Two crystal structures of the glucocorticoid receptor DNA-binding domain complexed with DNA are reported. The domain has a globular fold which contains two Zn-nucleated substructures of distinct conformation and function. When it binds DNA, the domain dimerizes, placing the subunits in adjacent major grooves. In one complex, the DNA has the symmetrical consensus target sequence; in the second, the central spacing between the target's half-sites is larger by one base pair. This results in one subunit interacting specifically with the consensus target half-site and the other nonspecifically with a noncognate element. The DNA-induced dimer fixes the separation of the subunits' recognition surfaces so that the spacing between the half-sites becomes a critical feature of the target sequence's identity.

Low reproducibility rates within life science research undermine cumulative knowledge production and contribute to both delays and costs of therapeutic drug development. An analysis of past studies indicates that the cumulative (total) prevalence of irreproducible preclinical research exceeds 50%, resulting in approximately US$28,000,000,000 (US$28B)/year spent on preclinical research that is not reproducible—in the United States alone. We outline a framework for solutions and a plan for long-term improvements in reproducibility rates that will help to accelerate the discovery of life-saving therapies and cures.

The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3, acting through its cognate nuclear receptor (vitamin D3 receptor, VDR) will induce myeloid leukemic cell lines to terminally differentiate into monocytes/macrophages. Because VDR acts by transcriptionally regulating responsive genes in a ligand-dependent manner, we sought target genes of the receptor that initiate the differentiation process in response to ligand. We screened a cDNA library prepared from the myelomonocytic U937 cell line with probes generated from either 1,25-dihydroxyvitamin D3-treated or untreated cells. We report here that a candidate clone that hybridized differentially is the Cdk inhibitor p21 war1' cn, l. Furthermore, we show that p21 is transcriptionally induced by 1,25-dihydroxvitamin D 3 in a VDR-dependent, but not p53-dependent, manner, and we identify a functional vitamin D response element in the p21 promoter. Transient overexpression of p21 and/or the related Cdk inhibitor p27 in U937 cells in the absence of 1,25-dihydroxvitamin D3 results in the cell-surface expression of monocyte/macrophage-specific markers, suggesting that ligand-modulated transcriptional induction of the p21 gene facilitates the induced differentiation of this monoblastic cell line. We believe that this is the first report demonstrating that the ectopic overexpression of a Cdk inhibitor such as p21 or p27 directly leads to a terminal differentiation program.

Nuclear receptors modulate the transcription of genes in direct response to small lipophilic ligands. Binding to ligands induces conformational changes in the nuclear receptors that enable the receptors to interact with several types of cofactor that are critical for transcription activation (transactivation). We previously described a distinct set of ligand-dependent proteins called DRIPs, which interact with the vitamin D receptor (VDR); together, these proteins constitute a new cofactor complex. DRIPs bind to several nuclear receptors and mediate ligand-dependent enhancement of transcription by VDR and the thyroid-hormone receptor in cell-free transcription assays. Here we report the identities of thirteen DRIPs that constitute this complex, and show that the complex has a central function in hormone-dependent transactivation by VDR on chromatin templates. The DRIPs are almost indistinguishable from components of another new cofactor complex called ARC, which is recruited by other types of transcription activators to mediate transactivation on chromatin-assembled templates. Several DRIP/ARC subunits are also components of other potentially related cofactors, such as CRSP, NAT, SMCC and the mouse Mediator, indicating that unique classes of activators may share common sets or subsets of cofactors. The role of nuclear-receptor ligands may, in part, be to recruit such a cofactor complex to the receptor and, in doing so, to enhance transcription of target genes.

Steroid, retinoid, vitamin D 3 , and thyroid hormones signal through ligand-dependent transcription factors that collectively comprise a superfamily of intracellular, soluble receptors (hereafter collectively called nuclear receptors) that reside in the nucleus or translocate there in response to hormonal signals. As the largest known family of eukaryotic transcriptional regulators, nuclear receptors are implicated via the target genes they modulate in the control of cell growth and differentiation, homeostasis, development, and several physiological processes (for review, see Freedman 1997 and references therein). Moreover, because they are regulated tightly by small lipophilic molecules, they are extremely attractive as pharmacologic targets.Nuclear receptors all share a common organization in functional domains and extensive homologies in structure. A DNA-binding domain allows the receptors to bind as homodimers, or heterodimers with a common partner, retinoid X receptor (RXR), to specific DNA response elements typically composed of two hexameric half-sites organized as direct or inverted repeats. The carboxy-terminal half of the prototype nuclear receptor includes a ligand-binding domain (LBD) with a superimposed dimerization surface, and a ligand-dependent transcriptional activation function called AF-2, located at the extreme carboxyl terminus of the receptor (Danielian et al. 1992;Barettino et al. 1994;Durand et al. 1994). Crystallographic analyses have revealed that binding of a specific ligand, all-trans retinoic acid (ATRA), to retinoic acid receptor ␥ (RAR␥) induces a conformational change in its structure that modifies the orientation of the AF-2 core motif, contained within the last of 12 ␣-helices that

, is a seco-steroid hormone that binds with high affinity to a nuclear receptor, the vitamin D 3 receptor (VDR). This receptor selectively associates with recognition sequences in the promoter region of target genes, thereby regulating the transcription of those genes. The principal functions of 1,25(OH) 2 D 3 are the stimulation of intestinal calcium and phosphorus absorption, mediation of bone remodeling, and conservation of minerals in the kidney (for reviews, see references 46 and 63). In addition to its action in these tissues, however, 1,25(OH) 2 D 3 has been found in skin, testes, breast, muscle, pancreas, endocrine glands, thymus, and bone marrow, suggesting additional regulatory functions for the hormone. Notably, 1,25(OH) 2 D 3 appears to play an important role in modulating the growth of cells of the immune system: the hormone can induce the differentiation of myeloid leukemia cells along a monocyte/ macrophage lineage (1, 4, 6, 49) and can inhibit T-lymphocyte proliferation and activation both in vivo and in vitro (8,10,37,43,44). Interleukin-2 (IL-2), gamma interferon, and granulocytemacrophage colony-stimulating factor (GM-CSF) mRNA levels all decrease after T cells are exposed to 1,25(OH) 2 D 3 , but the molecular mechanisms mediating these changes have not been fully described (9,58,(66)(67)(68)(69)79).T-cell activation is a key step in the initiation of an immunological response. Upon receipt of the appropriate stimulus, a complex signaling cascade is initiated, resulting in cell proliferation and secretion of cytokines that enhance the immune response. One of the first genes to be expressed postactivation is the IL-2 gene. The lymphokine IL-2 exerts its influence by interacting with the IL-2 receptor on the surface of activated T cells, and this interaction is required for progression through the cell cycle (transition from G 1 to S phase). The kinetics of IL-2 induction are quite rapid, with transcripts detectable within 30 to 45 min after activation. A complex enhancer, which includes 275 bp just downstream from the transcription start site (Ϫ52 to Ϫ326), has been exhaustively studied (16,24,75). Binding sites for several ubiquitous and T-cell-specific transcription factors were defined in this region, and the proteins that bind to these sites, including Oct-1, AP1, NF-B, and NFATp/c, have been identified (65).Several agents, including the drugs cyclosporin A and FK506 (18, 51), as well as glucocorticoids (25,80,81) and retinoids (13, 19) appear to act as immunosuppressors by targeting IL-2 expression. 1,25(OH) 2 D 3 inhibits the entry of activated T cells into S phase (68); similar blocks at this point in the cell cycle have been demonstrated for other inhibitors of IL-2 synthesis, such as the synthetic glucocorticoid dexamethasone (7), further implicating IL-2 as a target for vitamin D 3 's immunosuppressive effect.These observations demonstrate an important role for 1,25(OH) 2 D 3 in the immunomodulation of T lymphocytes but not the actual mechanism by which this regulation is carried out....

Estrogen deficiency in menopause is a major cause of osteoporosis in women. Estrogen acts to maintain the appropriate ratio between bone-forming osteoblasts and bone-resorbing osteoclasts in part through the induction of osteoclast apoptosis. Recent studies have suggested a role for Fas ligand (FasL) in estrogen-induced osteoclast apoptosis by an autocrine mechanism involving osteoclasts alone. In contrast, we describe a paracrine mechanism in which estrogen affects osteoclast survival through the upregulation of FasL in osteoblasts (and not osteoclasts) leading to the apoptosis of pre-osteoclasts. We have characterized a cell-type-specific hormone-inducible enhancer located 86 kb downstream of the FasL gene as the target of estrogen receptor-alpha induction of FasL expression in osteoblasts. In addition, tamoxifen and raloxifene, two selective estrogen receptor modulators that have protective effects in bone, induce apoptosis in pre-osteoclasts by the same osteoblast-dependent mechanism. These results demonstrate that estrogen protects bone by inducing a paracrine signal originating in osteoblasts leading to the death of pre-osteoclasts and offer an important new target for the prevention and treatment of osteoporosis.

receptor LBDs converged on a family of related proteins that are collectively termed the p160 coactivators. They are represented by SRC-1/NCoA-1, TIF2/GRIP1/NCoA-2, and pCIP/ACTR/AIB1 (see Xu et al., 1999 and references therein). LBDs were used as baits since an essential