Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging

Journal of Biological Chemistry

Kent

Brown

et al. 2019

Self Cite

282

280

Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region for compaction of chromatin in vitro, promote the condensate formation both in vitro and in vivo. We showed that the CBX2 condensates concentrate DNA and nucleosomes. Using genetic engineering, we report that trimethylation of Lys-27 at histone H3 (H3K27me3), a marker of heterochromatin formation produced by PRC2, had minimal effects on the CBX2 condensate formation. We further demonstrated that the CBX2 condensate formation does not require CBX2–PRC1 subunits; however, the condensate formation of CBX2–PRC1 subunits depends on CBX2, suggesting a mechanism underlying the assembly of CBX2–PRC1 condensates. In summary, our results reveal that PcG condensates assemble through liquid–liquid phase separation (LLPS) and suggest that phase-separated condensates can organize PcG-bound chromatin.

Section: Cbx2 Forms Condensates In Cellsmentioning

confidence: 62%

Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation

Journal of Biological Chemistry

Kent

Brown

et al. 2019

Self Cite

282

280

“…During evolution, the number of genes encoding Cbx proteins has increased, which has resulted in structural and functional diversification ( Cheng et al, 2014 ; Klauke et al, 2013 ; Morey et al, 2012 ; Ren and Kerppola, 2011 ; Ren et al, 2008 ; Tatavosian et al, 2015 ; Vincenz and Kerppola, 2008 ; Whitcomb et al, 2007 ; Zhen et al, 2014 ). At the single-molecule level, we quantified the kinetic fractions of the Cbx proteins within living mES cells and revealed that ∼ 30% of Cbx2, Cbx7, and Cbx8 associate with chromatin at a given time period while ∼10–15% of Cbx4 and Cbx6 bind to chromatin.…”

Section: Discussionmentioning

confidence: 99%

“…These mES cells were maintained in mES medium (DMEM (D5796; Sigma-Aldrich, St Louis, MO) supplemented with 15% FBS (F0926; Sigma-Aldrich, St Louis, MO), 2 mM glutamine (25030–081; Life Technologies, Carlsbad, CA), 100 units/ml penicillin-streptomycin (15140–122; Life Technologies, Carlsbad, CA), 0.1 mM β-mercaptoethanol (21985–023; Life Technologies, Carlsbad, CA), 10 3 units/ml leukemia inhibitor factor (LIF) and 0.1 mM non-essential amino acids (1114050; Life Technologies, Carlsbad, CA)) at 37°C in 5% CO 2 . 4-hydroxytamoxifen (OHT; H7904; Sigma-Aldrich Inc, St Louis, MO) was administrated to deplete the Ring1b alleles of the Ring1a −/− /Ring1b fl/fl ; Rosa26::CreERT2 mES cells ( Tatavosian et al, 2015 ; Zhen et al, 2014 ). HEK293T cells were maintained as described previously ( Tatavosian et al, 2015 ; Zhen et al, 2014 ).…”

Section: Methodsmentioning

confidence: 99%

“…4-hydroxytamoxifen (OHT; H7904; Sigma-Aldrich Inc, St Louis, MO) was administrated to deplete the Ring1b alleles of the Ring1a −/− /Ring1b fl/fl ; Rosa26::CreERT2 mES cells ( Tatavosian et al, 2015 ; Zhen et al, 2014 ). HEK293T cells were maintained as described previously ( Tatavosian et al, 2015 ; Zhen et al, 2014 ).…”

Section: Methodsmentioning

confidence: 99%

“…Single-molecule techniques have been widely applied to study DNA- and chromatin-templated processes in vitro and provide insights into genetic information flow in vivo ( Bell and Kowalczykowski, 2016 ; Dangkulwanich et al, 2014 ; Duzdevich et al, 2014 ; Geertsema and van Oijen, 2013 ; Harada et al, 2016 ; Herbert et al, 2008 ; Li et al, 2004 ; Ngo et al, 2015 ; Ren et al, 2003 ; Ren et al, 2006 ; Tatavosian et al, 2015 ). Recent advances in single-molecule imaging allow measuring the quantitative kinetics of gene control in living mammalian cells ( Chen et al, 2014 ; Coleman et al, 2015 ; Gebhardt et al, 2013 ; Grimm et al, 2015 ; Izeddin et al, 2014 ; Katz et al, 2016 ; Knight et al, 2015 ; Liu et al, 2015 , 2014 ; Mazza et al, 2012 , 2013 ; Morisaki et al, 2014 ; Normanno et al, 2015 ; Swinstead et al, 2016 ; Zhang et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin

Zhen

Huynh

et al. 2016

eLife

Self Cite

101

107

The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes.DOI: http://dx.doi.org/10.7554/eLife.17667.001

Sm-ChIPi: Single-Molecule Chromatin Immunoprecipitation Imaging

Chromatin Immunoprecipitation

Ren

2017

Epigenetic complexes regulate chromatin dynamics via binding to and assembling on chromatin. However, the mechanisms of chromatin binding and assembly of epigenetic complexes within cells remain incompletely understood, partly due to technical challenges. Here, we present a new approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) that enables to assess the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was developed based on chromatin immunoprecipitation followed by single-molecule fluorescence microscopy imaging. In this method, an epigenetic complex subunit fused with a gene coding for a fluorescent protein is stably expressed in its corresponding knockout cells. Nucleosomes associated with epigenetic complexes are isolated from cells at native conditions and incubated with biotinylated antibodies. The resulting complexes are immobilized on a quartz slide that had been passivated and functionalized with NeutrAvidin. Image stacks are then acquired by using single-molecule TIRF microscopy. The individual spots imaged by TIRF microscopy represent single protein-nucleosome complexes. The number of copies of the protein complexes on a nucleosome is inferred from the fluorescence photobleaching measurements. Sm-ChIPi is a sensitive and direct method that can quantify the cellular assembly stoichiometry of epigenetic complexes on chromatin.