Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells

Kent

Brown

et al. 2018

Preprint

Self Cite

17Polycomb group (PcG) proteins are master regulators of development and differentiation. 18 Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG 19 proteins form condensates in the nucleus of cells and these condensates are the physical sites 20 of PcG-targeted gene silencing. However, the physiochemical principles underlying the PcG 21 condensate formation remain unknown. Here we show that Polycomb repressive complex 1 22 (PRC1) protein Cbx2, one member of the Cbx family proteins, contains a long stretch of 23 intrinsically disordered region (IDR). Cbx2 undergoes phase separation to form condensates. 24Cbx2 condensates exhibit liquid-like properties and can concentrate DNA and nucleosomes. We 25 demonstrate that the conserved residues within the IDR promote the condensate formation in 26 vitro and in vivo. We further indicate that H3K27me3 has minimal effects on the Cbx2 27 condensate formation while depletion of core PRC1 subunits facilitates the condensate 28 formation. Thus, our results reveal that PcG condensates assemble through liquid-liquid phase 29 separation (LLPS) and suggest that PcG-bound chromatin is in part organized through phase-30 separated condensates. 31 decrease of YFP-Cbx2 through diffusion. We did not detect YFP-Cbx2 condensates in the 131 lysate. We expected that formaldehyde crosslinking would preserve Cbx2 condensates. After 132 sonication and centrifugation, YFP-Cbx2 condensates would be within the pellets. To test this 133 speculation, prior to lysis, we cross-linked cells with formaldehyde. Lysates were prepared from 134 the cross-linked cells and subjected to sonication. Using fluorescence microscopy, we observed 135

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Polycomb Cbx2 Condensates Assemble through Phase Separation

Kent

Brown

et al. 2018

Preprint

Self Cite

“…Labelling HaloTag Fusion Proteins is described in more detail at Bio-protocol ( Duc and Ren, 2017 ). 24 hr prior to imaging, mES cells stably expressing HaloTag fusion proteins were seeded to gelatin-coated cover glass dish.…”

Section: Methodsmentioning

confidence: 99%

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

“…After changing buffer once, dialysis was performed overnight. 10 l of the dialyzed sample were added to a coverglass dish made as described previously (82). After all condensates had settled down to the surface of the coverslip, DIC or fluorescence images of condensates were acquired using an Axio Observer D1 microscope (Zeiss, Germany) equipped with a 100ϫ/1.40 numerical aperture oil immersion objective with additional 2.5ϫ magnification and an Evolve electronmultiplying charge-coupled density camera (512 ϫ 512; Photometrics, Tucson, AZ).…”

Section: Polycomb Cbx2 Condensates By Phase Separation In Vitro Condementioning

confidence: 99%

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

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.