Showing papers by "Michael G. Rosenfeld published in 1998"
TL;DR: A general mechanism for the assembly of nuclear receptors with co-activators is suggested, based on the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-γ homodimer.
Abstract: The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-gamma ligand-binding domain (LBD), at 2.2 A resolution; this structure reveals a large binding pocket, which may explain the diversity of ligands for PPAR-gamma. We also describe the ternary complex containing the PPAR-gamma LBD, the antidiabetic ligand rosiglitazone (BRL49653), and 88 amino acids of human SRC-1 at 2.3 A resolution. Glutamate and lysine residues that are highly conserved in LBDs of nuclear receptors form a 'charge clamp' that contacts backbone atoms of the LXXLL helices of SRC-1. These results, together with the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-gamma homodimer, suggest a general mechanism for the assembly of nuclear receptors with co-activators.
1,852 citations
TL;DR: The data suggest that N-CoR- and SMRT-containing complexes act as rate-limiting components in the actions of specific nuclear receptors, and that their actions are regulated by multiple signal transduction pathways.
Abstract: Several lines of evidence indicate that the nuclear receptor corepressor (N-CoR) complex imposes ligand dependence on transcriptional activation by the retinoic acid receptor and mediates the inhibitory effects of estrogen receptor antagonists, such as tamoxifen, suppressing a constitutive N-terminal, Creb-binding protein/coactivator complex-dependent activation domain. Functional interactions between specific receptors and N-CoR or SMRT corepressor complexes are regulated, positively or negatively, by diverse signal transduction pathways. Decreased levels of N-CoR correlate with the acquisition of tamoxifen resistance in a mouse model system for human breast cancer. Our data suggest that N-CoR- and SMRT-containing complexes act as rate-limiting components in the actions of specific nuclear receptors, and that their actions are regulated by multiple signal transduction pathways.
686 citations
TL;DR: Different classes of mammalian transcription factors functionally require distinct components of the coactivator complex, including CREB-binding protein (CBP/p300), nuclear receptor coactivators (NCoAs), and p300/CBP-associated factor (p/CAF).
Abstract: Different classes of mammalian transcription factors—nuclear receptors, cyclic adenosine 3′,5′-monophosphate–regulated enhancer binding protein (CREB), and signal transducer and activator of transcription-1 (STAT-1)—functionally require distinct components of the coactivator complex, including CREB-binding protein (CBP/p300), nuclear receptor coactivators (NCoAs), and p300/CBP-associated factor (p/CAF), based on their platform or assembly properties. Retinoic acid receptor, CREB, and STAT-1 also require different histone acetyltransferase (HAT) activities to activate transcription. Thus, transcription factor–specific differences in configuration and content of the coactivator complex dictate requirements for specific acetyltransferase activities, providing an explanation, at least in part, for the presence of multiple HAT components of the complex.
642 citations
TL;DR: Data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coActivator complexes required for nuclear receptor function.
Abstract: Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3′) in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPARγ activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function.
616 citations
TL;DR: The identification of these limiting co-repressor and co-activator complexes and their specific interaction motifs, in concert with solution of the structures of the receptor ligand-binding domain in apo (empty) and ligand bound forms, indicates a common molecular mechanism by which these factors activate and repress gene transcription.
609 citations
TL;DR: The results identify a major cause of CPHD in humans and suggest a direct or indirect role for PROP1 in the ontogenesis of pituitary gonadotropes, as well as somatotrope, lactotropes and caudomedial thyrotropes.
Abstract: Combined pituitary hormone deficiency (CPHD) in man denotes impaired production of growth hormone (GH) and one or more of the other five anterior pituitary hormones. Mutations of the pituitary transcription factor gene POU1F1 (the human homologue of mouse Pit1) are responsible for deficiencies of GH, prolactin and thyroid stimulating hormone (TSH) in Snell and Jackson dwarf mice and in man, while the production of adrenocorticotrophic hormone (ACTH), luteiniz-ing hormone (LH) and follicle stimulating hormone (FSH) is preserved. The Ames dwarf (df) mouse displays a similar phenotype, and appears to be epistatic to Snell and Jackson dwarfism. We have recently positionally cloned the putative Ames dwarf gene Prop1 (ref. 1)f which encodes a paired-like homeodomain protein that is expressed specifically in embryonic pituitary and is necessary for Pit1 expression. In this report we have identified four CPHD families with homozy-gosity or compound heterozygosity for inactivating mutations of PROP1. These mutations in the human PROP1 gene result in a gene product with reduced DNA-binding and transcriptional activation ability in comparison to the product of the murine df mutation. In contrast to individuals with POU1F1 mutations, those with PROP1 mutations cannot produce LH and FSH at a sufficient level and do not enter puberty spontaneously. Our results identify a major cause of CPHD in humans and suggest a direct or indirect role for PROP1 in the ontogenesis of pituitary gonadotropes, as well as somatotropes, lactotropes and caudomedial thyrotropes.
516 citations
TL;DR: Pitx2 seems to serve as a critical downstream transcription target that mediates left–right asymmetry in vertebrates and changes in Pitx2 expression are evident in mouse mutants with laterality defects.
Abstract: The handedness of visceral organs is conserved among vertebrates and is regulated by asymmetric signals relayed by molecules such as Shh, Nodal and activin. The gene Pitx2 is expressed in the left lateral plate mesoderm and, subsequently, in the left heart and gut of mouse, chick and Xenopus embryos. Misexpression of Shh and Nodal induces Pitx2 expression, whereas inhibition of activin signalling blocks it. Misexpression of Pitx2 alters the relative position of organs and the direction of body rotation in chick and Xenopus embryos. Changes in Pitx2 expression are evident in mouse mutants with laterality defects. Thus, Pitx2 seems to serve as a critical downstream transcription target that mediates left-right asymmetry in vertebrates.
509 citations
TL;DR: The molecular basis of generating diverse pituitary cell phenotypes from a common precursor is investigated, providing in vivo and in vitro evidence that their development involves three sequential phases of signaling events and the action of a gradient at an ectodermal boundary.
Abstract: During development of the mammalian pituitary gland specific hormone-producing cell types, critical in maintaining homeostasis, emerge in a spatially and temporally specific fashion from an ectodermal primordium. We have investigated the molecular basis of generating diverse pituitary cell phenotypes from a common precursor, providing in vivo and in vitro evidence that their development involves three sequential phases of signaling events and the action of a gradient at an ectodermal boundary. In the first phase, the BMP4 signal from the ventral diencephalon, expressing BMP4, Wnt5a, and FGF8, represents a critical dorsal neuroepithelial signal for pituitary organ commitment in vivo. Subsequently, a BMP2 signal emanates from a ventral pituitary organizing center that forms at the boundary of a region of oral ectoderm in which Shh expression is selectively excluded. This BMP2 signal together with a dorsal FGF8 signal, appears to create opposing activity gradients that are suggested to generate overlapping patterns of specific transcription factors underlying cell lineage specification events, whereas Wnt4 is needed for the expansion of ventral pituitary cell phenotypes. In the third phase, temporally specific loss of the BMP2 signal is required to allow terminal differentiation. The consequence of these sequential organ and cellular determination events is that each of the hormone-producing pituitary cell types-gonadotropes, thyrotropes, somatotropes, lactotropes, corticotropes, and melanotropes-appear to be determined, in a ventral-to-dorsal gradient, respectively.
467 citations
TL;DR: It is demonstrated that ETO interacts with the nuclear receptor corepressor N-CoR, the mSin3 corepressors, and histone deacetylases, suggesting that it is a component of one or more core pressor complexes.
Abstract: t(8;21) is one of the most frequent translocations associated with acute myeloid leukemia. It produces a chimeric protein, acute myeloid leukemia-1 (AML-1)-eight-twenty-one (ETO), that contains the amino-terminal DNA binding domain of the AML-1 transcriptional regulator fused to nearly all of ETO. Here we demonstrate that ETO interacts with the nuclear receptor corepressor N-CoR, the mSin3 corepressors, and histone deacetylases. Endogenous ETO also cosediments on sucrose gradients with mSin3A, N-CoR, and histone deacetylases, suggesting that it is a component of one or more corepressor complexes. Deletion mutagenesis indicates that ETO interacts with mSin3A independently of its association with N-CoR. Single amino acid mutations that impair the ability of ETO to interact with the central portion of N-CoR affect the ability of the t(8;21) fusion protein to repress transcription. Finally, AML-1/ETO associates with histone deacetylase activity and a histone deacetylase inhibitor impairs the ability of the fusion protein to repress transcription. Thus, t(8;21) fuses a component of a corepressor complex to AML-1 to repress transcription.
447 citations
TL;DR: Allosteric inhibition of RXR results from a rotation of the RXR AF-2 helix that places it in contact with the RAR coactivator-binding site, allowing RXR ligands to bind and promote the binding of a second LXXLL motif from the same SRC-1 molecule.
Abstract: Retinoic-acid receptor-α (RAR-α) and peroxisome proliferator-activated receptor-γ (PPAR-γ) are members of the nuclear-receptor superfamily that bind to DNA as heterodimers with retinoid-X receptors (RXRs)1,2. PPAR–RXR heterodimers can be activated by PPAR or RXR ligands3, whereas RAR–RXR heterodimers are selectively activated by RAR ligands only, because of allosteric inhibition of the binding of ligands to RXR by RAR4,5. However, RXR ligands can potentiate the transcriptional effects of RAR ligands in cells6. Transcriptional activation by nuclear receptors requires a carboxy-terminal helical region, termed activation function-2 (AF-2) (refs 7,8,9), that forms part of the ligand-binding pocket and undergoes a conformational change required for the recruitment of co-activator proteins, including NCoA-1/SRC-1 (refs 10,11,12,13,14,15,16,17). Here we show that allosteric inhibition of RXR results from a rotation of the RXR AF-2 helix that places it in contact with the RAR coactivator-binding site. Recruitment of an LXXLL motif of SRC-1 to RAR in response to ligand displaces the RXR AF-2 domain, allowing RXR ligands to bind and promote the binding of a second LXXLL motif from the same SRC-1 molecule. These results may partly explain the different responses of nuclear-receptor heterodimers to RXR-specific ligands.
345 citations
TL;DR: It is proposed that cross-talk between the p65 component of NF-κB and glucocorticoid receptors is due, at least in part, to nuclear competition for limiting amounts of the coactivators CBP and SRC-1, thus providing a novel mechanism for decreasing expression of genes involved in the inflammatory response.
TL;DR: There is a switch in specific requirements for histone acetyltransferase functions of CBP, or p/CAF are required for Pit-1 function that is stimulated by cyclic AMP or growth factors, respectively, showing the effects of different signal-transduction pathways on specific DNA-bound transcription factors.
Abstract: POU-domain proteins, such as the pituitary-specific factor Pit-1, are members of the homeodomain family of proteins which are important in development and homeostasis, acting constitutively or in response to signal-transduction pathways to either repress or activate the expression of specific genes1. Here we show that whereas homeodomain-containing repressors such as Rpx2 seem to recruit only a co-repressor complex, the activity of Pit-1 (ref. 3) is determined by a regulated balance between a co-repressor complex that contains N-CoR/SMRT4,5, mSin3A/B6,7,8 and histone deacetylases6,7,8 and a co-activator complex that includes the CREB-binding protein (CBP)9 and p/CAF10. Activation of Pit-1 by cyclic AMP or growth factors depends on distinct amino- and carboxy-terminal domains of CBP, respectively. Furthermore, thehistone acetyltransferase functions of CBP11,12 or p/CAF10 are required for Pit-1 function that is stimulated by cyclic AMP or growth factors, respectively. These data show that there is a switch in specific requirements for histone acetyltransferases and CBP domains in mediating the effects of different signal-transduction pathways on specific DNA-bound transcription factors.
TL;DR: It was found that the C/H3 domain is not required for retinoic acid receptor (RAR) function, nor is it involved in E1A inhibition, revealing differences in required CBP domains for transcriptional activation by RAR and STAT1.
Abstract: CREB binding protein (CBP) functions as an essential coactivator of transcription factors that are inhibited by the adenovirus early gene product E1A. Transcriptional activation by the signal transducer and activator of transcription-1 (STAT1) protein requires the C/H3 domain in CBP, which is the primary target of E1A inhibition. Here it was found that the C/H3 domain is not required for retinoic acid receptor (RAR) function, nor is it involved in E1A inhibition. Instead, E1A inhibits RAR function by preventing the assembly of CBP-nuclear receptor coactivator complexes, revealing differences in required CBP domains for transcriptional activation by RAR and STAT1.
TL;DR: A novel component of the m Sin3 complex, SAP30, binds to mSin3 and is capable of mediating transcriptional repression via histone deacetylases, suggesting that SAP30 is involved in the functional recruitment of themSin3-histone de acetylase complex to a specific subset of N-CoR corepressor complexes.
TL;DR: [DPhe11, His12]Svg(11-40), named antisauvagine-30, was the most potent and selective ligand to suppress agonist-induced adenylate cyclase activity in HEK cells expressing mCRFR2beta.
Abstract: Different truncated and conformationally constrained analogs of corticotropin-releasing factor (CRF) were synthesized on the basis of the amino acid sequences of human/rat CRF (h/rCRF), ovine CRF (oCRF), rat urocortin (rUcn), or sauvagine (Svg) and tested for their ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1), or mouse CRF receptor, type 2β (mCRFR2β). Furthermore, the potency of CRF antagonists to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRFR1 (HEK-rCRFR1 cells) or mCRFR2β (HEK-mCRFR2β cells) was determined. In comparison with astressin, which exhibited a similar affinity to rCRFR1 (Kd = 5.7 ± 1.6 nM) and mCRFR2β (Kd = 4.0 ± 2.3 nM), [dPhe11,His12]Svg(11–40), [dLeu11]Svg(11–40), [dPhe11]Svg(11–40), and Svg(11–40) bound, respectively, with a 110-, 80-, 68-, and 54-fold higher affinity to mCRFR2β than to rCRFR1. The truncated analogs of rUcn displayed modest preference (2- to 7-fold) for binding to mCRFR2β. In agreement with the results of these binding experiments, [dPhe11,His12]Svg(11–40), named antisauvagine-30, was the most potent and selective ligand to suppress agonist-induced adenylate cyclase activity in HEK cells expressing mCRFR2β.
TL;DR: The expression of β-gal, a surrogate marker of Tst-1/SCIP/Oct-6, peaks at the same stage of Schwann cell development at which development is arrested intst-4-chloro-3-indolyl-β-d-galactoside– (Bluo-gal) stained nerves showed that promyelinating Schwann cells express the highest levels ofβ-Gal, both in developing and in regenerating nerves.
Abstract: Tst-1/SCIP/Oct-6, a POU domain transcription factor, is transiently expressed by developing Schwann cells and is required for their normal development into a myelinating phenotype. Intst-1/scip/oct-6–null sciatic nerves, Schwann cells are transiently arrested at the “promyelinating” stage, when they have a one-to-one relationship with an axon but before they have elaborated a myelin sheath. To determine when Schwann cells express Tst-1/SCIP/Oct-6, we examined β-galactosidase (β-gal) expression in heterozygous tst-1/scip/oct-6 mice, in which one copy of the tst-1/scip/oct-6 gene has been replaced with the LacZ gene. β-Gal expression from the LacZ gene seems to parallel Tst-1/SCIP/Oct-6 expression from the endogenoustst-1/scip/oct-6 gene in developing and regenerating sciatic nerves. Furthermore, electron microscopic examination of 5bromo-4-chloro-3-indolyl-β-d-galactopyranoside– (X-gal) and halogenated indolyl-β-d-galactoside– (Bluo-gal) stained nerves showed that promyelinating Schwann cells express the highest levels of β-gal, both in developing and in regenerating nerves. Thus, the expression of β-gal, a surrogate marker of Tst-1/SCIP/Oct-6, peaks at the same stage of Schwann cell development at which development is arrested intst-1/scip/oct-6–null mice, indicating that Tst-1/SCIP/Oct-6 has a critical role in promyelinating Schwann cells.
TL;DR: The conserved LIM-interaction domain of Clim coregulators was used to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3.
Abstract: Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, we used the conserved LIM-interaction domain of Clim coregulators to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, we used LMO-4 as a bait to screen for such DNA-binding proteins in epidermis and isolated the mouse homologue of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix-loop-helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain.
TL;DR: resolution of the crystal structures of Oct-1 and Pit-1 POU domains bound to DNA as a monomer and homodimer confirmed several of the in vitro findings regarding interactions of this bipartite DNA binding domain with DNA and has provided important information regarding the flexibility and versatility of POU domain proteins.
Abstract: Transcription factors serve critical roles in the progressive development of general body plan, organ commitment, and finally, specific cell types. Comparison of the biological roles of a series of individual members within a family permits some generalizations to be made regarding the developmental events that are likely to be regulated by a particular class of transcription factors. Here, we evidence that the developmental functions of the family of transcription factors characterized by the POU DNA binding motif exerts roles in mammalian development.
Patent•
12 Jun 1998TL;DR: In this paper, a substantially purified nucleic acid molecule encoding a p/CIP polypeptide, which regulates the activity of CBP/p300-dependent transcription factors, is presented.
Abstract: The present invention provides a substantially purified nucleic acid molecule encoding a p/CIP polypeptide, which regulates the activity of CBP/p300-dependent transcription factors. The invention also provides a substantially purified p-CIP polypeptide and active fragments thereof. In addition, the invention provides methods of identifying an effective agent that alters the association of a p/CIP polypeptide with a second protein. Further provided herein are methods of selectively inhibiting signal transduction pathways using an active fragment of a p/CIP polypeptide or a nucleic acid molecule encoding such an acive fragment.
Journal Article•
01 Jul 1998-Journal of gravitational physiology : a journal of the International Society for Gravitational Physiology
TL;DR: Researchers studied the effect of 2G exposure on body temperature in Wild type and BRN 3.1 Knockout mice to determine the feasibility to using the animals as an animal model of the effects of altered gravitational fields on vestibular system physiology.
Abstract: Researchers studied the effect of 2G exposure on body temperature in Wild type and BRN 3.1 Knockout mice to determine the feasibility to using BRN 3.1 Knockout mice as an animal model of the effects of altered gravitational fields on vestibular system physiology.