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Interactions of the crustacean nuclear receptors HR3 and E75 in the regulation of gene transcription.

ABSTRACT: Endocrine signal transduction occurs through cascades that involve the action of both ligand-dependent and ligand-independent nuclear receptors. In insects, two such nuclear receptors are HR3 and E75 that interact to transduce signals initiated by ecdysteroids. We have cloned these nuclear receptors from the crustacean Daphnia pulex to assess their function as regulators of gene transcription in this ecologically and economically important group of organisms. Both nuclear receptors from D. pulex (DappuHR3 (group NR1F) and DappuE75 (group NR1D)) exhibit a high degree of sequence similarity to other NR1F and NR1D group members that is indicative of monomeric binding to the RORE (retinoid orphan receptor element). DappuE75 possesses key amino acid residues required for heme binding to the ligand-binding domain. Next, we developed a gene transcription reporter assay containing a luciferase reporter gene driven by the RORE. DappuHR3, but not DappuE75, activated transcription of the luciferase gene in this system. Co-transfection experiments revealed that DappuE75 suppressed DappuHR3-dependent luciferase transcription in a dose-dependent manner. Electrophoretic mobility shift assays confirmed that DappuHR3 bound to the RORE. However, we found no evidence that DappuE75 similarly bound to the response element. These experiments further demonstrated that DappuE75 prevented DappuHR3 from binding to the response element. In conclusion, DappuHR3 functions as a transcriptional activator of genes regulated by the RORE and DappuE75 is a negative regulator of this activity. DappuE75 does not suppress the action of DappuHR3 by occupying the response element but presumably interacts directly with the DappuHR3 protein. Taken together with the previous demonstration that daphnid HR3 is highly induced by 20-hydroxyecdysone, these results support the premise that HR3 is a major component of ecdysteroid signaling in some crustaceans and is under the negative regulatory control of E75.

SUBMITTER: Hannas BR

PROVIDER: S-EPMC2873136 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Interactions of the crustacean nuclear receptors HR3 and E75 in the regulation of gene transcription.

Hannas Bethany R BR Wang Ying H YH Baldwin William S WS Li Yangchun Y Wallace Andrew D AD LeBlanc Gerald A GA

General and comparative endocrinology 20100324 2

Endocrine signal transduction occurs through cascades that involve the action of both ligand-dependent and ligand-independent nuclear receptors. In insects, two such nuclear receptors are HR3 and E75 that interact to transduce signals initiated by ecdysteroids. We have cloned these nuclear receptors from the crustacean Daphnia pulex to assess their function as regulators of gene transcription in this ecologically and economically important group of organisms. Both nuclear receptors from D. pulex ...[more]

PMID: 20346363

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Project description:Ecdysteroids initiate signaling along multiple pathways that regulate various aspects of development, maturation, and reproduction in arthropods. Signaling often involves the induction of downstream transcription factors that either positively or negatively regulate aspects of the pathway. We tested the hypothesis that crustaceans express the nuclear receptors HR3 (ortholog to vertebrate ROR) and E75 (ortholog to vertebrate rev-erb) in response to ecdysteroid signaling. HR3 and E75 cDNAs were cloned from the crustacean Daphnia magna. The DNA-binding domain and ligand-binding domain of the daphnid HR3 were 95% and 61% identical to those of Drosophila melanogaster. The DNA-binding domain and ligand-binding domain of the daphnid E75 were 100% and 71% identical to those of D. melanogaster. Both receptors exhibited structural characteristics of binding to DNA as a monomer. The expression of these receptor mRNAs was evaluated through the adult molt cycle and during embryo development. E75 levels were relatively constant throughout the adult molt cycle and through embryo development. HR3 levels were comparable to those of E75 during the initial phases of the adult molt cycle but were elevated approximately 30-fold at a time in the cycle co-incident with the pre-molt surge in ecdysteroid levels. HR3 mRNA levels in embryos also varied co-incident with ecdysteroid levels. To substantiate a role of ecdysteroids in the expression of HR3, daphnids were continuously exposed to 20-hydroxyecdysone and changes in gene expression were measured. HR3 levels were significantly induced by 20-hydroxyecdysone; while E75 levels were minimally affected. These results are consistent with the premise that transcription of HR3 is regulated by ecdysteroids in the crustacean D. magna and that HR3 likely serves as a mediator of ecdysteroid regulatory action in crustaceans. The marginal induction of E75 by 20-hydroxyecdysone may represent limited, tissue or cell-type-specific induction of this transcription factor.

Dataset Information

Interactions of the crustacean nuclear receptors HR3 and E75 in the regulation of gene transcription.

Publications

Interactions of the crustacean nuclear receptors HR3 and E75 in the regulation of gene transcription.

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