Fig. 2: Strategies to study the role of transcriptional condensates on transcriptional activity.

a Correlative strategies generally involve using fluorescence microscopy to compare the spatial distributions of the condensates of a TF of interest (green) with those of other biomolecules with a known role in transcription, such as coactivators or Pol II (red). The merged images illustrate different degrees of colocalization (yellow): i) ‘total colocalization’, where both biomolecules colocalize in the same condensates, and ii) ‘partial colocalization’, where only a subset of condensates colocalize with the condensates formed by the other biomolecule. b Causal strategies involve performing perturbations of TF structure and analyzing their impact on certain properties in the cells. Top panels: TFs usually present a modular structure with a DNA-binding domain (DBD), and an IDR responsible for transactivation, target searching, and, often, condensate formation. TF structure can be experimentally altered by different approaches, including i) pharmacological targeting (oval), ii) deletions/truncations (scissors), and iii) point-mutations (cross). Analyzing the TF spatial distribution and the TF-driven transcriptional activity after these perturbations allows for exploring the contribution of the specific TF structural features to condensate formation and activity. Bottom panels: Synthetic TFs (green-purple) can be designed by fusing domains of the TF of interest to moieties with known properties. iv) Designed DNA binding: replacing the TF DBD with a well-studied, bacterial TF DBD, or RNA-guided dCas9 (not illustrated), and introducing a specific transcription reporter (cyan) driven by this DBD allow studying how the TF IDR contributes to transcriptional activity at these designed DNA targets. v) Designed nucleation: replacing the TF DBD with a responsive module capable of light- or chemical-controlled oligomerization helps to understand the contribution of the TF IDR to condensate formation. vi) Designed condensation: this strategy includes analyzing the effects of replacing the TF IDR with an exogenous IDR with enhanced condensation propensity, helping to understand the role of condensation on the TF activity at its natural DNA targets. These strategies can also be combined or modified to analyze more complex scenarios, according to the specific system.