Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657, Japan.
Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657, Japan.
Insect Biochem Mol Biol. 2019 Jan;104:30-38. doi: 10.1016/j.ibmb.2018.12.003. Epub 2018 Dec 7.
The Masculinizer (Masc) gene encodes a novel lepidopteran-specific protein that controls both masculinization and dosage compensation in the silkworm Bombyx mori. The Masc protein possesses two CCCH-type zinc finger domains (ZFs), a nuclear localization signal, and an 11-amino-acid region that is highly conserved among lepidopteran insects. Using a cell-based assay system, we revealed that two cysteine residues localized in the conserved region, but not ZFs, are required for masculinization. In addition, nuclear localization of the Masc protein is not associated with masculinizing activity. Because dosage compensation is considered to occur in the nucleus, we inferred that the two ZFs play a role in the establishment of dosage compensation. To investigate this hypothesis at the organism level, we utilized the CRISPR/Cas9 system and established three B. mori strains whose Masc is partially deleted at different regions. The strain lacking the 210 C-terminal amino acids of the Masc protein showed male-specific embryonic lethality due to its low abundance and/or instability. The male embryos of this strain expressed the female-type splice variants of B. mori doublesex and did not express the male-type mRNA of B. mori IGF-II mRNA-binding protein. Furthermore, mRNA levels of Z-linked genes were abnormally enhanced only in male embryos. In contrast, the strain lacking both ZFs grew normally and did not show any defective phenotypes including sexual differentiation and the expression of Z-linked genes, demonstrating that the two CCCH-type ZFs, which are conserved in lepidopteran Masc homologs, are dispensable for masculinization and dosage compensation.
雄化基因（Masc）编码一种新型鳞翅目特异性蛋白，该蛋白可控制家蚕（Bombyx mori）的雄化和剂量补偿。Masc 蛋白含有两个 CCCH 型锌指结构域（ZFs）、一个核定位信号和一个在鳞翅目昆虫中高度保守的 11 个氨基酸区域。我们使用基于细胞的测定系统发现，位于保守区域但不是 ZFs 中的两个半胱氨酸残基对于雄化是必需的。此外，Masc 蛋白的核定位与雄化活性无关。由于剂量补偿被认为发生在核内，我们推断两个 ZFs 在建立剂量补偿中发挥作用。为了在生物体水平上验证这一假设，我们利用 CRISPR/Cas9 系统建立了三个 Masc 部分缺失不同区域的家蚕品系。缺乏 Masc 蛋白 210 个 C 末端氨基酸的品系由于其丰度低和/或不稳定而表现出雄性特异性胚胎致死。该品系的雄性胚胎表达了雌性型 B. mori doublesex 的剪接变体，而不表达雄性型 B. mori IGF-II mRNA 结合蛋白的 mRNA。此外，仅在雄性胚胎中 Z 连锁基因的 mRNA 水平异常增强。相比之下，缺乏两个 ZFs 的品系正常生长，没有表现出任何缺陷表型，包括性分化和 Z 连锁基因的表达，表明两个 CCCH 型 ZFs 在鳞翅目 Masc 同源物中是保守的，对于雄化和剂量补偿是可有可无的。
