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Fig. 3. A putative TRE in Xenopus Sox3 gene can mediate transcriptional activation by T3-bound TR/RXR in frog oocyte. (A) Putative TREs and promoter constructs. Sox3 gene sequence was obtained from Xenopus laevis genome sequences at http://xenopus.lab.nig.ac.jp/blast.php and searched for TREs by using NHR Scan at http://nhrscan.genereg.net/cgi-bin/nhr_scan.cgi?rm=advanced. Three putative TREs were found and are listed above the schematic diagram of the Sox3 gene with their positions relative to the first nucleotide of the start codon (designated as “ + 1”). Three promoter constructs, the full length Sox3 promoter including the putative TRE2 and TRE3 (Psox3), a truncated version of Sox3 promoter (Psox3δ), and the truncated version of Sox3 promoter with the TRE2 (Psox3δ+TRE) inserted immediately upstream of it, were generated to drive the firefly luciferase expression in pGL4 vector (Promega). TRE: thyroid hormone response element; F-luc: firefly luciferase gene. (B) TRE2 binds to TR/RXR in vitro. Double strand DNA oligos containing the putative TREs shown in (A) were used in competitive electrophoretic mobility shift assay (EMSA) against the infrared (IR) dye IR700 (LI-COR, Lincoln, NE)-labeled, well-characterized TRE of Xenopus laevis TRβ gene in the presence of in vitro translated TR/RXR proteins. Unlabeled TRE of the Xenopus laevis TRβ gene (TRE) and a mutant version of TRE of Xenopus laevis TRβ gene (mTRE) known to lack binding to TR/RXR were used as the positive and negative control, respectively [147] and [148]. All unlabeled oligos were used in 100 times excess over the labeled IR700-TRE. Note that only TRE and TRE2 competed effectively, suggesting that TRE2, but not TRE1 or TRE3, binds to TR/RXR specifically. (C) Sox3 promoter can be activated by liganded TR/RXR in frog oocyte. Transcription assay was done in Xenopus laevis oocytes where the cytoplasm of stage VI oocytes were injected with 460 pg per oocyte of TR and RXR mRNA mixture or GFP mRNA. 2 hours later, the firefly luciferase reporter constructs shown in (A) (34.5 pg per oocyte) and the phRG-TK (Promega) expressing Renilla luciferase as an internal control (34.5 pg per oocyte) were coinjected into the nuclei of the oocytes. After incubation at 18 °C overnight in the presence or absence of 100 nM T3, 5 oocytes were collected per sample and lysed in 75 μl of 1 × Passive Lysis Buffer (Promega) for dual luciferase assay by following the manufacturer’s protocol. The relative expression of firefly luciferase to Renilla luciferase (F/R) was determined with the F/R value for oocytes injected with GFP mRNA instead of TR/RXR in the absence of T3 set as 1. Note that the full-length promoter was activated by TR/RXR in the presence of T3. This activation was drastically reduced when the TRE sequences were deleted and was restored when TRE2 was inserted into the truncated promoter, suggesting that TRE2 is capable of mediating T3 induction. Each data point shows the average of 5 samples with the standard error. Statistical analysis was done through ANOVA with Tukey’s Multiple Component Test. *: p<0.05. (L. Fu and Y.-B. Shi, unpublished observations). Image published in: Fu L and Shi YB (2017) Copyright © 2017. This image is reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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