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PubMed2004

Mapping activation and repression domains of the vnd/NK-2 homeodomain protein.

A transient transfection assay using Drosophila S2 tissue culture cells and WT and mutant Drosophila vnd/NK-2 homeobox cDNAs was used to localize repression and activation domains of vnd/NK-2 homeodomain protein. A repression domain was identified near the N terminus of vnd/NK-2 homeodomain protein (amino acid residues 154-193), which contains many hydrophobic amino acid residues. The major determinants of the repression domain were shown to be amino acid residues F155, W158, I161, L162, L163, and W166. Truncated protein consisting of the N-terminal repression domain and the DNA-binding homeodomain repressed transcription as efficiently as WT vnd/NK-2 protein. An activation domain was identified between the tinman domain and the homeodomain (amino acid residues 277-543), which consists of a glutamine-rich subdomain and two acidic subdomains. No effect was detected of the tinman domain or the NK-2-specific domain on either activation or repression of a beta-galactosidase reporter gene.

Amino Acid Sequence↗
PubMed2000

Stra13 expression is associated with growth arrest and represses transcription through histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms.

Stra13 is a transcriptional repressor related within its basic helix-loop-helix domain with the Drosophila Hairy, Enhancer of Split, and the mouse Hes1 proteins that interact with the corepressor Groucho. Because Stra13 lacks the conserved WRPW motif for interaction with Groucho, we examined the function and mechanism of transcriptional repression mediated by Stra13 that exhibits several distinctive features. Here, we report that Stra13 expression is closely associated with cell growth arrest induced by several triggers such as retinoic acid and trichostatin A (TSA; a specific histone deacetylase inhibitor) as well as by serum starvation. Stra13 expression is transcriptionally repressed and maintained at a low level in cells through a negative autoregulatory mechanism that is brought about by its interaction with the corepressor histone deacetylase (HDAC1). This interaction requires the Stra13 C-terminal domain containing three alpha-helices, which are also functionally critical to its repressive activity. Thus, inhibition of HDAC activity by TSA abrogates Stra13-mediated repression of its promoter, resulting in induction of Stra13 expression that is coincident with TSA-induced growth arrest. Further, once induced, Stra13 strongly represses the expression of the cell proliferation-associated gene c-Myc through an HDAC1-independent pathway that involves its interaction with the basal transcription factor TFIIB. Our studies indicate that Stra13 may play a key role in signaling pathways that lead to growth arrest and terminal differentiation by repression of target genes via HDAC-dependent and HDAC-independent mechanisms.

3T3 Cells↗
PubMed2002

Alternate surfaces of transcriptional coregulator GRIP1 function in different glucocorticoid receptor activation and repression contexts.

Members of the mammalian p160 family, such as GRIP1, are known as glucocorticoid receptor (GR) coactivators; at certain glucocorticoid response elements (GREs), however, GRIP1 acts as a GR corepressor. We characterized functional interactions of GR and GRIP1 in a repression complex where GR tethers to DNA-bound activator protein-1 (AP-1), as at the human collagenase-3 gene, and tested whether the identified interactions were similar or different at other response elements. At the AP-1 tethering GRE, we mapped the GRIP1 corepressor activity to a domain distinct from the two known GRIP1 activation domains; it exhibited intrinsic GR-independent repression potential when recruited to DNA via Gal4 DNA-binding domain. Interestingly, neither the domain nor the activity was detected in the other two p160 family members, SRC1 and RAC3. The same GRIP1 corepression domain was required for GR-mediated repression at the nuclear factor-kappaB (NF-kappaB) tethering GRE of the human IL-8 gene. In contrast, at the osteocalcin gene GRE, where GR represses transcription by binding to a DNA site overlapping the TATA box, both GRIP1 and SRC1 corepressed, and the GRIP1-specific repression domain was dispensable. Thus, in a single cell type, GR and GRIP1 conferred one mode of activation and two modes of repression by selectively engaging distinct surfaces of GRIP1 in a response element-specific manner.

Carrier Proteins↗
PubMed1991

Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression.

Glucose (catabolite) repression is mediated by multiple mechanisms that combine to regulate transcription of the GAL genes over at least a thousandfold range. We have determined that this is due predominantly to modest glucose repression (4- to 7-fold) of expression of GAL4, the gene encoding the transcriptional activator of the GAL genes. GAL4 regulation is affected by mutations in several genes previously implicated in the glucose repression pathway; it is not dependent on GAL4 or GAL80 protein function. GAL4 promoter sequences that mediate glucose repression were found to lie downstream of positively acting elements that may be "TATA boxes." Two nearly identical sequences (10/12 base pairs) in this region that may be binding sites for the MIG1 protein were identified as functional glucose-control elements. A 4-base-pair insertion in one of these sites causes constitutive GAL4 synthesis and leads to substantial relief (50-fold) of glucose repression of GAL1 expression. Furthermore, promoter deletions that modestly reduce GAL4 expression, and therefore presumably the amount of GAL4 protein synthesized, cause much greater reductions in GAL1 expression. These results suggest that GAL4 works synergistically to activate GAL1 expression. Thus, glucose repression of GAL1 expression is due largely to a relatively small reduction of GAL4 protein levels caused by reduced GAL4 transcription. This illustrates how modest regulation of a weakly expressed regulatory gene can act as a sensitive genetic switch to produce greatly amplified responses to environmental changes.

Base Sequence↗
PubMed1994

A Drosophila gene promoter is subject to glucose repression in yeast cells.

Previous work has shown that the alpha-amylase gene of Drosophila melanogaster is subject to repression by dietary glucose. Moreover, glucose repression of this gene is mediated by promoter elements that lie upstream of the transcriptional start site. In this study, we examined the activity of the glucose-repressible Drosophila promoter in transformed yeast cells. We show that the amylase promoter region can mediate glucose repression of a heterologous reporter gene in yeast. The implication of this result is that the yeast regulatory machinery can recognize the Drosophila promoter signals. This, in turn, implies an unexpectedly high degree of evolutionary conservation in the mechanism of glucose repression among eukaryotes. It also shows that genes that have acquired complex patterns of developmental regulation-e.g., the Drosophila amylase gene, can still retain, intact, more primitive forms of regulation, such as glucose repression.

Actins↗
PubMed1995

Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein.

The SSN6-TUP1 protein complex represses transcription of diversely regulated genes in the yeast Saccharomyces cerevisiae. Here we present evidence that MIG1, a zinc-finger protein in the EGR1/Zif268 family, recruits SSN6-TUP1 to glucose-repressed promoters. DNA-bound LexA-MIG1 represses transcription of a target gene in glucose-grown cells, and repression requires SSN6 and TUP1. We also show that MIG1 and SSN6 fusion proteins interact in the two-hybrid system. Unexpectedly, we found that LexA-MIG1 activates transcription strongly in an ssn6 mutant and weakly in a tup1 mutant. Finally, LexA-MIG1 does not repress transcription in glucose-deprived cells, and MIG1 is differentially phosphorylated in response to glucose availability. We suggest a role for phosphorylation in regulating repression.

Bacterial Proteins↗
PubMed1997

Translational coupling by modulation of feedback repression in the IF3 operon of Escherichia coli.

A pseudoknot formed by a long-range interaction in the mRNA of the initiation factor 3 (IF3) operon is involved in the translational repression of the gene encoding ribosomal protein L35 by another ribosomal protein, L20. The nucleotides forming the 5' strand of the key stem of the pseudoknot are located within the gene for IF3, whereas those forming the 3' strand are located 280 nt downstream, immediately upstream of the Shine-Dalgarno sequence of the gene for L35. Here we show that premature termination of IF3 translation at a nonsense codon introduced upstream of the pseudoknot results in a substantial enhancement of L20-mediated repression of L35 expression. Conversely, an increase of IF3 translation decreases repression. These results, in addition to an analysis of the effect of mutations in sequences forming the pseudoknot, indicate that IF3 translation decreases L20-mediated repression of L35 expression. We propose that ribosomes translating IF3 disrupt the pseudoknot and thereby attenuate repression. The result is a novel type of translational coupling, where unfolding of the pseudoknot by ribosomes translating IF3 does not increase expression of L35 directly, but alleviates its repression by L20.

Bacterial Proteins↗
PubMed1996

Structure/functional properties of the yeast dual regulator protein NGG1 that are required for glucose repression.

NGG1p/ADA3p is a yeast dual function regulator required for the complete glucose repression of GAL4p-activated genes (Brandl, C. J., Furlanetto, A. M., Martens, J. A., and Hamilton, K. S. (1993) EMBO J. 12, 5255-5265). Evidence for a direct role for NGG1p in regulating activator function is supported by the finding that NGG1p is also required for transcriptional activation by GAL4p-VPl6 and LexA-GCN4p (Pina, B., Berger, S. L., Marcus, G. A., Silverman, N., Agapite, J., and Guarente, L. (1993) Mol. Cell. Biol. 13, 5981-5989). By analyzing deletion derivatives of the 702-amino acid protein, we identified a region essential for glucose repression within residues 274-373. Essential sequences were further localized to a segment rich in Phe residues that is predicted to be an amphipathic alpha helix. As well as finding mutations within this region that reduced glucose repression, we identified mutations that made NGG1p a better repressor. In addition, NGG1p probably represses GAL4p activity as part of a complex containing ADA2p because single and double disruptions of ngg1 and ada2 had comparable effects on glucose repression. We also localized a transcriptional activation domain within the amino-terminal amino acids of NGG1p that is proximal or overlapping the region required for glucose repression. Activation by GAL4p-NGG1p(1-373) requires ADA2p; however, activation by GAL4p-NGG1p(1-308), is ADA2p-independent. This suggests that a site required for ADA2p interaction lies between amino acids 308 and 373 and that ADA2p has a regulatory role in activation by GAL4p-NGG1p(1-373).

Alleles↗
PubMed1996

Translational repression dependent on the interaction of the Xenopus Y-box protein FRGY2 with mRNA. Role of the cold shock domain, tail domain, and selective RNA sequence recognition.

We have examined the determinants of the translational repression of mRNA by the Xenopus oocyte-specific Y-box protein FRGY2 using in vitro and in vivo assays. In vitro reconstitution of messenger ribonucleoprotein (mRNP) complexes demonstrates that the sequence-specific RNA-binding cold shock domain is not required for translational repression, whereas the RNA-binding C-terminal tail domain is essential. However, microinjection of reconstituted mRNPs into Xenopus oocytes demonstrates that although translational repression occurs in the absence of consensus RNA binding sequences for FRGY2, the presence of FRGY2 recognition elements within mRNA potentiates translational repression. Analysis of the in vivo assembly of mRNP shows that the cold shock domain alone is not stably incorporated into mRNP, whereas the C-terminal tail domain is sufficient for stable incorporation. We suggest that translational repression of mRNA by FRGY2 is favored by sequence-selective recognition of RNA sequences by the cold shock domain. However, translational repression in vitro and the assembly of mRNP in vivo requires the relatively nonspecific interaction of the C-terminal tail domain with mRNA. Thus two distinct domains of FRGY2 are likely to contribute to translational control.

Animals↗
PubMed1998

Identification of amino acid residues of Gsalpha critical to repression of adipogenesis.

Gsalpha regulates the differentiation of 3T3-L1 mouse embryonic fibroblasts to adipocytes, a process termed adipogenesis. Through the expression of chimera created by substituting regions of Gsalpha with corresponding regions of the G protein Gialpha2, the domain of Gsalpha involved in repression of adipogenesis was localized to sequence 146-235 of the molecule (Wang, H-y., Johnson, G. L., Liu, X. , Malbon, C. C. (1996) J. Biol. Chem. 271, 22022-22029). As a prelude to alanine-scanning mutagenesis, chimeras in Gsalpha constructed from trisection of the sequence 125-213 of Gialpha2 were expressed stably, and clones were evaluated for the ability of the chimera to repress adipogenesis in response to the inducers, dexamethasone and methylisobutylxanthine, in combination. The chimera containing sequence 150-177 of Gialpha2 repressed adipogenesis, whereas the chimeras with either sequence 125-149 or 178-213 of Gialpha2 failed to repress induction of adipogenesis. Alanine-scanning mutagenesis of these two critical domains was performed first in clusters and then confirmed by analysis of single mutations. Six residues unique to Gsalpha were identified as critical to repression of adipogenesis, Asn167, Cys200, Leu203, Ser205, Val214, and Lys216. Leu203 and Ser205 are required in tandem, as mutagenesis to alanine of either one alone was without effect on repressor activity. The remaining four residues are required for repressor activity; mutation of any one of these abolishes the ability of Gsalpha to repress adipogenesis, although not affecting the ability of the mutant form of Gsalpha to regulate adenylylcyclase. Using conserved landmarks found in the crystal structures of Gialpha1 and Gsalpha, the Leu203 and Ser205 cluster appears to be exposed, closely aligned and located in switch I region. Asn167, Val214, and Lys216 project to regions on Gsalpha that are exposed in the GTPgammaS-liganded state of the alpha subunit. We speculate that these residues constitute an important contact domain between Gsalpha and the effector controlling adipogenesis, which is yet to be identified.

3T3 Cells↗
PubMed1999

Identification of a surface of FNR overlapping activating region 1 that is required for repression of gene expression.

A library of Escherichia coli fnr mutants has been screened to identify FNR (regulator of fumarate and nitrate reduction) variants that are defective repressors, but competent activators. All but one of seventeen variants had substitutions close to or within the face of FNR that contains activating region 1 (AR1). Activating region 1 is known to contact the alpha subunit of RNA polymerase to facilitate transcription activation. It is now evident that this face also has a role in FNR-mediated repression. Single amino acid substitutions at Lys54, Gly74, Ala95, Met147, Leu193, Arg197, or Leu239, and double substitutions at Ser13 and Ser145, Cys16 and Ile45, Tyr69 and Ser133, or Lys164 and Phe191, impaired FNR-mediated repression of ndh without greatly affecting activation from model Class I (FNR site at -71.5) and Class II (FNR site at -41.5) FNR-activated promoters. Although repression was impaired in a second group of FNR variants with substitutions at Leu34, Arg72 and Leu193, Phe92, or Ser178, transcription activation from the simple FNR-dependent promoters was severely reduced. However, expression from pyfiD (FNR sites at -40.5 and -93.5) and a derivative lacking the site at -93.5, pyfiD-/+, remained relatively high indicating that this second group have a context-dependent activation defect as well as a repression defect. The prediction that the substitutions affecting repression were likely to be in solvent exposed regions of FNR was supported by analysis of peptides produced by partial proteolysis of FNR. Thus, FNR-mediated repression at promoters with multiple FNR sites requires regions of FNR that are different from, but overlap, AR1.

Amino Acid Substitution↗
PubMed1999

Multiple modes of repression by the Smad transcriptional corepressor TGIF.

TGIF is a DNA-binding homeodomain protein that has been demonstrated to play a role in transforming growth factor beta-regulated transcription and implicated in the control of retinoid-responsive transcription. We investigated the intrinsic transcriptional activity of TGIF fused to a heterologous DNA-binding domain. Our results demonstrate that TGIF is a transcriptional repressor able to repress transcription from several different promoters. Repression by TGIF is insensitive to the distance at which it is bound from the promoter. Moreover, the wild type TGIF effectively represses transcription when bound to its cognate DNA-binding site via its homeodomain. Deletion analysis revealed the presence of at least two separable repression domains within TGIF. Repression by one of these is dependent on the activity of histone deacetylases (HDACs), whereas the other appears not to require HDAC activity. Finally, we demonstrate that TGIF interacts with HDACs via its carboxyl-terminal repression domain. Together, these results suggest that TGIF is a multifunctional transcriptional repressor, which acts in part by recruiting HDAC activity.

Animals↗
PubMed2000

Bile acid induction of cytokine expression by macrophages correlates with repression of hepatic cholesterol 7alpha-hydroxylase.

In the studies reported herein, we show that two complementary experimental models: inbred strains of mice (i.e. C57BL/6 and C3H/HeJ), and a differentiated line of rat hepatoma cells (i.e. L35 cells), require the activation of cytokines by monocyte/macrophages to display bile acid negative feedback repression of cholesterol 7alpha-hydroxylase (CYP7A1). Feeding a bile acid-containing atherogenic diet for 3 weeks to C57BL/6 mice led to a 70% reduction in the expression of hepatic CYP7A1 mRNA, whereas no reduction was observed in C3H/HeJ mice. The strain-specific response to repression of CYP7A1 paralleled the activation of hepatic cytokine expression. Studies using cultured THP-1 monocyte/macrophages showed that the hydrophobic bile acid chenodeoxycholate, a well established potent repressor of CYP7A1, induced the expression of mRNAs encoding interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFalpha). In contrast, the hydrophilic bile acid ursodeoxycholate, which does not repress CYP7A1, did not induce cytokine mRNA expression by THP-1 cells. Chenodeoxycholate activation of cytokines by THP-1 cells was blocked by the peroxisome proliferator-activated receptor gamma agonist rosiglitazone. The expression of cytokines (e.g. IL-1 and TNFalpha) by THP-1 cells paralleled with the ability of these cells to produce conditioned medium that when added to rat L35 hepatoma cells, repressed CYP7A1. Moreover, rosiglitazone, which blocks cytokine activation by macrophages, also blocked the repression of CYP7A1 normally exhibited by C57BL/6 mice fed the bile acid-containing atherogenic diet. The combined data indicate that the activation of cytokines may mediate CYP7A1 repression caused by feeding mice an atherogenic diet containing bile acids.

Animals↗
PubMed2000

Ikaros interactions with CtBP reveal a repression mechanism that is independent of histone deacetylase activity.

We have previously shown that Ikaros can repress transcription through the recruitment of histone deacetylase complexes. Here we provide evidence that Ikaros can also repress transcription through its interactions with the co-repressor, C-terminal binding protein (CtBP). CtBP interacts with Ikaros isoforms through a PEDLS motif present at the N terminus of these proteins but not with homologues like Aiolos which lack this motif. Mutations in Ikaros that prevent CtBP interactions reduce its ability to repress transcription. CtBP interacts with Sin3A but not with the Mi-2 co-repressor and it represses transcription in a manner that is independent of histone deacetylase activity. These data strongly suggest that CtBP contributes to a histone deacetylase activity independent mechanism of repression by Ikaros. Finally, we show that the viral oncoprotein E1A, which binds to CtBP, also shows a strong association with Ikaros. This Ikaros-E1A interaction may underlie Ikaros's decreased ability to repress transcription in E1A transformed cells.

3T3 Cells↗
PubMed2000

The minimal repression domain of MBD2b overlaps with the methyl-CpG-binding domain and binds directly to Sin3A.

Different mechanisms mediating methylation-dependent repression have been demonstrated. Two of these mechanisms play a role in the context of the granulocyte/macrophage-specific lysozyme gene: direct interference with DNA binding of the transcription factor GA-binding protein and deacetylation of histones. Besides enhancement in the unmethylated state, and transcriptional repression upon DNA methylation, the lysozyme downstream enhancer confers tissue-specific demethylation. Because both demethylation activity and repression ability have been attributed to the methyl-CpG-binding domain-containing protein MBD2, we analyzed this protein. The short form MBD2b binds to the methylated lysozyme enhancer and mediates transcriptional repression. MBD2b is capable of binding to the transcriptional repressor Sin3A. The interaction domain of Sin3A required for binding to MBD2b contains the paired amphipathic helix 3. We identified a minimal functional domain that confers both transcriptional repression as well as the interaction to Sin3A. In contrast to the functionally related proteins MeCP2 and MBD1, the repression domain of MBD2b overlaps with the methyl-CpG-binding domain.

Cell Line↗
PubMed2001

Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.

Hairy/Enhancer of split 1 (Hes1) is a mammalian transcriptional repressor that plays crucial roles in the regulation of several developmental processes, including neuronal differentiation. The aim of this study was to elucidate the molecular mechanisms that regulate the transcription repression activity of Hes1. It is shown here that Hes1 associates with the nuclear matrix, the ribonucleoprotein network of the nucleus that plays important roles in transcriptional regulation. Nuclear matrix binding is mediated by the same Hes1 C-terminal domain that is also required for transcriptional repression. This domain contains the WRPW motif that acts as a binding site for the transcriptional corepressor Groucho, which also localizes to the nuclear matrix. Both the nuclear matrix association and transcription repression activity of Hes1 are inhibited by deletion of the WRPW motif, indicating that Groucho acts as a transcriptional corepressor for Hes1. This corepressor role is not modulated by the Groucho-related gene product Grg5. In contrast, the Runt-related protein RUNX2, which localizes to the nuclear matrix and interacts with Groucho and Hes1, can inhibit both the Groucho.Hes1 interaction and the transcription repression ability of Hes1. Together, these observations suggest that transcriptional repression by Hes1 requires interactions with Groucho at the nuclear matrix and that RUNX proteins act as negative regulators of the repressive activity of Groucho.Hes1 complexes.

Amino Acid Sequence↗
PubMed2001

p53 amino acids 339-346 represent the minimal p53 repression domain.

The p53 tumor suppressor protein functions as an activator and also as a repressor of gene transcription. Currently, the mechanism of transcriptional repression by p53 remains poorly understood. To help clarify this mechanism, we carried out studies designed to identify the minimal repression domain that inhibits p53 transcriptional activities. We found only eight amino acids (339) of the COOH-terminal domain (termed P53MRD) that possess activities of repression. The exact location of this minimal domain is on the E6-binding region, and it lacks the ability of tetramerization. P53MRD is able to repress the transcription of p53 while not affecting VP16. The mutants (amino acids M340P and F341D) of native p53 also lost transcriptional repression of the thymidine kinase chloramphenicol acetyltransferase promoter. These results suggest that this eight-amino acid element is required for the repression of p53.

Binding Sites↗
PubMed2001

Requirement for high mobility group protein HMGI-C interaction with STAT3 inhibitor PIAS3 in repression of alpha-subunit of epithelial Na+ channel (alpha-ENaC) transcription by Ras activation in salivary epithelial cells.

Previously, we have demonstrated that oxidative stress or Ras/ERK activation leads to the transcriptional repression of alpha-subunit of epithelial Na(+) channel (ENaC) in lung and salivary epithelial cells. Here, we further investigated the coordinated molecular mechanisms by which alpha-ENaC expression is regulated. Using both stable and transient transfection assays, we demonstrate that the overexpression of high mobility group protein I-C (HMGI-C), a Ras/ERK-inducible HMG-I family member, represses glucocorticoid receptor (GR)/dexamethasone (Dex)-stimulated alpha-ENaC/reporter activity in salivary epithelial cells. Northern analyses further confirm that the expression of endogenous alpha-ENaC gene in salivary Pa-4 cells is suppressed by an ectopic HMGI-C overexpression. Through yeast two-hybrid screening and co-immunoprecipitation assays from eukaryotic cells, we also discovered the interaction between HMGI-C and PIAS3 (protein inhibitor of activated STAT3 (signal transducer and activator of transcription 3)). A low level of ectopically expressed PIAS3 cooperatively inhibits GR/Dex-dependent alpha-ENaC transcription in the presence of HMGI-C. Reciprocally, HMGI-C expression also coordinately enhances PIAS3-mediated repression of STAT3-dependent transactivation. Moreover, overexpression of antisense HMGI-C construct is capable of reversing the repression mediated by Ras V12 on GR- and STAT3-dependent transcriptional activation. Together, our results demonstrate that Ras/ERK-mediated induction of HMGI-C is required to effectively repress GR/Dex-stimulated transcription of alpha-ENaC gene and STAT3-mediated transactivation. These findings delineate a network of inhibitory signaling pathways that converge on HMGI-C.PIAS3 complex, causally associating Ras/ERK activation with the repression of both GR and STAT3 signaling pathways in salivary epithelial cells.

Animals↗