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PubMed2001

Repression domains of class II ERF transcriptional repressors share an essential motif for active repression.

We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif (L)/(F)DLN(L)/(F)(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.

Amino Acid Motifs↗

PubMed1996

Mapping of the transcriptional repression domain of the lymphoid-specific transcription factor oct-2A.

The lymphoid-specific transcription factor Oct-2a is implicated in B cell-specific transcriptional activity via the octamer motif. Structure/function analysis of various Oct-2a effector regions in the context of the GAL4 DNA-binding domain revealed that Oct-2a contains two functionally different activation domains at the N and the C termini. The transcriptional activity of both domains is strongly potentiated by interactions with distinct B cell-specific coactivators. Recently, we have identified a repression domain located within the N terminus of Oct-2a (amino acids 2-99). When this domain was transferred to a potent activator, transcription was strongly inhibited. In this study we present a deletion analysis of the N-terminal region of Oct-2a to determine the minimal repression domain. We identified a stretch of 23 amino acids, rich in serine and threonine residues, which was responsible for most of the repression activity. We show that repression is strongly dependent on the type of enhancer present in the reporter plasmid as well as on the cell line tested. The possibility that Oct-2a can act as an activator and/or a repressor may have important consequences for the function of Oct-2a in B cell differentiation and other developmental processes.

Amino Acid Sequence↗

PubMed1999

Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and Gli3 as primary mediators of Shh signaling.

Gli family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling in vertebrates. The question remains unanswered, however, as to how these Gli proteins participate in the Shh signaling pathway. In this study, regulatory activities associated with the Gli2 protein were investigated in relation to the Shh signaling. Although Gli2 acts as a weak transcriptional activator, it is in fact a composite of positive and negative regulatory domains. In cultured cells, truncation of the activation domain in the C-terminal half results in a protein with repressor activity, while removal of the repression domain at the N terminus converts Gli2 into a strong activator. In transgenic mouse embryos, N-terminally truncated Gli2, unlike the full length protein, activates a Shh target gene, HNF3beta, in the dorsal neural tube, thus mimicking the effect of Shh signal. This suggests that unmasking of the strong activation potential of Gli2 through modulation of the N-terminal repression domain is one of the key mechanisms of the Shh signaling. A similar regulatory mechanism involving the N-terminal region was also found for Gli3, but not for Gli1. When the Shh signal derived from the notochord is received by the neural plate, the widely expressed Gli2 and Gli3 proteins are presumably converted to their active forms in the ventral cells, leading to activation of transcription of their target genes, including Gli1.

Amino Acid Sequence↗

PubMed2002

The SUPERMAN protein is an active repressor whose carboxy-terminal repression domain is required for the development of normal flowers.

SUPERMAN was identified as a putative regulator of transcription that acts in floral development, but its function remains to be clarified. We demonstrate here that SUPERMAN is an active repressor whose repression domain is located in the carboxy-terminal region. Ectopic expression of SUPERMAN that lacked the repression domain resulted in a phenotype similar to that of superman mutants, demonstrating that the repression activity of SUPERMAN is essential for the development of normal flowers. Constitutive expression of SUPERMAN resulted in a severe dwarfism but did not affect cell size, indicating that SUPERMAN might regulate genes that are involved in cell division.

Amino Acid Motifs↗

PubMed1997

The adenovirus E1A repression domain disrupts the interaction between the TATA binding protein and the TATA box in a manner reversible by TFIIB.

The human adenovirus E1A 243 amino acid oncoprotein possesses a transcription repression function that appears to be linked with its ability to induce cell cycle progression and to inhibit cell differentiation. The molecular mechanism of E1A repression has been poorly understood. Recently, we reported that the TATA binding protein (TBP) is a cellular target of E1A repression. Here we demonstrate that the interaction between TBP and the E1A repression domain is direct and specific. The TBP binding domain within E1A 243R maps to E1A N-terminal residues approximately 1 to 35 and is distinct from the TBP binding domain within conserved region 3 unique to the E1A 289R transactivator. An E1A protein fragment consisting of only the E1A N-terminal 80 amino acids (E1A 1-80) and containing the E1A repression function was found to block the interaction between TBP and the TATA box element as shown by gel mobility and DNase protection analysis. Interestingly, a preformed TBP-TATA box promoter complex can be dissociated by E1A 1-80. Further, TFIIB can prevent E1A disruption of TBP-TATA box interaction. TFIIB, like TBP, can overcome E1A repression of transcription in vitro. The ability of the E1A repression domain to block TBP interaction with the TATA box and the ability of TFIIB to reverse E1A disruption of the TBP-TATA box complex implies a mechanism for E1A repression distinct from those of known cellular repressors that target TBP.

Adenovirus E1A Proteins↗

PubMed1998

Human EZF, a Krüppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains.

Members of the erythroid Krüppel-like factor (EKLF) multigene family contain three C-terminal zinc fingers, and they are typically expressed in a limited number of tissues. EKLF, the founding member, transactivates the beta-globin promoter by binding to the CACCC motif. EKLF is essential for expression of the beta-globin gene as demonstrated by gene deletion experiments in mice. Using a DNA probe from the zinc finger region of EKLF, we cloned a cDNA encoding a member of this family from a human vascular endothelial cell cDNA library. Sequence analysis indicated that our clone, hEZF, is the human homologue of the recently reported mouse EZF and GKLF. hEZF is a single-copy gene that maps to chromosome 9q31. By gel mobility shift analysis, purified recombinant hEZF protein bound specifically to a probe containing the CACCC core sequence. In co-transfection experiments, we found that sense but not antisense hEZF decreased the activity of a reporter plasmid containing the CACCC sequence upstream of the thymidine kinase promoter by 6-fold. In contrast, EKLF increased the activity of the reporter plasmid by 3-fold. By fusing hEZF to the DNA-binding domain of GAL4, we mapped a repression domain in hEZF to amino acids 181-388. We also found that amino acids 91-117 of hEZF confer an activation function on the GAL4 DNA-binding domain.

Amino Acid Sequence↗

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↗

PubMed2003

Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis.

The redundancy of genes for plant transcription factors often interferes with efforts to identify the biologic functions of such factors. We show here that four different transcription factors fused to the EAR motif, a repression domain of only 12 amino acids, act as dominant repressors in transgenic Arabidopsis and suppress the expression of specific target genes, even in the presence of the redundant transcription factors, with resultant dominant loss-of-function phenotypes. Chimeric EIN3, CUC1, PAP1, and AtMYB23 repressors that included the EAR motif dominantly suppressed the expression of their target genes and caused insensitivity to ethylene, cup-shaped cotyledons, reduction in the accumulation of anthocyanin, and absence of trichomes, respectively. This chimeric repressor silencing technology (CRES-T), exploiting the EAR-motif repression domain, is simple and effective and can overcome genetic redundancy. Thus, it should be useful not only for the rapid analysis of the functions of redundant plant transcription factors but also for the manipulation of plant traits via the suppression of gene expression that is regulated by specific transcription factors.

Amino Acid Motifs↗

PubMed1997

A G-Box-Binding Protein from Soybean Binds to the E1 Auxin-Response Element in the Soybean GH3 Promoter and Contains a Proline-Rich Repression Domain.

The E1 promoter fragment (-249 to -203) is one of three auxin-response elements (AuxREs) in the soybean (Glycine max L.) GH3 promoter (Z.-B. Liu, T. Ulmasov, X. Shi, G. Hagen, T.J. Guilfoyle [1994] Plant Cell 6: 645-657). Results presented here further characterize and delimit the AuxRE within the E1 fragment. The E1 fragment functioned as an AuxRE in transgenic tobacco (Nicotiana tabacum L.) plants, as well as in transfected protoplasts. The AuxRE within E1 contains a G-box, and this G-box was used to clone a G-box-binding factor (GBF) from soybean (SGBF-2). This 45-kD GBF contains an N-terminal proline-rich domain and a C-terminal basic/leucine zipper DNA-binding domain. Gel-mobility shift assays were used to characterize the binding specificity of SGBF-2. Antiserum raised against recombinant SGBF-2 was used to further characterize SGBF-2 and antigenically related GBFs in soybean nuclear extracts. Co-transfection assays with effector and reporter plasmids in carrot (Daucus carota L.) protoplasts indicated that the N-terminal proline-rich domain of SGBF-2 functioned as a repression domain in both basal and auxin-inducible transcription.

Journal Article↗

PubMed2005

Repressive domain of unliganded human estrogen receptor alpha associates with Hsc70.

Estrogen receptor (ER) is a hormone-inducible transcription factor as a member of the nuclear receptor gene superfamily. Unliganded ER is transcriptionally silent and capable of DNA binding; however, it is unable to suppress the basal activity of the target gene promoters, unlike non-steroid hormone receptors that associate with corepressors in the absence of their cognate ligands. To study the molecular basis of how unliganded human ERalpha is maintained silent in gene regulation upon the target gene promoters, we biochemically searched interactants for hERalpha, and identified heat shock protein 70 (Hsc70). Hsc70 appeared to associate with the N-terminal hormone binding E domain, that also turned out a transcriptionally repressive domain. Competitive association of Hsc70 with a best known coactivator p300 was observed. Thus, these findings suggest that Hsc70 associates with unliganded hERalpha, and thereby deters hERalpha from recruiting transcriptional coregulators, presumably as a component of chaperone complexes.

Cells, Cultured↗

PubMed2004

Aux/IAA proteins contain a potent transcriptional repression domain.

Aux/IAA proteins are short-lived nuclear proteins that repress expression of primary/early auxin response genes in protoplast transfection assays. Repression is thought to result from Aux/IAA proteins dimerizing with auxin response factor (ARF) transcriptional activators that reside on auxin-responsive promoter elements, referred to as AuxREs. Most Aux/IAA proteins contain four conserved domains, designated domains I, II, III, and IV. Domain II and domains III and IV play roles in protein stability and dimerization, respectively. A clear function for domain I had not been established. Results reported here indicate that domain I in Aux/IAA proteins is an active repression domain that is transferable and dominant over activation domains. An LxLxL motif within domain I is important for conferring repression. The dominance of Aux/IAA repression domains over activation domains in ARF transcriptional activators provides a plausible explanation for the repression of auxin response genes via ARF-Aux/IAA dimerization on auxin-responsive promoters.

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↗

PubMed2005

LeuO protein delimits the transcriptionally active and repressive domains on the bacterial chromosome.

LeuO protein relieves bacterial gene silencer AT8-mediated transcriptional repression as part of a promoter relay mechanism found in the ilvIH-leuO-leuABCD gene cluster. The gene silencing activity has recently been characterized as a nucleoprotein filament initiated at the gene silencer. In this gene locus, the nucleoprotein filament cis-spreads toward the target leuO promoter and results in the repression of the leuO gene. Although the cis-spreading nature of the transcriptionally repressive nucleoprotein filament has been revealed, the mechanism underlying LeuO-mediated gene silencing relief remains unknown. We have demonstrated here that LeuO functions analogously to the eukaryotic boundary element that delimits the transcriptionally active and repressive domains on the chromosome by blocking the cis-spreading pathway of the transcriptionally repressive heterochromatin. Given that one LeuO-binding site is positioned between the gene silencer and the target promoter, the simultaneous presence of a second LeuO-binding site synergistically enhances the blockade, resulting in a cooperative increase in LeuO-mediated gene silencing relief. A known DNA loop-forming protein, the lac repressor (LacI), was used to confirm that cooperative protein binding via DNA looping is responsible for the blocking synergy. Indeed, a distal LeuO site located downstream cooperates with the LeuO sites located upstream of the leuO gene, resulting in synergistic relief for the repressed leuO gene via looping out the intervening DNA between LeuO sites in the ilvIH-leuO-leuABCD gene cluster.

Binding Sites↗

PubMed2001

The speckling domain of the Wilms tumor suppressor WT1 overlaps with the transcriptional repression domain.

The Wilms tumor suppressor gene WT1 encodes a zinc finger protein, expressed as different splicing variants, that has all the hallmarks of a transcription factor. The -KTS form of WT1 displays a homogeneous localization within the nucleus and has been shown to activate or repress the activity of various target genes. In contrast, the WT1(+KTS) variant demonstrates a speckled pattern of expression within the nucleus. This and its association with factors of the splicing machinery has led to the hypothesis that WT1(+KTS) might play a role in post-transcriptional processes. By the generation of a series of deletion constructs and subsequent immunofluorescence analysis, we have identified and characterized the domain which is responsible for the localization of WT1 variants in nuclear speckles. The speckling domain comprises amino acids 76-120 within the N-terminus of WT1 and is sufficient to target other proteins into distinct nuclear domains. Interestingly the WT1 speckling domain does not overlap with the domain required for interaction with the splicing factor U2AF65 but overlaps with the transcriptional repression domain. Thus our data challenge the view that association of WT1 with spliceosomes is responsible for the speckling phenotype.

Animals↗

PubMed2006

Structure/function of KRAB repression domains: structural properties of KRAB modules inferred from hydrodynamic, circular dichroism, and FTIR spectroscopic analyses.

The abundant zinc finger proteins (ZFPs) sharing the KRAB motif, a potent transcription repression domain, direct the assembly on templates of multiprotein repression complexes. A pivotal step in this pathway is the assembly of a KRAB domain-directed complex with a primary corepressor, KAP1/KRIP-1/TIF1beta. The structure/function dependence of KRAB/TIF1beta protein-protein interaction and properties of the complex, therefore, play pivotal roles in diverse cellular processes depending on KRAB-ZFPs regulation. KRAB domains are functionally bipartite. The 42 amino acid-long KRAB-A module, indeed, is necessary and sufficient for transcriptional repression and for the interaction with the tripartite RBCC region of TIF1beta, while the KRAB-B motif seems to potentiate the assembly of the complex. The structural properties of KRAB-A and KRAB-AB domains from the human ZNF2 protein have been investigated by characterizing highly purified lone (A) and composite (AB) modules. Hydrodynamic and spectroscopic features, investigated by means of gel filtration, circular dichroism, and infrared spectroscopy, provide evidence that both KRAB-A and KRAB-AB domains present low compactness, structural disorder, residual secondary structure content, flexibility, and tendency to molecular aggregation. Comparative analysis among KRAB-A and KRAB-AB modules suggests that the presence of the -B module may influence the properties of lone KRAB-A by affecting the structural flexibility and stability of the conformers. The combined experimental data and the intrinsic features of KRAB-A and KRAB-AB primary structures indicate a potential role of specific subregions within the modules in driving structural flexibility, which is proposed to be of importance for their function.

Amino Acid Sequence↗

PubMed1997

Two evolutionarily conserved repression domains in the Drosophila Kruppel protein differ in activator specificity.

To identify biologically functional regions in the product of the Drosophila melanogaster gene Kruppel, we cloned the Kruppel homolog from Drosophila virilis. Both the previously identified amino (N)-terminal repression region and the DNA-binding region of the D. virilis Kruppel protein are greater than 96% identical to those of the D. melanogaster Kruppel protein, demonstrating a selective pressure to maintain the integrity of each region during 60 million to 80 million years of evolution. An additional region in the carboxyl (C) terminus of Kruppel that was most highly conserved was examined further. A 42-amino-acid stretch within the conserved C-terminal region also encoded a transferable repression domain. The short, C-terminal repression region is a composite of three subregions of distinct amino acid composition, each containing a high proportion of either basic, proline, or acidic residues. Mutagenesis experiments demonstrated, unexpectedly, that the acidic residues contribute to repression function. Both the N-terminal and C-terminal repression regions were tested for the ability to affect transcription mediated by a variety of activator proteins. The N-terminal repression region was able to inhibit transcription in the presence of multiple activators. However, the C-terminal repression region inhibited transcription by only a subset of the activator proteins. The different activator specificities of the two regions suggest that they repress transcription by different mechanisms and may play distinct biological roles during Drosophila development.

Amino Acid Sequence↗

PubMed1994

Krüppel-associated boxes are potent transcriptional repression domains.

The Krüppel-associated box (KRAB) is a highly conserved, 75-aa region containing two predicted amphipathic alpha-helices. The KRAB domain is present in the amino-terminal regions of more than one-third of all Krüppel-class Cys2His2 zinc finger proteins and is conserved from yeast to man; however, its function is unknown. Here it is shown that the KRAB domain functions as a DNA binding-dependent transcriptional repressor when fused to a heterologous DNA-binding domain from the yeast GAL4 protein. A 45-aa segment containing one of the predicted KRAB amphipathic helices was necessary and sufficient for repression. Amino acid substitutions in the predicted helix abolished the repression function. These results assign a function, transcriptional repression, to the highly conserved KRAB box and define a minimal repression domain which may aid in identifying mechanisms of repression.

3T3 Cells↗

PubMed1995

Masking of the CBF1/RBPJ kappa transcriptional repression domain by Epstein-Barr virus EBNA2.

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) is a transcriptional activator that is essential for EBV-driven B cell immortalization. EBNA2 is targeted to responsive promoters through interaction with a cellular DNA binding protein, C promoter binding factor 1 (CBF1). A transcriptional repression domain has been identified within CBF1. This domain also interacts with EBNA2, and repression is masked by EBNA2 binding. Thus, EBNA2 acts by countering transcriptional repression. Mutation at amino acid 233 of CBF1 abolishes repression and correlates with a loss-of-function mutation in the Drosophila homolog Su(H).

Antigens, Viral↗