Showing papers on "Corepressor published in 1996"

Nuclear Receptor Coactivators and Corepressors

Abstract: The nuclear receptors belong to a superfamily of proteins, many of which are ligand-regulated, that bind to specific DNA sequences and control specific gene transcription. Recent data show that, in addition to contacting the basal transcription machinery directly, nuclear receptors inhibit or enhance transcription by recruiting an array of coactivator or corepressor proteins to the transcription complex. In this review we define the properties of these putative coregulatory factors; we describe the basal and coregulatory factors that are currently known to interact with nuclear receptors; we suggest various mechanisms by which coactivators and corepressors act; we discuss issues that are raised by the presence of multiple, perhaps competing, coregulatory factors; and we speculate how these additional regulatory layers may explain the heterogeneity of hormone responses that are observed in normal and malignant tissues.

KAP-1, a novel corepressor for the highly conserved KRAB repression domain.

Abstract: The KRAB repression domain is one of the most widely distributed transcriptional effector domains yet identified, but its mechanism of repression is unknown. We have cloned a corepressor, KAP-1, which associates with the KRAB domain but not with KRAB mutants that have lost repression activity. KAP-1 can enhance KRAB-mediated repression and is a repressor when directly tethered to DNA. KAP-1 contains a RING finger, B boxes, and a PHD finger; the RING-B1-B2 structure is required for KRAB binding and corepression. We propose that KAP-1 may be a universal corepressor for the large family of KRAB domain-containing transcription factors.

Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3

Abstract: YY1 is a mammalian zinc-finger transcription factor with unusual structural and functional features. It has been implicated as a positive and a negative regulatory factor that binds to the CCATNTT consensus DNA element located in promoters of many cellular and viral genes. A mammalian cDNA that encodes a YY1-binding protein and possesses sequence homology with the yeast transcriptional factor RPD3 has been identified. A Gal4 DNA binding domain-mammalian RPD3 fusion protein strongly represses transcription from a promoter containing Gal4 binding sites. Association between YY1 and mammalian RPD3 requires a glycine-rich region on YY1. Mutations in this region abolish the interaction with mammalian RPD3 and eliminate transcriptional repression by YY1. These data suggest that YY1 negatively regulates transcription by tethering RPD3 to DNA as a cofactor and that this transcriptional mechanism is highly conserved from yeast to human.

NAB2, a corepressor of NGFI-A (Egr-1) and Krox20, is induced by proliferative and differentiative stimuli.

Abstract: Previous work had identified a corepressor, NAB1, which represses transcriptional activation mediated by NGFI-A (also known as Egr-1, zif268, and Krox24) and Krox20. These zinc finger transcription factors are encoded by immediate-early genes and have been implicated in a wide variety of proliferative and differentiative processes. We have isolated and characterized another corepressor, NAB2, which is highly related to NAB1 within two discrete domains. The first conserved domain of NAB2 mediates an interaction with the R1 domain of NGFI-A. NAB2 represses the activity of both NGFI-A and Krox20, and its expression is regulated by some of the same stimuli that induce NGFI-A expression, including serum stimulation of fibroblasts and nerve growth factor stimulation of PC12 cells. The human NAB2 gene has been localized to chromosome 12ql3.3-14.1, a region that is rearranged in several solid tumors, lipomas, uterine leiomyomata, and liposarcomas. Sequencing of the Caenorhabditis elegans genome has identified a gene that bears high homology to both NAB1 and NAB2, suggesting that NAB molecules fulfill an evolutionarily conserved role.

The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain.

Abstract: Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.

SMRT isoforms mediate repression and anti-repression of nuclear receptor heterodimers.

Abstract: Transcriptional repression represents an important component in the regulation of cell differentiation and oncogenesis mediated by nuclear hormone receptors. Hormones act to relieve repression, thus allowing receptors to function as transcriptional activators. The transcriptional corepressor SMRT was identified as a silencing mediator for retinoid and thyroid hormone receptors. SMRT is highly related to another corepressor, N-CoR, suggesting the existence of a new family of receptor-interacting proteins. We demonstrate that SMRT is a ubiquitous nuclear protein that interacts with unliganded receptor heterodimers in mammalian cells. Furthermore, expression of the receptor-interacting domain of SMRT acts as an antirepressor, suggesting the potential importance of splicing variants as modulators of thyroid hormone and retinoic acid signaling.

A nuclear hormone receptor corepressor mediates transcriptional silencing by receptors with distinct repression domains.

Abstract: Ligand-independent transcriptional repression is an important function of nuclear hormone receptors. An interaction screen with the repression domain of the orphan receptor RevErb identified N-CoR, the corepressor for thyroid hormone receptor (TR) and retinoic acid receptor (RAR). N-CoR is likely to be a bona fide transcriptional corepressor for RevErb because (i) RevErb interacts with endogenous N-CoR, (ii) ectopic N-CoR potentiates RevErb-mediated repression, and (iii) transcriptional repression by RevErb correlates with its ability to bind N-CoR. Remarkably, a region homologous to the CoR box which is necessary for TR and RAR to interact with N-CoR is not required for RevErb. Rather, two short regions of RevErb separated by approximately 200 amino acids are required for interaction with N-CoR. The primary amino acid sequence of the N-terminal region of RevErb essential for N-CoR interaction is not homologous to that of TR or RAR, whereas similarities exist among the C-terminal domains of the receptors. N-CoR contains two adjacent but distinct interaction domains, one of which binds tightly to both RevErb and TR whereas the other binds more weakly and differentially interacts with the nuclear receptors. These results indicate that multiple nuclear receptors, utilizing different primary amino acid sequences, repress transcription by interacting with N-CoR.

Two receptor interacting domains in the nuclear hormone receptor corepressor RIP13/N-CoR.

Abstract: The thyroid hormone receptor (TR) and the retinoic acid receptor (RAR) act as transcriptional repressors when they are not occupied by their cognate ligands. This repressor function is mediated by proteins called corepressors. One of the nuclear hormone receptor corepressors, N-CoR, was originally isolated as a retinoid X receptor-interacting protein called RIP13. We have isolated a new potential variant of RIP13/N-CoR that is missing previously described transcriptional repressor domains but is similar in structure to the related corepressor termed SMRT or TRAC-2. Detailed analysis of the interaction with TR and RAR demonstrates that RIP13/N-CoR contains a new receptor interaction domain, termed ID-II, in addition to the previously described domain, referred to here as ID-I. Both ID-I and ID-II are capable of interacting independently with either TR or RAR, as assessed by the yeast two-hybrid system, by a mammalian two-hybrid system, or by direct in vitro binding. Results with all three approaches confirm that RIP13/N-CoR also interacts with retinoid X receptor, but this interaction is weaker than that with TR or RAR. Together, these results demonstrate that RIP13/N-CoR can interact with several different nuclear hormone receptors via two separate receptor interaction domains. Differences between the interactions observed in the different systems suggest that corepressor function may be modified by additional factors present in various cell types.

Requirement of a corepressor for Dr1-mediated repression of transcription.

Abstract: A Dr1-associated polypeptide (DRAP1) was isolated from HeLa cells and found to function as a corepressor of transcription. Corepressor function requires an interaction between DRAP1 and Dr1. Heterodimer formation was dependent on a histone fold motif present at the amino terminus of both polypeptides. Association of DRAP1 with Dr1 results in higher stability of the Dr1-TBP-TATA motif complex and precluded the entry of TFIIA and/or TFIIB to preinitiation complexes. DRAP1 was found to be expressed in all tissues analyzed with higher levels in tissues with a low mitotic index. Analysis of DRAP1 in the developing brain of rat demonstrated undetectable levels of DRAP1 in actively dividing cells but high levels of DRAP1 expression in differentiated non dividing cells. Dr1 was immunodetected in all cells analyzed. A model for DRAP1-dependent, Dr1-mediated repression of transcription is proposed.

The Cyc8 (Ssn6)-Tup1 corepressor complex is composed of one Cyc8 and four Tup1 subunits.

Abstract: The Cyc8 (Ssn6)-Tup1 corepressor complex is required for repression in several important regulatory systems in yeast cells, including glucose repression and mating type. Cyc8-Tup1 is recruited to target genes by interaction with diverse repressor proteins that bind directly to DNA. Since the complex has a large apparent molecular mass of 1,200 kDa on nondenaturing gels (F. E. Williams, U. Varanasi, and R. J. Trumbly, Mol. Cell. Biol. 11:3307-3316, 1991), we used a variety of approaches to determine its actual subunit composition. Immunoprecipitation of epitope-tagged complex and reconstitution of the complex from in vitro-translated proteins demonstrated that only the Cyc8 and Tup1 proteins were present in the complex. Hydrodynamic properties showed that these proteins have unusually large Stokes radii, low sedimentation coefficients, and high frictional ratios, all characteristic of asymmetry which partly accounts for the apparent high molecular weight. Calculation of native molecular weights from these properties indicated that the Cyc8-Tup1 complex is composed of one Cyc8 subunit and four Tup1 subunits. This composition was confirmed by reconstitution of the complex from Cyc8 and Tup1 expressed in vitro and analysis by one- and two-dimensional gel electrophoresis.

The extreme C terminus of progesterone receptor contains a transcriptional repressor domain that functions through a putative corepressor.

Abstract: Binding of a hormone agonist to a steroid receptor leads to the dissociation of heat shock proteins, dimerization, specific DNA binding, and target gene activation. Although the progesterone antagonist RU486 can induce most of these events, it fails to activate human progesterone receptor (hPR)-dependent transcription. We have previously demonstrated that a conformational change is a key event leading to receptor activation. The major conformational distinction between hormone- and antihormone-bound receptors occurs within the C-terminal portion of the molecule. Furthermore, hPR mutants lacking the C terminus become transcriptionally active in the presence of RU486. These results suggest that the C terminus contains a repressor domain that inhibits the transcriptional activity of the RU486-bound hPR. In this study, we have defined a 12 amino acid (12AA) region in the C terminus of hPR that is necessary and sufficient for the repressor function when fused to the C-terminal truncated hPR or to the GAL4 DNA-binding domain. Mutations in the 12AA domain (aa 917-928) generate an hPR that is active in the presence of RU486. Furthermore, overexpression of the 12AA peptide activates the RU486-bound wild-type hPR without affecting progesterone-dependent activation. These results suggest that association of the 12AA repressor region with a corepressor might inactivate hPR activity when it is bound to RU486. We propose that binding of a hormone agonist to the receptor changes its conformation in the ligand-binding domain so that association with coactivator is promoted and activation of target gene occurs.

Two Receptor Interaction Domains in the Corepressor, N-CoR/RIP13, Are Required for an Efficient Interaction with Rev-erbAα and RVR: Physical Association is Dependent on the E Region of the Orphan Receptors

Abstract: Rev-erbA alpha and RVR/Rev-erb beta/BD73 are orphan steroid receptors that have no known ligands in the 'classical sense', These 'orphans' do not activate transcription, but function as dominant transcriptional silencers, The thyroid hormone receptor (TR) and the retinoic acid receptor (RAR) act as transcriptional silencers by binding corepressors (e.g. N-CoR/RIP13 and SMRT/TRAC-2) in the absence of ligands, The molecular basis of repression by orphan receptors, however, remains obscure, and it is unclear whether these corepressors mediate transcriptional silencing by Rev-erbA alpha and RVR, Recently, two new variants of N-CoR have been described, RIP13a and RIP13 Delta 1. The characterisation of these splice variants has identified a second receptor interaction domain (ID-II), in addition to the previously characterised interaction domain (ID-I), This investigation utilised the mammalian two hybrid system and transfection analysis to demonstrate that Rev-erbA alpha and RVR will not efficiently interact with either ID-I or ID-II separately from RIP13a or RIP13 Delta 1. However, they interact efficiently with a domain composed of ID-I and ID-II from RIP13a, Interestingly, the interaction of Rev-erbA alpha and RVR is strongest with ID-I and ID-II from RIP13 Delta 1. Detailed deletion analysis of the orphan receptor interaction with RIP13/N-CoR rigorously demonstrated that the physical association was critically dependent on an intact E region of Rev-erbA alpha and RVR, Over-expression of the corepressor interaction domains (i.e. dominant negative forms of N-CoR/RIP13) could alleviate orphan receptor-mediated repression of transactivation by GALVP16, This demonstrated that these regions could function as anti-repressors. In conclusion, these data from two independent approaches demonstrate that repression by Rev-erbA alpha and RVR is mediated by an interaction of ID-I and ID-II of N-CoR, RIP13a and Delta 1 with the putative ligand binding domain of the orphan receptors.

Cell Cycle-Regulated Repression of B-myb Transcription: Cooperation of an E2F Site with a Contiguous Corepressor Element

Abstract: B-myb belongs to a group of cell cycle genes whose transcription is repressed in G0/early G1 through a binding site for the transcription factor E2F. Here, we show that the B-myb repressor element is specifically recognised by heterodimers consisting of DP-1 and E2F-1, E2F-3 or E2F-4. Surprisingly, E2F-mediated repression is dependent on a contiguous corepressor element that resembles the CHR previously established as a corepressor of the CDE in cell cycle genes derepressed in S/G2, such as cyclin A, cdc2 and cdc25C. A factor binding to the B-myb CHR was identified in fractionated HeLa nuclear extract and found to interact with the minor groove, as previously shown by in vivo footprinting for the cyclin A CHR. The B-myb and cdc25C CHRs are related with respect to protein binding but are functionally clearly distinct. Our results support a model where both E2F- and CDE-mediated repression, acting at different stages in the cell cycle, are dependent on promoter-specific CHR elements.

Sin3 corepressor function in Myc-induced transcription and transformation

Abstract: Many basic-helix-loop-helix-leucine zipper (b-HLH-LZ) proteins, including the Myc family and non-Myc family, bind a common DNA sequence CACGTG, yet have quite different biological actions. Myc binds this sequence as a heterodimer with Max in the activation of both transcription and transformation. The Myc family members Mad and Mxi1 are known to suppress Myc-induced transcription and transformation and to dimerize with Max to form ternary complexes with the mammalian Sin3 transcriptional corepressor (mSin3). The b-HLH-LZ domain of TFEB, which cannot heterodimerize within the Myc family, does not suppress Myc-induced transcription or transformation. However, transfer of a 25- to 36-aa region from Mad or Mxi1, which interacts with mSin3, to the b-HLH-LZ of TFEB, mediated profound suppression of Myc-induced transcription and transformation. These results suggest that the DNA binding specificities of the Myc family and non-Myc family b-HLH-LZ proteins, in the context of the cellular genes involved in Myc-induced transformation, are shared. The results also demonstrate that targeting mSin3 to CACGTG sites via a non-Myc family DNA binding domain is sufficient to oppose Myc activity in growth regulation.

Transcriptional silencing by unliganded thyroid hormone receptor beta requires a soluble corepressor that interacts with the ligand-binding domain of the receptor

Abstract: Unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of genes bearing thyroid hormone response elements in their promoters. Binding of hormonal ligand to the receptor releases the transcriptional silencing and leads to gene activation. Previous studies showed that the silencing activity of TR is located within the C-terminal ligand-binding domain (LBD) of the receptor. To dissect the role of the LBD in receptor-mediated silencing, we used a cell-free transcription system containing HeLa nuclear extracts in which exogenously added unliganded TRbeta repressed the basal level of RNA polymerase II-driven transcription from a thyroid hormone response element-linked template. We designed competition experiments with a peptide fragment containing the entire LBD (positions 145 to 456) of TRbeta. This peptide, which lacks the DNA-binding domain, did not affect basal RNA synthesis from the thyroid hormone response element-linked promoter when added to a cell-free transcription reaction mixture. However, the addition of the LBD peptide to a reaction mixture containing TRbeta led to a complete reversal of receptor-mediated transcriptional silencing in the absence of thyroid hormone. An LBD peptide harboring point mutations, which severely impair receptor dimerization, also inhibited efficiently the silencing activity of TR, indicating that the relief of repression by the LBD was not due to the sequestration of TR or its heterodimeric partner retinoid X receptor into inactive homo- or heterodimers. We postulate that the LBD peptide competed with TR for a regulatory molecule, termed a corepressor, that exists in the HeLa nuclear extracts and is essential for efficient receptor-mediated gene repression. We have identified the region from positions 145 to 260 (the D domain) of the LBD as a potential binding site of the putative corepressor. We observed further that a peptide containing the LBD of retinoic acid receptor (RAR) competed for TR-mediated silencing, suggesting that the RAR LBD may bind to the same corepressor activity as the TR LBD. Interestingly, the RAR LBD complexed with its cognate ligand, all-trans retinoic acid, failed to compete for transcriptional silencing by TRbeta, indicating that the association of the LBD with the corepressor is ligand dependent. Finally, we provide strong biochemical evidence supporting the existence of the corepressor activity in the HeLa nuclear extracts. Our studies demonstrated that the silencing activity of TR was greatly reduced in the nuclear extracts preincubated with immobilized, hormone-free glutathione S-transferase-LBD fusion proteins, indicating that the corepressor activity was depleted from these extracts through protein-protein interactions with the LBD. Similar treatment with immobilized, hormone-bound glutathione S-transferase-LBD, on the other hand, failed to deplete the corepressor activity from the nuclear extracts, indicating that ligand binding to the LBD disrupts its interaction with the corepressor. From these results, we propose that a corepressor binds to the LBD of unliganded TR and critically influences the interaction of the receptor with the basal transcription machinery to promote silencing. Ligand binding to TR results in the release of the corepressor from the LBD and triggers the reversal of silencing by allowing the events leading to gene activation to proceed.

Dual-function regulators: the cAMP receptor protein and the CytR regulator can act either to repress or to activate transcription depending on the context.

Abstract: Studies of gene regulation have revealed that several transcriptional regulators can switch between activator and repressor depending upon both the promoter and the cellular context. A relatively simple prokaryotic example is illustrated by the Escherichia coli CytR regulon. In this system, the cAMP receptor protein (CRP) assists the binding of RNA polymerase as well as a specific negative regulator, CytR. Thus, CRP functions either as an activator or as a corepressor. Here we show that, depending on promoter architecture, the CRP/CytR nucleoprotein complex has opposite effects on transcription. When acting from a site close to the DNA target for RNA polymerase, CytR interacts with CRP to repress transcription, whereas an interaction with CRP from appropriately positioned upstream binding sites can result in formation of a huge preinitiation complex and transcriptional activation. Based on recent results about CRP-mediated regulation of transcription initiation and the finding that CRP possesses discrete surface-exposed patches for protein-protein interaction with RNA polymerase and CytR, a molecular model for this dual regulation is discussed.

Transcriptional Repression by Rev-erbAα is Dependent on the Signature Motif and Helix 5 in the Ligand Binding Domain: Silencing Does not Involve an Interaction With N-CoR

Abstract: Rev-erbA alpha is an orphan nuclear receptor that functions as a dominant transcriptional repressor. Tissue culture and in situ hybridisation studies indicated that Rev-erbA alpha plays an important role in mammalian differentiation and development. Previous studies have localised the silencing domain of Rev-erbA alpha to the D/E region of the orphan receptor. This study utilised the GAL4 hybrid system to demonstrate that efficient repression is mediated by 34 amino acids (aa) between aa 455 and 488 in the E region of the receptor. This domain contains the ligand binding domain (LBD)-signature motif [(F/W)AKxxxxFxxLxxxDQxxLL] and a region that, according to the recently published crystal structures of steroid receptors, would be predicted to form helix 5 of the canonical LBD structure. Fine deletions and site-specific mutagenesis indicated that both the LBD signature motif and helix 5 were necessary for efficient silencing. Utilising mammalian two hybrid technology, we have also demonstrated that Rev-erbA alpha does not associate with the interaction domain (aa 2218-2451) of the nuclear receptor corepressor, N-CoR, that is known to interact with the thyroid hormone and retinoic acid receptors. This suggested that transcriptional repression by Rev-erbA alpha is not mediated through an interaction with N-CoR. In conclusion, we have identified and characterised the minimal domain of Rev-erbA alpha, that mediates transcriptional repression by this orphan receptor.

NMR studies of the mode of binding of corepressors and inducers to Escherichia coli trp repressor.

Abstract: The binding of the corepressors tryptophan and 5-methyltryptophan and of the inducers .?;-indolepropionate, 3-indoleacrylate and 5-methylindole to the E.wherichia cnli trp repressor have been studied by 'HNMR spectroscopy. Identification of the resonances of the protons of bound ligands and their NOES to protons of the protein (measured as transferred NOE) was greatly facilitated by the use of saqlcs of the protein in which the hydrogens of all residues excepl alanine, isoleucine and threonine were repluced by deuterium. Chemical-shift changes of protein-backbone resonances and side-chain-amide resonances on ligand binding were measured with generally or seleclively "N-labelled protein. The patterns of changes in the chemical shifts of protein resonances and, particularly, ligand resonances distinguish the corepressors from the inducers, indicating, in agreement with earlier work, that corepressvrs and inducers bind to the protein in different ways. The NOES observed for the bound ligands have been uscd to deterrnine the position of the ligands in the crystallographically determined binding site, by means of a siniulaterlannealing molecular-dynamics protocol. The structures obtai tied show that the oricntation in the binding site of the indole rings of tryptophan and 5-mcthyltryptophan and of 3-intlolepropionate and 3-indoleacrylate differ by approximately 180" in solution (in agreement wilh the ci-ystiillographic data for complexes of the trp repressor with tryptophan or with 3-indolepropionate). The value and limitations of calculating ligand positions based on transferred NOE are discussed. Keyworrls: NMR ; trp repressor; corepressor binding; inducer binding; tryptophan.

NMR Studies of the Escherichia coli Trp Repressor ·trpRs Operator Complex

Abstract: To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 1H-NMR spectroscopy. We report here the 1H-NMR chemical shifts of a 20-bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor. Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37-kDa complex with the Trp holorepressor. Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence. The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules. We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex. The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA. This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator.