Abstract
Genetic screens have identified many novel components of various biological processes, such as components required for cell cycle and cell division. While forward genetic screens typically generate unstructured ‘hit’ lists, genetic interaction mapping approaches can identify functional relations in a systematic fashion. Here, we discuss a recent study by our group demonstrating a two-step approach to first screen for regulators of the mitotic cell cycle, and subsequently guide hypothesis generation by using genetic interaction analysis. The screen used a high-content microscopy assay and automated image analysis to capture defects during mitotic progression and cytokinesis. Genetic interaction networks derived from process-specific features generate a snapshot of functional gene relations in those processes, which follow a temporal order during the cell cycle. This complements a recently published approach, which inferred directional genetic interactions reconstructing hierarchical relationships between genes across different phases during mitotic progression. In conclusion, this strategy leverages unbiased, genome-wide, yet highly sensitive and process-focused functional screening in cells.
Highlights
During cell division, a cell undergoes several consecutive events to replicate and divide its genome and distribute it to two daughter cells
Microscopy techniques illustrate the localization of cellular components [3,4,5] and cell cycle specific factors or the presence of cell cycle markers such as residue-specific phosphorylation of histone H3 [6, 7]
Image analysis can extract multiple phenotypic features from a cell population, which allows for simultaneously following distinct biological processes
Summary
A cell undergoes several consecutive events to replicate and divide its genome and distribute it to two daughter cells. To identify regulators of the cell cycle in a systematic fashion, model systems such as budding yeast, cultured Drosophila or human cells have been exploited in genome-scale functional screens [8,9,10]. An RNAi screen with live imaging of human HeLa cells has exploited a stably expressed GFP-labeled histone H2B to identify genes required for proper chromosome segregation and cell cycle propagation [13]. While such studies have identified cell cycle regulators with high sensitivity, genetic interaction analysis approaches have been able to define functional and epistatic relations between genes [14]. This study characterized novel modulators by genome-wide high-content imaging RNAi screening, and structured the resulting ‘hit’ list using mitotic index- and nuclear area-focused genetic interaction analysis
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