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PubMed1992

Repression and activation of the Drosophila dopa decarboxylase gene in glia.

Glial expression of the Drosophila dopa decarboxylase gene (Ddc) is repressed by a regulatory region located approximately 1 kb upstream of the transcriptional start site. We have used in vitro mutagenesis and germ line transformation to determine which elements within the Ddc promoter mediate repression. Our evidence suggests that the hypodermal cell activator elements IIA and IIB play a major role in the transcriptional regulation of Ddc in glial cells. A variety of mutations demonstrate that element IIA is a strong glial activator element and that element IIB is necessary for glial repression. Although these two regulatory elements are nearly identical in sequence, our data suggest that they are not redundant. Altering the wild-type number and spacing of elements IIA and IIB indicates that the wild-type arrangement of this repeat is critical for repression. We conclude that these key elements of the Ddc promoter regulate both activation and repression in glia.

Animals↗

PubMed1992

Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid.

The gene coding for apolipoprotein AI (apoAI), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine. Liver-specific expression is controlled by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located between nucleotides -222 and -110 upstream of the apoAI gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXR alpha over the previously characterized RA receptors RAR alpha and RAR beta. In this study we investigated the combined effects of ARP-1 and RXR alpha on apoAI gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXR alpha-mediated transactivation of the apoAI gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXR alpha bind to site A with similar affinities. These assays also revealed that ARP-1 and RXR alpha bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXR alpha alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAI gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXR alpha has very little effect on the levels of expression regardless of the presence or absence of RA. However, while ARP-1 alone or ARP-1 and RXR alpha together dramatically repress expression in the absence of RA, the repression by ARP-1 and RXR alpha together, but not ARP-1 alone, is almost completely alleviated in the presence of RA. These results indicate that transcriptional repression by ARP-1 sensitizes apoAI gene responsiveness to RXR alpha and RA and suggest that the magnitude of this responsiveness is regulated by the intracellular ratio of ARP-1 to RXR alpha. These observations raise the possibility that transcriptional repression is a general mechanism for switching gene transcription between alternative transcription activation pathways.

Apolipoprotein A-I↗

PubMed1993

The yeast alpha 2 protein can repress transcription by RNA polymerases I and II but not III.

The alpha 2 protein of the yeast Saccharomyces cerevisiae normally represses a set of cell-type-specific genes (the a-specific genes) that are transcribed by RNA polymerase II. In this study, we determined whether alpha 2 can affect transcription by other RNA polymerases. We find that alpha 2 can repress transcription by RNA polymerase I but not by RNA polymerase III. Additional experiments indicate that alpha 2 represses RNA polymerase I transcription through the same pathway that it uses to repress RNA polymerase II transcription. These results implicate conserved components of the transcription machinery as mediators of alpha 2 repression and exclude several alternate models.

Base Sequence↗

PubMed1994

Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation.

Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.

Animals↗

PubMed1994

Nucleosomal location of the STE6 TATA box and Mat alpha 2p-mediated repression.

It has been proposed that yeast MATa cell-specific genes are repressed in MAT alpha cells by the Mat alpha 2p repressor-directed placement of a nucleosome in a position that incorporates the TATA box of the MATa-specific gene close to the nucleosomal pseudodyad. In this study, we address this proposal directly with a series of plasmids designed to place the MATa-specific STE6 TATA box at different locations in a nucleosome and in the internucleosomal linker. These plasmids contain different lengths of synthetic random DNA between the Mat alpha 2p operator and the TATA box of the STE6 promoter, which is located upstream of a lacZ reporter gene in a multicopy plasmid. We show that in MAT alpha cells, a nucleosome is retained in an identical translational frame relative to the Mat alpha 2p operator in all the constructs investigated, irrespective of the sequence of the DNA wrapped onto the histone octamer. This result shows that the nucleosomal organization of the STE6 promoter in MAT alpha cells is not conferred by the sequence of the promoter itself. No expression of the lacZ reporter gene was detectable in MAT alpha cells in any of the constructs, even with the TATA box located in a short internucleosomal linker. These data indicate that repression of MATa-specific genes in MAT alpha cells does not require the precise translational placement of the TATA box close to the nucleosomal pseudodyad; the gene remains repressed when the TATA box is located within the investigated 250-bp region in the organized chromatin domain abutting the Mat alpha 2p operator in MAT alpha cells and may remain repressed with the TATA box located anywhere within this organized repression domain.

ATP-Binding Cassette Transporters↗

PubMed1995

Two domains of p53 interact with the TATA-binding protein, and the adenovirus 13S E1A protein disrupts the association, relieving p53-mediated transcriptional repression.

The tumor suppressor gene product p53 can activate and repress transcription. Both transcriptional activation and repression are thought to involve the direct interaction of p53 with the basal transcriptional machinery. Previous work has demonstrated an in vitro interaction between p53 and the TATA-binding protein that requires amino acids 20 to 57 of p53 and amino acids 220 to 271 of the TATA-binding protein. The present results show that a 75-amino-acid segment from the carboxy terminus of p53 also can bind to the TATA-binding protein in vitro, and this interaction requires amino acids 217 to 268 of the TATA-binding protein, essentially the same domain that is required for interaction with the amino-terminal domain of p53. A carboxy-terminal segment of p53 can mediate repression when bound to DNA as a GAL4-p53 fusion protein. The amino- and carboxy-terminal p53 interactions occur within the domain on the TATA-binding protein to which the adenovirus 13S E1A oncoprotein has previously been shown to bind. The 13S E1A oncoprotein can dissociate the complex formed between the carboxy-terminal domain of p53 and the TATA-binding protein and relieve p53-mediated transcriptional repression. These results demonstrate that two independent domains of p53 can potentially interact with the TATA-binding protein, and they define a mechanism--relief of repression--by which the 13S E1A oncoprotein can activate transcription through the TATA motif.

Adenovirus E1A Proteins↗

PubMed1995

Helix-loop-helix transcription factors mediate activation and repression of the p75LNGFR gene.

Sequence analysis of rat and human low-affinity nerve growth factor receptor p75LNGFR gene promoter regions revealed a single E-box cis-acting element, located upstream of the major transcription start sites. Deletion analysis of the E-box sequence demonstrated that it significantly contributes to p75LNGFR promoter activity. This E box has a dual function; it mediates either activation or repression of the p75LNGFR promoter activity, depending on the interacting transcription factors. We showed that the two isoforms of the class A basic helix-loop-helix (bHLH) transcription factor ME1 (ME1a and ME1b), the murine homolog of the human HEB transcription factor, specifically repress p75LNGFR promoter activity. This repression can be released by coexpression of the HLH Id2 transcriptional regulator. In vitro analyses demonstrated that ME1a forms a stable complex with the p75LNGFR E box and likely competes with activating E-box-binding proteins. By using ME1a-overexpressing PC12 cells, we showed that the endogenous p75LNGFR gene is a target of ME1a repression. Together, these data demonstrate that the p75LNGFR E box and the interacting bHLH transcription factors are involved in the regulation of p75LNGFR gene expression. These results also show that class A bHLH transcription factors can repress and Id-like negative regulators can stimulate gene expression.

Animals↗

PubMed1995

Identification of a silencer module which selectively represses cyclic AMP-responsive element-dependent gene expression.

The cyclic AMP (cAMP)-inducible promoter from the rat lactate dehydrogenase A subunit gene (LDH A) is associated with a distal negative regulatory element (LDH-NRE) that represses inherent basal and cAMP-inducible promoter activity. The element is of dyad symmetry, consisting of a palindromic sequence with two half-sites, 5'-TCTTG-3'. It represses the expression of an LDH A/chloramphenicol acetyltransferase (CAT) reporter gene in a dose-dependent, orientation- and position-independent fashion, suggesting that it is a true silencer element. Uniquely, it selectively represses cAMP-responsive element (CRE)-dependent transcription but has no effect on promoters lacking a CRE sequence. The repressing action of LDH-NRE could be overcome by cotransfection with LDH A/CAT vector oligonucleotides containing either the LDH-NRE or CRE sequence. This suggests that the reversal of repression was caused by the removal of functional active, limiting transacting factors which associate with LDH-NRE as well as with CRE. Gel mobility shift, footprinting, and Southwestern blotting assays demonstrated the presence of a 69-kDa protein with specific binding activity for LDH-NRE. Additionally, gel supershift assays with anti-CREB and anti-Fos antibodies indicate the presence of CREB and Fos or antigenically closely related proteins with the LDH-NRE/protein complex. We suggest that the LDH-NRE and CRE modules functionally interact to achieve negative modulation of cAMP-responsive LDH A transcriptional activity.

Animals↗

PubMed1995

Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro.

The neuron-specific N1 exon of the mouse c-src transcript is normally skipped in nonneuronal cells. In this study, we examined the sequence requirements for the exclusion of this exon in nonneuronal HeLa cell nuclear extracts. We found that the repression of the N1 exon is mediated by specific intron sequences that flank the N1 exon. Mutagenesis experiments identified conserved CUCUCU elements within these intron regions that are required for the repression of N1 splicing. The addition of an RNA competitor containing the upstream regulatory sequence to the HeLa extract induced splicing of the intron downstream of N1, indicating that the competitor sequence binds to splicing repressor proteins. The similarities between this mechanism for src splicing repression and the repression of other regulated exons point to a common role of exon-spanning interactions in splicing repression.

Alternative Splicing↗

PubMed1995

Transcriptional repression by Msx-1 does not require homeodomain DNA-binding sites.

This study investigates the transcriptional properties of Msx-1, a murine homeodomain protein which has been proposed to play a key role in regulating the differentiation and/or proliferation state of specific cell populations during embryogenesis. We show, using basal and activated transcription templates, that Msx-1 is a potent repressor of transcription and can function through both TATA-containing and TATA-less promoters. Moreover, repression in vivo and in vitro occurs in the absence of DNA-binding sites for the Msx-1 homeodomain. Utilizing a series of truncated Msx-1 polypeptides, we show that multiple regions of Msx-1 contribute to repression, and these are rich in alanine, glycine, and proline residues. When fused to a heterologous DNA-binding domain, both N- and C-terminal regions of Msx-1 retain repressor function, which is dependent upon the presence of the heterologous DNA-binding site. Moreover, a polypeptide consisting of the full-length Msx-1 fused to a heterologous DNA-binding domain is a more potent repressor than either the N- or C-terminal regions alone, and this fusion retains the ability to repress transcription in the absence of the heterologous DNA site. We further show that Msx-1 represses transcription in vitro in a purified reconstituted assay system and interacts with protein complexes composed of TBP and TFIIA (DA) and TBP, TFIIA, and TFIIB (DAB) in gel retardation assays, suggesting that the mechanism of repression is mediated through interaction(s) with a component(s) of the core transcription complex. We speculate that the repressor function of Msx-1 is critical for its proposed role in embryogenesis as a regulator of cellular differentiation.

3T3 Cells↗

PubMed1995

FER-1, an enhancer of the ferritin H gene and a target of E1A-mediated transcriptional repression.

Ferritin, the major intracellular iron storage protein of eucaryotic cells, is regulated during inflammation and malignancy. We previously reported that transcription of the H subunit of ferritin (ferritin H) is negatively regulated by the adenovirus E1A oncogene in mouse NIH 3T3 fibroblasts (Y. Tsuji, E. Kwak, T. Saika, S. V. Torti, and F. M. Torti, J. Biol. Chem. 268:7270-7275, 1993). To elucidate the mechanism of transcriptional repression of the ferritin H gene by E1A, a series of deletions in the 5' flanking region of the mouse ferritin H gene were constructed, fused to the chloramphenicol acetyltransferase (CAT) gene, and transiently cotransfected into NIH 3T3 cells with an E1A expression plasmid. The results indicate that the E1A-responsive region is located approximately 4.1 kb 5' to the transcription initiation site of the ferritin H gene. Further analyses revealed that a 37-bp region, termed FER-1, is the target of E1A-mediated repression. This region also serves as an enhancer, augmenting ferritin H transcription independently of position and orientation. FER-1 was dissected into two component elements, i.e., a 22-bp dyad symmetry element and a 7-bp AP1-like sequence. Insertion of these DNA sequences into a ferritin H-CAT chimeric gene lacking an E1A-responsive region indicated that (i) the 22-bp dyad symmetry sequence by itself has no enhancer activity, (ii) the AP1-like sequence has moderate enhancer activity which is repressed by E1A, and (iii) the combination of the dyad symmetry element and the AP1-like sequence is required for maximal enhancer activity and repression by E1A. Gel retardation assays and cotransfection experiments with c-fos and c-jun expression vectors suggested that members of the Fos and Jun families bind to the AP1-like element of FER-1 and contribute to its regulation. In addition, gel retardation assays showed that E1A reduces the ability of nuclear proteins to bind to the AP1-like sequence without affecting the levels of nuclear factors that recognize the 22-bp dyad symmetry element. Taken together, these results demonstrate that FER-1 serves as both an enhancer of ferritin H transcription and a target for E1A-mediated repression.

Adenovirus E1A Proteins↗

PubMed1996

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

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.

Amino Acid Sequence↗

PubMed1996

Functional analysis of histones H2A and H2B in transcriptional repression in Saccharomyces cerevisiae.

The presence of H2A-H2B dimers in nucleosomes can inhibit the binding of transcription factors to chromatin templates. To study the roles of histones H2A and H2B in transcriptional repression in vivo, mutant forms of these histones were analyzed in two different assay systems. Two repression domains were identified in H2A. One domain includes residues that fall in the beginning of the H2A-H2B dimerization region, and the second is in the H2A N terminus, a region of potential interactions with nonhistone proteins. The function of H2A and H2B in one repression assay was found to be dependent on three SPT (suppressor of Ty) genes whose products are important for chromatin-mediated repression. These results suggest that repressive chromatin structure may be established through the interactions of the Spt proteins with these histones. In contrast, other proteins, the products of the HIR (histone regulation) genes, may function to direct H2A and H2B to specific promoters.

Base Sequence↗

PubMed1996

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

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

Amino Acid Sequence↗

PubMed1996

Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells.

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.

3T3 Cells↗

PubMed1997

Functional domains of the human orphan receptor ARP-1/COUP-TFII involved in active repression and transrepression.

The orphan receptor ARP-1/COUP-TFII, a member of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of nuclear receptors, strongly represses transcriptional activity of numerous genes, including several apolipoprotein-encoding genes. Recently it has been demonstrated that the mechanism by which COUP-TFs reduce transcriptional activity involves active repression and transrepression. To map the domains of ARP-1/COUP-TFII required for repressor activity, a detailed deletion analysis of the protein was performed. Chimeric proteins in which various segments of the ARP-1/COUP-TFII carboxy terminus were fused to the GAL4 DNA binding domain were used to characterize its active repression domain. The smallest segment confering active repressor activity to a heterologous DNA binding domain was found to comprise residues 210 to 414. This domain encompasses the region of ARP-1/COUP-TFII corresponding to helices 3 to 12 in the recently published crystal structure of other members of the nuclear receptor superfamily. It includes the AF-2 AD core domain formed by helix 12 but not the hinge region, which is essential for interaction with a corepressor in the case of the thyroid hormone and retinoic acid receptor. Attachment of the nuclear localization signal from the simian virus 40 large T antigen (Flu tag) to the amino terminus of ARP-1/COUP-TFII abolished its ability to bind to DNA without affecting its repressor activity. By using a series of Flu-tagged mutants, the domains required for transrepressor activity of the protein were mapped. They include the DNA binding domain and the segment spanning residues 193 to 399. Transcriptional activity induced by liver-enriched transactivators such as hepatocyte nuclear factor 3 (HNF-3), C/EBP, or HNF-4 was repressed by ARP-1/COUP-TFII independent of the presence of its cognate binding site, while basal transcription or transcriptional activity induced by ATF or Sp1 was not perturbed by the protein. In conclusion, our results demonstrate that the domains of ARP-1/COUP-TFII required for active repression and transrepression do not coincide. Moreover, they strongly suggest that transrepression is the predominant mechanism underlying repressor activity of ARP-1/COUP-TFII. This mechanism most likely involves interaction of the protein with one or several transcriptional coactivator proteins which are employed by various liver-enriched transactivators but not by ubiquitous factors such as Sp1 or ATF.

Apolipoproteins B↗

PubMed1998

Rapamycin induces the G0 program of transcriptional repression in yeast by interfering with the TOR signaling pathway.

The macrolide antibiotic rapamycin inhibits cellular proliferation by interfering with the highly conserved TOR (for target of rapamycin) signaling pathway. Growth arrest of budding yeast cells treated with rapamycin is followed by the program of molecular events that characterizes entry into G0 (stationary phase), including the induction of polymerase (Pol) II genes typically expressed only in G0. Normally, progression into G0 is characterized by transcriptional repression of the Pol I and III genes. Here, we show that rapamycin treatment also causes the transcriptional repression of Pol I and III genes. The down-regulation of Pol III transcription is TOR dependent. While it coincides with translational repression by rapamycin, transcriptional repression is due in part to a translation-independent effect that is evident in extracts from a conditional tor2 mutant. Biochemical experiments reveal that RNA Pol III and probably transcription initiation factor TFIIIB are targets of repression by rapamycin. In view of previous evidence that TFIIIB and Pol III are inhibited when protein phosphatase 2A (PP2A) function is impaired, and that PP2A is a component of the TOR pathway, our results suggest that TOR signaling regulates Pol I and Pol III transcription in response to nutrient growth signals.

Antifungal Agents↗

PubMed1998

Methyl-CpG-binding protein MeCP2 represses Sp1-activated transcription of the human leukosialin gene when the promoter is methylated.

Human leukosialin (CD43) is expressed in a cell lineage-specific as well as a differentiation stage-specific fashion. The leukosialin promoter, made up of an Sp1 binding site and a sequence similar to that of an initiator, possesses high transcriptional potential. Previous data have demonstrated that the leukosialin gene is down-regulated in nonproducing cells by DNA methylation. In this paper the repressive mechanism of DNA methylation in expression systems is reported. In vitro DNA methylation with SssI (CpG) methylase of leukosialin-chloramphenicol acetyltransferase (CAT) constructs drastically reduced transcriptional activities in stable transfection systems with the human HeLa and Jurkat cell lines. On the other hand, the transcriptional repression by in vitro methylation was less pronounced in Drosophila melanogaster cells, which lack genomic methylation. In these cells, Sp1 could transactivate equally well both the unmethylated and methylated leukosialin promoter. In order to test whether one of the methyl-CpG-binding proteins, MeCP2, is responsible for transcriptional repression of the leukosialin gene, I isolated the human MeCP2 cDNA (encoding 486 amino acid residues) and expressed it in Drosophila cells. I found that MeCP2 substantially inhibited Sp1-activated transcription when the leukosialin promoter was methylated. The level of repression was directly proportional to the amount of MeCP2 expression vector transfected. Analysis of C-terminal deletion mutants of MeCP2 showed that repressive activity of Sp1 transactivation is localized to the N-terminal region consisting of amino acid residues 1 to 193, which encompass the methyl-binding domain. These results suggest that interference with Sp1 transactivation by MeCP2 is an important factor in the down-regulation of leukosialin gene expression by DNA methylation.

Antigens, CD↗