Limb-specific Shh expression is regulated by the (~1 Mb distant) ZRS enhancer. In the mouse, limb bud restricted spatiotemporal expression of Shh occurs from ~E10-E11.5 at the distal posterior margin and is essential for correct autopod formation. Here, we have analysed the higher-order chromatin conformation of Shh in expressing and non-expressing tissues, both by fluorescence in situ hybridisation (FISH) and by chromosome conformation capture (5C). Conventional and super-resolution light microscopy identified significantly elevated frequencies of Shh/ZRS co-localisation only in the Shh expressing regions of the limb bud.However, Shh-ZRS spatial distances were consistently shorter than intervening distances to a neural enhancer in all tissues and developmental stages analysed. 5C identified a topologically associating domain (TAD) over the Shh/ZRS genomic region and enriched interactions between Shh and ZRS throughout E11.5 embryos. Shh/ZRS co-localisation, therefore, correlates with the spatiotemporal domain of limb bud-specific Shh expression, but close Shh/ZRS proximity in the nucleus occurs regardless of whether the gene or enhancer is active. We suggest that this constrained chromatin configuration optimises the opportunity for the active enhancer to locate and instigate Shh expression.

Polycomb repressive complex 1 (PRC1) strongly influences 3D genome organization, mediating local chromatin compaction and clustering of target loci. Several PRC1 subunits have the capacity to form biomolecular condensates through liquid–liquid phase separation in vitro and when tagged and over-expressed in cells. Here, we use 1,6-hexanediol, which can disrupt liquid-like condensates, to examine the role of endogenous PRC1 biomolecular condensates on local and chromosome-wide clustering of PRC1-bound loci. Using imaging and chromatin immunoprecipitation, we show that PRC1-mediated chromatin compaction and clustering of targeted genomic loci—at different length scales—can be reversibly disrupted by the addition and subsequent removal of 1,6-hexanediol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to reduced PRC1 occupancy detected by chromatin immunoprecipitation following 1,6-hexanediol treatment as the addition of 2,5-hexanediol has similar effects on binding despite this alcohol not perturbing PRC1-mediated 3D clustering, at least at the sub-megabase and megabase scales. These results suggest that weak hydrophobic interactions between PRC1 molecules may have a role in polycomb-mediated genome organization.

Polycomb-repressive complex 1 (PRC1) has a strong influence on 3D genome organization, mediating local chromatin compaction as well as localized and chromosome-wide clustering of target loci. Several subunits of PRC1 have been shown to have the capacity to form biomolecular condensates through liquid-liquid phase separation in vitro and when tagged and over-expressed in cells. Here, we use 1,6-hexandiol, which disrupts liquid-like condensates, to examine the role of endogenous PRC1 biomolecular condensates on local and chromosome-wide clustering of PRC1-bound loci. Using imaging and chromatin immunoprecipitation combined with deep sequencing analyses, we show that PRC1-mediated chromatin compaction and clustering of targeted genomic loci - at megabase and tens of megabase length scales - can be reversibly disrupted by the addition and subsequent removal of 1,6-hexandiol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to the reduction of PRC1 binding following 1,6-hexandiol treatment as the addition of 2,5-hexandiol has similar effects on binding despite this alcohol not perturbing PRC1-mediated 3D clustering, at least at the sub-megabase and megabase scales. These results suggest that weak hydrophobic interactions between PRC1 molecules, characteristic of liquid-liquid phase separation, have a role in polycomb-mediated genome organization.