A Genomic and Genetic Analysis of Doublesex Targets and Function in Drosophila Sexual Dimorphism
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Date
2015-07-28
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Johns Hopkins University
Abstract
Sex determination pathways are diverse throughout the animal kingdom, but converge upon conserved genes that encode products that regulate sexual dimorphism. One such downstream factor across many diverged sex determination pathways is the Drosophila doublesex (dsx) gene. The role of doublesex is highly conserved in different insects and dsx homologs (dsx, mab-3 related transcription factors, DMRTs) play roles in sexual differentiation in a diverse array of metazoans. In Drosophila, nearly all manifestations of sexual dimorphism between males and females are regulated by doublesex, yet there are only three known direct targets of DSX, which cannot account for the differences in regulation by DSX in sexually dimorphic tissues.
To gain a comprehensive understanding of DSX targets, we undertook multiple experimental approaches that allowed us to identify genes that were bound by DSX, genes whose expression changed in response to DSX perturbation, and genes that function in dsx-expressing cells. DSX protein binding was assayed by ChIP-seq and DamID-seq on S2 cells expressing tagged DSX-M or DSX-F. We also examined DSX occupancy in adult fat body and gonads using DamID-seq or DamID-chip. These experiments identified 3,717 genes bound by DSX in at least one occupancy dataset. Strikingly, we found that genes with the highest levels of DSX occupancy were bound by DSX in all occupancy data sets. This suggests one main mechanism of DSX action would be binding to potential targets in all tissues/contexts rather than having context-dependent targets. In this model of DSX action, additional inputs (such as segmental identity) would be needed to enact transcriptional regulation of bound genes in the appropriate context. Further strengthening this model, although 2,668 genes are bound by DSX in our adult
fat body occupancy data, less than 1% of these occupied genes show large and robust transcriptional changes in response to acute changes in DSX isoform.
We found that predicted DSX targets are significantly enriched in genes that yield phenotypes in sexually dimorphic tissues after RNAi knockdown in dsx-expressing cells (p=0.002). 41 (70.7%) of high probability DSX targets had phenotypes in at least one sexual dimorphic tissue compared to 7 (31.8%) of low probability targets. Altogether, the occupancy, transcriptional profiling, and functional testing have provided a detailed description of how dsx regulates sexual development.
New dsx-interacting genes include genes involved in insect hormone signaling. We have identified the Ecdysone receptor gene as a target of DSX. Since the Drosophila gonad represents an excellent model to dissect how DSX acts on a particular time and place to promote development of a sexually dimorphic tissue, we examined the Ecdysone receptor gene, which is involved in ecdysteroid signaling, for roles in gonad sexual development. My data supports the hypothesis that the steroid hormone ecdysone elicits a different response in the male vs. female gonad and that this difference is regulated by DSX and may be important for proper formation of the ovary vs. the testis. Rather than being strictly a genetic process, results from our experiments may demonstrate that sexual differentiation in the gonad occurs through a combination of signals that include sex specific hormone signaling. Since the formation of the gonad may represent processes that are conserved from flies to man, this research will provide insight into conserved genes that regulate developmentally similar pathways whose outcome generates major differences observed between the sexes.
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Keywords
sex-determination, gonad development, drosophila gonad, doublesex, doublesex targets, genomics, sexual dimorphism, ecdysteroid hormone, hormonal signaling