Illuminating biomolecular assemblies in gene regulation.

The nucleus is a highly compartmentalized organelle and this spatial organization reflects gene-regulatory environments. Chromatin exists in two distinct forms: transcriptionally active, euchromatin and silenced, compacted heterochromatin. The spatial organization of chromatin along with its transcriptional activity is governed by biomolecular assemblies (BAs). Gene regulatory assemblies form and operate through highly dynamic protein-protein and protein-DNA interactions often established via their recruitment by non-coding RNAs. The formation of BAs is essential for retaining diffusible regulatory proteins at specific genomic regions, enabling local confinement and precise gene regulation. Phase separation, particularly in the form of liquid-liquid condensation, is suggested to play a crucial role in transcriptional regulation, serving as a key driver of biomolecular assembly formation. However, some studies indicate that phase separation may also be a non-essential byproduct of the crowded nuclear environment or may not be involved in certain BAs. Despite extensive investigations into these macromolecular crowding phenomena, the precise mechanisms underlying both the formation of gene-regulatory BAs and how these localized protein concentrations function to regulate chromatin structure and gene expression remain unclear. This review highlights progress made in elucidating the mechanisms of chromatin-modifying BAs, highlighting how super-resolution microscopy and single-molecule technologies are proving essential for probing these nuclear structures in situ, within their native cellular context.