斑馬魚肌肉調控因子 Myf5 啟動子活性之負調控分子機制

Abstract

Myf5 是屬於肌肉發育調控因子之一，在肌肉細胞的特化與分化上都扮演著相當重要的角色。而myf5 的活化與抑制則受到嚴謹的調控，因此使得myf5在體節的表現具有時間與位置的特異性。我的研究發現斑馬魚 myf5 的intron I含有一個新的intronic microRNA (miR-In300)，可以抑制myf5啟動子表現的能力，並透過抑制myf5的表現達到調控肌肉的發育。然而，對於miR-In300是透過抑制哪些基因轉譯成蛋白質，而調控myf5在體節的表現。利用LAMP這個我所開發新技術找到一個可能是miR-In300目標基因 Dickkopf-3 (dkk-3)。whole-mount in situ hybridization實驗結果顯示dkk-3在體節的表現和myf5有相似的情形。這也暗示說dkk-3 和myf5在體節的表現有關。利用胚胎發育抑制劑抑制 (morpholino, MO) knock down dkk-3 的表現會造成myf5在體節的表現下降但在presomitic mesoderm的表現則不受影響。而且注射dkk-3 MO胚胎的表型和注射myf5 morphant的結果也很類似。同時，注射dkk-3 MO到myf5綠螢光轉殖魚胚胎可以明顯觀察到在體節部分的綠螢光表現下降。顯示說dkk-3會影響myf5啟動子在體節的活性。從dual-luciferase assay與Western blot的結果，證實miR-In300確實能夠抑制dkk-3的蛋白質表現。因此miR-In300是透過抑制目標基因 dkk-3蛋白質的表現，進而抑制myf5啟動子在體節的活性，形成一個自我負回饋的調控機制。

Although a strong, negative cis-regulatory motif located at the first intron +502/+835 (I300) of zebrafish myf5 was reported, the detailed molecular mechanism underlying the negative modulation remains unclear. To understand this repression network, we microinjected zebrafish eggs with RNA corresponding to the sense strand of I300, resulting in the dramatic reduction of luciferase reporter activity driven by the myf5 promoter. Within this I300 segment, we identified an intronic microRNA located at +610/+632 (miR-In300) and observed that it appeared predominately at the mature somites, but weakly at newly formed somites, which were opposed to the expression patterns of zebrafish myf5. By using the novel method we developed, labeled miRNA pull-down assay system (LAMP), we subsequently screened the Dickkopf-3 gene (dkk3) and identified it as the target gene of miR-In300. Inhibition of the dkk3 translation by microinjection of the dkk3-specific antisense oligonucleotide (MO) did result in down-regulation of myf5 transcripts in somites, whereas co-injection of myf5 mRNA with dkk3-MO enabled rescue of the defects induced by microinjection of dkk3-MO alone. In addition, microinjection of the miR-In300-MO enhanced myf5 transcripts in somites and Dkk3 protein level in zebrafish embryos. We concluded that miR-In300 binds to the dkk3, which, in turn, inhibits the translation of the dkk3 mRNA and results in suppressing the promoter activity of zebrafish myf5.