Med2: A ... - Cell Press

Molecular Cell, Vol. 14, 675–683, June 4, 2004, Copyright 2004 by Cell Press

Structure and Function of CRSP/Med2: A Promoter-Selective Transcriptional Coactivator Complex Dylan J. Taatjes1 and Robert Tjian* Howard Hughes Medical Institute Department of Molecular and Cell Biology 401 Barker Hall University of California, Berkeley Berkeley, California 94720

Summary The multi-subunit, human CRSP coactivator—also known as Mediator (Med)—regulates transcription by mediating signals between enhancer-bound factors (activators) and the core transcriptional machinery. Interestingly, different activators are known to bind distinct subunits within the CRSP/Med complex. We have isolated a stable, endogenous CRSP/Med complex (CRSP/Med2) that specifically lacks both the Med220 and the Med70 subunits. The three-dimensional structure of CRSP/Med2 was determined to 31 A˚ resolution using electron microscopy and single-particle reconstruction techniques. Despite lacking both Med220 and Med70, CRSP/Med2 displays potent, activator-dependent transcriptional coactivator function in response to VP16, Sp1, and Sp1/SREBP-1a in vitro using chromatin templates. However, CRSP/Med2 is unable to potentiate activated transcription from a vitamin D receptor-responsive promoter, which requires interaction with Med220 for coactivator recruitment, whereas VDR-directed activation by CRSP/Med occurs normally. Thus, it appears that CRSP/Med may be regulated by a combinatorial assembly mechanism that allows promoter-selective function upon exchange of specific coactivator targets. Introduction The human CRSP/Mediator coactivator (CRSP/Med) serves as an adaptor for directing transactions between enhancer-bound factors and the core transcriptional machinery (Malik and Roeder, 2000; Naä¨r, 2001; Rachez and Freedman, 2001). CRSP/Med is a large, multi-subunit complex that is partially conserved in eukaryotes; purification of the complex has been achieved by a number of different methods, each yielding an ⵑ1 MDa complex that potentiates activator-dependent transcription (Boyer et al., 1999; Fondell et al., 1996; Gu et al., 1999; Malik et al., 2000; Naä¨r et al., 1999; Rachez et al., 1999; Ryu et al., 1999; Sun et al., 1998). Its large size (CRSP/ Med consists of approximately 15 subunits) is thought to accommodate myriad protein-protein interactions. Indeed, the various subunits of CRSP/Med are targeted by a variety of promoter-selective transcription factors, many of which are involved in diverse signaling pathways (Taatjes et al., 2004a). Interestingly, however, these *Correspondence: [email protected] 1 Present addess: Department of Chemistry and Biochemistry, Campus Box 215, University of Colorado, Boulder, Colorado 80309.

Short Article

regulatory proteins, which are important in providing specificity in gene expression patterns, bind different subunits in the CRSP/Med complex. The implications of this are unknown. Med220 is the largest subunit in the 1.2 MDa human CRSP/Med complex and is targeted by a number of nuclear receptors (TR, VDR, RAR␣, RXR␣, PPAR␣, PPAR␥, ER, GR, HNF4) (Burakov et al., 2000; Fondell et al., 1996; Hittelman et al., 1999; Malik et al., 2002; Rachez et al., 1999; Yuan et al., 1998). This large subunit of CRSP/Med is essential for embryonic viability in mice (Ito et al., 2000; Zhu et al., 2000); MEF cells derived from Med220 (⫺/⫺) embryos revealed defects in transcription activation in response to TR (which binds Med220), but not other activators (Ito et al., 2000). Further, these Med220 (⫺/⫺) MEFs showed specific defects in PPARstimulated adipogenesis, but not MyoD-dependent myogenesis (Ge et al., 2002). Deletion of Med130, another CRSP/Med subunit, is also embryonic lethal in mice. Again, however, a cell line could be established from these knockout mice. As with Med220, the Med130 (⫺/⫺) ES cells showed impaired activation at a specific subset of promoters that required Med130 for its activator-CRSP/Med interaction (E1A and Elk-1) (Stevens et al., 2002). Similarly, in Xenopus, depletion of Med105, a CRSP/Med subunit that interacts with Smad2/3-Smad4, specifically inhibited activation of its target genes; by contrast, signaling and transcription activation by Smad1-Smad4 (which does not interact with Med105) was unaltered (Kato et al., 2002). Importantly, in each of these cases (Med220, Med130, and Med105), transcription activation remained robust at promoters responsive to activators that recruited CRSP/ Med via alternate subunits. Such studies suggest that CRSP/Med may adopt promoter-selective function by simple loss or exchange of activator-targeted subunits in the complex. However, each of these cases involved artificial ablation of the given subunit (via knockout or antisense oligonucleotide treatment). Moreover, each null mutation was lethal. Thus, despite implications that CRSP/Med could function in a promoter-selective manner, there was little evidence that such alternative CRSP/Med complexes actually existed as stable, endogenous species. Here we describe the isolation and characterization of an endogenous CRSP/Med complex, CRSP/Med2, which specifically lacks the Med220 and Med70 polypeptides. Structural features of this complex, when compared with “intact” CRSP/Med, allowed us to identify a region likely to contain the two missing subunits. Further, in vitro transcription experiments indicate that CRSP/Med2 possesses promoter-selective coactivator function that can be directly attributed to its altered subunit composition. Results Purification and Characterization of CRSP/Med2 Although CRSP/Med was originally isolated from the P1M fraction (Ryu and Tjian, 1999; Ryu et al., 1999;

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Figure 1. Purification and Subunit Composition of CRSP/Med2 (A) Purification scheme used for isolation of CRSP/Med2. (B) Silver stained, gradient (5%–15%) acrylamide gel of CRSP/Med2, purified as shown in (A). Polypeptides present in the complex are listed according to their molecular weight to the right of the silver stained bands. Where applicable, the nomenclature system proposed by Rachez and Freedman (2001) is used here and throughout the text. Additional protein bands corresponding to breakdown products of the GST-VP16 fusion protein are indicated with asterisks. (C) Western blot analysis of CRSP/Med2 against the subunits shown.

Taatjes et al., 2002), immunoblot analysis revealed that at least some CRSP/Med components were also present in the P0.5M fraction (data not shown). Therefore, we subjected this material to further purification, as outlined in Figure 1A. Following affinity purification with GSTVP16, it was evident that subunits of both CRSP/Med and the larger, related ARC-L complex were present and bound to the VP16 affinity resin (data not shown). Elution of this material followed by a glycerol gradient sedimentation step allowed isolation of a multi-subunit complex that was similar, but not identical, to CRSP/Med isolated from the P1M fraction. In particular, the Med220 and Med70 subunits appeared to be absent from the P0.5Mderived complex. To substantiate this, glycerol-gradient purified fractions were concentrated by TCA precipitation and analyzed by gradient (5%–15%) SDS-PAGE (Figure 1B). This provided a better assessment of the subunit composition and confirmed that Med220 and Med70 were indeed absent from the P0.5M-derived complex. Furthermore, subunit stoichiometry was shown to be 1:1 based upon SYPRO Ruby staining (Molecular

Probes, Inc., data not shown). Immunoblots confirmed the loss of Med220 and Med70, while detecting the presence of other CRSP/Med subunits (Figure 1C). Importantly, ARC-L specific subunits (cdk8, cyclin C, Med230, Med240) were also absent from this P0.5M “CRSP/Med-like” complex. Given that this P0.5M-derived complex resembled CRSP/Med, we will provisionally call it CRSP/Med2 to distinguish it from the highly related, previously characterized CRSP/Med coactivator complex. Since CRSP/ Med2 was isolated using a similar series of purification steps as CRSP/Med and migrated on a glycerol gradient in a similar manner, CRSP/Med2 most likely represents a stable, endogenous multi-subunit complex. Structural Analysis of CRSP/Med2 We had previously determined the 3D structure of CRSP/Med to approximately 30 A˚ resolution using electron microscopy and single particle reconstruction techniques (Taatjes et al., 2002). Now, having isolated a multi-subunit complex, CRSP/Med2, highly related to

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Figure 2. EM Analysis of CRSP/Med2 (A) Untilted electron micrograph of negatively-stained CRSP/Med2 sample at ⫻30,000 magnification shows uniform staining and a homogeneous field of particles. Scale bar, 800 A˚. (B) Angular distribution of 2826 experimental single particle images comprising the final CRSP/Med2 data set. The size of each circle correlates with the number of experimental images representing that particular set of Euler angles (larger circle, more images). (C) Plot showing the Fourier shell correlation (FSC) function of two independent CRSP/Med2 reconstructions and the 3␴ noise curve (noise). Resolution is estimated at 31 A˚ using the 0.5 correlation coefficient cutoff. (D) 3D reconstruction of CRSP/Med2 at 31 A˚ resolution. The structure is rendered to a mass of 1.0 MDa, the approximate molecular weight of the complex. Four different views of CRSP/Med2 are shown, resulting from rotations of the complex as indicated. Arrows (A)–(C) indicate prominent, solvent-exposed regions in the complex (see text). Scale bar, 75 A˚.

CRSP/Med, we endeavored to determine its 3D structure. In particular, we wanted to compare this new complex to CRSP/Med to assess not only its structural simi-

larities, but also to potentially identify the location of the missing subunits. Electron micrographs of CRSP/ Med2 revealed that the sample was homogeneous and

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slightly elongated in shape, similar to CRSP/Med. A typical micrograph is shown in Figure 2A; micrographs were obtained in both tilted and untilted orientations to allow for 3D reconstruction via the random conical tilt method (Radermacher et al., 1987). The experimental projection images provide an excellent representation of angular space, although preferred orientations are evident (Figure 2B). Resolution of the final reconstruction was 31 A˚, based upon 0.5 Fourier Shell Correlation criteria (Figure 2C) (Harauz and van Heel, 1986). Details regarding the single particle reconstruction of CRSP/Med2 are provided in the Experimental Procedures section. The 3D structure of CRSP/Med2, shown in Figure 2D, has been rendered to a mass of 1.0 MDa, corresponding to the approximate mass estimated for a CRSP/Med complex lacking Med220 and Med70. The refined structure is shown from four viewing angles in Figure 2D. CRSP/Med2 is somewhat elongated (approximate dimensions 315 ⫻ 155 ⫻ 155 A˚) and resembles previously characterized CRSP/Med complexes in terms of structural organization (defined head, body, and leg regions) (Naä¨r et al., 2002; Taatjes et al., 2002). Its large size, extended structure, and somewhat “open” conformation (note channels [arrow a and b] and hollow region [arrow c] in Figure 2D) maximize the surface area of CRSP/Med2, potentially facilitating diverse protein-protein interactions. Structural Comparison of CRSP/Med2 and CRSP/Med Given its high degree of subunit similarity with CRSP/ Med, we expected the structure of CRSP/Med2 to closely resemble CRSP/Med. However, CRSP/Med can adopt multiple distinct conformations dependent upon the binding of specific ligands, such as the activation domains of transcription factors (Taatjes et al., 2002). Thus, it was important to compare CRSP/Med and CRSP/Med2 structures that were bound to the same activator. Figure 3 shows a comparison of CRSP/Med2 and CRSP/Med structures, both bound to VP16, from two different viewing angles. As expected, the two distinct VP16-bound structures represent the same conformational state, based upon both visual comparison and cross-correlation of the structures (see Experimental Procedures). Only a slight conformational distortion is evident at the “top” of the CRSP/Med2 complex (denoted by the red “X” in Figure 3), which may be predicated by the loss of Med220 and Med70. CRSP/Med2 is considerably shorter than CRSP/Med (315 A˚ versus 360 A˚), likely a manifestation of the missing subunits in CRSP/Med2. Further, a clear region of protein density is absent from the central “lower” portion of the CRSP/ Med2 complex (arrows in Figure 3). We speculate that this missing density (which remains even if CRSP/Med2 is rendered to a mass of 1.2 MDa) corresponds to the Med220 site, in part because it is the only region in CRSP/Med2 that is clearly missing protein mass (see Discussion). CRSP/Med2 Potentiates ActivatorDependent Transcription The CRSP/Med complex, which contains Med220 and Med70, was previously shown to possess potent coacti-

Figure 3. Comparison of VP16-CRSP/Med2 and VP16-CRSP/Med Structures Side-by-side comparison of CRSP/Med2 and CRSP/Med reveals clear similarities in conformation. Structures are shown from two viewing angles, rotated as shown. Also evident is a defined region of missing protein density in the CRSP/Med2 complex, indicated by the arrows, which represents the location of Med220 (see text). The red “X” denotes an isolated region where protein density has apparently shifted in CRSP/Med2. Scale bar, 75 A˚.

vator function on chromatin templates in vitro (Naä¨r et al., 2002; Taatjes et al., 2002). By contrast, the larger, highly related ARC-L complex was shown to be inactive (Taatjes et al., 2002). While ARC-L specifically contains four subunits not present in CRSP/Med (cdk8, cyclin C, Med230, Med240), it also lacks Med70, which has thus far been found to be specific for the transcriptionally active CRSP/Med complex. Consequently, Med70 may be critical for CRSP/Med coactivator function. Given that CRSP/Med2 lacks Med70, we wanted to determine whether it could still potentiate transcription in an activator-dependent manner. To test this, we performed in vitro transcription assays and measured activity as a function of CRSP/Med or CRSP/Med2. Transcription activation was examined in response to a number of activators, including VP16 (Figure 4A), SREBP with Sp1 (Figure 4B), and Sp1 alone (not shown). In each case, CRSP/Med2 activated transcription to a level similar to that of CRSP/Med, suggesting that Med70 may not be required for CRSP/Med transactivation in vitro, at least at the promoters tested (but see below).


Figure 4. CRSP/Med2 Is a Promoter-Selective Coactivator In vitro transcription on chromatin–assembled templates. Purified and recombinant basal transcription factors (TFIIA, -IIB, -IID, -IIE, -IIF, -IIH, and RNA polymerase II) were added to assembled chromatin templates for each transcription reaction shown (see Experimental Procedures). Activators (VP16, SREBP-1a and Sp1, or VDR/RXR) and coactivators (CRSP/Med or CRSP/Med2) were added as indicated. (A and B) Both CRSP/Med and CRSP/Med2 activate transcription in response to VP16 (A) or SREBP/Sp1 (B) on chromatin templates. (C) CRSP/Med, but not CRSP/Med2, potentiates activator-dependent transcription in response to VDR/RXR. VDR ligand (1,25-dihydroxyvitamin D3) was added as shown.

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CRSP/Med2 Is a Promoter-Selective Coactivator The Med220 subunit is targeted by a variety of regulatory proteins, including many nuclear receptors (Burakov et al., 2000; Fondell et al., 1996; Hittelman et al., 1999; Malik et al., 2002; Rachez et al., 1999; Yuan et al., 1998). Consequently, loss of this subunit could result in loss of activation at some promoters (e.g., those responsive to nuclear receptors) but not others. To address this question directly, we tested the activity of CRSP/Med2 using a promoter template responsive to VDR, which has been well documented to interact with the Med220 subunit in a ligand-dependent manner (Rachez et al., 1999, 2000; Ren et al., 2000). In contrast to the in vitro transcription experiments comparing CRSP/Med and CRSP/Med2 described above, only CRSP/Med, but not CRSP/Med2, was able to mediate transcriptional activation at the VDR-responsive promoter (Figure 4C). Furthermore, CRSP/Med2, when titrated into the transcription reaction, did not affect CRSP/Med activation from the VDR template (data not shown). These results suggest that CRSP/Med2 is unable to serve as a coactivator at a subset of promoters—minimally, those requiring activator interaction with the Med220 subunit. Because CRSP/Med2 lacks both Med220 and Med70, it is possible that both subunits are required for the coactivator function of CRSP/Med at VDR-dependent promoters. And since CRSP/Med2 was isolated as an endogenous complex from human cells, this suggests that CRSP/ Med may be regulated in part by its subunit composition. Such a simple, yet elegant regulatory mechanism may allow a more dynamic transcriptional response by the CRSP/Med coactivator. Discussion To date, different transcriptional activators have been shown to bind at least 7 distinct subunits in the CRSP/ Med complex (Asada et al., 2002; Burakov et al., 2000; Fondell et al., 1996; Hittelman et al., 1999; Ito et al., 1999; Kato et al., 2002; Malik et al., 2002; Mittler et al., 2003; Rachez et al., 1999; Stevens et al., 2002; Yang et al., 2004; Yuan et al., 1998). Despite the apparent selectivity of activator targets, the identity of the CRSP/Med subunit and its specific activator interface may not be critical for transcriptional activation. Indeed, the activator may simply provide a means to recruit and tether CRSP/Med to the promoter. However, recent structural analysis has revealed that distinct activators that target different subunits in CRSP/Med can actually induce significant structural changes in the CRSP/Med complex upon binding (Taatjes et al., 2002). Thus, these activator-induced conformational shifts may help provide specificity in transcriptional response. Another potential regulatory implication of these different activator targets may be to allow promoter-selective recruitment and function of the coactivator. Specifically, subunit exchange within the CRSP/Med complex could generate derivatives with unique functional specificity. This appears to be true in the case of the CRSP/Med2 complex described here, where subunit loss imparts promoter-selective transcriptional activation in vitro. The structural comparison of CRSP/Med2 and CRSP/ Med indicates that VP16 binding can still induce the

same conformational state despite the absence of the Med220 and Med70 subunits. Structural comparison of CRSP/Med and CRSP/Med2 also identifies one prominent region in the head/body domain that is the likely location of Med220 in the CRSP/Med complex. We speculate this region corresponds to the Med220 site because the missing protein density in this domain does not result from the “shortening” of the complex relative to CRSP/Med. More importantly, a similar region was identified from difference maps comparing antibodylabeled CRSP/Med complexes bound to nuclear receptors, which are known to bind Med220 (Taatjes et al., 2004b). However, localization of Med220 to this region does not exclude the possibility that the subunit may also extend to other regions (e.g., the head domain) of the complex. Indeed, with a mass of 220 kDa, Med220 represents about 20% of the total protein density of the 1.2 MDa CRSP/Med complex. Since both Med220 and Med70 are absent from CRSP/Med2, these two polypeptides may be associated with each other in the CRSP/ Med complex. If so, Med70 would also reside in the same region as Med220; however, we cannot rule out the location of Med70 in the leg or head regions of the CRSP/Med complex. The Med70 subunit is particularly intriguing because it is the only known CRSP/Med subunit that is not present in the larger, transcriptionally inactive ARC-L complex (Taatjes et al., 2002). Thus, it was possible that Med70 might play a key role in potentiating transcription activation by the CRSP/Med coactivator. In fact, recent experiments involving Ras-inducible C/EBP␤ show that in its repressive state, C/EBP␤ associates with Med complexes containing cdk8 but lacking Med70 (similar to ARC-L), whereas upon activation, C/EBP␤ associates with Med complexes that contain Med70 but lack cdk8 (similar to CRSP/Med) (Mo et al., 2004). To our surprise, however, CRSP/Med2, which lacks Med70, was fully capable of potentiating activator-dependent transcription at a variety of promoter templates in vitro. Thus, Med70 may instead be required for activation only at a subset of promoters, and since Med70 is present only in metazoans, its regulatory functions may be specific for multi-cellular organisms. Although the in vitro transcriptional activity of CRSP/ Med and CRSP/Med2 were indistinguishable at several of the promoters tested, significant differences in coactivator function were observed when we assayed promoters that were under the control of nuclear receptors, such as the VDR/RXR heterodimers. It has been well documented that VDR requires Med220 to bind the CRSP/Med coactivator (Rachez et al., 1999, 2000; Ren et al., 2000); thus, activation was most likely inhibited in the presence of CRSP/Med2 (but not CRSP/Med) because CRSP/Med2 could not be recruited to the promoter by VDR, even in the presence of ligand. Indeed, CRSP/Med2 also cannot interact with TR (D.T. and R.T., unpublished data), which binds Med220 in a manner similar to VDR (Ren et al., 2000). The VDR/RXR heterodimer (and other NR/RXR heterodimers, including TR/ RXR) interacts specifically with two closely spaced LXXLL motifs within the Med220 polypeptide (Yuan et al., 1998), and the spacing of these motifs in Med220 appears to be critical for VDR/RXR recognition (Ren et al., 2000). Incidentally, LXXLL motifs are also present in


other CRSP/Med subunits, including Med150 and Med100; however, VDR evidently does not interact with these other CRSP/Med subunits, suggesting that this motif per se is not sufficient for NR recognition (Rachez et al., 1999; Yuan et al., 1998). Indeed, sequences flanking the LXXLL motif can strongly influence NR binding (Coulthard et al., 2003; Darimont et al., 1998; McInerney et al., 1998; Ren et al., 2000; Rochel et al., 2000; Warnmark et al., 2001). Since CRSP/Med2 was not completely inactive on VDRE templates, however, its residual activity might reflect a very weak VDR/RXR-CRSP/Med2 interaction. Alternately, this weak activity may result from a very small amount of CRSP/Med present in our CRSP/ Med2 samples. The large size of the CRSP/Med complex, coupled with the fact that it possesses multiple activator targets (Med220, Med150, Med130, Med105, Med100, Med97, Med78) may allow simultaneous, even cooperative, binding of several distinct activators. This tandem binding of the CRSP/Med (or CRSP/Med2) complex may aid in its recruitment and stability at select promoters, and may provide a means for synergistic activation at certain genes. Given the diversity of enhancer and promoter regions in humans (Levine and Tjian, 2003), with multiple binding sites for diverse sequence-specific regulatory proteins, it is possible that CRSP/Med2, which evidently cannot interact with nuclear receptors (at least via Med220), may promote synergistic activation mediated by certain regulatory proteins (e.g., SREBP-1a, Sp1) while “locking out” signals from others (e.g., nuclear receptors). Such an activation mechanism would combine regulatory elements inherent in both core promoter and enhancer structures as well as those intrinsic to the CRSP/Med coactivator. Such finely tuned regulatory mechanisms may be essential to establish the dynamic gene expression networks required by metazoan organisms. Experimental Procedures Purification of CRSP/Med2 HeLa nuclear extract was loaded onto a phosphocellulose column at 0.1M KCl HEMG (20 mM HEPES, 10 mM EDTA, 2 mM MgCl2, and 10% glycerol). Protein fractions were eluted at 0.1 M, 0.3 M, 0.5 M, and 1.0 M KCl HEMG. The protein fraction eluted at 0.5 M KCl (P0.5M fraction) was dialyzed against 0.1 M KCl HEMG and applied to a GST-VP16 affinity column. The P0.5M fraction was incubated over the immobilized affinity resin for 3h at 4⬚C, at which time the resin was washed 5 times with 50 column volumes of 0.5M KCl HEGN (20 mM HEPES, 10 mM EDTA, 10% glycerol, and 0.1% NP-40), and then once with 0.15 M KCl HEGN (0.02% NP-40). Bound material was eluted with 30 mM glutathione (in Tris buffer [pH 7.9]) and applied to a 2 ml glycerol gradient (15%–40% glycerol in 0.15 M KCl HEG). Some CRSP/Med2 preparations were incubated over an anti-Med220 resin for 1 hr at 4⬚C prior to running the glycerol gradient. The glycerol gradient was run at 55K RPM for 6 hr at 4⬚C. Fractions were collected in 100 ␮l aliquots and analyzed for the presence of CRSP/Med2 by SDS-PAGE. Electron Microscopy Micrographs were obtained with a Tecnai 12 (FEI Company, the Netherlands) TEM at 30,000⫻ magnification. CRSP/Med2 samples were dialyzed versus a 5% trehalose solution (20 mM HEPES, 0.1 mM EDTA, and 0.1 M KCl) and applied to a glow-discharged, carboncoated EM grid and stained with 4% uranyl acetate in water. Micrographs were obtained in both untilted (0⬚) and tilted (30–45⬚) orientations to allow single particle reconstruction using random conical

tilt. Micrographs were developed on Kodak SO163 film and digitized with a scan step of 13.3 ␮m, which corresponds to a 4.4 A˚ pixel size. Single Particle Reconstruction of CRSP/Med2 A total of 36 tilt-pair micrographs (72 total) were used for image processing, yielding 1588 single-particle tilt-pair images (3176 total). Image processing was completed using the SPIDER and WEB software package (Frank et al., 1996). Untilted images (1588) were subjected to reference-free alignment and merged into 20 distinct classes defined by K-means clustering (Frank, 1990). Three-dimensional structures for each class were then calculated by back-projection using the corresponding tilted images. The 3D structures were then cross-correlated against each other to establish a homogeneous data set. Of the 20 classes, 17 showed excellent crosscorrelation (R ⬎ 0.83) and were merged into a single data set (1413 images, representing 89% of the data set). These tilted, single particle images were used to generate an initial 3D structure to be used for angular refinement (Penczek et al., 1994). Angular refinement commenced with the generation of 83 noise-free reference projections (15⬚ angular step) generated from the initial volume. Both tilted and untilted images were used for this analysis, providing a data set of 2826 images; based upon highest cross-correlation, these images were matched to a given reference projection following inplane shifts and rotations. This provided a new set of Euler angles, which were implemented in the calculation of a refined 3D volume. This procedure was repeated until the Euler angles did not change upon subsequent refinement steps (4 additional cycles). Final angular refinement utilized 798 reference projections, corresponding to an angular step of 5 degrees. The final structure was rendered to 1.0 MDa, the predicted molecular mass of the CRSP/Med2 complex. Resolution was assessed by Fourier shell correlation (Harauz and van Heel, 1986). Based upon quantitation of CRSP/Med and CRSP/Med2 polypeptide bands using Coomassie and/or SYPRO Ruby (Molecular Probes, Inc.), the stoichiometry of each large subunit is 1:1, confirming our estimation of the molecular mass of each complex. Cross-Correlation Analysis Structures of VP16-CRSP/Med2 and VP16-CRSP/Med were translationally and rotationally aligned based upon highest cross-correlation coefficient. The best alignment between the structures (shown in Figure 3) resulted in a cross-correlation coefficient of 0.85. As a reference for conformational similarity, classes within the CRSP/ Med2 data set have an average cross-correlation value of 0.86. The cross-correlation value between CRSP/Med2 and CTD-CRSP/Med (which also resembles the structure of VP16-CRSP/Med) is 0.84. Significantly, cross-correlation coefficients between CRSP/Med2 and CRSP/Med conformers structurally distinct from VP16-CRSP/ Med—SREBP and “unliganded” structures—were much lower (0.78 for each), indicative of their different conformations (Naä¨r et al., 2002; Taatjes et al., 2002). In Vitro Transcription Supercoiled template plasmids (gal4, Sp1/SREBP, Sp1, or VDRE) were assembled into chromatin using Drosophila embryo cytosolic extract (S-190), purified Drosophila core histones, and an ATPregenerating system as described (Naä¨r et al., 1998). Chromatin assembly was performed at 27⬚C for 5 hr, followed by the addition of activators (0-5 nM Sp1 and SREBP, or gal4-VP16; 0-20 nM VDR/ RXR and ligand). After 45 min incubation at 27⬚C, purified and recombinant general transcription factors (TFIIA (40 nM), TFIIB (5 nM), TFIID (0.4 nM), TFIIE (20 nM), TFIIF (20 nM), TFIIH (0.2 nM), and 2 nM RNA polymerase II) were added, with and without non-limiting amounts of CRSP/Med or CRSP/Med2 (1-5 nM). Fifteen minutes later, rNTPs (0.5 nM final concentration) were added and transcription was allowed to proceed for 30 min, at which point transcription was terminated by addition of 3 volumes “stop” solution (20 mM EDTA, 0.2 M NaCl, 1% SDS, with 0.13 mg/mL glycogen and proteinase K). RNA was then isolated and analyzed by primer extension. Acknowledgments We thank M. Marr, K. Wright, and E. Nogales for helpful comments on the manuscript. We also thank C. Inouye for providing purified

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GTFs used for the in vitro transcription system and E. Nogales for the use of her electron microscope. This work was funded by grants from the NIH and Howard Hughes Medical Institute. Received: February 6, 2004 Revised: May 6, 2004 Accepted: May 19, 2004 Published: June 3, 2004 References Asada, S., Choi, Y., Yamada, M., Wang, S.C., Hung, M.C., Qin, J., and Uesugi, M. (2002). External control of Her2 expression and cancer cell growth by targeting a Ras-linked coactivator. Proc. Natl. Acad. Sci. USA 99, 12747–12752. Boyer, T.G., Martin, M.E.D., Lees, E., Riccardi, R.P., and Berk, A.J. (1999). Mammalian Srb/Mediator complex is targeted by adenovirus E1a protein. Nature 399, 276–279. Burakov, D., Wong, C.W., Rachez, C., Cheskis, B.J., and Freedman, L.P. (2000). Functional interactions between the estrogen receptor and DRIP205, a subunit of the heteromeric DRIP coactivator complex. J. Biol. Chem. 275, 20928–20934. Coulthard, V.H., Matsuda, S., and Heery, D.M. (2003). An extended LXXLL motif sequence determines the nuclear receptor binding specificity of TRAP220. J. Biol. Chem. 278, 10942–10951. Darimont, B.D., Wagner, R.L., Apriletti, J.W., Stallcup, M.R., Kushner, P.J., Baxter, J.D., Fletterick, R.J., and Yamamoto, K.R. (1998). Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev. 12, 3343–3356. Fondell, J.D., Ge, H., and Roeder, R.G. (1996). Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. Proc. Natl. Acad. Sci. USA 93, 8329–8333. Frank, J. (1990). Classification of macromolecular assemblies studied as “single particles.” Q. Rev. Biophys. 23, 281–329. Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y.H., Ladjadj, M., and Leith, A. (1996). SPIDER and WEB: Processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199. Ge, K., Guermah, M., Yuan, C.X., Ito, M., Wallberg, A.E., Spiegelman, B.M., and Roeder, R.G. (2002). Transcription coactivator TRAP220 is required for PPAR gamma 2-stimulated adipogenesis. Nature 417, 563–567. Gu, W., Malik, S., Ito, M., Yuan, C.X., Fondell, J.D., Zhang, X., Martinez, E., Qin, J., and Roeder, R.G. (1999). A novel human SRB/MEDcontaining cofactor complex, SMCC, involved in transcription regulation. Mol. Cell 3, 97–108. Harauz, G., and van Heel, M. (1986). Exact filters for general geometry three dimensional reconstruction. Optik 73, 146–153. Hittelman, A.B., Burakov, D., Iniguez-Lluhi, J.A., Freedman, L.P., and Garabedian, M.J. (1999). Differential regulation of glucocorticoid receptor transcriptional activation via AF-1 associated proteins. EMBO J. 18, 5380–5388. Ito, M., Yuan, C., Malik, S., Gu, W., Fondell, J.D., Yamamura, S., Fu, Z., Zhang, X., Qin, J., and Roeder, R.G. (1999). Identity between TRAP and SMCC complexes indicates novel pathways for the function of nuclear receptors and diverse mammalian activators. Mol. Cell 3, 361–370. Ito, M., Yuan, C.X., Okano, H.J., Darnell, R.B., and Roeder, R.G. (2000). Involvement of the TRAP220 component of the TRAP/SMCC coactivator complex in embryonic development and thyroid hormone action. Mol. Cell 5, 683–693.

Malik, S., Gu, W., Wu, W., Qin, J., and Roeder, R.G. (2000). The USAderived transcriptional coactivator PC2 is a submodule of TRAP/ SMCC and acts synergistically with other PCs. Mol. Cell 5, 753–760. Malik, S., Wallberg, A.E., Kang, Y.K., and Roeder, R.G. (2002). TRAP/ SMCC/mediator-dependent transcriptional activation from DNA and chromatin templates by orphan nuclear receptor hepatocyte nuclear factor 4. Mol. Cell. Biol. 22, 5626–5637. McInerney, E.M., Rose, D.W., Flynn, S.E., Westin, S., Mullen, T.M., Krones, A., Inostroza, J., Torchia, J., Nolte, R.T., Assa-Munt, N., et al. (1998). Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. Genes Dev. 12, 3357– 3368. Mittler, G., Stu¨hler, T., Santolin, L., Uhlmann, T., Kremmer, E., Lottspeich, F., Berti, L., and Meisterernst, M. (2003). A novel docking site on Mediator is critical for activation by VP16 in mammalian cells. EMBO J. 22, 6494–6504. Mo, X., Kowenz-Leutz, E., Xu, H., and Leutz, A. (2004). Ras induces mediator complex exchange on C/EBP␤. Mol. Cell 13, 241–250. Naä¨r, A.M., Beaurang, P.A., Robinson, K.M., Oliner, J.D., Avizonis, D., Scheek, S., Zwicker, J., Kadonaga, J.T., and Tjian, R. (1998). Chromatin, TAFs, and a novel multiprotein coactivator are required for synergistic activation by Sp1 and SREBP-1a in vitro. Genes Dev. 12, 3020–3031. Naä¨r, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W., and Tjian, R. (1999). Composite co-activator ARC mediates chromatindirected transcriptional activation. Nature 398, 828–832. Naä¨r, A.M., Lemon, B.D., and Tjian, R. (2001). Transcriptional coactivator complexes. Annu. Rev. Biochem. 70, 475–501. Naä¨r, A.M., Taatjes, D.J., Zhai, W., Nogales, E., and Tjian, R. (2002). Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation. Genes Dev. 16, 1339–1344. Penczek, P.A., Grassucci, R.A., and Frank, J. (1994). The ribosome at improved resolution: new techniques for merging and orientation refinement in 3D cryo-electron microscopy of biological particles. Ultramicroscopy 53, 251–270. Rachez, C., and Freedman, L.P. (2001). Mediator Complexes and Transcription. Curr. Opin. Cell Biol. 13, 274–280. Rachez, C., Lemon, B.D., Suldan, Z., Bromleigh, V., Gamble, M., Naar, A.M., Erdjument-Bromage, H., Tempst, P., and Freedman, L.P. (1999). Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 398, 824–828. Rachez, C., Gamble, M., Chang, C.B., Atkins, G.B., Lazar, M.A., and Freedman, L.P. (2000). The DRIP complex and SRC-1/p160 coactivators share similar nuclear receptor binding determinants but constitute functionally distinct complexes. Mol. Cell. Biol. 20, 2718–2726. Radermacher, M., Wagenknecht, T., Verschoor, A., and Frank, J. (1987). Three-dimensional reconstruction from a single-exposure random conical tilt series applied to the 50s ribosomal subunit of Eschericia coli. J. Microsc. 146, 113–136. Ren, Y., Behre, E., Ren, Z., Zhang, J., Wang, Q., and Fondell, J.D. (2000). Specific structural motifs determine TRAP220 interactions with nuclear hormone receptors. Mol. Cell. Biol. 20, 5433–5446. Rochel, N., Wurtz, J.M., Mitschler, A., Klaholz, B., and Moras, D. (2000). The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol. Cell 5, 173–179. Ryu, S., and Tjian, R. (1999). Purification of transcription cofactor complex CRSP. Proc. Natl. Acad. Sci. USA 96, 7137–7142. Ryu, S., Zhou, S., Ladurner, A.G., and Tjian, R. (1999). The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp1. Nature 397, 446–450.

Kato, Y., Habas, R., Katsuyama, Y., Naar, A., and He, X. (2002). A component of the ARC/Mediator complex required for TGF beta/ Nodal signalling. Nature 418, 641–646.

Stevens, J.L., Cantin, G.T., Wang, G., Shevchenko, A., Shevchenko, A., and Berk, A.J. (2002). Transcription control by E1A and MAP kinase pathway via Sur2 mediator subunit. Science 296, 755–758.

Levine, M., and Tjian, R. (2003). Transcription regulation and animal diversity. Nature 424, 147–151.

Sun, X., Zhang, Y., Cho, H., Rickert, P., Lees, E., Lane, W., and Reinberg, D. (1998). NAT, a human complex containing Srb polypeptides that functions as a negative regulator of activated transcription. Mol. Cell 2, 213–222.

Malik, S., and Roeder, R.G. (2000). Transcriptional regulation through Mediator-like coactivators in yeast and metazoan cells. Trends Biochem. Sci. 25, 277–283.

Taatjes, D.J., Naä¨r, A.M., Andel, F., Nogales, E., and Tjian, R. (2002).


Structure, function, and activator-induced conformations of the CRSP coactivator. Science 295, 1058–1062. Taatjes, D.J., Marr, M.T., and Tjian, R. (2004a). Regulatory diversity among metazoan co-activator complexes. Nat. Rev. Mol. Cell Biol. 5, 403–410. Taatjes, D.J., Schneider-Poetsch, T., and Tjian, R. (2004b). Distinct conformational states of nuclear receptor-bound CRSP-Med complexes. Nat. Struct. Mol. Biol., in press. Warnmark, A., Almlof, T., Leers, J., Gustafsson, J.A., and Treuter, E. (2001). Differential recruitment of the mammalian mediator subunit TRAP220 by estrogen receptors ERalpha and ERbeta. J. Biol. Chem. 276, 23397–23404. Yang, F., DeBeaumont, R., Zhou, S., and Naä¨r, A.M. (2004). The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator. Proc. Natl. Acad. Sci. USA 101, 2339–2344. Yuan, C., Ito, M., Fondell, J.D., Fu, Z., and Roeder, R.G. (1998). The TRAP220 component of a thyroid hormone receptor-associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion. Proc. Natl. Acad. Sci. USA 95, 7939–7944. Zhu, Y., Qi, C., Jia, Y., Nye, J.S., Rao, M.S., and Reddy, J.K. (2000). Deletion of PBP/PPARBP, the gene for nuclear receptor coactivator peroxisome proliferator-activated receptor-binding protein, results in embryonic lethality. J. Biol. Chem. 275, 14779–14782.