The central dogma of biology, the genetic information flowing from DNA to RNA to protein, involves numerous processes such as transcription, RNA processing, translation and protein activity control, all of which can be regulated. The first and the most important regulatory step is the regulation of transcription, performed mainly by DNA binding transcription factors (TFs). Complex, multilayer regulation system of TFs includes not only direct binding of TFs in target gene's binding sites, but also the complicated network of interactions between TFs and TF binding proteins. These include interactions with cofactors and other proteins in mediator complex, interactions with TF modulating proteins such as phosphatases and kinases and interactions with dimerization partners, subunits and inhibitory proteins.
As the TFs act in the context of large protein complexes formed by protein-protein interactions, an optimized workflow is required for efficient research. Affinity purification coupled with mass spectrometry (AP-MS) is the most widely used methodology to analyze protein complexes. We will use a novel high-throughput AP-MS that allows rapid generation of transgenic epitope tagged cell lines based on Gateway cloning and Flp-mediated cell transfection followed by fast pull-down purification and efficient mass spec-analysis.
Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy.
J Allergy Clin Immunol. 2019 Nov;144(5):1364-1376. DOI