A cis-regulatory element promoting increased transcription at low temperature in cultured ectothermic Drosophila cells.

BACKGROUND: Temperature change affects the myriad of concurrent cellular processes in a non-uniform, disruptive manner. While endothermic organisms minimize the challenge of ambient temperature variation by keeping the core body temperature constant, cells of many ectothermic species maintain homeostatic function within a considerable temperature range. The cellular mechanisms enabling temperature acclimation in ectotherms are still poorly understood. At the transcriptional level, the heat shock response has been analyzed extensively. The opposite, the response to sub-optimal temperature, has received lesser attention in particular in animal species. The tissue specificity of transcriptional responses to cool temperature has not been addressed and it is not clear whether a prominent general response occurs. Cis-regulatory elements (CREs), which mediate increased transcription at cool temperature, and responsible transcription factors are largely unknown. RESULTS: The ectotherm Drosophila melanogaster with a presumed temperature optimum around 25 °C was used for transcriptomic analyses of effects of temperatures at the lower end of the readily tolerated range (14-29 °C). Comparative analyses with adult flies and cell culture lines indicated a striking degree of cell-type specificity in the transcriptional response to cool. To identify potential cis-regulatory elements (CREs) for transcriptional upregulation at cool temperature, we analyzed temperature effects on DNA accessibility in chromatin of S2R+ cells. Candidate cis-regulatory elements (CREs) were evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. This CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D. CONCLUSION: Beyond a rich data resource for future analyses of transcriptional control within the readily tolerated range of an ectothermic animal, a novel reporter assay permitting quantitative characterization of CRE temperature dependence was developed. Our identification and functional dissection of the pst_E1 enhancer demonstrate the utility of resources and assay. The functional characterization of this CoolUp enhancer provides initial mechanistic insights into transcriptional upregulation induced by a shift to temperatures at the lower end of the readily tolerated range.

Variable Dose Analysis: A Novel High-throughput RNAi Screening Method for Drosophila Cells.

Genetic screens are a powerful approach to identify previously uncharacterized genes involved in specific biological processes. Several technologies have been developed for high-throughput screens using reagents such as RNAi or CRISPR, and each approach is associated with specific advantages and disadvantages. Variable Dose Analysis (VDA), is an RNAi-based method developed in Drosophila cells that improves signal-to-noise ratio compared to previous methods. VDA assays are performed by co-transfecting cells with a plasmid expressing shRNA, (a type of RNAi that can be easily expressed from a DNA plasmid) against a gene of interest and a second plasmid expressing a fluorescent reporter protein. Fluorescent protein expression, can be used as an indirect readout of shRNA expression and therefore target gene knockdown efficiency. Using this approach, we can measure phenotypes over a range of knockdown efficiencies in a single sample. When applied to genetic interaction screens, VDA results in improved consistency between screens and reliable detection of known interactions. Furthermore, because phenotypes are analyzed over a range of target gene knockdown efficiencies, VDA allows the detection of phenotypes and genetic interactions involving essential genes at sub-lethal knockdown efficiency. This therefore represents a powerful approach to high-throughput screening applicable to a wide range of biological questions.

Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells.

A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015).