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Department of Molecular and Integrative Physiology (J.N.W., G.D.H.) and Department of Internal Medicine (G.D.H.), Division of Metabolism, Endocrinology and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan 48109-0678
Address all correspondence and requests for reprints to: Gary D. Hammer, M.D., Ph.D., 1150 West Medical Center Drive, Office 5560A MSRB II, Ann Arbor, Michigan 48109-0678. E-mail: ghammer{at}umich.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase (P450c17) (12, 13, 14), 3ß-hydroxysteroid dehydrogenase (15), 21-hydroxylase (16, 17), 11ß-hydroxylase (3) as well as several additional genes involved in the steroid biosynthetic pathway including the melanocortin 2 receptor (Mc2r) (18, 19, 20) and the steroidogenic acute regulatory protein (21, 22), both early determinants of steroidogenic potential.
Nuclear receptors comprise a large family of related transcription factors that share a conserved structure and mechanism of activation. Classically, steroid receptors are DNA-dependent transcription factors that are regulated by small lipophilic molecules including adrenal steroids (mineralocorticoids and glucocorticoids), vitamin D3, retinoids (all-trans and 9-cis), gonadal and placental steroids (androgens, progestins, and estrogens), as well as thyroid hormone (23). In addition to these well-characterized hormone receptors, there are a significant number of receptors, referred to as orphan receptors, for which no regulatory ligand has been identified (24). Structural analysis of ligand-binding domains (LBDs) of classical as well as orphan nuclear receptors has provided insight into novel mechanisms by which some orphan receptors adopt active conformations similar to those observed for ligand-bound receptors. The presence of empty LBDs (liver receptor homolog-1, retinoid-related orphan receptor ß 
, estrogen-related receptor ß ), constitutive binding of ubiquitous structural ligand (hepatocyte nuclear factor-), or the complete absence of an LBD (nuclear growth factor I-B, Nor1, Nurr1) suggests that the regulation of some orphan receptors in response to physiological cues, particularly those involved in endocrine homeostasis, may be conferred by acting as downstream effectors of peptide hormone-mediated signaling cascades downstream of cell surface receptors (23, 25, 26). In support of this view, several posttranslational modifications of SF-1 have been observed, including phosphorylation, sumoylation, and acetylation, which regulate diverse aspects of SF-1 function (27, 28, 29, 30, 31, 32).
The proopiomelanocortin-derived peptide ACTH represents the primary hormone responsible for activation of steroid biosynthesis in the adrenal cortex (33). The binding of ACTH to its cognate G protein-coupled receptor promotes the activation of protein kinase A (PKA) and MAPK-dependent signaling cascades that collectively initiate adrenal-specific, steroidogenic transcriptional programs (27, 28, 34, 35, 36, 37). The pharmacological inhibition of either pathway is capable of inhibiting the ACTH-dependent transcriptional response, suggesting a critical role for PKA and MAPK in ACTH-dependent signaling (38, 39). Despite active investigation attempting to identify the downstream signaling events that couple PKA activation with steroidogenic enzyme gene transcription, no adequate mechanism has been proposed that would account for this phenomenon. Studies examining the ACTH-dependent MAPK pathway have implicated the downstream kinases, ERK 1/2, in the regulation of SF-1 phosphorylation (27, 28). Interestingly, the phosphorylation of SF-1 leads to enhanced transcriptional activation of target genes, presumably the effect of phosphorylation-induced association of cofactors with the LBD of SF-1. However, the dephosphorylation of SF-1 through the actions of the dual-specificity, mitogen-activated kinase phosphatase-1 (MKP-1) has also been proposed to activate SF-1 via MKP-1-mediated dephosphorylation of ERK 1/2 (40). Specifically, ACTH has been shown to elicit a robust increase in MKP-1 transcript and protein expression as well as the dephosphorylation of SF-1 and subsequent transcriptional activation of P450c17 in mouse Y1 adrenocortical cells (40, 41). Taken together, these potentially disparate conclusions suggest that context-specific phosphorylation and dephosphorylation of SF-1 both serve as positive influences on target gene transcription. Moreover, a temporal coordination of the phosphorylation and dephosphorylation of SF-1 may together contribute to the dynamic assembly of SF-1 complexes and ultimate transcriptional output.
The transcriptional activation of genes by nuclear hormone receptors requires a coordinated, temporal recruitment of a variety of factors concomitant with alterations of the local chromatin environment, which involves posttranslational alterations of core histones and ATP-dependent nucleosome remodeling (42). SF-1 has been shown to physically and/or functionally interact with a number of transcription factors and transcriptional cofactors on adrenal-specific promoters including ubiquitously expressed transcription factors (transcription factor IIB, surfactant protein 1, CCAAT enhancer binding protein-ß, nuclear factor 1, and Jun) (43, 44), general coactivators [p160/steroid receptor coactivator (SRC) family, receptor-interacting protein 140, cAMP response element-binding protein (CREB)-binding protein, and p300] (39, 45, 46, 47), and the adrenal/gonad-specific transcriptional corepressor, Dax-1(48). To date, the participation of these factors in SF-1-mediated transcription has largely been inferred from in vitro binding assays as well as studies utilizing reporter constructs that cannot faithfully recapitulate the in vivo transcriptional regulation of SF-1 target-genes within the endogenous, chromatinized environment.
In this study, we have performed chromatin immunoprecipitation (ChIP) assays over a 3-h time course after ACTH stimulation to determine the temporal dynamics of regulatory factor recruitment and transcriptional activation of the proximal region of the Mc2r gene promoter that is responsible for conferring ACTH responsiveness (49, 50). In an attempt to correlate the temporal activation of upstream signaling events with recruitment and/or occupancy of factors with the endogenous promoter, we have examined the activation of the PKA and MAPK-dependent pathways and addressed their relative contributions in SF-1-mediated transcriptional activation through the use of specific pharmacological inhibitors of both signaling cascades. In addition, we have examined the global role of histone deacetylases (HDACs) and serine/threonine phosphatases in the transcriptional activation of the Mc2r gene promoter. These studies have revealed that distinct enzymatic activities regulated downstream of ACTH-dependent pathways are capable of drastically altering the amplitude and frequency of transcriptional activation, thereby establishing the overall tone of the transcriptional response.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-amanitin-containing media followed by treatment with or without ACTH. We first assessed the activation of Mc2r gene transcription by performing ChIP assays at baseline and after ACTH stimulation using antibodies recognizing the phosphorylated heptad repeat (YSPTSPS) within the C-terminal domain of RNA polymerase II (RNA Pol II) as a surrogate for transcriptional initiation (54, 55). These experiments revealed a dramatic increase in phospho-RNA Pol II occupancy of the Mc2r proximal promoter region after 80 min of ACTH treatment (Fig. 1
). To validate the observed results, the same experimental samples were subjected to PCR amplification using a primer pair located approximately 6 kb from the transcriptional start site (distal Mc2r) and nonspecific primers amplifying a region of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene promoter. As expected, PCR performed with both primer pairs amplified products from genomic DNA, but failed to amplify products in experimental samples.
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-acetyl H4 antibodies to precipitate genomic fragments. Interestingly, two transient yet distinct periods of H4 hyperacetylation were observed with peak acetylation occurring at the 40- and 120-min time points (Fig. 2A). To verify that histone H4 hyperacetylation correlated with active transcription and an overall accumulation of Mc2r gene transcript, quantitative RT-PCR was performed at the 0- and 180-min time points. As expected, ACTH elicited a significant increase in Mc2r transcript levels (1.00 ± 0.073 vs. 1.32 ± 0.034; fold of baseline; P < 0.005), suggesting that productive rounds of transcriptional activation occur during this initial period after ACTH stimulation (Fig. 2B, left panel). As anticipated, each round of histone acetylation was followed by maximally active periods of transcriptional initiation at the 80- and 160-min time points as assessed by phospho-RNA Pol II promoter occupancy (Fig. 2B, right panel). Similar patterns of dynamic recruitment were also observed on the steroidogenic acute regulatory protein and P450scc promoters after ACTH stimulation (data not shown), suggesting a common mechanism regulating the ACTH-dependent transcriptional activation of several SF-1-dependent target genes. In contrast, real-time PCR analysis of the same samples using promoter-specific primers for the ACTH-independent GAPDH gene promoter demonstrate the relative absence of phospho-RNA Pol II occupancy, thereby confirming the specificity of the observed results (Fig. 2C). Collectively, these data suggest that the activation of ACTH-dependent signaling cascades is capable of evoking a cyclic transcriptional response similar to that observed after ligand binding and activation of classic steroid receptors.
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-HA or -myc antibodies. Epitope fusion protein was readily observed in stable cell lines using -myc (Fig. 3A) or -HA antibodies (data not shown). To establish that the epitope-tagged SF-1 fusion protein retained function, luciferase assays were performed in the presence of a mouse Mc2r-luc (–1 kb) reporter and increasing amounts of MycHA-SF-1 expression plasmid in the SF-1-deficient cell line, JEG 3 (Fig. 3B). Transfection of the reporter plasmid alone did not lead to significant activation. In contrast, a dose-dependent increase in MycHA-SF-1 expression resulted in a concomitant increase in luciferase activity. Using this cell line, the pattern of SF-1 recruitment after ACTH stimulation was assessed by performing ChIP assays using -HA antibodies. Interestingly, two distinct periods of maximal SF-1 recruitment were observed at the 40- and 120-min time points, consistent with the transient association of SF-1 with the Mc2r promoter (Fig. 3C). As anticipated, a similar pattern of SF-1 recruitment was observed using -myc antibodies (data not shown).
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). Lastly, the recruitment of SF-1 to the Mc2r promoter in an in vivo setting was assessed. Wild-type C57BL/6J mice were administered dexamethasone (2 µg/g body weight) at 2300 h followed by stimulation with ACTH (1 µg/g body weight) 12 h later. Adrenals were removed at the indicated time points, soluble chromatin extracts were prepared, and ChIP assays were performed using -SF-1 antibodies. Once again, SF-1 was actively recruited to the proximal promoter with maximal occupancy at the 60-min time point (Fig. 4b). The association of SF-1 with the proximal promoter returned to baseline levels by the 90-min time point; however, these results may reflect diminished serum ACTH levels due to hormone clearance rather than the regulated removal of SF-1 from the endogenous Mc2r promoter. Collectively, these data demonstrate that SF-1 is actively recruited to the Mc2r promoter within the native chromatinized environment.
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, A–C). Interestingly, the dynamics of SRC occupancy for each family member differed slightly after the initial recruitment event. For example, the second round of SRC-1 recruitment occurred later than the patterns observed for SRC-2 and SRC-3. Additionally, SRC-2 recruitment exhibited a moderate recruitment in the first round of transcription, with a more prominent second round of occupancy. In contrast, although the first recruitment of SRC-3 was robust, subsequent recruitment was diminished, potentially indicating a more pronounced role for SRC-3 in the initial activation events. These data support a specific role for each SRC family member in the transcriptional activation of nuclear receptor target genes.
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). As anticipated, SF-1 recruitment preceded the recruitment of the SRC family members and GCN5, suggesting that the recruitment of SF-1 is required as an initial step in complex formation. Furthermore, the clearance of SF-1 from the promoter coincided with the reduced occupancy of coactivators on the Mc2r promoter, further supporting the contention that the binding of SF-1 to DNA supports secondary cofactor recruitment and establishment of a transcriptionally competent promoter. Although the timing of SF-1 and GCN5 promoter occupancy were observed to temporally overlap, only an inferred association between these coactivators and SF-1 on the Mc2r gene promoter could be supported. Therefore, we performed re-ChIP experiments to directly examine whether SF-1 and one of these coregulatory proteins, GCN5, existed in separate or shared complexes on the Mc2r gene promoter. As expected, stimulation of Y1 cells with vehicle or ACTH for 40 min promoted the recruitment of both SF-1 and GCN5 to the Mc2r promoter as previously observed (Fig. 6, lanes 1–4). Interestingly, when SF-1 or GCN5 primary immunoprecipitations were eluted and immunoprecipitated with reciprocal antibodies (Fig. 6, lanes 5–8), an ACTH-dependent interaction between SF-1 and GCN5 was readily observed, demonstrating the coexistence of SF-1 and a known coregulatory factor in an ACTH-responsive, promoter-bound complex.
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). Immunoblots performed with -ERK 1/2 antibodies confirmed equal loading of proteins. Quantification of multiple experiments reveals a statistically significant biphasic pattern of transient ERK activation that closely paralleled the temporal dynamics of recruitment and activation observed on the Mc2r promoter (Fig. 7A and Table 1). The ERKs have been shown to phosphorylate SF-1 on a single serine residue (Ser-203) within a major activation domain (activation function 1), leading to alterations in the ability of SF-1 to interact with cofactors (27). Therefore, we used a phospho-specific antibody to assess SF-1 phosphorylation on this residue. Once again, the pattern of SF-1 phosphorylation mimicked the temporal pattern of ERK activation with two distinct rounds of SF-1 phosphorylation (Fig. 7B). Immunoblots were performed using -SF-1 antibodies to confirm that SF-1 levels were unchanged over the indicated time course. Although SF-1 serine 203 resides within a consensus ERK 1/2 site and is phosphorylated in vitro and in response to MAPK activation, it is feasible that alternative kinases mediate this ACTH-dependent phosphorylation event in vivo.
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). Next, we examined the downstream activation of PKA by performing in vitro kinase assays using kemptide as substrate. Activation of PKA was observed within 20 min after ACTH treatment with sustained levels of activation detected throughout the remainder of the time course without any observed pattern of cyclic activation (Fig. 8B).
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). The apparent absence of histone acetylation led us to examine whether pharmacological inhibition of ACTH-dependent signaling cascades altered the recruitment and association of SF-1 with the Mc2r gene promoter. Interestingly, pharmacological blockade of the MAPK or PKA pathways precluded the ability of SF-1 to associate with the Mc2r promoter, suggesting an essential role for both pathways in the regulation of early SF-1-dependent transcriptional events, namely, recruitment and physical association with regulated promoter elements (Fig. 9B). Consistent with the lack of H4 hyperacetylation and SF-1 recruitment, no occupancy of phosphorylated RNA Pol II was observed after pharmacological inhibition compared with saline-treated controls (Fig. 9C). Collectively, these data suggest that the MAPK and PKA pathways coordinately regulate critical processes culminating in SF-1 recruitment and binding to cognate response elements.
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) and protein (Fig. 10B) abundance over the extended time course to determine whether up-regulation of MKP-1 levels coincided with ERK 1/2 inactivation. Increases in MKP-1 mRNA were quantified by performing real-time PCR using MKP-1-specific, intron-spanning primers. GAPDH mRNA levels were used as an internal standard for data normalization. As anticipated, a significant increase (P < 0.01) in MKP-1 mRNA (380.05 ± 92.35) was detected at the 20-min time point when compared with baseline (100.00 ± 0.059), with a slow diminution over the remainder of the time course (40, 41). An increase in MKP-1 protein levels lagged MKP-1 transcription by approximately 20 min, with increased protein levels clearly observed at the 40-min time point. MKP-1 protein levels remained elevated through the 100-min time point with protein levels slowly decreasing through the remainder of the time course. Interestingly, the maximal accumulation of MKP-1 immediately preceded the inactivation of ERK 1/2, consistent with the known role of MKP-1 in the modulation of ERK activation and the observed dephosphorylation of ERK 1/2 in response to ACTH stimulation.
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). RT-PCR performed using RNA obtained from embryonic d 18 mice served as a positive control. HDAC 1 and 7 expression in the Y1 cell line was also confirmed by immunoblotting using specific antibodies (data not shown). ChIP assays performed over the extended time course using -HDAC 1 and -HDAC 7 antibodies revealed a single, distinct period of recruitment, spanning the 100- to 140-min time period (Fig. 11, B and C). Interestingly, the timing of HDAC recruitment coincided with RNA Pol II promoter clearance, which follows hypoacetylation of core histones within the proximal promoter region of the Mc2r gene.
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-PP1 antibodies to precipitate genomic fragments. Surprisingly, PP1 recruitment was biphasic with 10-fold and 22-fold increases in promoter occupancy at the 60- and 140-min time points, concomitant with the loss of H4 acetylation observed in the proximal promoter region (Fig. 12A). To determine whether ACTH stimulation altered the association of HDAC 1 with PP1, microcystin agarose was used to precipitate the catalytic subunit of PP1 and PP2A protein phosphatases before or after stimulating Y1 cells with ACTH for 120 min. Associated proteins were resolved by PAGE and immunoblotted with -HDAC antibodies. ACTH specifically induced the association of HDAC 1 but not HDAC 7 with PP1/PP2A (Fig. 12B), suggesting the potential of a functional interaction of these proteins on the Mc2r promoter.
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; refer to Fig. 1). In contrast, TSA pretreatment resulted in a dramatic increase in the acetylation of H4 on the Mc2r proximal promoter with a 40- to 50-fold increase in H4 acetylation. Interestingly, although the amplitude of this response was drastically higher after TSA pretreatment, no changes were observed in the dynamic periodicity of H4 acetylation over the entire time course. To determine the functional outcome of heightened H4 acetylation, -amanitin synchronized Y1 cells were treated with or without TSA 20 min after ACTH stimulation (before any recruitment) and phospho-RNA Pol II occupancy of the Mc2r gene promoter was assessed. Surprisingly, despite an exaggerated acetylation of histone H4, a state normally associated with heightened gene transcription, there was 1) a complete absence of transcriptional initiation as assessed by phospho-RNA Pol II occupancy of the Mc2r promoter (Fig. 13B); and 2) absence of mRNA accumulation as assessed by quantitative RT-PCR (data not shown). Therefore, although HDAC activity determines the magnitude of the transcriptional response, it is not solely responsible for promoter clearance after ACTH stimulation. Nonetheless, it is absolutely required for any productive transcriptional initiation. These data suggest the presence of nonhistone targets of histone acetyltransferase and deacetylase action, which are required for effective control of gene transcription.
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), the ability to examine the functional role of specific signaling pathways in the temporal regulation of promoter-specific events is limited. To circumvent this limitation, -amanitin-synchronized Y1 cells were treated with serine-threonine phosphatase inhibitor 20 min after ACTH stimulation (before any recruitment), and phosho-Pol II occupancy of the Mc2r gene promoter was assessed. Surprisingly, similar to HDAC inhibition, pharmacological inhibition of serine/threonine phosphatases before recruitment abolishes 1) transcriptional initiation as assessed by phospho-Pol II occupancy of the Mc2r gene promoter (Fig. 14A); and 2) mRNA accumulation as assessed by quantitative RT-PCR (data not shown). To assess the putative role of serine/threonine phosphatases in the regulation of the temporal dynamics observed for transcriptional initiation, pharmacological inhibition of serine/threonine phosphatases was initiated coincident with the first cycle of phospho-Pol II promoter occupancy, allowing for the initiation of a single round of transcription before the blockade of phosphatase action. ChIP assays were performed using -phospho-Pol II antibodies to examine the effect of serine/threonine phosphatase inhibition on the temporal pattern of recruitment and subsequent transcriptional activation of the Mc2r gene. These studies revealed a profound effect on the dynamic periodicity of phospo-RNA Pol II occupancy on the Mc2r promoter with the second round of recruitment occurring 20 min earlier than the normal recruitment of phospho-RNA Pol II (Fig. 14B). This effect upon the periodicity of recruitment was quite specific, with no changes in the amplitude of recruitment observed. Therefore, these data suggest that the temporal dynamics of serine/threonine phosphorylation/dephosphorylation of (an) unknown protein(s) is capable of modulating both transcriptional initiation and the ultimate periodicity of transcriptional initiation. In addition, these results elucidate an important role for serine/threonine phosphatase activity in the early stages of gene transcription.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Despite the high degree of structural conservation and shared mechanisms used by both ligand-dependent and orphan members of the nuclear receptor superfamily, a similar mode of cyclic transcriptional activation has not been established for the ligand-independent activation of orphan receptor-mediated transcriptional programs initiated by cell surface, peptide hormone receptors. We hypothesized that such a system would be controlled by the temporal activation of upstream signaling pathways capable of regulating the dynamic recruitment and assembly of transcriptional complexes through enzymatic modification of the nuclear receptor and associated complex components.
In this study, we demonstrate that activation of ACTH-dependent signaling cascades leads to the cyclical recruitment of SF-1, active remodeling of the local chromatin environment characterized by coactivator recruitment, and core histone acetylation followed by the subsequent initiation of multiple rounds of transcription. An assessment of PKA and MAPK signaling dynamics over the same time course indicates that both pathways are essential for SF-1 recruitment and the establishment of transcriptional competence, highlighting the potential existence of cross talk between, or indispensable contributions provided by, each signaling cascade in ACTH-mediated transcriptional activation. Although the absolute requirement for PKA activation in the ACTH-dependent transcriptional response has long been established, the critical effectors downstream of PKA activation have not been identified (75, 76). Therefore, considerable effort has been focused upon identification of downstream effectors of PKA action. More recently, a role for the MAPK signaling pathway has been substantiated in this coordinated transcriptional response in addition to its known role in partially mediating the weak mitogenic actions of ACTH (27, 28, 29, 37, 40, 78). Undoubtedly, the cyclical pattern of transcriptional initiation involves inherent recycling of transcriptional components as well as the modulation of intracellular signaling pathways, which ultimately dictate the formation of coregulatory complexes through a variety of posttranslational modifications. Therefore, the temporal regulation of signaling pathways responsible for regulating the posttranslational modification status of nuclear receptors and associated coregulatory proteins is an important area of active investigation.
We demonstrate that ACTH induces a cyclic phosphorylation of both ERK 1/2 and SF-1 on serine 203, coincident with the cyclic recruitment of SF-1 and ultimate transcriptional activation. This is of interest because we had previously shown that ERK 1/2 mediated phosphorylation of SF-1 on serine 203 served to enhance the recruitment of transcriptional coregulators and subsequent transcriptional output in transient transcription assays. On the contrary, Sewer and Waterman (79) subsequently revealed that the dephosphorylation of SF-1 on serine 203 correlated with an enhanced transcription of P450c17 in similar assays. The studies presented here indicate that coordinated phosphorylation and dephosphorylation of SF-1 may represent a potential determinant affecting transcriptional activation in the context of a chromatinized template. Mechanistically, MKP-1 has previously been implicated as the phosphatase responsible for reducing the phosphorylation of SF-1 on serine 203 (27, 79). We show that ACTH results in de novo synthesis of MKP-1 with maximal protein accumulation at the time of ERK 1/2 and SF-1 dephosphorylation and promoter clearance. Moreover, PKA has previously been shown to directly phosphorylate MKP-1 in vitro, suggesting that MKP-1 may be a putative downstream effector of PKA that is capable of modulating the dynamics of MAPK activation (40, 41, 79) and subsequent clearance of the SF-1 complex(s) from the promoter. Whereas loss of SF-1 recruitment and subsequent transcriptional activation after pretreatment with U0126 and H-89 are consistent with the proposed roles of PKA and the ERKs, it is certainly possible that other signaling pathways inhibited by these compounds may also be involved. We propose the following model to account for the intrinsic cyclicity of SF-1 recruitment. ACTH-stimulated ERK 1/2 activation leads to the phosphorylation of SF-1, resulting in SF-1 binding and coregulator recruitment. Concomitantly, ACTH-mediated activation of PKA results in the generation of MKP-1 leading to the inactivation of ERK 1/2 and subsequent reduction of SF-1 phosphorylation resulting in ultimate promoter clearance between transcriptional cycles (Fig. 15).
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Phylogenetic analysis of the nuclear hormone receptor superfamily suggests that common ancestral nuclear receptors may have been orphans, with ligand-binding capacity acquired independently for each receptor during evolution (24). In support of this view, several classical as well as orphan receptors can be activated in a ligand-independent manner, suggesting the retention of evolutionarily conserved mechanisms of transcription factor activation predating ligand acquisition (82, 83, 84, 85, 86). Coincident with the development of endocrine systems predicated on feed-forward mechanisms of activation and feed-back mechanisms of inhibition was the need to couple peptide-hormone-mediated signaling cascades to nuclear receptor function in endocrine organs. Whereas some nuclear receptors assumed the responsibility of responding to the steroid ligands synthesized in these endocrine glands, other orphans such as SF-1 became responsible for gene activation of the enzymes responsible for steroid production and gained the ability to become activated through posttranslational modifications mediated by the peptide hormones responsible for the feed-forward stimulation of an endocrine organ in a unique endocrine system. Activation of nuclear receptors by peptide hormone-dependent posttranslational modification and ligand binding appear to follow an ancient convention of transcriptional cyclicity. The recent observation of phospholipids molecules in the SF-1 binding pocket of bacterially derived SF-1 protein leaves open the possibility that peptide hormone stimulation might additionally modulate SF-1 transcriptional activity in mammalian systems by regulating ligand availability (87, 88). The common mechanisms by which these two modes of activation functionally coordinate the transcriptional cyclicity and interpeak promoter clearance should reveal fundamental mechanisms that determine the dynamic assembly and disassembly of nuclear receptor transcription complexes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-Amanitin was purchased from Sigma Chemical Co. (St. Louis, MO). All tissue culture reagents, protein A-agarose, and Trizol were purchased from Invitrogen (Carlsbad, CA). Human ACTH(1–24) was from Calbiochem (La Jolla, CA). Antibodies used were as follows. SF-1, acetylated-H4, and RNA polymerase II (Upstate Biotechnology, Inc., Lake Placid, NY); CTD4H8 (Upstate Biotechnology Inc., Charlottesville, VA); HA Y-11 and MKP-1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); ERK 1/2, phospho-ERK 1/2 (Thr202/Tyr204), and HDAC 1 and 7 antibodies (Cell Signaling Technology Inc., Beverly, MA). Luciferase activity was measured using the Luciferase Assay System (Promega Corp., Madison, WI), and ß-galactosidase chemiluminescence reagents were from Tropix (Applied Biosystems, Foster City, CA).
Plasmid Construction
The pCMVTag3c-HASF-1 expression plasmid was created by directional cloning of the BamHI/XhoI fragment from pciNeo-HASF-1 (27) into pCMVTag3c (Stratagene, La Jolla, CA). The resultant plasmid was linearized and transfected into Y1 adrenocortical cells and selected in G418 (500 µg/ml) containing media. G418-resistant colonies were isolated and screened for HASF-1 expression. The luciferase reporter plasmid (pGL3-mACTHR) containing 1 kb of the mouse melanocortin 2 receptor promoter was a kind gift of F. Beuschlein (University of Freiburg, Freiburg, Germany).
ChIP Assay
Y1 adrenocortical cells were maintained in DMEM supplemented with 7.5% horse serum, 2.5% fetal bovine serum, and antibiotics at 37 C under a humidified atmosphere of 5% CO2. Assay was performed with minor modifications of the procedure described by Shang et al. (57). Cells were grown to 90% confluence and serum deprived for 48 h in DMEM supplemented with 0.05% BSA followed by treatment with 2.5 µM -amanitin for 2 h. Cells were washed twice with PBS, and fresh serum-free medium supplemented with or without inhibitors was added 30 min before ACTH (1 x 10–8 M) stimulation over the indicated time course. Chromatin was cross-linked by addition of formaldehyde at a final concentration of 1% and incubation at 37 C for 10 min. Cells were washed two times in ice-cold PBS followed by collection in PBS containing 1x protease inhibitor cocktail (Sigma). Crude nuclei were prepared as previously described (89). Nuclei were resuspended in 150 µl of lysis buffer [1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), 1x protease inhibitor cocktail] per 2 x 106 cells and sonicated four times for 10 sec each at maximum setting (Fisher Sonic Dismembrator, model 300; Fisher Scientific, Hampton, NH) followed by centrifugation at 4 C for 15 min. Supernatants were collected and diluted in buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl, 1x protease inhibitor cocktail] followed by immunoclearing with 2 µg sheared salmon sperm DNA (Invitrogen), 15 µl preimmune serum, and 80 µl protein A-Sepharose [50% slurry in 10 mM Tris-HCl (pH 8.1), 1 mM EDTA, 0.5 mg/ml BSA, 0.05% sodium azide, 200 µg/ml sheared salmon sperm DNA) for 2 h at 4 C. Immunoprecipitation was performed overnight with specific antibodies. Immune complexes were recovered by addition of 40 µl protein A-Sepharose and incubation at 4 C for an additional hour. Precipitates were washed sequentially for 5 min each in buffer TSE I [0.1% SDS, 1%Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl], twice in TSE II [0.1% SDS, 1%Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl], and buffer III [0.25 M LiCl,1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)], followed by three washes in Tris-EDTA. Immunoprecipitates were extracted two times with 1% SDS, 0.1 M NaHCO3. Eluates were combined and heated overnight at 65 C to reverse cross-linking. DNA fragments were isolated by performing a phenol-chloroform extraction and subsequent precipitation of DNA overnight at –20 C. Primer pairs used for ChIP assays are listed in Table 1.
Re-ChIP
Soluble chromatin was prepared from -amanitin-synchronized Y1 cells treated with vehicle or ACTH (1 x 10–8) for 40 min. Complexes were eluted from primary immunoprecipitations by incubation in 10 mM dithiothreitol for 30 min at 37 C, diluted 1:50 in re-ChIP buffer [1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.1)] followed by immunoprecipitation with the second antibodies. Results were quantified by performing quantitative real-time PCR using Mc2r promoter-specific primers. Data are presented as percent of unstimulated for each experimental condition and were normalized to 1% input values.
cAMP Accumulation and PKA Activity
Y1 adrenocortical cells were treated exactly as mentioned for ChIP studies. cAMP accumulation was determined using an immunoassay kit (Sigma). PKA activity was measured in whole-cell lysates using a commercially available kit (Upstate Biotechnology). All assays were performed in triplicate. and results are expressed as mean ± SEM.
Immunoblotting
Total cell lysates were prepared in lysis buffer [50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, and protease inhibitor cocktail]. Lysates were allowed to rotate at 4 C for 30 min, and protein contents of the high-speed supernatant were measured using the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Equivalent quantities of protein (20–45 µg) were resolved on polyacrylamide-SDS gels, transferred to nitrocellulose membrane (Bio-Rad), and immunoblotted with specific antibodies. Results were visualized using the Supersignal West Dura Extended Duration Substrate kit (Pierce Chemical Co., Rockford, IL.).
RT-PCR and Real-Time Quantitative PCR
Total RNA was isolated from Y1 cells using the Trizol reagent (Invitrogen) according to the manufacturer’s directions. Isolated RNA was DNase treated using the DNase-Free kit (Ambion, Inc., Austin, TX). One microgram of total RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad). Real-time quantitative PCR was performed using the QuantiTect SYBR Green PCR kit (QIAGEN, Chatsworth, CA) and the Opticon 2 real-time PCR system (MJ Research, Beverly, MA). All data were normalized to GAPDH as an internal standard. The specific oligonucleotide sequences for MKP-1 and GAPDH are provided in Table 1. Results are expressed as the mean ± SEM of three independent experiments. All primer pairs were validated using delta C(T) analysis as previously described (90).
Luciferase Assay
Y1 adrenocortical cells (2 x 104/well) were plated in 24-well plates and transiently transfected 24 h later with the mouse Mc2r-Luc reporter plasmid and pCMVTag3c-HASF-1 by calcium phosphate coprecipitation. Cotransfection with CMV-ß-gal was used to normalize for transfection efficiency. Cells were lysed for measurement of luciferase and ß-galactosidase activity 48 h after transfection using a Veritas microplate luminometer (Turner Biosystems, Sunnyvale, CA). Data represent the mean ± SEM obtained from three independent experiments, each performed in triplicate.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online August 18, 2005
Abbreviations: ChIP, Chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; GCN5, general control of amino acid synthesis 5-like 1; HDAC, histone deacetylase; LBD, ligand-binding domain; Mc2r, melanocortin 2 receptor; MKP-1, mitogen-activated kinase phosphatase-1; P450c17, cytochrome P450 17-hydroxylase; PKA, protein kinase A; PP1, protein phosphatase 1; RNA Pol II, RNA polymerase II; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor 1; SRC, steroid receptor coactivator; TSA, trichostatin.
Received for publication May 30, 2005. Accepted for publication August 8, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase/c17–20 lyase). Mol Endocrinol 10:147–158
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