Insights to transcriptional networks by using high throughput RNAi strategies

Abstract

RNA interference (RNAi) is a powerful method to unravel the role of a given gene in eukaryotic cells. The development of high throughput assay platforms such as fluorescence plate readers and high throughput microscopy has allowed the design of genome wide RNAi screens to systemically discern members of regulatory networks around various cellular processes. Here we summarize the different strategies employed in RNAi screens to reveal regulators of transcriptional networks. We focus our discussion in experimental approaches designed to uncover regulatory interactions modulating transcription factor activity.

Figure 1:

The use of one promoter-reporter construct does not faithfully represent the activity of a given signaling pathway. (A) Transcription from a reporter promoter can reflect the integration of signals from several unknown signaling pathways. Hence, a proportion of the total reporter protein concentration is derived from unknown signal activities, which may lead to the identification of positive hits that are not related to the pathway/transcription factor under investigation. (B) A signaling pathway may employ several different effectors, i.e. transcription factors, with different target promoters (genes X, Y and reporter). Hence, the reporter protein might only partially reflect the signal activity, which may result in the accumulation of false negative hits.

Figure 2:

Localization as a tool to predict transcription factor activity. (A) The subcellular localization of a transcription factor is usually correlated with its post-translational modification state (i.e. phosphorylation, asterisk) priming the protein to chaperone binding and nuclear exclusion. High throughput microscope images can be used to measure GFP tagged or immunostained transcription factor subcellular localization (images below). (B) TF phosphorylation can also be detected as a band shift from SDS–PAGE gels with a specific antibody. Here, the band shift of FOXO protein as a result of insulin signaling activation is shown.