High-throughput assessment of multi-protein complexes demonstrates rules governing transcription regulation
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Abstract
Regulation of the spatiotemporal expression of genes is vital for the survival of cells in dynamic environments and for the existence of multicellular organisms comprising multiple distinct cell types. Thus, there is a need for high-throughput approaches to characterize the gene regulatory mechanisms that define distinct cell types and states. Gene expression changes are primarily coordinated by transcription factors (TFs) that bind throughout the genome and recruit cofactors (COFs) to establish and maintain the epigenome. These TF–COF interactions can be cell type-specific, can change in response to signals, and are dysregulated in many diseases. To investigate how TF–COF interactions control transcriptional and epigenetic differences across cell types and states, we developed several protein-binding microarray (PBM)-based approaches to characterize TF–COF complexes active in cell extracts.We developed CoRec (Cofactor Recruitment) as a high-throughput method for characterizing TF–COF complexes and demonstrated that it can be used to profile diverse TF–COF complexes across different cell types. To examine TF–COF interactions central to histone acetylation and gene expression, we profiled seven lysine acetyltransferases (KATs) in resting and activated Jurkat T cells. We demonstrated that TF–COF networks are highly dynamic, with 35% of interactions identified in resting T cells altered after 45 minutes of T-cell receptor (TCR) stimulation. Additionally, we found that heterotypic clusters of TF binding sites that recruit the KATs P300 and CBP are associated with increased promoter H3K27ac levels.
To examine how TFs work together in the recruitment of COFs, we developed the Cooperative Cofactor Recruitment (CCR) assay to profile synergistic recruitment of COFs by pairs of TF binding sites (TFBSs). We applied the CCR assay to four KATs in resting Jurkat T cells and found that 45% of profiled TFBS pairs exhibit cooperative KAT recruitment, but only 1% of these configurations cooperatively recruit more than one KAT. We also demonstrated that cooperative recruitment of the KAT P300 is associated with increased reporter gene expression, suggesting that combinatorial TF logic may play an important role in regulating gene expression.
Finally, we used the CoRec method to investigate the concept of indirect TF recruitment where a TF is recruited to DNA by another DNA-bound TF, much like a COF is recruited to DNA. We examined indirect recruitment of 12 TFs in K562 cells that we predicted might operate by both direct DNA binding and indirect recruitment. We found numerous examples of indirect TF recruitment, including several previously unreported interactions. We also determined that the presence of indirect recruitment sites in addition to a TF’s own binding site is predictive of increased TF occupancy at genomic loci. Overall, this work describes new methodological approaches to examine TF–COF and TF–TF interactions and lead to insights into the mechanisms of gene regulation, emphasizing the dynamic nature of TF–COF interactions and highlighting the complexity of combinatorial TF logic.
Description
2026
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Attribution 4.0 International