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PubMed1997

Transcriptional and posttranscriptional mechanisms of glucocorticoid-mediated repression of phosphoenolpyruvate carboxykinase gene expression in adipocytes.

Glucocorticoids exert pleiotropic effects, among which negative regulation of transcription has been recognized as of crucial importance. While glucocorticoids induce phosphoenolpyruvate carboxykinase (PEPCK) gene expression in liver cells, it represses gene activity in adipose cells. We used the 3T3-F442A adipocytes to analyze the underlying mechanisms in these cells, the synthetic glucocorticoid dexamethasone exerts a dominant repression either on basal or on beta-agonist stimulation of PEPCK gene expression. To determine whether glucocorticoid action required protein synthesis, we employed cycloheximide, anisomycin, and puromycin, three different translation inhibitors. None of these affected induction by isoprenaline or repression by dexamethasone of isoprenaline stimulation. In contrast, dexamethasone inhibitory action on basal PEPCK mRNA was totally prevented by the three translation inhibitors. Time courses of glucocorticoid action on basal and on induction by beta-agonist were similar. Half-maximal effect of dexamethasone on isoprenaline-induced PEPCK mRNA was obtained at about 10 nM, a tenfold higher concentration than that observed for the reduction of basal mRNA. Using the transcription inhibitor DRB, we showed that dexamethasone did not alter mRNA half-life, while isoprenaline strongly stabilized mRNA. In a 3T3-F442A stable transfectant bearing -2,100 base pairs of the PEPCK promoter fused to the chloramphenicol acetyltransferase (CAT) gene, isoprenaline stimulated CAT activity, whereas dexamethasone reduced basal and isoprenaline-induced CAT expression. Hence, beta-agonists exert both transcriptional and posttranscriptional regulation, while glucocorticoid action is purely transcriptional. However, mechanisms of glucocorticoid repression of basal and of beta-agonist stimulation appear different.

Adipocytes↗
PubMed1998

Retinoblastoma protein tethered to promoter DNA represses TBP-mediated transcription.

The retinoblastoma (RB) tumour suppressor protein negatively regulates cell proliferation by modulating transcription of growth-regulatory genes. Recruitment of Rb to promoters, by association with E2F complex or by fusion with heterologous DNA-binding domains, demonstrated that Rb represses directly transcription. Recent studies also suggest that the RB protein is able to repress gene transcription mediated by the RNA polymerase I and III. Since the TATA-binding protein (TBP) is an important component for transcription mediated by all three RNA polymerases, we have analysed the functional interaction between Rb and TBP in vivo in the context of RNA pol II-driven transcription. We demonstrated that in mammalian cells Rb tethered to promoter represses TBP-mediated activation in vivo, and Rb-mediated repression is reversed in the presence of the inhibition of histone deacetylase activity by trichostatin A (TSA).

Cell Line↗
PubMed1995

Effects of interferon-alpha on human B cells: repression of apoptosis and prevention of cell growth are independent responses of Burkitt lymphoma lines.

We have previously shown that interferon-alpha (IFN-alpha) can repress apoptosis in Burkitt lymphoma (BL) cells. In this study, we have compared this protective response with a further, well-established effect of IFN-alpha on BL cells, that of growth arrest. Of a panel of BL lines comprising (i) EBV-positive and -negative lines that retain the phenotype of the parental tumour cells and (ii) the prototype IFN-alpha-growth-inhibited line, Daudi, only Daudi cells were found to undergo substantial growth inhibition in response to the cytokine. By contrast, all lines, with the notable exception of Daudi, were protected by IFN-alpha from high-rate apoptosis initiated by the Ca2+ ionophore ionomycin. Ionomycin failed to elicit an IFN-alpha-repressible apoptotic response in either wild-type Daudi cells or IFN-resistant sublines that were refractory to the growth-arresting effects of the cytokine. Analysis of c-myc protein levels confirmed previous observations that repression of apoptosis in IFN-alpha-rescuable BL cells was associated with an early inhibition of myc that was followed by a return to high-level expression. Significantly, ionomycin alone induced a comparable transient inhibition of myc protein in Daudi cells. In Daudi cells, but not in IFN-alpha-rescuable BL cells, renewed expression of myc observed after the early, transient down-regulation was followed by sustained down-regulation of the protein, which paralleled growth arrest. Our results indicate that long-term growth arrest and repression of apoptosis in BL are distinct cellular responses to IFN-alpha.

Apoptosis↗
PubMed1983

Differential repression of specific mRNA in erythroblast cytoplasm: a possible role for free mRNP proteins.

Two types of in vivo untranslated 'free' mRNA-protein particles (mRNP) were isolated from duck erythroblast cytoplasm and characterised. Both types, namely the highly purified globin mRNA-specific '20S' mRNP and the '35S' mRNP containing a heterogenous non-globin mRNA population, are not translatable in rabbit reticulocyte lysates, but yield active mRNA upon deproteinisation. In vivo, 90% of globin mRNA is translated, but the majority of mRNA types are found in the inactive mRNP fraction, including fully repressed mRNA species. Searching for the factors controlling differential mRNA repression, we characterised and compared the protein composition of globin and '35S' mRNP using two dimensional gel electrophoresis, in vivo labelling with [35S]methionine and in vivo phosphorylation. The major proteins ubiquitously bound to globin or any other mRNA in the polyribosomes (e.g., the 73 K mol. wt. poly(A) binding protein) were not detected in purified inactive mRNP. In the latter some polypeptides appear to be associated with only one of the two inactive mRNA types while some others are common to both mRNPs. Furthermore, different rates of synthesis and phosphorylation characterize the protein populations of the two types of repressed mRNP. The specificity in composition and metabolism of the populations of polypeptides associated with different subpopulations of inactive cytoplasmic mRNA, as shown here, argues in favour of a role of mRNP proteins in mRNA recognition and selective translational repression, possibly in association with the ScRNA previously found as components of the free mRNP and able to inhibit protein synthesis.

Animals↗
PubMed1986

DNA-protein recognition: demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor.

The sequences required for full repression of the Escherichia coli deoP1 and P2 promoters by the deoR repressor (DeoR) have been analyzed in vivo. Using recombinant techniques, we have constructed a set of deo-lacZ fusions which contain different parts of the sequences involved in the regulation of deo expression on low copy number fusion vectors. Since these vectors are present in only one copy per chromosome at temperatures below 37 degrees C, this vector system allows very accurate studies of gene control signals. Our results show that three DeoR operator sites exist in the deoP1-P2 regulatory region. Two of these loci overlap the initiation sites for deoP1 (O1) and deoP2 (O2) transcription located 599 bp apart, whereas the third site (OE) is present approximately 270 bp upstream of P1. DeoR repression of both P1 and P2 transcription is weak on promoter fragments which only contain one operator site (O1 or O2). Enhanced repression by deoR is observed on promoter fragments containing two operator sites. However, all three sites are needed for full repression. These findings are discussed with respect to upstream and downstream control regions of eukaryotic genes.

Aldehyde-Lyases↗
PubMed1988

Roles of two DNA-binding factors in replication, segregation and transcriptional repression mediated by a yeast silencer.

The HMR E silencer is required for SIR-dependent transcriptional repression of the silent mating-type locus, HMR. The silencer also behaves as an origin of replication (ARS element) and allows plasmids to replicate autonomously in yeast. The replication and segregation properties of these plasmids are also dependent on the four SIR genes. We have previously characterized two DNA-binding factors in yeast extracts that recognize specific sequences at the HMR E silencer. These proteins, called ABFI (ARS-Binding Factor) and GRFI (General Regulatory Factor), are not encoded by any of the SIR genes. To investigate the biological roles of these factors, single-base-pair mutations were constructed in both binding sites at the HMR E silencer that were no longer recognized by the corresponding proteins in vitro. Our results indicate that the GRFI-binding site is required for the efficient segregation of plasmids replicated by the HMR E silencer. SIR-dependent transcriptional repression requires either an intact ABFI-binding site or GRFI-binding site, although the GRFI-binding site appears to be more important. A double-mutant silencer that binds neither ABFI nor GRFI does not mediate transcriptional repression of HMR. The replacement of HMR E with a chromosomal origin of replication (ARS1) allows partial SIR-dependent transcriptional repression of HMR, indicating a role for replication in silencer function. Together, these results suggest that the SIR proteins influence the properties of the HMR E silencer through interactions with other DNA-binding proteins.

Base Sequence↗
PubMed1991

Superhelical stress and nucleosome-mediated repression of 5S RNA gene transcription in vitro.

Nucleosomes were assembled on a plasmid carrying a Xenopus somatic 5S RNA gene prepared at different superhelix densities. The gene was preferentially assembled into a positioned nucleosome which was stable to superhelical stress. No evidence for a conformational change in the nucleosome was found, even under extreme negative superhelical stress. Transcription in an extract from Xenopus oocyte nuclei was repressed to a degree which depended on the number of nucleosomes assembled. Topoisomerase activity in the extract was effectively inhibited by camptothecin, which had no effect on transcription. Transcription of reconstitutes remained repressed relative to naked plasmids, and was independent of superhelix density. Transcripts from reconstitutes were derived solely from nucleosome-free genes. Thus, a histone octamer positioned on the gene was sufficient to block its transcription. Tryptic removal of the core histone tail domains had no effect on transcription at any superhelix density. Transcription of reconstitutes containing H3/H4 tetramers was also repressed, but not eliminated (unlike reconstitutes containing octamers), and repression was independent of superhelix density. We suggest that removal of histones H2A/H2B from the nucleosome facilitates activation of transcription in the extract. We conclude that superhelical stress alone does not activate transcription of a 5S RNA gene assembled into a nucleosome in vitro.

Animals↗
PubMed1993

Characterization of NGG1, a novel yeast gene required for glucose repression of GAL4p-regulated transcription.

The GAL1-10 genes of Saccharomyces cerevisiae are regulated by the interaction of cis- and trans-acting factors which facilitate activated transcription in galactose but not in glucose medium. By selecting mutations that allow expression of a defective gal1-10-his3 hybrid promoter, we have identified a novel gene, NGG1, which is required for glucose repression of the GAL10-related his3-G25 promoter. ngg1 was identified as a recessive null mutation that in the presence of a gal80 background resulted in a 300-fold relief of glucose repression for the his3-G25 promoter. This compared with a 20-fold and negligible relief of repression in gal80 and ngg1 strains, respectively. Deletion analysis of the his3-G25 promoter showed a correlation between the number of GAL4p binding sites and the relative level of NGG1p activity. Relief of glucose repression by NGG1 was dependent on the presence of GAL4, but was independent of the GAL4 promoter. In addition, NGG1p activity was seen for a promoter construct containing independent GAL4p binding sites. These results suggest that NGG1p acts to inhibit GAL4p function in glucose medium. We have cloned NGG1 by complementation and found that it contains an open reading frame of 2106 bp which could encode a protein with a molecular weight of 79,230.

Amino Acid Sequence↗
PubMed1994

Transcriptional repression by nucleosomes but not H1 in reconstituted preblastoderm Drosophila chromatin.

Chromatin reconstituted in an extract from preblastoderm Drosophila embryos represses transcription by RNA polymerase II. We have assembled regularly spaced nucleosomes on DNA attached to paramagnetic beads enabling the efficient purification of chromatin templates for transcription studies. We have used diagnostic salt extractions to establish that transcriptional repression of immobilized chromatin was largely due to nucleosome cores. When purified H1 was incorporated into chromatin, resulting in increased repeat lengths to 200-220 bp, the contribution of H1 to transcriptional repression was negligible. If more H1 was added no regularly spaced chromatin was obtained and only under these conditions was transcriptional inhibition by H1 apparent. We conclude that efficient repression of transcription by polymerase II in this system does not require the presence of histone H1.

Animals↗
PubMed1994

A distinct modulating domain in glucocorticoid receptor monomers in the repression of activity of the transcription factor AP-1.

Steroid receptors activate and repress genes. An important class of genes that they repress is controlled by the transcription factor AP-1. The activity of AP-1 is inhibited by the receptor, a mechanism exploited for the therapy of various forms of pathological hyperproliferation in humans. We show here by point mutations in the DNA binding domain and by the choice of steroid ligands that repression of AP-1 activity and transactivation functions of the glucocorticoid receptor (GR) are separable entities. While DNA binding and activation of glucocorticoid-regulated promoters require GR dimerization, we present data that suggest that repression is a function of GR monomers.

Amino Acid Sequence↗
PubMed1994

SPARC and thrombospondin genes are repressed by the c-jun oncogene in rat embryo fibroblasts.

The sequence-specific transcription factor c-Jun displays oncogenic potential in mammalian cells either in cooperation with activated Ras in primary embryonic fibroblasts or alone in established cell lines. Although pathways for signal transduction leading to activation of c-Jun proteins have been extensively studied, little is known about the events downstream of c-Jun stimulation. We isolated cellular genes that are targets of c-Jun by differential screening of a cDNA library from primary rat embryo fibroblasts. Two transcripts with sequences similar to known genes were repressed following transitory expression of a c-Jun-encoding vector. They correspond to the SPARC and thrombospondin 1 (TS1) genes, encoding extracellular matrix proteins. These genes are tightly regulated during embryogenesis and in adult tissues and are involved in the control of cell growth. c-Jun transitory repression of these two genes was demonstrated both in primary cells and in FR3T3, an established fibroblast cell line. The repression was also detected in FR3T3 derivatives stably transformed by c-Jun or Ras. Although c-Jun regulation of the TS1 gene was found at the promoter level, preliminary results strongly suggest that repression of SPARC and TS1 gene expression are mediated by a secreted factor. In contrast, expression of these genes was unaffected by transformation with oncogenes from DNA viruses. Our results identify new, specific, probably indirect c-Jun target genes and suggest previously unsuspected regulatory roles for SPARC and thrombospondin in the control of cell growth.

Animals↗
PubMed2006

Distinct localizations and repression activities of MM-1 isoforms toward c-Myc.

MM-1 was identified as a c-Myc-binding protein and has been reported to repress the E-box-dependent transcription activity of c-Myc by recruiting HDAC1 complex via TIF1 beta/KAP1. In this study, originally isolated MM-1 was found to be a fusion protein comprised of the N-terminal 13 amino acids from the sequence of chromosome 14 and of the rest of the amino acids from that of chromosome 12 and was found to be expressed ubiquitously in all human tissues. Four splicing isoforms of MM-1, MM-1alpha, MM-1beta, MM-1gamma, and MM-1delta, which are derived from the sequence of chromosome 12, were then identified. Of these isoforms, MM-1alpha, MM-1gamma, and MM-1delta were found to be expressed in tissue-specific manners and MM-1beta was found to be expressed ubiquitously. Although all of the isoforms potentially possessed c-Myc- and TIF1beta-binding activities, MM-1beta and MM-1delta were found to be mainly localized in the cytoplasm and MM-1alpha and MM-1gamma were found to be localized in the nucleus together with both c-Myc and TIF1beta. Furthermore, when repression activities of MM-1 isoforms toward c-Myc transcription activity were examined by reporter gene assays in HeLa cells, MM-1alpha, MM-1gamma, and MM-1gamma, but not MM-1beta, were found to repress transcription activity of c-Myc, and the degrees of repression by MM-1gamma and MM-1delta were smaller than those by MM-1 and MM-1alpha. These results suggest that each MM-1 isoform distinctly regulates c-Myc transcription activity in respective tissues.

Base Sequence↗
PubMed2006

A promoter element of the CD-RAP gene is required for repression of gene expression in non-cartilage tissues in vitro and in vivo.

The cartilage-derived retinoic acid-sensitive protein (CD-RAP) gene is expressed predominately in cartilage. Previous studies in transgenic mice have shown that the DNA promoter segment from -2,251 bp to -2,068 bp of the CD-RAP gene contains elements critical for gene expression. Subsequent studies revealed both positive and negative regulatory motifs in this 183 bp element. Here we show that this element demonstrates activation or repression of gene expression in vitro and in vivo based on cell type and content of transcription factors. The distribution of Sox (positive) and C/EBP (negative) transcription factors in cell lines and in mouse tissues is consistent with their positive and negative roles. In transgenic mice, when the 183-bp element was removed from a 3,345-bp cartilage-specific CD-RAP promoter, expression of the reporter gene became widespread, being observed in muscle, bone, lung, and liver in addition to cartilage. In vitro, mutation of the C/EBP site activated the inactive 3,345-bp CD-RAP gene promoter in myoblastic cells, suggesting that this site is responsible for (-2,079 bp) repression. These results indicate that the 183-bp element plays an important role in cartilage-specific gene expression by acting as a chondrocyte-regulatory module repressing transcription in non-chondrocytes and contributing to activation in chondrocytes. This is the first report of a functional DNA element necessary for repression in non-cartilage tissues in vivo.

Animals↗
PubMed2003

SIR-dependent repression of non-telomeric genes in Saccharomyces cerevisiae?

The proteins Sir2, Sir3 and Sir4 repress transcription of the silent mating-type loci HML and HMR and of reporter genes inserted at telomeres. Previous microarray analyses suggested that additional non-telomeric genes exist which are repressed by the Sir proteins. In this study, we tested the expression of 12 such genes by Northern analysis and RT-PCR. However, we were unable to verify their SIR-dependent regulation, which suggests that SIR-mediated repression may be restricted to the known repressed regions.

Blotting, Northern↗
PubMed1995

Further definition of the sequence and position requirements of the arginine control element that mediates repression and induction by arginine in Saccharomyces cerevisiae.

Repression or induction of the genes involved in arginine biosynthesis or catabolism, respectively, both require participation of the ArgRp/Mcm1p regulatory complex. Our previous work showed that those opposite effects were mediated by a similar arginine-responsive element of 23 nucleotides (that we now call ARC, for ARginine Control) situated close to the start of transcription in the repressed promoters and far upstream of the TATA-element in the induced promoters. To define more precisely the sequence and position requirements of the ARC element, we have now characterized by mutagenesis the promoter elements of the arginine-repressible ARG1 and ARG8 genes. We also identify a functional ARC in the CPA1 promoter, thereby confirming, in agreement with our previous mRNA pulse-labelling data, the participation of a transcriptional component in the arginine regulation of that gene otherwise submitted to a translational regulation. From the 12 ARC elements now characterized, we have derived a consensus sequence and show that such a synthetic element is able to mediate ArgRp/Mcm1p-dependent arginine regulation. An important new finding illustrated by ARG1 and CPA1, is that contrary to what all the previous data suggested, repression can be mediated by ARC elements located far upstream of the TATA-box. The new data suggest that the arginine repressor might inhibit transcription in an active process.

Arginine↗
PubMed1996

Multiple domains participate in distance-independent LAZ3/BCL6-mediated transcriptional repression.

The LAZ3/BCL6 gene implicated in diffuse large cell lymphomas encodes a transcriptional repressor containing Krüppel-fike zinc fingers. It harbours at its N-terminus a conserved protein/protein interaction motif, the BTB/POZ domain, which is also an autonomous transcriptional repression domain. We demonstrate here using several GAL4-LAZ3/BCL6 chimeras that the BTB/POZ domain plays an important but not exclusive role as its deletion gives rise to a GAL4 chimera that mediates significant, albeit reduced, transcriptional repression. Moreover, the repressive effect mediated either by LAZ3/BCL6 or by the isolated domains occurs with unaltered efficiency even at long distance (1.6 Kbp), ruling out steric hindrance mechanisms. Finally, though the absence of a TATA box appears to weaken this activity, it is largely promoter independent. Taken together, our results demonstrate that multiple domains participate in the promoter and distance-independent LAZ3/BCL6-mediated transcriptional repression.

Animals↗
PubMed1999

MBP-1 physically associates with histone deacetylase for transcriptional repression.

MBP-1, a c-myc promoter binding protein, is a mammalian transcription factor with intriguing properties including transcriptional repression of cellular genes. Recently, we have identified and characterized two different repressor domains of MBP-1. In this report, we have demonstrated that MBP-1 physically associates with histone deacetylase (HDAC), thus promoting formation of neucleosomes that inhibit transcription. Trichostatin A, an inhibitor of histone deacetylase, significantly reduces MBP-1-mediated transcriptional repression. However, MBP-1-mediated repression on c-myc promoter is resistant to histone deacetylase activity. Our results suggest that MBP-1 represses transcription by recruiting histone deacetylase as one of the mechanisms, whereas the other mechanism is resistant to HDAC activity and probably related to direct binding of promoter sequences or interaction through yet unidentified factor.

3T3 Cells↗
PubMed1999

Transcription factor NF1 mediates repression of the GLUT4 promoter by cyclic-AMP.

Prolonged treatment of 3T3-L1 adipocytes with 8-Br-cAMP decreases expression of GLUT4, the insulin-responsive glucose transporter. Expression of a promoter-reporter gene construct that contained 785 base pairs of 5'-flanking region of the murine GLUT4 gene was down regulated by 8-Br-cAMP (p < 0.001), whereas expression of constructs that contained 641 or 469 base pairs of 5'-flanking region was not. A reporter gene construct in which bases -706 to -676 were deleted was not repressed by 8-Br-cAMP, thereby identifying a 30 bp region as necessary for repression of the GLUT4 promoter by 8-Br-cAMP. Mutations in this regulatory element that disrupt binding of the transcription factor NF1 abolish the 8-Br-cAMP-induced repression of the gene. Although insulin and cAMP both repress the GLUT4 promoter through this cis-element, they appear to do this through different mechanisms, as treatment with 8-Br-cAMP does not induce the phosphorylation of NF1 that is induced by insulin treatment.

3T3 Cells↗