Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Hypermethylation in cancer often occurs in CpG islands that span the promoter regions of tumor suppressor genes. However, it is not clear if hypermethylation is limited to single target genes or if multiple genes are simultaneously methylated. To understand the extent of aberrant de novo methylation, we have analyzed the methylation pattern of a number of tumor-related genes in leukemia from the same cohort of patients. We used bisulfite genomic sequencing to characterize the methylation pattern of the CpG islands associated with the calcitonin, estrogen receptor, E-cadherin, p15, p16, Rb, GST-Pi, and HIC1 genes in the bone marrow from 9 normal and 20 patients with acute myeloid leukaemia (AML). All of the normal control samples were essentially unmethylated for each of the eight tumor-related genes studied. In contrast, 19 of 20 (95%) of the AML patients had an abnormal methylation pattern in at least one gene, and 15 of 20 (75%) had abnormal methylation patterns in two or more of the target genes. We conclude that there is a general deregulation of CpG island methylation in leukemia and that hypermethylation is not limited to single genes, but a number of genes are methylated concurrently. Moreover, the subset of genes that are commonly methylated in leukemia appear to be cancer type specific.
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PMID:Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia. 1044 89

Estrogen-responsive genes are regulated by altering the balance of estrogen receptor (ER) interaction with transcription activators and inhibitors. Here we examined the role of ER ligand on ER interaction with the Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) orphan nuclear receptor. COUP-TF binding to half-site estrogen response elements (EREs) was increased by the addition of estradiol (E2) -liganded ER (E2-ER), but not by ER liganded with the antiestrogens 4-hydroxytamoxifen (4-OHT-ER) or tamoxifen aziridine (TAz-ER). ER did not bind to single half-sites. Conversely, COUP-TF enhanced the ERE binding of purified E2-ER, but did not affect TAz-ER-ERE binding. In contrast, only antiestrogens enhanced direct interaction between ER and COUP-TF as assessed by GST pull-down assays. Identical results were obtained using either purified bovine or recombinant human ERalpha. Co-immunoprecipitation assays showed that ER and COUP-TF interact in extracts from MCF-7 and ERalpha-transfected MDA-MB-231 cells. Here we document that ER ligand impacts COUP-TF-ER interaction. COUP-TF interaction is mediated by the DNA binding and ligand-binding domains of ER. We suggest that changes in ER conformation induced by DNA binding reduce ER-COUP-TF interaction. Transient transfection of human MCF-7 breast cancer cells with a COUP-TFI expression vector repressed E2-induced luciferase reporter gene expression from single or multiple tandem copies of a consensus ERE. COUP-TFI stimulated 4-OHT-induced luciferase activity from a minimal ERE. Alone, COUP-TFI increased transcription from ERE half-sites or a single ERE in a sequence-dependent manner. These data provide evidence that the ERE sequence and its immediate flanking regions influence whether COUP-TF enhances, inhibits, or has no effect on ER ligand-induced ERE reporter gene expression and that COUP-TFI activates gene transcription from ERE half-sites. We suggest that COUP-TFI plays a role in mitigating estrogen-responsive gene expression.
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PMID:Role of estrogen receptor ligand and estrogen response element sequence on interaction with chicken ovalbumin upstream promoter transcription factor (COUP-TF). 1061 53

Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the reason for this requirement is not fully understood. In this study, we found that modified Ras, which is purified from Sf9 cells, could activate Raf-1 in a cell-free system, when incorporated into liposome. Using a bifunctional cross-linker and a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells, respectively. These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1. To support this, we found that, when fused to glutathione S-transferase (GST), unprocessed Ras expressed in Escherichia coli could bypass the requirement for liposome. A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide, H., and Kaziro, Y. (1998) Oncogene 16, 2781-2786), was still required for Raf-1 activation by GST-Ras. Furthermore, an enforced dimerization of unmodified oncogenic Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation of Raf-1. From these results, we conclude that membrane localization allows Ras to form a dimer, which is essential, although not sufficient, for Raf-1 activation.
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PMID:Formation of the Ras dimer is essential for Raf-1 activation. 1066 May 19

A zebrafish Ftz-F1 homologue, zFF1A (zebrafish Ff1a or Nr5a2, a member of nuclear receptor superfamily) and its C-terminally truncated variant (zFF1B) were previously identified. Due to lack of the identity box (I-box) and activation function 2 (AF-2) domain, zFF1B lacks transactivation function and fails to synergize with estrogen receptor (ER) in regulating promoters. It was speculated that the I-box might be involved in the zFF1A/ER interaction. In the present study, the function of the I-box was examined. In the absence of the I-box or with an altered heptad 9, the AF-2 of zFF1A was not functional, either in the presence or absence of ER. The GST pull-down assay showed that zFF1A and its mutants exerted similar physical contacts with ER-LBD, suggesting that the "dimerization" domain (I-box) is essential for the transcriptional activity of zFF1A. Moreover, nuclear receptor coactivator selectively activated zFF1 with the I-box but exerted no effect on zFF1B, indicating that the I-box is able to interact with the coactivators. By deletion study and analysis of the identified domains in GAL4-DNA binding domain, other regions of zFF1A critical for its AF were also delineated. Consistent with the mutation analysis, AF-2 was active only in the presence of the I-box. We also identified a novel AF domain (AF-3) located in the hinge region (amino acids 155-267), although the activity of AF-3 was inhibited by its flanking region. We suggest that the D and E regions of zFF1A possess both positive and negative transactivation functions, and interdomain "cross-talk" may confer the full transcriptional activity of the protein.
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PMID:A zebrafish ftz-F1 (Fushi tarazu factor 1) homologue requires multiple subdomains in the D and E regions for its transcriptional activity. 1074 75

Glutathione S-transferase pi (GST-pi) is a phase II detoxification enzyme whose expression is increased in estrogen receptor (ER)-poor breast cancers and in breast cancers resistant to certain chemotherapeutic agents. The aim of this study was to investigate the immunohistochemical expression of GST-pi in invasive breast carcinoma and to correlate the findings with those of nuclear morphometry. Formalin-fixed paraffin-embedded tissue specimens obtained from 21 invasive breast cancers and 16 adjacent (benign) tissues were immunohistochemically stained using polyclonal anti-human GST-pi antibody. There was positive (defined as >10% immunoreactive tumor cells) but variable expression of GST-pi in 10 (48%) cases. Nuclear morphometry in these 10 tumors revealed immunoreactive malignant cells to be larger (mean area 41.7+/-1.0 microm2) and more rounded in form when compared with non-staining cancer cells (mean area 28.7+/-0.7 microm2). It was also observed that GST-pi immunonegative tumor cells in GST-pi expressing tumors had different morphologies from malignant cells in the remaining 11 (52%) cancers that were regarded as GST-pi negative. Increased GST-pi expression determined by the percentage of positively staining tumor cells, was found to be significantly correlated with increased variability in nuclear area and perimeter (Spearman's rho=0.821, p=0.044 for both) in the subset of node-positive tumors. Our findings suggest that there exists two sub-populations of cancer cells with distinct nuclear morphologies in GST-pi positive tumors; factors other than GST-pi expression are likely to have a phenotypic effect on breast cancer cells; and there may be a special significance of this enzyme in axillary node-positive breast tumors.
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PMID:Nuclear morphometry and glutathione S-transferase pi expression in breast cancer. 1076 77

The human estrogen receptor-related receptor (ERRalpha1, NR3B1a) was shown to bind a steroidogenic factor binding element (SFRE), TCAAGGTCATC, 26 base pairs upstream from the estrogen response element (ERE) of the human lactoferrin gene promoter. A mutation made at SFRE significantly reduced estrogen-dependent transcription from the lactoferrin ERE in human endometrial cells. In this study, we demonstrated that ERRalpha1 binds both SFRE and ERE elements and constitutively transactivates the lactoferrin gene promoter. In DNase I footprinting protection analysis, both SFRE and ERE regions were protected by glutathione S-transferase-ERRalpha1 fusion protein. The receptor formed two protein-DNA complexes with either SFRE or ERE in electrophoresis mobility shift assay. Homodimerization of ERRalpha1 was confirmed with the mammalian two-hybrid system. ERRalpha1 activates reporter constructs containing various types of estrogen response elements in endometrial and non-endometrial cells in transient transfection experiments. Overexpressing the coactivator, SRC1a or GRIP1, further enhances ERRalpha1-induced transcriptional activity. We demonstrated that the AF2 domain of ERRalpha1 is essential for the transactivation function and that deletion or mutation at this region abrogates the activation capability. Protein-protein interaction between the SRC1a and ERRalpha1 C terminus was confirmed with a GST glutathione S-transferase "pull-down" assay. When comparing ERRalpha1 and the estrogen receptor alpha (ERalpha) in many of the experiments, we found that ERalpha can also bind SFRE of the lactoferrin gene and transactivate the promoter activity in a ligand-dependent manner. The present study demonstrated that ERRalpha1 binds similar DNA elements as ERalpha and confers its transactivation function constitutively. Therefore, ERRalpha1 may actively modulate the estrogen response of lactoferrin gene as well as other estrogen-responsive genes.
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PMID:Estrogen receptor-related receptor alpha 1 interacts with coactivator and constitutively activates the estrogen response elements of the human lactoferrin gene. 1077 8

We find that prothymosin alpha (PTalpha) selectively enhances transcriptional activation by the estrogen receptor (ER) but not transcriptional activity of other nuclear hormone receptors. This selectivity for ER is explained by PTalpha interaction not with ER, but with a 37-kDa protein denoted REA, for repressor of estrogen receptor activity, a protein that we have previously shown binds to ER, blocking coactivator binding to ER. We isolated PTalpha, known to be a chromatin-remodeling protein associated with cell proliferation, using REA as bait in a yeast two-hybrid screen with a cDNA library from MCF-7 human breast cancer cells. PTalpha increases the magnitude of ERalpha transcriptional activity three- to fourfold. It shows lesser enhancement of ERbeta transcriptional activity and has no influence on the transcriptional activity of other nuclear hormone receptors (progesterone receptor, glucocorticoid receptor, thyroid hormone receptor, or retinoic acid receptor) or on the basal activity of ERs. In contrast, the steroid receptor coactivator SRC-1 increases transcriptional activity of all of these receptors. Cotransfection of PTalpha or SRC-1 with increasing amounts of REA, as well as competitive glutathione S-transferase pulldown and mammalian two-hybrid studies, show that REA competes with PTalpha (or SRC-1) for regulation of ER transcriptional activity and suppresses the ER stimulation by PTalpha or SRC-1, indicating that REA can function as an anticoactivator in cells. Our data support a model in which PTalpha, which does not interact with ER, selectively enhances the transcriptional activity of the ER but not that of other nuclear receptors by recruiting the repressive REA protein away from ER, thereby allowing effective coactivation of ER with SRC-1 or other coregulators. The ability of PTalpha to directly interact in vitro and in vivo with REA, a selective coregulator of the ER, thereby enabling the interaction of ER with coactivators, appears to explain its ability to selectively enhance ER transcriptional activity. These findings highlight a new role for PTalpha as a coregulator activity-modulating protein that confers receptor specificity. Proteins such as PTalpha represent an additional regulatory component that defines a novel paradigm enabling receptor-selective enhancement of transcriptional activity by coactivators.
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PMID:Prothymosin alpha selectively enhances estrogen receptor transcriptional activity by interacting with a repressor of estrogen receptor activity. 1093 99

Retinoid receptor-related testis-associated receptor (RTR)/germ cell nuclear factor is a nuclear orphan receptor that plays an important role in the control of gene expression during early embryonic development and gametogenesis. It has been shown to repress transcriptional activation. In this study, we further characterize this repressor function. We demonstrate that RTR can suppress the transcriptional activation induced by the estrogen receptor related-receptor alpha1 through its response element. The latter is at least in part due to competition for binding to the same response element. In addition, RTR inhibits basal transcriptional activation, indicating that it functions as an active repressor. Mammalian two-hybrid analyses showed that RTR interacts with the co-repressor nuclear co-repressor (N-CoR) but is unable to interact with the co-repressor SMRT or RIP140. Pull-down analyses with glutathione S-transferase-RTR fusion protein demonstrated that RTR physically interacts with N-CoR in vitro, suggesting a potential role for N-CoR in the transcriptional repression by RTR. To identify the regions in RTR essential for the binding of RTR to N-CoR, the effect of various deletion and point mutations on this interaction was examined. This analysis revealed that this interaction requires the hinge domain, helix 3 as well as the helix 12 region of RTR. The residues Ser(246)-Tyr(247) in the hinge domain, Lys(318) in helix 3, and Lys(489)-Thr(490) in helix 12 are identified as being critical in this interaction. Our results demonstrate that RTR can function as an active transcriptional repressor and that this repression can be mediated through interactions with the co-repressor N-CoR. We show that this interaction exhibits several characteristics unique to RTR. Through its repressor function, RTR can suppress the induction of transcriptional activation by other nuclear receptors. These repressor activities may provide important mechanisms by which RTR regulates gene expression during development and gametogenesis.
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PMID:Characterization of the repressor function of the nuclear orphan receptor retinoid receptor-related testis-associated receptor/germ cell nuclear factor. 1094 Mar 6

The transcriptional activity of nuclear hormone receptors is known to be modulated by coregulator proteins. We found that the repressor of estrogen receptor activity (REA), a protein recruited to the hormone-occupied estrogen receptor (ER), decreased the transcriptional activity of ER, both when ER was acting directly through DNA response elements as well as when it was tethered to other transcription factors. Administration of antisense REA resulted in a 2-4-fold increase in ER transactivation, implying that endogenous REA normally dampens the stimulatory response to estradiol. To define the interaction regions between ER and REA, we used glutathione S-transferase pull-down assays. We found that REA bound to the ligand-binding domain (E domain) of ER, but not to other regions of ER, and that REA interaction with ER involved a region in the C-terminal half of REA. REA and the coactivator SRC-1 were involved in a functional competition for regulation of ER transcriptional activity, which we show results from competition between these two coregulators for interaction with ER. REA contains an LXXLL motif near its N terminus, but this motif was not involved in its binding to ER. Rather, this sequence was required for the competitive binding of REA and SRC-1 to ER and thus for optimal repression of ER activity. Our findings show that the regions of REA required for its interaction with ER and for its repression of ER activity are different.
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PMID:Analysis of estrogen receptor interaction with a repressor of estrogen receptor activity (REA) and the regulation of estrogen receptor transcriptional activity by REA. 1096 Apr 70


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