Direct activation of Xenopus iodotyrosine deiodinase by thyroid hormone receptor in the remodeling intestine during amphibian metamorphosis

Abstract

Thyroid hormone (TH) plays critical roles during vertebrate postembryonic development. TH production in the thyroid involves incorporating inorganic iodide into thyroglobulin. The expression of iodotyrosine deiodinase (IYD; also known as iodotyrosine dehalogenase 1) in the thyroid gland ensures efficient recycling of iodine from the byproducts of TH biosynthesis: 3'-monoiodotyrosine and 3', 5'-diiodotyrosine. Interestingly, IYD is known to be expressed in other organs in adult mammals, suggesting iodine recycling outside the thyroid. On the other hand, the developmental role of iodine recycling has yet to be investigated. Here, using intestinal metamorphosis as a model, we discovered that the Xenopus tropicalis IYD gene is strongly up-regulated by TH during metamorphosis in the intestine but not the tail. We further demonstrated that this induction was one of the earliest events during intestinal metamorphosis, with IYD being activated directly through the binding of liganded TH receptors to a TH response element in the IYD promoter region. Because iodide is mainly taken up from the diet in the intestine and the tadpole stops feeding during metamorphosis when the intestine is being remodeled, our findings suggest that IYD transcription is activated by liganded TH receptors early during intestinal remodeling to ensure efficient iodine recycling at the climax of metamorphosis when highest levels of TH are needed for the proper transformations of different organs.

Fig. 1.

The IYD gene is highly conserved in vertebrates. Amino acid sequence comparison among human, mouse, X. tropicalis, and X. laevis IYD gene. The amino acid residues identical to the human IYD sequence are shaded in gray. The conserved iodotyrosine dehalogenase domain of IYD is boxed.

Fig. 2.

The expression of IYD is strongly up-regulated in the intestine but remains low in tail upon T₃ treatment of premetamorphic X. tropicalis tadpoles. Premetamorphic tadpoles were treated with 10 nm T₃ for 2 d, and total RNA was isolated from the intestine and tail. qRT-PCR was performed to examine IYD expression in the intestine (A) and tail (B). The IYD mRNA level was normalized to EF1α expression. Error bars indicate sem (n = 3). *, P < 0.05.

Fig. 3.

Developmental expression profiles of IYD in the intestine and tail during natural metamorphosis of X. tropicalis. Total RNA was isolated from the intestine and tail of X. tropicalis tadpoles at the indicated stages, and qRT-PCR was carried out to determine IYD expression. The IYD mRNA level was normalized to EF1α expression (Supplemental Fig. 1, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org). Note that in the intestine, the expression of IYD mRNA was dramatically up-regulated by stage 60 and then reduced by the end of metamorphosis (A). In contrast, in the tail, the IYD mRNA expression remained low throughout development (B). Error bars indicate sem (n = 3). *, P < 0.05.

Fig. 4.

TR binds to the TRE in the IYD gene in vitro. A, Schematic diagram of the promoter region of the X. tropicalis IYD gene. The IYD TRE is shown as a white box with arrow, indicating the orientation of TRE. Black box shows the first exon. B, Comparison of the sequences of the TRE of the IYD gene and the mutated TRE (mTRE) to the consensus TRE. The TRE half-sites are shown in bold letters. The mutated nucleotides in the mTRE used for the gel mobility shift assay are underlined. C, The IYD TRE binds to TR/RXR heterodimers in vitro. A gel mobility shift assay was done with TR/RXR heterodimers translated in reticulocyte lysate in vitro and labeled X. laevis TRβ promoter TRE, a well-characterized TRE (51), in the presence or absence of 4-, 20-, or 100-fold of unlabeled wild-type or mutant IYD TRE as the competitor. Unprogrammed reticulocyte lysate (URL) was used as a negative control. The arrowhead indicates the complex of TRβ TRE with TR/RXR. Note that the complex was competed away efficiently by an excess of unlabeled wild-type IYD TRE but not mutated IYD TRE, indicating specific binding of the IYD TRE to TR/RXR.

Fig. 5.

The TRE in X. tropicalis IYD promoter mediates activation by liganded TR. A, Schematic diagrams show the reporter constructs of the wild-type and mutant of X. tropicalis IYD promoter. B, Mutating the TRE eliminates the induction of the promoters by T₃. Wild-type or mutant (mTRE) promoter construct was coinjected with the control Renilla luciferase construct phRG-tk into the nuclei of the oocytes with or without prior cytoplasmic injection of mRNA for X. tropicalis TRα and RXRβ. The oocytes were incubated at 18 C overnight in the presence or absence of 100 nm T₃ and then used for dual-luciferase assays. The relative activities of the firefly luciferase to Renilla luciferase were plotted. Error bars indicate sem (n = 3).

Fig. 6.

TR strongly associates specifically with the TRE in the IYD promoter in the intestine but not the tail in vivo. Stage 54 premetamorphic tadpoles were treated with or without T₃ for 2 d, and the intestine (A) and tail (B) were isolated for ChIP assay with the anti-TR antibody (top panel) or anti-Id14 antibody (bottom panel), which served as a negative control for antibody specificity. The immunoprecipitated DNA was analyzed by qPCR for the presence of the TRE region of IYD promoter. A region of IYD exon 3 with no TRE was analyzed as a negative control for binding specificity. Note that little or no TR was bound to the TRE of the IYD gene in the absence of T₃ in premetamorphic tadpoles. In the presence of T₃, TR binding to the TRE in the intestine was strongly increased in the intestine, but not the tail, in agreement with the specific, strong regulation of IYD mRNA expression by T₃ in the intestine but not the tail. There was no TR binding to IYD exon 3 in the presence or absence of T₃. Only background signals were observed with the anti-ID14 antibody. Error bars indicate sem (n = 3). *, P < 0.05.