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At least 433 records · Page 24Linked to original sources
PubMed2000

Probing the structure of RNAIII, the Staphylococcus aureus agr regulatory RNA, and identification of the RNA domain involved in repression of protein A expression.

RNAIII, a 514-nt RNA molecule, regulates the expression of many Staphylococcus aureus genes encoding exoproteins and cell-wall-associated proteins. We have studied the structure of RNAIII in solution, using a combination of chemical and enzymatic probes. A model of the secondary structure was derived from experimental data with the help of computer simulation of RNA folding. The model contains 14 hairpin structures connected by unpaired nucleotides. The data also point to three helices formed by distant nucleotides that close off structural domains. This model was generally compatible with the results of in vivo probing experiments with dimethylsulfate in late exponential-phase cultures. Toe-printing experiments revealed that the ribosome binding site of hld, which is encoded by RNAIII, was accessible to the Escherichia coli 30S ribosomal subunit, suggesting that the in vitro structure represented a translatable form of RNAIII. We also found that, within the 3' end of RNAIII, the conserved hairpin 13 and the terminator form an intrinsic structural domain that exerts specific regulatory activity on protein A gene expression.

Base Sequence↗
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↗
PubMed2000

The glucocorticoid receptor inhibits NFkappaB by interfering with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain.

Glucocorticoids repress NFkappaB-mediated activation of proinflammatory genes such as interleukin-8 (IL-8) and ICAM-1. Our experiments suggest that the glucocorticoid receptor (GR) confers this effect by associating through protein-protein interactions with NFkappaB bound at each of these genes. That is, we show that the GR zinc binding region (ZBR), which includes the DNA binding and dimerization functions of the receptor, binds directly to the dimerization domain of the RelA subunit of NFkappaB in vitro and that the ZBR is sufficient to associate with RelA bound at NFkappaB response elements in vivo. Moreover, we demonstrate in vivo and in vitro that GR does not disrupt DNA binding by NFkappaB. In transient transfections, we found that the GR ligand binding domain is essential for repression of NFkappaB but not for association with it and that GR can repress an NFkappaB derivative bearing a heterologous activation domain. We used chromatin immunoprecipitation assays in untransfected A549 cells to infer the mechanism by which the tethered GR represses NFkappaB-activated transcription. As expected, we found that the inflammatory signal TNFalpha stimulated preinitiation complex (PIC) assembly at the IL-8 and ICAM-1 promoters and that the largest subunit of RNA polymerase II (pol II) in those complexes became phosphorylated at serines 2 and 5 in its carboxy-terminal domain (CTD) heptapeptide repeats (YSPTSPS); these modifications are required for transcription initiation. Remarkably, GR did not inhibit PIC assembly under repressing conditions, but rather interfered with phosphorylation of serine 2 of the pol II CTD.

Cell Line↗
PubMed2006

Despite its strong transactivation domain, transcription factor FOXM1c is kept almost inactive by two different inhibitory domains.

FOXM1c (MPP2) is an activating transcription factor with several nuclear localization signals, a forkhead domain for DNA binding, and a very strong acidic transactivation domain. Despite its very strong transactivation domain, FOXM1c is kept almost inactive by two different independent inhibitory domains, the N-terminus and the central domain. The N-terminus as a specific negative-regulatory domain directly binds to and thus inhibits the transactivation domain completely. However, it lacks any transrepression potential. In contrast, the central domain functions as a strong RB-independent transrepression domain and as an RB-recruiting negative-regulatory domain. The N-terminus alone is sufficient to eliminate transactivation, while the central domain alone represses the transactivation domain only partially. This hierarchy of the two inhibitory domains offers the possibility to activate the almost inactive wild type in two steps in vitro: deletion of the N-terminus results in a strong transactivator, while additional deletion of the central domain in a very strong transactivator. We suggest that the very high potential of the transactivation domain has to be tightly controlled by these two inhibitory domains because FOXM1 stimulates proliferation by promoting G1/S transition, as well as G2/M transition, and because deregulation of such potent activators of proliferation can result in tumorigenesis.

Amino Acid Sequence↗
PubMed2002

Heat shock factor 1 contains two functional domains that mediate transcriptional repression of the c-fos and c-fms genes.

Heat shock factor 1 (HSF1), in addition to its pivotal role as a regulator of the heat shock response, functions as a versatile gene repressor. We have investigated the structural domains involved in gene repression using mutational analysis of the hsf1 gene. Our studies indicate that HSF1 contains two adjacent sequences located within the N-terminal half of the protein that mediate the repression of c-fos and c-fms. One region (NF) appears to be involved in quenching transcriptional activation factors on target promoters and binds to the basic zipper transcription factor NF-IL6 required for activation of c-fms and IL-1beta. The NF domain encompasses the leucine zipper 1 and 2 sequences as well as the linker domain between the DNA binding and leucine zipper regions. The function of this domain in gene repression is highly specific for HSF1, and the homologous region from conserved family member HSF2 does not restore repressive function in HSF2/HSF1 chimeras. In addition, HSF2 is not capable of binding to NF-IL6. The NF domain, although necessary for repression, is not sufficient, and a second region (REP) occupying a portion of the regulatory domain is required for repression. Neither domain functions independently, and both are required for repression. Furthermore, we constructed dominant inhibitors of c-fos repression by HSF1, which also blocked the repression of c-fms and IL-1beta, suggesting a shared mechanism for repression of these genes by HSF1. Our studies suggest a complex mechanism for gene repression by HSF1 involving the binding to and quenching of activating factors on target promoters. Mapping the structural domains involved in this process should permit further characterization of molecular mechanisms that mediate repression.

Animals↗
PubMed1995

Repression of transcriptional activity by heterologous KRAB domains present in zinc finger proteins.

We report the characterization of three novel members of the KRAB-domain containing C2-H2 zinc finger family (ZNF133, 136 and 140). KRAB (Krüppel-associated box) is an evolutionarily conserved protein domain found N-terminally with respect to the zinc finger repeats that encodes the DNA binding domain. ZNF133 and ZNF140 have both the KRAB A- and KRAB B-boxes present at their N-terminus, whereas ZNF136 contains only the KRAB A-box. We have previously demonstrated that the KRAB domains derived from ZNF133 and ZNF140 are potent transcriptional repression domains [Margolin et al. (1994) Proc. Natl. Acad. Sci. USA 91, 4509-4513]. The KRAB domain from ZNF136, containing only subdomain A, is a considerable weaker suppression domain; however, when fused to the heterologous KRAB B subdomain of ZNF10 (KOX1) the two subdomains from a KRAB domain which induces repression as potently as previously reported KRAB domains.

Amino Acid Sequence↗
PubMed1996

A unique transactivation sequence motif is found in the carboxyl-terminal domain of the single-strand-binding protein FBP.

The far-upstream element-binding protein (FBP) is one of several recently described factors which bind to a single strand of DNA in the 5' region of the c-myc gene. Although cotransfection of FBP increases expression from a far-upstream element-bearing c-myc promoter reporter, the mechanism of this stimulation is heretofore unknown. Can a single-strand-binding protein function as a classical transactivator, or are these proteins restricted to stabilizing or altering the conformation of DNA in an architectural role? Using chimeric GAL4-FBP fusion proteins we have shown that the carboxyl-terminal region (residues 448 to 644) is a potent transcriptional activation domain. This region contains three copies of a unique amino acid sequence motif containing tyrosine diads. Analysis of deletion mutants demonstrated that a single tyrosine motif alone (residues 609 to 644) was capable of activating transcription. The activation property of the C-terminal domain is repressed by the N-terminal 107 amino acids of FBP. These results show that FBP contains a transactivation domain which can function alone, suggesting that FBP contributes directly to c-myc transcription while bound to a single-strand site. Furthermore, activation is mediated by a new motif which can be negatively regulated by a repression domain of FBP.

Amino Acid Sequence↗
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↗
PubMed1997

Groucho-dependent and -independent repression activities of Runt domain proteins.

Runt domain proteins are transcriptional regulators that specify cell fates for processes extending from pattern formation in insects to leukemogenesis in humans. Runt domain family members are defined based on the presence of the 128-amino-acid Runt domain, which is necessary and sufficient for sequence-specific DNA binding. We demonstrate an evolutionarily conserved protein-protein interaction between Runt domain proteins and the corepressor Groucho. The interaction, however, is independent of the Runt domain and can be mapped to a 5-amino-acid sequence, VWRPY, present at the C terminus of all Runt domain proteins. Drosophila melanogaster Runt and Groucho interact genetically; the in vivo repression of a subset of Runt-regulated genes is dependent on the interaction with Groucho and is sensitive to Groucho dosage. Runt's repression of one gene, engrailed, is independent of VWRPY and Groucho, thus demonstrating alternative mechanisms for repression by Runt domain proteins. Unlike other transcriptional regulatory proteins that interact with Groucho, Runt domain proteins are known to activate transcription. This suggests that the Runt domain protein-Groucho interaction may be regulated.

Amino Acid Sequence↗
PubMed2001

The DEAD box protein DP103 is a regulator of steroidogenic factor-1.

The nuclear receptor steroidogenic factor-1 (SF-1) is essential for development of the gonads, adrenal gland, and the ventromedial hypothalamic nucleus. It also regulates the expression of pivotal steroidogenic enzymes and other important proteins in the reproductive system. We sought to elucidate the mechanisms that govern the transcriptional activity of SF-1. We demonstrate here that a previously uncharacterized domain, located C-terminal to the DNA binding domain of SF-1, exhibits transcriptional repression function. Point mutations in this domain markedly potentiate the transcriptional activity of native SF-1. Using an SF-1 region that spans this proximal repression domain as bait in a yeast two-hybrid system, we cloned an SF-1 interacting protein that is homologous to human DP103, a member of the DEAD box family of putative RNA helicases. DP103 directly interacts with the proximal repression domain of SF-1, and mutations in this domain abrogate its interaction with DP103. DP103 is expressed predominantly in the testis and is also expressed at a lower level in other steroidogenic and nonsteroidogenic tissues. Functionally, DP103 exhibits a native transcriptional repression function that localizes to the C-terminal region of the protein and represses the activity of wild-type, but not mutant, SF-1. Together, the physical and functional interaction of DP103 with a previously unrecognized repression domain within SF-1 represents a novel mechanism for regulation of SF-1 activity.

Amino Acid Sequence↗
PubMed1998

Involvement of negative cofactor NC2 in active repression by zinc finger-homeodomain transcription factor AREB6.

The transcription factor AREB6 contains a homeodomain flanked by two clusters of Krüppel type C2H2 zinc fingers. AREB6 binds to the E-box consensus sequence, CACCTGT, through either the N- or the C-terminal zinc finger cluster. To gain insights into the molecular mechanism by which AREB6 activates and represses gene expression, we analyzed the domain structure of AREB6 in the context of a heterologous DNA-binding domain by transient-transfection assays. The C-terminal region spanning amino acids 1011 to 1124 was identified as a conventional acidic activation domain. The region containing amino acids 754 to 901, which was identified as a repression domain, consists of 40% hydrophobic amino acids displaying no sequence similarities to other known repression domains. This region repressed transcription in vitro in a HeLa nuclear extract but not in reconstituted transcription systems consisting of transcription factor IID (TFIID), TFIIB, TFIIE, TFIIH/F, and RNA polymerase II. The addition of recombinant negative cofactor NC2 (NC2alpha/DRAP1 and NC2beta/Dr1) to the reconstituted transcription system restored the activity of the AREB6 repression domain. We further demonstrated interactions between the AREB6 repression domain and NC2alpha in yeast two-hybrid assay. Our findings suggest a mechanism of transcriptional repression that is mediated by the general cofactor NC2.

Animals↗
PubMed2006

Deciphering the roles of the histone H2B N-terminal domain in genome-wide transcription.

Histone N-terminal domains are frequent targets of posttranslational modifications. Multiple acetylated lysine residues have been identified in the N-terminal domain of H2B (K6, K11, K16, K17, K21, and K22), but little is known about how these modifications regulate transcription. We systematically mutated the N-terminal domain of histone H2B, both at known sites of lysine acetylation and elsewhere, and characterized the resulting changes in genome-wide expression in each mutant strain. Our results indicate that known sites of lysine acetylation in this domain are required for gene-specific transcriptional activation. However, the entire H2B N-terminal domain is principally required for the transcriptional repression of a large subset of the yeast genome. We find that the histone H2B repression (HBR) domain, comprised of residues 30 to 37, is necessary and sufficient for this repression. Many of the genes repressed by the HBR domain are located adjacent to telomeres or function in vitamin and carbohydrate metabolism. Deletion of the HBR domain also confers an increased sensitivity to DNA damage by UV irradiation. We mapped the critical residues in the HBR domain required for its repression function. Finally, comparisons of these data with previous studies reveal that a surprising number of genes are coregulated by the N-terminal domains of histone H2B, H3, and H4.

Acetylation↗
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↗
PubMed2005

Molecular genetic analysis of the yeast repressor Rfx1/Crt1 reveals a novel two-step regulatory mechanism.

In Saccharomyces cerevisiae, the repressor Crt1 and the global corepressor Ssn6-Tup1 repress the DNA damage-inducible ribonucleotide reductase (RNR) genes. Initiation of DNA damage signals causes the release of Crt1 and Ssn6-Tup1 from the promoter, coactivator recruitment, and derepression of transcription, indicating that Crt1 plays a crucial role in the switch between gene repression and activation. Here we have mapped the functional domains of Crt1 and identified two independent repression domains and a region required for gene activation. The N terminus of Crt1 is the major repression domain, it directly binds to the Ssn6-Tup1 complex, and its repression activities are dependent upon Ssn6-Tup1 and histone deacetylases (HDACs). In addition, we identified a C-terminal repression domain, which is independent of Ssn6-Tup1 and HDACs and functions at native genes in vivo. Furthermore, we show that TFIID and SWI/SNF bind to a region within the N terminus of Crt1, overlapping with but distinct from the Ssn6-Tup1 binding and repression domain, suggesting that Crt1 may have activator functions. Crt1 mutants were constructed to dissect its activator and repressor functions. All of the mutants were competent for repression of the DNA damage-inducible genes, but a majority were "derepression-defective" mutants. Further characterization of these mutants indicated that they are capable of receiving DNA damage signals and releasing the Ssn6-Tup1 complex from the promoter but are selectively impaired for TFIID and SWI/SNF recruitment. These results imply a two-step activation model of the DNA damage-inducible genes and that Crt1 functions as a signal-dependent dual-transcription activator and repressor that acts in a transient manner.

Chromatin↗
PubMed2005

Negative regulation of AAA + ATPase assembly by two component receiver domains: a transcription activation mechanism that is conserved in mesophilic and extremely hyperthermophilic bacteria.

Only a few transcriptional regulatory proteins have been characterized in extremely hyperthermophilic organisms, and most function as repressors. Structural features of the NtrC1 protein from the hyperthermophilic bacterium Aquifex aeolicus suggested that this protein functions similarly to the sigma(54)-polymerase activator DctD of Sinorhizobium meliloti. Here, we demonstrate that NtrC1 is an enzyme that hydrolyzes ATP to activate initiation of transcription by sigma(54)-holoenzyme. New structural data, including small-angle solution scattering data and the crystal structure of the phosphorylated receiver domain, show that NtrC1 uses a signal transduction mechanism very similar to that of DctD to control assembly of its AAA+ ATPase domain. As for DctD, the off-state of NtrC1 depends upon a tight dimer of the receiver domain to repress oligomerization of an intrinsically competent ATPase domain. Activation of NtrC1 stabilizes an alternative dimer configuration of the receiver domain that is very similar to the on-state dimers of the DctD and FixJ receiver domains. This alternative dimer appears to relieve repression of the ATPase domain by disrupting the off-state dimerization interface along the helical linker region between receiver and ATPase domains. Bacterial enhancer binding proteins typically have two linker sequences, one between N-terminal regulatory and central ATPase domains, and one between the central ATPase and C-terminal DNA binding domains. Sequence analyses reveal an intriguing correlation between the negative regulation mechanism of NtrC1 and DctD, and a structured N-terminal linker and unstructured C-terminal one; conversely, the very different, positive mechanism present in NtrC protein occurs in the context of an unstructured N-terminal linker and a structured C-terminal one. In both cases, the structured linkers significantly contribute to the stability of the off-state dimer conformation. These analyses also raise the possibility that a structured linker between N-terminal regulatory and central output domains is used frequently in regulatory proteins from hyperthermophilic organisms.

Adenosine Triphosphatases↗
PubMed2003

The nucleotides responsible for the direct physical contact between the chromatin insulator protein CTCF and the H19 imprinting control region manifest parent of origin-specific long-distance insulation and methylation-free domains.

The repression of the maternally inherited Igf2 allele has been proposed to depend on a methylation-sensitive chromatin insulator organized by the 11 zinc finger protein CTCF at the H19 imprinting control region (ICR). Here we document that point mutations of the nucleotides in physical contact with CTCF within the endogenous H19 ICR lead to loss of CTCF binding and Igf2 imprinting only when passaged through the female germline. This effect is accompanied by a significant loss of methylation protection of the maternally derived H19 ICR. Because CTCF interacts with other imprinting control regions, it emerges as a central factor responsible for interpreting and propagating gamete-derived epigenetic marks and for organizing epigenetically controlled expression domains.

Animals↗
PubMed1997

Copper-mediated repression of the activation domain in the yeast Mac1p transcription factor.

The expression of a number of genes encoding products involved in copper ion uptake in yeast is specifically inhibited by copper ions. We show here that copper metalloregulation occurs through Cu-dependent repression of the transactivation activity of Mac1p. A segment of the yeast transcription factor Mac1p was identified that activated transcription in vivo in a heterologous system using fusion polypeptides with the yeast Gal4 DNA-binding domain. The Gal4/Mac1p hybrid exhibits transactivation activity that is repressed in cells cultured in the presence of copper salts and derepressed in cells with reduced copper uptake. The repressive effect is specific for copper ions. The concentration dependency of the Cu-inactivation of Gal4/Mac1p is similar to that of Cu-inhibition of CTR1 expression, a known Cu-regulated gene in vivo. Copper inhibition of gene expression is not observed with a Gal4/Mac1p chimera containing the MAC1(up1) substitution within the transactivation domain. Cells harboring the MAC1(up1) allele fail to attenuate FRE1 and CTR1 expression in a Cu-dependent manner. Additional MAC1(up) alleles exist within the first of two cysteine-rich sequence motifs adjacent to the His --> Gln MAC1(up1) encoded substitution. Thus, Cu-regulation of Mac1p function arises from a novel Cu-specific repression of the transactivation domain function. Models for the mechanism of Cu-repression of Mac1p function will be discussed.

Amino Acid Sequence↗
PubMed1994

Repression of transcriptional activity at a distance by the evolutionarily conserved KRAB domain present in a subfamily of zinc finger proteins.

Sub-families of related zinc finger protein genes have been defined on the basis of evolutionarily conserved structural features found outside the C2-H2 finger repeats. Such elements include the FAX domain found in a large number of Xenopus ZFPs, the evolutionarily conserved KRAB (Krüppel-associated box) and the ZiN (zinc finger N-terminal) domains. Here we describe a new evolutionarily conserved motif within zinc finger proteins which we have named the leucine rich region (LeR). Since conserved modules in regulatory proteins may specify properties relevant to their action we have determined the functional capabilities of LeR and the KRAB domains in the regulation of gene transcription by fusing relevant regions to a heterologous DNA-binding domain (GAL4 DNA-binding domain). We found that the KRAB-A domain tethered to RNA polymerase II promoters by a GAL4 DNA-binding domain actively represses transcription in a distance-independent manner. KRAB-mediated repression is dependent on the dose of the GAL4-KRAB-A fusion protein and on the presence of GAL4 binding sites on the DNA. Conversely, the LeR domain did not modulate significantly the transcription. Our results indicate that the KRAB domain present in the non-finger region of many ZFP genes quenches transcription possibly due to specific protein-protein interactions between the KRAB-A domain and components of the proximal transcriptional apparatus.

Amino Acid Sequence↗