Sparse CBX2 nucleates many Polycomb proteins to promote facultative heterochromatinization of Polycomb target genes.

Facultative heterochromatinization of genomic regulators by Polycomb repressive complex (PRC) 1 and 2 is essential in development and differentiation; however, the underlying molecular mechanisms remain obscure. Using genetic engineering, molecular approaches, and live-cell single-molecule imaging, we quantify the number of proteins within condensates formed through liquid-liquid phase separation (LLPS) and find that in mouse embryonic stem cells (mESCs), approximately 3 CBX2 proteins nucleate many PRC1 and PRC2 subunits to form one non-stoichiometric condensate. We demonstrate that sparse CBX2 prevents Polycomb proteins from migrating to constitutive heterochromatin, demarcates the spatial boundaries of facultative heterochromatin, controls the deposition of H3K27me3, regulates transcription, and impacts cellular differentiation.

Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation.

Polycomb repressive complex 1 (PRC1) undergoes phase separation to form Polycomb condensates that are multi-component hubs for silencing Polycomb target genes. In this study, we demonstrate that formation and regulation of PRC1 condensates are consistent with the scaffold-client model, where the Chromobox 2 (CBX2) protein behaves as the scaffold while the other PRC1 proteins are clients. Such clients induce a re-entrant phase transition of CBX2 condensates.

Quantifying the Binding and Target-Search Kinetics of Transcriptional Regulatory Factors by Live-Cell Single-Molecule Tracking.

Eukaryotic transcriptional regulatory factors, such as transcription factors and epigenetic regulatory factors, must locate, bind, and assemble at specific genomic regions to execute functions within the complex and crowded environment of the nucleus. These dynamic processes are typically at nonequilibrium, so quantifying their binding and target-search processes within the native environment is essential for understanding transcriptional mechanisms. Live-cell single-molecule tracking (SMT) is an emerging technique that can be utilized to observe molecular trajectories of individual transcriptional regulatory complexes within the nucleus.

Nuclear condensates of p300 formed though the structured catalytic core can act as a storage pool of p300 with reduced HAT activity.

The transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core components, including the histone acetyltransferase (HAT) domain, the autoinhibition loop, and bromodomain.

Single-molecule imaging of epigenetic complexes in living cells: insights from studies on Polycomb group proteins.

Chromatin-associated factors must locate, bind to, and assemble on specific chromatin regions to execute chromatin-templated functions. These dynamic processes are essential for understanding how chromatin achieves regulation, but direct quantification in living mammalian cells remains challenging. Over the last few years, live-cell single-molecule tracking (SMT) has emerged as a new way to observe trajectories of individual chromatin-associated factors in living mammalian cells, providing new perspectives on chromatin-templated activities.