Yoshimoto S et al. (2006), Expression and promoter analysis of Xenopus DMR...

XB-ART-34942

Dev Growth Differ 2006 Dec 01;489:597-603. doi: 10.1111/j.1440-169X.2006.00894.x.

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Expression and promoter analysis of Xenopus DMRT1 and functional characterization of the transactivation property of its protein.

Yoshimoto S , Okada E , Oishi T , Numagami R , Umemoto H , Tamura K , Kanda H , Shiba T , Takamatsu N , Ito M .

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The doublesex and mab-3-related transcription factor 1 (DMRT1) is involved in testis formation in a variety of vertebrates. In the teleost fish, Medaka, DMY/DMRT1Y on the Y chromosome, a duplicate of the autosomal DMRT1 gene, is characterized as a sex-determining gene. We report here the characterization of the Xenopus DMRT1 genes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that X. laevis DMRT1 was expressed throughout the embryo during early development and was restricted to the primordial gonads after embryogenesis. Whole-mount in situ hybridization analysis of the gene confirmed its specific expression in the primordial gonads. To study the transcriptional control of DMRT1 gene expression, we isolated the predicted promoter region of X. tropicalis DMRT1 using databases for this species. Analysis of transgenic tadpoles with a green fluorescence protein (GFP) reporter showed that approximately 3 kb of the 5'-flanking sequence of the DMRT1 gene was implicated in DMRT1 expression in the primordial gonads. We also showed that the C-terminal region of DMRT1 functioned as a transactivation domain in cultured cells, by a luciferase reporter assay using fusion proteins with the DNA-binding domain of GAL4. These findings suggest that DMRT1 functions as an activator of one or more genes involved in sex determination or gonadal differentiation.

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Species referenced: Xenopus
Genes referenced: dmrt1 lgals4.2 prl.2 tbx2

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	Fig. 1. Structure of Xenopus DMRT1. (A) Amino acid sequence comparison among X. laevis, X. tropicalis, Rana rugosa and human DMRT1. The sequence of X. laevis DMRT1 we obtained was identical to that reported by Osawa et al. (2005). The Rana and human sequences of DMRT1 were reported by Shibata et al. (2002) and Raymond et al. (1998), respectively. The protein sequence of X. tropicalis DMRT1 was predicted by analyses of the X. tropicalis EST and genome databases. Multiple alignments were performed using the ClustalW program. Identical amino acids to X. laevis DMRT1 are shown in white against black. Xl, Xenopus laevis; Xt, Xenopus tropicalis; R, Rana rugosa; H, human. (B) Predicted intronexon structure of the X. tropicalis DMRT1 gene. Detailed information about the genome and EST databases and their analyses is provided in the Results section.
	Fig. 2. Expression of DMRT1 during development in X. laevis. (A) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of DMRT1 and elongation factor-1α (EF-1α) and ornithine decarboxylase (ODC) as a control in unfertilized eggs and embryos from stage (st.) 2-52. Each sample contained 0.25 μg of total RNA. L, H, P, M and G show liver, heart, pronephros, mesonephros and primordial gonads, respectively. (B) Specific expression of DMRT1 in the primordial gonad. Whole-mount in situ hybridization using the antisense or sense RNA probe for DMRT1 (nucleotides 598–1287) was performed in tadpoles at stage 52 during sex determination. f and h show forelimb and hindlimb. Scale bars, 0.5 mm.
	Fig. 3. Promoter reporter analysis of X. tropicalis DMRT1 in transgenic animals. Decondensed sperm nuclei were introduced into unfertilized eggs of X. laevis together with a plasmid bearing the green fluorescence protein (GFP) gene downstream of about 3 kb of the 5′-flanking sequence of X. tropicalis DMRT1. GFP expression was observed by a stereoscopic fluorescence microscope. Typical GFP expression in embryos at stage (st.) 17 and 30 (A) and in tadpoles at stage 52 (B) are shown. Scale bars, 0.5 mm.
	Fig. 4. Transactivation activity of the C-terminal region of X. laevis DMRT1. (A) In vitro transcription/translation was carried out using <35S>-methionine and pcDNA3 FLAG-GAL4BD, pcDNA3 FLAG-GAL4BD-xDMRT1(full), pcDNA3 FLAG-GAL4BD-xDMRT1(1–134), or pcDNA3 FLAG-GAL4BD-xDMRT1(130–334), and the <35S>-labeled proteins were detected by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. (B) P19 cells were transfected with 0.2 μg of the GAL4- responsive luciferase reporter plasmid, pFR-luc, and 10 ng or 30 ng of effector plasmid encoding a fusion protein with the DNA-binding domain of GAL4 (pcDNA3 FLAG-GAL4BD, pcDNA3 FLAG-GAL4BD-xDMRT1(full), pcDNA3 FLAG-GAL4BD-xDMRT1(1– 134), or pcDNA3 FLAG-GAL4BD-xDMRT1(130–334)), as indicated. The Renilla luciferase plasmid pRL-SV (5 ng) was used as a control for transfection efficiency in each experiment. Twenty-four hours after transfection, the luciferase activities were measured by a dual luciferase reporter system. Data represent the mean ± SE from three independent experiments.