Factor recruitment and TIF2/GRIP1 corepressor activity at a collagenase-3 response element that mediates regulation by phorbol esters and hormones
2001; Springer Nature; Volume: 20; Issue: 21 Linguagem: Inglês
10.1093/emboj/20.21.6071
ISSN1460-2075
AutoresInez Rogatsky, Kol A. Zarember, Keith R. Yamamoto,
Tópico(s)Inflammatory mediators and NSAID effects
ResumoArticle1 November 2001free access Factor recruitment and TIF2/GRIP1 corepressor activity at a collagenase-3 response element that mediates regulation by phorbol esters and hormones Inez Rogatsky Inez Rogatsky Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 513 Parnassus Avenue, HSW1201, San Francisco, CA, 94143-0450 USA Search for more papers by this author Kol A. Zarember Kol A. Zarember Present address: Department of Molecular Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Keith R. Yamamoto Corresponding Author Keith R. Yamamoto Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 513 Parnassus Avenue, HSW1201, San Francisco, CA, 94143-0450 USA Search for more papers by this author Inez Rogatsky Inez Rogatsky Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 513 Parnassus Avenue, HSW1201, San Francisco, CA, 94143-0450 USA Search for more papers by this author Kol A. Zarember Kol A. Zarember Present address: Department of Molecular Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Keith R. Yamamoto Corresponding Author Keith R. Yamamoto Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 513 Parnassus Avenue, HSW1201, San Francisco, CA, 94143-0450 USA Search for more papers by this author Author Information Inez Rogatsky1, Kol A. Zarember2 and Keith R. Yamamoto 1 1Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 513 Parnassus Avenue, HSW1201, San Francisco, CA, 94143-0450 USA 2Present address: Department of Molecular Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:6071-6083https://doi.org/10.1093/emboj/20.21.6071 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info To investigate determinants of specific transcriptional regulation, we measured factor occupancy and function at a response element, col3A, associated with the collagenase-3 gene in human U2OS osteosarcoma cells; col3A confers activation by phorbol esters, and repression by glucocorticoid and thyroid hormones. The subunit composition and activity of AP-1, which binds col3A, paralleled the intracellular level of cFos, which is modulated by phorbol esters and glucocorticoids. In contrast, a similar AP-1 site at the collagenase-1 gene, not inducible in U2OS cells, was not bound by AP-1. The glucocorticoid receptor (GR) associated with col3A through protein–protein interactions with AP-1, regardless of AP-1 subunit composition, and repressed transcription. TIF2/GRIP1, reportedly a coactivator for GR and the thyroid hormone receptor (TR), was recruited to col3A and potentiated GR-mediated repression in the presence of a GR agonist but not antagonist. GRIP1 mutants deficient in GR binding and coactivator functions were also defective for corepression, and a GRIP1 fragment containing the GR-interacting region functioned as a dominant-negative for repression. In contrast, repression by TR was unaffected by GRIP1. Thus, the composition of regulatory complexes, and the biological activities of the bound factors, are dynamic and dependent on cell and response element contexts. Cofactors such as GRIP1 probably contain distinct surfaces for activation and repression that function in a context-dependent manner. Introduction Eukaryotic transcriptional regulation is carried out by multiprotein complexes that assemble at genomic response elements close to regulated promoters. Many specific factors and general cofactors capable of participating in such regulatory complexes have been described (Freedman, 1999; Goodman and Smolik, 2000; Lemon and Tjian, 2000; Malik and Roeder, 2000), but the precise determinants of the composition and action of regulatory complexes are not well understood. At a given response element, specific cellular or physiological contexts can promote the assembly of complexes that differ in composition and regulatory activity. Closely related members of a regulator protein family may exert distinct effects on transcription or elicit similar effects by different mechanisms of action. Depending on the context, a single factor may recruit different cofactors or, alternatively, utilize the same cofactors in functionally distinct ways. It is apparent that response elements profoundly influence the activities of the bound components (Lefstin and Yamamoto, 1998); thus, regulatory complexes are fundamentally DNA–protein machines. Three classes of response elements have been described (Yamamoto et al., 1998): 'simple', at which a single regulatory factor binds; 'composite', at which two or more different regulators recognize specific DNA sequences; and 'tethering', at which one regulator binds to the element, and a second regulatory factor associates with the first through protein–protein interactions. In each case, the prevailing view is that the response element–regulatory factor complexes then recruit cofactors that alter chromatin structure, or contact or modify components of the transcription machinery ultimately leading to enhancement or repression. Intracellular receptors, such as the glucocorticoid receptor (GR) and the thyroid hormone receptor (TR), function at all three classes of response elements (Chandler et al., 1983; Karin et al., 1984; Diamond et al., 1990; Schule et al., 1990; Yang-Yen et al., 1990; Desbois et al., 1991; Konig et al., 1992). Hormone binding by the ligand binding domains (LBDs) of these receptors triggers changes in their conformations and regulatory activities. In some cases, hormone binding has also been demonstrated to alter the association of receptors with coactivators or corepressors (Fondell et al., 1996; Hanstein et al., 1996; Blanco et al., 1998; Kraus and Kadonaga, 1998; Naar et al., 1999; Rachez et al., 1999). For example, the p160 coactivators (SRC1, TIF2/GRIP1 and ACTR/Rac3/pCIP/AIB1) (Onate et al., 1995; Hong et al., 1996; Voegel et al., 1996; Yao et al., 1996; Anzick et al., 1997; Chen et al., 1997; Li et al., 1997; Torchia et al., 1997) interact with the activation function-2 (AF-2) region within the GR and TR LBDs; the interactions occur in conjunction with agonist, but not antagonist binding. The p160 molecules interact with the receptors through a domain that contains three LxxLL sequence motifs; these sequences, termed NR boxes (Darimont et al., 1998; Ding et al., 1998; Kalkhoven et al., 1998), are differentially recognized by different receptors. Among the three p160 factors, distinct expression patterns and phenotypes of p160-deficient mice (Xu et al., 1998, 2000; Qi et al., 1999; Weiss et al., 1999) suggest selective interactions between particular receptors and individual p160s, but neither the specificity of these interactions in vivo nor the mechanisms of coactivation by the p160s are well understood. As with activation, transcriptional repression also involves multicomponent protein–DNA complexes. For example, unliganded TR (apoTR) bound to a simple thyroid hormone response element (TRE) can recruit co-repressors N-CoR, SMRT or Alien (Horlein et al., 1995; Chen et al., 1996; Dressel et al., 1999), which in turn bind Sin3A and histone deacetylases (HDACs) (Heinzel et al., 1997; Nagy et al., 1997); the corepressors are released from TR, in favor of coactivators, upon hormone binding at simple TREs. In contrast, hormone-bound TR (holoTR) can repress transcription at tethering response elements, such as certain AP-1 sites (Desbois et al., 1991; Saatcioglu et al., 1993). These complexes are distinct from those at simple TREs but their activity is nevertheless sensitive to HDAC inhibitor trichostatin A (TSA) (M.Cronin and K.R.Yamamoto, unpublished data). GR also represses transcription in an agonist-dependent fashion at tethering response elements such as the AP-1 sites associated with the collagenase-3/MMP13 and collagenase-1/MMP1 genes (Jonat et al., 1990; Yang-Yen et al., 1990; Pendas et al., 1997; Tuckermann et al., 1999) or the NF-κB sites of the IL-8 and ICAM-1 genes (Nissen and Yamamoto, 2000). However, repression by GR appears to require neither HDAC activity (M.Cronin, R.M.Nissen and K.R.Yamamoto, unpublished data) nor corepressors N-CoR or SMRT; indeed, no functional corepressors have been identified for steroid receptors (Chen and Evans, 1995; Horlein et al., 1995; Dressel et al., 1999). Although activation and repression complexes are clearly distinct, there are several indications that they may be more closely related than appears at first glance: (i) many transcriptional regulators activate transcription in some contexts and repress in others (Roy et al., 1998); (ii) single base pair mutations in composite glucocorticoid response elements (GREs) of the prolactin and proliferin genes (Sakai et al., 1988; Starr et al., 1996) convert them from GR-repressible to GR-inducible elements; conversely, a single amino acid change in the GR DNA-binding domain (DBD) produces a receptor that activates under conditions in which the wild-type GR represses (Starr et al., 1996); (iii) transcriptional repression by GR is agonist dependent and antagonist sensitive, suggesting that repression is mediated by the receptor conformation that binds p160 coactivators (Jonat et al., 1990; Fryer et al., 2000; B.Darimont and K.R.Yamamoto, unpublished data); (iv) overexpression of a p160, TIF2/GRIP1, potentiates estrogen receptor (ER)-mediated repression of tumor necrosis factor-α (TNF) transcription, whereas ER antagonists and ER mutations disrupting the ER–p160 interaction decrease repression (An et al., 1999). To examine these apparent context-specific effects on regulatory factor assembly and activity, we analyzed the composition and function in vivo of regulatory complexes at a response element, col3A, that governs the expression of the collagenase-3 gene in a human osteosarcoma cell line. The col3A element binds AP-1 and confers phorbol 12-myristate 13-acetate (PMA) inducibility as well as repression by glucocorticoid and thyroid hormones. We monitored factor recruitment and activity at col3A under conditions of activation and repression. We assessed receptor and TIF2/GRIP1 association and function during repression, and determined whether GR and TR, two receptors from the same gene family, repress transcription using similar or distinct components. Results AP-1 subunit composition at the collagenase-3 AP-1 element The collagenase-3 gene is expressed in U2OS human osteosarcoma cells (Jimenez et al., 1999) stably transfected with rat GR [U2OS.G (Rogatsky et al., 1997)]; it can be induced by phorbol esters (PMA) and cytokines IL-6 and IL-1β (Borden et al., 1996; Solis-Herruzo et al., 1999) and repressed by glucocorticoids such as dexamethasone (Pendas et al., 1997; Tuckermann et al., 1999). A single AP-1 binding site positioned at −50/−44 relative to the collagenase-3 transcription start site contributes to basal expression, and is responsible for both PMA inducibility and hormonal repression (Porte et al., 1999; data not shown). Thus, regulation of the collagenase-3 gene emanates from a compact, well defined response element, which we denote col3A. The cFos component of the AP-1 protein family is critical for collagenase-3 promoter activation: cFos-overexpressing transgenic mice exhibit enhanced expression of collagenase-3, whereas cFos−/− knockout mice are collagenase-3 deficient (Gack et al., 1994; Borden et al., 1996). Furthermore, in fibroblasts cultured from cFos−/−cJun−/− double knockout mice, collagenase-3 is not inducible by PMA and other stimuli (Hu et al., 1994; Schreiber et al., 1995). The expression of the AP-1 family members themselves, particularly cFos, is regulated by AP-1 and GR (Kruijer et al., 1984; Treisman, 1985; Lamph et al., 1988; Subramaniam et al., 1992; Luo and Jackson, 1998; Boudreau et al., 1999). We therefore monitored the effects of PMA and dexamethasone, i.e. inducing and repressing conditions for collagenase-3, on the intracellular levels of cFos and on the subunit composition of AP-1 bound at col3A. As expected, cFos levels were increased by 1 h, and strongly elevated by 4–6 h of PMA treatment compared with untreated U2OS.G cells (Figure 1A); as controls, TNF and bacterial lipopolysaccharide (LPS) had no effect (left panel). Interestingly, during the first hour of PMA treatment, dexamethasone inhibited PMA-induced cFos accumulation, whereas it potentiated cFos levels at 4 or 6 h of treatment. In contrast, cJun expression was not significantly stimulated by PMA and was mildly inhibited by dexamethasone at all time points tested (data not shown). Figure 1.Occupancy of the coll3 AP-1 element by cFos and cJun in vivo. (A) Effect of GR on PMA induction of cFos expression. U2OS.G cells were untreated (con), or treated with PMA, TNF (T) or LPS (L), in the presence or absence of dex, for 1 or 6 h, as indicated, and cFos expression in whole cell extracts was examined by immunoblotting with N-terminal cFos [(N)cFos] polyclonal antibodies (left, upper panel). Equal loading into each lane was verified by blotting with anti-ERK polyclonal antibodies (left, lower panel). cFos expression in U2OS.G cells treated with PMA in the presence or absence of dex for the time indicated was assessed by blotting with C-terminal cFos [(C)cFos] rabbit polyclonal antibodies (right panel). (B and C) Composition of AP-1 subunits within activation and repression complexes at col3A. U2OS.G cells were untreated (−) or cultured in the presence of PMA (P) or PMA+dex (P/d) for 1 (B) or 5 (C) h and chromatin immunoprecipitations were performed with control rabbit serum (con), cJun, (N)cFos and (C)cFos rabbit polyclonal antibodies. In each case, a +153/+423 fragment of hsp70 gene, and the −150/+68 (coll3) and −1179/−897 (coll3−900) fragments of collagenase-3 gene were PCR amplified. Equal amounts of total genomic DNA (input) were used for immunoprecipitations in each treatment condition. 32P incorporation into the coll3 product was quantified on a Storm 860 PhosphorImager and normalized to the signal obtained with control rabbit serum in untreated cells (shown below the gel). (D) Dynamics of cFos recruitment to col3A under conditions of PMA induction and dex repression. U2OS.G cells were untreated, or exposed to PMA or PMA+dex for the times indicated and chromatin immunoprecipitations were performed with control rabbit serum (con) or (N)cFos antibody. coll3 and hsp70 sequences were PCR amplified, quantified and expressed as a coll3:hsp70 ratio. The ratio obtained with (N)cFos antibody in untreated cells was arbitrarily set as 1. Download figure Download PowerPoint To monitor AP-1 binding to col3A in vivo, we carried out chromatin immunoprecipitation assays using polyclonal antibodies to cFos or cJun (or normal rabbit serum as a negative control), and primers amplifying a 218 bp PCR product from −150 to +68 bp relative to the collagenase-3 transcription start site (coll3), which includes the col3A element. As controls, we amplified a fragment denoted coll3−900, which extends from −897 to −1179 bp upstream of the collagenase-3 transcription unit, and a +153/+423 bp fragment of the hsp70 gene. Cells were untreated, or were incubated for 1 h with PMA or PMA+dex; chromatin fragments containing identical amounts of total genomic DNA (input) were used for the immunoprecipitations (Figure 1B). Normalized to control serum, the cJun antibodies yielded an ∼8-fold enrichment of the col3A-containing fragment; occupancy by cJun appeared constitutive, unaffected by PMA or PMA+dex treatment (Figure 1B). In contrast, a 1 h exposure to PMA induced a 9-fold enrichment of cFos at col3A relative to untreated cells using antisera against either cFos N-terminal [(N)cFos; Figure 1B] or C-terminal [(C)cFos; data not shown] segments. Strikingly, the PMA-induced reconfiguration of the AP-1 subunits was not observed when the cells were co-treated with PMA+dex. These results suggest that basal AP-1 activity is provided by cJun, that a 1 h exposure to PMA induces cFos expression and binding to the AP-1 site, probably replacing cJun–cJun homodimers with cFos–cJun heterodimers, and that dexamethasone antagonizes this effect. In contrast, when the U2OS.G cells were treated with PMA+dex for 5 h, cFos occupancy of the coll3 fragment was selectively enhanced rather than inhibited, relative to PMA alone or to untreated cells (Figure 1C). The coll3−900 and hsp70 control fragments were not enriched under inducing or repressing conditions. In a time course experiment, we found that 1, 2 or 4 h of PMA treatment provoked a 3.5- to 5.5-fold increase in cFos occupancy of the coll3 fragment, normalized to the hsp70 control (Figure 1D). Consistent with the hormonal effect on cFos expression (Figure 1A), 1 h with PMA+dex resulted in lower cFos occupancy than with PMA alone, whereas 2 h of hormone treatment had little net effect, and 4 h produced an increase in cFos occupancy (Figure 1D). Thus, cFos and cJun binding at col3A correlated closely with the relative intracellular levels of these proteins in U2OS.G cells, suggesting that cFos occupancy is driven by synthesis and that AP-1 at col3A is dynamic, turning over within the time-frame examined. GR represses AP-1-dependent transcription independent of the composition of AP-1 subunits Consistent with the observed PMA-induced increase in cFos expression and col3A occupancy, we found that PMA stimulated the accumulation of collagenase-3 mRNA (measured by quantitative RT–PCR/Taqman) at 1 h (data not shown), and more strongly at 2 and 8 h (Figure 2A) of treatment. In all cases, the induction was completely blocked by dexamethasone, suggesting that collagenase-3 transcription is repressed by GR whether or not cFos is recruited to the repression complex. Figure 2.GR represses AP-1 activity independent of the AP-1 subunit composition at col3A. (A) Regulation of collagenase-3 mRNA expression by PMA and dex. U2OS.G cells were treated as shown for the times indicated, total RNA was isolated and reverse-transcribed. Resulting cDNA was subjected to real-time quantitative RT–PCR/Taqman with collagenase-3- and collagenase-1-specific primer/probe sets, in triplicate, using a primer/probe set for G3PDH as an internal control. (B) Binding of cFos to the colA element of the collagenase-1 gene. U2OS.G cells were treated and processed for chromatin immunoprecipitations as described in Figure 1C. A −150/+68 (coll3) region of collagenase-3 gene and a −229/+18 (coll1) region of collagenase-1 gene were co-amplified with +153/+423 and +153/+485 fragments of the hsp70 gene, respectively. 32P incorporation into each PCR product was quantified and expressed as coll3:hsp70 and coll1:hsp70 ratios; in each case, ratios obtained with control serum in untreated cells were arbitrarily set as 1 (shown below the gel). (C) Collagenase-3 induction is not required for repression. U2OS.G cells were treated for 1 h as indicated, total RNA was isolated and analyzed as described in (A). (D) GR-mediated repression of the AP-1-luc reporter activity in U2OS.G cells. Cells were transfected in 24-well plates with 40 ng/well of the AP-1-luc reporter and 50 ng/well of the β-actin-LacZ plasmid expressing β-galactosidase (β-gal). Where indicated, cells were treated with PMA (25 or 50 ng/ml), TNF (5 or 10 ng/ml) or LPS (0.5 or 1 μg/ml), in the absence or presence of 100 nM dex. The AP-1-luc reporter luciferase activity is normalized to β-gal activity and expressed as relative luminescence units (RLU). Download figure Download PowerPoint Unexpectedly, human fibroblast collagenase-1 (MMP1), which like collagenase-3 is a member of the matrix metalloproteinase family, appeared to be refractory at 2 h and only weakly induced by 8 h of PMA treatment of the U2OS.G cells (Figure 2A). The collagenase-1 gene shares substantial sequence similarity with the collagenase-3 gene, extending through the 5′ flanking region (Vu and Werb, 2000) and including an AP-1 element (termed colA, at −72/−66 bp relative to the transcription start site) that mediates PMA induction and dexamethasone repression in other cell contexts (Angel et al., 1987a; Schule et al., 1990; Yang-Yen et al., 1990). Consistent with the relative inactivity of the collagenase-1 gene in U2OS.G cells, 1 h (data not shown) or 5 h (Figure 2B) of PMA or PMA+dex produced little effect on cFos occupancy of colA. Conceivably, the collagenase-1 gene is packaged in an unfavorable chromatin structure in U2OS.G cells. Regard less, our findings identify collagenase-1 as a negative control for collagenase-3 mRNA expression and AP-1 recruitment in this cell line, and underscore the importance of context differences for gene regulation. As cJun occupancy of col3A was constitutive (Figure 1B) and cJun–GR interactions have been described (Diamond et al., 1990; Yang-Yen et al., 1990), we next examined whether collagenase-3 expression is dexamethasone-repressible even in the absence of PMA stimulation. As shown in Figure 2C, we found that uninduced collagenase-3 (but not collagenase-1) mRNA accumulation, generated at least in part by cJun homodimer occupancy of col3A (Figure 1B), was reduced in the presence of dex by ∼40%. We also addressed this issue in the context of an AP-1 site as the only regulatory element by monitoring the activity of an AP-1-luc reporter (Rogatsky et al., 1998) transiently transfected into U2OS.G cells. As expected, PMA, but not TNF or LPS, stimulated AP-1-luc reporter activity, and dexamethasone conferred repression (Figure 2D). Importantly, AP-1-luc reporter activity without PMA treatment was also repressed by dexamethasone. Similar effects of PMA and glucocorticoids were observed with several AP-1-containing reporters, including those derived from the bona fide human promoters (data not shown). We conclude that GR represses AP-1-mediated transcription independently of AP-1 subunit composition, consistent with findings that at tethering response elements, GR can repress transcription stimulated by heterologous activation domains (Nissen and Yamamoto, 2000). GR associates with the AP-1-bound col3A element in vivo in a hormone-dependent manner Indirect evidence supports the notion that GR represses AP-1-activated transcription by tethering to the bound AP-1 factor (Konig et al., 1992; M.Cronin and K.R.Yamamoto, unpublished data); AP-1 elements such as col3A do not serve as direct binding sites for GR. As a direct test of tethering, we carried out chromatin immunoprecipitation assays using anti-GR polyclonal antibodies. As shown in Figure 3A, a 1 h co-treatment of U2OS.G cells with PMA+dex produced a modest but reproducible enrichment of the coll3 fragment compared with untreated or PMA-treated cells. We speculate that the rather weak signals obtained in the GR immunoprecipitations may reflect limited epitope accessibility within the AP-1–GR complex or low efficiency of crosslinking. Notably, GR was similarly recruited to col3A in cells co-treated with TNF+dex (Figure 3A). As TNF had little effect on AP-1 activity (Figure 2D), cFos expression (Figure 1A) or recruitment (Figure 3A), this finding is consistent with the notion that GR is recruited to col3A regardless of the AP-1 subunit composition at the site. Under the same treatment conditions, control U6 gene sequences were not enriched. Thus, GR is recruited to the col3A element in a hormone-dependent manner. Figure 3.GR and TIF2 are recruited to col3A in a dex-dependent, PMA-independent manner. (A) GR recruitment to col3A. U2OS.G cells were untreated (−) or treated with PMA (P), PMA+dex (P/d), TNF (T) or TNF+dex (T/d) for 1 h and chromatin immunoprecipitations were performed with (N)cFos and GR polyclonal antibodies. coll3 and control U6 sequences were amplified, 32P incorporation into coll3 product was quantified and normalized to the untreated sample. (B) TIF2 occupancy of col3A. U2OS.G cells were treated as in Figure 1B and chromatin immunoprecipitations were performed with (N)cFos, (N)TIF2 polyclonal antibodies or normal rabbit serum (con). coll3 and hsp70 sequences were co-amplified, 32P incorporation into coll3 product was quantified and normalized to the untreated sample in each case. (C) AP-1 activation with PMA is not required for TIF2 recruitment. U2OS.G cells were untreated (−) or treated with dex (d), PMA (P) or PMA+dex (P/d) for 1 h and chromatin immunoprecipitations were performed with (C)cFos polyclonal and (C)TIF2 monoclonal antibodies. Quantitation was performed as described in (B). (D) TIF2 protein expression is unaffected by PMA or dexamethasone. U2OS.G cells were untreated (con) or treated with PMA or PMA/IO, in the presence or absence of dex, for 1 or 6 h, as indicated, and TIF2 expression in whole cell extracts was examined by immunoblotting with anti-TIF2 polyclonal antibodies (upper panel). Equal loading into each lane was verified by blotting with anti-ERK polyclonal antibodies (lower panel). Download figure Download PowerPoint TIF2/GRIP1 associates with GR at the col3A element in vivo The most well-documented agonist-dependent protein–protein interaction of intracellular receptors is with the members of the p160 family (Onate et al., 1995; Hong et al., 1996; Voegel et al., 1996; Chen et al., 1997; Li et al., 1997; Torchia et al., 1997; Darimont et al., 1998). Therefore, we tested the possibility that the p160 proteins, originally defined as coactivators, might participate in agonist-dependent repression by GR at col3A. We began by analyzing the expression of the three p160 family members, TIF2/GRIP1, SRC1 and ACTR/Rac3, in U2OS.G cells; TIF2 was readily observed by immunoblotting, whereas SRC1 and ACTR/Rac3 were barely detectable (data not shown). We therefore examined in chromatin immunoprecipitation assays whether TIF2 accumulates in col3A repression complexes under conditions of PMA induction or dexamethasone repression. As shown in Figure 3B, antibodies to TIF2/GRIP1 selectively precipitated the coll3 fragment but not the hsp70 control when cells were co-treated for 1 h with PMA+dex relative to untreated or PMA-treated cells. Importantly, dexamethasone alone promoted the association of TIF2 with the coll3 fragment (Figure 3C), whereas PMA alone had little or no effect (Figure 3B and C). These results suggest that TIF2 associates with repression complexes at col3A in a GR- and dexamethasone-dependent manner and that this association is independent of AP-1 activation by PMA. The finding that epitopes near the N- and C-terminus of TIF2 were both accessible (Figure 3B and C; see Materials and methods) supported the interpretation that TIF2 is not merely masked by AP-1, but fails to associate with the col3A regulatory complex in the absence of GR. To examine whether TIF2 recruitment to col3A repression complexes reflects increased TIF2 expression, we analyzed TIF2 protein levels in U2OS.G cells treated (for 1 or 6 h) with PMA or PMA+ionomycin [IO, a calcium ionophore that commonly enhances the effects of PMA (Truneh et al., 1985; Davies et al., 1990)], in the presence or absence of dexamethasone. TIF2 levels in all cases were indistinguishable from those in untreated control cells (Figure 3D) indicating that TIF2 is recruited into the col3A repression complexes from pre-existing intracellular pools. GRIP1 potentiates GR-dependent transcriptional repression To determine whether TIF2 plays a role in transcriptional repression, or is merely a non-functional 'passenger' recruited to the GR LBD as a result of agonist binding, we examined the effects of introducing GRIP1, the murine homolog of human TIF2, into U2OS.G cells by transient transfection. In control experiments, we first confirmed that transfected p160s would enhance the level of glucocorticoid induction of a luciferase reporter gene linked to a simple GRE (XG46TL; Rogatsky et al., 1998). Indeed, SRC1 and ACTR/Rac3 potentiated GR-mediated activation in a dose-dependent manner, maximally 5- to 10-fold (data not shown). GRIP1 enhanced both basal and hormone-dependent expression at low input doses of transfected DNA (Figure 4A), and squelched it at higher doses (data not shown). The modest effect of GRIP1 on GR activation (∼2-fold) probably reflected the high background of endogenous TIF2 protein in U2OS.G cells. Similar results were obtained in two other TIF2-expressing cell lines, A549 and SAOS2.G, whereas GRIP1 had a more robust effect in MCF7 cells, which express relatively low levels of TIF2 (data not shown). Hence, the magnitude of the effect of a given transfected p160 is inversely proportional to the level of that p160 expressed endogenously in the recipient cell line. Figure 4.GRIP1 overexpression potentiates GR-mediated activation and repression of AP-1 activity in an NR box 3-dependent manner. U2OS.G cells were transfected in 24-well plates with indicated amounts per well of pCDNA3-GRIP1 (wt, S2A, S3A or 4X). Total amount of transfected DNA was equalized with pCDNA3 empty vector. Transcriptional activation of an XG46TL [(A), 40 ng/well] or repression of AP-1-luc [(B) and (C), 40 ng/well] reporter was measured in the absence or presence of 100 (A and C) or 1 (B) nM dex. Endogenous AP-1 activity in (B) and (C) was stimulated with 25 ng/ml PMA. Luciferase activity is normalized to the β-gal activity of a co-transfected β-actin-LacZ plasmid (50 ng/well) and expressed as RLU. The y-axis in (C) is broken to visualize the effect of GRIP1 on GR-mediated repression at saturating
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