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Most vertebrate organs use adult stem cells to maintain homeostasis and ensure proper repair when damaged. How such organ-specific stem cells are formed during vertebrate development is largely unexplored. We have been using the thyroid hormone (T3)-dependent amphibian metamorphosis to address this issue. Early studies in Xenopus laevis have shown that intestinal remodeling involves complete degeneration of the larval epithelium and de novo formation of adult stem cells through dedifferentiation of some larval epithelial cells. We have further discovered that the histidine ammonia-lyase (HAL; also known as histidase or histidinase)-2 gene is strongly and specifically activated by T3 in the proliferating adult stem cells of the intestine during metamorphosis, implicating a role of histidine catabolism in the development of adult intestinal stem cells. To determine the mechanism by which T3 regulates the HAL2 gene, we have carried out bioinformatics analysis and discovered a putative T3 response element (TRE) in the HAL2 gene. Importantly, we show that this TRE is bound by T3 receptor (TR) in the intestine during metamorphosis. The TRE is capable of binding to the heterodimer of TR and 9-cis retinoic acid receptor (RXR) in vitro and mediate transcriptional activation by liganded TR/RXR in frog oocytes. More importantly, the HAL2 promoter containing the TRE can drive T3-dependent reporter gene expression to mimic endogenous HAL2 expression in transgenic animals. Our results suggest that the TRE mediates the induction of HAL2 gene by T3 in the developing adult intestinal stem cells during metamorphosis.
Figure 2. The HAL2 TRE binds to TR/RXR in vitro. (a) Intronâexon organization of the HAL2 gene. The +1 indicates putative transcription start site based on reported cDNA sequence. A putative TRE is present at â3091 to â3076 with the half TRE repeat shown in capital letters. The exons are shown in filled boxes whereas the introns and upstream sequences are shown as lines. The open box represents the HAL2 TRE. (b) Sequences of wild-type and mutant (mt) TRE of X. tropicalis HAL2 and X. laevis TRβ genes. The sequences of the TRE half sites are shown in capital letters with the mutated residues in the mutant TREs underlined. (c) Wild-type (wt) but not mutant (mt) HAL2 TRE (H2 TRE) competes effectively for binding to TR/RXR in vitro. Gel mobility shift assay was carried out with labeled wild-type X. laevis TRβ TRE and in vitroâtranslated TR/RXR in the presence or absence of 10 to 100à excess unlabeled wild-type or mutant TREs as indicated. Note that as expected, the complex formed between the labeled, well-characterized X. laevis TRβ TRE and TR/RXR was competed effectively by the unlabeled wild-type but not the mutant TRβ TRE. Similarly, the wild-type but not the mutant HAL2 TRE was able to compete effectively, indicating that HAL2 TRE binds to TR/RXR with similar affinity as the TRβ TRE.
Figure 3. The HAL2 TRE mediates transcriptional activation by T3 in frog oocytes. (a) Schematic diagram of promoter constructs pHAL2-TRE-luc (HAL2-TRE) and pHAL2-mtTRE-luc (HAL2-mtTRE) containing the wild-type and mutated HAL2 TRE, respectively. (b) The luciferase reporter construct pHAL2-TRE-luc (HAL2-TRE) or pHAL2-mtTRE-luc (HAL2-mtTRE) was coinjected with the control Renilla luciferase construct phRG-tk into the nuclei of Xenopus oocytes with or without prior cytoplasmic injection of X. laevis TRα and RXRα mRNAs. 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. Note that the reporter with the wild-type but not the mutated HAL2 TRE responded to T3 in the presence of TR/RXR.
Figure 4. The HAL2 TRE is bound by TR in tadpoleintestine. Stage 54 premetamorphic tadpoles were treated with or without T3 for 2 days, and the intestine was isolated for ChIP assay with the anti-TR antibody (top) or anti-ID14 antibody (bottom), which served as a negative control for antibody specificity. The immunoprecipitated DNA was analyzed by qPCR for the presence of the TRE region of HAL2 promoter. A region of the exon 5 in the Dot1L gene with no TRE (41) was analyzed as a negative control for binding specificity. Note that TR binding to the HAL2 TRE was increased upon T3 treatment. There was no TR binding to Dot1L exon 5 in the presence or absence of T3 and only background signals were observed with the anti-ID14 antibody.
Figure 5. The transgenic HAL2 promoter, driving EGFP in transgenic tadpoles, respond to T3 similarly as the endogenous HAL2 gene in the intestine. (a) The double promoter transgenic construct used to study the HAL2 promoter in vivo. The HAL2 promoter fragment flanked by the KpnI and AgeI restriction sites [with the numbers indicating the position relative to the transcription start site in Fig. 1(a)] was cloned to drive the expression of EGFP in a double promoter transgenic construct, which also contains an eye-specific promoter, the γ-crystallin promoter, driving the expression of the GFP3 (36, 37). The HAL2 TRE in the promoter is shown as an open box. (b and c) Stage 54 premetamorphic wild-type or transgenic tadpoles were treated with 10 nM T3 for 0 to 5 days and total intestinal RNA was isolated for expression analysis of the endogenous HAL2 mRNA (b) or transgenic EGFP mRNA (c). Note that the endogenous HAL2 expression was upregulated significantly after 3 to 5 days of treatment in both wild-type and transgenic tadpoles. The expression of the transgenic EGFP mimicked that of the endogenous HAL2 in the transgenic tadpoles but was, as expected, absent in the wild-type tadpoles.
Figure 6. The expression of the transgenic EGFP under the control of the HAL2 promoter mimics that of the endogenous gene in the intestine during natural metamorphosis. (a and b) RT-PCR analysis shows similar temporal expression patterns of the endogenous HAL2 and transgenic EGFP during development. Total intestinal RNA was isolated from wild-type or sibling transgenic tadpoles at indicated stages throughout metamorphosis and subjected to qRT-PCR analysis for expression of the endogenous HAL2 mRNA (a) or transgenic EGFP mRNA (b). Note that a similar pattern was observed for the EGFP and endogenous HAL2 expression in the transgenic tadpoles. (c and d). In situ hybridization confirms the epithelium-specific expression of the transgenic EGFP. The intestinal cross-sections from transgenic tadpoles at the climax of metamorphosis were used for in situ hybridization with an antisense (c) or sense probe (d) for EGFP which was visualized with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium staining (magnification ×200). Note that the transgenic EGFP was detected in the epithelium with an antisense RNA probe for EGFP (c) but not with the sense probe (d). Ct, connective tissue; Ep, epithelium; Lu, lumen; M, muscle.
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