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At least 217 records · Page 12Linked to original sources
PubMed2000

Translational control of cyclin B1 mRNA during meiotic maturation: coordinated repression and cytoplasmic polyadenylation.

Translational control is prominent during meiotic maturation and early development. In this report, we investigate a mode of translational repression in Xenopus laevis oocytes, focusing on the mRNA encoding cyclin B1. Translation of cyclin B1 mRNA is relatively inactive in the oocyte and increases dramatically during meiotic maturation. We show, by injection of synthetic mRNAs, that the cis-acting sequences responsible for repression of cyclin B1 mRNA reside within its 3'UTR. Repression can be saturated by increasing the concentration of reporter mRNA injected, suggesting that the cyclin B1 3'UTR sequences provide a binding site for a trans-acting repressor. The sequences that direct repression overlap and include cytoplasmic polyadenylation elements (CPEs), sequences known to promote cytoplasmic polyadenylation. However, the presence of a CPE per se appears insufficient to cause repression, as other mRNAs that contain CPEs are not translationally repressed. We demonstrate that relief of repression and cytoplasmic polyadenylation are intimately linked. Repressing elements do not override the stimulatory effect of a long poly(A) tail, and polyadenylation of cyclin B1 mRNA is required for its translational recruitment. Our results suggest that translational recruitment of endogenous cyclin B1 mRNA is a collaborative effect of derepression and poly(A) addition. We discuss several molecular mechanisms that might underlie this collaboration.

3' Untranslated Regions↗
PubMed2001

Role of CtBP in transcriptional repression by the Drosophila giant protein.

The giant protein is a short-range transcriptional repressor that refines the expression pattern of gap and pair-rule genes in the Drosophila blastoderm embryo. Short-range repressors including knirps, Krüppel, and snail utilize the CtBP cofactor for repression, but it is not known whether a functional interaction with CtBP is a general property of all short-range repressors. We studied giant repression activity in a CtBP mutant and find that this cofactor is required for giant repression of some, but not all, genes. While targets of giant such as the even-skipped stripe 2 enhancer and a synthetic lacZ reporter show clear derepression in the CtBP mutant, another giant target, the hunchback gene, is expressed normally. A more complex situation is seen with regulation of the Krüppel gene, in which one enhancer is repressed by giant in a CtBP-dependent manner, while another is repressed in a CtBP-independent manner. These results demonstrate that giant can repress both via CtBP-dependent and CtBP-independent pathways, and that promoter context is critical for determining giant-CtBP functional interaction. To initiate mechanistic studies of the giant repression activity, we have identified a minimal repression domain within giant that encompasses residues 89-205, including an evolutionarily conserved region bearing a putative CtBP binding motif.

Alcohol Oxidoreductases↗
PubMed1989

Glucose repression of LAC gene expression in yeast is mediated by the transcriptional activator LAC9.

In the yeast Kluyveromyces lactis the beta-galactosidase gene is induced by lactose or galactose. As shown here it can also be repressed by glucose but only in some strains. When the LAC9 gene of a repressible strain is substituted by an allele of a non-repressible strain, the beta-galactosidase gene is no longer glucose repressed. LAC9 codes for a regulatory protein homologous to GAL4 which activates transcription in the presence of the inducer. Since the LAC9 product is also present in the repressed strain and binds to DNA in vitro, as shown by DNA footprinting, glucose repression cannot be caused by repression of LAC9 gene expression. Instead, our results demonstrate that glucose repression is mediated by the LAC9 gene product, and is separable from the ability of LAC9 to activate transcription.

Enzyme Induction↗
PubMed1984

Upstream repression and CRP stimulation of the Escherichia coli L-arabinose operon.

Repression of the Escherichia coli araBAD promoter, PBAD, was studied using a mutant PBAD promoter (cip-5) that is expressed in the absence of the two proteins required for PBAD induction, AraC protein and the cyclic AMP receptor protein (CRP-cAMP). Like the wild type promoter, cip-5 was repressed by AraC protein, and this repression required a site well upstream of the transcriptional start site. cip-5 was used to determine whether repression results from interference with the functioning of either AraC protein at araI and/or CRP-cAMP. Repression of cip-5 was eliminated by a point mutation within the AraC protein binding site araI but was not affected in the absence of CRP-cAMP. These results suggest that repression involves an interaction between two AraC protein binding sites located over 200 nucleotides apart. Our results also suggest that the majority of the CRP requirement for PBAD is a result of PBAD repression. When repression was abolished by deletion of the araO2 site, the requirement for CRP-cAMP in PBAD induction was greatly reduced.

AraC Transcription Factor↗
PubMed1989

trans-repression of the mouse c-fos promoter: a novel mechanism of Fos-mediated trans-regulation.

Fos protein can trans-activate AP-1-dependent gene expression and trans-repress the c-fos promoter. Although we find that trans-repression is enhanced by coexpression of c-Jun, it does not require any of the AP-1 or ATF sites in the mouse c-fos promoter. A major target for repression is the serum response element (SRE). Fos mutants with an impaired leucine zipper are defective in trans-repression and transformation, suggesting that these functions involve the formation of Fos protein complexes. In contrast, mutations that abolish DNA binding of Fos enhance trans-repression but destroy the transforming potential of Fos. In addition, v-Fos protein efficiently transforms but is unable to trans-repress. These findings point to different mechanisms involved in trans-activation and trans-repression and suggest that trans-repression of the type described here is neither sufficient nor required for Fos-induced transformation.

Animals↗
PubMed2004

PIASy-mediated repression of the androgen receptor is independent of sumoylation.

PIASy, a member of the protein inhibitor of activated STAT (PIAS) family, represses the transcriptional activity of the androgen receptor (AR). In this report, we investigate the mechanism of PIASy-mediated repression of AR. We show that AR binds to the RING-finger like domain of PIASy. PIASy contains two transcriptional repression domains, RD1 and RD2. RD1, but not RD2, is required for PIASy-mediated repression of AR. We show that the RD1 domain binds HDAC1 and HDAC2 and that HDAC activity is required for PIASy-mediated AR repression. PIAS proteins possess small ubiquitin-related modifier (SUMO) E3 ligase activity. Conjugation of SUMO-1 to AR has been implicated in the regulation of AR activity. We examine if the SUMO ligase activity of PIASy is required for PIASy to repress AR. We show that a mutant PIASy, defective in promoting sumoylation, retains the ability to repress AR transcription. In addition, mutation of all the known sumoylation acceptor sites of AR does not affect the transrepression activity of PIASy on AR. Our results suggest that PIASy may repress AR by recruiting histone deacetylases, independent of its SUMO ligase activity.

Binding Sites↗
PubMed2006

RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription.

RUNX1 (AML1) is a gene that is frequently disrupted by chromosomal translocations in acute leukemia. Like its Drosophila homolog Runt, RUNX1 both activates and represses transcription. Both Runt and RUNX1 are required for gene silencing during development and a central domain of RUNX1, termed repression domain 2 (RD2), was defined as being required for transcriptional repression and for the silencing of CD4 during T-cell maturation in thymic organ cultures. Although transcriptional co-repressors are known to contact other repression domains in RUNX1, the factors that bind to RD2 had not been defined. Therefore, we tested whether RD2 contacts histone-modifying enzymes that may mediate both repression and gene silencing. We found that RD2 contacts SUV39H1, a histone methyltransferase, via two motifs and that endogenous Suv39h1 associates with a Runx1-regulated repression element in murine erythroleukemia cells. In addition, one of these SUV39H1-binding motifs is also sufficient for binding to histone deacetylases 1 and 3, and both of these domains are required for full RUNX1-mediated transcriptional repression. The association between RUNX1, histone deacetylases and SUV39H1 provides a molecular mechanism for repression and possibly gene silencing mediated by RUNX1.

Animals↗
PubMed1997

Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite-responsive elements.

Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-Lacl family of bacterial regulatory proteins, and the seryl-phosphorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the cre in the first gene (gntR) of the gnt operon (credown), this operon contains another cre located in the promoter region (creup). A translational gntR'-'lacZ fusion expressed under the control of various combinations of wild-type and mutant credown and creup was integrated into the chromosomal amyE locus, and then catabolite repression of beta-galactosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by creup was probably independent of catabolite repression exerted by credown; both creup and credown catabolite repression involved CcpA. Catabolite repression exerted by creup was independent of P-ser-HPr, and catabolite repression exerted by credown was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize creup, in contrast to its specific recognition of credown. However, CcpA complexed with glucose-6-phosphate specifically recognized creup as well as credown, but the physiological significance of this complexing is unknown.

Bacillus subtilis↗
PubMed1991

Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product.

The aberrant overexpression of interleukin 6 (IL-6) is implicated as an autocrine mechanism in the enhanced proliferation of the neoplastic cell elements in various B- and T-cell malignancies and in some carcinomas and sarcomas; many of these neoplasms have been shown to be associated with a mutated p53 gene. The possibility that wild-type (wt) p53, a nuclear tumor-suppressor protein, but not its transforming mutants might serve to repress IL-6 gene expression was investigated in HeLa cells. We transiently cotransfected these cells with constitutive cytomegalovirus (CMV) enhancer/promoter expression plasmids overproducing wt or mutant human or murine p53 and with appropriate chloramphenicol acetyltransferase (CAT) reporter plasmids containing the promoter elements of human IL-6, c-fos, or beta-actin genes or of porcine major histocompatibility complex (MHC) class I gene in pN-38 to evaluate the effect of the various p53 species on these promoters. Murine and human wt p53 derived from pCMVNc9 and pC53-SN3, respectively, strongly repressed the IL-6 (promoter position -225 to +13), c-fos (-711 to +42), beta-actin (-3400 to +912), and MHC (-528 to -38) promoters in serum-induced HeLa cells; additionally, IL-6 promoter/CAT transcription unit constructs induced by IL-1, phorbol ester, or pseudorabies virus were also repressed by wt human and murine p53. The murine transforming mutant p53 (pCMVc5) was less active in repressing the IL-6, c-fos, beta-actin, and MHC promoter constructs. The human p53 mutant derived from pC53-SCX3 was also less active than the wt protein in repressing the IL-6, c-fos, beta-actin, and MHC promoters, except that serum-induced IL-6/CAT expression was equally repressed by both human wt and mutant p53. In similar transient transfection experiments in HeLa cells, overexpression of the wt human retinoblastoma susceptibility gene product, RB, was found to repress the serum-induced IL-6 (-225 to +13), c-fos (-711 to +42), and beta-actin (-3400 to +912) promoters but not the PRV-induced IL-6 (-110 to +13) or the serum-induced MHC (-528 to -38) promoters. These observations identify transcriptional repression as a property of p53 and suggest that p53 and RB may be involved as transcriptional repressors in modulating IL-6 gene expression during cellular differentiation and oncogenesis.

Chloramphenicol O-Acetyltransferase↗
PubMed1996

Regulatory elements that control transcription activation and unsaturated fatty acid-mediated repression of the Saccharomyces cerevisiae OLE1 gene.

In Saccharomyces cerevisiae, unsaturated fatty acids are formed from saturated acyl-CoA precursors by Ole1p, a delta-9 fatty acid desaturase. OLE1 mRNA levels are differentially regulated by the addition of saturated or unsaturated fatty acids to the growth medium. One component of this regulation system involves the control of OLE1 transcription. Saturated fatty acids induce a 1.6-fold increase in transcription activity, whereas a large family of unsaturated fatty acids repress OLE1 transcription as much as 60-fold. A deletion analysis of OLE1 promoter::lacZ fusion reporter genes identified a 111-base pair (bp) fatty acid-regulated (FAR) region approximately 580 bp upstream of the start codon that is essential for transcription activation and unsaturated fatty acid repression. Deletion of an 88-bp sequence within that region resulted in a complete loss in transcription activation and unsaturated fatty acid regulation. The 111-bp FAR element strongly activates transcription and confers unsaturated fatty acid regulation on a heterologous CYC1 promoter test plasmid. Essential elements required for unsaturated fatty acid repression of OLE1 were found in the 5 and 3 region of the 111-bp sequence. The FAR element-mediated activation and fatty acid repression of transcription was found to be closely tied to fatty acyl-CoA metabolism. Two fatty acid activation genes, FAA1 and FAA4, were found to be essential for unsaturated fatty acid repression of OLE1 through the FAR sequences. Disruption of either gene results in reduced levels of unsaturated fatty acid repression; disruption of both genes completely blocks the regulatory response. Acyl-CoA binding protein (ACBP) plays a role in determining the level of FAR element activated transcription. Disruption of the ACBP gene causes a >5-fold activation of OLE1 transcription and a similar increase in OLE1 mRNA levels. Unsaturated fatty acid repression of OLE1 transcription, however, is not affected by the disrupted ACBP gene. These studies show that 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.

Base Sequence↗
PubMed1997

Transcriptional repression by zinc finger peptides. Exploring the potential for applications in gene therapy.

A series of studies were performed to determine whether zinc finger peptides could efficiently repress transcription from RNA polymerase II promoters in vivo and to determine how such repression might depend on the position of the zinc finger binding site with respect to those of the TATA box or the initiator element. Promoter constructs were prepared with Zif268 binding sites inserted at various positions, and the activity of a reporter gene was measured in transfection studies. We found that the peptide containing the three zinc fingers of Zif268 could efficiently repress activated transcription when bound to a site near the TATA box (19-fold repression) or when bound to a site near the initiator element (18-fold repression). Repression was even more effective when the zinc finger peptide was bound to both of these sites (63-fold repression). Novel zinc finger peptides that had been selected via phage display also served as repressors of activated transcription, but repression with these proteins was somewhat less efficient than with the Zif268 peptide.

Amino Acid Sequence↗
PubMed1998

Glucose repression in Saccharomyces cerevisiae is related to the glucose concentration rather than the glucose flux.

Glucose plays an important regulatory role in the yeast Saccharomyces cerevisiae, which is mostly reflected at the transcriptional level by glucose repression. The signal that initiates glucose repression is unknown, but data indicate that it is located at or above the level of glucose 6-phosphate, suggesting the involvement of either the intracellular or extracellular glucose concentration or the glucose flux in triggering glucose repression. We have investigated the role of the glucose flux and the extracellular glucose concentration in glucose repression by growing the cells in continuous culture under nitrogen limitation. By a step-wise increase in the glucose feed concentration, the glucose flux and extracellular glucose concentrations were modulated in an accurate way. Furthermore, the glucose flux and glucose concentrations were modulated independently of each other by increasing the dilution rate or by the use of fructose as a substrate. Using these approaches we demonstrate that glucose repression is related to the extracellular (or intracellular) glucose concentration rather than the glucose flux. At external glucose concentrations lower than 14 mM, glucose repression of SUC2 gene transcription was not triggered, whereas glucose repression of this gene was activated when the glucose concentration exceeded 18 mM. A comparable effect was observed for the glucose-repressible carbon source fructose.

Dose-Response Relationship, Drug↗
PubMed1998

Repression of cyclooxygenase-2 and prostaglandin E2 release by dexamethasone occurs by transcriptional and post-transcriptional mechanisms involving loss of polyadenylated mRNA.

The two cyclooxygenase (COX) isoforms convert arachidonic acid to precursor prostaglandins (PGs). Up-regulation of COX-2 is responsible for increased PG production in inflammation and is antagonized by corticosteriods such as dexamethasone. In human pulmonary A549 cells, interleukin-1beta (IL-1beta) increases prostaglandin E2 (PGE2) synthesis via dexamethasone-sensitive induction of COX-2. Nuclear run-off assays showed that COX-2 transcription rate was repressed 25-40% by dexamethasone, while PGE2 release, COX activity, and COX-2 protein were totally repressed. At the mRNA level, complete repression of COX-2 was only observed at later (6 h) time points. Preinduced COX-2 mRNA was also potently repressed by dexamethasone, yet suppression of transcription by actinomycin D showed little effect. This dexamethasone-dependent repression involved a reduced COX-2 mRNA half-life, was blocked by actinomycin D or cycloheximide, and was antagonized by the steroid antagonist RU38486. Repression of IL-1beta-induced PGE2 release, COX activity, and COX-2 protein by actinomycin D was only effective within the first hour following IL-1beta treatment, while dexamethasone was effective when added up to 10 h later, suggesting a functional role for post-transcriptional mechanisms of repression. Following dexamethasone treatment, shortening of the average length of COX-2 mRNA poly(A) tails was observed. Finally, ligation of the COX-2 3'-UTR to a heterologous reporter failed to confer dexamethasone sensitivity. In conclusion, these data indicate a major role for post-transcriptional mechanisms in the dexamethasone-dependent repression of COX-2 that require de novo glucocorticoid receptor-dependent transcription and translation. This mechanism involves shortening of the COX-2 poly(A) tail and requires determinants other than just the 3'-UTR for specificity.

Cycloheximide↗
PubMed1999

Two novel Krüppel-associated box-containing zinc-finger proteins, KRAZ1 and KRAZ2, repress transcription through functional interaction with the corepressor KAP-1 (TIF1beta/KRIP-1).

We have isolated two novel Krüppel-like zinc finger proteins containing the evolutionarily conserved Krüppel-associated box (KRAB), KRAZ1 and KRAZ2, and demonstrated that they repress transcription when heterologously targeted to DNA. Their repression activity appeared to be mediated by the putative corepressor KAP-1 (KRAB-associated protein-1), because KRAZ1/2 bind to KAP-1, but KRAB mutants of KRAZ1/2 that are unable to interact with KAP-1 lack repression activity, and KAP-1 has intrinsic repressor activity and potentiates KRAZ1/2-mediated repression. We dissected the KAP-1 protein into a KRAB-interacting domain and a region necessary for repression. Using a mammalian two-hybrid assay, we further demonstrated that KAP-1 deletions lacking repression activity fused to the VP16 transactivation domain strongly activated transcription when coexpressed with KRAZ1. In contrast, VP16-KAP-1 fusions retaining repression activity resulted in repression. These results provide the first evidence that KAP-1 functionally interacts with KRAB in mammalian cells and seems to exert repressor activity in the DNA-bound KRAB-KAP-1 complex, and they further support the hypothesis that KAP-1 functions as a corepressor for the large class of KRAB-containing zinc finger proteins.

3T3 Cells↗
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↗
PubMed2001

The POU domain factor Skin-1a represses the keratin 14 promoter independent of DNA binding. A possible role for interactions between Skn-1a and CREB-binding protein/p300.

The genes encoding keratin 5 and 14 are highly expressed in the basal cell layer keratinocytes of the epidermis, but both genes are silenced when keratinocytes move into the suprabasal compartment. The POU homeodomain factors Skn-1a and Tst-1, which are expressed in epidermis, may play a role in the suprabasal repression of the keratin 5 and 14 genes because keratin 14 mRNA expression persists in suprabasal cells in Skn-1/Tst-1 double knockout mice. In transfection experiments, both Skn-1a and Tst-1 repress the keratin 14 promoter, with the POU domain being sufficient for repression. The region of the keratin 14 gene sufficient and required for repression by Skn-1a is a 100-base pair sequence lacking POU-binding sites adjacent to the transcription start site. DNA-binding defective mutants of Skn-1a and Tst-1 are as effective at mediating repression as the wild type proteins, suggesting that protein-protein interactions rather than direct DNA binding are important for repression. We also show that CREB-binding protein (CBP)/p300 co-activators are strong activators of keratin 14 gene expression, acting through sequences close to the keratin 14 promoter. Further, CBP interacts directly with the POU domain of Skn-1a, and increasing concentrations of CBP can overcome Skn-1a-mediated repression, suggesting that POU domain factors may repress keratin 14 gene expression by interfering with the activity of co-activators such as CBP/p300.

Animals↗
PubMed2002

Dermo-1, a multifunctional basic helix-loop-helix protein, represses MyoD transactivation via the HLH domain, MEF2 interaction, and chromatin deacetylation.

Dermo-1 is a multifunctional basic helix-loop-helix (bHLH) transcription factor that has been shown to be a potent negative regulator for gene transcription and apoptosis. To understand the molecular mechanisms that mediate the function of Dermo-1, we generated a series of Dermo-1 mutants and used a MyoD-mediated transcriptional activation model to characterize the roles of its N-terminal, bHLH, and C-terminal structural domains in transcriptional repression. Both the C-terminal and HLH domains of Dermo-1 were essential for its repression of MyoD-mediated transactivation. Dermo-1 repressed, in a dose-dependent fashion, the transactivation activity of myocyte enhancer factor 2 (MEF2), a protein known to cooperate with MyoD in activating E-box-dependent gene expression. Both the N- and C-terminal domains of Dermo-1, but not the bHLH domain, were required for the inhibition of MEF2, suggesting that Dermo-1 inhibits both MyoD- and MEF2-dependent transactivation but through different mechanisms. Dermo-1 interacted directly with MEF2 and selectively repressed the MEF2 transactivation domain. An overall increase of histone acetylation induced by trichostatin A treatment reduced Dermo-1 transcriptional repression activity, suggesting that histone deacetylation is involved in Dermo-1-mediated transcriptional repression. Together, these results suggest that MEF2 is an important target in Dermo-1-mediated transcriptional repression and provide initial evidence of the involvement of histone acetylation in Dermo-1 transcriptional repression.

Acetylation↗
PubMed2002

Murine Sall1 represses transcription by recruiting a histone deacetylase complex.

The multi-zinc finger proteins of the Sal family regulate organogenesis. Genetic evidence from Drosophila has shown that spalt (sal) can alter gene expression in a cell autonomous fashion, but Sal proteins have never been directly analyzed for their ability to activate or repress transcription. In this report, we show that a member of the Sal family, mouse Sall1, is a potent transcriptional repressor. When fused to a heterologous DNA-binding domain, Sall1 represses transcription of a luciferase reporter by over 100-fold. Expression of the N terminus alone is sufficient for dose-responsive repression that, as shown by deletion analysis, requires the extreme N-terminal amino acids of the protein. The N terminus of Sall1 can repress at both short and long range relative to the promoter, and treatment with the histone deacetylase (HDAC) inhibitor, trichostatin A, alleviates repression by 3-fold. The same regions of the protein that are required for repression physically interact with components of chromatin remodeling complexes, HDAC1, HDAC2, RbAp46/48, MTA-1, and MTA-2. Finally, we demonstrate that Sall1 is localized to discrete nuclear foci and this localization depends on the N-terminal repression domain. Together, these results suggest that the N terminus of mouse Sall1 can recruit HDAC complexes to mediate transcriptional repression.

Amino Acid Sequence↗