Unstructured transcription factor interactions enable emergent specificity.
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| Title: | Unstructured transcription factor interactions enable emergent specificity. |
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| Authors: | Abidi, Abrar A. (AUTHOR), Cattoglio, Claudia (AUTHOR), Tang, Natalie N. (AUTHOR), Fan, Vinson B. (AUTHOR), Dailey, Gina M. (AUTHOR), Hay, Amir D. (AUTHOR), Kunamaneni, Prasanthi (AUTHOR), Milkie, Daniel E. (AUTHOR), Darzacq, Xavier (AUTHOR), Betzig, Eric (AUTHOR), Tjian, Robert (AUTHOR), Graham, Thomas G. W. (AUTHOR) |
| Source: | Science. 5/28/2026, Vol. 392 Issue 6801, p1-15. 15p. |
| Subjects: | Transcription factors, Chromatin, Cell imaging, Genetic regulation, Protein-protein interactions, Proteins, Protein domains |
| Abstract: | How intrinsically disordered regions (IDRs) shape chromatin binding and nuclear organization of transcription factors (TFs) remains unclear. We used proximity-assisted photoactivation (PAPA), a single-molecule protein-protein interaction sensor, to investigate how IDRs might influence TF interactions with each other and with chromatin in live cells. We found that the Sp1 DNA binding domain (DBD) interacted poorly with chromatin and did not colocalize with Sp1. Weak interaction of the isolated IDR with full-length Sp1 was enhanced by fusion to various unrelated DBDs. Live imaging of Drosophila polytene chromosomes confirmed that an IDR could confer sharp locus specificity on an otherwise nonspecific DBD. These findings suggest that TF specificity emerges on chromatin when ensembles of diverse, unstructured interactions are scaffolded by transient DNA contacts. Editor's summary: Transcription factors (TFs) are proteins that control the expression of genes. Textbooks describe TFs as recognizing specific DNA sequences through structured DNA-binding surfaces. However, these sequences are typically too short to account for the specificity of TF binding across large genomes, creating a longstanding paradox. Using single-molecule interaction measurements in living cells, Abidi et al. showed that regions within TFs that do not fold into stable three-dimensional structures play a central role in resolving this question. The authors propose that TF specificity emerges as a collective property of many weak and transient unstructured interactions among proteins and nucleic acids. —Di Jiang INTRODUCTION: Transcription factors (TFs) bind chromosomes and selectively influence when and where genes are activated. For decades, TF specificity has been explained by a model in which structured DNA binding domains (DBDs) locate genomic targets by recognizing well-defined DNA sequence motifs. Yet mounting evidence has unsettled this picture. Eukaryotic genomes contain vast numbers of potential binding sites, most of which remain unoccupied, and TFs typically recognize short and often degenerate motifs. TFs with identical sequence preferences regulate different genes within the same cell, and many apparently occupied sites lack recognizable motifs altogether. Moreover, genomic binding profiles often correlate poorly with transcriptional output. These findings expose paradoxes that trouble the conventional model of TF specificity—paradoxes that existing assays have not resolved—which leaves a central question unanswered: How do such underspecified TFs selectively regulate genes? The standard picture of TF binding is largely derived from bacterial regulators, which recognize longer and more specific sequences on much smaller genomes. Eukaryotic TFs also differ in that they contain large intrinsically disordered regions (IDRs) that do not fold into distinct three-dimensional structures. Although these regions have been proposed to alter genomic localization, how they do so remains mysterious. We therefore investigated whether IDR interactions among TFs shape their genomic localization patterns under physiological conditions. RATIONALE: Addressing this question requires observing TF interactions in nuclei of living cells, where chromatin binding and protein interactions are transient. Most existing approaches rely on fixation, permeabilization, or artificial multimerization, precluding observation of these interactions under native conditions. We therefore sought a method capable of directly detecting TF colocalization and chromatin association at single-molecule resolution in live cells while preserving physiological expression levels. RESULTS: We developed a live-cell single-molecule imaging strategy combining proximity-assisted photoactivation (PAPA) with high-throughput oblique line-scan (OLS) microscopy to measure TF colocalization on chromatin under native conditions. Whereas the human TF Sp1 extensively colocalized with itself on chromatin, the isolated Sp1 DBD showed severely reduced chromatin binding and failed to colocalize with full-length Sp1, despite identical sequence preferences. Analysis of IDR mutants revealed that aromatic residues are required for colocalization with endogenous Sp1, whereas basic residues flanking the DBD are essential for chromatin binding. Grafting the Sp1 IDR onto unrelated DBDs caused them to colocalize with endogenous Sp1, independent of their intrinsic DNA sequence preferences. A standard sequencing-based assay, cleavage under targets and release using nuclease (CUT&RUN), did not capture these behaviors, instead reporting similar genomic profiles for constructs that differed sharply in live-cell colocalization. This discrepancy suggests that CUT&RUN and other perturbative genomic methods may overrepresent DBD-dominated interactions while poorly capturing the IDR-dependent localization patterns that prevail in live cells. Complementary live-tissue imaging of Drosophila polytene chromosomes demonstrated that fusing an IDR to a nonspecific DBD was sufficient to confer a dynamic, locus-specific enrichment. CONCLUSION: Together, these results indicate that TF specificity in living cells cannot be explained primarily by DNA sequence recognition. Instead, genomic localization patterns emerge from collective interactions mediated by IDRs and stabilized by weak DNA contacts. A framework where specificity arises from ensembles of individually underspecified interactions, rather than from deterministic binding by structured domains, may help address discrepancies between observed TF binding, the genomic distribution of sequence motifs, and transcriptional output. Locus specificity of eukaryotic TFs as an emergent property.: Diverse IDR interactions (right) enable locus-specific collective DNA binding of TFs whose isolated IDR (left) and DBD (center) interactions are both insufficient and underspecified. [ABSTRACT FROM AUTHOR] |
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| Database: | Psychology and Behavioral Sciences Collection |
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