Project description:The Drosophila sex determination hierarchy consists of a splicing cascade with sex-specific transcription directing somatic sexual dimorphism. Our understanding of this pathway, and many others, is incomplete. Here we pioneer an approach to expand our knowledge of gene regulatory networks (GRNs) by leveraging natural genetic variation. This approach is generalizable to any natural population, including humans. Two studies from Drosophila female head tissue were used – the DSPR collection (alleles from 15 natural variants) and F1-hybrid collection (alleles from heterozygotes of 75 isogenic lines crossed to w1118) – in a structural equation model (SEM) analysis. We expanded the sex hierarchy GRN by adding novel links among genes in the pathway and by adding novel genes to the pathway. A link from fruitless (fru) to Sex-lethal (Sxl) was found in both populations, which is supported by the presence of fru binding sites in the Sxl locus. The splicing factors male-specific lethal 2 and Rm62 were correctly identified as downstream targets of Sxl. There were 754 additional candidate genes for an expanded sex hierarchy GRN. These candidates were enriched for genes with sex-biased splicing and many components of the spliceosome were placed in the GRN. As with other population-genetic analyses, the number of alleles limits the number of observable interactions. Network expansion was only clear in the F1-hybrid population, which has an average of twice as many alleles as the DSPR population. Independent studies of doublesex and transformer mutants support many novel connections, including evidence for a link between the sex hierarchy and metabolism, with the inclusion of Insulin-like receptor in the sex hierarchy GRN. RNA sequencing was performed on mRNA derived from adult male or female heads, for a total of 9 samples. These samples included females that produce the male isoform of dsx [w/+;DsxD/dsxm+r15 (XX)], and two dsx mutants: females [w/+; dsxm+r15/dsxd+r3 (XX)] and males [w;dsxm+r15/dsxd+r3 (XY)]. Two wild type genotypes (Berlin and Canton-S) were sequenced at the same time, but have previously been published as part of GSE50515. There were at least 3 replicates from biological samples.

Project description:Chromatin modifications have essential roles in directing nervous system development and behavior. Drosophila courtship is an ideal model for understanding the relationships between chromatin modifications, gene expression, sexual dimorphisms in neuroanatomy, and reproductive behaviors. These behaviors are largely innate and are under the control of sex determination hierarchy genes doublesex (dsx) and fruitless (fru) that encode sex-specific transcription factors. Transcripts from the fru P1 promoter are sex-specifically spliced, resulting in male-specific proteins, FruM, which are required for male courtship behaviors. FruM has been shown to form a complex with Bonus/TIF1, a transcriptional cofactor, and interact with chromatin modifying proteins. This indicates that sex-differential modification of the chromatin landscape contributes to creating and maintaining the potential for sexually dimorphic behavior. To examine chromatin modifications genome-wide in fru P1-expressing neurons, we have developed a method to purify chromatin in a cell-type-specific manner using a tagged histone H2B under UAS control, “Chromatag”, paired with sequential ChIP-seq. Here, we evaluate five chromatin modifications: H3K27ac, H3K4me3, H3K36me3, H3K9me3, and H3K27me3 to investigate fru P1-expressing neurons at three developmental stages in both sexes. These modifications have been well characterized for their roles in transcriptional activation or repression. We observe greater changes in chromatin modification profiles across developmental stages than between sexes. In addition, we have generated cell-type specific RNA-seq data sets, using Translating Ribosome Affinity Purification (TRAP), to examine gene expression in fru P1-expressing neurons of both sexes. Informed by these data sets, we have identified several genes with roles in neuronal function and examined their expression in fru P1-expressing neurons, revealing sexually dimorphic expression patterns. Altogether, this work offers insights into stage and sex-differences in chromatin modifications and gene expression in fru P1-expressing neurons.

Project description:N6-methyladenosine (m6A) has recently emerged as a widespread and conserved RNA modification that modulates messenger RNA (mRNA) processing and activity. As most m6A studies have been conducted in cultured cells, we established Drosophila as a model system that combines molecular and genomic analyses of m6A with investigations of organismal biology. We first apply miCLIP sequencing to map m6A modifications at single-nucleotide resolution during Drosophila embryogenesis. We next characterize Drosophila components of the m6A "writer" methyltransferase complex [MTC factors, METTL3/IME4, METTL14, FL(2)D and Nito], and characterize m6A-binding properties of the YTH domain-containing proteins, YT521-B and CG6422. We complement this by generating a collection of mutants in m6A writer and reader components. While Drosophila orthologs of mammalian MTC factors that have additional roles in splicing are lethal, our mutants in METTL and YTH factors are viable but present specific developmental and behavioral defects. We note that several mammalian m6A factors were originally identified as Drosophila regulators of Sxl splicing, which generates the master determinant of female identity. While our mutants in dedicated writer and reader machinery are not required to accumulate Sxl protein in the ovary, maternal loss of m6A writers and readers collaborates with reduction of Sxl to induce female lethality. Our data indicate Sxl is directly regulated by the m6A pathway, since miCLIP data show Sxl is a substantial target of intronic m6A in early embryos, in addition to bearing exonic m6A. Consistent with this, female-specific Sxl splicing is defective in m6A pathway mutants. YT521-B appears to be the major effector of m6A for Sxl regulation, as it shows much stronger genetic interactions with Sxl than CG6422 and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit for studying the m6A pathway in Drosophila reveals a major biological utilization in establishing sex-specific splicing.

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The Wright Stuff: Reimagining Path Analysis Reveals Novel Components of the Sex Determination Hierarchy in Drosophila Melanogaster

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