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Promoter generation for the chimeric sex-determining gene dm-W in Xenopus frogs.
Hayashi S
,
Tamura K
,
Tsukamoto D
,
Ogita Y
,
Takamatsu N
,
Ito M
.
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Many sex-determining genes (SDGs) were generated as neofunctionalized genes through duplication and/or mutation of gonadal formation-related genes. We previously identified dm-W as an SDG in the African clawed frog Xenopus laevis and found that a partial duplication of the masculinization gene dmrt1 created the neofunctionalized dm-W after allotetraploidization by interspecific hybridization. The allotetraploid Xenopus species have two dmrt1 genes, dmrt1.L and dmrt1.S. Xenopus laevis dm-W has four exons: two dmrt1.S-derived exons (exons 2 and 3) and two other exons (noncoding exon 1 and exon 4). Our recent work revealed that exon 4 originated from a DNA transposon, hAT-10. Here, to clarify when and how the noncoding exon 1 and its coexisting promoter evolved during the establishment of dm-W after allotetraploidization, we newly determined nucleotide sequences of the dm-W promoter region from two other allotetraploid species, X. largeni and X. petersii, and performed an evolutionary analysis. We found that dm-W acquired a new exon 1 and TATA-type promoter in the common ancestor of the three allotetraploid Xenopus species, resulting in the deletion of the dmrt1.S-derived TATA-less promoter. In addition, we demonstrated that the TATA box contributes to dm-W promoter activity in cultured cells. Collectively, these findings suggest that this novel TATA-type promoter was important for the establishment of dm-W as a sex-determining gene, followed by the degeneration of the preexisting promoter.
Fig. 1.
Intron 1-derived TATA-less promoter of dmrt1 for gonadal somatic cell expression. (A) Phylogenetic relationship among four Xenopus species used in this study. Ploidy and gene information are shown on the right. (B) mVISTA plots of X. laevis (Xl) dmrt1.L (upper) or Xl dm-W (lower) with the other three subfamily genes, using the genomic regions covering the noncoding Ex1 and Ex2. Dark and light blue colors indicate protein-coding and noncoding exons, respectively. Pink shows conserved noncoding sequences. (C) Sequence alignment of the corresponding regions to the upstream region and Ex2 of Xl dmrt1.L among the four. Purple and yellow indicate GC and CAAT boxes, respectively. Arrows indicate the 5′ site of Xl dm-W Ex2 or the transcription start site (TSS) of Xl dmrt1.L/S, and asterisks indicate identical nucleotides among all the sequences. Red shows a deletion in dm-W.
Fig. 2.
Noncoding Ex1 (ncEx1)-derived TATA-type promoter of dm-W for gonadal somatic cell expression. (A) Schematic diagram of a somatic TATA-less promoter region from X. laevis dmrt1.S and the corresponding regions from X. laevis and X. largeni dm-Ws. Red letters correspond to the translation initiation codon AUG. (B) Sequence comparison of TATA-type promoter regions among X. laevis, X. petersii and X. largeni dm-Ws. Purple, green and blue boxes indicate the GC box, TATA box and DPE, respectively. Red letters and arrows show tandem repeats. (C) Distribution of TEs in and around the ncEx1 of X. laevis, identified using the GIRI CENSOR program. Blue and orange boxes indicate DNA transposon- and retrotransposon-derived fragments, respectively. Noncoding and coding exons are shown as white and gray boxes, respectively. The 325-bp sequence containing the ncEx1, which was not recognized as possessing TEs, includes the core promoter elements, GC box (purple) and TATA box (green).
Fig. 3.
Core promoter activity through the TATA and GC boxes of X. laevis dm-W in transfected cultured cells. (A) Schematic diagram of the luciferase reporter plasmids containing the dm-W sequence from −176 to +149 (wild-type) and its mutants of the TATA box (TATA box MT) and GC box (GC box MT). (B–D) Promoter assay of X. laevis dm-W was performed by transfecting the luciferase reporters and wild-type, TATA box MT or GC box MT into human embryonic kidney (HEK293T) cells (B), X. laevis A6 cells (C) or X. laevis XL-B4 cells (D). Transfections were done three times in each cell line; error bars represent the standard errors of the means. a–d: means with different letters are significantly different from each other, tested using one-way ANOVA, followed by the Tukey–Kramer HSD test (P < 0.05).
Fig. 4.
Proposed model for promoter evolution of Xenopus dmrt1 subfamily genes. (A) Promoter variation in Xenopus dmrt1 subfamily genes. Although one dmrt1.S and two dmrt1.L promoters contain no TATA box (Fig. 1C and Supplementary Fig. S5), dm-W acquired a TATA-type promoter for gonadal somatic cell expression (Fig. 2B). (B) An evolutionary model for the emergence of dm-W through three independent insertions.
Supplementary Fig. S1. Analysis related to Fig. 1B: mVISTA plots of the four Xenopus dmrt1 subfamily genes (X. laevis (Xl) dmrt1.L, Xl dmrt1.S, Xl dm-W and X. tropicalis (Xt) dmrt1) using the genomic regions covering the noncoding Ex1 (ncEx1) and Ex2. Xt dmrt1 (top), Xl dmrt1.L (middle; the same as Fig. 1B) or Xl dmrt1.S (bottom) is shown as the query.
Supplementary Fig. S2. Sequence comparison of the X. laevis dmrt1.L somatic promoter region and its corresponding regions from X. laevis and X. largeni dm-Ws. Purple and red boxes indicate GC boxes and deletions, respectively. Red letters correspond to the translation initiation codon AUG. Arrows indicate the 5 site of Xl dm-W Ex2 and the transcription start site (TSS) of Xl dmrt1.L.
Supplementary Fig. S3. Detailed description of the middle drawing in Fig. 2C: TE distribution in and around the noncoding exon 1 (ncEx1) of X. laevis. TE distribution was analyzed using the GIRI CENSOR program and BLAST searches; blue and orange boxes indicate DNA transposon-derived and retrotransposon-derived fragments, respectively. Noncoding exons and coding exons are shown using gray boxes.
Supplementary Fig. S4.
kbp of the transposon-rich region upstream of the TSS of dm-W. Binding sites for six transcription factors were searched for as described in Supplementary materials and methods. The sites were selected with a P-value below 0.001 in MEME FIMO analysis. Blue and orange boxes indicate DNA transposons and RNA transposons containing LTR and non-LTR type, respectively. A green box corresponds to Repeat I in Supplementary Fig. S3.
Supplementary Fig. S5. Sequence comparison of the genomic regions covering the germ cell-specific promoter between X. laevis (Xl) dmrt1.L and X. tropicalis (Xt) dmrt1. Pink shows conserved noncoding sequences. Purple, yellow and brown boxes indicate the GC box, CAAT box and INR. Arrows indicate transcription start sites (TSSs).
