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

Abstract

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.

Opposite roles ofDMRT1and its W-linked paralogue,DM-W, in sexual dimorphism ofXenopus laevis: implications of a ZZ/ZW-type sex-determining system

“…The Nterminal region (amino acids 1-123), including the DNA-binding domain of DM-W, which is encoded by exons 2 and 3, has relatively high sequence identity (82%) with the corresponding region of DMRT1. By contrast, DM-W has no homologous region to exons 4-6 of DMRT1, which includes the sequence encoding the transactivation domain (Yoshimoto et al, 2006;Yoshimoto et al, 2008). The sequence of the DM-W exon 4, including an in-frame stop codon, contained no significant homology among genomic and cDNA sequences derived from vertebrate species, including X.…”

“…Transgenic tadpoles or frogs were produced using restriction-enzymemediated integration (REMI), as described previously (Yoshimoto et al, 2006). To detect the transgenes, PCR was performed using the genomic DNA from the liver of an individual frog or tadpole as the template, and specific primer pairs as follows: pcDNA3-FLAG-DMRT1, 5Ј-CAGCCAGGATTCTGGAGTTA-3Ј and 5Ј-CAACTAGAAGGC ACA -GTCG-3Ј; psh-DM-W1/2, 5Ј-CCCGTGTGGCAGATG ATT GTT ACCT-3Ј and 5Ј-TTTAGAAGGCACAGTCGAGG-3Ј.…”

“…The Cterminal region of DMRT1 contains a transactivation domain (Yoshimoto et al, 2006), suggesting that DM-W functions as a competitor of DMRT1 for its target genes in the primordial ZW gonads. Interestingly, DM-W mRNA is more abundant than DMRT1 mRNA in the ZW gonads early in sex determination (Yoshimoto et al, 2008).…”

Genome-wide analysis of DNA binding and transcriptional regulation by the mammalian Doublesex homolog DMRT1 in the juvenile testis

“…MAB-3 acts as a transcriptional repressor, as does the male isoform of DSX, but the female isoform of DSX can activate transcription (9,14,16). A fusion of the C-terminal domain of Xenopus DMRT1 to the yeast GAL4 DNA-binding domain can activate reporter gene transcription in transfected cells, whereas in the fish Medaka Dmrt1a and its paralog Dmrt1bY/DMY can repress reporter transcription in transfected cells (17,18). Finding the in vivo targets of DMRT1 and determining how their expression is regulated are critical to a better understanding of how DMRT1 directs testicular development and function.…”

Cloning and expression of candidate sexual development genes in the cane toad (Bufo marinus)

“…Dmrt1 was the only gene whose expression was restricted to the gonadal tissue in both male and female, and the Bidder's organ. This gonad specific expression of Dmrt1 is also observed in X. laevis, R. rugosa, as well as in other taxa Yoshimoto et al, 2006;Lei et al, 2007). The conserved gonad-specific expression pattern in many vertebrate lineages reflects its ancient association with sex-determination and sexual differentiation within both invertebrates and vertebrates (Raymond et al, 1998.…”