Automated protein-DNA interaction screening of Drosophila regulatory elements.

Drosophila melanogaster has one of the best characterized metazoan genomes in terms of functionally annotated regulatory elements. To explore how these elements contribute to gene regulation, we need convenient tools to identify the proteins that bind to them. Here we describe the development and validation of a high-throughput yeast one-hybrid platform, which enables screening of DNA elements versus an array of full-length, sequence-verified clones containing over 85% of predicted Drosophila transcription factors. Using six well-characterized regulatory elements, we identified 33 transcription factor-DNA interactions of which 27 were previously unidentified. To simultaneously validate these interactions and locate the binding sites of involved transcription factors, we implemented a powerful microfluidics-based approach that enabled us to retrieve DNA-occupancy data for each transcription factor throughout the respective target DNA elements. Finally, we biologically validated several interactions and identified two new regulators of sine oculis gene expression and hence eye development.

A yeast one-hybrid and microfluidics-based pipeline to map mammalian gene regulatory networks.

The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein-DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF-DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers.

A Comprehensive Drosophila melanogaster Transcription Factor Interactome.

Combinatorial interactions among transcription factors (TFs) play essential roles in generating gene expression specificity and diversity in metazoans. Using yeast 2-hybrid (Y2H) assays on nearly all sequence-specific Drosophila TFs, we identified 1,983 protein-protein interactions (PPIs), more than doubling the number of currently known PPIs among Drosophila TFs. For quality assessment, we validated a subset of our interactions using MITOMI and bimolecular fluorescence complementation assays. We combined our interactome with prior PPI data to generate an integrated Drosophila TF-TF binary interaction network. Our analysis of ChIP-seq data, integrating PPI and gene expression information, uncovered different modes by which interacting TFs are recruited to DNA. We further demonstrate the utility of our Drosophila interactome in shedding light on human TF-TF interactions. This study reveals how TFs interact to bind regulatory elements in vivo and serves as a resource of Drosophila TF-TF binary PPIs for understanding tissue-specific gene regulation.

PDF Signaling Is an Integral Part of the Drosophila Circadian Molecular Oscillator.

Circadian clocks generate 24-hr rhythms in physiology and behavior. Despite numerous studies, it is still uncertain how circadian rhythms emerge from their molecular and neural constituents. Here, we demonstrate a tight connection between the molecular and neuronal circadian networks. Using fluorescent transcriptional reporters in a Drosophila ex vivo brain culture system, we identified a reciprocal negative regulation between the master circadian regulator CLK and expression of pdf, the main circadian neuropeptide. We show that PDF feedback is required for maintaining normal oscillation pattern in CLK-driven transcription. Interestingly, we found that CLK and neuronal firing suppresses pdf transcription, likely through a common pathway involving the transcription factors DHR38 and SR, establishing a direct link between electric activity and the circadian system. In sum, our work provides evidence for the existence of an uncharacterized CLK-PDF feedback loop that tightly wraps together the molecular oscillator with the circadian neuronal network in Drosophila.

A high-throughput gateway-compatible yeast one-hybrid screen to detect protein-DNA interactions.

In recent years, new techniques have spurred the discovery of cis-regulatory DNA elements. These stretches of noncoding DNA contain combinations of recognition sites to which transcription factors (TFs) bind, and in doing so, these TFs can activate or repress transcription. These protein-DNA interactions form the core of gene regulatory networks (GRNs) that are responsible for the differential gene expression that allow diversification of cell types, developmental programs, and responses to the environment. The yeast one-hybrid system is a genetic assay to identify direct binding of proteins to DNA elements of interest and is, therefore, instrumental in uncovering these GRNs.