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At least 955 records · Page 53Linked to original sources
PubMed2002

Promyelocytic leukemia protein PML inhibits Nur77-mediated transcription through specific functional interactions.

The promyelocytic leukemia protein PML is a tumor and growth suppressor and plays an important role in a multiple pathways of apoptosis and regulation of cell cycle progression. Our previous studies and others also documented a role of PML in transcriptional regulation through its association with transcription coactivator CBP and transcription corepressor HDAC. Here, we showed that PML is a potent transcriptional repressor of Nur77, an orphan receptor and a member of the steroid receptor superfamily of proteins. We found that PML represses Nur77-mediated transactivation through a physical and functional interaction between the two proteins. PML interacts with Nur-77 in vitro in a GST-pull down assay and in vivo by coimmunoprecipitation assay. PML/Nur77 colocalized in vivo in a double immunofluorescent staining and confocal microscopic analysis. Our study further showed that the coiled-coil domain of PML interacts with the DNA-binding domain of Nur77 (amino acids 267-332). Electrophoretic mobility shift assay demonstrated that PML interferes with Nur77 DNA binding in a dose-dependent manner. This study indicates that PML interacts with the DNA-binding domain of Nur77 and represses transcription by preventing it from binding to the target promoter. This study supports a role of PML/Nur77 interaction in regulating cell growth and apoptosis.

Apoptosis↗
PubMed1999

Coding region intron/exon organization, alternative splicing, and X-chromosome inactivation of the KRAB/FPB-domain-containing human zinc finger gene ZNF41.

ZNF41 belongs to a cluster of human zinc finger genes residing within a gene-rich region at Xp11.23. ZNF41 encodes a KRAB/FPB (Krüppel-associated/finger preceding box) domain, a potent transcription repression motif present in hundreds of vertebrate zinc finger protein genes, composed of two protein modules, A and B. Three introns, placed at identical positions in paralogous genes, interrupt four exons encoding the ZNF41 N-terminal amino acids, the KRAB/FPB-A and KRAB/FPB-B modules, and the remaining coding region adjoined to the C-terminal zinc finger domain. Since the KRAB/FPB-A and KRAB/FPB-B modules are encoded by dedicated exons in ZNF41 and paralogous genes, exon skipping may lead to differential usage of these modules in alternative gene products. RT-PCR analysis of ZNF41 mRNAs showed that, while skipping of the KRAB/FPB-A and/or KRAB/FPB-B exons was not detected, the use of alternative donor/acceptor sites upstream of the KRAB/FPB-A exon generates multiple ZNF41 transcripts potentially encoding polypeptides differing in the N-terminal region and expressed in different tissues. The expression pattern in cell hybrids containing either active or inactive X chromosomes indicates that ZNF41, which resides within a region of the X chromosome that includes genes that are both subject to and escape X-inactivation, is susceptible to X-chromosome inactivation.

Alternative Splicing↗
PubMed2002

Cross-repression, a functional consequence of the physical interaction of non-liganded nuclear receptors and POU domain transcription factors.

Nuclear receptors (NRs) and POU domain factors form two important transcription factor families for which several levels of functional interference have been described. In this study, the adopted orphan receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) were found to perform direct protein-protein interactions with Pit-1, a representative POU domain factor. The ligand-dependent interaction profile of Pit-1 with CAR, PXR, and the vitamin D receptor in solution was shown to be that of a corepressor. In the absence of receptor agonist Pit-1 inhibited the complex formation of NRs with the retinoid X receptor on DNA. Also in living cells, Pit-1 and Oct-1, another POU domain factor, behaved like corepressors of NR signaling, and Pit-1-mediated repression was found to involve histone deacetylases. Conversely vitamin D receptor, CAR, and PXR were shown to act as repressors of Pit-1 signaling in different cell lines (MCF-7, HaCaT, and GH4C1). This repression was found to be independent of histone deacetylases and seems to be based on a competition of NRs with coactivator and corepressor proteins for overlaying interaction interfaces on the surface of Pit-1. Taken together this study suggests that cross-repression should occur in all tissues in which POU domain factors and non-liganded NRs meet each other.

Amino Acid Sequence↗
PubMed1999

Modulation of TEL transcription activity by interaction with the ubiquitin-conjugating enzyme UBC9.

The E-26 transforming specific (ETS)-related gene TEL, also known as ETV6, is involved in a large number of chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. The encoded protein contains two functional domains: a helix-loop-helix (HLH) domain (also known as pointed domain) located at the N terminus and a DNA-binding domain located at the C terminus. The HLH domain is involved in protein-protein interaction with itself and other members of the ETS family of transcription factors such as FLI1. TEL is a transcription factor, and we and others have shown that it is a repressor of gene expression. To understand further the role of TEL in the cell, we have used an in vivo interaction system to identify proteins that interact with TEL. We show that a protein, UBC9, interacts specifically with TEL in vitro and in vivo. UBC9 is a member of the family of ubiquitin-conjugating enzymes. These enzymes usually are involved in proteosome-mediated degradation; however, our data suggest that interaction of TEL with UBC9 does not lead to TEL degradation. Our studies show that UBC9 binds to TEL exclusively through the HLH domain of TEL. We also show that TEL expressed as fusion to the DNA-binding domain of Gal4 completely represses a Gal4-responsive promoter, but that the coexpression of UBC9 in the same system restores the activity of the promoter. Targeted point mutation of conserved amino acids in UBC9 essential for enzymatic ubiquitination of proteins does not affect interaction nor transcriptional activity. Based on our data, we conclude that UBC9 physically interacts with TEL through the HLH domain and that the interaction leads to modulation of the transcription activity of TEL.

DNA-Binding Proteins↗
PubMed1995

Positive and negative effects of nuclear receptors on transcription activation by AP-1 of the human choline acetyltransferase proximal promoter.

We have examined the 5'-flanking region (944 bp) of the human choline acetyltransferase (hChAT) gene for sequences that modulate its transcriptional activity and identified a sequence 5'-TGACCCA-3' which confers c-Jun/c-Fos (AP-1) inducibility of homologous and heterologous promoters. Using transient transfections in neuroblastoma NE-1-115 and COS-1 cells, we show that ligand-activated estrogen receptor (HEGo) represses the transcriptional activation by c-Fos/c-Jun. Testing HEGo mutants in transfection assays reveals that the ligand-binding domain is crucial for this repression, whereas the N-terminal (A/B) region and the DNA-binding domain are not essential. Gel retardation assays show that the hChAT AP-1 recognition sequence binds in vitro baculovirus-produced c-Jun/c-Fos proteins. This binding is inhibited by addition of baculovirus-produced HEGo. In contrast to HEGo, ligand-activated glucocorticoid, androgen, and retinoic acid receptors (RARs) enhance the transcription activation induced by c-Jun/c-Fos. All three types of RARs--RAR alpha, beta, gamma--and RXR alpha are able to stimulate AP-1 activity on the proximal hChAT promoter. Several mechanism possibilities involving protein-protein interaction are discussed to explain the phenomena.

Cells, Cultured↗
PubMed2006

A novel mechanism of transcriptional repression of p27kip1 through Notch/HRT2 signaling in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation occurs in vascular obstructive events such as atherosclerosis and restenosis. We previously showed that Notch receptors are induced in smooth muscle cells during vascular remodeling. Our goal was to determine the mechanisms employed by Notch signaling to regulate proliferation. Activation of Notch1 and Notch4 induced the VSMC-selective target genes HRT1 and HRT2, promoted cell cycle transit in smooth muscle cells, and led to loss of density-dependent growth inhibition. This was associated with a reduction in levels of the cyclin-dependent kinase inhibitor (cdk) p27(kip1). Over-expression of p27(kip1) resulted in a dose-dependent rescue of the Notch-induced phenotype and exit from the cell cycle. In addition, HRT2 expression was sufficient to promote S-phase entry, and we demonstrate that HRT2 interacts directly with the p27(kip1) promoter to repress transcription. Transcriptional repression occurred within the approximately 774 bp minimal p27(kip1) promoter region and mutational analysis demonstrated that repression is largely dependent on a conserved class-C domain. Our data show that Notch signaling acts to promote a proliferative phenotype in VSMC by modulation of the G1/S-phase checkpoint. In addition, we define a novel mechanism by which the Notch effector, HRT2, interacts directly with the class-C domain of the p27(kip1) promoter, repressing its expression. These studies identify a novel transcriptional target of HRT2, and show that Notch effectors directly control cell cycle regulatory components. We suggest that this mechanism is relevant to hyperproliferative states in VSMC seen during vascular remodeling and repair.

Adenoviridae↗
PubMed1997

Cytosolic domain of the type I interleukin-1 receptor spontaneously recruits signaling molecules to activate a proinflammatory gene.

Immediate postreceptor events activated by IL-1-IL-1R interaction remain undefined. We have initiated studies to identify candidate signal transducers that associate with the cytosolic domain (cd) of the IL-1R. Immunocomplex kinase assays demonstrated an IL-1-activated myelin basic protein kinase activity that coprecipitated with the IL-1R from rat mesangial, mouse EL-4, and HeLa cells. Using glutathione-S-transferase (GST) fusion proteins, HeLa cell lysates next were assayed for kinases that associated with IL-1R cytoplasmic sequences. A GST-IL-1R fusion protein containing the entire cd (amino acids 369-569; GST-IL-1Rcd) recruited a kinase activity in the absence and presence of IL-1 stimulation. In contrast, a GST-IL-1R membrane-proximal region mutant (amino acids 369-501; GST-IL-1RcdDelta), which lacks COOH-terminal amino acid residues required for nuclear factor-kappaB activation, poorly phosphorylated MBP. In gel, kinase assays demonstrated 63-, 83-, and 100-kD kinases that specifically coprecipitated with the HeLa IL-1R and the GST-IL-1Rcd, but not GST-IL-1RcdDelta. 35S-labeled proteins, with Mrs identical to the kinase activities, stably associated with GST-IL-1Rcd. Transient transfection assays of 293 cells were used to evaluate the functional significance of these findings. Simply increasing IL-1cd expression in 293 cells stimulated 5'-IL-6 flanking region-regulated CAT activity threefold above control, an effect blocked by the kinase inhibitors staurosporine and calphostin C. In summary, we have identified two previously unrecognized 63- and 83-kD kinases as well as a protein with an Mr similar to the recently cloned IL-1R-associated kinase, all of which associate spontaneously with the IL-1Rcd. Ectopic IL-1Rcd expression was sufficient to trigger cellular activation, suggesting that the extracellular domain of the intact receptor represses signal transduction until IL-1 is bound. Given that the IL-1Rcd signaling domain has been conserved in a functionally diverse group of transmembrane receptors, further characterization of this signaling process may define novel molecular mechanisms controlling cellular function and differentiation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

The requirement for the p53 proline-rich functional domain for mediation of apoptosis is correlated with specific PIG3 gene transactivation and with transcriptional repression.

Wild-type p53 is a tumor suppressor gene which can activate or repress transcription, as well as induce apoptosis. The human p53 proline-rich domain localized between amino acids 64 and 92 has been reported to be necessary for efficient growth suppression. This study shows that this property mainly results from impaired apoptotic activity. Although deletion of the proline-rich domain does not affect transactivation of several promoters, such as WAF1, MDM2 and BAX, it does alter transcriptional repression, reactive oxygen species production and sequence-specific transactivation of the PIG3 gene, and these are activities which affect apoptosis. Whereas gel retardation assays revealed that this domain did not alter in vitro the specific binding to the p53-responsive element of PIG3, this domain plays a critical role in transactivation from a synthetic promoter containing this element. To explain this discrepancy, evidence is given for a proline-rich domain-mediated cellular activation of p53 DNA binding.

Antibodies, Monoclonal↗
PubMed1993

A leucine zipper domain of the suppressor of Hairy-wing protein mediates its repressive effect on enhancer function.

The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effect of the gypsy retrotransposon by repressing the function of transcriptional enhancers controlling the expression of the mutant gene. A structural and functional analysis of su(Hw) was carried out to identify domains of the protein responsible for its negative effect on enhancer action. Sequence comparison among the su(Hw) proteins from three different species allows the identification of evolutionarily conserved domains with possible functional significance. An acidic domain located in the carboxy-terminal end of the Drosophila melanogaster protein is not present in su(Hw) from other species, suggesting a nonessential role for this part of the protein. A second acidic domain located in the amino-terminal region of su(Hw) is present in all species analyzed. This domain is dispensable in the D. melanogaster protein when the carboxy-terminal acidic domain is present, but the protein is nonfunctional when both regions are simultaneously deleted. Mutations in the zinc fingers result in su(Hw) protein unable to interact with DNA in vivo, indicating a functional role for this region of the protein in DNA binding. Finally, a region of su(Hw) homologous to the leucine zipper motif is necessary for the negative effect of this protein on enhancer function, suggesting that su(Hw) might exert this effect by interacting, directly or indirectly, with transcription factors bound to these enhancers.

Amino Acid Sequence↗
PubMed1999

A conserved motif in goosecoid mediates groucho-dependent repression in Drosophila embryos.

Surprisingly small peptide motifs can confer critical biological functions. One example is the WRPW tetrapeptide present in the Hairy family of transcriptional repressors, which mediates recruitment of the Groucho (Gro) corepressor to target promoters. We recently showed that Engrailed (En) is another repressor that requires association with Gro for its function. En lacks a WRPW motif; instead, it contains another short conserved sequence, the En homology region 1 (eh1)/GEH motif, that is likely to play a role in tethering Gro to the promoter. Here, we characterize a repressor domain from the Goosecoid (Gsc) developmental regulator that includes an eh1/GEH-like motif. We demonstrate that this domain (GscR) mediates efficient repression in Drosophila blastoderm embryos and that repression by GscR requires Gro function. GscR and Gro interact in vitro, and the eh1/GEH motif is necessary and sufficient for the interaction and for in vivo repression. Because WRPW- and eh1/GEH-like motifs are present in different proteins and in many organisms, the results suggest that interactions between short peptides and Gro represent a widespread mechanism of repression. Finally, we investigate whether Gro is part of a stable multiprotein complex in the nucleus. Our results indicate that Gro does not form stable associations with other proteins but that it may be able to assemble into homomultimeric complexes.

Animals↗
PubMed1996

Mutational analysis of the C-terminal region of AREA, the transcription factor mediating nitrogen metabolite repression in Aspergillus nidulans.

In Aspergillus nidulans the positive-acting, wide domain regulatory gene areA mediates nitrogen metabolite repression. Previous analysis demonstrated that the C-terminal 153 residues of the areA product (AREA) are inessential for at least partial expression of most genes subject to regulation by areA. Paradoxically, areAr2, a -1 frameshift replacing the wild-type 122 C-terminal residues with a mutant peptide of 117 amino acids, leads to general loss of function. To determine the basis for the areAr2 mutant phenotype, and as a means of delineating functional domains within the C-terminal region of AREA, we have selected and characterised areAr2 revertants. Deletion analysis, utilising direct gene replacement, extended this analysis. A mutant areA product truncated immediately after the last residue of the highly conserved GATA (DNA-binding) domain retains partial function. The areAr2 product retains some function with respect to the expression of uaZ (encoding urate oxidase) and the mutant allele is partially dominant with respect to nitrate reductase levels. Consistent with the areAr2 product having a debilitating biological activity, we have demonstrated that a polypeptide containing both the wild-type DNA-binding domain and the mutant C-terminus of AREA2 is able to bind DNA in vitro but no longer shows specificity for GATA sequences.

Amino Acid Sequence↗
PubMed2003

Investigating the role of the latch in the positive supercoiling mechanism of reverse gyrase.

Reverse gyrase is the only topoisomerase known to positively supercoil DNA and the only protein unique to hyperthermophiles. The enzyme comprises an N-terminal ATPase domain and a C-terminal topoisomerase I domain, which interact to couple the hydrolysis of ATP to the overwinding of DNA. The part of the ATPase domain termed the "latch" represses topoisomerase activity in the absence of nucleotide. Here I provide evidence that the latch, in addition to its regulatory role, participates in the supercoiling mechanism during the DNA cleavage and religation steps. The latch also contributes to the coordination of ATP hydrolysis and positive supercoiling by inhibiting ATPase activity in the absence of supercoiling. The latch therefore plays an important role in the communication between the two domains of reverse gyrase.

Adenosine Triphosphatases↗
PubMed1992

A negative retinoic acid response element in the rat oxytocin promoter restricts transcriptional stimulation by heterologous transactivation domains.

Retinoic acid receptors are ligand-dependent transcription factors that stimulate gene transcription from promoters containing retinoic acid or thyroid hormone response elements. We describe a high-affinity binding site from the rat oxytocin promoter that mediates negative transcriptional regulation by the retinoic acid receptor. To examine whether strong, constitutive transactivation domains would be capable of stimulating gene transcription when bound to this DNA binding site that normally mediates transcriptional repression, we fused the transactivation domain of the herpes simplex viral protein VP16 to the amino terminus of the retinoic acid receptor and tested the activity of the chimeric protein on the negative retinoic acid response element. This chimeric retinoic acid receptor acted as a strong, constitutive transactivator when bound to promoters containing palindromic thyroid hormone/retinoic acid response elements but surprisingly it still repressed gene transcription when bound to promoters containing the oxytocin-negative retinoic acid response element. These results suggest that a negative DNA binding site itself can inhibit the function of even potent constitutive transactivation domains, and provide evidence that tethering of a constitutive transactivation domain to DNA is insufficient to activate gene transcription.

Animals↗
PubMed1998

A role for Groucho tetramerization in transcriptional repression.

The Drosophila Groucho (Gro) protein is a corepressor required by a number of DNA-binding transcriptional repressors. Comparison of Gro with its homologues in other eukaryotic organisms reveals that Gro contains, in addition to a conserved C-terminal WD repeat domain, a conserved N-terminal domain, which has previously been implicated in transcriptional repression. We determined, via a variety of hydrodynamic measurements as well as protein cross-linking, that native Gro is a tetramer in solution and that tetramerization is mediated by two putative amphipathic alpha-helices (termed leucine zipper-like motifs) found in the N-terminal region. Point mutations in the leucine zipper-like motifs that block tetramerization also block repression by Gro, as assayed in cultured Drosophila cells with Gal4-Gro fusion proteins. Furthermore, the heterologous tetramerization domain from p53 fully substitutes for the Gro tetramerization domain in transcriptional repression. These findings suggest that oligomerization is essential for Gro-mediated repression and that the primary function of the conserved N-terminal domain is to mediate this oligomerization.

Amino Acid Sequence↗
PubMed2001

Structure and specificity of GATA proteins in Th2 development.

Development of Th2 subset of CD4+ T cells involves the interleukin-4 (IL-4)- and Stat6-dependent increase in GATA-3 expression during primary activation. Recently we reported that the phenotypic stability and factor independence of Th2 cells involves acquisition of an intracellular pathway that maintains GATA-3 expression. Evidence from retroviral expression studies implied that this pathway involved an autoactivation of GATA-3 expression, since Stat6-deficient T cells induced endogenous GATA-3 when infected with GATA-3-expressing retroviruses. That study left unresolved the issue of whether GATA-3 autoactivation was direct or indirect. Several other Th2-specific transcription factors have been described, including c-Maf and JunB. We therefore examined the ability of these other transcription factors to induce GATA-3 expression and promote Th2 development. Neither c-Maf nor JunB induced Th2 development in Stat6-deficient CD4+ T cells, in contrast to GATA-3. Consistent with this indication of a possible direct autoactivation pathway, we also observed that heterologous GATA family proteins GATA-1, GATA-2, and GATA-4 were also capable of inducing GATA-3 expression in developing Stat6-deficient T cells and promote Th2 development. Mutational analysis revealed evidence for two distinct mechanisms of GATA-3 action. IL-4 induction by GATA-3 required each of the functional domains to be present, whereas repression of gamma interferon could occur even when mutants of GATA-3 lacking the second transactivation domain, TA2, were expressed. The GATA-dependent induction of the GATA-3 but not the other GATA genes in T cells suggests that T-cell-specific cis elements within the GATA-3 locus likely cooperate with a general GATA recognition motif to allow GATA-3-dependent autoactivation.

Animals↗
PubMed2004

Derepression by depolymerization; structural insights into the regulation of Yan by Mae.

Yan, an ETS family transcriptional repressor, is regulated by receptor tyrosine kinase signaling via the Ras/MAPK pathway. Phosphorylation and downregulation of Yan is facilitated by a protein called Mae. Yan and Mae interact through their SAM domains. We find that repression by Yan requires the formation of a higher order structure mediated by Yan-SAM polymerization. Moreover, a crystal structure of the Yan-SAM/Mae-SAM complex shows that Mae-SAM specifically recognizes a surface on Yan-SAM that is also required for Yan-SAM polymerization. Mae-SAM binds to Yan-SAM with approximately 1000-fold higher affinity than Yan-SAM binds to itself and can effectively depolymerize Yan-SAM. Mutations on Mae that specifically disrupt its SAM domain-dependent interactions with Yan disable the derepression function of Mae in vivo. Depolymerization of Yan by Mae represents a novel mechanism of transcriptional control that sensitizes Yan for regulation by receptor tyrosine kinases.

Amino Acid Sequence↗
PubMed2002

GALLEX, a measurement of heterologous association of transmembrane helices in a biological membrane.

Whereas a variety of two-hybrid systems are available to measure the interaction of soluble proteins, related methods are significantly less developed for the measurement of membrane protein interactions. Here we present a two-hybrid system to follow the heterodimerization of membrane proteins in the Escherichia coli inner membrane. The method is based on the repression of a reporter gene activity by two LexA DNA binding domains with different DNA binding specificities. When coupled to transmembrane domains, heterodimeric association is reported by repression of beta-galactosidase synthesis. The LexA-transmembrane chimeric proteins were found to correctly insert into the membrane, and reproducible signals were obtained measuring the homodimerization as well as heterodimerization of wild-type and mutant glycophorin A transmembrane helices. The GALLEX data were compared with data recently gained by other methods and discussed in the general context of heteroassociation of single TM helices. Additionally, the formation of heterodimers between the TM domains of the alpha(4) and the beta(7) integrin subunits were tested. The results show that both homo- and heterodimerization of membrane proteins can be measured accurately using the GALLEX system.

Bacterial Proteins↗
PubMed1997

Interference with the expression of a novel human polycomb protein, hPc2, results in cellular transformation and apoptosis.

Polycomb (Pc) is involved in the stable and heritable repression of homeotic gene activity during Drosophila development. Here, we report the identification of a novel human Pc homolog, hPc2. This gene is more closely related to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1). However, the hPc2 and CBX2/hPc1 proteins colocalize in interphase nuclei of human U-2 OS osteosarcoma cells, suggesting that the proteins are part of a common protein complex. To study the functions of the novel human Pc homolog, we generated a mutant protein, delta hPc2, which lacks an evolutionarily conserved C-terminal domain. This C-terminal domain is important for hPc2 function, since the delta hPc2 mutant protein which lacks the C-terminal domain is unable to repress gene activity. Expression of the delta hPc2 protein, but not of the wild-type hPc2 protein, results in cellular transformation of mammalian cell lines as judged by phenotypic changes, altered marker gene expression, and anchorage-independent growth. Specifically in delta hPc2-transformed cells, the expression of the c-myc proto-oncogene is strongly enhanced and serum deprivation results in apoptosis. In contrast, overexpression of the wild-type hPc2 protein results in decreased c-myc expression. Our data suggest that hPc2 is a repressor of proto-oncogene activity and that interference with hPc2 function can lead to derepression of proto-oncogene transcription and subsequently to cellular transformation.

3T3 Cells↗