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Review
. 2021 Jul 9;49(12):6621-6637.
doi: 10.1093/nar/gkab304.

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

Kyle Brown  1 , Haralambos Andrianakos  1 , Steven Ingersoll  1 , Xiaojun Ren  1
Affiliations
Review

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

Kyle Brown et al. Nucleic Acids Res. .

Abstract

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.

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Figures

Figure 1.
Figure 1.
Schematic description of the two major PcG complexes. (A) Two major classes of PRC1 complexes: CBX-PRC1 and RYBP-PRC1. The PRC1 complexes assemble around the catalytic core, one copy of the six PCGF1–6 paralogs and one of the E3 ubiquitin ligase RING1A/B. They are divided into canonical PRC1 (CBX-PRC1) or variant PRC1 (RYBP-PRC1). The CBX-PRC1 complexes contain the RING1A/B-PCGF2/4 core, one copy of the CBX2/4/6/7/8 paralogs, and one copy of the PHC1–3 subunits, giving rise to the two canonical PCGF2- and PCGF4-PRC1 complexes, respectively. The RYBP-PRC1 complexes contain RYBP/YAF2 instead of one of the CBX proteins. RYBP and YAF2 are able to recognize the H2AK119Ub1 mark, which promotes RYBP-PRC1 ubiquitin ligase activity. The catalytic core is colored and additional subunits are gray. (B) Two major classes of PRC2 complexes: PRC2.1 and PRC2.2. The minimally catalytic core of PRC2 is comprised of one copy of EZH1/2, EED and SUZ12.The core stoichiometric PRC2 complex consists of the catalytic core and one copy of RBBP4/7. The PRC2 complexes are divided into two major subcomplexes, PRC2.1 and PRC2.2. PRC2.1 contains one copy of PCL paralogs (PCL1/PHF1, PCL2/MTF2 or PCL3/PHF19) and one of two auxiliary proteins, EPOP or PALI1. PRC2.2 contains the JARID2 and AEBP2 auxiliary proteins. These auxiliary proteins within PRC2.1 and PRC2.2 cooperate to deposit H3K27me3 via synergetic and independent mechanisms. The catalytic core is colored and auxiliary proteins are gray.
Figure 2.
Figure 2.
Schematic representation of the quantification of the target-search process. (A) Schematic description of live-cell SMT, including labelling, imaging, and data processing. (B) The ‘fast-tracking’/short interval time stroboscopic SMT experiments, consisting of a short camera exposure time followed by a short camera dark time, typically with a total interval time of 5–30 ms, are performed to extract fractions and diffusion coefficients (F1 (D1), F2 (D2) and F3 (D3)), which represent chromatin-bound, confined, and free diffusion populations of total molecules within cells, respectively. The displacement histogram is fitted through kinetic modelling. A long interval time of 200–1000 ms and low illumination laser intensities (to minimize photobleaching) are carried out to measure residence times (τtb and τsb) as well as stable and transient bound fractions (f1tb and f1sb) of total chromatin-bound molecules. The dwell-time distribution is typically fitted by a two-component decay function. The stable and transient bound fractions (F1tb and F1sb) of total molecules within cells can be described as formula imageand formula image, respectively. formula image is the average number of non-specific interactions by which one molecule undergoes before encountering a specific site ( formula image) and τ3D is the average free time between two binding events ( formula image). The target-search time needed to find a specific site is described as formula image. The relationship among the specific-bound fraction (F1sb), the residence time on specific site (τsb) and the target-search time (τsearch) is characterized as formula image.
Figure 3.
Figure 3.
Binding mechanisms of PRC1 and PRC2. (A) Binding of PRC1 to chromatin. The RYBP and YAF2 subunits of RYBP-PRC1 recognize H2AK119Ub1, which promotes RYBP-PRC1 ubiquitin ligase activity. PCGF1-PRC1, PCGF3/5-PRC1, and PCGF6-PRC1 are stabilized at chromatin through interaction with DNA (BCOR and KDM2B for PCGF1-PRC1, USF1 for PCGF3/5-PRC1, and DP-1, E2F6, MAX and MGA for PCGF6-PRC1). CBX2-PRC1 binds to chromatin mainly through CBX2 interaction with DNA and H3K27me3 makes a minor contribution to the binding (not shown). CBX7- and CBX8-PRC1 co-recognize H3K27me3 and DNA. The molecular mechanisms underlying CBX4- and CBX6-PRC1 binding remain to be characterized. The arrowhead curves indicate that complexes recognize specific features of chromatin. The dashed arrowhead curves indicate that the molecular mechanisms underlying complex stabilization remain unclear. (B) Binding of PRC2 to chromatin. The core PRC2 complex (dashed circle) has intrinsic chromatin-binding activity, mainly through interaction with DNA. The EED subunit of PRC2 recognizes H3K27me3, which promotes PRC2 methyltransferase activity. PRC2.1 is stabilized at chromatin by PCL interaction with CpG-rich DNA. JARID2 and AEBP2 recognize both CpG-rich DNA and H2AK119Ub1, which stabilizes PRC2.2 at chromatin and promotes its activity.
Figure 4.
Figure 4.
LLPS accelerates the target-search kinetics of CBX2. Phase separated Polycomb condensates speed up CBX2 locating its stable sites by reducing the 3D free diffusion time (the major time factor in target-search) and the number of non-specific trials between specific binding events, thereby enhancing its genomic occupancy.
Figure 5.
Figure 5.
A scaffold-adapter-client phase separation model for organizing Polycomb genes in mES cells. Within the model, CBX2-PRC1 is the scaffold, CBX7-PRC1 is the adapter, and H3K27me3-marked chromatin is the client. CBX2-PRC1 phase separates to form condensates, which is driven by CBX2. CBX7-PRC1 recruits H3K27me3-marked regions into CBX2-PRC1 condensates through the interactions between CBX7 and H3K27me3 and PHC polymerization between CBX2-PRC1 and CBX7-PRC1, thereby bringing the client (H3K27me3-marked chromatin) into the scaffold (CBX2-PRC1 condensates). This model is based on observations from mES cells in which CBX4 and CBX8 are not expressed. CBX6 occupies a small fraction of Polycomb targets. At this stage, we cannot exclude the possibility of some clients having weak phase separation capacities. Thus, both direct interactions and composition-dependent phase separation among Polycomb subunits (PRC1 and PRC2) and Polycomb targets could contribute to the biogenesis of Polycomb condensates. The current model implies a sequential binding event, but it is possible that the order of binding events is different from the proposed model.
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