Ishihara A et al. (2012), The genomic structure and the expression profil...

XB-ART-45940

Gene 2012 Dec 01;5102:126-32. doi: 10.1016/j.gene.2012.09.002.

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The genomic structure and the expression profile of the Xenopus laevis transthyretin gene.

Ishihara A , Nishiyama N , Makita Y , Yamauchi K .

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Transthyretin (TTR) is a major thyroid hormone-binding protein in the amphibian tadpole whose plasma mRNA and protein levels are altered during metamorphosis. While the temporal and spatial expression patterns and genomic structure of the TTR gene are well studied in higher vertebrates, detailed expression pattern in the extrahepatic tissues, the transcriptional regulation, and the genomic structure have not yet been identified in amphibians. In this study, we attempted to elucidate these mechanisms. Here, we determined the genomic structure of the Xenopus laevis TTR gene including 5'-flanking regions, and examined TTR expression patterns in several tissues. The TTR gene of X. laevis is composed of 4 exons and 3 introns, and the nucleotide sequence of intron 1 is not similar to that previously reported. This suggests that the TTR gene of X. laevis was duplicated and the gene cloned in this study was the other copy of previously reported gene. We also found that TTR was primarily transcribed in the liver of both tadpoles and adults. In the adult liver, TTR transcripts were more abundant in males than females. In higher vertebrates, the expression of TTR is controlled by several transcription factors including forkhead box A2 (FoxA2). However, in the X. laevis liver, FoxA2 expression patterns were not similar to TTR. We also found that exogenous FoxA2 increased the X. laevis TTR promoter-driven luciferase activity. These results suggest that, in amphibian, the expression of TTR is regulated partially by FoxA2, and that another system may exist to control TTR expression.

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Species referenced: Xenopus laevis
Genes referenced: foxa2 ttr
GO keywords: thyroid hormone receptor binding [+]

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	Fig. 1. Southern blot hybridization. (A) Restriction map of the xTTR gene. The map shows the cleavage sites of EcoRI, PstI, XbaI, HindIII, and BamHI. The characters in the figure are E, EcoRI; P, PstI; X, XbaI; H, HindIII; B, BamHI. This restriction map displays only relevant restriction endonuclease sites. EcoRI fragment and PstI fragment are represented as Clone#1 and Clone#2, respectively. Intron 1 fragment cloned by Prapunpoj et al. is presented as Clone#3. (B) Southern hybridization. Ten micrograms of adult X. laevis liver genomic DNA was digested with BamHI, EcoRI, HindIII, PstI, and XbaI and subjected to electrophoresis on 1% agarose gel. Southern blot analysis was performed using xTTR cDNA (left panel) or intron 3 fragment (right panel) as probes. The positions of the DNA molecular size markers are indicated on the left of each panel. Arrowheads represent the fragments for cloning.
	Fig. 2. The expression of xTTR in several tissues during metamorphosis or in adult stages. Total RNA was extracted from the tadpole tissues at NF52, 54, 56, 58, 60, and 62 or adult tissues. The vertical axis represents the ratio of the amount of xTTR transcripts in each sample to those in NF52 liver sample as a magnitude of the induction (fold) after normalization with the housekeeping gene GAPDH. Each value is the mean ± SEM of triplicate determinations. (A) Stage-dependent expression of xTTR in tadpole liver (open bar), kidney (shaded bar), and eye (solid bar). NF stage is the developmental stages outlined by Nieuwkoop and Faber. (B) Tissue-dependent expression of xTTR in adult male (open bar) and female (solid bar) tissues. The characters in the figure are L, liver; I, intestine; K, kidney; B, brain; E, eye; S, skin.
	Fig. 3. The expression of xFoxA2 in several tissues during metamorphosis and in adult stages. Total RNA was extracted from the tadpole tissues at NF52, 54, 56, 58, 60, and 62 or adult tissues. The vertical axis represents the ratio of the amount of xFoxA2 transcripts in each sample to those in NF52 liver sample as a magnitude of the induction (fold) after normalization with the housekeeping gene GAPDH. Each value is the mean ± SEM of triplicate determinations. (A) Stage-dependent expression of xFoxA2 in tadpole liver (open bar), kidney (shaded bar), and eye (solid bar). NF stage is the developmental stages outlined by Nieuwkoop and Faber. (B) Tissue-dependent expression of xFoxA2 in adult male (open bar) and female (solid bar) tissues. The characters in the figure are L, liver; I, intestine; K, kidney; B, brain; E, eye; S, skin.
	Fig. 4. Luciferase activity in X. laevis cultured cell lines transfected with xTTR promoter-luciferase reporter vector and endogenous FoxA2 expression in X. laevis cultured cell lines. Four cell lines, KR (Rafferty, 1969), XTC-2 (Pudney et al., 1973), XL58 (Li et al., 1998), and XL177 (Miller and Daniel, 1977) were used. The left vertical axis represents luciferase activity (open bar). Each value is the mean ± SEM of triplicate determinations. The right vertical axis represents the ratio of the amount of xFoxA2 transcripts in each sample to that in XL177 sample as a magnitude of the induction (fold) after normalization with the housekeeping gene GAPDH (solid bar). Each value is the mean ± SEM of triplicate determinations.
	Fig. 5. Luciferase activity in X. laevis cultured cell lines transfected with full-length or deleted xTTR promoter-luciferase reporter vector. (A) Nucleotide sequence of 5′-flanking region of xTTR. Predicted FoxA2-binding sequences and TATA-box are color-coded. The start or end position of each deletion construct is shown by arrow (Pro#1, 2, 3, 4, 5, 6, 7, and 8). (B) The 1.1 kb 5′-flanking region of the xTTR is truncated from its 5′-end and cloned upstream of the luciferase reporter gene followed by transient transfection into KR cells. Each promoter-luciferase constructs are represented as promoter #1 (Pro#1) to promoter #8 (Pro#8) in the figure. The luciferase activity of each construct was normalized to βGal activity. Each value is the mean ± SEM of triplicate determinations. *p < 0.05.
	Fig. 6. The effects of exogenous xFoxA2 on xTTR promoter activity. The xTTR promoter-reporter construct was cotransfected with an empty expression plasmid or various concentrations of xFoxA2 cDNA expression construct (0, 0.35, 3.5, 35 ng/well). The luciferase activity of each construct was normalized to Renilla reniformis luciferase activity. Each value is shown as the mean ± SEM of triplicate determinations. The letters on top of the bars denote values of statistical significance (p < 0.05; one-way analysis of variance by Fisher's least significant difference test for multiple comparisons).