Past exam of the systems biology course of the University of Cambridge / 2024 / Paper 4 / Section II / 2.3 / Solution

Model and central question. Use Saccharomyces cerevisiae as a tractable eukaryotic system with a well-annotated genome and established DNA repair and checkpoint biology. The aim is to identify the UV-responsive transcriptional programme and separate direct regulation from secondary changes caused by cell-cycle arrest, altered growth or loss of viability. The resulting network is a yeast model; conservation in other eukaryotes remains a hypothesis to test.

Discovery design. Compare UV-exposed and mock-handled cultures at baseline and several recovery stages, using independent biological replicates and balanced processing. Include matched handling controls and record growth, viability and cell cycle state. Choose conditions yielding an interpretable damage response rather than treating terminally damaged cells as a normal regulatory trajectory. Biological replication and randomization must occur at the culture level, not merely by sequencing the same material repeatedly.

Measure transcript abundance using RNA sequencing and assess sample quality before testing differences. Model condition, time, their interaction and recorded batch factors, using an appropriate count-based expression model. Report effect sizes, uncertainty and false discovery rate, not only a list passing a significance cutoff. Where changes in total RNA per cell could invalidate relative normalization, include a suitable external reference strategy and distinguish relative composition from absolute expression.

Candidate regulators and direct targets. Cluster well-supported expression trajectories to nominate coordinated modules, then ask whether their promoters are enriched for known transcription-factor motifs. Combine motif evidence with regulator expression, localization or activity measurements and prior knowledge. Motif occurrence and coexpression alone are insufficient to establish a direct regulatory edge: a factor can be activated without changing its own transcript abundance, and many coexpressed genes share an upstream cause.

For prioritized factors, compare chromatin association before and after damage using chromatin immunoprecipitation with genome-wide readout and appropriate input and specificity controls. Binding near a gene is additional evidence, not proof that the binding event regulates it. A targeted regulator perturbation and rescue comparison should test the predicted expression response while monitoring viability and checkpoint effects. Reporter or promoter-element comparisons can further test a specific cis-regulatory mechanism without assuming that every enriched motif is functional.

Temporal and causal integration. Use timing, perturbation effects and occupancy jointly. A plausible direct target changes after regulator activation, shows relevant occupancy evidence and responds to a selective perturbation. Secondary effects may appear later or depend on cell-cycle redistribution. Where transcript abundance cannot separate synthesis from RNA stability, a focused measurement of newly synthesized RNA can test the proposed transcriptional component.

Build a gene regulatory network whose edges retain sign, condition, confidence and evidence type. Predict responses to a held-out perturbation or recovery condition, and assess those predictions using new biological samples. Release the design, metadata, code and qualified edge list. Success is a validated account of important regulatory modules and their limits, rather than an unsupported claim to have measured every possible interaction in the eukaryotic damage response.