# Doublesex and GATAβ4 synergistically regulate the sex-dimorphic expression of storage protein 1 in Bombyx mori

## TL;DR

This study reveals how the genes doublesex and GATAβ4 work together to control the sex-specific expression of a storage protein in silkworms.

## Contribution

The novel finding is that dsxM interacts with GATAβ4 to indirectly regulate SP1 expression in males, rather than directly repressing it.

## Key findings

- Mutation of the doublesex gene leads to upregulated SP1 expression in male silkworms.
- GATAβ4 interacts with both dsxF and dsxM to regulate SP1 expression differently in females and males.
- dsxM inhibits GATAβ4 binding to the SP1 promoter in males, preventing SP1 upregulation.

## Abstract

Sexually dimorphic traits are widespread in organisms and are crucial for reproduction and behavior. These traits are typically controlled by sex-specific genes. However, their regulatory mechanisms are complex and incompletely understood. In Bombyx mori, a group of sex-differential storage proteins (SPs) exists, with storage protein 1 (SP1) expressed exclusively in females. In this study, we used the CRISPR/Cas9 system to knock out the doublesex gene and found that SP1 expression was sharply upregulated in male doublesex mutants and downregulated in female doublesex mutants, which suggests that doublesex is a key factor in the sex-differential expression of SP1. Then, we revealed that the female-specific doublesex isoform (dsxF) bound to and activated the SP1 promoter more strongly than the male-specific isoform (dsxM). Meanwhile, a transcription factor named GATAβ4 was found to be involved in the regulation by doublesex. Overexpression of GATAβ4 in Bombyx mori larvae affected adult reproductive behavior and dramatically upregulated SP1 expression in males. Furthermore, GATAβ4 interacted with both dsxF and dsxM, promoting nuclear translocation of dsxM, which in turn inhibited GATAβ4 binding to the SP1 promoter. In total, we found that dsxM did not directly repress SP1 expression in males but instead cooperated with other transcription factors to regulate downstream gene expression. These findings provide new insights into the regulation of sex-specific genes and the mechanisms controlling dimorphic traits.

Sex-specific expressed genes are widely present across various organisms and play a crucial role in reproduction and evolution. However, the regulatory mechanisms governing their expression remain relatively poorly understood. In this study, we used the female-specific expression gene SP1 in the silkworm as a model and demonstrated that mutation of the sex-determining transcription factor doublesex or transgenic overexpression of the nutritional transcription factor GATAβ4 successfully induced high SP1 expression in male silkworms. Our findings indicate that in female silkworms, both dsxF and GATAβ4 directly regulate the high expression of SP1. In contrast, in male silkworms, the direct regulatory effect of dsxM on SP1 is weak. Instead, dsxM primarily interacts with GATAβ4, inhibiting its binding to the SP1 promoter and thereby blocking GATAβ4-mediated SP1 upregulation, ultimately leading to low SP1 expression. These findings suggest a potential mechanism regulating sex-specific gene expression.