Distinct chromatin states partition the genome to locally enrich associated activities for transcription, DNA replication, repair and recombination in a cell-type-specific manner. Revealing the underlying structure-function relationships is challenging due to the complex hierarchical organization of the nucleosome chain that ranges from clusters of a few nucleosomes to chromosome territories with largely different dynamic properties [1]. Confined translocations of individual nucleosomes with a mass of 240 kDa for a nucleosome core particle occur on the nanometer and second time scale [2••]. In contrast, micrometer-scale translocations of chromatin loci to form CTCF-mediated loops take place on the 10 min to hour scale [3,4]. Translocations of whole chromosomes during interphase are hardly detectable even on the hour scale with human chromosome 1 comprising 0.25 Gb DNA and 1.2 million nucleosomes with a total mass of 520 GDa or 0.87 pg [5]. Thus, the assignment of chromatin material properties and the resulting implications for the enrichment of factors and/or access to the genome will be dependent on the length and time scales studied. Accordingly, the generalized description of chromatin as “liquid” and “solid” is fraught with difficulties as apparent from the ongoing discussions in the field [2••,6, 7, 8, 9]. Likewise, identifying subcompartment properties that inform about the contribution of liquid–liquid phase separation (LLPS) for chromatin organization and to distinguish it from alternative mechanisms is critically dependent on the time and length scales studied as discussed previously [10].

Here we review recent approaches to measure features of chromatin subcompartments (CSCs) that have dimensions on the 100 nm to μm scale and involve the assembly of protein/RNA macromolecules around certain chromatin loci. They include for example the nucleolus, constitutive heterochromatin domains, clusters of active RNA polymerase II or complexes of PML bodies at telomeres [10, 11, 12] (Figure 1). Measuring the material properties or other features of these CSCs in the endogenous environment of the cell nucleus is challenging but a crucial step to infer structure-function relationships. We discuss recent advancements in the approaches to dissect organizational principles of CSCs and their associated functional implications with respect to the local enrichment of protein and RNA factors as well as regulating access to the genome.