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At least 523 records · Page 29Linked to original sources
PubMed1998

Characterization of tTA and its functional domain in tetracycline repressor-mediated gene repression system.

To elucidate of role(s) of tTA as a repressor in the tTA-mediated gene repression system, we introduced mutations into the acidic domain of VP16 and examined the effects of such various mutations. In the transient repression experiment, a region containing 34 amino acids of the activation domain of VP16 (412-456) which interacts with TFIIB was found to be necessary and sufficient for the tTA-mediated repression of gene expression. However, in the experiment to investigate the fact that tTA-regulated repression is related to the activation function of VP 16, we found that the repression abilities of tTA derivatives did not correlate exactly with their activation abilities. Therefore, we conclude that increased mass of VP16 in tTA might be also important for efficient repression in addition to functional domain of VP16.

Blotting, Western↗
PubMed1999

Transcriptional repression by the Drosophila giant protein: cis element positioning provides an alternative means of interpreting an effector gradient.

Early developmental patterning of the Drosophila embryo is driven by the activities of a diverse set of maternally and zygotically derived transcription factors, including repressors encoded by gap genes such as Krüppel, knirps, giant and the mesoderm-specific snail. The mechanism of repression by gap transcription factors is not well understood at a molecular level. Initial characterization of these transcription factors suggests that they act as short-range repressors, interfering with the activity of enhancer or promoter elements 50 to 100 bp away. To better understand the molecular mechanism of short-range repression, we have investigated the properties of the Giant gap protein. We tested the ability of endogenous Giant to repress when bound close to the transcriptional initiation site and found that Giant effectively represses a heterologous promoter when binding sites are located at -55 bp with respect to the start of transcription. Consistent with its role as a short-range repressor, as the binding sites are moved to more distal locations, repression is diminished. Rather than exhibiting a sharp 'step-function' drop-off in activity, however, repression is progressively restricted to areas of highest Giant concentration. Less than a two-fold difference in Giant protein concentration is sufficient to determine a change in transcriptional status of a target gene. This effect demonstrates that Giant protein gradients can be differentially interpreted by target promoters, depending on the exact location of the Giant binding sites within the gene. Thus, in addition to binding site affinity and number, cis element positioning within a promoter can affect the response of a gene to a repressor gradient. We also demonstrate that a chimeric Gal4-Giant protein lacking the basic/zipper domain can specifically repress reporter genes, suggesting that the Giant effector domain is an autonomous repression domain.

Animals↗
PubMed1997

Transcriptional activation and repression by RORalpha, an orphan nuclear receptor required for cerebellar development.

Mutation of the orphan nuclear receptor RORalpha results in a severe impairment of cerebellar development by unknown mechanisms. We have found that RORalpha activates transcription from only a subset of sites to which it binds strongly as a monomer. RORalpha also selectively binds as a homodimer to a direct repeat of this monomer site with a 2-bp spacing between the AGGTCA sequences (Rev-DR2 site) and is a much more potent transcriptional activator on this site than on monomer sites or other direct repeats. To better understand the transcriptional regulatory functions of RORalpha, we fused its C terminus to a heterologous DNA-binding domain. Mutational analysis revealed that RORalpha contains both transcriptional activation and transcriptional repression domains, with the repression domain being more active in some cell types. The abilities of RORalpha polypeptides to repress transcription correlate with their abilities to interact with the nuclear receptor corepressors N-CoR and SMRT in vitro. However, the AF2 region of RORalpha inhibits corepressor interaction on DNA, consistent with the lack of repression by the full-length receptor. Thus, transcriptional regulation by RORalpha is complex and likely to be regulated in a cell type- and target gene-specific manner.

Animals↗
PubMed2004

The CCAAT enhancer-binding protein (C/EBP)beta and Nrf1 interact to regulate dentin sialophosphoprotein (DSPP) gene expression during odontoblast differentiation.

Terminal differentiation of odontoblasts, the principal cells in dentin formation, proceeds by synthesis of type I collagen and noncollagenous proteins. DSP and DPP are specific markers for terminally differentiated odontoblasts and are encoded by a single gene DSPP (dentin sialophosphoprotein). In an attempt to understand the molecular mechanisms required for tissue-specific expression of the DSPP gene, we have identified a novel interaction between two bZIP transcription factors, Nrf1 and the CCAAT enhancer-binding protein (C/EBP)beta. This interaction was confirmed by both immunoprecipitation and chromatin immunoprecipitation assays. In undifferentiated odontoblasts, Nrf1 and C/EBPbeta repress DSPP promoter activity individually and synergistically by cooperatively interacting with each other. This mutual interaction is facilitated by the bZIP domains in both the proteins. The repression domain in both Nrf1 and C/EBPbeta was determined, and deletion of this domain abolished transcriptional repression. In fully differentiated odontoblasts, the loss of interaction between Nrf1 and C/EBPbeta results in an increased DSPP transcription. Further, this interaction was found to be dependent on phosphorylation at Ser(599) of Nrf1. Thus, the physical interaction between Nrf1 and C/EBPbeta provide a novel mechanism for the transcriptional regulation of DSPP in odontoblasts.

Animals↗
PubMed1999

Transcriptional repression by the Caenorhabditis elegans germ-line protein PIE-1.

In the early Caenorhabditis elegans embryo, maternally expressed PIE-1 protein is required in germ-line blastomeres to inhibit somatic differentiation, maintain an absence of mRNA transcription, and block phosphorylation of the RNA polymerase II large subunit (Pol II) carboxy-terminal domain (CTD). We have determined that PIE-1 can function as a transcriptional repressor in cell culture assays. By fusing PIE-1 sequences to the yeast GAL4 DNA-binding domain, we have identified a PIE-1 repression domain that appears to inhibit the transcriptional machinery directly. A sequence element that is required for this repressor activity is similar to the Pol II CTD heptapeptide repeat, suggesting that the PIE-1 repression domain might target a protein complex that can bind the CTD. An alteration of this sequence element that blocks repression also impairs the ability of a transgene to rescue a pie-1 mutation, suggesting that this repressor activity may be important for PIE-1 function in vivo.

Amino Acid Sequence↗
PubMed1998

p53 suppresses cytokine induced, Stat5 mediated activation of transcription.

p53 is a tumor suppressor which exerts its function through the regulation of genes mediating cell cycle arrest and the induction of apoptosis. Cellular survival and proliferation can be positively regulated through the action of cytokines. These signals act through the activation of cell surface receptors, and the phosphorylation of intracellular signaling components, e.g. members of the Stat family (signal transducers and activators of transcription). The signaling effects of p53 and the cytokine receptors on the cellular phenotype are counteracting. We investigated the influence of p53 on the transactivation potential of Stat5. p53 repressed the prolactin induction of the Stat5 mediated transcription of the beta-casein promoter-luciferase reporter gene, but did not affect IFN-gamma induced, Stat1 dependent transcription of the IRF-1 promoter. The inhibition was not due to a decrease in the cellular concentration of Stat5 or to interference with its specific DNA binding activity. No repression of the basal transcriptional activity of the beta-casein promoter was observed. p53 mutants defective in their DNA binding or oligomerization functions had only weak inhibitory effects, but a mutant of p53 in the transactivation domain, efficiently repressed Stat5 dependent induction. The repressive function of p53 on Stat5 activity is independent of the amino-terminal transactivation domain, but requires a functional DNA binding domain and the carboxyl-terminal domain. Our experiments show that p53 counteracts Stat5 mediated cytokine induction of gene transcription. The effect is specific for Stat5 and independent of p53 induced apoptosis.

Animals↗
PubMed1994

Functional dissection of the yeast Cyc8-Tup1 transcriptional co-repressor complex.

DNA-binding repressor proteins mediate regulation of yeast genes by cell type (Mcm1/alpha 2 and a1/alpha 2), glucose (Mig1) and oxygen (Rox1) (refs 1-4 respectively). An unusual feature of all these regulatory pathways is that transcriptional repression requires two physically associated proteins that do not bind DNA Cyc8(Ssn6) and Tup1. The Cyc8-Tup1 complex has been proposed to be a co-repressor that is recruited to target promoters by pathway-specific DNA-binding proteins, but the specific functions of the individual proteins are unknown. Here we show that when it is bound upstream of a functional promoter through the LexA DNA-binding domain, Tup1 represses transcription in the absence of Cyc8. Deletion analysis indicates that Tup1 contains at least two non-overlapping transcriptional repression regions with minimal primary sequence similarity, and a separable Cyc8-interaction domain. These Tup1 domains, which do not include the beta-transducin motifs, are necessary and partially sufficient for Tup1 function. We suggest that Tup1 performs the repression function of the Cyc8-Tup1 co-repressor complex, and that Cyc8 serves as a link with the pathway-specific DNA-binding proteins.

Amino Acid Sequence↗
PubMed1998

Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-kappaB downregulation in transcriptional repression of the p40 gene.

Interleukin 12 (IL-12), produced by myelomonocytic cells, plays a pivotal role in the development of T helper 1 (Th1) cells, which are involved in the pathogenesis of chronic inflammatory autoimmune disorders. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] inhibits IL-12 production by activated macrophages and dendritic cells, thus providing a novel interpretation to its immunosuppressive properties. 1,25(OH)2D3 significantly inhibits mRNA expression for both IL-12 p35 and p40 subunits acting at the transcriptional level. The effect of 1,25(OH)2D3 on p40 promoter activation was analyzed by cotransfecting monocytic RAW264.7 cells with p40 promoter/reporter constructs and expression vectors for vitamin D3 receptor (VDR) and/or retinoid X receptor (RXRalpha). We observed transcriptional repression of the p40 gene by 1,25(OH)2D3, which required coexpression of VDR with RXR and an intact VDR DNA-binding domain. The repressive effect maps to a region in the p40 promoter containing a binding site for NF-kappaB (p40-kappaB). Deletion of the p40-kappaB site abrogates part of the inhibitory effect on the p40 promoter, confirming the functional relevance of this site. Activation of monocytic THP-1 cells in the presence of 1,25(OH)2D3 results in reduced binding to the p40-kappaB site. Thus, 1,25(OH)2D3 may negatively regulate IL-12 production by downregulation of NF-kappaB activation and binding to the p40-kappaB sequence.

Animals↗
PubMed1998

The t(3;21) fusion product, AML1/Evi-1, interacts with Smad3 and blocks transforming growth factor-beta-mediated growth inhibition of myeloid cells.

The t(3;21)(q26;q22) chromosomal translocation associated with blastic crisis of chronic myelogenous leukemia results in the formation of the AML1/Evi-1 chimeric protein, which is thought to play a causative role in leukemic transformation of hematopoietic cells. Here we show that AML1/Evi-1 represses growth-inhibitory signaling by transforming growth factor-beta (TGF-beta) in 32Dcl3 myeloid cells. The activity of AML1/Evi-1 to repress TGF-beta signaling depends on the two separate regions of the Evi-1 portion, one of which is the first zinc finger domain. AML1/Evi-1 interacts with Smad3, an intracellular mediator of TGF-beta signaling, through the first zinc finger domain, and represses the Smad3 activity, as Evi-1 does. We also show that suppression of endogenous Evi-1 in leukemic cells carrying inv(3) restores TGF-beta responsiveness. Taken together, AML1/Evi-1 acts as an inhibitor of TGF-beta signaling by interfering with Smad3 through the Evi-1 portion, and both AML1/Evi-1 and Evi-1 repress TGF-beta-mediated growth suppression in hematopoietic cells. Thus, AML1/Evi-1 may contribute to leukemogenesis by specifically blocking growth-inhibitory signaling of TGF-beta in the t(3;21) leukemia.

Animals↗
PubMed1992

DNA binding by c-Ets-1, but not v-Ets, is repressed by an intramolecular mechanism.

The E26 avian retrovirus causes an acute leukemia in chickens and transforms both myeloid and erythroid cells. The virus encodes a 135 kDa fusion protein which contains amino acid sequences derived from the viral Gag protein and the two cellular transcription factors c-Myb and c-Ets-1p68. Previously we have shown that like v-myb, v-ets on its own is also active in transformation, but only within the erythroid lineage. To understand better the mechanisms involved in the oncogenic activation of c-Ets-1p68, we used the polyoma PEA3 element, a known Ets binding site, to compare the sequence-specific DNA binding and transactivating properties of v-Ets and c-Ets-1p68. Using Ets protein synthesized in rabbit reticulocyte lysate in gel retardation assays, we detected little binding of c-Ets-1p68 to an oligonucleotide containing the PEA3 motif whereas v-Ets bound strongly. However, in transient cotransfection assays in chicken embryo fibroblasts both c-Ets-1p68 and v-Ets transactivated transcription from a heterologous promoter linked to PEA3 elements. Interestingly, fragments of c-Ets-1p68 with strong DNA binding activity could be produced by limited proteolysis, indicating that the DNA binding domain is repressed within the full-length molecule. By deletion mapping the DNA binding domain was localized to the most highly conserved region of the Ets-related proteins known as the ETS domain. The C-terminus as well as a region in the middle of the polypeptide chain are involved in repression of DNA binding in c-Ets-1p68. Significantly, v-Ets contains a 16 amino acid substitution at the C-terminus. Our results suggest that intramolecular repression of DNA binding is a regulatory mechanism in c-Ets-1p68 which is lost in v-Ets.

Animals↗
PubMed2000

PRH represses transcription in hematopoietic cells by at least two independent mechanisms.

PRH (proline-rich homeodomain protein) is strongly expressed in the hematopoietic compartment. Here we show that PRH is a repressor of transcription in hematopoietic cells. A fragment of PRH that includes the homeodomain can bind to TATA box sequences in vitro and can also bind to the TATA box-binding protein. PRH represses transcription from TATA box-containing promoters in intact cells but does not repress transcription from a promoter lacking a TATA box. A mutation in the PRH homeodomain that blocks binding to DNA but that has little or no effect on binding to the TATA box-binding protein significantly reduces the ability of the protein to repress transcription and provides the first clear demonstration that a homeodomain can bring about transcriptional repression in vivo by binding to a TATA box. However, we also show that mutation of the PRH homeodomain does not block the ability of PRH to repress transcription when this protein is tethered upstream of the TATA box via a heterologous DNA-binding domain. PRH also contains an N-terminal proline-rich repression domain that is separate from the homeodomain. Deletion mapping suggests that this repression domain contains at least two regions that both independently contribute to transcriptional repression.

Binding Sites↗
PubMed2000

Nuclear pore complexes in the organization of silent telomeric chromatin.

The functional regulation of chromatin is closely related to its spatial organization within the nucleus. In yeast, perinuclear chromatin domains constitute areas of transcriptional repression. These 'silent' domains are defined by the presence of perinuclear telomere clusters. The only protein found to be involved in the peripheral localization of telomeres is Yku70/Yku80. This conserved heterodimer can bind telomeres and functions in both repair of DNA double-strand breaks and telomere maintenance. These findings, however, do not address the underlying structural basis of perinuclear silent domains. Here we show that nuclear-pore-complex extensions formed by the conserved TPR homologues Mlp1 and Mlp2 are responsible for the structural and functional organization of perinuclear chromatin. Loss of MLP2 results in a severe deficiency in the repair of double-strand breaks. Furthermore, double deletion of MLP1 and MLP2 disrupts the clustering of perinuclear telomeres and releases telomeric gene repression. These effects are probably mediated through the interaction with Yku70. Mlp2 physically tethers Yku70 to the nuclear periphery, thus forming a link between chromatin and the nuclear envelope. We show, moreover, that this structural link is docked to nuclear-pore complexes through a cleavable nucleoporin, Nup145. We propose that, through these interactions, nuclear-pore complexes organize a nuclear subdomain that is intimately involved in the regulation of chromatin metabolism.

Antigens, Nuclear↗
PubMed1999

Solution structure of the receptor tyrosine kinase EphB2 SAM domain and identification of two distinct homotypic interaction sites.

The sterile alpha motif (SAM) is a protein interaction domain of around 70 amino acids present predominantly in the N- and C-termini of more than 60 diverse proteins that participate in signal transduction and transcriptional repression. SAM domains have been shown to homo- and hetero-oligomerize and to mediate specific protein-protein interactions. A highly conserved subclass of SAM domains is present at the intracellular C-terminus of more than 40 Eph receptor tyrosine kinases that are involved in the control of axonal pathfinding upon ephrin-induced oligomerization and activation in the event of cell-cell contacts. These SAM domains appear to participate in downstream signaling events via interactions with cytosolic proteins. We determined the solution structure of the EphB2 receptor SAM domain and studied its association behavior. The structure consists of five helices forming a compact structure without binding pockets or exposed conserved aromatic residues. Concentration-dependent chemical shift changes of NMR signals reveal two distinct well-separated areas on the domains' surface sensitive to the formation of homotypic oligomers in solution. These findings are supported by analytical ultracentrifugation studies. The conserved Tyr932, which was reported to be essential for the interaction with SH2 domains after phosphorylation, is buried in the hydrophobic core of the structure. The weak capability of the isolated EphB2 receptor SAM domain to form oligomers is supposed to be relevant in vivo when the driving force of ligand binding induces receptor oligomerization. A formation of SAM tetramers is thought to provide an appropriate contact area for the binding of a low-molecular-weight phosphotyrosine phosphatase and to initiate further downstream responses.

Amino Acid Motifs↗
PubMed1995

WT1, the Wilms' tumor suppressor gene product, represses transcription through an interactive nuclear protein.

The Wilms' tumor suppressor gene, wt1, encodes a transcription factor of the zinc finger family. Mutations in WT1 have been detected in subsets of Wilms' tumor and in patients with the Denys-Drash Syndrome. In order to determine how WT1 regulates transcription and perhaps the consequences that mutations in WT1 may have, we established that residues 85-124 and 181-250 of WT1 constitute domains that function independently with a DNA binding domain to repress or activate transcription, respectively, and function equally effectively with heterologous promoters, suggesting the activator and repressor domains interact with nuclear components of general importance. To seek evidence for such components, increasing concentrations of WT1 repressor domain without a zinc finger DNA binding domain were co-transfected with fixed concentrations of wild-type (wt) WT1 and PDGF A-chain promoter/reporter gene constructs. As levels of the repressor domain were increased, a progressive loss of wt WT1 repressor activity and a progressive increase in its activation were observed, suggesting that the repressor domain of WT1 competes with wt WT1 for an interactive protein that is an essential component of the repressor activity of wt WT1. Because the most common mutation associated with Denys-Drash Syndrome disrupts the zinc finger domains of WT1, the results also suggest that the mutant WT1 may have aberrant DNA binding activity and perhaps function as a dominant negative effector of wt WT1.

3T3 Cells↗
PubMed2005

CHES1/FOXN3 interacts with Ski-interacting protein and acts as a transcriptional repressor.

Checkpoint Suppressor 1 (CHES1; FOXN3) encodes a member of the forkhead/winged-helix transcription factor family. The human CHES1 cDNA was originally identified by its ability to function as a high-copy suppressor of multiple checkpoint mutants of Saccharomyces cerevisiae. Accumulating expression profile data suggest that CHES1 plays a role in tumorigenicity and responses to cancer treatments, though nothing is known regarding the transcriptional function of CHES1 or other FOXN proteins in human cells. In this report, we find that the carboxyl terminus of CHES1 fused to a heterologous DNA binding domain consistently represses reporter gene transcription in cell lines derived from tumor tissues. Using a cytoplasmic two-hybrid screening approach, we find that this portion of CHES1 interacts with Ski-interacting protein (SKIP; NCoA-62), which is a transcriptional co-regulator known to associate with repressor complexes. We verify this interaction through co-immunoprecipitation experiments performed in mammalian cells. Further analysis of the CHES1/SKIP interaction indicates that CHES1 binds to a region within the final 66 hydrophobic residues of SKIP thus defining a new protein-protein interaction domain of SKIP. These data suggest that CHES1 recruits SKIP to repress genes important for tumorigenesis and the response to cancer treatments.

Blotting, Western↗
PubMed1994

Overlapping domains on the p53 protein regulate its transcriptional activation and repression functions.

Wild-type p53 has been shown to inhibit transcription from several viral and cellular promoters without known p53-binding sites, while transactivating promoters with p53-binding sites. Using a series of N- and C-terminal p53 deletion mutants and wild-type p53, we have defined the domains on p53 responsible for its transcriptional functions. To test transcriptional activation by p53 we have used a promoter-chloramphenicol acetyltransferase (CAT) construct containing synthetic p53-binding sites. To check transcriptional inhibition by p53 we have used a human cytomegalovirus immediate-early promoter construct, CMV-CAT. Using transient transfection-transcription assays in Saos-2 cells, we determined that the p53 transcriptional activation and repression domains overlap at the N-terminus. This suggests the possibility that the same transcriptional machinery is involved in both functions. A C-terminal deletion up to amino acid 327 (del 393-327) eliminated repression of CMV-CAT, while preserving the transactivation function to a large extent. Using gluteraldehyde cross-linking experiments, we observed that the mutant del 393-327, which is transactivation-competent, but repression-defective, could not oligomerize. Thus, oligomerization of p53 is not required for transactivation, but may be essential for repression. Interestingly, transactivation by the oligomerization-defective mutant could be inhibited by cotransfection with a plasmid expressing the transforming mutant p53-175H.

Gene Deletion↗
PubMed1991

Regulation of smooth muscle myosin light chain kinase by calmodulin.

The mutagenesis work described in this paper has been instrumental in furthering our understanding of how CaM binds to and activates MLCK. Figure 2 schematically represents this interaction. The inactive MLCK appears to have a catalytic domain that is repressed by a substrate inhibitory domain that overlaps with the CaM binding domain, a basic amphipathic helix. In the presence of Ca2+, CaM undergoes a conformational change that exposes two hydrophobic pockets, one in each globular lobe, that are important for binding to MLCK. Upon binding CaM, MLCK undergoes a conformational change that derepresses the catalytic site, allows substrate access and light chain phosphorylation. Calmodulin antagonist drugs intercalate within these hydrophobic pockets to interfere with target enzyme binding. The total loss of activity if W800 is altered to A illustrates the importance of these hydrophobic interactions within the enzyme. The basic residues are also important; most of the basic residues in the binding domain of MLCK appear to aid in CaM binding but are not in themselves crucial, this includes the RRK triad. However, a specific electrostatic interaction between R812 of MLCK and CaM is suggested by the complete failure in MLCK activation if this residue is changed to an A. Electrostatic interactions between MLCK and CaM are also indicated by the TaM-BM1 mutant. This mutant can bind to but not activate MLCK. It is hypothesized that TaM-BM1 will bind to the basic amphipathic helix of MLCK but that the alterations in the surface charges (especially E14 and T34) and/or hydrophobicity (S38) prevent the proper conformational change in MLCK necessary for light chain phosphorylation. The resulting MLCK-CaM complex is therefore, inactive but can bind TaM-BM1. The exact interaction of these amino acids in CaM with MLCK will have to await the elucidation of a CaM-MLCK co-crystal.

Amino Acid Sequence↗
PubMed1987

Colocalization of DNA-binding and transcriptional activation functions in the human glucocorticoid receptor.

Using a combination of a transient expression assay and in vitro mutagenesis, we showed previously that the human glucocorticoid receptor (hGR) is composed of a series of discrete functional domains. Here we report the effects of selective deletion of each of these domains on hGR ability to activate transcription of the MTV-CAT fusion gene. Deletion of the immunogenic domain or the entire amino-terminal half of the protein reduces but does not abolish the ability of the hGR to induce transcriptional activation. Somewhat surprisingly, deletion of the steroid-binding domain engenders a constitutively active receptor, revealing that this domain normally represses receptor function. However, the central, cysteine-rich region contains all the information required for both DNA binding and trans-activation. Taken together, these data delineate a core domain in the hGR spanning 88 amino acids that determines both DNA-binding and transcriptional activation functions. This physical linkage distinguishes the glucocorticoid receptor from other described eukaryotic regulatory proteins, where these two functions have been shown to be separable.

Animals↗