Cloning and nextPBM analysis of the mediator and BRG1 associated factor complexes

• Main Author: Buckshaw II, Robert S.
• Other Authors: Siggers, Trevor W
• Language: en_US
• Published: 2020
• Subjects: Systematic biology; BAF; Epigenetic regulation; Immunology; Mediator; PBM; SWI/SNF
• Online Access: https://hdl.handle.net/2144/41179

Coordination of gene expression within the cell requires the integrated actions of various multi-protein, gene regulatory complexes. The Mediator and BRG1 Associate Factor (BAF) complexes are large, dynamic regulatory cofactors (COF) that are made up of multiple different submodules, and play key roles in regulating gene expression. Gene-specific regulation requires that transcription factors (TFs) recruit these COF complexes to gene promoters. How separate subdomains in each complex interact with distinct sets of TFs in each cell remains an important question. In this study, to address this question, we sought to apply the nuclear extract protein-binding microarray (nextPBM) technology being developed in our lab to study interactions between TFs and subunits of the Mediator and BAF complexes. To facilitate this, we cloned, expressed and purified subdomains of proteins from the Mediator and BAF complexes. We then used the nextPBM technology to study the interactions of our subdomains with TFs in human macrophages. We identified several new interactions with TFs, and demonstrate the utility of this approach to student TF-COF interaction.