Paired yeast one-hybrid assays to detect DNA-binding cooperativity and antagonism across transcription factors
Date
2024
DOI
Authors
Version
Embargo Date
2027-03-11,2027-03-11
OA Version
Citation
Abstract
Transcription factors (TFs) are proteins that bind DNA in a sequence-specific manner to regulate target gene expression. TF-DNA binding can be positively or negatively regulated by other TFs in the nucleus; specifically, pairs of TFs can cooperatively bind DNA in either a mutual/simultaneous or stepwise manner, or can interact antagonistically by competing for binding sites or sequestering one another to prevent DNA binding. Methods to detect co-binding of TF-pairs to specific DNA regions are limited, and thus far cooperative and antagonistic relationships between TFs have mostly been detected using low throughput approaches. Here, paired yeast one-hybrid (pY1H) assays were developed to detect DNA-binding cooperativity and antagonism between hundreds of TF-pairs at DNA regions of interest in a high throughput manner. Based on enhanced yeast one-hybrid (eY1H) assays, this reporter-based approach can identify interactions between hundreds of human TFs or TF-pairs and human gene promoters in yeast. It was observed that cooperative and antagonistic relationships between TFs are widespread across and between TF families, are DNA region-specific, and can be affected by alternative TF isoform usage. pY1H assays were used to reveal a broad network of cooperative and antagonistic interactions at the promoters of cytokine genes, which encode tightly regulated immune signaling proteins with diverse functions. These interactions dramatically expand the cytokine gene regulatory network, and are often facilitated or antagonized by multiple TF-partners with diverse transcriptional activities and expression profiles. Cytokine genes were found to be widely targeted by TFs that can be modulated at both the expression and activity levels and that are largely resistant to antagonism by TF partners. Furthermore, clusters of potentially coregulated cytokines and the TFs that preferentially target them were identified. Overall, this study demonstrates the widespread role of cooperativity and antagonism in regulating protein-DNA interactions, provides a tool to study these relationships across a variety of systems, and identifies widespread patterns of cytokine gene targeting by TFs and TF-pairs.
Description
2024