In multicellular organisms, the same genetic code gives rise to a large diversity of cell types. A fully stretched out version of this code measures in the size range of meters, which needs to be compacted into a spheroid nucleus with a diameter of ∼10 micrometers for each cell. Considerable efforts have been made to understand how this code is organized within the nuclear space to enable chromosome segregation during cell division and the proper regulation of each gene throughout developmental stages [1]. Some of the organizational principles have been well-established, beginning with the nucleosome, where a complex of four histone pairs are wrapped around by a short segment (∼147 base pairs) of genomic DNA [2]. Nucleosomes assemble to further compact DNA into heterogeneous groups [3], such as nucleosome clutches with sizes between 30 and 100 nm [4], followed by chromatin nanodomains with sizes around 200–300 nm 5, 6. These nucleosome groups further assemble into larger domains of higher genomic contact frequency known as topologically associated domains [7], A and B compartments [8], and even chromosome-specific territories [9]. These organizational domains are thought to arise from multiple mechanisms. One such mechanism is the phase separation of chromatin regions, such as with nuclear speckles and heterochromatin 10, 11, or phase separation of chromatin into liquid-like condensed domains of active chromatin 12, 13. Loop extrusion 14, 15, as commonly caused by the interplay of CCCTC-binding factor (CTCF) and cohesin, has also been shown to play a key role in genome organization 7, 16. Finally, changes in physical association conferred by posttranslational modifications (PTMs) on histones or DNA and the reader proteins that bind to these PTMs further help organize chromatin [17]. Each of these mechanisms have been shown to play a role in either repressing or facilitating gene expression. However, to date, these mechanisms have been studied, for the most part, as separate pieces of the same puzzle. As with any puzzle, each piece alone does not provide the full picture, and how each piece fits together to collectively regulate the activation and repression of different genes is an open question (Figure 1). Observing all these mechanisms in action simultaneously comes with a set of unique challenges compounded by the number of targeted features. It is therefore imperative to both advance and develop novel multimodal methods for visualizing the chromatin and epigenetic features surrounding a gene before, during, and after transcription to delineate more clearly the mechanisms of gene regulation.

The need to visualize the interplay between chromatin features has been highlighted before [18], underscoring the complexity of gene-regulatory systems where no single mechanism can fully explain a gene’s transcriptional state. For example, multimodal imaging of DNA structure and RNA transcripts has revealed that 3D chromatin structure changes in the context of gene transcription during development 19, 20. The presence of histone PTMs is also implicated in changes of chromatin accessibility [21] and transcriptional state [22]. These same histone marks are also capable of phase- separating into condensates, a potential mechanism for epigenetic organization within nuclei [23]. Polymerase II (PolII) also forms condensates and gains PTMs implicated in different stages of transcription [24]. However, the cause-and-effect relationship between histone marks and transcription is debated [25], and histone marks associated with silenced chromatin can also be found in active regions and vice versa [26]. The principles and relevance of phase separation in chromatin regulation are both novel and controversial [27]. In other contexts, chromatin loop formation has been shown to guide transcription [28], be influenced by transcription 29, 30••, insulate genes into different topological domains 7, 31, or not impact transcription when disrupted [32], making the cause-and-effect relationship between genome folding, enhancer–promoter proximity, and transcription unclear. A resolution to these contradictions likely requires a more comprehensive view of gene architecture, dynamics, PTMs, and gene activation.

In this review, we aim to highlight recent advancements in imaging methods that enable the co-visualization of structural and epigenetic factors that drive gene activation. These advancements include live-cell microscopy approaches, fine structure super-resolution imaging techniques, and in situ labeling of genomic regions in the context of transcriptional features. Last, we suggest how these technological advancements could be combined to provide a comprehensive view of active gene architecture.