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. Furthermore, we show that LLPS of CBX2 is required for the demarcation and deposition of H3K27me3 and is essential for cellular differentiation. Our findings uncover new functional roles of LLPS in the formation of facultative heterochromatin and unravel a new mechanism by which low-abundant proteins nucleate many other proteins to form compartments that enable them to execute their functions.

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. The composition of the multi-component PRC1 condensates (1) determines the dynamic properties of the scaffold protein; (2) selectively promotes the formation of CBX4-PRC1 condensates while dissolving condensates of CBX6-, CBX7-, and CBX8-PRC1; and (3) controls the enrichment of CBX4-, CBX7-, and CBX8-PRC1 in CBX2-PRC1 condensates and the exclusion of CBX6-PRC1 from CBX2-PRC1 condensates. Our findings uncover how multi-component PRC1 condensates are assembled via an intricate scaffold-client mechanism whereby the properties of the PRC1 condensates are sensitively regulated by its composition and stoichiometry.

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. Here, we describe the use of live-cell SMT to observe trajectories of individual transcriptional regulatory complexes. We delineate the imaging analysis to obtain chromatin-bound fraction and residence time. Finally, we elaborate on the kinetic modeling to estimate target-search parameters. These binding and target-search parameters facilitate the understanding of how transcription is spatially and temporally regulated under physiological and pathological conditions.

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. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased due to occlusion of the active site in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.

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. Here, we discuss the relative merits of live-cell SMT techniques currently in use. We provide new insights into how Polycomb group (PcG) proteins, master regulators of development and cell differentiation, decipher genetic and epigenetic information to achieve binding stability and highlight that Polycomb condensates facilitate target-search efficiency. We provide perspectives on liquid-liquid phase separation in organizing Polycomb targets. We suggest that epigenetic complexes integrate genetic and epigenetic information for target binding and localization and achieve target-search efficiency through nuclear organization.