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Gen Comp Endocrinol
2020 Dec 01;299:113590. doi: 10.1016/j.ygcen.2020.113590.
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Thyroid hormone directly activates mitochondrial fission process 1 (Mtfp1) gene transcription during adult intestinal stem cell development and proliferation in Xenopus tropicalis.
???displayArticle.abstract??? Thyroid hormone (T3) regulates vertebrate development via T3 receptors (TRs). T3 level peaks during postembryonic development, a period around birth in mammals or metamorphosis in anurans. Anuran metamorphosis offers many advantages for studying T3 and TR function in vivo largely because of its total dependent on T3 and the dramatic changes affecting essentially all organs/tissues that can be easily manipulated. Earlier studies have shown that TRs are both necessary and sufficient for mediating the metamorphic effects of T3. Many candidate TR target genes have been identified during Xenopus tropicalis intestinal metamorphosis, a process that involves apoptotic degeneration of most of the larval epithelial cells and de novo development of adult epithelial stem cells. Among these putative TR target genes is mitochondrial fission process 1 (Mtfp1), a nuclear-encoded mitochondrial gene. Here, we report that Mtfp1gene expression peaks in the intestine during both natural and T3-induced metamorphosis when adult epithelial stem cell development and proliferation take place. Furthermore, we show that Mtfp1 contains a T3-response element within the first intron that is bound by TR to mediate T3-induced local histone H3K79 methylation and RNA polymerase recruitment in the intestine during metamorphosis. Additionally, we demonstrate that the Mtfp1 promoter can be activated by T3 in a reconstituted frog oocyte system in vivo and that this activation is dependent on the intronic TRE. These findings suggest that T3 activates Mtfp1 gene directly via the intronic TRE and that Mtfp1 in turn facilitate adult intestinal stem cell development/proliferation by affecting mitochondrial fission process.
Fig. 1. Xenopus tropicalis Mtfp1 is upregulated during natural and T3-induced metamorphosis. (a) The mRNA levels of Mtfp1 during T3-induced metamorphosis. Premetamorphic stage 54 tadpoles were treated for 2 days with 10 nM T3 and total RNA was isolated from the intestine for qRT-PCR analysis (n = 3). (b) Mtfp1 mRNA level during natural metamorphosis. Total RNA was isolated from the intestine of tadpoles at indicated stages and subjected to qRT-PCR analysis (n = 3). Diagramed below the expression data are schematics of the intestine at indicated stages. The intestine at stage 54 has a simple structure with a single epithelial fold, the typhlosole, surrounded by thin layers of connective tissue and muscles. At the metamorphic climax stages 60–62, the larval epithelial cells undergo apoptosis, as indicated by the open circles, and adult stem cells, as indicated by black dots, are formed and proliferating. By stage 66, the newly formed adult epithelium (EP) has many folds, surrounded by thick layers of connective tissue (CT) and muscles (MU). L: intestinal lumen. All data are shown in mean ± S.E.M. (***P ≤ 0.005).
Fig. 2. The MTP18 domain of Mtfp1 is highly conserved and Xenopus tropicalis Mtfp1 promoter is regulated by TR via an intronic TRE in vivo. (a) Amino acid alignment of Mtfp1 from X. tropicalis, X. laevis, H. sapiens and M. musculus. The boxed region is the MTP18 domain. Shaded amino acids indicate conserved residues. (b) Schematic diagram of the Mtfp1gene with a putative TRE (white box) in the first intron. Gray box shows the putative 5′ UTR and black boxes are exons. The putative TRE is located at + 371 bp from the putative transcription start site. The TRE of the TRβ gene is shown for comparison. (c) Activation of the Mtfp1 promoter by liganded TR in the reconstituted oocytes. The reporter construct had the Mtfp1 promoter, including the TRE in the first intron, driving the expression of firefly luciferase. The internal control was the Renilla luciferase reporter phRG-TK vector. pGL4 was a negative control for promoter activity, and TRβ promoter construct was a positive T3-response promoter control. GFP was used as a control for TR/RXR mRNA. The oocytes were injected with indicated mRNA and reporter construct and harvested for luciferase assay (n = 3). The data are shown mean ± S.E.M. (***P ≤ 0.005).
Fig. 3. TR activates Mtfp1 transcription though putative TRE when T3 is present. (a) Schematic diagrams of the Mtfp1 promoter construct containing wild type TRE (Mtfp1 TRE) or a mutant TRE (Mtfp1 mTRE). The TRE sequences are shown in capital letters with the mutated position in red. (b) The promoter with wild type Mtfp1 TRE but not mTRE is activated by T3 in the presence of T3 when TR and RXR were expressed in Xenopus laevis oocytes. The oocytes were injected as indicated and harvested for luciferase assay (n = 3). The data are shown mean ± S.E.M. (***P ≤ 0.005; n.s. not significant).
Fig. 4. The TRE in the first intron of Mtfp1 gene is bound by TR in the intestine of premetamorphic tadpoles treated with T3, accompanied by increased RNA polymerase II recruitment and histone H3K79 methylation. ChIP assay were done on the intestine of stage 54 tadpoles with or without treatment with T3 with the antibody against the antigen indicated on each panel or control IgG (n = 3). (a) TR binds the Mtfp1 TRE region in the intestine and T3 treatment increases this binding. (b) Di-methylation of histone H3K79, a transcription activation histone mark, is increased in the TRE region by T3 treatment. (c) The level of RNA polymerase II recruitment at TRE region of Mtfp1 is increased after T3 treatment. (d) The negative control IgG ChIP yields only background signals. The data are shown in mean ± S.E.M. (***P ≤ 0.005. n.s. not significant).
Fig. 5. TR binding to the TRE region peaks in the intestine at the climax of metamorphosis, accompanied by peak levels of RNA polymerase II and H3K79 methylation during natural metamorphosis. ChIP assay were done on tadpole intestine at different stages with the antibody against the antigen indicated on each panel or control IgG (n = 3). (a) TR binding to Mtfp1 TRE increases at metamorphic climax (stage 60 and 62) before dropping to a lower level by the end of metamorphosis. (b)/(c) The changes in the levels of dimethylated H3K79 (b) and RNA polymerase II (c) at the TRE region parallel that in TR binding. (d) The negative control IgG ChIP yields only background signals throughout metamorphosis. The data are shown in mean ± S.E.M. (***P ≤ 0.005. n.s. not significant).
