Mechanisms of gene regulation by vitamin D3 receptor: a network of coactivator interactions

Gene 246 (2000) 9–21 www.elsevier.com/locate/gene

Review

Mechanisms of gene regulation by vitamin D receptor: 3 a network of coactivator interactions Christophe Rachez, Leonard P. Freedman * Cell Biology Program Memorial Sloan-Kettering Cancer Center 1275 York Avenue, New York, NY 10021, USA Received 12 November 1999; accepted 26 January 2000 Received by A.J. van Wijnen

Abstract The vitamin D receptor regulates transcription in direct response to its cognate hormonal ligand, 1,25(OH ) D . Ligand 3 2 3 binding leads to the recruitment of coactivators. Many of these factors, acting in large complexes, have emerged as chromatin remodelers partly through intrinsic histone modifying activities. In addition, other ligand-recruited complexes appear to act more directly on the transcriptional appartus, suggesting that transcriptional regulation by VDR and other nuclear receptors may involve a process of both chromatin alterations and direct recruitment of key initiation components at regulated promoters. © 2000 Elsevier Science B.V. All rights reserved. Keywords: CBP/p300; DRIP; 1,25(OH ) D ; SRC-1; Transcription 2 3

1. Introduction Vitamin D , via its active metabolite 1a,253 dihydroxyvitamin D [1,25(OH ) D ], is a major compo3 2 3 nent in the regulation of calcium and phosphorus metabolism. It plays a key role in the adsorption of these essential minerals in the intestine, and in their mobilization in bone tissues. Besides these ‘classical’ effects of 1,25(OH ) D , the hormone also regulates growth inhibi2 3 tion and differentiation of a number of cell types, including hematopoietic cells and keratinocytes, and also has immunosuppressive effects on activated B-and T-lymphocytes. Most of the known biological effects of 1,25(OH ) D occur through the direct transcriptional 2 3 regulation of specific target genes. These regulatory effects of 1,25(OH ) D are mediated through the 2 3 ligand’s specific and high affinity binding to a nuclear receptor, the vitamin D receptor ( VDR) (Baker et al., 1988). In this review, we will focus on the structure and functional properties of VDR as they relate to its role as a transcriptional activator, and describe the recently * Corresponding author. Tel.: +1-212-639-2976; fax: +1-212-717-3298. E-mail address: [email protected] (L.P. Freedman)

characterized coactivators and coactivator complexes involved in mediating its transcriptional effects.

2. Target gene regulation by vitamin D

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VDR shares a common organization in functional domains and extensive homologies in structure with other members of the superfamily of nuclear receptors. In addition to vitamin D , this family of transcription 3 factors includes receptors for retinoids, thyroid and steroid hormones, fatty acids, oxysterols, and farnesoids, together with orphan receptors (Mangelsdorf et al., 1995; Freedman, 1998). 2.1. VDR-regulated promoters and vitamin D response 3 elements VDR regulates gene expression by binding to specific DNA response elements in the promoter region of target genes. Together with the receptors for thyroid hormone ( TR) and all-trans retinoic acid (RAR), VDR forms a subfamily of nuclear receptors that bind DNA predominantly as heterodimers with a common partner, the retinoid X receptor, RXR (Glass, 1994), on response

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elements typically composed of two hexameric half-sites organized as direct repeats (DR). The binding specificity is provided by the spacing between each half-site ( Umesono and Evans, 1989). Following this rule, the consensus vitamin D response element ( VDRE ) has a half-site spacing of three nucleotides (DR3; Umesono et al., 1991; Towers et al., 1993). However, some response elements for VDR and other receptors do not follow this rule, and VDR can also regulate gene expression by binding in different configurations on palindromic or composite vitamin D response elements 3 (Candeliere et al., 1996; Koszewski et al., 1996). A handful of key target genes identified for VDR transcription activation have been known for several years. They include genes involved in the regulation of calcium metabolism and bone formation (for a review, see DeLuca, 1992). More recently, genes involved in the control of cell growth and differentiation by vitamin D have been identified in the monomyelocytic cell line 3 U937 using a modified differential screen (Liu et al., 1996). These target genes include p21Waf1/Cip1, the cell surface differentiation marker CD14, transcription factors like HoxA-10 and Mad1, as well as Ki antigen and several unknown genes. These genes are induced by vitamin D to different extents and reinforce a central 3 role for vitamin D in the myeloid differentiation process 3 (Rots et al., 1998). Several genes have also been identified as targets of transrepression by various steroid and nuclear receptors (reviewed in Herrlich and Gottlicher, 1998). Genes that are downregulated in response to 1,25(OH ) D include 2 3 those encoding human and chick parathyroid hormone (PTH ) rat a1(I ) collagen, human atrial natriuretic peptide (ANP), interleukin-2 and -12 (IL-2 and IL-12), interferon-c (IFNc), GMCSF, and the rat bone sialoprotein (BSP) (reviewed in Christakos et al., 1996; Cippitelli and Santoni, 1998; D’Ambrosio et al., 1998). For the chick PTH and BSP genes, imperfect DR3 elements have been identified at promoter proximal sites through which 1,25(OH ) D -mediated repression 2 3 occurs. However, the promoters for the human PTH, human ANP, and human IL-2 genes do not appear to contain canonical DR3-type response elements. Nevertheless, direct VDR binding has been demonstrated within these promoters. In the IL-2 and GMCSF promoters, the minimal sequence required to confer repression consists of a composite sequence known to bind the T-cell transcription factor NFAT-1 and JunFos (Alroy et al., 1995; Towers et al., 1999). Included in this sequence is an extended imperfect VDRE halfsite. VDR DNA binding was shown to be necessary, but not sufficient, to confer repression. These results provide a molecular explanation for how 1,25(OH ) D 2 3 acts as a modest immunosuppressing agent in T-lymphocytes (Rigby, 1988; Lemire, 1995). Interestingly, these phenomena imply that vitamin D 3

as an immunosuppressant might be effective in combination with multiple signalling pathways, such as NF-AT and AP-1. In the context of a whole mouse, however, genetic disruption of VDR has major effects on growth, bone formation and female reproduction, but the immune system of these mice does not seem to be impaired ( Yoshizawa et al., 1997). 2.2. Structure and function of VDR The three-dimensional structure of VDR has not yet been solved1, but it can be described by comparison with known structures of several nuclear receptors, on the basis of its extensive sequence homology with several other members of the nuclear receptor superfamily. The DNA- and ligand-binding domains (DBD and LBD, respectively) are two separable globular domains whose structures have been described in different configurations. The DBD structures of the glucocorticoid receptor (GR) and the estrogen receptor ( ER) were the first solved on DNA by crystallography (Luisi et al., 1991; Schwabe et al., 1993). Later, the structure of TR-DBD was solved in its prototypic heterodimer configuration with RXR on a DR-4 DNA element (Rastinejad et al., 1995). This structure provides a visualization of the determinants of the spacing rule for nuclear receptor binding on DR-type response elements ( Kurokawa et al., 1993; Perlmann et al., 1993). It was also the basis for modeling the DNA-binding configuration of VDRDBD as a heterodimer with RXR-DBD on its response element VDRE (DR3) (Rastinejad et al., 1995), confirming earlier predictions that VDR bound a DR3 as an asymmetric, head-to-tail dimer ( Towers et al., 1993). The LBD contributes to ligand binding, dimerization, and transcriptional activity. Functional studies have revealed that the ligand-dependent transcription activity of nuclear receptors is supported by a single short amino acid sequence called AF-2 (activation function-2), located at the extreme C-terminus of the receptor (Danielian et al., 1992; Barettino et al., 1994; Durand et al., 1994). Glutamic acid and leucine residues in this segment are highly conserved among all members of the nuclear receptor family, and point mutations of residues within the AF-2 core motif impair transcription of nuclear receptors, including VDR (Durand et al., 1994). The LBD structures of several nuclear receptors have been solved and provide insights into how the different functions of ligand-binding, dimerization and transactivation are supported by a single domain. The structures of RXR and RAR LBDs are globular domains comprising 12 a-helices (Bourguet et al., 1995; Renaud et al., 1995). Comparisons between the RXR-LBD without 1 Since acceptance of this review, the crystal structure of VDR ligand binding domain bound to its ligand has been published by the group of D. Moras (N. Rochel et al. (2000) Mol. Cell 5: 173–179).

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ligand (apo-RXR) and the RAR-LBD bound by its specific ligand, all-trans retinoic acid, (holo-RAR) reveal a key role of the ligand in mediating transcriptional activity. These structures show that the LBD undergoes a major conformational change upon ligand binding that modifies the orientation of the AF-2 core motif (within helix 12) on the surface of the LBD, thereby creating a change in solvent-exposed amino acids. This ligand-induced conformational change presumably results in the formation of a new binding interface that allows protein–protein contacts between the AF-2 and additional factors central to transactivation. The LBD structures of several other nuclear receptors (including TR, PR, ER, PPARc; Wagner et al., 1995; Brzozowski et al., 1997; Nolte et al., 1998; Williams and Sigler, 1998) confirm the structural homology between many, if not all, members of the family. The analysis of the ER-LBD structure demonstrates the effect of ligand on the allosteric positioning of the AF-2 helix in considerable detail. An antagonist ER ligand induces a positioning of the AF-2 in a conformation that does not create the same binding interface as an agonistbound ER (Brzozowski et al., 1997; Pike et al., 1999). In fact, these results suggest that the AF-2 helix, in the presence of an antagonist, masks the binding site for a transcription target, such as a helical motif of a coactivator, as will be discussed later. The tertiary structure for the VDR-LBD was recently modelled, based on crystallographic data from the TR-LBD and sequence conservation between the two receptors (Norman et al., 1999). Not surprisingly, the structure appears to be very similar to all other nuclear receptor-LBDs, except that the VDR-LBD contains an extra loop structure between helices 2 and 3 that does not exist in any other receptor structure studied thus far. Another interesting characteristic of VDR is the absence of a ligand-independent AF-1 transactivation motif, identified in the N-terminus domain of steroid receptors and a number of nuclear receptors. The very short A/B domain of VDR (18 amino acids) does not leave much space for an AF-1.

3. Coactivators for VDR and other nuclear receptors 3.1. Direct contacts with the transcription machinery Transcriptional activation of genes regulated by nuclear receptors and other transcription factors is currently understood as a multi-step process that is initiated at the promoter region of expressed genes. It is catalyzed by RNA polymerase II (RNA Pol II ) and requires the assembly of general transcription factors (GTFs) including TFIIA, -B, -D, -E, -F, -H, at the promoter (illustrated in Fig. 2; for a review, see Zawel and Reinberg, 1995; Roeder, 1996). This process is regulated

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by a combination of transcription factors recruited on a given promoter by direct DNA binding to their specific response elements (as described previously for VDR on its VDRE ). DNA-bound factors mediate protein–protein interactions with components of the transcription machinery, ultimately targeting the recruitment and/or control of RNA Pol II. Consistent with this model, several direct contacts have been identified between VDR or other nuclear receptors and the basal transcription apparatus. Interaction of VDR with TFIIB has been documented as occuring through a direct, specific binding to the VDR-LBD that nevertheless occurs in a ligand-independent fashion (MacDonald et al., 1995). Alternatively, TFIIB has been shown to synergize with liganded VDR in vivo, but this effect is dependent on the cell type (Blanco et al., 1995). A more recent analysis also demonstrates that TFIIB binding is actually disrupted by addition of 1,25(OH ) D in both in-vivo 2 3 and in-vitro assays (Masuyama et al., 1997). Interestingly, the VDR binding site lies within the region of the LBD that does not include the AF-2 domain (Blanco et al., 1995). This last result is also confirmed by the fact that point mutations (L417A and E420A) within the AF2 that abolish VDR-mediated transcription activation do not prevent binding of TFIIB (Jurutka et al., 1997). Taken together, these data suggest that the VDR–TFIIB interaction may not be the determining interaction for transactivation regulated by 1,25(OH ) D in vivo, but also may 2 3 suggest the requirement of additional targets for VDR transcription activity. For example, another basal factor, TFIIA, has also been shown to bind VDR. This effect is strongly stimulated by ligand and occurs in the context of VDR bound to a promoter DNA template (Lemon et al., 1997). VDR and other nuclear receptors have also been found to bind several TBP-associated factors, TAFs, [ TATA box-binding protein ( TBP) associated factors] that comprise the basal complex TFIID. A 135 kDa subunit, TAF 135, binds and enhances the activities of II VDR, RAR, TR, but not those of RXR and ER (Mengus et al., 1997). TAF 55 has been shown to be a II direct target of VDR and TRa, by binding to a 40-amino-acid region spanning a-helices H3–H5 of the VDR and TRa LBDs (Lavigne et al., 1999). TAF 28 II stimulates transcription by RXR, but has a potentiating or repressive effect on VDR and ER activity, depending on the cell type (Cos versus HeLa cells, respectively, May et al., 1996). Finally, VDR binds a number of newly discovered factors that appear to bridge or recruit other activities important for the activation process, as described at length below. 3.2. SRC/p160 family of coactivators 3.2.1. Gene regulation by chromatin remodelling Coactivators can be loosely defined as proteins that potentiate the activity of specific transcription factors.

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Their functionality appears upon recruitment onto a promoter through protein–protein interactions with a DNA-bound activator (a ligand-activated nuclear receptor, for example). Over the last few years, a growing number of proteins have been identified as coactivators for various nuclear receptors (for reviews, see Torchia et al., 1998; Freedman, 1999; Lemon and Freedman, 1999). Many of these putative coactivators have been cloned by yeast two-hybrid or GST pull-down assays by virtue of their interaction with members of the nuclear receptor family and their ability to potentiate transcriptional activity. Among the many nuclear receptor coactivators characterized so far, a homologous family of proteins has emerged. This family has been alternatively named SRC, NCoA, or more generically, p160, based on one of its first identified members, the 160 kDa protein steroid receptor coactivator-1 (SRC-1) (Onate et al., 1995). The SRC/p160 family comprises three types of factors, on the basis of their homologies, including SRC-1/NCoA-1, GRIP1/TIF2/NCoA-2, and pCIP/RAC3/ACTR/AIB1/ TRAM-1 (for reviews, see McKenna et al., 1999; Xu et al., 1999). The original enzymatic activity found to be common to these factors is histone acetyl-transferase (HAT ) activity, which catalyzes the acetylation of lysine residues at the N-terminal tails of histones (Chen et al., 1997; Spencer et al., 1997). Acetylation is thought to destabilize the interactions between DNA and the histone cores that form its nucleosomal structure in the nucleus. The SRC/p160 family members would thereby act as coactivators by loosening the repressive effect of chromatin on gene expression. In the original publications describing these proteins, ligand-dependent binding to VDR was only documented with the coactivator ACTR, but subsequent analyses have shown that VDR is also the target of GRIP1/TIF-2 and SRC-1 (Hong et al., 1997; Masuyama et al., 1997b ; our observations). Specific inactivation of SRC-1 by gene targeting shows that there may be at least partial functional redundancy between the different SRC/p160 family members, since only a partial resistance to hormonal response is observed in SRC-1(−/−) mice. This phenomenon is concomitant to increased mRNA levels of other coactivators like TIF2, perhaps compensating for the loss of SRC-1 ( Xu et al., 1998). The coactivator effects of SRC-1 on nuclear receptors have also been demonstrated in vitro in the presence of chromatin assembled templates (Liu et al., 1999). Under these conditions, SRC-1 strongly potentiates the liganddependent activity of the progesterone receptor (PR-B). Interestingly, this potentiation also occurs to a certain extent on naked DNA, in the absence of chromatin. These results may suggest a dual effect of SRC-1, both on chromatin remodelling and on other activities or interactions yet to be identified.

3.2.2. Nuclear receptor–coactivator interactions Besides the identification of HAT function, nuclear receptor binding motifs have been delineated within the sequence of the SRC/p160 coactivators. These binding motifs, alternatively called signature motifs, NIDs, NR boxes, or LXDs, are composed of stretches of leucines (defined by a LXXLL consensus sequence) that forms an a-helix (Heery et al., 1997; Torchia et al., 1997). In the presence of its ligand, the receptor-LBD undergoes a change in its conformation that modifies the position of its AF-2 helix. The combination of helix 12 and a-helices 3 and 5 together form a ‘charge clamp’ (Nolte et al., 1998). This structure creates a docking site for a coactivator NR box a-helix. The receptor binding motifs in SRC-1 and GRIP1/TIF2 are arranged as sets of three NR boxes each separated by 50–60 amino acids. Several combinations of pairs of these motifs direct coactivator interaction with nuclear receptor heterodimers (Darimont et al., 1998; Ding et al., 1998; McInerney et al., 1998; Voegel et al., 1998). As depicted in Fig. 1, interaction of GRIP1 with RXR/TR and RXR/RAR heterodimers requires primarily the second and third motifs (NR boxes 2 and 3), whereas SRC-1 interaction with RXR/PPAR is driven mainly by NR boxes 1 and 2 (McInerney et al., 1998), and estrogen receptor interaction requires essentially NR box 2 (McInerney et al., 1998; Mak et al., 1999). Moreover, additional amino acids in the N- and C-termini of these motifs are required for binding specificity (Darimont et al., 1998; McInerney et al., 1998; Mak et al., 1999). Recent crystal structure analysis of a complex of liganded PPARc-LBD with a peptide encompassing two LXXLL motifs of SRC-1 generated a model where NR boxes 2 and 3 are each binding one receptor of the heterodimer, yielding a stoichiometry of one SRC-1 molecule per receptor dimer (Nolte et al., 1998).

Fig. 1. Coactivator/nuclear receptor interactions occur through NR boxes. NR boxes are helical motifs containing a signature LXXLL sequence. Several combinations of NR boxes 1, 2, and 3 of SRC/p160 coactivators are differentially required for interaction with nuclear receptors (See Section 3.2.2). According to the crystal structure analysis of PPARc-LBD with SRC-1 NR boxes (Nolte et al., 1998), a single coactivator binds both subunits of a nuclear receptor heterodimer.

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3.2.3. Larger coactivator assemblies — multifunctional HAT activity The SRC/p160 family of coactivators form complexes with CBP/p300 (Chen et al., 1997). The co-integrators CBP and p300 bind a large panel of transcription factors (Goldman et al., 1997). They bind nuclear receptors, but much more weakly than do SRC/p160. They also have HAT activity (Bannister and Kouzarides, 1996; Ogryzko et al., 1996), and they appear to interact with nuclear receptors cooperatively with SRC/p160 and other components like p/CIP and PCAF, together forming a larger co-activator complex (McKenna et al., 1998; Torchia et al., 1998). Alternatively, CBP and SRC-1 may be stabilized by a specific RNA coactivator, SRA (steroid receptor RNA activator). Interestingly, SRA has been found to be part of a 600–700 kDa ribonucleoprotein structure that includes SRC-1 (Lanz et al., 1999). The fact that different components of the complex possess HAT activity suggests that they have a cooperative effect or/and an increased array of specificities. Recent reports present additional functions for CBP’s HAT activity besides chromatin remodelling. CBP/p300 is able to acetylate non-histone proteins, such as the transcription factor p53, whose acetylation promotes its DNA-binding activity (Gu and Roeder, 1997). Components of the basal machinery ( TFIIEa, TFIIF ) are also acetylated by p300, PCAF and TAFII250, but the effects of this modification are not yet understood (Imhof et al., 1997). Intriguingly, CBP/p300 can regulate the association between ACTR and the estrogen receptor by directly acetylating ACTR at two lysines close to one of its NR boxes, thereby disrupting its association with ER (Chen et al., 1999). Currently, our view of how SRC/p160 functions is primarily to act as a means of recruiting CBP/p300 to a nuclear receptor. It is CBP/p300 rather than SRC/p160 that appears to be the primary source of HAT activity. 3.3. Corepressors SMRT, and NCoR have been identified as corepressors of RAR and TR by binding to these receptors in the absence of their respective ligand (Chen and Evans, 1995; Horlein et al., 1995). These corepressors provide a link between unliganded receptors and histone deacetylase complexes that stabilize chromatin by deacetylation of the histones N-termini. This suggests a mechanism for the repressive activity of unliganded TR and RAR. However, there is no firm evidence of direct binding to and co-repression by SMRT and NCoR on VDR. Alternatively, VDR silencing may occur through different mechanisms, such as competition for binding to a VDRE, as shown for the multifunctional regulator YY1 associated with the nuclear matrix (Gu et al., 1997; McNeil et al., 1998), or ligand-dependent inhibition of VDR-RXR heterodimerization, as shown for the ribosomal protein L7a, which has been proposed to act as

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a corepressor of VDR and RXR activities (BerghoferHochheimer et al., 1998). 3.4. Other coactivators Several proteins that have been found to act as coregulators of VDR cannot be classified in any of the previous categories. NCoA-62, identified by yeast twohybrid, coactivates VDR in a ligand-dependent fashion, but no function has been assigned to this protein (Baudino et al., 1998). TIF1 was found to interact with VDR and other nuclear receptors (Le Douarin et al., 1996). TIF1 also interacts with heterochromatin-associated proteins and has a kinase activity that can phosphorylate several general transcription factors ( TFIIEa, TAFII28 and TAFII55), suggesting a novel mechanism of transcription regulation for nuclear receptors ( Fraser et al., 1998).

4. DRIP complex and other complexes 4.1. Identification of new cofactor complexes for transcription regulation Besides the SRC/p160 coactivators, VDR-mediated transcription also occurs through a distinct coactivator complex called DRIP (vitamin D Receptor Interacting Proteins; Rachez et al., 1998; Rachez et al., 1999). Several laboratories, including our own, have recently identified a novel type of nuclear receptor coactivator complex, alternatively called DRIP, TRAP, ARC, NAT, or mammalian Mediator ( Table 1), depending on the purification process, and the activators tested as targets. These complexes are required for activation of transcription in vitro, in different transcription assays involving purified components of the transcription machinery. At the time of their respective discoveries, each one of these complexes was thought to be specific for a distinct transcription factor. DRIP and ARC (Activator Recruited Cofactor; Naar et al., 1998, 1999) complexes were purified out of nuclear extracts using GST fusions with VDR-LBD, or with the activation motifs of several transcription factors (SREBP-1a, NF-kB and VP16), respectively. The TRAP complex ( TR Associated Proteins; Fondell et al., 1996; Ito et al., 1999) was isolated by co-immunoprecipitation of epitope-tagged TR stably expressed in HeLa cells. TRAP was later identified as identical to the SMCC complex (Srb/Medcontaining cofactor complex; Gu et al., 1999), a complex purified by co-immunoprecipitation with antibodies directed against epitope-tagged Srb10 (see below). The cloning of these component subunits by independent groups revealed the near-identity of their sequences for many of them, which now suggests that these different complexes might actually constitute a single, universal one ( Fig. 2). Importantly, some subunits do

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Table 1 Subunit composition of general coactivator complexesa

a Subunits in bold with shading are equivalent in the different complexes, or homologs between mammalian and yeast complexes. Subunits in the same lanes have similar molecular weights regardless of any homology. See Section 4 for references and comments.

differ from complex to complex. For example, TRAP150 has no homology with any of its candidate counterparts in other complexes. In addition, DRIP/ARC/CRSP130, also identified as hSur2 (Boyer et al., 1999), has not been identified in the TRAP complex. The various subunit compositions are summarized in Table 1. DRIP, ARC and TRAP complexes appear to have broader target specificities by their close identities with other complexes recently identified: CRSP, NAT, and mammalian Mediator. CRSP (Cofactor Required for Sp1 Activation; Ryu et al., 1999), purified by multiple chromatographic steps, appears to be a subset of nine subunits of the DRIP/ARC complex and might represent a stable core of subunits or a conserved subcomplex among various functionally related complexes. The

CRSP complex differs, however, by its two unrelated 34 and 70 kDa subunits ( Table 1). The homology of CRSP70’s N-terminus with the elongation factor TFIIS is a unique feature among all the complexes described so far. Despite its limited number of subunits, CRSP potentiates the activity of the transcription factor Sp1 in vitro. The NAT complex (Negative regulator of Activated Transcription) was identified by co-immunoprecipitation out of HeLa nuclear extracts with an antibody against hSrb10/CDK8 (Sun et al., 1998). It shares many common subunits with DRIP, ARC and TRAP. However, when the NAT complex was tested in vitro in a purified transcription assay in the presence of RNA pol II, general transcription factors ( TFIIA, to -H ),

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Fig. 2. Network of interactions between coactivators and transcription factors. SRC/p160 coactivators are specific for nuclear receptors, but the cointegrators CBP and p300 also bind a large number and classes of transcription factors. The DRIP complex and its analogous complexes ARC, TRAP/SMCC, NAT, CRSP, and Mediator are the target of nuclear receptors and many other transcription factors that bind via specific subunits. The SRC/CBP complex appears to act, although perhaps not exclusively, through histone acetyltransferase activity (HAT ) on histone and nonhistone proteins. The DRIP and analogous complexes may provide RNA pol II with the ability to respond to activators through its direct recruitment to the basal machinery on the promoter, and may also regulate the initiation of transcription by phosphorylation of the RNA pol II C-terminal repeat domain (CTD) and one of its cofactors PC4 (see Section 4 for details).

and a cofactor activity PC4 (see below), it exhibited a repressing effect on transcription driven by various activators, without any influence on basal transcription. This unexpected result suggests that these complexes may have not only an activation potential but also repressive activities on transcription in vitro, as will be discussed later. The mammalian Mediator was identified (Jiang et al., 1998) through biochemical purification out of nuclear extracts of murine cells. Its name reflects its homologies with a yeast Mediator counterpart ( Table 1; see below). 4.2. Functional studies of RNA pol II in yeast — analogy with the mammalian cofactor complexes The identification of DRIP and other mediator-like complexes in human cells provides a functional link between them and a series of previous studies in yeast (for reviews, see Myer and Young, 1998; Parvin and Young, 1998). RNA polymerase II (core polymerase) in yeast, tested in an in-vitro transcription assay together with general transcription factors, directs only basal transcription. The yeast Mediator complex can interact with the RNA pol II core to form a holoenzyme that is then responsive to activation by transcription factors. Genetic and biochemical analyses in yeast revealed the importance of several types of factors for transcription of target genes by RNA pol II in response to activators

(Myer and Young, 1998). They include the products of SRB genes, identified in a genetic screen as suppressors of mutations (truncations) in the CTD of RNA pol II ( Koleske and Young, 1994). Srb proteins point to the importance of CTD phosphorylation in the transition between transcription initiation at the promoter and elongation of the RNA transcript (Hengartner et al., 1998, and references therein). Biochemical fractionation resulted in the isolation of Mediator as a multi-subunit complex ( Kim et al., 1994) that contained Srb proteins, and another set of components named Med proteins (Lee et al., 1997; Myers et al., 1998). Evidence for the essential role of Mediator subunits has recently been demonstrated through the disruption of SRB7 in mice, which leads to embryonic lethality ( Tudor et al., 1999) consistent with SRB7 being essential for cell viability Several other factors (Gal11, Rgr1, Sin4, Pgd1 and Rox3) originally identified in various genetic studies in yeast, form a discrete subcomplex of Mediator/ holoenzyme, and affect both positive and negative regulation of transcription (Li et al., 1995 ; Myers et al., 1999). Detailed functional studies in yeast revealed the importance of both Srb and Med components of the Mediator complex in either general transcription (Srb4; Thompson and Young, 1995), or activation of transcription by specific factors both in vivo and in vitro, such as Gcn4, Gal4, or VP16 (observed for Med2, Med6,

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Pgd1/Hrs1, and Sin4; Lee et al., 1997; Myers et al., 1998). This last observation led the authors to define the yeast Mediator as a ‘global transcription coactivator’ (Myers et al., 1999). The existence of human homologs of a number of yeast Mediator proteins suggested the existence of a corresponding complex in higher organisms. Mammalian Mediator was identified in mice by Kornberg’s group (Jiang et al., 1998). Its homology to the yeast Mediator is also suggested by some of its functional characteristics: the complex is able to bind the CTD of RNA pol II, and it stimulates in-vitro CTD phosphorylation catalyzed by TFIIH. However, ‘mammalian Mediator’, as isolated, has not yet been shown to function in transcription assays. Some of its subunits have yeast conterparts (Rgr1, Med6, Med7, Srb7), but several other subunits have not matched any yeast homologs. Some, but not all, of the mouse Mediator subunits are in fact also present within DRIP, ARC, TRAP/SMCC, and NAT (see Table 1). That DRIP, ARC and TRAP/SMCC complexes all contain Med components (Gu et al., 1999; Naar et al., 1999; Rachez et al., 1999) may suggest that these complexes could be human homologs of the Mammalian Mediator. However, a side-by-side comparison of the respective subunits within human and mouse complexes favors a scenario where ‘mammalian Mediator’ is distinct from DRIP, ARC and TRAP/SMCC complexes ( Table 1). A larger diversity of complexes in higher eukaryotes relative to yeast was previously suggested for the RNA Pol II holoenzyme on the basis of its different subunit compositions in mammalian preparations (i.e. variations in a subset of components) (Parvin and Young, 1998). The potential differences in composition of the DRIP, ARC, TRAP/SMCC, NAT and mammalian Mediator complexes may reflect this diversity. Whatever the case, the presence of Srb/Med subunits in DRIP, ARC, and TRAP/SMCC strongly suggests that this complex functions at least partly through recruitment of RNA pol II. 4.3. Functional activities of the mammalian complexes As mentioned previously, activities of all these complexes were tested in highly purified transcription assays in vitro, in response to specific activators. The DRIP complex strongly potentiated ligand-dependent VDRRXR transcription on DNA templates assembled into chromatin, but interestingly had little or no effect on the same transcription in the absence of chromatin (naked templates). None of the previously identified SRC/p160 coactivators has been found to be part of the purified DRIP complex (Rachez et al., 1999), and the absence of any HAT activity in the DRIP complex suggests that it may contain distinct chromatin remodelling activities, or may recruit them. The ARC complex tested on chromatin-assembled templates exhibits co-

operativity between different activators, such as Sp1 and SREBP-1a, on the same template promoter, in conditions where ARC has no effect on the same activators tested individually (Naar et al., 1999). This is an interesting demonstration of the ability of these complexes to integrate multiple transcription pathways into a synergistic effect on gene activation. TRAP/SMCC, whose activity was tested only in the absence of chromatin, also enhances activator-dependent transcription in a purified in-vitro system with RNA pol II, GTFs, and PC4, but only in the absence, or limiting concentrations, of TFIIH (Gu et al., 1999). In a transcription system including both PC4 and TFIIH, the SMCC complex repressed transcription. This effect was specific for the presence of activators like Gal4-AH, since no strong effect was observed on basal transcription. By analogy with the effect of Mediator in yeast on phosphorylation of RNA pol II CTD, the kinase activity of SMCC was tested in vitro. Although the CTD was phosphorylated, the major substrate was the cofactor PC4, a singlestranded DNA binding protein that is required for activated transcription (Ge et al., 1994; Kretzschmar et al., 1994a). PC4 has been shown to interact in vitro with TFIIA and VP16 (Ge et al., 1994). More importantly, PC4 binding and transcription activities are lost upon phosphorylation (Ge et al., 1994; Kretzschmar et al., 1994a). The conservation of PC4 in yeast (as Tsp1) highlights its general requirement among eukaryotic systems (Henry et al., 1996). Interestingly, the repression effect by SMCC was also observed when a CTD-less form of RNA pol II was used. Based on this, the authors suggested that regulation of transcription by SMCC could be mediated in concert with PC4, but independently of modifications of the RNA pol II CTD (Gu et al., 1999). More recently, Roeder’s group demonstrated that TRAP/SMCC complex activity, at limiting amounts of TFIIH, can be synergistically stimulated by addition of PC4 and a distinct cofactor, PC2 (an unresolved positive cofactor activity; Kretzschmar et al., 1994b). However, it appears that a further increase in the amounts of PC4 in this assay led to repression of the initial transcription effect. This might reflect the sensitivity of the highly purified assay to subtle variations in its individual components, like PC4. It could also explain why two highly similar complexes like SMCC and NAT have been independently characterized as an activator and repressor of activator-dependent transcription, depending on the equilibrium between constituents of the assays. These observations, however, could still reflect real and decisive variations in the subunit compositions of the two complexes that are biologically relevant.

5. Integration of signalling pathways The SRC-1/p160 family and the DRIP complex represent two unrelated protein complexes, together carrying

C. Rachez, L.P. Freedman / Gene 246 (2000) 9–21

Fig. 3. Two models for coactivator action. (A) In the sequential model, a histone acetyltransferase activity (HAT )-containing complex of SRC/p160 and CBP/p300 is recruited first by VDR in response to ligand. Dissociation of this complex occurs through direct acetylation of the p160 coactivator by CBP. This, in turn, would lead to promoter accessibility that would then allow binding of the DRIP complex to VDR (step 2) and direct recruitment of RNA pol II. (B) In the combinatorial/cooperative model, both complexes could be recruited separately on the same promoter by two bound receptor heterodimers, leading to simultaneous chromatin remodelling and basal machinery recruitment.

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by the DRIP/ARC/TRAP complex. Definitive experimental analysis has yet to confirm this view ( Fondell et al., 1999; Freedman, 1999). In vitro, however, the SRC/p160 and DRIP coactivators appear to have no real intrinsic difference in their ability to interact with nuclear receptors. They both utilize the AF-2 of nuclear receptors and bind with similar affinities [ TRAP220 vs. TIF2 for TR (Treuter et al., 1999); DRIP205 and GRIP1 for VDR (our unpublished observation)]. Thus, we could envision a cooperative model where both CBP/p160 and DRIP complexes simultaneously occupy a promoter, and through their combined actions facilitate activation of transcription ( Fig. 3B). The observation that CBP can acetylate ACTR, leading to the latter’s dissociation from liganded ER (Chen et al., 1999), suggests a mechanism for the sequential model, whereby the first complex functions to acetylate histones and disrupt chromatin structure, whereupon it itself dissociates from the receptor, allowing the DRIP complex to bind and act at the level of direct recruitment ( Fig. 3A). Recent studies have revealed multiple binding motifs for coactivators within the DRIP and TRAP complexes. For example, GR interacts with the DRIP complex via both AF-1 and AF-2 motifs with DRIP150 and DRIP205, respectively (Hittelman et al., 1999) (Fig. 2). These results define the GR-binding motif in the DRIP complex as a combination of two subunits, and might explain the presence of two unrelated activation functions in the same receptor. In the TRAP/SMCC complex, both p53 and VP16 interact via TRAP80, but binding of VP16 and TR (which binds TRAP220) to the TRAP complex are not mutually exclusive, suggesting that this complex may support activation by a combination of distinct transcription factors (Fig. 2). The transactivation domain (conserved region 3, CR3) of E1A interacts directly with hSur-2, which corresponds to DRIP130 (Boyer et al., 1999). This defines yet another novel and distinct target motif for an activator within the complexes.

6. Conclusion several distinct activities, that have been shown to potentiate VDR (and other nuclear receptors) transcription activity. These activities may function together to provide a synergistic effect of 1,25(OH ) D -mediated 2 3 activation, or to provide specificity of targeting to its regulation. VDR transcription regulation may require a combination of chromatin remodeling activities, as well as efficient recruitment of the RNA pol II machinery, via several of its basal factors. We have suggested a step-wise model that combines these distinct activities (Fig. 3A), where a CBP/p160 type coactivator complex might be required for chromatin remodelling, followed by the direct recruitment of the transcription machinery

Transcriptional regulation by 1,25(OH ) D can be 2 3 dissected into several functional activities that are mediated by VDR. Generally, transactivation requires that a repressed state of chromatin has to be disrupted in a given target gene’s regulatory regions, together with the ability to promote productive elongation of RNA products by the RNA pol II machinery from the site of transcription initiation. Several candidates for such activities that are recruited by nuclear receptors in direct response to ligand binding have been recently identified, as has been described in this review. The HAT activitycontaining coactivators (SRC/p160 family, CBP/p300,

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PCAF, etc.) at face value appear to act primarily in the disruption of chromatin through histone modifications, although a series of additional, provocative targets corresponding to ATP-dependent chromatin remodellers have more recently been identified. The regulation of RNA pol II and its ability to respond to activators appear to be mediated by a distinct type of cofactor complex (DRIP/ARC/TRAP/SMCC/Mediator) whose functions are not yet completely elucidated. Given that many classes of activators interact with DRIP beyond VDR and nuclear receptors, this complex must be considered as a regulatory panel for RNA pol II rather than an exclusive target for nuclear receptors. DRIP/ARC/TRAP/SMCC may therefore be viewed as a downstream target of multiple transcription activators, perhaps conferring to RNA pol II the ability to simultaneously integrate multiple signalling pathways onto a single promoter in vivo. This model may provide a key for an interpretation of some unresolved mechanisms involving the simultaneous cross-talk of several signalling systems.

Acknowledgements This work was supported by grants from the NIH and Human Frontiers Science Program. The authors thank J. Ward, T. Staeva, K. Wassmann and S. Larochelle for critical comments on the manuscript. The authors also thank members of the Freedman lab, B.D. Lemon, A. Na¨a¨r, R. Tjian, M. Garabedian, M. Lazar, and J. Parvin, for their input and contributions.

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