Characterization of transcription factor response kinetics in parallel

Betul Bilgin, Aritro Nath, Christina Chan, S. Patrick Walton

Transcription factors (TFs) are effectors of cell signaling pathways that regulate gene expression. TF networks are highly interconnected; one signal can lead to changes in many TF levels, and one TF level can be changed by many different signals. TF regulation is central to normal cell function, with altered TF function being implicated in many disease conditions. Thus, measuring TF levels in parallel, and over time, is crucial for understanding the impact of stimuli on regulatory networks and on diseases. Here, we report the parallel analysis of temporal TF level changes due to multiple stimuli in distinct cell types. We have analyzed short-term dynamic changes in the levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), signal transducer and activator of transcription 3 (Stat3), cAMP response element-binding protein (CREB), glucocorticoid receptor (GR), and TATA binding protein (TBP), in breast and liver cancer cells after tumor necrosis factor-alpha (TNF-α) and palmitic acid (PA) exposure. In response to both stimuli, NF-kB and CREB levels were increased, Stat3 decreased, and TBP was constant. GR levels were unchanged in response to TNF-α stimulation and increased in response to PA treatment. Our results show significant overlap in signaling initiated by TNF-α and by PA, with the exception that the events leading to PA-mediated cytotoxicity likely also include induction of GR signaling. These results further illuminate the dynamics of TF responses to cytokine and fatty acid exposure, while concomitantly demonstrating the utility of parallel TF measurement approaches in the analysis of biological phenomena.