Direct activation of tRNA methyltransferase-like 1 (Mettl1) gene by thyroid hormone receptor implicates a role in adult intestinal stem cell development and proliferation during Xenopus tropicalis metamorphosis

Abstract

Background: Thyroid hormone (T3) plays an important role in vertebrate development. Compared to the postembryonic development of uterus-enclosed mammalian embryos, T3-dependent amphibian metamorphosis is advantageous for studying the function of T3 and T3 receptors (TRs) during vertebrate development. The effects of T3 on the metamorphosis of anurans such as Xenopus tropicalis is known to be mediated by TRs. Many putative TR target genes have been identified previously. Among them is the tRNA methyltransferase Mettl1.

Results: We studied the regulation of Mettl1 gene by T3 during intestinal metamorphosis, a process involves near complete degeneration of the larval epithelial cells via apoptosis and de novo formation of adult epithelial stem cells and their subsequent proliferation and differentiation. We observed that Mettl1 was activated by T3 in the intestine during both natural and T3-induced metamorphosis and that its mRNA level peaks at the climax of intestinal remodeling. We further showed that Mettl1 promoter could be activated by liganded TR via a T3 response element upstream of the transcription start site in vivo. More importantly, we found that TR binding to the TRE region correlated with the increase in the level of H3K79 methylation, a transcription activation histone mark, and the recruitment of RNA polymerase II by T3 during metamorphosis.

Conclusions: Our findings suggest that Mettl1 is activated by liganded TR directly at the transcriptional level via the TRE in the promoter region in the intestine during metamorphosis. Mettl1 in turn regulate target tRNAs to affect translation, thus facilitating stem cell formation and/or proliferation during intestinal remodeling.

Keywords: Anuran metamorphosis; Intestine; Methyltransferase; Stem cell; Thyroid hormone receptor; Xenopus tropicalis; tRNA.

Fig. 1

Expression of Xenopus tropicalis Mettl1 increases during T3-induced and natural metamorphosis. a The expression of Mettl1 was analyzed during T3-induced metamorphosis. Stage 54 tadpoles were treated with 10 nM T3 for 2 days and total RNA was isolated from the intestine for RT-PCR analysis. b During natural metamorphosis, Mettl1 expression gradually increased from premetamorphic period to peak at the metamorphic climax. Total RNA was isolated from the intestine of tadpoles at indicated stages for RT-PCR analysis. Shown below the expression data are schematic diagrams of the intestine at different stages. In premetamorphic tadpoles at stage 54, the intestine is a simple structure with a single epithelial fold, the typhlosole, and thin layers of connective tissue and muscles. At the metamorphic climax around stage 61, the larval epithelial cells begin to undergo apoptosis, as indicated by the open circles. Concurrently, the proliferating adult stem cells are formed de novo via dedifferentiation of some larval epithelial cells, as indicated by black dots. By the end of metamorphosis at stage 66, the newly developed adult epithelium (EP) has multiple folds, surrounded by thick layers of connective tissue (CT) and muscles (MU). L: intestinal lumen. All data represent mean ± S.E.M. Significance value was ***P ≤ 0.005

Fig. 2

Mettl1 methyltransf_4 domain is highly conserved evolutionally and liganded TR enhances Xenopus tropicalis Mettl1 promoter activity in vivo. a Amino acid alignment of Mettl1 from X. tropicalis, X. laevis, H. sapiens and M. musculus. The boxed region is the methyltransf_4 domain. Shaded amino acids indicate conserved residues. b Schematic representation of Mettl1 promoter and the first two exons. The putative TRE is shown as a white box. Gray box indicates the putative 5′ UTR of Mettl1 and black boxes indicate exons. The putative TRE is located at − 1128 bp from the predicted transcription start site of Mettl1 gene. c The Mettl1 promoter is activated by liganded TR in Xenopus oocytes. pGL4 was used as a negative control, and TRβ promoter construct was used as a positive control. The oocytes were injected with indicated mRNAs and reporter and harvested for luciferase assay. All data represent mean ± S.E.M. Significance value was ***P ≤ 0.005

Fig. 3

Liganded TR activates the Mettl1 promoter through the putative TRE. a Schematic diagrams of the Mettl1 promoter construct with the putative TRE of Mettl1 (Mettl1 TRE) or a mutant TRE (Mettl1 mTRE). The TRE sequence is shown below the TRE with the mutated residues shown in red. The Mettl1 TRE and Mettl1 mTRE fragments were cloned into pGL4.10 luciferase reporter vector. b The Mettl1 wild type but not the mutant promoter construct is activated by liganded TR in Xenopus laevis oocytes. The oocytes were injected with indicated mRNAs and reporter and harvested for luciferase assay. All data represent mean ± S.E.M. Significance value was ***P ≤ 0.005. n.s. not significant

Fig. 4

TR binds to the TRE in the Mettl1 promoter region in the intestine during T3-induced metamorphosis. ChIP assays were performed with indicated antibodies on the intestine of premetamorphic tadpoles treated with or with T3. a Liganded TR is present in the TRE region of Mettl1 promoter in the intestine of premetamorphic tadpoles and the binding is increased by T3 treatment. b The level of di-methylated H3K79, a histone mark for transcription activation, increases in the TRE region after T3 treatment. c RNA polymerase II is recruited to the Mettl1 TRE region after T3 treatment. d Only background ChIP signal is detected with the negative control IgG ChIP in the presence or absence of T3 treatment. All data represent mean ± S.E.M. Significance value was ***P ≤ 0.005. n.s. not significant

Fig. 5

Peak levels of TR binding at the climax of intestinal metamorphosis correlates with peak levels of RNA polymerase II recruitment and H3K79 methylation in the intestine during natural metamorphosis. ChIP assays were performed with indicated antibodies on the intestine of tadpoles at different metamorphic stages. a TR binding to the TRE region of Mettl1 gene at metamorphic climax stages (stage 60 and 62) is increased compared to that at premetamorphic stage 54. After climax, TR binding is reduced in TRE region of Mettl1 gene. b The level of dimethylated H3K79 in the TRE region of Mettl1 gene peaks in the intestine at metamorphic climax. c RNA polymerase II recruitment to the TRE region of Mettl1 promoter also peaks at metamorphic climax period. d Only background ChIP signal is detected with the negative control IgG ChIP throughout metamorphosis. n.s. not significant