Naama Barkai

Transcription factors (TFs) regulate gene expression by binding to cis -regulatory regions that contain their specific binding motifs. In budding yeast, intrinsically disordered regions (IDRs) have emerged as key effectors of genome preferences; however, whether this principle holds in the complex mammalian genomes remains unclear. Here, we profile the binding of three nuclear steroid receptors (SRs) containing long disordered N-terminal domains (NTDs), confirming that the NTDs direct SR binding across genomes. Motif enrichment, coupled with footprint analysis, reveals that SR mutants lacking their NTDs favor binding in proximity to the AP-1 motif, whereas the NTD permits binding at the canonical SR motif also when distant from AP-1 sites. Furthermore, profiling SRs and their mutants in budding yeast confirms that the NTD can direct genome targeting also in the absence of its mammalian-specific interactors. Our results provide new insights into the specificity of SR target selection, opening new avenues for the potential modulation of their activities.

Transcription factors (TFs) bind DNA through sequence-specific DNA-binding domains (DBDs), yet genome-wide analyses show that TFs occupy only a small fraction of their motif occurrences. This raises the question of how TFs distinguish specific targets from the many potential sites in the genome. To investigate determinants of binding specificity beyond the cognate motif and cofactor influences, we measured the binding of 60 human TFs across the budding yeast genome. Although human TFs robustly recognized their motifs, they displayed strong selectivity in site occupancy. Nucleosome abundance explained this selectivity only in part: among the 5-20% of motif sites that were bound, a substantial fraction remained nucleosome covered. Furthermore, TFs recognizing similar motif sequences independently localized to distinct subsets of sites within different promoters. Despite the absence of human-specific cofactors in yeast, both binding stability and genomic preferences depended on largely disordered non-DBD regions. These findings suggest intrinsically disordered regions (IDRs) may therefore direct genome binding TF target recognition across evolutionarily distant genomes. Graphical

Transcription factors (TFs) are proteins crucial for regulating gene expression. Effective regulation requires the TFs to rapidly bind to their correct target, enabling the cell to respond efficiently to stimuli such as nutrient availability or the presence of toxins. However, the search process is hindered by slow diffusive movement and the presence of ‘false’ targets – DNA segments that are similar to the true target. In eukaryotic cells, most TFs contain an intrinsically disordered region (IDR), which is commonly assumed to behave as a long, flexible polymeric tail composed of hundreds of amino acids. Recent experimental findings indicate that the IDR of certain TFs plays a pivotal role in the search process. However, the principles underlying the IDR’s role remain unclear. Here, we reveal key design principles of the IDR related to TF binding affinity and search time. Our results demonstrate that the IDR significantly enhances both of these aspects. Furthermore, our model shows good agreement with experimental results, and we propose further experiments to validate the model’s predictions.

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.

Transcription factors (TFs) contribute to organismal development and function by regulating gene expression. Despite decades of research, the factors determining the specificity and speed at which eukaryotic TFs detect their target binding sites remain poorly understood. Recent studies have pointed to intrinsically disordered regions (IDRs) within TFs as key regulators of the process by which TFs find their target sites on DNA (the TF target search). However, IDRs are challenging to study because they can confer specificity despite low sequence complexity and can be functionally conserved despite rapid sequence divergence. Nevertheless, emerging computational and experimental approaches are beginning to elucidate the sequence–function relationship within the IDRs of TFs. Additional insights are informing potential mechanisms underlying the IDR-directed search for the DNA targets of TFs, including incorporation into biomolecular condensates, facilitating TF co-localization, and the hypothesis that IDRs recognize and directly interact with specific genomic regions.

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.

RNA-binding proteins (RBPs) are composed of RNA-binding domains (RBDs) often linked via intrinsically disordered regions (IDRs). Structural and biochemical analyses have shown that disordered linkers contribute to RNA binding by orienting the adjacent RBDs and also characterized certain disordered repeats that directly contact the RNA. However, the relative contribution of IDRs and predicted RBDs to the in vivo binding pattern is poorly explored. Here, we upscaled the RNA-tagging method to map the transcriptome-wide binding of 16 RBPs in budding yeast. We then performed extensive sequence mutations to distinguish binding determinants within predicted RBDs and the surrounding IDRs in eight of these. The majority of the predicted RBDs tested were not individually essential for mRNA binding. However, multiple IDRs that lacked predicted RNA-binding potential appeared essential for binding affinity or specificity. Our results provide new insights into the function of poorly studied RBPs and emphasize the complex and distributed encoding of RBP–RNA interaction in vivo.

Activation domains (ADs) within transcription factors (TFs) induce gene expression by recruiting coactivators such as the Mediator complex. Coactivators lack DNA binding domains (DBDs) and are assumed to passively follow their recruiting TFs. This is supported by direct AD-coactivator interactions seen in vitro but has not yet been tested in living cells. To examine that, we targeted two Med15-recruiting ADs to a range of budding yeast promoters through fusion with different DBDs. The DBD-AD fusions localized to hundreds of genomic sites but recruited Med15 and induced transcription in only a subset of bound promoters, characterized by a fuzzy-nucleosome architecture. Direct DBD-Med15 fusions shifted DBD localization towards fuzzy-nucleosome promoters, including promoters devoid of the endogenous Mediator. We propose that Med15, and perhaps other coactivators, possess inherent promoter preference and thus actively contribute to the selection of TF-induced genes.