Vitamin D receptor and nuclear receptor coactivators: crucial interactions in vitamin D-mediated transcription

Steroids 66 (2001) 171–176

Vitamin D receptor and nuclear receptor coactivators: crucial interactions in vitamin D-mediated transcription Paul N. MacDonald*, Troy A. Baudino, Hisashi Tokumaru, Diane R. Dowd, Chi Zhang Department of Pharmacology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA

Abstract The nuclear actions of 1,25-dihydroxyvitamin D3 [1␣,25(OH)2D3] are mediated by the vitamin D receptor (VDR). Binding of ligand induces conformational changes in the VDR which promote heterodimerization with retinoid X receptor (RXR) and recruitment of a number of nuclear receptor coactivator proteins including the steroid receptor coactivator (SRC) family members, select SMAD proteins, a novel coactivator complex referred to as DRIP, and a variety of other putative factors. We recently described a novel nuclear receptor coactivator termed NCoA-62 that interacts with the VDR to enhance 1␣,25(OH)2D3-activated transcription. NCoA-62 is unrelated to the SRC family, the DRIP complex, as well as other nuclear receptor coactivators described thus far. The molecular mechanisms involved in NCoA-62 coactivator function are poorly understood, but protein-protein interactions are likely to play an important role. The purpose of this paper is to briefly review salient features of the coactivators involved in VDR-activated transcription and to focus on our current understanding of NCoA-62 and its interplay with other nuclear receptor coactivator proteins. It is clear from the studies described here that a concerted series of interactions with multiple coactivator proteins are essential for high order transactivation by 1␣,25(OH)2D3 and the VDR. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Vitamin D receptor; Nuclear receptor; Transcription regulation; Coactivators; Steroid hormones

1. Vitamin D-mediated transcription The biologic effects of 1␣,25(OH)2D3 are mediated through the VDR, a member of the superfamily of nuclear receptors for steroid hormones, thyroid hormone, and the fat-soluble vitamins A and D. The VDR functions as a ligand-activated transcription factor that binds to specific DNA sequence elements (VDREs) in vitamin D responsive genes and ultimately influences the rate of RNA polymerase II-mediated transcription [1–3]. The precise DNA sequence elements that mediate vitamin D responsiveness have been identified in a variety of genes [4 –10]. In general, VDREs are imperfect direct repeats of the sequence GGGTGA separated by a 3 nucleotide spacer. By serving as specific enhancer binding sites for VDR, VDREs confer 1␣,25(OH)2D3-responsiveness to their native promoters as well as to heterologous promoters that are normally unresponsive. As with other members of the nuclear receptor superfamily, the VDR can be molecularly dissected into at least * Corresponding author. Tel.: ⫹1-216-368-2466; fax: ⫹1-216-3683395. E-mail address: [email protected] (P.N. MacDonald).

two discrete domains, the DNA-binding domain (DBD) and the hormone-binding domain (HBD). The DBD consists of 2 zinc finger motifs which are located within the first 100 residues of the amino terminus. The remaining 300 residues comprise the HBD and a poorly conserved hinge region. The HBD binds 1␣,25(OH)2D3 with high affinity and is also important for protein-protein contacts and for transactivation function. Binding of the 1␣,25(OH)2D3 to VDR HBD induces heterodimerization of VDR with RXR and high affinity binding of the VDR-RXR heterodimer to the VDRE promoter sequences. These initial macromolecular interactions initiate a communication process that ultimately influences the rate of RNA polymerase II-directed transcription. The question of how distal trans-acting factors, such as the VDR, function to alter transcription is central to understanding eukaryotic gene expression. One critical facet in understanding the communication process linking the VDR to transcriptional preinitiation complex is the nuclear receptor co-modulatory proteins (i.e. the coactivators and corepressors) (Fig. 1). Nuclear receptor coactivator proteins have emerged as central players in the communication process connecting ligand-activated receptors to the preinitiation complex [11, 12]. The general functional properties of these transcrip-

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Fig. 1. General properties of putative nuclear receptor co-modulatory proteins in vitamin D-mediated transcription.

tional cofactors are their ability to interact with nuclear receptors and modulate their transcriptional responsiveness to the ligand. Thus far, the best characterized coactivators are the SRC family of nuclear receptor coactivators [12] which includes three members at present: SRC-1 (NCoA-1), SRC-2 (GRIP-1, TIF2, NCoA-2), and SRC-3 (pCIP, RAC3, ACTR, AIB-1, TRAM-1). The mechanisms through which coactivators function in VDR- and other nuclear receptormediated transcriptional pathways are largely unknown. Nuclear receptor coactivators may function as bridging proteins that link the receptor to RNA polymerase II and the basal transcription machinery and possibly recruit limiting components into preinitiation complex assembly. Indeed, SRC-1 interacts with general transcription factors, such as transcription factor IIB (TFIIB) and TATA-binding protein (TBP) [13], as well as with other coactivators, such as CBP/p300 [14,15]. Coactivator proteins such as SRC-1 and CBP/p300 also possess intrinsic histone acetyltransferase activity. Thus, ligand-activated receptors may function to recruit coactivators that remodel chromatin structure and permit greater accessibility of the transcriptional machinery to DNA [16 –19].

as well as SRC interaction with the VDR illustrating the importance of coactivator contacts with the AF-2 domain in the mechanism [24,25]. Structural studies of VDR and of related receptors provide insight into the mechanism of ligand-induced interaction of the VDR AF-2 domain with the NR-boxes of SRC coactivators [26 –29]. It is hypothesized that the 1␣,25(OH)2D3 ligand promotes coactivator interaction by inducing a repositioning of the AF-2 activation helix (helix H12). In the unliganded state, the AF-2 domain (helix H12) projects out away from the globular core of the LBD and in the liganded state the AF-2 domain is folded over onto the LBD globular core domain. One outcome of helix H12 folding is the creation of a platform or protein interaction surface through which nuclear receptor coactivator proteins effectively dock with the VDR [26,27]. Scanning mutagenesis of the thyroid hormone receptor (TR) defined a coactivator interaction surface composed of helices H12, H3, H4, and H5 [30] and structural analysis of the estrogen, thyroid hormone, and PPAR␥ receptors complexed to NRbox peptides confirmed this model [31–33]. We and others reported that a similar surface may exist on the VDR since select mutations within helices H12 and H3 disrupt SRC coactivator interaction and VDR-activated transcription [24,25,34 –36]. For example, a helix H3 mutant VDR [VDR(Y236A)] binds 1␣,25(OH)2D3 with high affinity and it heterodimerizes with RXR. However, this H3 mutant did not interact with SRC-1 or GRIP1 and it did not activate 1␣,25(OH)2D3-mediated transcription {}. Based on our data and the structural data of others, it is likely that this region of the VDR (helix H3) together with the AF-2 domain (helix H12) forms a coactivator binding surface or platform through which proteins such as SRCs bind and mediate the transactivation properties of the VDR.

2. The SRC family of nuclear receptor coactivator proteins 3. NCoA-62/SKIP, a novel nuclear receptor coactivator The SRC family members interact in a ligand-dependent manner with the nuclear receptors [20 –22]. This interaction is mediated through three leucine-rich motifs located in the central region of SRC [23] which have the consensus sequence, LXXLL. These leucine-rich regions are termed nuclear receptor boxes or NR-boxes. Mutations of the NRbox abolish interaction with nuclear receptors and their coactivator activity. The key domain of the receptor that mediates interaction with the coactivator NR-box is the activation function-2 (AF-2) domain. The AF-2 domain is an amphipathic ␣-helical region at the carboxyl terminus of the LBD. Deletion of or mutations within the AF-2 domain selectively abolish ligand-activated transcription by disrupting receptor interaction with the SRC or other NR-box coactivators [22,24]. The VDR AF-2 domain consists of a centrally conserved glutamic acid residue (E420 in the hVDR sequence) flanked on either side by hydrophobic residues. Mutation of residues in the hydrophobic or hydrophilic faces abolish 1␣,25(OH)2D3-activated transcription

P160 coactivator proteins, such as GRIP-1 [21,37] and SRC-1 [20] interact with nuclear receptors in a liganddependent manner through the AF-2 domain and this interaction is important in modulating the transcriptional response of the liganded receptor. However, a number of nuclear receptor coactivators are distinct from the p160 family and may function through alternate pathways. Other important coactivators that function in VDR-mediated transcription include the VDR-interacting protein complex or DRIP/TRAP complex [38], CBP/P300 [14], SMAD3 [39], and NCoA-62 [40]. The latter coactivator was cloned in our laboratory as a protein that interacted with the VDR [40]. It was independently isolated by another group as a protein that interacts with the v-Ski oncogene and was termed SKIP, for Ski-interacting protein [41]. This group suggested a role for NCoA-62/SKIP in cellular differentiation pathways including steroid hormone-mediated cellular differentiation. With regard to the nuclear receptor interaction,

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Fig. 2. NCoA-62 interacts with various nuclear receptors in an in vitro interaction assay. Purified GST-NCoA-62 was incubated with radiolabeled nuclear receptors and protein-protein complexes were visualized following SDS-PAGE analysis. Protein-protein complexes were examined in the absence or presence of the individual cognate ligands for each receptor.

NCoA-62 interacts directly with VDR as well as other nuclear receptors tested including the retinoid X receptor (RXR), estrogen receptor (ER), and glucocorticoid receptor (GR) (Fig. 2). In addition to interacting directly with various nuclear receptors, NCoA-62 functions to modestly augment nuclear receptor-mediated transactivation as assessed in hormone responsive reporter gene assays. Coexpression of NCoA-62 in vitamin D, retinoic acid, estrogen, and glucocorticoid reporter gene expression assays augments ligand-activated transcription in each system by a factor of 2 or 3-fold [40]. Based on its deduced amino acid sequence, NCoA-62 is a novel protein that is not related to p160 coactivators. None the less, it is classified as a nuclear receptor coactivator since it interacts directly with nuclear receptors to augment ligand-activated transcription. NCoA-62 is highly related to BX42, a Drosophila melanogaster nuclear protein putatively involved in ecdysone-stimulated transcription [42,43]. Related proteins also exist in yeast and in C. elegans, but their function is unknown. Sequence comparisons between these related proteins identified three domains with varying degrees of conservation, the SNW domain, the SH2-like domain [44] and the highly charged (HC) carboxyl-terminal domain [40,44], but the function of these three domains is not well characterized (Fig. 3). Deletion studies of NCoA-62 demonstrated that the highly charged COOH-terminal domain is essential for coactivator activity [40]. Our more recent studies indicate that this region is a transactivation domain that we have desig-

Fig. 4. The highly charged COOH-terminus of NCoA-62 contains a transactivation domain. Various lengths of the NCoA-62 C-terminus were fused to the Gal4 DNA-binding domain and the fusions were tested for the ability to transactivate a gal4-responsive growth hormone reporter gene in COS-7 cells.

nated TAD-1 for Transactivation Domain-1. Several lines of evidence support the definition of TAD-1 as a transactivation domain. First, deletion of the TAD-1 domain abolished coactivator function in VDR-, ER-, RAR-, and GRmediated transcription without compromising NCoA-62 interaction with nuclear receptors (data not shown). Second, removal of TAD-1 from the context of full-length NCoA-62 and fusing it to a heterologous DNA-binding domain [GAL4 (1–147)] was sufficient to confer transactivation activity to that fusion protein (Fig. 4). Finally, an R449A mutant within the TAD-1 domain abrogated coactivator activity of full-length NCoA-62 in nuclear receptor-mediated transcription and similarly reduced the autonomous transactivation by the minimal TAD-1 domain. Thus, TAD-1 is a bona-fide transactivation domain that is both necessary and sufficient for transcriptional activation and for the coactivator activity of NCoA-62 in nuclear receptormediated transcription. The centrally conserved SNW domain of NCoA-62 seems to be essential for mediating interactions with the nuclear receptors including the VDR. Two-hybrid interaction studies showed that elimination of the SNW domain between residues 220 and 388 completely eliminated interaction with the VDR [40]. Moreover, this domain alone was

Fig. 3. Putative domains in the NCoA-62 protein. The SNW domain is the highly conserved mid-region of NCoA-62 that may be important for nuclear receptor contacts. The TAD-1 region is a modest transactivation domain in which over 50% of the residues are charged. An SH-2-like region exists between the SNW and TAD-1 regions.

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Fig. 5. The central SNW region of NCoA-62 is sufficient to mediate protein-protein interaction with the VDR. GST-VDR was incubated with radiolabeled gal4-NCoA62 (218 –277) and protein-protein complexes were analyzed by SDS-PAGE and autoradiography (lane 3). Gal4 DBD did not interact with GST-VDR in this system (data not shown). Radiolabeled full-length NCoA-62 was examined in lane 6.

sufficient to form a specific protein-protein complex with VDR in GST-pulldown assays (Fig. 5). Thus, this highly conserved central domain is critical for mediating interactions with nuclear receptors. This region also contains two serine residues that are highly phosphorylated. The functional role of the phosphorylation in NCoA-62 action is still under investigation.

4. NCoA-62 and P160 coactivators interact via distinct domains of the VDR Importantly, NCoA-62 interaction with VDR does not require the AF-2 domain. Deletion of or mutations within the VDR AF-2 domain abolish ligand-dependent interaction with SRC coactivators. However, NCoA-62 retains interactions with these AF-2 mutants. This property is highlighted in Fig. 6, in which the VDR AF-2 deletion mutant [VDR(C403STOP)] abolishes ligand-dependent interaction with the SRC coactivator family, but it retains strong interaction with NCoA-62. Moreover, NCoA-62 does not contain canonical NR-box motifs (LXXLL) indicating that that this novel coactivator may contact other important domains within the VDR LBD. Thus, NCoA-62 belongs to a growing class of AF-2 independent coactivators. The likelihood exists that distinct classes of coactivator proteins may function through different mechanisms to cooperatively enhance VDR-activated transcription. NCoA-62 interacts with unliganded receptors and ligand addition generally enhances formation of the complex by only two- or three-fold. Thus, an intriguing paradox arises. How is the modest effect of ligand on VDR/NCoA-62 interaction (2-fold) translated into the dramatic effect of

Fig. 6. NCoA-62 and SRC-1 interact with distinct domains on VDR. GST-NCoA-62 or GST-SRC-1 were incubated with radiolabeled wt VDR or with an AF-2 deletion mutant of VDR (C403STOP) and protein-protein complexes were visualized following SDS-PAGE and autoradiography. Note that ligand-dependent interaction between VDR and SRC-1 is ablated in the VDR(C403STOP) mutant while NCoA-62 retains interaction with this mutant VDR.

ligand on VDR-activated transcription (generally over 20fold). The answer likely resides in the cooperative interplay and requirement of multiple coactivator proteins in the nuclear receptor complex. To test this possibility, SRC-1 and GRIP-1, were examined in the vitamin D-responsive reporter gene assay in COS-7 cells. GRIP-1 or SRC-1 expression augmented VDR-activated transcription by approximately 2-fold, a level of enhancement similar to that observed with NCoA-62 expression under these sub-optimal conditions. However, coexpression of NCoA-62 and either GRIP-1 or SRC-1 resulted in a synergistic enhancement in 1␣,25(OH)2D3-activated expression of the reporter gene construct. As expected, coexpression of GRIP-1 and SRC-1, two related coactivators resulted in an additive effect of vitamin D-mediated transcription compared to each coactivator alone. The synergism observed in these studies suggests that these two distinct coactivators function together to regulate nuclear receptor-mediated transcription. As discussed above, NCoA-62 interacts with the VDR through domains that are distinct from those involved in p160 coactivator interaction. If distinct domains of VDR mediate NCoA-62 interaction compared to P160 coactivator interaction, then the possibility exists that NCoA-62 and p160 coactivators may simultaneously contact the VDR to form a ternary complex. As illustrated in Fig. 7, in vitro GST pulldown studies demonstrated the formation of such a complex between NCoA-62, liganded VDR, and GRIP-1 or SRC-1 thus, providing strong support for this possibility. This ternary complex is completely dependent on the 1␣,25(OH)2D3 ligand in the binding assay indicating that a ligand-dependent interaction of SRC-1 or GRIP-1 with the VDR/NCoA-62 complex occurs. Thus, while the 1␣,25(OH)2D3 ligand may have only a modest effect on NCoA-62/VDR complex formation, it does have a profound effect on the formation of the NCoA-62/VDR/p160 ternary complex. It is likely that all

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Fig. 7. P160 coactivators, NCoA-62, and liganded VDR form a ternary complex in an in vitro GST pulldown assay. GST-NCoA-62 was incubated with radiolabeled GRIP-1 or SRC-1 in the absence or presence of purified baculovirus-expressed VDR (BEVS-VDR). Note in lane 3 that labeled GRIP-1 does not interact appreciably with GST-NCoA-62 in this system. Addition of unliganded VDR has no affect on NCoA-GRIP complexes (lane 4). However, addition of 10⫺8 M 1␣,25(OH)2D3 to this binding assay promotes a ternary complex between GST-NCoA-62, GRIP, and liganded VDR (lane 5).

three proteins within this complex are essential for the full transactivation potential of the system.

5. Conclusions The mechanisms through which coactivators function are diverse. Some coactivators may function as macromolecular bridges between the nuclear receptor and the PIC. Their interaction with the PIC (either direct or indirect) may promote PIC assembly or enhance the stability of the PIC, thereby leading to activated transcription. An emerging property of several other coactivator proteins including CREB binding protein (CBP) and SRC-1 is that these coactivators possess intrinsic histone acetyltransferase (HAT). Histone acetylation may result in a disruption or loosening of the chromatin structure making promoters more accessible to the transcription machinery and ultimately leading to an increase in the rate of transcription. Thus, nuclear receptors may interact in a ligand-dependent manner to recruit enzymes that modify chromatin structure at a particular promoter. Thus, different classes of nuclear receptor coactivators may function through different mechanisms to augment ligand-activated transcription. This appears to be the case for NCoA-62 and the SRC coactivator family. Clearly, the effects are cooperative and coexpression in mammalian cells leads to synergistic effects on VDR-activated transcription. This is likely do to the fact that SRCs and NCoA-62 interact with distinct domains on the VDR. SRCs interact in a ligand-dependent manner with the H3, H4, H5, H12 coactivator interaction surface on VDR. Although the precise domain of VDR that mediates interactions with NCoA-62 is unknown, it is evident that this interaction is mediated exclusively through the VDR ligand binding domain and that it is independent of the AF-2 helix (helix H12). That the SRC and NCoA-62 interaction occurs via distinct domains on VDR is also evident in the observation of a ternary complex between NCoA-62, liganded VDR,

and the SRC coactivator family members. We propose that this ternary complex explains the cooperative, synergistic effects on NCoA-62 and SRC coactivators in VDR-activated transcription. Further studies are ongoing to test this model of coactivator action.

References [1] Haussler MR, Haussler CA, Jurutka PW, Thompson PD, Hsieh JC, Remus LS, Selznick SH, Whitfield GK. The vitamin D hormone and its nuclear receptor: molecular actions and disease states. J Endocrinol 1997;154(Suppl):S57–S73. [2] Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, Selznick SH, Dominguez CE, Jurutka PW. The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 1998;13(3):325– 49. [3] Kraichely DM, MacDonald PN. Transcriptional activation through the vitamin D receptor in osteoblasts [In Process Citation]. Front Biosci 1998;3:D821–D833. [4] Morrison NA, Shine J, Fragonas J-C, Verkest V, McMenemy L, Eisman JA. 1,25-Dihydroxyvitamin D-responsive element and glucocorticoid repression in the osteocalcin gene. Science 1989;246: 1158 – 61. [5] Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, Denhardt DT. Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (Spp-1 or osteopontin) gene expression. Proc Natl Acad Sci USA 1990;87:9995–9. [6] Ozono K, Liao J, Kerner SA, Scott RA, Pike JW. The vitamin D-responsive element in the human osteocalcin gene. J Biol Chem 1990;265:21881– 8. [7] Darwish HM, DeLuca HF. Identification of a 1,25-dihydroxyvitamin D3-response element in the 5⬘-flanking region of the rat calbindin D-9k gene. Proc Natl Acad Sci USA 1992;89(January, 1992):603–7. [8] Demay MB, Gerardi JM, DeLuca HF, Kronenberg HM. DNA sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 receptor and confer responsiveness to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 1990;87:369 –73. [9] Gill RK, Christakos S. Identification of sequence elements in mouse calbindin-D28k gene that confer 1,25-dihydroxyvitamin D3- and butyrateinducible responses. Proc Natl Acad Sci USA 1993;90:2984 – 8. [10] Cao X, Ross FP, Zhang L, MacDonald PN, Chappel J, Teitelbaum SL. Cloning of the promoter for the avian integrin b3 subunit gene

176

[11]

[12] [13]

[14]

[15]

[16]

[17]

[18]

[19] [20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

P.N. MacDonald et al. / Steroids 66 (2001) 171–176 and its regulation by 1,25-dihydroxyvitamin D3. J Biol Chem 1993; 268:27371– 80. Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L. Nuclear receptor coactivators and corepressors. Mol Endocrinol 1996;10:1167–77. McKenna NJ, Lanz RB, O’Malley BW. Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 1999;20(3):321– 44. Takeshita A, Yen PM, Misiti S, Cardona GR, Liu Y, Chin WW. Molecular cloning and properties of a full-length putative thyroid hormone receptor coactivator. Endocrinology 1996;137(8):3594 –7. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin S-C, Heyman RA, Rose DW, Glass CK, Rosenfeld MG. A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 1996;85:403–14. Yao TP, Ku G, Zhou N, Scully R, Livingston DM. The nuclear hormone receptor coactivator SRC-1 is a specific target of p300. Proc Natl Acad Sci USA 1996;93(20):10626 –31. Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA, McKenna NJ, Onate SA, Tsai SY, Tsai MJ, O’Malley BW. Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 1997; 389(6647):194 – 8. Orgryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 1996;87:953–9. Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML, Nakatani Y, Evans RM. Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 1997;90(3):569 – 80. Bannister AJ, Kouzarides T. The CBP co-activator is a histone acetyltransferase. Nature 1996;384(6610):641–3. Onate SA, Tsai SY, Tsai M-J, O’Malley BW. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 1995;270:1354 –7. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR. GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 1996;93(10):4948 –52. Voegel JJ, Heine MJ, Zechel C, Chambon P, Gronemeyer H. TIF2, a 160 kDa transcriptional mediator for the ligand-dependent activation function AF-2 of nuclear receptors. Embo J 1996;15(14):3667–75. Heery DM, Kalkhoven E, Hoare S, Parker MG. A signature motif in transcriptional co-activators mediates binding to nuclear receptors [see comments]. Nature 1997;387(6634):733– 6. Masuyama H, Brownfield CM, St-Arnaud R, MacDonald PN. Evidence for ligand-dependent intramolecular folding of the AF-2 domain in vitamin D receptor-activated transcription and coactivator interaction. Mol Endocrinol 1997 (in press). Jurutka PW, Hsieh JC, Remus LS, Whitfield GK, Thompson PD, Haussler CA, Blanco JC, Ozato K, Haussler MR. Mutations in the 1,25-dihydroxyvitamin D3 receptor identifying C-terminal amino acids required for transcriptional activation that are functionally dissociated from hormone binding, heterodimeric DNA binding, and interaction with basal transcription factor IIB, in vitro. J Biol Chem 1997;272(23):14592–9. Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D. Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-␣. Nature 1995;375:377– 82. Renaud J-P, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer H, Moras D. Crystal structure of the RAR-␥ ligand-binding domain bound to all-trans retinoic acid. Nature 1995;378:681–9. Wagner RL, Apriletti JW, McGrath ME, West BL, Baxter JD, Fletterick RJ. A structural role for hormone in the thyroid hormone receptor. Nature 1995;378:690 –7.

[29] Wurtz JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D, Gronemeyer H. A canonical structure for the ligand-binding domain of nuclear receptors [see comments] [published erratum appears in Nat Struct Biol 1996 Feb;3(2):206]. Nat Struct Biol 1996;3(1):87–94. [30] Feng W, Ribeiro RC, Wagner RL, Nguyen H, Apriletti JW, Fletterick RJ, Baxter JD, Kushner PJ, West BL. Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors. Science 1998;280(5370):1747–9. [31] Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD, Fletterick RJ, Yamamoto KR. Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev 1998;12(21): 3343–56. [32] Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV. Ligand binding and co-activator assembly of the peroxisome proliferatoractivated receptor-␥. Nature 1998;395(6698):137– 43. [33] Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 1998;95(7):927–37. [34] Kraichely DM, Nakai YD, MacDonald PN. Identification of an autonomous transactivation domain in helix H3 of the vitamin D receptor. J Cell Biochem 1999;75(1):82–92. [35] Kraichely DM, Collins J, DeLisle RK, MacDonald PN. The autonomous transactivation domain in helix H3 of the vitamin D receptor is required for transactivation and coactivator interaction. J Biol Chem 1999;274(20):14352– 8. [36] Jimenez-Lara AM, Aranda A. Lysine 246 of the vitamin D receptor is crucial for ligand-dependent interaction with coactivators and transcriptional activity. J Biol Chem 1999;274(19):13503–10. [37] Hong H, Kohli K, Garabedian MJ, Stallcup MR. GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 1997;17(5):2735– 44. [38] Rachez C, Suldan Z, Ward J, Chang CP, Burakov D, ErdjumentBromage H, Tempst P, Freedman LP. A novel protein complex that interacts with the vitamin D3 receptor in a ligand-dependent manner and enhances VDR transactivation in a cell-free system. Genes Dev 1998;12(12):1787– 800. [39] Yanagisawa J, Yanagi Y, Masuhiro Y, Suzawa M, Watanabe M, Kashiwagi K, Toriyabe T, Kawabata M, Miyazono K, Kato S. Convergence of transforming growth factor-␤ and vitamin D signaling pathways on SMAD transcriptional coactivators. Science 1999; 283(5406):1317–21. [40] Baudino TA, Kraichely DM, Jefcoat SC, Winchester SK, Partridge NC, MacDonald PN. Isolation and characterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription. J Biol Chem 1998;273(26):16434 – 41. [41] Dahl R, Wani B, Hayman MJ. The Ski oncoprotein interacts with Skip, the human homolog of Drosophila Bx42. Oncogene 1998; 16(12):1579 – 86. [42] Wieland C, Mann S, Besser H, Saumweber H. The Drosophila nuclear protein Bx42, which is found in many puffs on polytene chromosomes, is highly charged. Chromosoma 1992;101:517–25. [43] Saumweber H, Frasch M, Korge G. Two puff-specific proteins bind within the 2.5 kb upstream region of the Drosophila melanogaster Sgs-4 gene. Chromosoma 1990;99:52– 60. [44] Folk P, Puta F, Krpejsova L, Blahuskova A, Markos A, Rabino M, Dottin RP. The homolog of chromatin binding protein Bx42 identified in Dictyostelium. Gene 1996;181(1–2):229 –31.