Background Gene regulation is critical for proper cellular function. Next-generation sequencing technology has revealed the presence of regulatory networks that regulate gene expression and essential cellular functions. Studies investigating the epigenome have begun to uncover the complex mechanisms regulating transcription. Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is quickly becoming the assay of choice for many epigenomic investigations. However, whether intervention-mediated changes in accessible chromatin determined by ATAC-seq can be harnessed to generate intervention-inducible reporter constructs has not been systematically assayed. Results We used the insulin signaling pathway as a model to investigate chromatin regions and gene expression changes using ATAC- and RNA-seq in insulin-treated Drosophila S2 cells. We found correlations between ATAC- and RNA-seq data, especially when stratifying differentially-accessible chromatin regions by annotated feature type. In particular, our data demonstrated a weak but significant correlation between chromatin regions annotated to enhancers (1-2 kb from the transcription start site) and downstream gene expression. We cloned candidate enhancer regions upstream of luciferase and demonstrate insulin-inducibility of several of these reporters. Conclusions Insulin-induced chromatin accessibility determined by ATAC-seq reveals enhancer regions that drive insulin-inducible reporter gene expression.

Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is rapidly becoming the assay of choice to investigate chromatin-mediated gene regulation, largely because of low input requirements, a fast workflow, and the ability to interrogate the entire genome in an untargeted manner. Many studies using ATAC-seq use mammalian or human-derived tissues, and established protocols work well in these systems. However, ATAC-seq is not yet widely used in Drosophila. Vinegar flies present several advantages over mammalian systems that make them an excellent model for ATAC-seq studies, including abundant genetic tools that allow straightforward targeting, transgene expression, and genetic manipulation that are not available in mammalian models. Because current ATAC-seq protocols are not optimized to use flies, we developed an optimized workflow that accounts for several complicating factors present in Drosophila. We examined parameters affecting nuclei isolation, including input size, freezing time, washing, and possible confounds from retinal pigments. Then, we optimized the enzymatic steps of library construction to account for the smaller Drosophila genome size. Finally, we used our optimized protocol to generate ATAC-seq libraries that meet ENCODE quality metrics. Our optimized protocol enables extensive ATAC-seq experiments in Drosophila, thereby leveraging the advantages of this powerful model system to understand chromatin-mediated gene regulation.

The Drosophila brain contains tens of thousands of distinct cell types. Thousands of different transgenic lines reproducibly target specific neuron subsets, yet most still express in several cell types. Furthermore, most lines were developed without a priori knowledge of where the transgenes would be expressed. To aid in the development of cell type-specific tools for neuronal identification and manipulation, we developed an iterative assay for transposase-accessible chromatin (ATAC) approach. Open chromatin regions (OCRs) enriched in neurons, compared to whole bodies, drove transgene expression preferentially in subsets of neurons. A second round of ATAC-seq from these specific neuron subsets revealed additional enriched OCR2s that further restricted transgene expression within the chosen neuron subset. This approach allows for continued refinement of transgene expression, and we used it to identify neurons relevant for sleep behavior. Furthermore, this approach is widely applicable to other cell types and to other organisms.

Gene regulation is critical for proper cellular function. Next-generation sequencing technology has revealed the presence of regulatory networks that regulate gene expression and essential cellular functions. Studies investigating the epigenome have begun to uncover the complex mechanisms regulating transcription. Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is quickly becoming the assay of choice for epigenomic investigations. Integrating epigenomic and transcriptomic data has the potential to reveal the chromatin-mediated mechanisms regulating transcription. However, integrating these two data types remains challenging. We used the insulin signaling pathway as a model to investigate chromatin regions and gene expression changes using ATAC- and RNA-seq in insulin-treated Drosophila S2 cells. We show that insulin causes widespread changes in chromatin accessibility and gene expression. Then, we attempted to integrate ATAC- and RNA-seq data to predict functionally-relevant chromatin regions that control the transcriptional response to insulin. We show that using differential chromatin accessibility can predict functionally-relevant genome regions, but that stratifying differentially-accessible chromatin regions by annotated feature type provides a better prediction of whether a chromatin region regulates gene expression. In particular, our data demonstrate a strong correlation between chromatin regions annotated to distal promoters (1-2 kb from the transcription start site). To test this prediction, we cloned candidate distal promoter regions upstream of luciferase and validated the functional relevance of these chromatin regions. Our data show that distal promoter regions selected by correlations with RNA-seq are more likely to control gene expression. Thus, correlating ATAC- and RNA-seq data can home in on functionally-relevant chromatin regions.

Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is rapidly becoming the assay of choice to investigate chromatin-mediated gene regulation, largely because of low input requirements, a fast workflow, and the ability to interrogate the entire genome in an unbiased manner. Many studies using ATAC-seq use mammalian or human-derived tissues and the established protocols work well in these systems. However, ATAC-seq is not yet widely used in Drosophila model systems. Vinegar flies present several key advantages over mammalian systems that make them an excellent model for ATAC-seq studies. For example, there are abundant genetic tools in flies that are not available in mammalian models that allow straightforward targeting, transgene expression, and genetic manipulation. Because current ATAC-seq protocols are not optimized to use fly tissue as input, we developed an optimized workflow that accounts for several complicating factors present in Drosophila. We examined several parameters of library construction, including input size, freezing time, and washing, which specifically address the fly cuticle and possible confounds from retinal pigments. Then, we further optimized the enzymatic steps of library construction to account for the smaller genome size in flies. Finally, we used our optimized protocol to generate ATAC-seq libraries that displayed metrics indicating excellent library quality. Our optimized protocol will enable extensive ATAC-seq experiments in Drosophila, thereby leveraging the advantages of this powerful model system to understand chromatin-mediated gene regulation.