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.

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.
Most animals have an XY sex chromosome system, in which males have X and Y sex chromosomes, whereas females have two X chromosomes. On the other hand, in most lepidopteran insects, females are the heterogametic sex. In this system, which is analogous to the one found in birds and some reptiles, males have two Z sex chromosomes and females have Z and W sex chromosomes. The silkworm Bombyx mori, a model species of lepidopteran insects, uses a WZ sex determination system, and the W chromosome is known to possess a dominant role in female determination [1,2].
Dosage compensation is a common mechanism that equilibrates X-or Z-linked gene expression with the autosomes. In Drosophila melanogaster, which uses an XY system, males compensate for reduced dosage of X-linked genes by hypertranscribing their hemizygous X chromosome through chromosome-wide hyperacetylation of H4K16 [3,4]. Sex-lethal, a master switch for sex determination in D. melanogaster, controls dosage compensation by inhibiting translation of male-specific lethal 2 (msl-2), and mutations in msl-2 lead to male-specific lethality [5]. These findings demonstrate that sex determination cascade is tightly coupled with dosage compensation system and a failure of dosage compensation results in sex-specific death.
In 2014, we reported that the Z-linked gene Masculinizer (Masc) determines maleness in the silkworm B. mori (Lepidoptera: Bombycidae) [6]. We found that depletion of Masc mRNA in early embryos results in abnormal upregulation of Z-linked genes and male-specific embryonic death. These results indicate that Masc plays essential roles in both masculinization and dosage compensation in B. mori embryos [6]. In females, Masc mRNA is cleaved by the PIWI protein complexed with the W chromosome-derived female-specific PIWIinteracting RNA (piRNA), Fem piRNA, which inhibits masculinization and leads to feminization [6].
Masc encodes a CCCH-tandem zinc finger (ZF) protein that is conserved among lepidopteran insects [6][7][8][9][10]. Transfection of Masc cDNA in B. mori ovaryderived BmN-4 cells leads to the expression of maletype variants of both B. mori doublesex (Bmdsx M ) and B. mori IGF-II mRNA-binding protein (BmIMP M ) [6,7]. Using this system, we attempted to search for functional residues required for the masculinizing activity of Masc and identified two cysteines in the highly conserved region of the lepidopteran Masc proteins, at residues 301 and 304 (Cys-301 and Cys-304), both of which are required for the masculinizing activity [7]. We also found that two ZFs of the Masc protein are not essential for masculinization in BmN-4 cells [7]. We generated several Masc knockout B. mori strains using the CRISPR/Cas9 system and confirmed that the two ZFs are not required for either masculinization or dosage compensation in B. mori [11]. However, due to several technical difficulties, we could not determine which amino acid residues are essential for dosage compensation in B. mori.
In the current study, we established a new in vitro assay system using RNA-seq data from BmN-4 cells transfected with Masc derivatives and successfully estimated the level of Masc-induced dosage compensation in BmN-4 cells. Using this system, we found that Masc Cys-301 is required for dosage compensation. In addition, we were also able to detect dosage compensation induced by other lepidopteran Masc proteins. We further investigated the relationships between Mascinduced cell growth inhibition, masculinizing activity, and the level of dosage compensation.

Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was prepared from transfected cells using TRIzol reagent (Invitrogen) and used for reverse transcription using avian myeloblastosis virus reverse transcriptase with an oligo-dT primer (TaKaRa, Kusatsu, Japan). PCR was performed using KOD FX-neo DNA polymerase (TOYOBO, Osaka, Japan). Sex-specific splicing of Bmdsx was examined with RT-PCR as reported previously [7]. Quantitative RT-PCR (RT-qPCR) of BmIMP M and rp49 was performed using a KAPA TM SYBR FAST qPCR kit (Kapa Biosystems, Wilmington, MA, USA), as previously described [7].

RNA-seq
Libraries for RNA-seq were generated from total RNA of transfected cells using the SureSelect Strand-Specific RNA library Prep Kit (Agilent, Santa Clara, CA, USA). The cDNAs were analyzed using the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) with 100-bp pairedend reads according to the manufacturer's protocol. RNAseq reads were mapped to 13 789 B. mori gene models (putative protein-coding genes whose chromosomal locations are identified), and the transcript abundance in each gene model was quantified as previously described [6,15].

Sequence deposition
The deep sequencing data obtained in this study are available under the accession number DRA008403 in the DNA Data Bank of Japan (DDBJ).

Establishment of a cell-based assay system for monitoring the Masc protein-induced dosage compensation
To estimate the level of dosage compensation governed by the Masc protein, we attempted to establish a cellbased assay system using B. mori cultured cells. We performed RNA-seq on two B. mori cell lines, BmN-4 and M1, both of which were transfected with B. mori Masc (BmMasc) or its derivative cDNAs. BmN-4 is an ovary-derived female cell line [12], and M1 is an embryo-derived male cell line [13]. We first used two BmMasc cDNAs, Fem piRNA-resistant BmMasc (Masc-R) and Cys-301 to Ser BmMasc-R (MR-CS) [5,7]. Because Cys-301 is completely conserved among lepidopteran Masc proteins and plays an essential role for the masculinizing activity of BmMasc in BmN-4 cells [7,10], we speculated that this cysteine residue is also required for dosage compensation. We measured the expression levels of B. mori genes in BmMasc cDNA-transfected cells by mapping RNA-seq reads onto the B. mori gene models. MA plots and boxplot of the mapping results clearly show that transfection of Masc-R cDNA decreased the expression of Z-linked genes (red dots in Fig. 1A) in both BmN-4 and M1 cells and that the suppression was more apparent in BmN-4 cells than in M1 cells (Fig. 1A,B). The expression levels of autosomal genes (black dots in Fig. 1A) were comparable in Masc-R cDNA-transfected cells to those in empty vector-transfected cells. On the other hand, transfection of MR-CS cDNA did not greatly affect the expression of autosomal and Z-linked genes in either BmN-4 or M1 cells (Fig. 1A,B). Combining these observations with our previous results [7,10], we concluded that Cys-301 is crucial for both masculinization and dosage compensation of the BmMasc protein. In addition, we observed that the genes on the chromosome 27 were relatively downregulated in M1 cells by transfection of either Masc-R or MR-CS cDNA (Fig. 1B). However, in BmN-4 cells, transfection of MR-CS cDNA, but not Masc-R cDNA, reduced their expression levels (Fig. 1B), indicating that this effect does not depend on the Masc activity. Taken together, we decided to use BmN-4 cells to monitor the levels of Masc-induced dosage compensation.

Comparison of the levels of dosage compensation induced by three lepidopteran Masc proteins
We have previously shown that two Masc proteins, OfMasc from O. furnacalis (Lepidoptera: Crambidae) and TvMasc from T. varians (Lepidoptera: Bombycidae), exhibit the masculinizing activity in BmN-4 cells [8,9]. We next assessed the levels of dosage compensation induced by these non-BmMasc proteins using a cell-based assay system. We performed RNA-seq of total RNA prepared from BmN-4 cells transfected with Masc-R, MR-CS, OfMasc, and TvMasc cDNAs and compared the expression levels of Z-linked and autosomal genes with those of empty vector-transfected cells. We reproducibly observed dosage compensation in Masc-R-transfected cells and little effect on gene expression in MR-CS-transfected cells (Fig. 2). We also detected repression of Zlinked genes, but not autosomal genes, in OfMasc-and TvMasc-transfected cells, indicating that these two non-BmMasc proteins also induce dosage compensation in BmN-4 cells. The repression levels of Z-linked genes in OfMasc-and TvMasc-transfected cells were comparable to those observed in BmMasc-transfected cells (Fig. 2). These results strongly suggest that OfMasc and TvMasc proteins are involved in dosage compensation of Z-linked genes.

Cell growth inhibition induced by the Masc proteins
In previous studies, we observed that transfection of Masc-R cDNA resulted in growth inhibition in BmN-4 cells, whereas transfection of MR-CS cDNA did not [6,10]. However, the mechanism of Masc-induced cell growth inhibition remains unknown. We next assessed the effect of transfection of OfMasc or TvMasc cDNA on cell growth of BmN-4 cells. As observed in Masc-R cDNA-transfected cells, expression of both OfMasc and TvMasc inhibited cell growth (Fig. 3A). The degree of growth inhibition decreased as follows, from highest to lowest: (a) OfMasc-transfected cells; (b) Masc-R-or TvMasc-transfected cells; and (c) MR-CSor empty vector-transfected cells. To examine whether this growth inhibition is associated with the apoptotic pathway, we measured caspase activity in Masc cDNA-transfected cells. As shown in Fig. 3B, the caspase activity was higher in OfMasc cDNA-transfected cells than in cells transfected with other Masc cDNAs or empty vector, indicating that the significant inhibition of cell growth observed in OfMasc-transfected cells is caused by apoptosis. Caspase activity in Masc-R and TvMasc cDNA-transfected cells was comparable to that in empty vector-transfected cells. Although caspase activity in transfected cells may be linked to the expression levels of the Masc proteins, this suggests that cell growth inhibition induced by these two Masc proteins is not associated with apoptosis.

Comparison of the masculinizing activities of the three Masc proteins
To compare the masculinizing activity of the lepidopteran Masc proteins, we investigated the splicing patterns of Bmdsx and the expression levels of BmIMP M in Masc cDNA-transfected cells. As previously reported [5][6][7][8][9], transfection of Masc cDNAs from three lepidopteran species induced the expression of Bmdsx M and BmIMP M (Fig. 4A,B). The band intensities of Bmdsx M were similar among the three Masc proteins, whereas the level of BmIMP M expression was~1.5 times higher in OfMasc cDNA-transfected cells than in cells transfected with other Masc cDNAs (Fig. 4B).

Discussion
In this study, we successfully established a cell-based system to monitor the levels of Masc-induced dosage compensation. Using this new system, we identified Cys-301 of BmMasc as an essential residue for both dosage compensation and masculinization and were able to estimate the levels of dosage compensation induced by non-BmMasc proteins. These results indicate that this system is capable of identifying residues or domains of the Masc proteins involved in dosage compensation, and of assessing the levels of dosage compensation induced by other lepidopteran Masc proteins. In addition, our findings strongly suggest that both roles of the Masc protein are conserved not only in the closely related species (T. varians) but also in the evolutionarily distant species (O. furnacalis).
We reported that transfection of Masc-R cDNA results in severe inhibition of cell growth in BmN-4 cells [6] and that transfection of MR-CS cDNA did not induce cell growth inhibition [10], indicating that BmMasc activity is required for this inhibition. In this study, we compared the levels of masculinization and dosage compensation in BmN-4 cells transfected with three lepidopteran Masc cDNAs and found that Mascinduced growth inhibition appears to be associated with its masculinizing activity (Fig. 4B). In addition, we showed that transfection of OfMasc, but not BmMasc or TvMasc, induces caspase activation, although growth inhibition was observed in BmN-4 cells transfected with each Masc cDNA. To know this reason, we attempted to identify differentially expressed genes (DEGs) using RNA-seq data from Masc cDNAtransfected cells. However, we did not identify DEGs common to all treatments or apoptosis-related DEGs in OfMasc cDNA-transfected cells (data not shown). Further experiments, such as analyzing RNA-seq data from samples taken at different time points after transfection, are needed to clarify the mechanism of Mascinduced cell growth inhibition in BmN-4 cells.
Recently, Zheng et al. reported that BmMasc induces the A and B isoforms of the RNA-binding protein gene BxRBP3, whose overexpression in female cultured cells (BmF cells) efficiently inhibits splicing of the exons 3 and 4 in Bmdsx and enhances Bmdsx M expression [16]. To confirm whether this signaling pathway is also utilized in BmN-4 cells, we examined the expression of BxRBP3 isoforms in BmN-4 cells using RNA-seq data. We found that the A and B isoforms were neither induced by transfection with Masc cDNAs nor detected in the transcriptome of BmN-4 cells (data not shown). RNA-seq analysis showed that the D isoform of BxRBP3 is predominant in BmN-4 cells. RT-PCR using published primers confirmed the results obtained from our RNA-seq data (data not shown), suggesting that Masc-induced masculinization signaling differs between BmN-4 and BmF. We are currently searching for the factors that are activated under the Masc signaling in silkworm cells.  supported by grants from JSPS KAKENHI (15H02482 to SK and TK) and MEXT KAKENHI (17H06431 to SK and TK and 16H06279 to YS).