How cells achieve the complex task of regulating gene expression and switching between transcriptional programs have puzzled researchers for decades. In the last few years, a wealth of evidence showed that many molecules involved in transcription, including diverse transcription factors (TFs), co-regulators, the RNA polymerase 2 and the Mediator complex,1, 2, 3, 4, 5 do not distribute homogeneously within the nucleus but concentrate in discrete foci referred to as transcriptional condensates.

Several authors claimed that a liquid-liquid phase separation process (LLPS) promotes the formation of many of these biomolecular condensates and particularly, of transcriptional condensates in the cell nucleus6, 7. In this framework, TFs and other transcription-related molecules would bind to chromatin regions generating an initial seed that would grow by recruiting different multivalent molecules usually containing intrinsic disordered regions8, 9. Others challenged the LLPS paradigm and proposed alternative models that may explain the formation of these condensates.10, 11, 12, 13 Given the ongoing debate in the field, we will use the term ‘transcriptional condensates’ in this work as an operational definition, without implying any mechanism for their formation.

Initial works suggested that transcriptional condensates stimulate gene expression as they concentrate molecules close to their DNA targets; however, the relationship between transcriptional condensates and gene expression seems to be more complex and far from being understood.14

Pluripotent stem cells can self-renew indefinitely and have the potential to generate all the cell types of an adult organism through specific commitment pathways in response to developmental cues. These cells are an excellent model to study the molecular mechanisms involved in gene regulation, embryo development, for drug screening and constitute an important promise in regenerative medicine.15, 16

Pluripotency is primarily dictated by the core pluripotency TFs OCT4, SOX2 and NANOG, which induce genes promoting self-renewal and repress those involved in differentiation17. Although these TFs collectively regulate numerous genes, their expression profiles differ during differentiation. Specifically, NANOG concentration rapidly decreases during the transition from the ground state to primed pluripotency, while OCT4 and SOX2 levels remain constant in both cultured embryonic stem (ES) cells and in the embryo in this time window.18

The preservation of pluripotency and transitions between states require a precise regulation of both, the absolute concentration of the pluripotency TFs and their relative expression levels19, 20, 21. Relevantly, ES cells exhibit intrinsic cell-to-cell heterogeneities in the levels of SOX2 and OCT4 that could affect cell commitment in contexts that promote differentiation22, 23. In addition, OCT4 and SOX2 form condensates in ES cells24, 25 that reorganize in response to differentiation cues at early stages that precede their downregulation24. In the case of OCT4, the condensates contribute to the reorganization of topologically associated domains (TADs) during reprogramming.25 These observations underscore the relevance of TF́s concentrations and their distributions within the nuclear space to cell fate decisions.

One of the most important limitations that constrains our understanding on transcription regulation and, more specifically, the role of compartmentalization in this process, lies in the difficulty of selectively manipulating the spatial distribution of a transcriptional player without affecting other functions.14

Different strategies have been used to analyze the involvement of condensates in transcription regulation, many of them aimed to perturb the interactions between molecules within condensates by either introducing mutations to proteins or using specific chemical agents. Alternatively, other strategies focused on generating artificial transcriptional condensates by using, for example, optogenetic tools (reviewed in.14

Here, we describe a simple method, serendipitously discovered, to generate light-induced clusters (LICs) nucleating TFs labeled with Janelia Fluor® (JF) probes via HaloTag in live cells. By simply irradiating a region of the nucleus with the appropriate laser line, the user can select the position and size of the LIC. We explored some characteristics of the mechanism of LIC formation and analyzed the interactions stabilizing these structures. We also showed that the pluripotency TFs OCT4 and SOX2 and the glucocorticoid receptor (GR) could concentrate in LICs but, in the case of GR, the process seems to require certain TF conformations and/or cellular contexts. Finally, we show that OCT4 recruitment to LICs affects the concentration of the TF in the nucleoplasm, its subnuclear distribution and the interactions with chromatin. This new method could serve as a valuable tool for exploring how the spatial organization of TFs shapes the landscape of interactions with chromatin and ultimately defines transcription patterns.