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At least 505 records · Page 28Linked to original sources
PubMed2003

Dynamic interplay of the SUMO and ERK pathways in regulating Elk-1 transcriptional activity.

The ETS domain transcription factor Elk-1 is a direct target of the MAP kinase pathways. Phosphorylation of the Elk-1 transcriptional activation domain by MAP kinases triggers its activation. However, Elk-1 also contains two domains with repressive activities. One of these, the R motif, appears to function by suppressing the activity of the activation domain. Here, we demonstrate that SUMO modification of the R motif is required for this repressive activity. A dynamic interplay exists between the activating ERK MAP kinase pathway and the repressive SUMO pathway. ERK pathway activation leads to both phosphorylation of Elk-1 and loss of SUMO conjugation and, hence, to the loss of the repressive activity of the R motif. Thus, the reciprocal regulation of the activation and repressive activities are coupled by MAP kinase modification of Elk-1.

Animals↗
PubMed2001

Activation and repression by the C-terminal domain of Dorsal.

In the Drosophila embryo, Dorsal, a maternally expressed Rel family transcription factor, regulates dorsoventral pattern formation by activating and repressing zygotically active fate-determining genes. Dorsal is distributed in a ventral-to-dorsal nuclear concentration gradient in the embryo, the formation of which depends upon the spatially regulated inhibition of Dorsal nuclear uptake by Cactus. Using maternally expressed Gal4/Dorsal fusion proteins, we have explored the mechanism of activation and repression by Dorsal. We find that a fusion protein containing the Gal4 DNA-binding domain fused to full-length Dorsal is distributed in a nuclear concentration gradient that is similar to that of endogenous Dorsal, despite the presence of a constitutively active nuclear localization signal in the Gal4 domain. Whether this fusion protein activates or represses reporter genes depends upon the context of the Gal4-binding sites in the reporter. A Gal4/Dorsal fusion protein lacking the conserved Rel homology domain of Dorsal, but containing the non-conserved C-terminal domain also mediates both activation and repression, depending upon Gal4-binding site context. A region close to the C-terminal end of the C-terminal domain has homology to a repression motif in Engrailed - the eh1 motif. Deletion analysis indicates that this region mediates transcriptional repression and binding to Groucho, a co-repressor known to be required for Dorsal-mediated repression. As has previously been shown for repression by Dorsal, we find that activation by Dorsal, in particular by the C-terminal domain, is modulated by the maternal terminal pattern-forming system.

Amino Acid Motifs↗
PubMed2003

Methyl-CpG-binding domain protein-2 mediates transcriptional repression associated with hypermethylated GSTP1 CpG islands in MCF-7 breast cancer cells.

GSTP1, encoding the pi-class glutathione S-transferase, is commonly inactivated by somatic CpGisland hypermethylation in cancers of the prostate, liver, and breast. We report here thathypermethylation of CpG dinucleotides at the 5' transcriptional regulatory region was sufficient to inhibit GSTP1 transcription in MCF-7 breast cancer cells and that repression of GSTP1 transcription was mediated in part by the methyl-CpG-binding domain (MBD) protein MBD2. MCF-7 breast cancer cells contained only hypermethylated GSTP1 CpG island alleles and failed to express GSTP1 mRNA or GSTP1 polypeptides. In contrast, MCF-7/ADR cells contained only unmethylated GSTP1 CpG island alleles and exhibited abundant GSTP1 expression. Chromatin immunoprecipitation analysis detected the presence of MBD2 and DNMT1 at the GSTP1 promoter in MCF-7 breast cancer cells but not in MCF-7/ADR breast cancer cells. In a test of the contribution of MBD2 to GSTP1 repression in MCF-7 breast cancer cells, transfection of small interference RNA complementary to MBD2 mRNA into MCF-7 cells both reduced MBD2 polypeptide levels and stimulated GSTP1 mRNA expression. These findings implicate MBD2 in GSTP1 silencing associated with somatic GSTP1 CpG island hypermethylation in breast cancer cells.

Alleles↗
PubMed2006

FOXM1c is activated by cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1, but repressed by GSK-3alpha.

Two different inhibitory domains, N-terminus and central domain, keep FOXM1c almost inactive despite its strong transactivation domain. Here, we demonstrate that cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1 activate FOXM1c. Cyclin E/Cdk2 does not target its transactivation domain or its DNA-binding domain. Instead, its activating effect strictly depends on the presence of either the central domain or the N-terminus of FOXM1c and thus is completely lost if both inhibitory domains are deleted. Cyclin E/Cdk2 activates FOXM1c by releasing its transactivation domain from the repression by these two inhibitory domains. However, it does not directly increase the transactivation potential of the TAD. The DNA-binding is not affected by cyclin E/Cdk2, neither directly nor indirectly. These two activating effects of cyclin E/Cdk2 via central domain and N-terminus are additive. Cyclin A/Cdk2 and cyclin A/Cdk1 show similar characteristics. GSK-3alpha, another proliferation-associated kinase, represses FOXM1c.

Cyclin A↗
PubMed2003

Functional cross-antagonism between transcription factors FLI-1 and EKLF.

FLI-1 is an ETS family transcription factor which is overexpressed in Friend erythroleukemia and contributes to the blockage of differentiation of erythroleukemic cells. We show here that FLI-1 represses the transcriptional activity of the beta-globin gene promoter in MEL cells and interacts with two of its critical transactivators, GATA-1 and EKLF. Unexpectedly, FLI-1 enhances the stimulating activity of GATA-1 on a GATA-1-responsive promoter but represses that of EKLF on beta-globin and an EKLF-responsive artificial promoters. This repressive effect of FLI-1 requires the ETS DNA binding domain and its association with either the N- or C-terminal domain, which themselves interact with EKLF but not with GATA-1. Furthermore, the FLI-1 ETS domain alone behaves as an autonomous repression domain when linked to the Gal4 DNA binding domain. Taken together, these data indicate that FLI-1 represses EKLF-dependent transcription due to the repression activity of its ETS domain and its indirect recruitment to erythroid promoters by protein-protein interaction with EKLF. Reciprocally, we also show that EKLF itself represses the FLI-1-dependent megakaryocytic GPIX gene promoter, thus further suggesting that functional cross-antagonism between FLI-1 and EKLF might be involved in the control of the erythrocytic versus megakaryocytic differentiation of bipotential progenitors.

Acetamides↗
PubMed2005

N-glycosylation influences the latency and catalytic properties of mammalian purple acid phosphatase.

Purple acid phosphatase (PAP), also known as tartrate-resistant acid phosphatase or uteroferrin, contains two potential consensus N-glycosylation sites at Asn(97) and Asn(128). In this study, endogenous rat bone PAP was found to possess similar N-glycan structures as rat recombinant PAP heterologously expressed in baculovirus-infected Sf9 insect cells. PAP from Sf9 cells was shown to contain two N-linked oligosaccharides, whereas PAP expressed by mammalian CHO-K1 cells was less extensively glycosylated. The extent of N-glycosylation affected the catalytic properties of the enzyme, as N97Q and N128Q mutants, containing a single oligosaccharide chain, exhibited a lower substrate affinity and catalytic activity compared to those of the fully glycosylated PAP in the native, monomeric state. The differences in substrate affinity and catalytic activity were abolished and partially restored, respectively, by proteolytic cleavage in the loop domain, indicating that the extent of N-glycosylation influences the interaction of the repressive loop domain with catalytically important residues.

Acid Phosphatase↗
PubMed2003

Nuclear localization and transcriptional repression are confined to separable domains in the circadian protein CRYPTOCHROME.

Circadian rhythms are driven by molecular clocks composed of interlocking transcription/translation feedback loops. CRYPTOCHROME (CRY) proteins are critical components of these clocks and repress the activity of the transcription factor heterodimer CLOCK/BMAL1. Unlike the homologous DNA repair enzyme 6-4 PHOTOLYASE, CRYs have extended carboxyl-terminal tails and cannot repair DNA damage (reviewed in ). Unlike mammals, Xenopus laevis contains both CRYs (xCRYs) and 6-4 PHOTOLYASE (xPHOTOLYASE), providing an excellent comparative tool to study CRY repressive function. We can extend findings to CRYs in general because xCRYs share high sequence homology with mammalian CRYs. We show here that deletion of xCRYs' C-terminal domain produces proteins that are, like xPHOTOLYASE, unable to suppress CLOCK/BMAL1 activation. However, these truncations also cause the proteins to be cytoplasmically localized. A heterologous nuclear localization signal (NLS) restores the truncation mutants' nuclear localization and repressive activity. Our results demonstrate that the CRYs' C termini are essential for nuclear localization but not necessary for the suppression of CLOCK/BMAL1 activation; this finding indicates that these two functions reside in separable domains. Furthermore, the functional differences between CRYs and PHOTOLYASE can be attributed to the few amino acid changes in the conserved portions of these proteins.

Amino Acid Sequence↗
PubMed1999

SMRTe, a silencing mediator for retinoid and thyroid hormone receptors-extended isoform that is more related to the nuclear receptor corepressor.

SMRT (silencing mediator for retinoid and thyroid hormone receptors) and N-CoR (nuclear receptor copressor) mediate transcriptional repression of important regulators that are involved in many signaling pathways. SMRT and N-CoR are related proteins that form complexes with mSin3A/B and histone deacetylases to induce local chromatin condensation and transcriptional repression. However, SMRT is substantially smaller than N-CoR, lacking an N-terminal domain of approximately 1,000 aa that are present in N-CoR. Here, we report the identification of SMRT-extended (SMRTe), which contains an N-terminal sequence that shows striking similarity with N-CoR. As in N-CoR, this SMRTe-N-terminal domain also represses basal transcription. We find that SMRTe expression is regulated during cell cycle progression and SMRTe transcripts are present in many embryonic tissues. These data redefine a structurally and functionally more related nuclear receptor corepressor family and suggest an additional role for SMRTe in the regulation of cycle-specific gene expression in diverse signaling pathways.

Amino Acid Sequence↗
PubMed2003

Structure of the sterile alpha motif (SAM) domain of the Saccharomyces cerevisiae mitogen-activated protein kinase pathway-modulating protein STE50 and analysis of its interaction with the STE11 SAM.

The sterile alpha motif (SAM) is a 65-70-amino acid domain found in over 300 proteins that are involved in either signal transduction or transcriptional activation and repression. SAM domains have been shown to mediate both homodimerization and heterodimerization and in some cases oligomerization. Here, we present the solution structure of the SAM domain of the Saccharomyces cerevisiae protein, Ste50p. Ste50p functions as a modulator of the mitogen-activated protein kinase (MAPK) cascades in S. cerevisiae, which control mating, pseudohyphal growth, and osmo-tolerance. This is the first example of the structure of a SAM domain from a MAPK module protein. We have studied the associative behavior of Ste50p SAM in solution and shown that it is monomeric. We have examined the SAM domain from Ste11p, the MAPK kinase kinase that associates with Ste50p in vivo, and shown that it forms dimers with a self-association K(d) of approximately 0.5 mm. We have also analyzed the interaction of Ste50p SAM with Ste11p SAM and the effects of mutations at Val-37, Asp-38, Pro-71, Leu-73, Leu-75, and Met-99 of STE50 on the heterodimerization properties of Ste50p SAM. We have found that L73A and L75A abrogate the Ste50p interaction with Ste11p, and we compare these data with the known interaction sites defined for other SAM domain interactions.

Amino Acid Sequence↗
PubMed1999

hairy stripe 7 element mediates activation and repression in response to different domains and levels of Krüppel in the Drosophila embryo.

The Drosophila gap gene Krüppel (Kr) encodes a zinc finger-type transcription factor required for controlling the spatial expression of other segmentation genes during early blastoderm stage. Here we show that two independent and transferable repressor domains of Krüppel act to control expression of the pair-rule gene hairy, and that the minimal cis-acting element of hairy stripe7 (h7) mediates either Krüppel-dependent activation or repression in different regions of the blastoderm embryo. The C-terminal region of Krüppel which encompasses the predominant repressor domain is not essential for activation, but is required to fully suppress h7-mediated transcription in response to high levels of Krüppel activity. This domain contains an interaction motif for dCtBP, a homologue of the human co-repressor CtBP. dCtBP activity is, however, dispensable for Krüppel-mediated repression in the embryo since Krüppel-mediated repression functions in the absence of dCtBP. Possible modes of h7-mediated gene regulation in response to the different domains and levels of Krüppel are discussed.

Alcohol Oxidoreductases↗
PubMed2005

Contextual interactions determine whether the Drosophila homeodomain protein, Vnd, acts as a repressor or activator.

At the molecular level, members of the NKx2.2 family of transcription factors establish neural compartment boundaries by repressing the expression of homeobox genes specific for adjacent domains [Muhr et al. (2001) Cell, 104, 861-873; Weiss et al. (1998) Genes Dev., 12, 3591-3602]. The Drosophila homologue, vnd, interacts genetically with the high-mobility group protein, Dichaete, in a manner suggesting co-operative activation [Zhao and Skeath (2002) Development, 129, 1165-1174]. However, evidence for direct interactions and transcriptional activation is lacking. Here, we present molecular evidence for the interaction of Vnd and Dichaete that leads to the activation of target gene expression. Two-hybrid interaction assays indicate that Dichaete binds the Vnd homeodomain, and additional Vnd sequences stabilize this interaction. In addition, Vnd has two activation domains that are typically masked in the intact protein. Whether vnd can activate or repress transcription is context-dependent. Full-length Vnd, when expressed as a Gal4 fusion protein, acts as a repressor containing multiple repression domains. A divergent domain in the N-terminus, not found in vertebrate Vnd-like proteins, causes the strongest repression. The co-repressor, Groucho, enhances Vnd repression, and these two proteins physically interact. The data presented indicate that the activation and repression domains of Vnd are complex, and whether Vnd functions as a transcriptional repressor or activator depends on both intra- and inter-molecular interactions.

Animals↗
PubMed1997

Transcriptional repression mediated by the PR domain zinc finger gene RIZ.

The RIZ (G3B or MTB-Zf) zinc finger gene is structurally related to the myeloid leukemia gene, MDS1-EVI1, and the transcription repressor/differentiation factor, PRDI-BF1/BLIMP1, through a conserved amino-terminal motif, the PR domain. Similar to MDS1-EVI1, RIZ gene normally produces two protein products that differ by the PR domain. The smaller protein RIZ2 lacks the PR domain of RIZ1 but is otherwise identical to RIZ1. Here we show that RIZ proteins bind to GC-rich or Sp-1-binding elements and repress transcription. Both RIZ1 and RIZ2 repressed the herpes simplex virus thymidine kinase (HSV-TK) promoter, one of the best characterized eukaryotic promoters. Recombinant RIZ1 proteins were able to bind to HSV-TK promoter. This binding was mediated by the GC-rich Sp-1 elements of the promoter and the first three zinc finger motifs of RIZ1. RIZ also encodes a repressor domain that was mapped to the central region of the protein. Fusion of this region to the GAL4 DNA-binding domain generated GAL4 site-dependent transcriptional repressors. We also show that RIZ1 protein can efficiently repress the simian virus 40 (SV40) early promoter, which primarily consists of Sp-1 sites; RIZ2, however, only weakly repressed this promoter, suggesting a role for PR in modulating RIZ protein function. The data have implications for a role of RIZ proteins in the regulation of cellular gene promoters, many of which are characterized by GC-rich elements.

3T3 Cells↗
PubMed1995

Developmentally regulated mouse gene NK10 encodes a zinc finger repressor protein with differential DNA-binding domains.

Using oligonucleotides complementary to the conserved inter-finger region of a variety of previously described zinc finger-encoding genes, a novel mouse gene was cloned and characterized. The gene is localized on chromosome 8 and comprises five exons. Its corresponding mRNA is developmentally regulated in various tissues and includes an open reading frame encoding a protein of 72,422 daltons. It shares amino-terminal homologies with human KRAB (or FPB) boxes, and contains 13 zinc fingers of the C2-H2 type. The NK10 KRAB domains exhibit repressing activity when tested in GAL4 fusion protein assays. Cloning of putative target sequences revealed that the individual domains differentially contribute to zinc-dependent target DNA binding.

Amino Acid Sequence↗
PubMed2002

The gibberellin signaling pathway is regulated by the appearance and disappearance of SLENDER RICE1 in nuclei.

The slender rice1 mutant (slr1) shows a constitutive gibberellin (GA) response phenotype. To investigate the mode of action of SLR1, we generated transgenic rice expressing a fusion protein consisting of SLR1 and green fluorescent protein (SLR1-GFP) and analyzed the phenotype of the transformants and the subcellular localization of GFP in vivo. SLR1-GFP worked in nuclei to repress the GA signaling pathway; its overproduction caused a dwarf phenotype. Application of GA(3) to SLR1-GFP overproducers induced GA actions such as shoot elongation, downregulation of GA 20-oxidase expression, and upregulation of SLR1 expression linked with the disappearance of the nuclear SLR1-GFP protein. We also performed domain analyses of SLR1 using transgenic plants overproducing different kinds of truncated SLR1 proteins. The analyses revealed that the SLR1 protein can be divided into four parts: a GA signal perception domain located at the N terminus, a regulatory domain for its repression activity, a dimer formation domain essential for signal perception and repression activity, and a repression domain at the C terminus. We conclude that GA signal transduction is regulated by the appearance or disappearance of the nuclear SLR1 protein, which is controlled by the upstream GA signal.

Arabidopsis Proteins↗
PubMed2001

The biological functions of the methyl-CpG-binding protein MeCP2 and its implication in Rett syndrome.

Methylation of DNA is essential for development in the mouse and plays an important role in inactivation of the X-chromosome, genomic imprinting and gene silencing. The properties of the methyl-CpG binding proteins (MeCPs) are being proved to be the key to interpreting the connection between DNA methylation and transcriptional repression. The founder member of the family, MeCP2, consists of a single polypeptide that contains both a methyl-CpG binding domain (MBD) and transcriptional repression domain (TRD). MBD binds to a single symmetrically methylated CpG site and is responsible for chromatin localization of the protein. NMR studies have revealed that the MBD adopts a wedge-shaped molecular structure. The TRD interacts with Sin3, which is known to form complexes with histone deacetylases. MeCP2-mediated transcriptional repression may involve two distinct mechanisms, one being dependent on chromatin modification by histone deacetylation and the other being chromatin independent. Mutations in MeCP2 gene cause the X-linked neurodevelopmental disease Rett syndrome. The spectrum of mutations reflects the importance of the MBD and TRD domains. We speculate that abnormal gene expression in Rett patients leads to dysfunction of the central nervous system. We propose a genetic therapeutic approach based on activation of the wild type copy of the MeCP2 gene located in the inactive X chromosome.

Animals↗
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↗
PubMed1999

The Wilms' tumor suppressor gene (wt1) product represses different functional classes of transcriptional activation domains.

We have studied the ability of the wt1 tumor suppressor gene product to repress different classes of activation domains previously shown to stimulate the initiation and elongation steps of RNA polymerase II transcription in vivo. Repression assays revealed that WT1 represses all three classes of activation domains: Sp1 and CTF, which stimulate initiation (type I), human immunodeficiency virus type I Tat fused to a DNA-binding domain, which stimulates predominantly elongation (type IIA), and VP16, p53 and E2F1, which stimulate both initiation and elongation (type IIB). WT1 is capable of exerting its repression effect over a significant distance when positioned approximately 1700 bp from the core promoter. Deletion analysis of WT1 indicates that the responsible domain resides within the first 180 N-terminal amino acids of the protein. Nuclear run-ons analyzing the effects of WT1 on initiation of transcription demonstrate inhibition of this process. Our observations imply that WT1 can repress activators that stimulate initiation and/or elongation.

Animals↗
PubMed1993

A novel repression module, an extensive activation domain, and a bipartite nuclear localization signal defined in the immediate-early transcription factor Egr-1.

Egr-1 is an immediate-early response gene induced transiently and ubiquitously by mitogenic stimuli and also regulated in response to signals that initiate differentiation. The Egr-1 gene product, a nuclear phosphoprotein with three zinc fingers of the Cys2His2 class, binds to the sequence CGCCCCCGC and transactivates a synthetic promoter construct 10-fold in transient-transfection assays. We have analyzed the structure and function of the Egr-1 protein in detail, delineating independent and modular activation, repression, DNA-binding, and nuclear localization activities. Deletion analysis, as well as fusions to the DNA-binding domain of GAL4, indicated that the activation potential of Egr-1 is distributed over an extensive serine/threonine-rich N-terminal domain. In addition, a novel negative regulatory function has been precisely mapped 5' of the zinc fingers: amino acids 281 to 314 are sufficient to confer the ability to repress transcription on a heterologous DNA-binding domain. Specific DNA-binding activity was shown to reside in the three zinc fingers of Egr-1, as predicted by homology to other known DNA-binding proteins. Finally, nuclear localization of Egr-1 is specified by signals in the DNA-binding domain and basic flanking sequences, as determined by subcellular fractionation and indirect immunofluorescence. Basic residues 315 to 330 confer partial nuclear localization on the bacterial protein beta-galactosidase. A bipartite signal consisting of this basic region in conjunction with either the second or third zinc finger, but not the first, suffices to target beta-galactosidase exclusively to the nucleus. Our work shows that Egr-1 is a functionally complex protein and suggests that it may play different roles in the diverse settings in which it is induced.

Animals↗