Masc-induced dosage compensation in silkworm cultured cells.

The Masculinizer (Masc) gene encodes a CCCH-tandem zinc finger protein that controls both masculinization and dosage compensation in the silkworm Bombyx mori. We previously measured the masculinizing activity of the lepidopteran Masc proteins using B. mori ovary-derived cell line BmN-4. Here, we established an RNA-seq data-based assay system in which the level of B. mori Masc (BmMasc)-induced dosage compensation can be estimated in BmN-4 cells. Using this system, we found that a cysteine residue at position 301, which was shown to be essential for the masculinizing activity of BmMasc, is also required for dosage compensation. We further investigated the relationships between Masc-induced cell growth inhibition, masculinizing activity, and the level of dosage compensation, using Masc genes from three lepidopteran insects. In summary, we have established a cell-based system to monitor levels of Masc-induced dosage compensation.

Two CCCH-type zinc finger domains in the Masc protein are dispensable for masculinization and dosage compensation in Bombyx mori.

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.

Identification of a bipartite nuclear localization signal in the silkworm Masc protein.

The silkworm Masculinizer (Masc) gene encodes a CCCH-tandem zinc finger protein that controls both masculinization and dosage compensation. Masc protein is a nuclear protein, but the mechanism underlying the transport of this protein into the nucleus has not yet been elucidated. Here, we identified a functional bipartite nuclear localization signal (NLS) located between residues 274 and 290 of the Masc protein. Sequence comparison revealed that this bipartite NLS is evolutionarily conserved in Masc proteins from other lepidopteran insects. Furthermore, we showed that the degree of nuclear localization is not associated with the masculinizing activity of the Masc protein.

Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein.

We have recently discovered that the Masculinizer (Masc) gene encodes a CCCH tandem zinc finger protein, which controls both masculinization and dosage compensation in the silkworm Bombyx mori. In this study, we attempted to identify functional regions or residues that are required for the masculinizing activity of the Masc protein. We constructed a series of plasmids that expressed the Masc derivatives and transfected them into a B. mori ovary-derived cell line, BmN-4. To assess the masculinizing activity of the Masc derivatives, we investigated the splicing patterns of B. mori doublesex (Bmdsx) and the expression levels of B. mori IGF-II mRNA-binding protein, a splicing regulator of Bmdsx, in Masc cDNA-transfected BmN-4 cells. We found that two zinc finger domains are not required for the masculinizing activity. We also identified that the C-terminal 288 amino acid residues are sufficient for the masculinizing activity of the Masc protein. Further detailed analyses revealed that two cysteine residues, Cys-301 and Cys-304, in the highly conserved region among lepidopteran Masc proteins are essential for the masculinizing activity in BmN-4 cells. Finally, we showed that Masc is a nuclear protein, but its nuclear localization is not tightly associated with the masculinizing activity.