Controlling for gene expression changes in transcription factor protein networks.

Area of Science:

• Molecular Biology
• Proteomics
• Genomics

Background:

• Affinity purification coupled with mass spectrometry (AP-MS) identifies protein-protein interactions and complex composition.
• Systematic analysis of transcription factor interactions using AP-MS is lacking.
• Potential for gene expression perturbation from ectopic transcription factor expression to cause false positives in AP-MS.

Purpose of the Study:

• To assess if ectopic expression of an affinity-tagged transcription factor perturbs gene expression, leading to false positive protein identifications in AP-MS.
• To validate the sufficiency of typical experimental controls for transcription factor AP-MS studies.
• To establish a framework for analyzing transcription factor protein interactions.

Main Methods:

• Affinity purification mass spectrometry (AP-MS) using an affinity-tagged transcription factor (RelA) as bait.
• Quantitative proteomics to analyze protein abundance changes.
• RNA sequencing to assess gene expression perturbations.
• Biochemical approaches to investigate novel interactions.

Main Results:

• Overexpression of transcription factor RelA did not cause non-specific co-purification of proteins due to gene expression changes.
• Standard experimental controls are sufficient for accurate identification of bait-associated proteins.
• Known RelA interactors (e.g., NFκB family members) were enriched.
• A novel interaction between RelA and the mitochondrial chaperone Tim13 was identified.

Conclusions:

• Ectopic transcription factor expression does not inherently lead to false positive protein identifications in AP-MS when using typical controls.
• Standard controls are adequate for comparing multiple baits in large-scale AP-MS studies.
• The study provides a validated framework for analyzing transcription factor interactomes.