The doublesex proteins of Drosophila melanogaster bind directly to a sex-specific yolk protein gene enhancer.
ABSTRACT The doublesex (dsx) gene of Drosophila melanogaster encodes both male-specific and female-specific polypeptides, whose synthesis is regulated by alternative sex-specific splicing of the primary dsx transcript. The alternative splicing of the dsx mRNA is the last known step in a cascade of regulatory gene interactions that involves both transcriptional and post-transcriptional mechanisms. Genetic studies have shown that the products of the dsx locus are required for correct somatic sexual differentiation of both sexes, and have suggested that each dsx product functions by repressing expression of terminal differentiation genes specific to the opposite sex. However, these studies have not shown whether the dsx gene products function directly to regulate the expression of target genes, or indirectly through another regulatory gene. We report here that the male- and female-specific DSX proteins, expressed in E.coli, bind directly and specifically in vitro to three DNA sequences located in an enhancer region that regulates female-specific expression of two target genes, the yolk protein genes 1 and 2. This result suggests strongly that dsx is a final regulatory gene in the hierarchy of regulatory genes controlling somatic sexual differentiation.
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ABSTRACT: Primary sex-determination ''switches'' evolve ra-pidly, but Doublesex (DSX)-related transcription fac-tors (DMRTs) act downstream of these switches to control sexual development in most animal species. Drosophila dsx encodes female-and male-specific isoforms (DSX F and DSX M), but little is known about how dsx controls sexual development, whether DSX F and DSX M bind different targets, or how DSX proteins direct different outcomes in diverse tissues. We undertook genome-wide analyses to identify DSX targets using in vivo occupancy, binding site predic-tion, and evolutionary conservation. We find that DSX F and DSX M bind thousands of the same targets in multiple tissues in both sexes, yet these targets have sex-and tissue-specific functions. Interestingly, DSX targets show considerable overlap with targets identified for mouse DMRT1. DSX targets include transcription factors and signaling pathway compo-nents providing for direct and indirect regulation of sex-biased expression. INTRODUCTIONDevelopmental Cell 12/2014; 31(6):761-773. DOI:10.1016/j.devcel.2014.11.021 · 10.37 Impact Factor
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ABSTRACT: Protein-DNA interactions play a significant role in gene regulation and expression. In order to identify transcription factor binding sites (TFBS) of double sex (DSX)-an important transcription factor in sex determination, we applied the DNA adenine methylation identification (DamID) technology to the fat body tissue of Drosophila, followed by deep sequencing (DamID-Seq). One feature of DamID-Seq data is that induced adenine methylation signals are not assured to be symmetrically distributed at TFBS, which renders the existing peak calling algorithms for ChIP-Seq, including SPP and MACS, inappropriate for DamID-Seq data. This challenged us to develop a new algorithm for peak calling. A challenge in peaking calling based on sequence data is estimating the averaged behavior of background signals. We applied a bootstrap resampling method to short sequence reads in the control (Dam only). After data quality check and mapping reads to a reference genome, the peaking calling procedure compromises the following steps: 1) reads resampling; 2) reads scaling (normalization) and computing signal-to-noise fold changes; 3) filtering; 4) Calling peaks based on a statistically significant threshold. This is a non-parametric method for peak calling (NPPC). We also used irreproducible discovery rate (IDR) analysis, as well as ChIP-Seq data to compare the peaks called by the NPPC. We identified approximately 6,000 peaks for DSX, which point to 1,225 genes related to the fat body tissue difference between female and male Drosophila. Statistical evidence from IDR analysis indicated that these peaks are reproducible across biological replicates. In addition, these peaks are comparable to those identified by use of ChIP-Seq on S2 cells, in terms of peak number, location, and peaks width.PLoS ONE 01/2015; 10(3):e0117415. DOI:10.1371/journal.pone.0117415 · 3.53 Impact Factor
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ABSTRACT: "Regulatory evolution," that is, changes in a gene's expression pattern through changes at its regulatory sequence, rather than changes at the coding sequence of the gene or changes of the upstream transcription factors, has been increasingly recognized as a pervasive evolution mechanism. Many somatic sexually dimorphic features of Drosophila melanogaster are the results of gene expression regulated by the doublesex (dsx) gene, which encodes sex-specific transcription factors (DSX(F) in females and DSX(M) in males). Rapid changes in such sexually dimorphic features are likely a result of changes at the regulatory sequence of the target genes. We focused on the Flavin-containing monooxygenase-2 (Fmo-2) gene, a likely direct dsx target, to elucidate how sexually dimorphic expression and its evolution are brought about. We found that dsx is deployed to regulate the Fmo-2 transcription both in the midgut and in fat body cells of the spermatheca (a female-specific tissue), through a canonical DSX-binding site in the Fmo-2 regulatory sequence. In the melanogaster group, Fmo-2 transcription in the midgut has evolved rapidly, in contrast to the conserved spermathecal transcription. We identified two cis-regulatory modules (CRM-p and CRM-d) that direct sexually monomorphic or dimorphic Fmo-2 transcription, respectively, in the midguts of these species. Changes of Fmo-2 transcription in the midgut from sexually dimorphic to sexually monomorphic in some species are caused by the loss of CRM-d function, but not the loss of the canonical DSX-binding site. Thus, conferring transcriptional regulation on a CRM level allows the regulation to evolve rapidly in one tissue while evading evolutionary constraints posed by other tissues.Proceedings of the National Academy of Sciences 02/2015; DOI:10.1073/pnas.1501192112 · 9.81 Impact Factor