Abstract
Modulators (Ms) are proteins that modify the activity of transcription factors (TFs) and influence expression of their target genes (TGs). To discover modulators of NF-κB/RelA, we first identified 365 NF-κB/RelA-binding proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We used a probabilistic model to infer 8349 (M, NF-κB/RelA, TG) triplets and their modes of modulatory action from our combined LC-MS/MS and ChIP-Seq (ChIP followed by next generation sequencing) data, published RelA modulators and TGs, and a compendium of gene expression profiles. Hierarchical clustering of the derived modulatory network revealed functional subnetworks and suggested new pathways modulating RelA transcriptional activity. The modulators with the highest number of TGs and most non-random distribution of action modes (measured by Shannon entropy) are consistent with published reports. Our results provide a repertoire of testable hypotheses for experimental validation. One of the NF-κB/RelA modulators we identified is STAT1. The inferred (STAT1, NF-κB/RelA, TG) triplets were validated by LC-selected reaction monitoring-MS and the results of STAT1 deletion in human fibrosarcoma cells. Overall, we have identified 562 NF-κB/RelA modulators, which are potential drug targets, and clarified mechanisms of achieving NF-κB/RelA multiple functions through modulators. Our approach can be readily applied to other TFs.