Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior

Abstract

Examining the role of chromatin modifications and gene expression in neurons is critical for understanding how the potential for behaviors are established and maintained. We investigate this question by examiningDrosophila melanogaster fru P1neurons that underlie reproductive behaviors in both sexes. We developed a method to purify cell-type-specific chromatin (Chromatag), using a tagged histone H2B variant that is expressed using the versatile Gal4/UAS gene expression system. Here, we use Chromatag to evaluate five chromatin modifications, at three life stages in both sexes. We find substantial changes in chromatin modification profiles across development and fewer differences between males and females. Additionally, we find chromatin modifications that persist in different sets of genes from pupal to adult stages, which may point to genes important for cell fate determination infru P1neurons. We generated cell-type-specific RNA-seq data sets, using translating ribosome affinity purification (TRAP). We identify actively translated genes infru P1neurons, revealing novel stage- and sex-differences in gene expression. We also find chromatin modification enrichment patterns that are associated with gene expression. Next, we use the chromatin modification data to identify cell-type-specific super-enhancer-containing genes. We show that genes with super-enhancers infru P1neurons differ across development and between the sexes. We validated that a set of genes are expressed infru P1neurons, which were chosen based on having a super-enhancer and TRAP-enriched expression infru P1neurons.

Single-cell transcriptome profiles ofDrosophila fruitless-expressing neurons from both sexes

“…There are 147 genes that are either male- or female-biased, depending on the cluster ( Supplemental Table 9 ). When we compare these sex-biased genes to genes identified as sex-biased in our previous cell-type-specific TRAP study at 48hrAPF, we find significant overlap between gene lists ( Supplemental Table 4 ) (Palmateer et al ., 2021).…”

“…In total, we find 3,582 marker genes in the male data set, 3,203 in the female data set, and 3,662 in the full data set ( Supplemental Table 4 ). When we determine the overlap of the marker genes in the three analyses, the majority are shared (2,573 genes; Figure 1C ), indicating that fru P1 neurons from males and females have overlapping gene expression profiles ( Figure 1 ), as we previously showed (Newell et al ., 2016; Palmateer et al ., 2021). However, each analysis reveals unique marker genes, with the most occurring in the male data set ( Figure 1C ).…”

“…The DIP s encode members of the Immunoglobulin superfamily (IgSF), a family that is highly enriched among the full marker gene list ( Supplemental Table 5 ). Previous studies have shown that the IgSFs are regulated by Fru M and/or have enriched expression in fru P1 neurons (Brovkina et al ., 2021; Dalton et al ., 2013; Goldman and Arbeitman, 2007; Neville et al ., 2014; Newell et al ., 2016; Palmateer et al ., 2021; Vernes, 2014). Additionally, our analysis of adult dpr / DIP -expressing neurons that also express fru P1 ( DIP ∩ fru P1 ), showed that DIP-alpha ∩ fru P1 and DIP-delta ∩ fru P1 neurons have region-specific dimorphism in CNS neuron number and projection pattern (Brovero et al, 2021).…”

“…Work from our lab and others have examined gene expression in fru P1 neurons, Fru M regulated expression, Fru M binding, chromatin modifications in fru P1 neurons, and open chromatin regions in fru P1 neurons (Brovkina et al, 2021; Dalton et al, 2013; Goldman and Arbeitman, 2007; Neville et al, 2014; Newell et al, 2016; Palmateer et al, 2021; Vernes, 2014). Among these studies, only two have evaluated the mid-pupal stage (Neville et al ., 2014; Palmateer et al ., 2021). However, these studies used bulk sequencing approaches, thereby lacking the resolution to examine differences in gene expression across individual neurons.…”

An alternative D. melanogaster 7SK snRNP

“…Visualization of reads density and the genomic environment is carried out with the JBROWSE genome browser [ 65 ]. RNA-Seq datasets (SRA reference PRJNA644503; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA644503 ) from wild type pupae are from [ 66 ] and PRO-seq data (GEO reference GSE131160; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131160 ) are from Ueberschar et al [ 37 ].…”