation of prolactin transcription, as shown for several other genes (33). Using chromatin immunoprecipitation (ChIP), we found that in untreated cells, acetylation of histone H3 and H4 was elevated in the promoter region, and in the case of histone H4, could be enhanced further by stimulatory hormones within 30 min. Inhibition by dopamine or disruption of ERK1/2 signaling was accompanied by a 2-fold decrease in histone acetylation. Our RNA interference data demonstrate a specific role for mSin3A in repression of the prolactin gene, and we show that this corepressor, together with HDAC2, can be rapidly recruited to the genomic prolactin promoter. Taken together, these findings give new insight to how epigenetic mechanisms determine prolactin gene expression and how they are regulated by inhibitory signaling pathways.
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RESULTS
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Stimulation of Prolactin Gene Expression by Inhibition of HDACs
Because transcription cofactors can regulate the level of histone acetylation on target genes, we examined how acetylation affects activity of the prolactin promoter and expression of the endogenous prolactin gene. Pharmacological agents, such as trichostatin A (TSA) and valproic acid (VPA), induce histone acetylation by inhibiting the activity of the HDACs. As shown in Fig. 1
, A and B, both TSA and VPA induced the transiently transfected prolactin promoter/reporter construct in a dose-dependent manner over a 6-h treatment period. TSA was especially potent, with significant induction at concentrations as low as 25 nM and reaching a maximum 6-fold induction at 200 nM (Fig. 1A
); VPA stimulated the promoter by 3-fold at 1 mM (Fig. 1B
). Control promoters, including mouse mammary tumor virus and rous sarcoma virus (RSV), were not stimulated by low concentrations of HDAC inhibitors (Fig. 1
, A and B, and data not shown). The slight induction of RSV, seen at higher concentrations, suggests a possible general effect of HDAC inhibition on transcription using episomal DNA templates. Consistent with regulation of the prolactin promoter construct, the endogenous prolactin gene was induced by HDAC inhibition after 24 h of TSA treatment, in contrast to a control, the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [Fig. 1
, C (mRNA) and D (protein)]. Interestingly, expression of prolactin-related GH gene was also induced by HDAC inhibition (Fig. 1D
).

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Fig. 1. Activation of Prolactin Gene Transcription by HDAC Inhibitors
A and B, GH4ZR7 cells were transiently transfected by electroporation with 1 µg/plate of 422 PRL or RSV-Luc reporter constructs. The cells were then treated with increasing concentrations of HDAC inhibitors: A, TSA at 25 nM, 50 nM, 100 nM, and 200 nM; or B, VPA at 100 µM, 250 µM, 500 µM, and 1 mM, for 6 h before harvest. Promoter activity was determined by measuring luciferase assay and the data are represented as fold increase against the nontreated control. The effect of TSA on endogenous prolactin expression was determined by measuring: C, prolactin (PRL) mRNA levels 24 h after 200 nM TSA treatment (*, P < 0.01) compared with GAPDH gene as a control; and D, Western blot showing the protein levels of PRL, GH, and GAPDH after 200 nM TSA treatment in time course experiments (4, 8, 16, and 24 h; *, P < 0.05). The levels of mRNA and proteins were quantified by densitometry. Results from three or more separate experiments are shown in each panel (±SE).
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A possible alternate explanation for the stimulatory effects of TSA and VPA on the prolactin gene is that these agents activate a signaling pathway that induces gene-specific transcription independently of effects on HDAC activity. Indeed, stimulation of ERK1/2 phosphorylation by VPA has been reported in neuronal cell cultures (34, 35). Because ERK1/2 signaling is strongly stimulatory for the prolactin gene promoter, we assessed changes in phospho-ERK levels in response to TSA and VPA treatment. As shown by Western blot (Fig. 2
), we did not detect any significant increase in phospho-ERK over a 2-h treatment period with HDAC inhibitors. During this time, levels of acetylated histone H3 and H4 were markedly increased, consistent with the deacetylase inhibitory activity of TSA and VPA. Our data, therefore, support a model in which the prolactin gene is sensitive to changes in histone acetylation, and HDACs contribute either indirectly or directly to the inhibition of prolactin gene transcription.

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Fig. 2. HDAC Inhibitors Do Not Affect Activated ERK Levels in GH4 Cells
TSA (200 nM) and VPA (500 µM) were used to treat GH4ZR7 cells for 10 min, 30 min, and 2 h before being harvested in RIPA buffer. Approximately 50 µg of total protein was loaded in each lane. Levels of phospho-ERK were determined by Western analysis. The blot was first probed with anti-phospho-ERK antibody (pERK) and then stripped and probed with total ERK1/2 antibody (tERK) to standardize for gel loading. The levels of histone acetylation were determined by immunoblot using antibodies against acetylated histone H3 (Ac-H3) and histone H4 (Ac-H4). Total ERK indicates even gel loading. This experiment was repeated twice with identical results.
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Prolactin Promoter Context and Responsiveness to HDAC Inhibitor, TSA
Pit-1 has been proposed as a target for several signaling pathways that regulate prolactin gene transcription, and multimerized 1P or 3P Pit-1 binding sites from the prolactin promoter are sufficient to confer responsiveness to TRH (26), cAMP (24), and dopamine (11). Given the robust stimulation of the 422 prolactin promoter by HDAC inhibitor, TSA, we tested whether a minimal prolactin promoter (36P) containing tandem repeats of the high-affinity 1P site could be regulated by TSA. As shown in Fig. 3
, the adenylate cyclase activator forskolin significantly stimulated both the 422 prolactin promoter and 3x1P promoter construct. However, TSA was unable to stimulate a minimal promoter containing only Pit-1 sites. An identical construct with 3xSp1 sites yielded a 10-fold response to TSA; hence, isolated DNA sites for certain transcription factors could confer TSA regulation to the minimal prolactin promoter. This suggests that Pit-1 alone may not be sufficient to sustain HDAC-mediated repression of the prolactin gene promoter, and that HDAC responsiveness requires coordinate interactions of several transcription factor-binding sites.

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Fig. 3. Regulation of HDAC Inhibitors on Prolactin Promoter Elements
422 PRL (1 µg/plate), 3x1P (5 µg/plate), and 3xSp1 (5 µg/plate)-Luc reporter constructs were transfected into GH4ZR7 cells. The cells were then treated with 10 µM forskolin (FSK) or 200 nM TSA for 6 h before harvest, and promoter activity is determined by luciferase assay. Data are presented as fold induction against nontreated control. Statistical analysis: a, P < 0.01; b, P < 0.001; c, P < 0.05; d, P < 0.01 (ANOVA). Results from three or more separate experiments are shown (±SE).
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Hyperacetylation of Histone H3 and H4 in Prolactin Genomic Enhancer and Promoter Regions
To determine how histone acetylation controls expression of the endogenous prolactin gene, and how it may be regulated by stimulatory and inhibitory signaling pathways, we used ChIP quantified by real time PCR. Three sets of primers (designed using Primer Express software by ABI, Foster City, CA) were used to monitor the distal enhancer region, the promoter region, and the 3'-end of the gene within the fourth intron (Fig. 4A
). Both the promoter and enhancer regions contain well-characterized transcription factor-binding sites and are required for transactivation of the gene, whereas, to date, regulatory elements have not been identified in the fourth intron. Using antibodies specific to acetylated histone H3 and H4, we observed that levels of acetylation of both histones are high at the distal enhancer and proximal promoter regions. This supports the notion that regions containing important regulatory elements are accessible during gene activation and therefore more heavily acetylated. On the other hand, only a low level of histone acetylation was detected at the 3'-end of the gene (Fig. 4B
). Our results suggest that within the prolactin gene, high levels of histone acetylation are associated with regions containing transcription factor-binding sites.

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Fig. 4. Hyperacetylation of Histone H3 and H4 in Prolactin Genomic Enhancer and Promoter Regions
A, Three sets of primers were used to monitor the enhancer (1.8 kb), promoter (200 bp), and fourth intron (+9KB) regions of the prolactin gene. B, GH4ZR7 cells were fixed with 1% formaldehyde and sonicated, and acetylated histones were precipitated using antibodies specific for acetylated histone H3 (Ac-H3) and histone H4 (Ac-H4). The precipitates were quantified by real-time PCR using primers specific for the three regions to determine the relative levels of histone acetylation along the prolactin gene. The results were represented as input-normalized gene copy number. Data from three separate experiments are shown (± SE).
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Dynamic Control of Histone Acetylation of the Genomic Prolactin Promoter by Stimulatory and Inhibitory Hormones
ChIP was used to determine how estrogen, a key activator of prolactin gene transcription, alters histone acetylation. 17ß-Estradiol increased the level of acetylation of histone H4 on the prolactin gene in a time-dependent manner (Fig. 5A
), with a maximum reached by 45 min (Fig. 5B
). The most significant increase in acetylation was seen on histone H4, with only a slight increase in H3 acetylation after 45 min (Fig. 5B
). The effects of other stimulatory hormones on histone acetylation were compared with estrogen; a modest induction of H4 acetylation was also seen in TRH-treated cells (data not shown). In contrast to these hormones, dopamine effectively reduced the level of acetylated histone H4 acetylation in the proximal promoter region (Fig. 6
). The reduction of H4 acetylation in dopamine-treated cells is rapiddetectable by 15 min and reaching a maximal effect (50% control level) by 45 min. Interestingly, no rapid changes in histone H4 acetylation were seen using another inhibitor of prolactin gene transcription, dexamethasone (Fig. 6
), suggesting that dopamine and dexamethasone evoke different mechanisms to repress the prolactin gene. The specificity of dopamine inhibition of the histone H4 acetylation on the prolactin gene provides new insight to the nuclear mechanisms of dopamine action.

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Fig. 5. 17ß-Estradiol-Induced Histone Acetylation of the Genomic Prolactin Promoter
A, GH4ZR7 cells were treated with 17ß-estradiol (E2, 20 ng/ml) for 30 min and fixed with 1% formaldehyde for ChIP analysis, side by side with the untreated control. The lysates were precipitated with nonimmunized rabbit Ig (Rabbit Ig) and antiacetyl-histone H4 (Ac-H4) antibody. Genomic DNA was isolated from each lysate to serve as input control (Input). Samples were amplified by PCR (25 cycles) using primers specific for the prolactin promoter. The PCR products were separated by gel electrophoresis, and the level of histone H4 acetylation (Ac-H4) was assessed by Southern analysis. This experiment was repeated three times with identical results. B, Real-time PCR was used to quantify ChIP samples to determine the levels of histone H3 (Ac-H3) and histone H4 (Ac-H4) acetylation during the estradiol time course. Statistical analysis: °, P < 0.05; *, P < 0.01 (ANOVA). Data are represented as input-normalized gene copy number, and the results from three separate experiments are shown (±SE).
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Fig. 6. Contrasting Actions of Dopamine, Dexamethasone (Dex), and U0126 on Histone Acetylation of the Prolactin Promoter
GH4ZR7 cells were treated with 1 µM dopamine (panel A), 100 nM dexamethasone (panel B), or 10 µM U0126 (panel C). Samples from ChIP assay were amplified by PCR (26 cycles) using primers specific for the prolactin promoter. The PCR products were separated by gel electrophoresis and the level of histone H3 and H4 acetylation (Ac-H3 and Ac-H4) was assessed by Southern analysis. Nonimmunized rabbit Ig (Rabbit Ig) and genomic DNA isolated from crude lysates (input) serve as controls. The quantification was done by real-time PCR to monitor the change of histone acetylation levels at indicated time points. Statistical analysis: °, P < 0.05; *, P < 0.01 (ANOVA). The data are presented as input-normalized gene copy number. Results from three or more separate experiments are shown (±SE).
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Epigenetic Effect of ERK1/2 Inhibition on the Genomic Prolactin Promoter
We demonstrated previously that D2R activation in GH4ZR7 cells inhibits ERK1/2 activity (12), raising the question of how this change in signaling contributes to transcriptional inhibition of the prolactin gene. ChIP analysis (Fig. 6
) shows that in U0126-treated cells the time course of histone H4 deacetylation at the prolactin promoter paralleled the deacetylation response to dopamine, with maximal 5060% loss of acetylation at about 1 h. Surprisingly, the MEK1 inhibitor also reduced histone H3 acetylation by about 60%, whereas dopamine was ineffective in this regard (Fig. 6
). The basis for the qualitatively different effects of dopamine and U0126 on histones H3 and H4 is not clear but may be due, in part, to the greater magnitude and duration of ERK1/2 inhibition in U0126-treated cells. Under experimental conditions matching those of the ChIP study, we found that the onset of ERK1/2 inhibition is equivalent in dopamine- and U0126-treated cells, but that levels of phospho-ERK are suppressed maximally by 7075% after dopamine, and more than 95% after U0126 (Fig. 7
). Moreover, dopamine suppression of ERK1/2 recovers by about 1 h, whereas U0126 suppression is sustained. The quantitative relationship between ERK1/2 activity in lactotroph cells and histone acetylation of the prolactin gene is currently being addressed.

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Fig. 7. Profiles of ERK Inhibition by Dopamine and U0126
The time-dependent inhibition of ERK by dopamine (DA, 1 µM) and U0126 (U0, 10 µM) was examined in GH4ZR7 cells. The cells were harvested at indicated time points after treatments of dopamine or U0126, and ERK activity was monitored by Western blotting. The blot was first probed with anti-phospho-ERK antibody (pERK) and then stripped and probed with total ERK1/2 antibody (tERK) to standardize for gel loading. The level of ERK activity was determined by densitometry and plotted against time. Data from three separate experiments are shown (±SE).
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Stable Binding of Pit-1 to the Genomic Prolactin Promoter
Because Pit-1 is a critical transactivator of the prolactin gene and its binding sites can function as hormone response elements, we investigated the effect of hormone treatments on Pit-1 binding to the genomic prolactin promoter. Several Pit-1 antibodies (generated in different rabbits and Pit-1 specific in Western analysis) were tested using the ChIP assay. The antibody-precipitated DNA was analyzed by PCR/Southern blot and real-time PCR using promoter-specific primers. From lysates of GH4ZR7 cells, we specifically precipitated prolactin promoter fragments in a dose-dependent manner (Fig. 8A
). Using P-2 antibody, we demonstrated specific association of Pit-1 with the prolactin promoter in GH4ZR7 cells but did not detect a significant signal in nonpituitary, rat-derived cell lines, such as mesangial cells (P17) and muscle cells (L6) (Fig. 8B
). Using conditions optimized for histone deacetylation analysis, we used the same Pit-1 antibody to study the effect of dopamine, U0126, TRH, and estrogen on Pit-1 binding to the prolactin promoter. As shown in Fig. 8C
, neither inhibitory nor stimulatory hormones/agents significantly altered Pit-1 binding to the prolactin gene promoter. These studies with intact cells are therefore consistent with previous in vitro studies of Pit-1 binding (using deoxyribonuclease footprinting and EMSA), which revealed no significant changes in Pit-1 binding activity after hormone treatment.

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Fig. 8. Stable Binding of Pit-1 to the Genomic Prolactin Promoter
A, Increasing amounts (1, 3, and 6 µl) of different Pit-1 antibodies, P-1, -2, -3, and -4, were used to precipitate prolactin gene promoter fragments by ChIP assay from GH4ZR7 cells. The results were quantified by real-time PCR and presented as input-normalized gene copy number. B, Control rabbit Ig (Rabbit Ig) and Pit-1 antibody P-2 (Pit-1) were used to precipitate prolactin promoter fragments from rat mesangial cells passage 17 (P17), rat muscle cells (L6), and GH4ZR7 cells (GH4) in ChIP assay, quantified by real-time PCR. The ratio of Pit-1 to control rabbit Ig (Pit-1/Ig ratio) is shown. C, Southern analysis and real-time PCR quantification of Pit-1 binding to prolactin promoter in response to hormone treatments. Dopamine (DA, 1 µM), U0126 (10 µM), TRH (100 nM), and 17ß-estradiol (E2, 20 ng/ml) were used to treat GH4ZR7 cells for 30 min and then harvested for ChIP pull down. The lysates were precipitated with nonimmunized rabbit Ig (Rabbit Ig) and P-2 Pit-1 (Pit-1) antibody. Genomic DNA was isolated from each lysate to serve as input control (Input). Samples were amplified by PCR (27 cycles) for Southern blotting (upper panel) and quantified by real-time PCR (lower panel) using primers against prolactin promoter. The real-time PCR data are presented as fold change compared with untreated control. Data from three separate experiments are shown (±SE).
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Recruitment of HDAC2/mSin3A to the Genomic Prolactin Promoter in Response to Dopamine
The rapid reduction of histone H4 acetylation by dopamine treatment suggested that increased assembly of corepressor/HDAC on the genomic prolactin promoter might be an early response to D2R activation. To identify potential components of this epigenetic regulatory pathway, we first used immunoblots to identify HDAC and corepressor proteins expressed in GH4ZR7 cells. As shown in Fig. 9A
, we observed expression of mSin3A, HDAC1, and HDAC2 in pituitary and in all cell lines tested, including GH4ZR7. Although, corepressor NcoR (nuclear receptor corepressor) has previously been shown to interact with Pit-1 (36), we were unable to detect NcoR in GH4ZR7 cells (Fig. 9B
). As HDAC1, HDAC2, and mSin3A can form a corepressor complex (37, 38), these proteins may play a significant role in regulating gene transcription in the pituitary lactotrophs. We next used the ChIP assay to monitor association of corepressor/HDAC proteins with the prolactin promoter in response to dopamine. Dopamine treatment caused a rapid recruitment of HDAC2 and mSin3A to the prolactin promoter with a concomitant reduction in histone H4 acetylation (Fig. 9C
). Peak association of HDAC/corepressor was observed by 15 min after dopamine treatment, and retention of the proteins was maintained significantly above basal levels over the 1-h test period. These data show that HDAC2 and mSin3A are two corepressor components recruited to the prolactin gene promoter after D2R activation that likely contribute to the deacetylation of histone H4.

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Fig. 9. Recruitment of HDAC2/mSin3A to the Genomic Prolactin Promoter in Response to Dopamine
A, Western analysis was used to determine the expression of corepressor complexes in lactotroph cells. Antibodies against HDAC1, HDAC2, mSin3A, and Pit-1 (P-2 antibody) were used to probe lysates of Jurkat, GH4ZR7 (GH4), Sprague Dawley rat pituitaries (Pituitary), L6, and NIH3T3 cells. B, NcoR antibody was used to probe Jurkat and GH4ZR7 (GH4) cell lysates. Approximately 50 µg of total protein was loaded in each lane. This experiment was repeated three times with identical results. C, GH4ZR7 cells were treated with dopamine (DA, 1 µM), and the level of histone H4 acetylation (Ac-H4) and the recruitment of mSin3A/HDAC2 were monitored by ChIP assay followed by real-time PCR. For HDAC2 and mSin3A measurements, the data are represented by fold induction compared with time zero (right y-axis). For histone H4 acetylation (Ac-H4), the data are presented as percent change from time zero (left y-axis). Data from three separate experiments are shown (±SE).
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The Effect of mSin3A Depletion on Prolactin Gene Expression
To assess the level of involvement of corepressor complexes on prolactin gene regulation, we used siRNA targeting mSin3A to reduce its expression in GH4ZR7 cells. As shown in Fig. 10A
, vectors expressing anti-mSin3A siRNA effectively and specifically depleted mSin3A expression by 70% (72 h after transfection), whereas the expression of GAPDH was not affected. Depletion of mSin3A in transfected cells may actually be greater than this, as our transfection efficiency is about 7075% (data not shown). Control vectors carrying random sequences had no effect on either mSin3A or GAPDH expression, demonstrating the specificity of siRNA-mediated knockdown. As shown in Fig. 10B
, loss of mSin3A in GH4ZR7 cells led to a significant increase in prolactin content. Interestingly, this increase is comparable to that seen in cells treated with the general HDAC inhibitor, TSA (Fig. 1D
), and further supports the model suggested by our corepressor recruitment experimentsthat mSin3A plays a direct role in basal and dopamine-regulated repression of the prolactin gene.

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Fig. 10. The Effect of mSin3A Depletion on Endogenous Prolactin Expression
A, GH4ZR7 cells were transfected with vectors expressing siRNA for mSin3A (Si-mSin3A) or random sequences (Si-Random). The levels of mSin3A and GAPDH in these cells were then monitored by Western analysis 24, 48, and 72 h after transfection. The representative of three experiments with identical results is shown. B, The levels of prolactin (PRL) and GAPDH proteins 72 h after Si-mSin3A or Si-Random transfections were shown by Western blotting and quantified by densitometry. Data from three separate experiments are shown (±SE; *, P < 0.05).
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DISCUSSION
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Dopaminergic control of prolactin release and synthesis is an important homeostatic mechanism that maintains endocrine and reproductive functions. D2R-dependent activation of several G protein-coupled signaling pathways leads to effective suppression of prolactin release and gene transcription. Dopamine reduces intracellular cAMP concentrations and inhibits ERK1/2 signaling, and here we show that at the nuclear level, activation of D2Rs also leads to histone deacetylation and recruitment of corepressor complex mSin3A/HDAC2 to the prolactin gene promoter (Fig. 11
). These results bring new insight to the distal signaling mechanisms that mediate dopamine regulation of a key endocrine target gene in the pituitary.

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Fig. 11. Epigenetic Mechanisms in Dopamine-Dependent Regulation of the Prolactin Gene
In the absence of dopamine, low levels of ERK activity in pituitary lactotroph cells maintain histone acetylation (AC) in the prolactin (PRL) promoter region, in part by antagonizing corepressor functions. Inhibition of ERK by U0126, or after activation of D2R/Go-dependent signaling (12 ), leads to histone deacetylation on the prolactin gene. The repression of HDAC activity by TSA results in the induction of prolactin gene expression, suggesting that the prolactin gene is partially silenced by epigenetic mechanisms. siRNA-mediated depletion of mSin3A induces the prolactin gene. The corepressor complex mSin3A/HDAC2, which represses prolactin expression, is recruited rapidly to the prolactin gene in response to dopamine. Promoter-bound transcription factors including Pit-1 and ETS proteins may support interaction of corepressor complexes with the prolactin gene.
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The level of acetylation of histone N-terminal tails typically shows a positive correlation with the state of gene activation (33, 39), and we show that prolactin genomic regions that exhibit deoxyribonuclease hypersensitivity, and confer hormone responsiveness, are most highly acetylated. Either stimulatory or inhibitory regulators of prolactin transcription can alter histone acetylation in the promoter region, and acetylation can be regulated in a qualitatively distinct manner. For example, estrogen, TRH, and dopamine regulate histone H4 acetylation/deacetylation with little or no effect on histone H3, whereas the MEK1 inhibitor U0126 targets both histones H3 and H4. The ability of U0126 to cause a more pronounced deacetylation of histones at the prolactin promoter, in comparison with dopamine, is noteworthy given its more potent effect on prolactin gene repression. Studies in yeast (40) and with recombinant chromatin assembly systems (41) have shown that lysine mutations that prevent acetylation of either histone H3 or H4 can reduce the response to transcriptional coactivators, but a simultaneous block of H3 and H4 acetylation causes severe or complete disruption of transactivation. Consistent with the well-established link between ERK1/2 activation and stimulation of prolactin gene transcription, our study provides an epigenetic basis for how inhibition of ERK1/2 signaling can lead to repression of the prolactin gene.
Although dopamine suppresses phospho-ERK levels in GH4ZR7 cells by about 75%, and within the same rapid time frame as U0126, it is unable to effect a U0126-like decrease in histone H3 acetylation. The difference in regulation of histone deacetylation may be related to 1) the greater effectiveness of U0126 in suppressing ERK1/2 activation, 2) the sustained suppression of ERK1/2 after U0126 treatment vs. transient suppression after dopamine treatment, or 3) the ability of dopamine to regulate not only ERK signaling, but also other signal transduction pathways. Unique effects on ERK inhibition or other pathways may result in the recruitment of functionally distinct corepressor/HDAC complexes, or alter the kinetics of their association/dissociation with the genomic prolactin promoter.
In contrast to dopamine and the MEK1 inhibitor U0126, the synthetic glucocorticoid dexamethasone failed to cause deacetylation of either histone H3 or H4, although it is a potent inhibitor of prolactin promoter activity and prolactin gene expression in GH4ZR7 cells (Baker, R., and H. Elsholtz, unpublished data). Because glucocorticoids have been shown to antagonize some inflammatory cytokine actions through gene-specific recruitment of HDACs (42), it is still possible that some epigenetic effects may be directed at the prolactin gene as well. For example, glucocorticoids may have delayed actions on histone deacetylation that were not detected under the short-term conditions of this study. Alternatively, because regulation of the histone deacetylation pattern can be complex, involving changes to specific lysines in H3 and H4 N-terminal tails (43, 44), it may be necessary to examine glucocorticoid-directed repression of the prolactin gene with antibodies that discriminate more subtle changes at individual residues. It is also possible that glucocorticoids inhibit prolactin transcription by mechanisms largely independent of histone modification, e.g. by blocking phosphorylation of RNA polymerase II and delaying promoter clearance (45).
The POU-homeodomain protein Pit-1 activates prolactin gene transcription, integrates responses to stimulatory and inhibitory signals, and, as shown recently with chromatin-assembled templates, may have a pivotal role in organizing nucleosomal structure in the promoter region (46). Chromatin-dependent functions of Pit-1 have been observed also for the human GH gene in which a set of Pit-1 sites specifies function of the 5'-locus control region (47). By ChIP analysis we did not detect changes in Pit-1 association/dissociation with the genomic prolactin promoter after treatment with various hormones or agents. These data support the view that Pit-1 functions, in part, as a promoter-bound docking site for coregulatory complexes recruited by diverse hormone signals (36, 48). Interestingly, our transient transfection data showed that the 422P prolactin promoter is strongly induced by HDAC inhibitor TSA, whereas multimerized high-affinity Pit-1 sites are unable to confer TSA responsiveness. Three tandemly arrayed Sp1-binding sites, in contrast, confer a 10-fold induction to TSA, in agreement with previous work in human embryonic kidney (HEK) 293 cells using episomal 3xSp1/ promoter constructs (49). In that study, coimmunoprecipitation and pull-down experiments confirmed that Sp1 binds directly to HDAC1, and may therefore facilitate HDAC interactions with the target gene independently of other transcription factors. Our transfection data suggest that Pit-1 alone is not sufficient for corepressor recruitment and may require other factors bound to prolactin promoter sequences to form stable interactions with corepressor/HDAC complexes. Good candidates for such a role might be ETS proteins that recognize multiple sites in the proximal promoter region of the gene. Although our site-directed mutation of the ETS core motif GGA(A/T) in two Pit-1/ ETS composite sites (212, 160) did not disrupt the stimulatory response of the 422P promoter to TSA (Liu, J., and H. Elsholtz, unpublished data), ETS sites nearer the promoter TATA box or possibly other DNA elements may be more critical for TSA responsiveness. Interestingly, ETS proteins Elk-1 (50) and PU.1 (51) have been shown to recruit HDACs to target genes. Protein pull-down studies and further chromatin analysis using sequential immunoprecipitation of transcription factors and corepressors is needed to demonstrate direct involvement of these factors in corepressor/HDAC recruitment. Of particular interest are the ETS factors ETS-1 and GA-binding protein, which are endogenously expressed in lactotrophs and have been shown to interact with specific elements of the prolactin promoter (29).
This study shows, using a loss-of-function approach, that the transcriptional regulator mSin3A contributes to repression of the endogenous prolactin gene. Sin3 is conserved in species from yeast to humans, and although commonly considered a corepressor it may also activate some genes and perform a range of transcription-independent functions, such as regulating DNA repair, replication timing, and retrotransposon transposition frequency (52). Mammalian cells contain two highly conserved paralogues, mSin3A and mSin3B, capable of forming multiprotein complexes with some unique functional characteristics. Remarkably, in GH4ZR7 cells, depletion of mSin3A using interfering RNA causes a 70% increase in prolactin gene expression. The magnitude of this induction is similar to the approximately 2-fold induction observed following TSA treatment, consistent with an obligatory role for Sin3A/HDACs in repression of the prolactin gene. Moreover, our demonstration that activation of dopamine D2Rs rapidly enhances association of the mSin3A corepressor with the endogenous prolactin gene promoter is consistent with its role as a terminal mediator of the dopamine response in GH4ZR7 cells. Interestingly, dopamine regulation of transiently transfected prolactin promoter constructs was not diminished significantly in mSin3A-depleted cells (Liu, J., and H. Elsholtz, unpublished). This suggests the role of mSin3A may be dependent on the chromatin context of the target gene. Although transfected episomal reporter genes can be regulated by overexpression of recombinant mSin3A (53, 54, 55) or v-ski, an oncoprotein that impairs Sin3/HDAC recruitment (56, 57), the transcriptional effects on nonchromatin promoters resulting from endogenous mSin3A depletion are not clear. Given stimulation of the transfected prolactin promoter by TSA (see Fig. 1
), it is possible that in mSin3A-deficient GH4 cells this construct is repressed by HDACs in a less stringent manner. This could include direct interaction of HDACs with episomal promoter-bound factors or association with corepressor complexes that do not contain mSin3A. Moreover, functional redundancy may also play a role; mSin3B is 60% identical to mSin3A with well-conserved protein interactive domains and may therefore compensate for mSin3A deficiency in some transcriptional contexts.
In conclusion, this study demonstrates that acetylation/deacetylation of the genomic prolactin promoter is dynamically controlled by stimulatory and inhibitory endocrine signals. Rapid histone deacetylation is observed after dopamine D2 receptor activation, but not after glucocorticoid receptor activation, suggesting that transcriptional repression of the prolactin gene by endocrine regulators may involve distinct mechanisms. Inhibition of ERK1/2 activity is a potent signal for histone deacetylation in the prolactin promoter region, providing a molecular basis for transcriptional repression of prolactin. Interestingly, whereas the endogenous prolactin gene and phylogenetically conserved Pit-1-dependent GH gene are both induced by HDAC inhibition in GH4 cells (this study), we have shown that only the prolactin gene is sensitive to inhibitors of ERK1/2 signaling (12). How the signaling events that follow ERK1/2 inhibition lead to selective epigenetic changes, differentially controlling transcription of these genes, remains to be determined.
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MATERIALS AND METHODS
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Reagents and Plasmid Constructs
Dexamethasone, dopamine, 17ß-estradiol, forskolin, TSA, and VPA were purchased from Sigma-Aldrich (St. Louis, MO). TRH was from Roche Molecular Biochemicals (Indianapolis, IN). MEK1 inhibitor, U0126, was purchased from Calbiochem (La Jolla, CA). Antibodies to HDAC1, HDAC2, mSin3A, phospho-ERK1/2, ERK1, NcoR, GAPDH. and control rabbit Ig were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to acetylated histone H3 and -H4 were from Upstate Biotechnology, Inc. (Lake Placid, NY). PRL and GH antibodies were obtained from A. F. Parlow (National Hormone and Peptide Program, Harbor-University of California Los Angeles Medical Center, Los Angeles, CA). Pit-1 antibodies were provided by S. J. Rhodes (Purdue University, Indianapolis, IN). ECL reagents were from Pierce Biotechnology, Inc. (Rockford, IL). The luciferase reporter plasmid 422 rPRL/Luc, which contains a 456-bp fragment of the prolactin promoter (positions 422 to +34, relative to the transcription start site), reporter constructs containing prolactin gene TATA box with multiple binding sites of Pit-1 (3x1P) or Sp1 (3xSp1), and the RSV/Luc reporter plasmid, used as a negative control, have been described previously (6, 11).
Cell Culture, Treatment of Cells, and Transfections
GH4ZR7 cells were cultured in F12 Hams (Sigma) supplemented with 12.5% horse serum and 2.5% fetal bovine serum and antibiotics (Pen/Strep) at 37 C, 5% CO2. L6 and NIH3T3 cells were cultured in DMEM containing 10% fetal bovine serum and antibiotics. Rat mesangial cells, passage 17 (P17), were incubated in DMEM with 14% fetal bovine serum and antibiotics. Jurkat cells were in RPMI 1640 medium with 2 mM L-glutamine, 10% fetal bovine serum, 4.5 g/liter glucose, and antibiotics. The cells were subcultured for experimentation onto 10-cm plates and grown to 80% confluence. They were given fresh culture media 12 h before treatments. Transfections of GH4ZR7 cells were done by electroporation as previously described (11).
Chromatin Immunoprecipitation Assays
Cells were first treated with hormones as indicated. Approximately 2 x 107 cells were fixed with formaldehyde (1% final concentration) at room temperature for 10 min. Cells were washed twice with ice-cold PBS, scraped, and collected in PBS. After centrifugation for 5 min at 1000 x g, the cell pellet was washed with ice-cold PBS. Soluble chromatin was prepared by resuspension of the pellet in 0.9 ml lysis buffer [1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), 1 µg/ml leupeptin, 1 ng/ml aprotinin, and 500 µM phenylmethyl-sulfonyl fluoride), followed by sonication of lysates (60 Sonic Dismembrator; Fisher Scientific, Pittsburgh, PA) to generate genomic fragment sizes of 500-1000 bp. Sonicates were centrifuged for 10 min at 12,000 rpm at 4 C. Supernatants were diluted 1:10 in immunoprecipitation dilution buffer (1% Triton X-100; 2 mM EDTA; 150 mM NaCl; 20 mM Tris-HCl, pH 8.1). Diluent (1 ml) was transferred to a microtube containing indicated antibody or control antibody and agitated for 2 h at room temperature. Protein-A-Sepharose (45 µl of a 50% slurry in Tris-HCl, pH 8.1, containing 2 µg salmon sperm DNA) was added to each sample, mixed, and incubated for 1 h at room temperature. Precipitates were washed sequentially for 10 min each using TSE-I (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 150 mM NaCl; 20 mM Tris-HCl, pH 8.1), TSE-II (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 500 mM NaCl; 20 mM Tris-HCl, pH 8.1), buffer III (1% Nonidet P-40; 1 mM deoxycholate; 1 mM EDTA; 0.25 M LiCl; 10 mM Tris-HCl, pH 8.1) and TE (1 mM EDTA; 10 mM Tris-HCl, pH 8.1). Residual TE was removed by pipetting, and beads were extracted with 350 µl of 1% SDS, 0.1 M NaHCO3 and incubated overnight at 65 C to reverse the formaldehyde cross-links. DNA fragments were recovered and purified using QIAEX II Gel Extraction Kit (QIAGEN, Chatsworth, CA), and then resuspended in 50 µl TE. Real-time PCR was used to quantify PRL gene sequences in 5 µl of the DNA sample; prolactin promoter-specific primers were 5'-CAAACGAGACTCAAGATGTCAGTCA-3' and 5'-CAGGAAGACATAGTGGCCAGAAA-3'; prolactin enhancer-specific primers were 5'-TGTGGAACTGGAAGCTGTAGAACT-3' and 5'-AGAGCAGGTCCTGGGAACTGA-3'; prolactin gene intron 4 primers were 5'-AGTTGGAGCCCTGGGTCAGT-3' and 5'-AGCTTGCAACCCATAAAACA-3'.
Southern Blot Analysis
PCR products were resolved on a 1% TBE agarose gel. The gel was soaked in denaturing buffer (0.5 M NaOH, 1.5 M NaCl) twice for 15 min each, and twice in neutralization buffer (0.5 M Tris-HCl; 1.5 M NaCl, pH 7.5) for 15 min each. The samples were transferred overnight onto nylon membrane (Nytran Supercharge; Schleicher & Schuell, Keene, NH) using a 5x SSC solution. The blot was fixed twice by UV-cross-linking and prehybridized at 55 C (4 h) in hybridization buffer (15% formamide; 0.5 M NaPO4, pH 7.0; 1 mM EDTA; 1% BSA; 7% SDS). Probes corresponding to the prolactin gene proximal promoter and intron 4 were synthesized using a [32P]dATP/ PCR reaction with primers as indicated above (ChIP assays). Probes were boiled for 5 min and added immediately to the hybridizing solution, overnight at 55 C. Blots were washed in 0.5x SSC-0.1% SDS for 30 min two times at 55 C. Autoradiography was performed using Kodak X-Omat Blue film (Eastman Kodak, Rochester, NY).
Western Analysis
Cells from 10-cm dishes were harvested in 0.25 ml of radioimmune precipitation assay buffer (1% Nonidet P-40; 0.1% SDS; 50 mM Tris-HCl, pH 8.0; 0.5% deoxycholate salt; 5 mM EDTA; 1 mM EGTA, pH 8.0; 1 µg/ml leupeptin; 1 µg/ml aprotinin; 5 mM NaF; 0.5 mM phenylmethyl-sulfonyl fluoride; 0.5 mM sodium orthovanadate) and stored at 80 C overnight. Extract protein was quantified by BCA protein assay (Pierce Biotechnology, Inc.), approximately 50 µg of whole-cell lysate per sample was resolved on SDS 12% polyacrylamide gels at 130 V, and proteins were transferred to nitrocellulose. Membranes were blocked for 12 h in 5% nonfat dry milk in 1x Tris-buffered saline with Tween 20. The blots were incubated overnight with indicated primary antibodies (1:1000) in fresh 5% nonfat dry milk in 1x Tris-buffered saline with Tween 20, followed by 1 h incubation with horseradish peroxidase-conjugated secondary antibody (1:2000) at room temperature. Protein bands were visualized using the ECL detection method and exposed to Kodak Blue X-Omat film.
siRNA Selective Gene Knockdown
Specific sequences targeting rat mSin3A mRNA were cloned into pSilencer 2.1 Hygro (Ambion, Inc., Austin, TX). The plasmid was transfected into the GH4ZR7 cells using Lipofectamin 2000 (Invitrogen). Lipofectamin 2000 (5 µl) was diluted first in 250 µl of serum-free, antibiotic-free, 1x Opti-MEM and incubated for 5 min at room temperature before mixing with 4 µg of plasmid DNA in 250 µl media. The mixture was then incubated at room temperature for 20 min before added dropwise into culture cells in 30-mm plates containing 2 ml Opti-MEM. After 6 h in the 37 C, 5% CO2 incubator, the cells were replated with normal growth media and harvested 24, 48, and 72 h later. The sequence targeting rat mSin3A is 5'-GATCCGAGTGATAGTCCTGCCATATTCAAGAGATATGGCAGGACTATCACTCTTTTTTGGAAA-3'.
Real-Time PCR Calculations and Statistical Analysis
The real-time PCR was carried out using ABI 7900HT Real-Time PCR system with SYBR Green Master Mix from Applied Biosystems (Foster City, CA). Thermal cycling was initiated with an initial denaturation at 50 C for 2 min and 95 C for 10 min. After this initial step, 40 cycles of PCR (95 C for 15 sec, 60 C for 1 min) were carried out. The gene copy number in ChIP precipitates was calculated using the absolute quantification method with standard curves of PCR threshold values generated by 0.1 ng, 1 ng, 5 ng, 20 ng, and 100 ng of GH4ZR7 cell genomic DNA as described (Applied Biosystems, User Bulletin no. 2, 4303859). The conversion of DNA mass into gene copy number is as follows: approximately 6.5 pg of genomic DNA is contained within one rat cell and carries two copies of the prolactin gene. Therefore in 1 ng of rat genomic DNA there are: (1 ng ÷ 6.5 pg x 2) = approximately 300 copies of gene per ng DNA. In the cases where relative quantification was presented, the (2
Ct) method was used with the background control IgG as the reference and the first ChIP sample at time point 0 as the calibrator (58). Duplicates were done for every sample to ensure that accurate value was obtained, and an additional dissociation step was used to confirm that only the desired fragment was amplified. Statistical analysis of data was performed using Statistical Packages for the Social Sciences. Multiple comparisons were done with ANOVA and the Bonferroni test for post hoc analysis. Differences between values were considered statistically significant at P < 0.05.
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ACKNOWLEDGMENTS
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We thank Dr. Simon Rhodes (Purdue University, Indianapolis, IN) for the gift of Pit-1 antibodies. We also thank Dr. Rod Bremner and Zuyao Ni (University of Toronto, Toronto, Ontario, Canada) for their help in setting up the ChIP assay.
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FOOTNOTES
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This work was supported by a grant from the Canadian Institutes of Health Research (to H.P.E.).
First Published Online February 24, 2005
Abbreviations: ChIP, Chromatin immunoprecipitation; D2R, D2-type receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase; MEK, MAPK kinase; NcoR, nuclear receptor corepressor; RSV, rous sarcoma virus; siRNA, small interfering RNA; SDS, sodium dodecyl sulfate; TE, 1 mM EDTA; 10 mM Tris-HCl, pH 8.1; TSA, trichostatin A; VPA, valproic acid.
Received for publication March 16, 2004.
Accepted for publication February 18, 2005.
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