Biomedical subjects

G Sathya

Publications and source records attributed to G Sathya.

5 recordsLinked to original sources

PubMed2001

Fusion estrogen receptor proteins: toward the development of receptor-based agonists and antagonists.

Estrogen-induced signaling mediated by estrogen receptors (ERs) is also affected by aberrant ERs that act as constitutively active or dominant negative modulators. Variant ERs can contribute to carcinogenesis and to the loss of estrogen responsiveness, rendering antiestrogen therapy ineffective. Determining target gene response during co-synthesis of different ER species is difficult, because dimers formed in the presence of more than one ER species are a heterogenous population of homo- or heterodimers. We engineered a homofusion ERalpha as a prototype single-chain receptor by genetically conjugating two ER monomers into a covalently fused single-chain protein to obtain a homogeneous population. This permits analysis of symmetrical or asymmetrical mutations that simulate variant homo- and heterodimers. Although a monomer, the homofusion receptor exhibited similar biochemical and functional properties to the dimeric ERalpha. We used activation function-2 (AF2) defective mutants as a model in either one or both receptor domains for a dominant-negative phenotype by suppressing the reporter activity induced by the WT receptor. When co-expressed with ERalpha, the fusion variant deficient in both AF2 functions suppressed the reporter activity effectively induced by ERalpha. These results show the utility of fusion receptors as models for generation of receptor-based agonists and antagonists.

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PubMed1999

An explanation for observed estrogen receptor binding to single-stranded estrogen-responsive element DNA.

Estrogen-inducible genes contain an enhancer called the estrogen response element (ERE), a double-stranded inverted repeat. The estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo.

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PubMed1998

Sequence requirements for estrogen receptor binding to estrogen response elements.

The estrogen receptor (ER) is a transcription factor that binds to a specific DNA sequence found in the regulatory regions of estrogen-responsive genes, called the estrogen response element (ERE). Many genes that contain EREs have been identified, and most of these EREs contain one or more changes from the core consensus sequence, a 13-nucleotide segment with 10 nucleotides forming an inverted repeat. A number of genes have multiple copies of these imperfect EREs. In order to understand why natural EREs have developed in this manner, we have attempted to define the basic sequence requirements for ER binding. To this end, we measured the binding of homodimeric ER to a variety of nonconsensus EREs. We discovered that an ERE containing even a single change from the consensus may be unable to bind ER. However, an ERE with two changes from the consensus may be capable of binding avidly to ER in the context of certain flanking sequences. We found that changes in the sequences flanking a nonconsensus ERE can greatly alter ER-ERE affinity, either positively or negatively. Careful study of sequences flanking a series of EREs made it possible to develop rules that predict whether ER binds to a given natural ERE and also to predict the relative amounts of binding when comparing two EREs.

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PubMed1997

Chicken ovalbumin upstream promoter-transcription factor interacts with estrogen receptor, binds to estrogen response elements and half-sites, and inhibits estrogen-induced gene expression.

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) was identified as a low abundance protein in bovine uterus that co-purified with estrogen receptor (ER) in a ligand-independent manner and was separated from the ER by its lower retention on estrogen response element (ERE)-Sepharose. In gel mobility shift assays, COUP-TF bound as an apparent dimer to ERE and ERE half-sites. COUP-TF bound to an ERE half-site with high affinity, Kd = 1.24 nM. In contrast, ER did not bind a single ERE half-site. None of the class II nuclear receptors analyzed, i.e. retinoic acid receptor, retinoid X receptor, thyroid receptor, peroxisome proliferator-activated receptor, or vitamin D receptor, were constituents of the COUP-TF.DNA binding complex detected in gel mobility shift assays. Direct interaction of COUP-TF with ER was indicated by GST "pull-down" and co-immunoprecipitation assays. The nature of the ER ligand influenced COUP-TF-ERE half-site binding. When ER was liganded by the antiestrogen 4-hydroxytamoxifen (4-OHT), COUP-TF-half-site interaction decreased. Conversely, COUP-TF transcribed and translated in vitro enhanced the ERE binding of purified estradiol (E2)-liganded ER but not 4-OHT-liganded ER. Co-transfection of ER-expressing MCF-7 human breast cancer cells with an expression vector for COUP-TFI resulted in a dose-dependent inhibition of E2-induced expression of a luciferase reporter gene under the control of three tandem copies of EREc38. The ability of COUP-TF to bind specifically to EREs and half-sites, to interact with ER, and to inhibit E2-induced gene expression suggests COUP-TF regulates ER action by both direct DNA binding competition and through protein-protein interactions.

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PubMed1997

Effects of multiple estrogen responsive elements, their spacing, and location on estrogen response of reporter genes.

Most highly estrogen-responsive genes possess multiple estrogen-responsive elements (EREs) that act synergistically to activate expression. Synergism between EREs appears to depend on structural features of the EREs and the promoter. To examine the activation process, we cloned single or multiple tandem copies of the consensus ERE into reporter plasmids. These plasmids contained either a chloramphenicol acetyl transferase reporter gene driven by a minimal promoter or a luciferase reporter gene driven by the Simian virus 40 (SV40) promoter. Using MCF-7 human breast cancer cells, we demonstrate that synergism among EREs depends on the number of EREs, their spacing, and the distance of the EREs from the promoter. The induction capacity of EREs falls off slowly with distance from the promoter. Remarkably, multiple EREs can induce effectively and synergize even when they are located more than 2000 nucleotides from the promoter. For EREs located immediately upstream of the promoter, both the distance separating the EREs and the distance to the promoter have to be optimal for synergy. Altering either distance changes the response from synergistic to additive. For distant EREs, presumed to interact by a looping mechanism at the promoter, the length of DNA between the EREs and the promoter is not critical. Synergy among closely spaced EREs that are far from the promoter only requires an optimal distance separating the ERE centers of symmetry. Interestingly, very widely separated EREs can also synergize, presumably also because of their ability to interact by looping. The estrogen response from single or multiple tandem copies of ERE half-palindromes near the SV40 promoter was also tested. The negligible induction capacity of a single half-site was not significantly increased in multiple sites. The biological role of half-EREs is not apparent in the system employed here.

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