Showing papers on "Psychological repression published in 1996"
TL;DR: It is proposed that KAP-1 may be a universal corepressor for the large family of KRAB domain-containing transcription factors and can enhance KRAb-mediated repression and is a repressor when directly tethered to DNA.
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.
633 citations
TL;DR: It is reported that Tup1 directly interacts with the amino-terminal tails of histones H3 and H4, providing a molecular basis for this connection and suggesting a mechanism whereby the organization of chromatin may be modulated in response to changing environmental signals.
Abstract: Repression of yeast a cell-specific genes by the global repressor Ssn6lTupl has been linked to a specific organization of chromatin. We report here that Tupl directly interacts with the amino-terminal tails of histones H3 and H4, providing a molecular basis for this connection. This interaction appears to be required for Tupl function because amino-terminal mutations in H3 and H4 that weaken interactions with Tupl cause derepression of both a cell-specific and DNA damage-inducible genes. Moreover, the Tupl histone-binding domain coincides with the previously defined Tupl repression domain. Tupllhistone interactions are negatively influenced by high levels of histone acetylation, suggesting a mechanism whereby the organization of chromatin may be modulated in response to changing environmental signals.
480 citations
TL;DR: It is suggested that hexose sensing in the secretory pathway is essential for mediating the activation of defense-related genes as well as repression of photosynthetic genes in vaclnv and cwlnv plants.
Abstract: Systemic acquired resistance (SAR) has been reported to be associated with lesion-mimic mutants. Tobacco plants expressing vacuolar and apoplastic yeast-derived invertase (vaclnv and cwlnv, respectively) develop spontaneous necrotic lesions similar to hypersensitive responses caused by avirulent pathogens. Therefore, SAR and metabolic alterations leading to the activation of defense-related responses were studied in these plants. Defense-related gene transcripts, callose content, peroxidase activities, and levels of salicylic acid were found to be elevated. The defense reactions were accompanied by increased resistance toward potato virus Y and were measured as decreased viral spreading and reduced multiplication in systemic leaves of the transgenic plants. Interestingly, the accumulation of pathogenesis-related (PR) protein transcripts (PR-Q) and repression of photosynthetic gene transcripts (chlorophyll a/b binding protein) were inversely correlated and required the same threshold level of hexoses for induction and repression. Expression of a cytosolic yeast-derived invertase in transgenic tobacco plants with equally increased levels of sugars neither displayed SAR responses nor showed decreased levels of photosynthetic genes. It is suggested that hexose sensing in the secretory pathway is essential for mediating the activation of defense-related genes as well as repression of photosynthetic genes in vaclnv and cwlnv plants.
365 citations
TL;DR: These findings suggest that the mutual repression of PR and RelA is due to a direct interaction between these proteins, and that NF-κB-regulating cytokine receptors are expressed in progesterone target tissues, like breast and endometrium.
352 citations
TL;DR: It is shown that the repression of MyoD-mediated E box (MyoD consensus) reporter activity by E1A is correlated with its interaction with p300, indicating that p300 participates in Myo D-dependent transactivation.
323 citations
TL;DR: The recent progress toward identification of molecular targets suggests several specific mechanisms for achieving active repression, including transcriptional inhibition, which is not mediated simply by steric hindrance mechanisms.
316 citations
TL;DR: In a linker histone H1 knockout strain (ΔH1) of Tetrahymena thermophila, the number of mature RNAs produced by genes transcribed by pol I and pol III and of most genes transcribes by pol II remains unchanged.
315 citations
TL;DR: Engrailed's active repression function in vivo is dependent on a highly conserved interaction that was established early in the evolution of the homeobox gene superfamily, and it is shown using rescue transgenes that the widely conserved in vivo repression domain is required for the normal function of engrailed in the embryo.
Abstract: The engrailed homeoprotein is a dominantly acting or ‘active’ transcriptional repressor both in cultured cells and in vivo. When retargeted via a homeodomain swap to the endogenous fushi tarazu gene (ftz), it actively represses it, resulting in a ftz mutant phenocopy. We have mapped functional regions of engrailed using this in vivo repression assay. In addition to a region containing an active repression domain identified in cell culture assays (K. Han and J. L. Manley (1993) EMBO J. 12, 2723–2733), we find that two evolutionarily conserved regions contribute to activity. The one of these that does not flank the HD is particularly crucial to repression activity in vivo. We find that this domain is present not only in all engrailed-class homeoproteins but also in all known members of several other classes, including goosecoid, Nk1, Nk2 and msh. Thus engrailed's active repression function in vivo is dependent on a highly conserved interaction that was established early in the evolution of the homeobox gene superfamily. We further show using rescue transgenes that the widely conserved in vivo repression domain is required for the normal function of engrailed in the embryo.
282 citations
TL;DR: It is shown here that a reporter gene flanked by two functional silencers is not repressed when integrated at >200 kb from a telomere, suggesting that all three factors are in limiting concentrations.
Abstract: Transcriptional repression at the silent mating-type loci in yeast requires the targeting of silent information regulator (Sir) proteins through specific interactions formed at cis-acting silencer elements. We show here that a reporter gene flanked by two functional silencers is not repressed when integrated at >200 kb from a telomere. Repression is restored by creation of a new telomere 13 kb from the integrated reporter or by elevated expression of SIRl, SIRS, and/or SIR4. Coupled expression represses in an additive manner, suggesting that all three factors are in limiting concentrations. When overexpressed, Sir3 and Sir4 are dispersed throughout the nucleoplasm, in contrast to wild-type cells where they are clustered in a limited number of foci together with telomeres. Efficient silencer function thus seems to require either proximity to a pool of concentrated Sir proteins, that is, proximity to telomeres, or delocalization of the silencing factors.
279 citations
TL;DR: The tetrameric Lac repressor can bind simultaneously to twolacoperators on the same DNA molecule, thereby including the formation of a DNA loop, and repression increased significantly with decreasing inter-operator DNA length, indicating that the local Lac repression concentration at O1 is crucial for tight repression.
272 citations
TL;DR: It is demonstrated that SMRT is a ubiquitous nuclear protein that interacts with unliganded receptor heterodimers in mammalian cells and 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.
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.
TL;DR: It is concluded that phosphorylation of the regulatory domain of HSf1 decreases the activity of HSF1 at control temperature, and a mechanism for modification ofHSF1 activity by growth control signals is proposed.
Abstract: Human heat shock transcription factor 1 (HSF1) is responsible for stress-induced transcription of heat shock protein genes. The activity of the HSF1 transcriptional activation domains is modulated by a separate regulatory domain, which confers repression at control temperature and heat inducibility. We show here that two specific proline-directed serine motifs are important for function of the regulatory domain: Mutation of these serines to alanine derepresses HSF1 activity at control temperature, and mutation to glutamic acid, mimicking a phosphorylated serine, results in normal repression at control temperature and normal heat shock inducibility. Tryptic mapping shows that these serines are the major phosphorylation sites of HSF1 at control temperature in vivo. Stimulation of the Raf/ERK pathway in vivo results in an increased level of phosphorylation of these major sites and the regulatory domain is an excellent substrate in vitro for the mitogen-activated MAPK/ERK. We conclude that phosphorylation of the regulatory domain of HSF1 decreases the activity of HSF1 at control temperature, and propose a mechanism for modification of HSF1 activity by growth control signals.
TL;DR: The results indicate that multiple nuclear receptors, utilizing different primary amino acid sequences, repress transcription by interacting with N-CoR.
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.
TL;DR: It is demonstrated that Rb can repress the level of transcription from pol III templates both in vitro and in vivo, and it is suggested that restraining protein biosynthesis may be important in the prevention of tumour development.
Abstract: Transcription by RNA polymerase (pol) III is under cell-cycle control, being higher in S and G2 than in G0 and early G1 phases. Many transformed cell types have elevated pol III activity, presumably to sustain sufficient protein synthesis for unrestrained growth. The retinoblastoma tumour-suppressor protein (Rb) restricts cellular proliferation, and is often found mutated in transformed cells. Here we demonstrate that Rb can repress the level of transcription from pol III templates both in vitro and vivo. Analysis of Rb-deficient SAOS2 cells and primary fibroblasts from Rb-/- mice demonstrates elevated levels of pol III activity in the absence of functional Rb protein. Rb-induced repression of pol III activity is alleviated by mutations in the Rb pocket domain that occur naturally in tumours, and by viral transforming proteins that bind and inactivate Rb. These results implicate repression of pol III transcription as a mechanism for Rb-induced growth arrest, and suggest that restraining protein biosynthesis may be important in the prevention of tumour development.
TL;DR: It is shown that a 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region and that SID repression is dominant over several distinct transcriptional activators.
Abstract: Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary complex with Max and mSin3A or mSin3B, the mammalian orthologs of the Saccharomyces cerevisiae transcriptional corepressor SIN3. The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A. Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity. Here we show that a 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region. A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites. In addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains. When fused to a full-length c-Myc protein, the Mad1 SID specifically represses both c-Myc's transcriptional and transforming activities. Fusions between the GAL DNA binding domain and full-length mSin3 were also capable of repression. We show that the association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices. Our results indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators.
TL;DR: The molecular basis of the absence of xylanase I formation on glucose was studied to postulate that basal transcription of xyn1 is repressed by glucose and mediated by an inverted repeat of the consensus motif for Cre1‐mediated carbon catabolite repression.
Abstract: The filamentous fungus Trichoderma reesei forms two specific, xylan-inducible xylanases encoded by xyn1 and xyn2 to degrade the beta-1,4-D-xylan backbone of hemicelluloses. This enzyme system is formed in the presence of xylan, but not glucose. The molecular basis of the absence of xylanase I formation on glucose was the purpose of this study. Northern blotting of the xyn1 transcript as well as the use of the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) as a reporter consistently showed that the basal expression of xyn1 was affected by glucose, whereas its induction by xylan remained uninfluenced. The repression of basal xyn1 transcription is mediated by the carbon catabolite repressor protein Cre1, which in vivo binds to two of four consensus sites (5'-SYG-GRG-3') in the xyn1 promoter, which occurred in the form of an inverted repeat. T. reesei strains, bearing a xyn1::hph reporter construct, in which four nucleotides from the middle of the inverted repeat had been removed, expressed hph on glucose at a level comparable to that observed during growth on a carbon catabolite derepressing carbon source. Northern analysis of xyn1 expression in a T. reesei mutant strain (RUT C-30), which contains a truncated, non-functional cre1 gene, also confirmed basal transcription of xyn1. In this strain, xyn1 transcription was still inducible by xylose or xylan to an even higher degree than in the wild-type strain, suggesting that induction overcomes glucose repression at the level of xyn1 expression. Based on these data, we postulate that basal transcription of xyn1 is repressed by glucose and mediated by an inverted repeat of the consensus motif for Cre1-mediated carbon catabolite repression.
TL;DR: Promoter elements responsible for unsaturated fatty acid-mediated transcription repression are tightly linked to OLE1 activation sequences and that Ole1 transcription levels are closely tied to acyl-CoA metabolism.
TL;DR: Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27c 3T3 cells mRNA, suggesting that the PDGF-regulated decrease inp27kIP1 expression occurred through a translational mechanism.
Abstract: We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.
TL;DR: A role is defined for the Msx-1 homeodomain, particularly the N-terminal arm residues in protein-protein interaction and transcriptional repression, and implicate a more complex role overall forHomeodomains in transcriptional regulation.
Abstract: In a previous study we showed that the murine homeodomain protein Msx-1 is a potent transcriptional repressor and that this activity is independent of its DNA binding function. The implication of these findings is that repression by Msx-1 is mediated through its association with certain protein factors rather than through its interaction with DNA recognition sites, which prompted investigation of the relevant protein factors. Here we show that Msx-1 interacts directly with the TATA binding protein (TBP) but not with several other general transcription factors. This interaction is mediated by the Msx-1 homeodomain, specifically through residues in the N-terminal arm. These same N-terminal arm residues are required for repression by Msx-1, suggesting a functional relationship between TBP association and transcriptional repression. This is further supported by the observation that addition of excess TBP blocks the repressor action of Msx-1 in in vitro transcription assays. Finally, DNA binding activity is separable from both TBP interaction and repression, which further shows that these other activities of the Msx-1 homeodomain are distinct. Therefore, these findings define a role for the Msx-1 homeodomain, particularly the N-terminal arm residues in protein-protein interaction and transcriptional repression, and implicate a more complex role overall for homeodomains in transcriptional regulation.
TL;DR: These studies establish short-range repression as a flexible form of gene regulation and suggest that the key distinction among repressors is their range of action.
Abstract: The early Drosophila embryo provides a unique system for the analysis of transcriptional repression since a broad spectrum of repressors are distributed in spatially distinct patterns. Kruppel (Kr) and snail (sna), two zinc finger repressors, are essential for segmentation and for the establishment of the mesoderm/neuroectoderm boundary, respectively. Both repressors were examined in the context of synthetic gene complexes containing modular promoters and divergently transcribed reporter genes. These studies indicate that Kr and sna function as short-range repressors, which can mediate either quenching or direct repression of the transcription complex, depending on the location of repressor sites. When located within an upstream enhancer, the repressor locally quenches nearby activators and permits other enhancers to interact with the transcription complex (enhancer autonomy). In contrast, when bound to promoter-proximal regions the repressor functions in a dominant fashion and blocks multiple enhancers. Local quenching and dominant repression require close linkage (<100 bp) of the repressor with either upstream activators or the transcription complex. These studies establish short-range repression as a flexible form of gene regulation and suggest that the key distinction among repressors is their range of action.
TL;DR: DRAP1 was found to be expressed in all tissues analyzed with higher levels in tissues with a low mitotic index and a model for DRAP1-dependent, Dr1-mediated repression of transcription is proposed.
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.
TL;DR: Results indicate that sugar sensing and establishment of catabolite repression are controlled by an interregulatory network, involving all three yeast sugar kinases and the Ras-cAMP pathway.
Abstract: Addition of rapidly fermentable sugars to cells of the yeast Saccharomyces cerevisiae grown on nonfermentable carbon sources causes a variety of short-term and long-term regulatory effects, leading to an adaptation to fermentative metabolism. One important feature of this metabolic switch is the occurrence of extensive transcriptional repression of a large group of genes. We have investigated transcriptional regulation of the SUC2 gene encoding repressible invertase, and of HXK1, HXK2 and GLK1 encoding the three known yeast hexose kinases during transition from derepressed to repressed growth conditions. Comparing yeast strains that express various combinations of the hexose kinase genes, we have determined the importance of each of these kinases for establishing the catabolite-repressed state. We show that catabolite repression involves two distinct mechanisms. An initial rapid response is mediated through any kinase, including Glk1, which is able to phosphorylate the available sugar. In contrast, long-term repression specifically requires Hxk2 on glucose and either Hxk1 or Hxk2 on fructose. Both HXK1 and GLK1 are repressed upon addition of glucose or fructose. However, fructose repression of Hxk1 is only transient, which is in line with its preference for fructose as substrate and its requirement for long-term fructose repression. In addition, expression of HXK1 and GLK1 is regulated through cAMP-dependent protein kinase. These results indicate that sugar sensing and establishment of catabolite repression are controlled by an interregulatory network, involving all three yeast sugar kinases and the Ras-cAMP pathway.
TL;DR: It is demonstrated that hTR alpha directly contacts the TATA-binding protein (TBP) and that preincubation of h TR alpha with TBP completely alleviates TR-mediated repression in vitro, and the repression domain of hTRalpha maps to the C-terminal ligand-binding region.
Abstract: Unliganded human thyroid hormone receptor alpha (hTR alpha) can repress transcription by inhibiting the formation of a functional preinitiation complex (PIC) on promoters bearing thyroid hormone receptor (TR)-binding elements. Here we demonstrate that hTR alpha directly contacts the TATA-binding protein (TBP) and that preincubation of hTR alpha with TBP completely alleviates TR-mediated repression in vitro. Using stepwise preassembled PICs, we show that hTR alpha targets either the TBP/TFIIA or the TBP/TFIIA/TFIIB steps of PIC assembly for repression. We also show that the repression domain of hTR alpha maps to the C-terminal ligand-binding region and that direct TR-TBP interactions can be inhibited by thyroid hormone. Together, these results suggest a model in which unliganded hTR alpha contacts promoter-bound TBP and interferes with later steps in the initiation of transcription.
TL;DR: Analysis of the YY1-mediated repression of the HPV type 16 (HPV-16) E6-E7 promoter revealed a cluster of five sites, located in the vicinity of an AP-1 motif, that were found to be responsible for repressing the HPV-16 P97 promoter.
Abstract: YY1 is a multifunctional transcription factor that has been shown to regulate the expression of a number of cellular and viral genes, including the human papillomavirus (HPV) oncogenes E6 and E7. In this study, we have analyzed the YY1-mediated repression of the HPV type 16 (HPV-16) E6-E7 promoter. A systematic analysis to identify YY1 sites present in the HPV-16 long control region showed that of 30 potential YY1 binding motifs, 24 bound purified recombinant YY1 protein, but only 10 of these were able to bind YY1 when nuclear extracts of HeLa cells were used. Of these, only a cluster of five sites, located in the vicinity of an AP-1 motif, were found to be responsible for repressing the HPV-16 P97 promoter. All five sites were required for repression, the mutation of any one site giving rise to a four- to sixfold increase in transcriptional activity. The target for YY1-mediated repression was identified as being a highly conserved AP-1 site, and we propose that AP-1 may represent a common target for YY1 repression. We also provide data demonstrating that YY1 can bind the transcriptional coactivator CREB-binding protein and propose a potentially novel mechanism by which YY1 represses AP-1 activity as a result of this YY1-CREB-binding protein interaction.
TL;DR: Functional assays showed that interleukin-2 regulation of BSAP concentration may provide a mechanism for controlling both repressor and activator functions of BS AP during a B cell immune response.
TL;DR: The carboxy-terminal region of mammalian Cut proteins was found to function as an active repression domain in a distance-independent manner, and this activity was narrowed to two subdomains that can independently repress activated transcription.
Abstract: By analogy with other homeodomain proteins conserved in evolution, mammalian Cut proteins are believed, as in Drosophila melanogaster, to play an important role in determining cell type specificity in several tissues. At the molecular level, Cut proteins appear to serve as transcriptional repressors. In this study, we have examined the mechanism by which the human Cut (hCut) protein down-regulates gene expression. The homeodomain and the three regions called Cut repeats are evolutionarily conserved and were previously shown to function as DNA binding domains. The carboxy-terminal region, although it does not show amino acid sequence homology per se, in all cases is enriched in alanine and proline residues, a distinctive feature of some transcriptional repression domains. Our results reveal two distinct modes of repression: competition for binding site occupancy and active repression. On one hand, the composite DNA binding domain formed by Cut repeat 3 and the Cut homeodomain was shown to bind to CCAAT and Sp1 sites within the tk gene promoter and to reduce gene expression, presumably by preventing activation by the corresponding transcription factors. On the other hand, the carboxy-terminal region of mammalian Cut proteins was found to function as an active repression domain in a distance-independent manner. We have further narrowed this activity to two subdomains that can independently repress activated transcription. Finally, we present a model to illustrate the two mechanisms by which Cut proteins repress gene expression.
TL;DR: It is shown that overexpression of Gfi-1 also inhibits cell death induced by cultivation of IL-2-dependent T-cell lines in IL- 2-deficient media and may inhibit apoptosis by means of its repression of multiple proapoptotic regulators.
Abstract: The Gfi-1 protooncogene encodes a nuclear zinc-finger protein that carries a novel repressor domain, SNAG, and functions as a position- and orientation-independent active transcriptional repressor. The Gfi-1 repressor allows interleukin 2 (IL-2)-dependent T cells to escape G1 arrest induced by IL-2 withdrawal in culture and collaborates with c-myc and pim-1 for the induction of retrovirus-induced lymphomas in animals. Here we show that overexpression of Gfi-1 also inhibits cell death induced by cultivation of IL-2-dependent T-cell lines in IL-2-deficient media. Similarly, induction of Gfi-1 in primary thymocytes from mice carrying a metal-inducible Gfi-1 transgene inhibits cell death induced by cultivation in vitro. The protein and mRNA levels of the proapoptotic regulator Bax are down-regulated by Gfi-1 in both immortalized T-cell lines and primary transgenic thymocytes. The repression is direct and depends on several Gfi-1-binding sites in the p53-inducible Bax promoter. In addition to Bax, Gfi-1 also represses Bak, another apoptosis-promoting member of the Bcl-2 gene family. Therefore, Gfi-1 may inhibit apoptosis by means of its repression of multiple proapoptotic regulators. The antiapoptotic properties of Gfi-1 provide a potential explanation for its strong collaboration with c-myc during oncogenesis.
TL;DR: It is shown that the expression of GSTP1 is regulated by the redox status of the cell, and the effect of NAC was shown to be mediated largely by the AP1 site, since mutation of this site abolished the induction by NAC.
Abstract: High levels of expression of GSTP1-1 are associated with cell proliferation, embryogenesis and malignancy. Given the role of glutathione S-transferase (GST) in detoxication, it is possible that GSTP1-1 evolved specifically to protect proliferating cells and share regulatory mechanisms with other cellular genes which are involved in cell division and tumorigenesis. We have previously shown that the expression of GSTP1 is suppressed by retinoic acid (RA) in the presence of the retinoic acid receptor (RAR) as a result of decreased transcription from its promoter. Through deletion analysis, we show here that the RA-RAR-dependent repression is mediated by the region -73 to +8. Further mutation analysis of this region indicates that the DNA sequence required for RA-RAR-dependent repression co-localizes with a consensus activator protein-1 (AP1) site essential for the promoter activity. The degree of repression correlates with the residual activity of the AP1 site. There are two adjacent G/C boxes. The one immediately downstream from the AP1 site is not essential for the promoter activity, but mutation of the second, further downstream, impairs the promoter. On the other hand, mutation of either of these two G/C boxes has little effect on RA-RAR suppression. We also show that the expression of GSTP1 is regulated by the redox status of the cell. Using the chloramphenicol acetyltransferase assay system, we have demonstrated that treatment with H2O2 induced transcription from the promoter and that this effect can be blocked by pre-incubation with N-acetylcysteine (NAC). It was shown that the induction by H2O2 is mediated by trans-acting factor NF-kappa B (nuclear factor kappa B), via a putative NF-kappa B site, 'GGGACCCTCC', located from -96 to -86. Co-transfection with an NF-kappa B (p65) expression construct increased the promoter activity, an effect which could be blocked by co-transfection with an I kappa B (MAD-3) expression construct. Deletion of the NF-kappa B site abolished the effect of both H2O2 and co-transfection of NF-kappa B. Interestingly, NAC is also an inducer for GSTP1. The effect of NAC was shown to be mediated largely by the AP1 site, since mutation of this site abolished the induction by NAC.
TL;DR: Analysis of the IE2-p53 interaction suggests that IE2 may function as a transcriptional repressor, which is recruited to p53's target genes by interacting with p53.
TL;DR: Analysis of interactions between the mating pheromone response and the osmoregulatory pathways in Saccharomyces cerevisiae suggests that the DPXDEP motif plays a role in the negative regulation of MAP kinases.
Abstract: Mitogen-activated protein (MAP) kinase cascades are conserved signal transduction pathways that are required for eukaryotic cells to respond to a variety of stimuli. Multiple MAP kinase pathways can function within a single cell type; therefore, mechanisms that insulate one MAP kinase pathway from adventitious activations by parallel pathways may exist. We have studied interactions between the mating pheromone response and the osmoregulatory (high-osmolarity glycerol response [HOG]) pathways in Saccharomyces cerevisiae which utilize the MAP kinases Fus3p and Hog1p, respectively. Inactivating mutations in HOG pathway kinases cause an increase in the phosphotyrosine content of Fus3p, greater expression of pheromone-responsive genes, and increased sensitivity to growth arrest by pheromone. Therefore, the HOG pathway represses mating pathway activity. In a HOG1+ strain, Fus3p phosphotyrosine increases modestly and transiently following an increase in the extracellular osmolarity; however, it increases to a greater extent and for a sustained duration in a hog1-delta strain. Thus, the HOG-mediated repression of mating pathway activity may insulate the mating pathway from activation by osmotic stress. A FUS3 allele whose gene product is resistant to the HOG-mediated repression of its phosphotyrosine content has been isolated. This mutant encodes an amino acid substitution in the highly conserved DPXDEP motif in subdomain XI. Other investigators have shown that the corresponding amino acid is also mutated in a gain-of-function allele of the MAP kinase encoded by the rolled locus in Drosophila melanogaster. These data suggest that the DPXDEP motif plays a role in the negative regulation of MAP kinases.