Nuclear receptors (NRs) comprise a family of ligand inducible transcription factors. To achieve transcriptional activation of target genes, DNA-bound NRs directly recruit general transcription factors (GTFs) to the preinitiation complex or bind intermediary factors, so-called coactivators. These coactivators often constitute subunits of larger multiprotein complexes that act at several functional levels, such as chromatin remodeling, enzymatic modification of histone tails, or modulation of the preinitiation complex via interactions with RNA polymerase II and GTFs. The binding of NR to coactivators is often mediated through one of its activation domains. Many NRs have at least two activation domains, the ligand-independent activation function (AF)-1, which resides in the N-terminal domain, and the ligand-dependent AF-2, which is localized in the C-terminal domain. In this review, we summarize and discuss current knowledge regarding the molecular mechanisms of AF-1- and AF-2-mediated gene activation, focusing on AF-1 and AF-2 conformation and coactivator binding.

The activation function 2/ligand-dependent interaction between nuclear receptors and their coregulators is mediated by a short consensus motif, the so-called nuclear receptor (NR) box. Nuclear receptors exhibit distinct preferences for such motifs depending both on the bound ligand and on the NR box sequence. To better understand the structural basis of motif recognition, we characterized the interaction between estrogen receptor ␣ and the NR box regions of the p160 coactivator TIF2. We have determined the crystal structures of complexes between the ligand-binding domain of estrogen receptor ␣ and 12-mer peptides from the Box 2 and Box 3 regions of TIF2. Surprisingly, the Box 3 module displays an unexpected binding mode that is distinct from the canonical LXXLL interaction observed in other ligand-binding domain/NR box crystal structures. The peptide is shifted along the coactivator binding site in such a way that the interaction motif becomes LXXYL rather than the classical LXXLL. However, analysis of the binding properties of wild type NR box peptides, as well as mutant peptides designed to probe the Box 3 orientation, suggests that the Box 3 peptide primarily adopts the "classical" LXXLL orientation in solution. These results highlight the potential difficulties in interpretation of protein-protein interactions based on cocrystal structures using short peptide motifs. The estrogen receptor ␣ (ER␣)1 is a ligand-activated transcription factor that mediates the biological effects of the steroid hormone estrogen. Like other nuclear receptors (NRs), ER␣ exhibits a characteristic modular domain organization that includes two autonomous transcriptional activation functions (AF1 and AF2) that regulates transcription through interactions with NR coregulators (1-3). AF1, which resides in the N-terminal region of ER␣, is constitutively active and regulated by growth factors (4 -6). In contrast, AF2, which is located in the C-terminal ligand-binding domain (LBD) of ER, is entirely dependent on ligand for its activity.In recent years, structural and functional studies of both the AF2 domain of ER␣ and associated coregulators have greatly enhanced our knowledge of ligand-dependent, ER-mediated transcriptional activation. A large number of coactivators have been isolated that primarily target the LBD of the receptor in a ligand-and AF2-dependent manner (7,8). The most widely studied group of AF2 coactivators includes the p160 family of proteins (steroid receptor coactivator 1, TIF2/glucocorticoid receptor-interacting protein 1, and steroid receptor coactivator 3/AIB1) (9 -13) and the p300/cAMP-responsive element-binding protein-binding protein (14, 15). These factors possess intrinsic histone acetyltransferase activity and/or function in complexes with other acetyltransferases such as p300/cAMPresponsive element-binding protein-binding protein-associated factor (16, 17). They act to remodel chromatin through the regulation of histone acetylation status (18) and are therefore believed to influence promoter accessibility. The...

The N-terminal regions of the estrogen receptor ␣ (ER␣-N) and ␤ (ER␤-N) were expressed and purified to homogeneity. Using NMR and circular dichroism spectroscopy, we conclude that both ER␣-N and ER␤-N are unstructured in solution. The TATA box-binding protein (TBP) has been shown previously to interact with ER␣-N in vitro and to potentiate ER-activated transcription. We used surface plasmon resonance and circular dichroism spectroscopy to confirm and further characterize The estrogen receptors (ERs)1 are ligand-inducible transcription factors that mediate the biological effects of estrogens. Two isoforms of human ER, encoded by two different genes, have been cloned and characterized, ER␣ and ER␤ (1-3). The ERs belong to a superfamily of nuclear receptors (NRs) that includes receptors for steroid hormones, thyroid hormones, and other hormones, as well as orphan receptors for which no ligand is known (4). All nuclear receptors share a similar modular structure; a variable N-terminal region, followed by a DNA binding domain (DBD), a hinge region, and a C-terminal ligand binding domain (LBD) (5). Transactivation regions have been localized to the LBD and in many cases also to the N-terminal region of the nuclear receptors (6, 7). The ligand-bound ERs bind as homodimers to specific DNA sequences termed estrogen response elements and regulate transcription through interaction with transcription modulators and recruitment of the general transcription machinery (8). ER␣ and ER␤ have also been shown to heterodimerize on estrogen response elements (9 -11).ER␣ contains two major transcription activation functions (AFs): one located in the N-terminal region (AF-1) and one in the C-terminal region of the LBD (AF-2) (12). A third activation function has been reported (AF-2a) residing in the boundary between the hinge and the LBD domains of ER␣ (13). The AF-1 function is hormone-independent, whereas the AF-2 function requires the presence of hormone (12). Full transcription activity of the ER␣ is thought to be achieved by synergism between the AFs. Further, the activities of the AFs are dependent on promoter and cellular context (14 -16). The ER␤ has a high homology to ER␣ in the DBD (96% amino acid identity) and in the LBD (58% amino acid identity) (2). In contrast, the Nterminal region of ER␤ is ϳ80 amino acids shorter than that of ER␣ and has also very poor sequence homology to that of ER␣. The N-terminal region in ER␤ is well conserved between different species such as rat, mouse, and human, which would imply functional importance.To date relatively little information has been available on the structure of the N-terminal regions of the NRs. The glucocorticoid receptor (GR) N-terminal transactivation region 1 (AF-1) and a shorter core fragment of 1, the 1core, have been shown by NMR and circular dichroism (CD) spectroscopy to be unstructured in aqueous solution (17). Furthermore, the isolated N-terminal transactivation region of the progesterone receptor A (PR A) has been shown to be sensitive to rapid degradation in limit...

Estrogen receptors (ERs) associate with distinct transcriptional coactivators to mediate activation of target genes in response to estrogens. Previous work has provided multiple evidence for a critical role of p160 coactivators and associated histone acetyltransferases in estrogen signaling. In contrast, the involvement of the mammalian mediator complex remains to be established. Further, although the two subtypes ER␣ and ER␤ appear to be similar in regard to principles of LXXLLmediated coactivator binding to the AF-2 activation domain, there are indications that the context-dependent transcriptional activation profiles of the two ERs can be quite distinct. Potentially, this could be attributed to differences with regard to coregulator recruitment. We have here studied the interactions of the nuclear receptor-binding subunit of the mammalian mediator complex, referred to as TRAP220, with ER␣ and ER␤. In comparison to the p160 coactivator TIF2, we find that TRAP220 displays ER␤ preference. Here, we show that this is a feature of the binding specificity of the TRAP220 LXXLL motifs and demonstrate that the ER subtype-specific F-domain influences TRAP220 interaction. Such differences with regard to coactivator recruitment indicate that the relative importance of individual coregulators in estrogen signaling could depend on the dominant ER subtype.

Transcriptional activation by nuclear receptors (NRs) involves the concerted action of coactivators, chromatin components, and the basal transcription machinery. Crucial NR coactivators, which target primarily the conserved ligand-regulated activation (AF-2) domain, include p160 family members, such as TIF2, as well as p160-associated coactivators, such as CBP/p300. Because these coactivators possess intrinsic histone acetyltransferase activity, they are believed to function mainly by regulating chromatin-dependent transcriptional activation. Recent evidence suggests the existence of an additional NR coactivator complex, referred to as the thyroid hormone receptor-associated protein (TRAP) complex, which may function more directly as a bridging complex to the basal transcription machinery. TRAP220, the 220-kDa NR-binding subunit of the complex, has been identified in independent studies using both biochemical and genetic approaches. In light of the functional differences identified between p160 and TRAP coactivator complexes in NR activation, we have attempted to compare interaction and functional characteristics of TIF 2 and TRAP220. Our findings imply that competition between the NR-binding subunits of distinct coactivator complexes may act as a putative regulatory step in establishing either a sequential activation cascade or the formation of independent coactivator complexes. Nuclear hormone and orphan receptors (NRs)1 comprise a large family of transcription factors and participate in multiple aspects of development and homeostasis of higher eucaryotic organisms but also in deregulation of normal cellular functions (for review, see Refs. 1 and 2). They can be categorized into different subfamilies according to characteristics such as nature of ligand, DNA response element, or oligomerization status. Usually, steroid hormone receptors, which mainly form homodimers, are distinguished from a large diverse subfamily of receptors for nonsteroid ligands, such as thyroid hormone (TR), retinoids (retinoic acid receptor and retinoid X receptor (RXR)), and eicosanoids (peroxisome proliferator-activated receptor (PPAR)), as well as many orphan receptors, for which ligands have not been identified yet or do not exist. Unlike steroid receptors, most of these NRs function as heterodimers with RXR and thus represent highly dynamic transcription factor complexes due to the association of two receptor subunits with distinct structural and functional features (3-6). The majority of NRs utilizes two distinct domains for transcription activation, located in the N and C termini, respectively: a constitutive AF-1 and a ligand-regulated AF-2 as part of the multifunctional ligand-binding domain (LBD). NRs function in concert with multiple transcriptional cofactors, including basal transcription factors, corepressors, and coactivators (for review, see Refs. 7 and 8). Substantial progress in structural and functional analysis has allowed a more detailed understanding of interactions between the AF-2 domain and associated cofactors a...