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

Ingersoll, Steven; Brown, Kyle L.; Ma, Brian; Ren, Xiaojun

doi:10.1007/978-1-0716-2847-8_11

Cited by 2 publications

(5 citation statements)

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“…For labeling of our mES or NPC cells with HaloTag fusion proteins, 50-500 pM Janelia Fluor® 549 (Tocris, 6147) dye was added to cells in culture medium such that 5-20 proteins are visible within each nucleus. Kinetic and chromatin bound population dynamics were analyzed as previously described 24,37,99,100 . Images were acquired using the configuration listed in “ Optical setup for live-Cell single-molecule tracking ” section.…”

Section: Star Methodsmentioning

confidence: 99%

“…For residence time and kinetic fraction analysis, image stacks without visible cell drift were analyzed. Tracking settings used in U-track algorithms 102 were applied in MATLAB as described previously 24,99,100 . To determine kinetic fractions, we constructed the displacement histograms and then carried out kinetic modeling of the measured displacements using Spot-On 103 , which quantitatively measures three kinetic fractions (populations) of total molecules within the nucleus.…”

Section: Star Methodsmentioning

confidence: 99%

“…The defined populations are F 1 (total chromatin bound), F 2 (confined motion), and F 3 (free diffusion). All Spot-On settings were described previously 24,37,99,100 . To determine stable chromatin-bound fraction (F s ) and long-residence time (τ l ), we determined the track length of individual particles whose D m is less than 0.032 μm 2 /s.…”

Section: Star Methodsmentioning

confidence: 99%

“…To determine stable chromatin-bound fraction (F s ) and long-residence time (τ l ), we determined the track length of individual particles whose D m is less than 0.032 μm 2 /s. Following correction for photobleaching, the normalized cumulative frequency distributions are fitted with a one- or two-component exponential decay function as described previously 24,37,99,100 , which generate stable chromatin-bound fraction (F s ) and long-residence time (τ l ). The target-search time (τ s ) was estimated from the long residence time (τ l ) and stable chromatin-bound fraction ( F s ) as described previously 24,37 .…”

Section: Star Methodsmentioning

confidence: 99%

“…Live-cell single-molecule tracking analysis: Kinetic and chromatin bound population dynamics were analyzed as previously described 24,37,99,100 . For residence time and kinetic fraction analysis, image stacks without visible cell drift were analyzed.…”

Section: Cellprofiler Analysis Of In Vitro and Live-cell Condensate P...mentioning

confidence: 99%

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

Ingersoll,

Trouth,

Luo

et al. 2024

Preprint

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SUMMARYFacultative 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.

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