The three-dimensional organization of the eukaryotic genome determines cellular function and is sustained by specific interactions between chromatin, proteins, and RNA molecules. These molecular interactions facilitate the assembly of macromolecular membrane-less organelles, also known as biological condensates, and result in the discrete nuclear spatial organization of active and repressive chromatin (euchromatin/heterochromatin, respectively).

Heterochromatin maintains genome integrity and function, and was traditionally considered to be in a stably repressed state. Instead, it is now established that heterochromatin is widely transcribed and that its transcripts scaffold heterochromatin assembly by recruiting chromatin-modifying enzymes and are responsive to developmental or pathological processes. Also, heterochromatin-associated proteins like HP1a exist in a dynamic equilibrium between chromatin-bound and diffuse forms that can be regulated by different biological contexts. It was also recently proposed that the assembly of heterochromatin condensates shows phase-separation properties. Thus, heterochromatin condensates are now considered as dynamic architectural features of the eukaryotic genome that can be modulated by developmental, environmental, and pathological stimuli to regulate genome expression and stability.

In light of this, novel methodologies have been developed to elucidate the biophysical properties that modulate the assembly, regulation, and functions of heterochromatin condensates. This methods collection gathers some of these new methodologies that focus on the molecular and biophysical interactions between heterochromatin, RNA, and heterochromatin-associated proteins to probe heterochromatin condensates.