Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Analysis of the 5'-flanking region of the gene encoding the 28-kDa glutathione S-transferase of Schistosoma mansoni (Sm28GST) indicated the presence of motifs identical to AP-1 and CCAAT-family transcription factor recognition sequences. Gel retardation experiments showed that nuclear extracts from adult S. mansoni bound to an oligodeoxynucleotide containing at CCAAT box. A DNA fragment corresponding to the region of Sm28GST containing the CCAAT motif was demonstrated to interact with schistosome nuclear proteins. This binding was dependent on the presence of the CCAAT pentanucleotide motif. Nuclear factor Y (NF-Y) is a member of the CCAAT transcription factor family that has absolute requirement for the CCAAT sequence and that is highly conserved throughout evolution. The results of a PCR-based strategy aimed at cloning the NF-YA protein of S. mansoni are presented.
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PMID:Schistosoma mansoni: interaction of nuclear extracts with the CCAAT-binding site revealed by the gel shift assay. 782 4

The CCAAT-binding factor NF-Y (CBF/CP1) is a heteromeric transcription factor involved in the regulation of a variety of eukaryotic genes. We identified NF-Y as the CCAAT activity binding to the promoter region of the gene coding for the 28-kDa glutathione S-transferase of the human parasite Schistosoma mansoni (Sm28GST). We isolated the NF-YA cDNA from S. mansoni (SmNF-YA): the complete 268 amino acid sequence harbors a region in its C-terminal part that shows homology with the subunit interaction and DNA-binding domains of the mammalian NF-YA; the N-terminal region has an amino acid composition reminiscent of the mammalian and echinoderm counterparts, rich in glutamine and hydrophobic residues, but shows no sequence similarity at the primary level. In vitro synthesized SMNF-YA is able to associate with mammalian NF-YB/C subunits in the absence of DNA and to bind to the Sm28GST CCAAT box. Surprisingly, a monoclonal antibody directed against the non-conserved Q-rich activation domain of mammalian NF-YA supershifts and immunoprecipitates SMNF-YA, strongly suggesting structure conservation in the activation domain between divergent species.
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PMID:Cloning of Schistosoma mansoni transcription factor NF-YA subunit: phylogenic conservation of the HAP-2 homology domain. 881 62

The regulation of the rat fatty acid synthase gene by mediators such as diet, hormones, cAMP, sterols or retinoic acid is controlled by three NF-Y binding sites. All three sites have a neighbouring Sp1-binding GC-box. This NF-Y/Sp1 motif is conserved in the FAS promoters of rat, human, goose and chicken. We have previously shown cooperative binding of NF-Y and Sp1 to the promoter region at -500 coincident with a diet-induced DNAse I-hypersensitive site. Here, we show an in-vivo interaction of NF-YA with Sp1 using the yeast two-hybrid system. The interacting domains are located between amino acids 55 and 139 of the NF-Y subunit NF-YA and between amino acids 139 and 344 of Sp1. In addition, we show by co-immunoprecipitation direct interaction of NF-Y subunit NF-YA with Sp1 in extracts of rat hepatoma cells H4IIE. Furthermore, we demonstrate by the GST pull-down assay that NF-YA interacts physically with Sp1 in-vitro in the absence of DNA. Therefore, NF-Y can be added to the list of transcription factors interacting with Sp1.
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PMID:Interaction between the two ubiquitously expressed transcription factors NF-Y and Sp1. 1039 39

17beta-Estradiol (E2) stimulated proliferation and DNA synthesis in MCF-7 human breast cancer cells, and this was accompanied by induction of E2F1 mRNA and protein levels. Analysis of the E2F1 gene promoter showed that the -146 to -54 region was required for E2-responsiveness in transient transfection assays, and subsequent deletion/mutation analysis showed that a single upstream GC-rich and two downstream CCAAT-binding sites were required for transactivation by E2. Gel mobility shift assays with multiple oligonucleotides and protein antibodies (for supershifts) showed that the -146 to -54 region of the E2F1 gene promoter bound Sp1 and NF-Y proteins in MCF-7 cells. The estrogen receptor (ER) protein enhanced Sp1 interactions with upstream GC-rich sites, and interactions of ER, Sp1, and ER/Sp1 with downstream DNA bound-NF-Y was investigated by kinetic analysis for protein-DNA binding (on- and off-rates), coimmunoprecipitation, and pulldown assays using wild-type and truncated glutathione S-transferase (GST)-Sp1 chimeric proteins. The results showed that Sp1 protein enhanced the Bmax of NF-Y-DNA binding by more than 5-fold (on-rate); in addition, the Sp1-enhanced NF-Y-DNA complex was further stabilized by coincubation with ER and the rate of dissociation (t1/2) was decreased by approximately 50%. Sp1 antibodies immunoprecipitated [35S]NF-YA after coincubation with unlabeled Sp1 protein. Thus, transcriptional activation of E2F1 gene expression in MCF-7 cells by E2 is regulated by multiprotein ER/Sp1-NF-Y interactions at GC-rich and two CCAAT elements in the proximal region of the E2F1 gene promoter. This represents a unique trans-acting protein complex in which ligand-dependent transactivation by the ER is independent of direct ER interactions with promoter elements.
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PMID:Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. 1044 10

We used the yeast two-hybrid system to show that the serum response factor (SRF) and zinc-fingers and homeobox 1 (ZHXI) proteins interact with the A subunit of nuclear factor-Y (NF-YA). GST pulldown assays revealed that both proteins interact specifically with NF-YA in vitro. Amino acids located between 272 and 564, a region that contains two homeodomains, are required for the interaction of ZHX1 with NF-YA. Two different domains of NF-YA, a glutamine-rich region and a serine/threonine-rich region, are necessary for the interactions with ZHX1 and SRF, respectively.
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PMID:Identification of proteins that interact with NF-YA. 1057 Oct 58

The three distal transcriptional regulatory elements of the rat pyruvate kinase M gene, referred to as boxes A, B, and C, are located around -270 base pairs upstream from the transcriptional initiation site. Electrophoretic mobility shift assays with specific competitors and antibodies show that both box A and box B bind to Sp1 and Sp3 and that box C binds nuclear factor-Y (NF-Y). Luciferase reporter assays revealed that although box A and box B alone have no independent effect on luciferase activities, box C alone stimulates transcription. However, the inclusion of all three elements lead to maximal activity because of a synergistic effect, mainly between box B and box C, suggesting that functional synergism between Sp1/Sp3 and NF-Y is critical for the pyruvate kinase M (PKM) gene distal promoter activity. In fact, co-transfection of a dominant negative mutant of NF-YA (NF-YA29) resulted in a decrease in reporter activity in a box C-dependent manner. In addition, the overexpression of Sp1 or Sp3 and NF-Y in Drosophila SL2 cells synergistically stimulated PKM gene distal promoter activity. Using a mammalian two-hybrid system in HeLa cells, it was shown that both Sp1 and Sp3 interacted with NF-YA but not NF-YB and NF-YC. Moreover, glutathione S-transferase pull-down assays revealed that only in vitro translated (35)S-labeled NF-YA interacted with both Sp1 and Sp3 in vitro. A subunit interaction domain of NF-YA, which forms a heterotrimer with NF-YB and NF-YC, is not required for these interactions with Sp1 or Sp3. Thus, we conclude that Sp1, Sp3, and NF-Y stimulate the transcription of the PKM gene via their interactions.
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PMID:Sp family members and nuclear factor-Y cooperatively stimulate transcription from the rat pyruvate kinase M gene distal promoter region via their direct interactions. 1074 33

The vasorelaxant and anti-mitogenic activities of the atrial and brain natriuretic peptides depend upon their binding to the type A natriuretic peptide receptor (NPR-A) expressed on the surface of vascular cells. Intervention strategies aimed at controlling NPR-A expression are limited by the paucity of studies in this area. Here we identify a sequence CCAAT between -141 and -137 of the NPR-A promoter that, when mutated, reduces promoter activity by 90% in rat aortic smooth muscle (RASM) cells. Protein/DNA cross-linking and immunoperturbation of electrophoretically shifted complexes formed between RASM nuclear extracts and an oligonucleotide surrounding the CCAAT sequence indicates that the heterotrimeric transcription factor NF-Y binds specifically to the wild-type, but not mutated, CCAAT element. Cotransfection of a dominant negative mutant of the NF-YA subunit results in a concentration-dependent decrease in the activity of the NPR-A promoter in RASM cells confirming that endogenous NF-Y is an activator of the promoter. Mutation of the CCAAT element, in conjunction with mutation of all three Sp1 sites previously shown to be involved in NPR-A promoter regulation, virtually eliminates NPR-A promoter activity in RASM cells. Coexpression of all three NF-Y subunits together with Sp1 in Drosophila cells deficient in these factors indicates that NF-Y and Sp1 act synergistically to reconstitute NPR-A promoter activity. A direct physical association between NF-Y and Sp1 can be demonstrated both in vitro by glutathione S-transferase pull-down assay and in the intact cell by coimmunoprecipitation and functional studies. Together, these studies show that NPR-A promoter activity is dominantly regulated through functional, and possibly physical, interactions of NF-Y and Sp1.
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PMID:Functional interaction of NF-Y and Sp1 is required for type a natriuretic peptide receptor gene transcription. 1102 37

c-Myc plays a key role in the cell cycle dependent control of the PDGF beta-receptor mRNA. The mouse platelet-derived growth factor (PDGF) beta-receptor promoter contains a CCAAT motif, and NF-Y plays an essential role in its transcription. Coexpression of c-Myc represses PDGF beta-receptor luciferase reporter activity, and the CCAAT motif in the promoter is indispensable for this repression. Here we show that c-Myc binds NF-Y subunits, YB and YC, by immunoprecipitation from cotransfected COS-1 cells. The in vitro-translated c-Myc also binds the glutathione S-transferase (GST)-NF-YB fusion protein and GST-NF-YC, but not GST-NF-YA. The most C-terminal region of HAP domains of NF-YB and NF-YC, and the Myc homology boxes, but not the C-terminal bHLHZip domain, are indispensable for the coimmunoprecipitation, and also for the repression of PDGF beta-receptor. c-Myc binds NF-Y complex without affecting the efficiency of NF-Y binding to DNA. However, the expression of Myc represses the transcriptional activation of NF-YC when fused to the GAL4 DNA binding domain. Furthermore, this repression was seen only when Myc homology boxes are present, and NF-YC contains the c-Myc binding region.
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PMID:Mechanism for the transcriptional repression by c-Myc on PDGF beta-receptor. 1128 29

We have previously characterized binding sites for the NF-Y transcription factor (-71/-52) and Brn-2 POU-domain protein (-92/-71) in the neuronal promoter of the human aromatic L-amino acid decarboxylase gene [Mol. Brain Res. 56 (1998) 227]. We have now explored the functional role of these binding sites in transfected SK-N-BE neuroblastoma cells. Mutations of the NF-Y site that abolish binding depressed expression of a luciferase reporter gene up to 25-fold. The overexpression of a dominant negative mutant of NF-YA subunit depressed expression by 60%. Promoter activity was increased by the overexpression of Brn-2. Mutations or deletion of the binding site of Brn-2 did not suppress transcriptional activation by overexpressed Brn-2, while promoters defective in NF-Y binding were not transactivated by Brn-2. A GST-pulldown experiment showed that recombinant human Brn-2 protein weakly interacts with recombinant NF-Y outside of DNA. Cooperative binding of recombinant NF-Y and GST--Brn-2 proteins on the neuronal promoter was evidenced by an electrophoretic mobility shift assay. The POU-domain of Brn-2 was sufficient for such interaction. The results thus suggest that the activation of the neuronal promoter of the aromatic L-amino acid decarboxylase gene requires a direct interaction between the ubiquitous NF-Y factor and a cell-specific POU-domain protein. The NF-Y, but not the Brn-2 binding site, is essential for the recruitment of the NF-Y/Brn-2 complex on the promoter.
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PMID:NF-Y binding is required for transactivation of neuronal aromatic L-amino acid decarboxylase gene promoter by the POU-domain protein Brn-2. 1131 76