Biology subjects

Manadre, W.

Publications and source records attributed to Manadre, W..

2 recordsLinked to original sources

bioRxiv2025

Protein massively parallel binding assay reveals transcription factor binding determinants

Transcription factors select their genomic binding sites in genomes depending on their DNA binding domain (DBD) but also on regions outside the DBD (nonDBD). However, it remains challenging to define these determinants within nonDBDs and reveal their mechanism of action. Towards this, we introduce here an in-vivo method for parallel analysis of thousands of designed peptides for binding a DNA sequence of interest (Protein Massively Parallel Binding Assay, pMPBA). We apply it to scan the full sequence space of budding yeast TFs and generate a detailed map of DNA localizing determinants. Within the set of predicted DBDs, we reveal a large variation in DNA binding affinities, depending on the family and on different sequence characteristics, including charge. Strong signals were not confined to predicted DBDs but included a considerable fraction of nonDBD peptides, most of which were predicted as intrinsically disordered. pMPBA opens new possibilities for high-throughput analysis of peptide-DNA binding within cells.

genomics↗

bioRxiv2024

Massively Parallel Binding Assay (MPBA) reveals limited transcription factor binding cooperativity, challenging models of specificity

DNA binding domains (DBDs) within transcription factors (TFs) recognize short sequence motifs that are highly abundant in genomes. In vivo, TFs bind only a small subset of motif occurrences, which is often attributed to the cooperative binding of interacting TFs at proximal motifs. However, large-scale testing of this model is still lacking. Here, we describe a novel method allowing parallel measurement of TF binding to thousands of designed sequences within yeast cells and apply it to quantify the binding of dozens of TFs to libraries of regulatory regions containing clusters of binding motifs, systematically mutating all motif combinations. With few exceptions, TF occupancies were well explained by independent binding to individual motifs, with motif cooperation being of only limited effects. Our results challenge the general role of motif combinatorics in directing TF genomic binding and open new avenues for exploring the basis of protein-DNA interactions within cells.

systems biology↗