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???displayArticle.abstract??? Thyroid hormone (T3) affects development and metabolism in vertebrates. We have been studying intestinal remodeling during T3-dependent Xenopus metamorphosis as a model for organ maturation and formation of adult organ-specific stem cells during vertebrate postembryonic development, a period characterized by high levels of plasma T3. T3 is believed to affect development by regulating target gene transcription through T3 receptors (TRs). While many T3 response genes have been identified in different animal species, few have been shown to be direct target genes in vivo, especially during development. Here we generated a set of genomic microarray chips covering about 8000 bp flanking the predicted transcription start sites in Xenopus tropicalis for genome wide identification of TR binding sites. By using the intestine of premetamorphic tadpoles treated with or without T3 and for chromatin immunoprecipitation assays with these chips, we determined the genome-wide binding of TR in the control and T3-treated tadpoleintestine. We further validated TR binding in vivo and analyzed the regulation of selected genes. We thus identified 278 candidate direct TR target genes. We further provided evidence that these genes are regulated by T3 and likely involved in the T3-induced formation of adult intestinal stem cells during metamorphosis.
Figure 1. ChIP-on-chip assay identified putative TR targets in the tadpoleintestine. (A) Schematic representation of a gene in the genomic chips. About 5500 bp upstream and 2500 bp downstream of the 5′-end of the cDNA sequence in the databank for each putative gene in the Xenopus tropicalis genome database were used to design a 60 bp oligonucleotide probe at an average tiling distance of 205 bp, covering the entire 8000 bp. The probes were custom-printed onto the genomic chips. PSS: putative transcription start site. (B) Venn-diagram showing the genes with TR binding sites as identified from the ChIP-on-chip assay with the intestine samples from control and T3-treated stage 54 premetamorphic tadpoles. Note that vast majority of the genes bound by TR in the control tadpoles were also found in the T3-treated animals.
Figure 2. The ChIP signals across the 8000 bp promoter region for PGPEP1 and TFG1 from the control and T3 treated tadpoleintestine. Both the control and T3-treated groups had two independent samples as shown. The arrow points to the putative start site (PSS).
Figure 3. ChIP confirmation of the TR binding to target sites identified from the ChIP-on-chip assay. The intestine from stage 54 tadpoles treated with or without T3 was isolated and subjected to anti-TR antibody ChIP assay and the region around the putative TR binding sites as identified from the ChIP-on-chip assay was PCR amplified. Note that no TR binding was found in the control gene (exon 5 of the TRβ gene) while all three newly identified target genes had TR binding in the absence of T3 and this binding was enhanced upon T3 treatment, in agreement with the ChIP-on-chip data. * indicates pairs of samples with significant differences (p < 0.05).
Figure 4. RT-PCR analysis confirming the regulation of newly identified TR targets as T3 response genes. The RNA was isolated from the intestine of stage 54 tadpoles treated with or without T3 was isolated and subjected to RT-PCR analysis for gene expression. Note that all three genes were found to be induced by T3 treatment. *Indicates pairs of samples with significant differences (p < 0.05).
Figure 5. The putative TREs in the candidate TR target genes can mediate transcriptional activation by T3 in frog oocytes. The luciferase reporter construct containing the TREs of Dot1L, MBD3, PPM1B, PGPEP1, JUNB, BEND7, respectively, was co-injected with the control Renilla luciferase construct phRG-TK into the nuclei of Xenopus oocytes with or without prior cytoplasmic injection of Xenopus laevis TRα and RXRα mRNAs or GFP mRNA as negative control. 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 all the reporters responded to T3 in the presence of TR/RXR.
Figure 6. Venn diagram showing overlap of the TR target genes identified by ChIP-on-chip assay with known developmentally regulated genes identified from an early expression microarray study. Of the 278 TR target genes identified by ChIP-on-chip assay, 95 or 34% were also found on the microarray used for the expression study (Note that the actually overlap might be higher as most genes may have different names on the microarray chips used in the expression study and on the genomic chips used here, therefore showing up as non-overlapping). Of the 95 genes present in the gene expression microarray, 38 were found to be regulated during metamorphosis in the intestine in either the epithelium (EP, 13 genes), non-epithelium (Non-EP, 14 genes) or both (11 genes), suggesting that about 40% of the genes identified here are regulated by T3 during intestinal metamorphosis.
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