Polycomb Cbx2 Condensates Assemble through Phase Separation

Abstract

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

Enhancer Features that Drive Formation of Transcriptional Condensates

“…Although our model explicitly incorporates enhancer DNA, TFs, and coactivators, the underlying mechanistic framework can be extended to understand diverse condensates that form at specific genomic loci. Examples may include condensates in heterochromatin organization (Larson et al, 2017;Strom et al, 2017), histone locus body assembly (Nizami et al, 2010), long non-coding RNA (lncRNA)-mediated paraspeckle formation (Fox et al, 2018;Yamazaki et al, 2018), nucleolar formation (Feric et al, 2016;Pederson, 2011), and in polycomb-mediated transcriptional silencing (Tatavosian et al, 2018). Recent advances in microscopy at the nano-scales (Li et al, 2019) can potentially shed light onto whether transcription-associated condensates form higher-order sub-structures, like the nucleolus (Feric et al, 2016).…”

Histone Modifications Regulate Chromatin Compartmentalization by Contributing to a Phase Separation Mechanism

“…A number of proteins that function in chromatin compartmentalization have an intrinsic capacity for LLPS. These include Agenet domain-containing protein 1 (ADCP1) (Zhao et al, 2019), bromodomain-containing protein 4 (Brd4) (Sabari et al, 2018), heterochromatin protein 1a (HP1a) (Larson et al, 2017), HP1a (Strom et al, 2017), and chromobox 2 (CBX2) (Tatavosian et al, 2018). Some proteins, such as CBX2 (Plys et al, 2019;Tatavosian et al, 2018), HP1a (Larson et al, 2017), and H1 (Turner et al, 2018), undergo LLPS when mixed with DNA in vitro.…”

“…These include Agenet domain-containing protein 1 (ADCP1) (Zhao et al, 2019), bromodomain-containing protein 4 (Brd4) (Sabari et al, 2018), heterochromatin protein 1a (HP1a) (Larson et al, 2017), HP1a (Strom et al, 2017), and chromobox 2 (CBX2) (Tatavosian et al, 2018). Some proteins, such as CBX2 (Plys et al, 2019;Tatavosian et al, 2018), HP1a (Larson et al, 2017), and H1 (Turner et al, 2018), undergo LLPS when mixed with DNA in vitro. Based on these observations, it has been suggested that chromatin compartments are assembled via LLPS (Hnisz et al, 2017;Larson et al, 2017;Sabari et al, 2018;Strom et al, 2017;Tatavosian et al, 2018).…”

“…Some proteins, such as CBX2 (Plys et al, 2019;Tatavosian et al, 2018), HP1a (Larson et al, 2017), and H1 (Turner et al, 2018), undergo LLPS when mixed with DNA in vitro. Based on these observations, it has been suggested that chromatin compartments are assembled via LLPS (Hnisz et al, 2017;Larson et al, 2017;Sabari et al, 2018;Strom et al, 2017;Tatavosian et al, 2018).…”

Chromatin state switching in a polymer model with mark-conformation coupling

“…Additional to the equilibrium phase transition scenario proposed in this Letter, nonequilibrium features of the chemical dynamics [19,20] and the phase separation properties of the key components in chromatin dynamics [8,[41][42][43][44] may play roles in enhancing or diminishing the switch-like behavior. Nevertheless, the fact that a simple mark-conformation coupling can lead to a discrete switch indicates that nonlinear dynamics and well-designed chemical networks may not be essential in explaining cell fate dynamics.…”