CoCoA, a Nuclear Receptor Coactivator which Acts through an N-Terminal Activation Domain of p160 Coactivators

Molecular Cell, Vol. 12, 1537–1549, December, 2003, Copyright 2003 by Cell Press

CoCoA, a Nuclear Receptor Coactivator which Acts through an N-Terminal Activation Domain of p160 Coactivators Jeong Hoon Kim,1 Hongwei Li,1 and Michael R. Stallcup1,2,* 1 Department of Pathology and 2 Department of Biochemistry and Molecular Biology University of Southern California Los Angeles, California 90089

Summary The p160 coactivators bind to and potentiate transcriptional activation by nuclear receptors by recruiting secondary coactivators such as the histone acetyltransferases p300 and CBP and the protein methyltransferase CARM1. The function of the highly conserved N-terminal basic-helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domain of p160 coactivators is unknown. This region is required for coactivator synergy among p160, p300, and CARM1 coactivators. We identified a coactivator, coiled-coil coactivator (CoCoA), which binds to this domain and thereby enhances transcriptional activation by the estrogen receptor and other nuclear receptors. Endogenous CoCoA was found simultaneously with p160 coactivators on the promoter of an endogenous estrogen-responsive gene. Reduction of endogenous cellular CoCoA levels inhibited the estrogenstimulated expression of transiently transfected and endogenous genes. Moreover, CoCoA cooperated synergistically with GRIP1, CARM1, and p300 to enhance ER-mediated transcription. Thus, the N-terminal region of p160 coactivators contains an additional activation domain which contributes to coactivator function by recruitment of CoCoA. Introduction Nuclear receptors (NRs) are ligand-regulated transcription factors which include receptors for steroid and thyroid hormones, retinoic acid, and vitamin D, as well as orphan receptors (Mangelsdorf and Evans, 1995; Tsai and O’Malley, 1994). NRs share a structural organization that consists of a variable N-terminal region, a conserved DNA binding domain (DBD) containing two zinc fingers, and a conserved ligand binding domain (LBD). NRs have two distinct transcriptional activation functions (AF), AF-1 in the N-terminal region and the hormone dependent AF-2 in the LBD. The overall structures of NR LBDs are similar, and ligand binding induces a conformational change which is necessary for binding of transcriptional coactivators (Moras and Gronemeyer, 1998). Coactivators contribute to transcriptional activation by helping to remodel chromatin structure and recruit RNA polymerase II and its associated basal transcription machinery. The p160 coactivator family has three genetically distinct but related family members: SRC-1, GRIP1/ TIF-2, and AIB1/ACTR/RAC3/pCIP/TRAM1 (McKenna et al., 1999; Westin et al., 2000). Functional and genetic *Correspondence: [email protected]

knockout experiments (Torchia et al., 1997; Xu et al., 1998, 2000; Gehin et al., 2002) as well as in vitro transcription systems (Liu et al., 2001) have demonstrated the critical roles of these coactivators in mediating NR function. p160 coactivators also bind to and enhance the activity of other classes of DNA binding transcriptional activator proteins, including AP-1, NF␬B, HNF-1, MEF2C, and TEF4 (Lee et al., 1998; Sheppard et al., 1999; Soutoglou et al., 2000; Belandia and Parker, 2000; Chen et al., 2000b). Endogenous p160 coactivators are recruited to the hormone-responsive promoters of native, chromosomally integrated genes through direct, hormone-dependent interactions with NRs (Shang et al., 2000). The promoter-bound p160 coactivators contribute to chromatin remodeling by bringing secondary coactivators (which bind to p160 coactivators but not directly to NRs) to the promoter. The 160 kDa p160 coactivators share several similar structural features: the central NR interaction domain consisting of three NR box motifs (LXXLL, where L is leucine and X is any amino acid), the C-terminal activation domains AD1 and AD2, and the N-terminal basic-helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domain (McKenna et al., 1999; Westin et al., 2000). The LXXLL motifs interact with a hydrophobic cleft in the NR LBD formed as a result of ligand-induced conformational changes. AD1 and AD2 recruit downstream secondary coactivators involved in chromatin remodeling: AD1 (amino acids 1040–1120 of GRIP1) recruits the histone acetyltransferases p300 and CBP (Chen et al., 1997; Torchia et al., 1997; Yao et al., 1996); AD2 at the C terminus (amino acids 1122–1462 of GRIP1) recruits protein arginine methyltransferases (PRMTs) such as coactivator-associated arginine methyltransferase 1 (CARM1) and PRMT1 (Chen et al., 1999; Koh et al., 2001). These histone-modifying enzymes act synergistically with p160 coactivators to enhance NR function (Chen et al., 2000a; Koh et al., 2001; Lee et al., 2002). Thus, the C-terminal ADs of p160 coactivators serve as platforms for recruitment of downstream/secondary coactivators and transmission of the transcriptional activation signal from NRs to these secondary coactivators. Among the three members of the p160 family, the N-terminal bHLH-PAS domain is the most highly conserved region (60% identity for amino acids 1–350) (Anzick et al., 1997), but the function of this region remains mostly unknown. PAS domains are multifunctional domains found on proteins from prokaryotes to humans (Gu et al., 2000). They serve as DNA binding, proteinprotein interaction, or ligand binding surfaces in various bHLH-PAS transcription factors, including aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT), and hypoxia-inducible factors (HIFs). ARNT uses its PAS domain to form heterodimers with several other bHLH-PAS proteins, but the bHLHPAS domain of p160 coactivators is not required for the interaction with ARNT and AHR (Beischlag et al., 2002). Because of their extensive size and high degree of sequence conservation, the bHLH-PAS domains of p160 coactivators presumably have multiple interaction sur-

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faces. While NRs bind to the central LXXLL motifs of p160 coactivators, the bHLH-PAS domain serves as a protein interaction surface for other types of DNA binding transcription factors, including myocyte enhancer factor 2C (MEF2C) and transcriptional enhancer factor 4 (TEF4) (Belandia and Parker, 2000; Chen et al., 2000b). It is also possible that this region could participate in intramolecular interactions to regulate the coactivator activity of p160 coactivators or intermolecular interactions with other coactivators. Here we address the hypothesis that the bHLH-PAS region may be important for interaction of p160 coactivators with unidentified downstream (i.e., secondary) coactivators which help p160 coactivators to mediate transcriptional activation by DNA-bound transcription factors such as NRs. To further elucidate the molecular role of the bHLH-PAS domain of p160 coactivators, we performed a yeast two-hybrid screen using the bHLHPAS domain of GRIP1 as bait. We demonstrate here that the bHLH-PAS domain plays a key role in the activity of the multisubunit p160 coactivator complex, and we report the isolation and characterization of a coiled-coil coactivator (CoCoA) as a secondary coactivator, which enhances NR-mediated transcription through its interaction with the bHLH-PAS domain of GRIP1.

Results The bHLH-PAS Domain of GRIP1 Is Required for Synergistic Activation of NR Function In previous studies the bHLH-PAS domain of p160 coactivators was not required for coactivator function in some transient transfection assays with relatively high NR levels (Belandia and Parker, 2000; Chen et al., 1997; Reiter et al., 2001). However, the bHLH-PAS domain was important for transcriptional activation by progesterone receptor on chromatin templates in vitro (Liu et al., 2001). The latter result suggests that the bHLH-PAS domain helps to transmit the activation signal to the transcription machinery and that the requirement for this domain may be assay system dependent. We first reexamined the effect of the deletion of the bHLH-PAS domain of GRIP1 on coactivator function at high level of NRs (using 2–5 ng of ER or AR expression vector). Deletion of the bHLH-PAS domain had no effect on the coactivator function of GRIP1 under these highNR conditions (Figures 1A and 1B). We have previously shown that similar transient transfection assays employing lower levels of NRs exhibit much more stringent requirements for multiple coactivators (Koh et al., 2001; Lee et al., 2002). Under these low-NR conditions (0.02 ng of ER vector or 0.5 ng of AR vector), three coactivators (GRIP1, CARM1, and p300) enhanced NR function in a synergistic manner (Lee et al., 2002; Figures 1C and 1D, assays 1–3). This synergy was completely abolished when the GRIP1 deletion mutant lacking the bHLH-PAS domain was used instead of wild-type GRIP1 (Figures 1C and 1D, assays 3 and 4). Thus, the bHLH-PAS domain of GRIP1 is necessary for three-coactivator synergy and required for NR function when NR is expressed at low levels.

Figure 1. Contribution of the bHLH-PAS Domain of GRIP1 to Coactivator Activity CV-1 cells were transfected with plasmids as indicated and grown in medium containing 100 nM E2 for ER or 20 nM DHT for AR. The luciferase activity results shown are representative of five independent experiments. Reporter plasmids (200 ng): MMTV(ERE)-LUC for ER, MMTV-LUC for AR. Expression vectors: pHE0 encoding ER␣, 2 ng for (A) and 0.02 ng for (C); pSV-AR0, 5 ng for (B) and 0.5 ng for (D); pSG5.HA-GRIP1 or pSG5.HA-GRIP1(563–1462), 200 ng; pSG5.HACARM1, 200 ng; pCMV-p300, 200 ng.

Isolation of CoCoA A yeast two-hybrid screen of a mouse 17 day embryo cDNA library, using amino acids 5–479 of GRIP1 (Figure 2A) as bait, identified a protein which we named CoCoA. Coiled-coils consist of two to five amphipathic ␣ helices that twist around one another to form a supercoil; they are known to be involved in protein-protein interactions and DNA-protein interactions (Lupas, 1996a, 1996b). The CoCoA clone has a predicted open reading frame of 691 codons encoding a protein with a predicted molecular mass of 77.3 kDa (Figure 2C); however, the expressed recombinant protein runs at approximately 100 kDa on SDS-PAGE (data not shown). The CoCoA cDNA contains a short 5⬘ (22 bp) and a long 3⬘ (626 bp) untranslated region. Hydropathy analysis of the predicted amino acid sequence of CoCoA indicated a central hydrophilic region with a high potential to form an ␣-helical struc-

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ture. Lupas’s algorithm (Lupas, 1996b) to detect coiledcoil regions indicated that the central hydrophilic region (amino acids 144–513) of CoCoA has a high probability to form a coiled-coil structure (Figure 2D). This central region also contains three leucine zipper motifs (amino acids 163–184, 198–219, and 475–496) within the predicted coiled-coil (Figures 2B and 2C). A BLAST (Zhang and Madden, 1997) search of the GenBank database revealed that CoCoA is identical (except for codon 301) to a mouse cDNA clone of unknown function (accession number AK007393) and similar (79% amino acid identity) to an anonymous complete human cDNA clone (KIAA1536).Two known coiled-coil proteins with significant homology to CoCoA were also identified. The coiled-coil domain of CoCoA has 25% identity and 49% similarity with the coiled-coil domain of Tax binding protein TXBP151 (Chun et al., 2000; De Valck et al., 1999) and34% identity and 52% similarity with the coiled-coil domain of Xenopus nuclear dot protein 52 (NDP52) (accession number AF312719). The N-terminal region of CoCoA (amino acids 1–149) has 58% identity and 71% similarity with the N-terminal region of NDP52, but no homolog of the C-terminal region of CoCoA (amino acids 501–691) was found. In a Northern blot analysis, a single CoCoA transcript of about 3.5 kb was observed in all mouse tissues examined except spleen, with high levels of expression in the heart and kidney, suggesting that CoCoA is widely expressed (Figure 2E). Interaction of CoCoA with GRIP1 The interaction between CoCoA and GRIP1 was confirmed in mammalian two-hybrid assays, using Gal4 DBD and Vp16 fusion proteins of CoCoA and GRIP1 to activate a luciferase reporter plasmid controlled by Gal4 response elements (Figure 3A, assays 1–5). The N-terminal domain of GRIP1 was necessary and sufficient for the interaction with CoCoA (assays 6–9). A smaller N-terminal fragment (containing only the bHLH-PAS sequences, amino acids 1–363) of another member of the p160 family, SRC-1, also bound to CoCoA (assays 10 and 11). Thus, the interaction with CoCoA was conserved among p160 coactivators. Full-length GRIP1 and an N-terminal fragment of GRIP1, but not GRIP1(563– 1462), bound to CoCoA in coimmunoprecipitation assays of extracts from transiently transfected COS-7 cells (Figure 3B). GST pull-down assays were used to determine which

Figure 2. Identification, Sequence, Secondary Structure, and Expression of CoCoA (A) Functional domains of GRIP1 include the basic-helix-loop-helix (bHLH) region, Per-Arnt-Sim (PAS) A and B regions, three NR binding motifs (NR Boxes), and activation domains (AD1 and AD2). The N-terminal GRIP1 fragment used as bait in the yeast two-hybrid screen is also indicated (bait).

(B) A schematic drawing of CoCoA structure indicates the location of the predicted coiled-coil structure, three leucine zipper motifs (LZ), and the autonomous activation domain. (C) The predicted amino acid sequence of CoCoA is shown. The predicted coiled-coil domain is boxed, and the three leucine zipper motifs are underlined. Amino acid 301, which is different (Q versus E) from the previously reported AK007393 protein, is indicated by an asterisk. (D) The probability plot for forming coiled-coils in CoCoA was produced by the COILS program using the MTIDK matrix (Lupas, 1996b). (E) CoCoA mRNA expression in various mouse tissues is shown in an autofluorogram of a mouse multiple tissue Northern blot, with sizes of RNA markers (Clontech) indicated on the left.

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Figure 3. Interaction of CoCoA Coiled-Coil Domain with bHLH-PAS Domain of GRIP1 In Vivo and In Vitro (A) For mammalian two-hybrid assays CV-1 cells were transfected with 200 ng of GK1-LUC reporter plasmid (controlled by Gal4 response elements) and 200 ng of each indicated expression plasmid. Luciferase activity results shown are representative of three independent experiments. (B) Coimmunoprecipitation of V5-epitope tagged CoCoA and HA-epitope tagged GRIP1 or its deletion mutants was performed with COS-7 cells in 100 mm dishes transfected with 2.5 ␮g each of pcDNA3.1-CoCoA.V5 and pSG5.HA-GRIP1, pSG5.HA-GRIP1N (amino acids 5–479), or pSG5.HA-GRIP1⌬N (amino acids 563–1462). Cell extracts were prepared 48 hr after transfection and immunoprecipitated with anti-V5 monoclonal antibody or control normal mouse IgG. Immunoprecipitated V5-CoCoA (ii and iv) and coprecipitated HA-GRIP1 proteins (i and iii) were detected by immunoblot analysis with antibodies against the epitope tags. A portion of the original cell extract was also examined for HA-GRIP1 and V5-CoCoA expression (5% input). (C) To test binding of full-length (FL) CoCoA or its fragments to GST-fused GRIP1 bHLH-PAS domain (GRIP1N) in vitro, pSG5.HA vectors (2.5 ␮g) encoding HA-epitope-tagged CoCoA fragments were transfected into COS-7 cells. Cell extracts were prepared 48 hr after transfection and incubated with glutathione-Sepharose beads bound with bacterially expressed GST-GRIP1N (amino acids 5–479) or GST. The bound proteins were analyzed by immunoblot with anti-HA antibody. A portion of the original cell extracts was also tested for HA-CoCoA expression (10% input). (D) CV-1 cells were transfected with 200 ng of GK1-LUC reporter plasmid and 200 ng of the indicated expression plasmids. Luciferase activity results shown are representative of three independent experiments.

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fragments of CoCoA, expressed by transient transfection in COS-7 cells, bound to GST-GRIP1N. The minimum region of CoCoA required for the interaction with GRIP1N was the central coiled-coil domain (amino acids 150–500) containing three leucine zipper motifs (Figure 3C); in contrast, N-terminal and C-terminal fragments containing the first and third leucine zipper motifs, respectively, did not interact with GRIP1N. Thus, the central coiled-coil domain of CoCoA is essential, and the first and third leucine zipper motifs were not sufficient for interaction with GRIP1. The interaction of the coiledcoil domain of CoCoA with GRIP1N in vivo was further confirmed in mammalian two-hybrid assays in CV-1 cells (Figure 3D). CoCoA Functions as a Secondary Coactivator CoCoA was tested as a coactivator for ER in transient transfection assays using relatively high-level ER conditions (see Figure 1), since these conditions are better for observing the contributions of single coactivators and pairs of coactivators. CoCoA alone had no effect on ER activity (Figure 4A, assays 5–8), but in the presence of GRIP1, CoCoA enhanced ER function in a dose-dependent and hormone-dependent manner (assays 9–12). The secondary coactivator activity of CoCoA was similar in magnitude to that of CARM1 (assays 3 and 4). CoCoA also enhanced transcriptional activation by GR, AR, and TR in a hormone-dependent and GRIP1-dependent manner (data not shown). The dependence of CoCoA coactivator function on GRIP1 indicates that CoCoA is a secondary coactivator for ER and is recruited to the promoter by binding to the GRIP1 N-terminal domain. In confirmation of this proposed relationship, coimmunoprecipitation experiments in extracts of COS-7 cells containing various combinations of transiently expressed ER, GRIP1, and CoCoA showed that CoCoA does not interact directly with ER but associates indirectly with ER through GRIP1 (Figure 4B). To test the functional importance of the interaction between CoCoA and the GRIP1 N-terminal region, we examined the ability of CoCoA to act as a coactivator in cooperation with GRIP1 deletion mutants lacking the N-terminal region, AD1, or AD2. All three GRIP1 mutants enhanced ER activity; CoCoA further enhanced the activity of full-length GRIP1 and the mutants lacking AD1 or AD2, but did not enhance the activity of the GRIP1 mutant lacking the N-terminal region (Figures 4C and 4D). Thus, the integrity of the bHLH-PAS domain of GRIP1, which is the binding site for CoCoA, is essential for the secondary coactivator function of CoCoA. Furthermore, the activity of CoCoA is independent of AD1 and AD2, suggesting that CoCoA functions as a coactivator by triggering a different activation pathway from those used by AD1 and AD2. An Autonomous Transcriptional Activation Domain in the C-Terminal Region of CoCoA Is Required for CoCoA Coactivator Function When various CoCoA fragments were fused to Gal4 DBD, three Gal4DBD-CoCoA fusion proteins containing the C-terminal region of CoCoA activated expression of a Gal4-responsive reporter gene (Figure 5A). In contrast, all of the other CoCoA fragments, including full-length

CoCoA, failed to activate transcription. All of the fusion proteins were expressed at similar levels (data not shown). The fact that progressive N-terminal deletions of CoCoA to residue 500 correspondingly increased the autonomous transcriptional activation activity suggests a possible role for the central coiled-coil region in regulating the transcriptional activation activity of the C-terminal domain. We next tested the effect of CoCoA or its fragments on the activation function of the bHLH-PAS domain of GRIP1 in a mammalian one-hybrid system (Figure 5B). The modest transcriptional activation function of the GRIP1 bHLH-PAS domain (assay 7) was strongly stimulated by full-length CoCoA (assay 8). However, deletion of the C-terminal activation domain (assay 11) or the coiled-coil domain (assays 9 and 10) completely abolished the CoCoA activity. Thus, both the ability to bind GRIP1 and the autonomous activation domain of CoCoA are required for its coactivator function. CoCoA Is Recruited to Estrogen-Regulated Promoters in a Hormone-Dependent Manner CoCoA is associated with the GRIP1 N-terminal domain in vivo (Figures 3A and 3B), and this association was important for enhancement of transcriptional activation by ER in transient transfection assays (Figure 4). To test whether CoCoA is involved in transcriptional activation of a native estrogen-regulated gene (i.e., a gene in its native chromosomal position) by ER, we employed chromatin immunoprecipitation (ChIP) assays to look for estrogen-dependent recruitment of CoCoA to the pS2 promoter in MCF-7 breast cancer cells. E2 treatment caused recruitment of ER␣, GRIP1, and CoCoA to the pS2 promoter region (Figure 6A). Control ChIP assays using normal IgG produced a weak signal that did not increase with hormone treatment. The ␤-actin coding region served as a negative control; similar levels of pS2 promoter and ␤-actin coding region fragment amplification were observed for the input chromatin samples taken before immunoprecipitation. By quantitative realtime PCR analysis we observed 4-, 3.7-, and 11-fold induction of pS2 promoter occupancy by ER␣, GRIP1, and CoCoA, respectively, with E2 treatment, whereas normal rabbit IgG did not show any significant hormonedependent change (Figure 6B). A very similar pattern of androgen-dependent recruitment of AR, GRIP1, and CoCoA to the PSA promoter was observed in LNCaP prostate cancer cells (data not shown). Our transient transfection results indicated that the coactivator activity of CoCoA with ER depends on its association with the p160 coactivator (Figure 4). To test whether CoCoA and p160 coactivators are associated with each other in the same complex on the pS2 promoter (rather than recruited separately or sequentially to pS2 promoters), we performed a chromatin immunoprecipitation with antibodies against CoCoA and then reimmunoprecipitated the crosslinked complexes with antibodies against the p160 coactivator AIB1 (ChIP and ReIP experiment). The reverse experiment was also performed with AIB1 antibodies first followed by CoCoA antibodies. In both cases, we observed a hormonedependent PCR signal with primers against the pS2 promoter, whereas no PCR signal was observed when

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Figure 4. Coactivator Function of CoCoA with NR Depends on the Presence of GRIP1 with an Intact bHLH-PAS Domain (A) CV-1 cells were transfected with MMTV(ERE)-LUC reporter plasmid (200 ng), pHE0 encoding human ER (2 ng), pSG5.HA-GRIP1 (100 ng), pSG5.HA-CARM1 (200 ng), and variable amounts (50, 100, 200, and 400 ng) of pSG5.HA-CoCoA, as indicated, and grown in medium containing or lacking E2. Luciferase activity results shown are representative of five independent experiments. (B) COS-7 cells were transfected with 2 ␮g each of pHEG0, pSG5.HA-CoCoA, and pSG5.HA-GRIP1, as indicated, and grown with E2. Cell extracts were immunoprecipitated (IP) with anti-ER␣ antibody or control normal rabbit IgG. Immunoprecipitated ER␣ (bottom panels) and coprecipitated HA-CoCoA (top panels) were detected by immunoblot (IB) analysis with anti-ER␣ antibody and anti-HA antibody. (C) CV-1 cells were transfected with 200 ng of MMTV(ERE)-LUC, 2 ng of ER expression vector, 100 ng of pSG5.HA-GRIP1 or pSG5.HAGRIP1(563–1462), and 200 ng of pSG5.HA-CoCoA, as indicated. Luciferase activity results shown are representative of three independent experiments. (D) Transient transfections using pSG5.HA-GRIP1⌬AD1 or pSG5.HA-GRIP1⌬AD2 were performed as in (C).

nonspecific IgG was substituted for the ReIP antibody (Figure 6C). Thus, E2 causes recruitment of CoCoA to the pS2 promoter, where it is associated with p160 coactivators. A modified ChIP experiment (reporter coimmunoprecipitation assay or reporter CoIP) was performed to test whether CoCoA participates in similar manners in the hormonal activation of transient reporter genes versus native (chromosomally integrated) genes. COS-7 cells were transfected with MMTV(ERE)-LUC reporter plasmid and expression vectors encoding ER␣, GRIP1, and CoCoA; cells were untreated or treated with E2 for 45 min and then subjected to the ChIP protocol with anti-

bodies against ER␣, GRIP1, and CoCoA. E2 treatment induced ER␣, GRIP1, and CoCoA occupancy of the MMTV(ERE) promoter (Figure 6D), indicating that the observed coactivator activity of CoCoA in transient transfection assays (Figure 4) involves the recruitment of CoCoA to the hormone-regulated promoter. Requirement for Endogenous CoCoA for Transcriptional Activation by the GRIP1 N-Terminal Domain and by ER As a further test of the physiological role of CoCoA in ER and GRIP1 function, a small interfering RNA (siRNA) was transfected into COS-7 cells to specifically reduce

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Figure 5. C-Terminal Activation Domain Is Required for CoCoA Coactivator Function (A) CV-1 cells were transfected with GK1-LUC reporter plasmid (200 ng) and a plasmid encoding either Gal4 DBD or Gal4 DBD fused to various CoCoA fragments (200 ng), as indicated. In the schematic diagrams, numbers indicate CoCoA amino acid positions. Luciferase activity results shown are representative of three independent experiments. (B) CV-1 cells were transfected with GK1-LUC (200 ng), plasmid encoding Gal4 DBD or Gal4 DBD fused to GRIP1N (amino acids 5–479) (200 ng), and 200 ng of pSG5.HA plasmid encoding full-length (FL) CoCoA or its fragments, as indicated. Luciferase activity results shown are representative of three independent experiments.

the level of endogenous CoCoA mRNA; a control siRNA with a scrambled sequence had no effect on CoCoA or ␤-actin mRNA levels (Figure 7A, inset). Reporter gene activation by Gal4DBD-GRIP1N was reduced more than 50% by the CoCoA-directed siRNA, but not by the scrambled-sequence siRNA (Figure 7A, assays 4–6). There was no effect on the basal activity of Gal4 DBD (assays 1–3). Thus, endogenous CoCoA was specifically

required for the autonomous transcriptional activation activity of the GRIP1 N-terminal domain. In MCF-7 cells the CoCoA-directed siRNA, but not the scrambled-sequence siRNA, reduced the level of endogenous CoCoA mRNA but not ␤-actin mRNA (Figure 7C, top and bottom panels). Estrogen-induced expression of a transiently transfected reporter gene was inhibited more than 60% by the CoCoA-directed siRNA,

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Figure 6. CoCoA Is Recruited to EstrogenResponsive Promoters in a Hormone-Dependent Manner In vivo binding of ER␣, GRIP1, and CoCoA to either the endogenous pS2 promoter or the transiently transfected MMTV (ERE) promoter was examined by ChIP (A–C) and reporter CoIP (D) assays. (A) Crosslinked, sheared chromatin from MCF-7 cells grown with or without E2 (45 min) was immunoprecipitated with the indicated antibodies. The following volumes of the coprecipitated DNA were analyzed by PCR using primers to amplify the pS2 promoter (opposing pair of arrows in the diagram) or the ␤-actin coding region: 1 ␮l of 1:10 diluted samples (input, ER, and CoCoA); 1 or 2 ␮l of the undiluted samples (IgG or GRIP1). Results shown are representative of ten independent experiments. (B) Real-time PCR analysis using primers for the pS2 promoter was performed with 1 ␮l of 1:10 diluted samples. The results are shown as percentage of input and are the mean and standard deviation from triplicate reactions. (C) ChIP and ReIP assays were performed for the pS2 promoter in MCF-7 cells untreated or treated with E2 for 45 min. First (ChIP) and second (ReIP) chromatin immunoprecipitations were performed with the indicated antibodies. Results shown are representative of three independent experiments. (D) Occupancy of the transiently transfected MMTV(ERE) promoter by ER and coactivators was examined by reporter CoIP assay as described in the Experimental Procedures. Transfected COS-7 cells were untreated or treated with E2 for 45 min before formaldehyde crosslinking. Chromatin immunoprecipitation was performed with the indicated antibodies; PCR analysis was performed with primers spanning the nucleosome B region of the MMTV promoter (pair of opposing arrows in the diagram). Results shown are representative of four independent experiments.

but not by the scrambled-sequence siRNA (Figure 7B). Estrogen-induced expression of the endogenous pS2 gene (in its native chromosomal position) was similarly inhibited by the CoCoA-specific siRNA but not by the nonspecific siRNA (Figure 7C, middle panel). Note that only endogenous ER and coactivators were involved in these experiments. Thus, endogenous CoCoA was required for efficient hormonal induction of these genes by ER.

addition of CoCoA as a fourth coactivator caused either no enhancement or a decrease in the reporter gene activity observed with the combination of GRIP1, CARM1, and p300 (data not shown). Thus, it is very important to titrate the level of NR vector used for various combinations of coactivators, and the amount of NR vector required to observe synergy generally decreases as the number of coactivators used increases. Discussion

Secondary Coactivator Function of CoCoA, CARM1, and p300 Is Synergistic and Dependent on GRIP1 Since CoCoA, CBP/p300, and CARM1 bind to separate activation domains of GRIP1 and apparently activate transcription by different mechanisms, we tested for coactivator synergy in transient transfection assays using low-ER conditions (see Figure 1). Under these conditions GRIP1 and CoCoA together modestly enhanced reporter gene activity, as did the combination of GRIP1, CARM1, and p300; coexpression of CoCoA along with the other three coactivators caused an additional 6-fold enhancement (Figure 7D, assays 4–6) which was entirely dependent on the presence of GRIP1 (assay 7), consistent with a role for GRIP1 as a platform for recruiting the three secondary coactivators. Note that, in order to observe the four-coactivator synergy, the level of ER expression vector used in Figure 7D (0.002 ng) had to be carefully titrated to a level below that used to observe optimal three-coactivator (GRIP1-p300-CARM1) synergy, which was demonstrated in Figures 1C and 1D (0.02 ng). At 0.2 or 0.02 ng of ER expression vector,

The Dual Roles of the bHLH-PAS Domain of p160 Coactivators in Signal Input and Signal Output Coactivators may be thought of as a mechanism for transmitting the transcriptional activation signal from a DNA-bound transcriptional activator protein to the transcription machinery. For example, p160 coactivators are recruited to the promoter by NRs and thereby receive the activating signal from NRs through direct contact of the coactivator LXXLL motifs with the NRs (Heery et al., 1997; Westin et al., 2000). The p160 coactivators transmit the signal through their activation domains. AD1 and AD2 recruit the secondary coactivators p300/ CBP and CARM1, respectively, which contribute to transcriptional activation by acetylation and methylation of histones and possibly other proteins in the transcription machinery (Chen et al., 1997, 1999, 2000a; Ma et al., 2001). The current study demonstrates that the bHLH-PAS domain of p160 coactivators can function as an additional activation domain (which we call AD3) by recruit-

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ing another secondary coactivator, CoCoA (Figure 7E). The role of the highly conserved bHLH-PAS domain in helping to mediate transcriptional activation by NRs was previously unknown. While some previous studies produced mixed verdicts as to whether the bHLH-PAS domain was necessary for p160 coactivator function (Liu et al., 2001; Belandia and Parker, 2000; Chen et al., 1997; Reiter et al., 2001), we show here that the bHLH-PAS domain is essential under low-NR transient transfection conditions which demonstrate more stringent coactivator requirements (Figure 1). Thus, GRIP1 binds directly to ER and provides an appropriate interaction surface for recruitment of CoCoA to the promoter (Figure 7E). CoCoA does not bind directly to ER but functions as a coactivator by binding to the bHLH-PAS domain of GRIP1. The activating function of the bHLH-PAS domain depends on CoCoA to amplify its activity and is independent of the AD1 and AD2 activities of GRIP1. In addition to its binding and cooperative coactivator function with the bHLH-PAS domain of GRIP1, CoCoA also has similar physical and functional interactions with other p160 coactivators (Figures 3A and 6C), suggesting a general role of CoCoA as a secondary coactivator for p160 coactivators. Three additional coactivators or basal transcription factors were previously shown to interact with the bHLHPAS region of p160 coactivators. hMMS19 (human methyl methanesulfonate 19) can act as a coactivator for the AF-1 activation domain of ER and also binds to subunits of TFIIH (Wu et al., 2001), suggesting a possible mechanism of hMMS19 coactivator function which remains to be tested. BAF57, a subunit of the mammalian SWI/SNF ATP-dependent chromatin remodeling complex, binds to both the bHLH-PAS domain of the p160 coactivators and to ER, and is necessary for the coactivator activity of p160s (Belandia et al., 2002). However, the functional importance of the p160-BAF57 binding interaction has not been determined. The bHLH-PAS region of p160 coactivators also binds cyclin T1, a component of p-TEFb (positive-acting transcription elongation factor b) (Kino et al., 2002), suggesting a possible link between p160 coactivators and the basal transcription machinery. Thus, the bHLH-PAS domains of p160 coactivators may have multiple mechanisms for transmitting activating signals to the transcription machinery: through CoCoA (as demonstrated here) by an as yet unknown mechanism (see discussion below), and possibly through hMMS19 or cyclin T1 to components of the basal transcriptional machinery or through BAF57 to the ATP-dependent chromatin remodeling complexes. Given the large size (more than 300 amino acids) and high degree of conservation (60% amino acid identity) of the bHLH-PAS domains of the three p160 proteins, it is reasonable to assume that there may be many protein interaction surfaces on the bHLH-PAS domain. In addition to the signal output functions discussed above, the bHLH-PAS domain of p160 coactivators has also been shown previously to serve as a signal input or binding site for other (i.e., non-NR) classes of DNA binding transcription factors, including MEF2C, TEF4, and Tat (HIV-1 transcriptional activator) (Chen et al., 2000b; Belandia and Parker, 2000; Kino et al., 2002).

CoCoA, a Coactivator with a Coiled-Coil Domain to Bind GRIP1 and an Activation Domain for Downstream Signaling A database search indicated that CoCoA has no homology with any known coactivators but has a predicted coiled-coil structure (Figure 2); thus, CoCoA represents a novel type of NR coactivator. While some bHLH-PAS proteins can form heterodimers through mutual interactions between their respective PAS domains (Taylor and Zhulin, 1999), this is clearly not the case for p160 binding to CoCoA, since CoCoA does not harbor a bHLH-PAS domain. The coiled-coil domain of CoCoA is responsible for binding the bHLH-PAS region of GRIP1 (Figure 3). There is a parallel previous example for this finding: the recently described ARNT-interacting protein AINT, another coiled-coil protein, interacts with the bHLH-PAS domain of ARNT through its highly structured coiledcoil domain (Sadek et al., 2000). These two examples suggest that a particular type of coiled-coil structure may serve as a bHLH-PAS recognition motif. The CoCoA C-terminal activation domain, which along with the coiled-coil domain is essential for coactivator function (Figure 5B), consists of 20% acidic amino acids and 30% hydrophobic amino acids (Figures 2B and 2C). The hydrophobic residues are interspersed with acidic residues in a manner reminiscent of the herpes simplex virus VP16 activation domain (Cress and Triezenberg, 1991). Physiological Relevance of CoCoA in Transcriptional Activation by ER and GRIP1 CoCoA acted synergistically in combination with two other GRIP1 binding proteins, p300/CBP and CARM1, in low-NR transient transfection assays (Figure 7D). In addition, endogenous CoCoA was required for efficient ER and GRIP1 function (Figures 7A–7C) and bound simultaneously with p160 coactivators in a hormonedependent manner to the promoters of steroid hormoneregulated genes in their native chromosomal locations (Figures 6A–6C). These findings demonstrate that CoCoA is a physiologically relevant part of the transcriptional activation process and is recruited to the endogenous promoter through its contact with the N-terminal region of p160 coactivators. Our further demonstration that CoCoA is similarly recruited in a hormone-dependent manner to a transiently transfected estrogen-responsive promoter (Figure 6D) suggests that CoCoA participates in a similar manner as a component of the p160 coactivator complex in the transcriptional activation of both native and transiently transfected target promoters. The correlation of findings with the transiently transfected and stably integrated reporter genes also provides an important validation of the results obtained in our low-NR transient transfection assay system, which demonstrate a requirement for the N-terminal region of GRIP1 (Figures 1C and 1D). Although the mechanism of the CoCoA-p300-CARM1 cooperation remains to be determined, the fact that their cooperation for NR-mediated transcription is not additive but synergistic suggests that each coactivator makes a distinct contribution to the transcriptional activation process and thus that CoCoA recruits different downstream targets or uses a different signaling pathway from those of CARM1 and p300.

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Figure 7. Requirement for Endogenous CoCoA for GRIP1 and ER Function, and Synergistic Enhancement of NR-Mediated Transcriptional Activation by Four Coactivators (GRIP1, CoCoA, CARM1, and p300) (A) COS-7 cells in 12-well dishes were transfected using Lipofectamine 2000 with GK1-LUC reporter plasmid (200 ng), plasmids encoding Gal4 or Gal4-GRIP1N (amino acids 5–479) (200 ng), and 80 pmole of either the CoCoA siRNA duplex or scramble siRNA duplex. Luciferase activity was measured 72 hr after transfection. Results shown are representative of four independent experiments. (Inset) Total RNA from the siRNA-transfected cells was analyzed by reverse transcriptase-PCR analysis using primers for CoCoA or ␤-actin mRNA. (B) MCF-7 cells were transfected using Lipofectamine 2000 with 2ERE-tk-LUC reporter (200 ng) and 40 (⫹) or 80 (⫹⫹) pmole of either the CoCoA siRNA duplex or scramble siRNA duplex, as indicated. Forty-eight hours after transfection, cells were treated with E2 or untreated and harvested after an additional 24 hr for luciferase assays. Results shown are representative of three independent experiments. Reverse transcriptase-PCR analysis was also performed to confirm the reduction of CoCoA mRNA levels (data not shown). (C) MCF-7 cells were transfected using Targefect-siRNA Transfection Kit with the indicated siRNA and treated or untreated with E2 as in (B).

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Experimental Procedures Yeast Two-Hybrid Screening GRIP1 cDNA encoding the bHLH-PAS domain (amino acids 5–479) was cloned into EcoRI and SmaI sites downstream of the Gal4 DBD in pGBT9 (Clontech) to generate a bait plasmid (pGBT9-GRIP1N). The yeast strain HF7c (containing his3 and lacZ genes controlled by Gal4 responsive elements) was sequentially transformed with the pGBT9-GRIP1N plasmid and a mouse 17 day embryo cDNA library in pGAD10 (Clontech). We performed HIS3 jump-start to improve the chances of detecting weak interactions. 2 ⫻ 106 transformants were first plated on synthetic complete media plates lacking leucine and tryptophan, incubated until colonies appeared, and harvested. The amplified transformants (approximately 2 ⫻ 107) were plated onto synthetic complete media plates lacking histidine, leucine, and tryptophan, and containing 50 mM 3-amino-1,2,4-triazole to suppress low-level expression of His3 due to autonomous transactivation activity of the bait. pGAD10 plasmids from colonies that survived without histidine and were LacZ-positive were rescued and sequenced. A total of approximately six million yeast transformants were screened in four independent screens. CoCoA was represented by four of the 52 LacZ-positive clones and was the most frequently isolated clone. Northern Blot Analysis The originally isolated (see above) cDNA fragment encoding CoCoA was labeled with fluorescein-UTP using a random priming labeling kit (Amersham Pharmacia). A mouse multiple tissue Northern blot (Clontech) containing 2 ␮g of poly(A) RNA from each tissue was probed with the fluorescein-labeled CoCoA cDNA according to the manufacturer’s instructions. Plasmids For expression of CoCoA, GRIP1, and their fragments in mammalian cells and in vitro, the following mammalian expression vectors were used: pSG5.HA (Chen et al., 1999) for expressing N-terminal hemagglutinin (HA)-tagged proteins, pM (Clontech) for N-terminal Gal4 DBD fusion proteins, pVP16 (Clontech) for N-terminal VP16 AD fusion proteins, and pcDNA 3.1/HIS-V5 (Invitrogen) for C-terminal V5-tagged CoCoA. PCR-amplified CoCoA cDNA fragments (with amino acid numbers shown in parentheses) were subcloned as follows: full-length CoCoA, CoCoA(1–500), CoCoA(150–500), and CoCoA(150–691) into XhoI and BglII sites of pSG5.HA and BamHI and MluI sites of pM and VP16; all other CoCoA fragments, EcoRI-XhoI fragments inserted into EcoRI and XhoI sites of pSG5.HA and EcoRI and SalI sites of pM and VP16. The full-length CoCoA fragment was also cloned into BamHI and XhoI sites of pcDNA 3.1/HIS-V5 and pGEX-5X1 (Amersham Pharmacia). Other plasmids were constructed using the indicated cDNA fragments and restriction sites: GRIP1(5–479), an EcoRI-XhoI fragment inserted into EcoRI-XhoI sites of pSG5.HA and into EcoRI and SalI sites of pM; GRIP1(563– 1462) into EcoRI-MluI sites of pM; full-length GRIP1(5–1462) into EcoRI site of VP16; GRIP1(563–1462) as an EcoRI-SalI fragment inserted into EcoRI and XhoI sites of pSG5.HA; human SRC-1a(1– 363) into BamHI and SalI sites of pM. The following plasmids were described previously as indicated: pGEX-4T1.GRIP1(5–479) (Huang and Stallcup, 2000); pSG5.HA vectors encoding GRIP1(5–1462), GRIP1⌬AD1, and GRIP1⌬AD2 (Chen et al., 2000a); pHEG0 (Metzger et al., 1995) encoding wild-type human ER␣ or pSG5-HE0 (Chen et

al., 1999) encoding the G400V ER mutant with lower background activity; pSV-AR0 encoding human AR, pSG5.HA-CARM1, and luciferase reporter plasmids MMTV(ERE)-LUC for ER, MMTV-LUC for AR, and GK1-LUC for Gal4 (Chen et al., 1999); pCMV-p300 (Lee et al., 2002). To generate the 2ERE-tk-LUC reporter plasmid, oligonucleotides containing two estrogen response elements (ERE) (5⬘GATCCAGGTCACAGTGACCTAGGTCACAGTGACCTC-3⬘ [sense] and 5⬘-TCGAGAGGTCACTGTGACCTAGGTCACTGTGACCTG-3⬘ [antisense]) and a fragment containing the herpes virus tk promoter (⫺109–⫹50) were cloned into the BglII site of the pGL3-basic vector (Promega).

Cell Culture and Transient Transfection CV-1 and COS-7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum. For reporter gene assays, CV-1 cells were plated at 1 ⫻ 105 cells/well in 12-well plates and transiently transfected by TargeFect F1 reagent (Targeting Systems). Total amount of plasmid DNA added to each well was adjusted to 1.0 ␮g by adding the necessary amount of pSG5.HA empty vector. After transfection, cells were incubated in phenol red-free DMEM containing 5% fetal bovine serum treated with dextrancoated charcoal (Gemini Bioproducts), 20 mM Na-HEPES (pH 7.2), penicillin, and streptomycin, with or without 100 nM E2 or 20 nM dihydrotestosterone (DHT). Forty-eight hours after transfection, cell extracts were prepared and assayed for luciferase activity as described previously (Lee et al., 2002). The results shown are the means and SD of triplicate points. Instead of using internal controls, results shown are representative of multiple independent experiments, as indicated in the figure legends.

Immunoblots and Coimmunoprecipitation Procedures were previously described (Lee et al., 2002). For immunoblots 50 ␮g of protein from cell lysate was analyzed with rat monoclonal antibody 3F10 against HA epitope (Roche), mouse monoclonal antibody R960-25 against V5 epitope (Invitrogen), or anti-ER antibody G-20 (Santa Cruz Biotechnology) and developed with horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology) which were visualized with the ECL system (Amersham Pharmacia). For each immunoprecipitation reaction, 1 mg of cell lysate was incubated with 1 ␮g of the anti-V5 antibody, anti-ER antibody HC-20 (Santa Cruz Biotechnology), or normal IgG (Santa Cruz Biotechnology), and 20 ␮l of protein A/G agarose suspension (Santa Cruz Biotechnology) overnight at 4⬚C. Immunoblots were then performed with the anti-HA antibody and, after stripping, with the anti-V5 antibody or the anti-ER antibody.

GST Pull-Down Assay HA epitope-tagged CoCoA fragments were expressed in COS-7 cells by transient transfection (see above) or translated in vitro using TNT-Quick Coupled Transcription/Translation System (Promega). GST fusion proteins were expressed in Echerichia coli BL21 and bound to glutathione-Sepharose-4B beads (Amersham Pharmacia). Each COS-7 cell extract (1 mg of total protein) or in vitro translation reaction (10 ␮l) was incubated for 4 hr with 1–2 ␮g of GST-fused protein bound to beads in NETN buffer (Koh et al., 2001). Bound proteins were analyzed by immunoblot with anti-HA antibody.

Total RNA was analyzed by reverse transcriptase-PCR to measure the levels of CoCoA, pS2, and ␤-actin mRNA. Results shown are representative of four independent experiments. (D) CV-1 cells were transfected with MMTV(ERE)-LUC reporter plasmid (200 ng), pHE0 (0.002 ng), pSG5.HA-GRIP1 (100 ng), pSG5.HA-CARM1 (200 ng), pCMV-p300 (200 ng), and pSG5.HA-CoCoA (200 ng), as indicated. Transfected cells were grown in medium containing E2. Luciferase activity results shown are representative of four independent experiments. (E) Model of the p160 transcription complex. The activated NR binds to the hormone response element (HRE) and recruits p160 coactivators to the promoter by directly binding to the NR interaction domain (NID) of the p160 coactivator. AD1 and AD2 in the C-terminal region of p160 coactivators transmit the transcriptional activation signals by recruiting downstream secondary coactivators p300/CBP and CARM1, respectively. AD3 activation domain in the N-terminal bHLH-PAS region of p160 coactivators also transmits the transcriptional activation signal by recruiting CoCoA. CoCoA mediates transcriptional activation through its C-terminal transcriptional activation domain by binding to currently unknown proteins or components of the transcription machinery.

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Chromatin Immunoprecipitation, Chromatin Immunoprecipitation and Reimmunoprecipitation, and Reporter Coimmunoprecipitation Rabbit antisera (Zymed) were raised against two CoCoA C-terminal peptides (amino acids 555–569 and 659–673) which have no homology to any other sequences in the databases. Each CoCoA antiserum produced a single major band at the appropriate molecular weight in immunoprecipitations or immunoblots of extracts from COS-7 cells transfected with a CoCoA expression vector; this band was missing from extracts of untransfected cells (data not shown). ChIP assays were performed largely as described previously (Ma et al., 2001, 2003). In brief, MCF-7 cells were grown in phenol red-free DMEM supplemented with 5% dextran/charcoal-stripped serum in 150 mm dishes for 3 days, and then treated with or without 100 nM E2 for 45 min. A small portion of the crosslinked, sheared chromatin solution was saved as input DNA, and the remainder was used for immunoprecipitation with 1 ␮g of anti-ER␣ antibody HC-20 (Santa Cruz Biotechnology), anti-GRIP1 antibody A300-025A (Bethyl Laboratories), anti-AIB1 antibody C-20 (Santa Cruz Biotechnology), or 10 ␮l of an equal mixture of the two rabbit antisera against CoCoA, and 30 ␮l of preblocked protein A/G plus-agarose with rotation overnight at 4⬚C. Immunoprecipitated DNAs were purified by phenolchloroform extraction, precipitated by ethanol, and resuspended in 100 ␮l of TE buffer. PCR amplifications were performed with 0.01–5 ␮l DNA using 30–35 cycles. The following primers were used: pS2 (⫺353/⫺31), 5⬘-GGCCATCTCTCACTATGAATCACTTCTGC-3⬘ (forward) and 5⬘-GGCAGGCTCTGTTTGCTTAAAGAGCG-3⬘ (reverse); ␤-actin (nucleotides 68–327 of GenBank file NM001101), 5⬘-CTCAC CATGGATGATGATATCGC-3⬘ (forward) and 5⬘-ATTTTCTCCATGTC GTCCCAGTTG-3⬘ (reverse). PCR was performed with a serial dilution of input and precipitated DNA samples to determine the linear range of the amplification, and results shown are from the linear range. PCR products were run on 1.8% agarose gels and analyzed by ethidium bromide staining. Quantitative real-time PCR reaction conditions were identical to the standard PCR reactions, except that each reaction contains 250 nM of each primer, 250 nM probe, and AmpliTaq Gold PCR Master Mix (Applied Biosystems). The following primers and probe were used: 5⬘-TGTCACGGCCAAGCCTTT-3⬘ (forward); 5⬘-CCCGCCAGG GTAAATACTGTAC-3⬘ (reverse); 5⬘-6FAM-CGGCCATCTCTCACTAT GAATCACTTCTGC-BHQ1-3⬘ (probe), which contains 6 carboxyfluorescein-aminohexylamidite (6FAM) and black hole quencher 1 (BHQ1) (Biosearch Technologies, Inc.). Real-time PCR reactions were performed using the ABI PRISM 7900HT sequence detector (Applied Biosystems) and 45 cycles of amplification. In ChIP and ReIP experiments (Shang et al., 2000; Ma et al., 2003), the initial immunoprecipitated complexes from the ChIP procedure were eluted by incubation with 10 mM DTT at 37⬚C for 30 min and diluted 1:50 in IP dilution buffer. The eluates were diluted and reimmunoprecipitated with the second antibodies. For reporter CoIP assay, COS-7 cells were transiently transfected with MMTV(ERE)-LUC reporter plasmid (2 ␮g) and pHEG0 encoding human ER␣ (1 ␮g), pSG5.HA-GRIP1 (1 ␮g), and pSG5.HA-CoCoA (1 ␮g) expression vectors. Two days after transfection, cells were treated with 100 nM E2 for 45 min. Soluble chromatin fraction was prepared and immunoprecipitated, and PCR was performed as described above. The following primers spanning the nucleosome B region of the MMTV promoter were used for PCR amplification: 5⬘ATTAGCCTTTATTTGCCCAACCTTG-3⬘ (forward) and 5⬘-CAGCACT CTTTTATATTATGGTTTAC-3⬘ (reverse).

RNA Interference and RT-PCR siRNA oligonucleotides for CoCoA were designed using the Target Finder program (Ambion), chemically synthesized by the USC Norris Comprehensive Cancer Center Microchemical Core, and annealed as follows: siCoCoA, 5⬘-CACCAAGGUGGAAUGUCACdTdT-3⬘ (sense) and 5⬘-GUGACAUUCCACCUUGGUGdTdT-3⬘ (antisense); Scramble (same nucleotide composition with scrambled sequence), 5⬘-UUC UCCGAACGUGUCACGUdTdT-3⬘ (sense) and 5⬘-ACGUGACACGUU CGGAGAAdTdT-3⬘ (antisense). For transfection of siRNA, MCF-7 or COS-7 cells were plated into 12-well plates, grown until reaching 70%–80% confluence, and transfected with 40 or 80 pmole of siRNA duplex using Targefect-siRNA transfection kit (Targeting Systems)

or Lipofectamine 2000 (Invitrogen) following the manufacturers’ instructions. Total RNA was extracted with Trizol reagent (Invitrogen). The reverse transcriptase-PCR analysis was performed with 0.1–2 ng of total RNA using the Access RT-PCR system (Promega). The primers used in reverse transcriptase-PCR reactions were as follows: CoCoA, 5⬘-CACACCAGTGTCCAGTTCCAA-3⬘ (forward) and 5⬘-CTTCGTCAGCACTTTCTCACT-3⬘ (reverse); pS2, 5⬘-CATGGAG AACAAGGTGATCTG-3⬘ (forward) and 5⬘-CTTCTGGAGGGACGTC GATGG-3⬘ (reverse); ␤-actin, 5⬘-CCTCGCCTTTGCCGATCC-3⬘ (forward) and 5⬘-GGATCTTCATGAGGTAGTCAGTC-3⬘ (reverse). Acknowledgments We thank Mr. Dan Gerke for expert technical assistance, Dr. Heng Hong for pGBT9.GRIP1(5–479), and Mr. David Y. Lee for advice on ChIP assays. Real-time PCR and confocal microscopy instrumentation and technical support were provided by the University of Southern California Research Center for Liver Diseases. This work was supported by grant DK43093 to M.R.S. from the National Institutes of Health. J.H.K. was supported by a predoctoral fellowship from the University of Southern California/Norris Breast Cancer Research Training Program. Received: April 29, 2003 Revised: October 21, 2003 Accepted: October 31, 2003 Published: December 18, 2003 References Anzick, S.L., Kononen, J., Walker, R.L., Azorsa, D.O., Tanner, M.M., Guan, X.Y., Sauter, G., Kallioniemi, O.P., Trent, J.M., and Meltzer, P.S. (1997). AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277, 965–968. Beischlag, T.V., Wang, S., Rose, D.W., Torchia, J., Reisz-Porszasz, S., Muhammad, K., Nelson, W.E., Probst, M.R., Rosenfeld, M.G., and Hankinson, O. (2002). Recruitment of the NCoA/SRC-1/p160 family of transcriptional coactivators by the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator complex. Mol. Cell. Biol. 22, 4319–4333. Belandia, B., and Parker, M.G. (2000). Functional interaction between the p160 coactivator proteins and the transcriptional enhancer factor family of transcription factors. J. Biol. Chem. 275, 30801–30805. Belandia, B., Orford, R.L., Hurst, H.C., and Parker, M.G. (2002). Targeting of SWI/SNF chromatin remodelling complexes to estrogenresponsive genes. EMBO J. 21, 4094–4103. Chen, H., Lin, R.J., Schiltz, R.L., Chakravarti, D., Nash, A., Nagy, L., Privalsky, M.L., Nakatani, Y., and Evans, R.M. (1997). Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90, 569–580. Chen, D., Ma, H., Hong, H., Koh, S.S., Huang, S.M., Schurter, B.T., Aswad, D.W., and Stallcup, M.R. (1999). Regulation of transcription by a protein methyltransferase. Science 284, 2174–2177. Chen, D., Huang, S.M., and Stallcup, M.R. (2000a). Synergistic, p160 coactivator-dependent enhancement of estrogen receptor function by CARM1 and p300. J. Biol. Chem. 275, 40810–40816. Chen, S.L., Dowhan, D.H., Hosking, B.M., and Muscat, G.E. (2000b). The steroid receptor coactivator, GRIP-1, is necessary for MEF2C-dependent gene expression and skeletal muscle differentiation. Genes Dev. 14, 1209–1228. Chun, A.C., Zhou, Y., Wong, C.M., Kung, H.F., Jeang, K.T., and Jin, D.Y. (2000). Coiled-coil motif as a structural basis for the interaction of HTLV type 1 Tax with cellular cofactors. AIDS Res. Hum. Retroviruses 16, 1689–1694. Cress, W.D., and Triezenberg, S.J. (1991). Critical structural elements of the VP16 transcriptional activation domain. Science 251, 87–90. De Valck, D., Jin, D.Y., Heyninck, K., Van de Craen, M., Contreras, R., Fiers, W., Jeang, K.T., and Beyaert, R. (1999). The zinc finger

Nuclear Receptor Coactivator CoCoA 1549

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