The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle1. Although processive ubiquitination might—in principle—arise from Bre1 and Rad6 travelling with RNA polymerase II2, the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid–liquid phase separation3 as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated ‘reaction chambers’, with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Data analysis software, scripts and parameters used to analyse ChIP-exo datasets can be accessed via https://github.com/CEGRcode/Gallego_2019.
Fuchs, G. & Oren, M. Writing and reading H2B monoubiquitylation. Biochim. Biophys. Acta 1839, 694–701 (2014).
Weake, V. M. & Workman, J. L. Histone ubiquitination: triggering gene activity. Mol. Cell 29, 653–663 (2008).
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
Batta, K., Zhang, Z., Yen, K., Goffman, D. B. & Pugh, B. F. Genome-wide function of H2B ubiquitylation in promoter and genic regions. Genes Dev. 25, 2254–2265 (2011).
Cucinotta, C. E., Young, A. N., Klucevsek, K. M. & Arndt, K. M. The nucleosome acidic patch regulates the H2B K123 monoubiquitylation cascade and transcription elongation in Saccharomyces cerevisiae. PLoS Genet. 11, e1005420 (2015).
Gallego, L. D. et al. Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6–Bre1. Proc. Natl Acad. Sci. USA 113, 10553–10558 (2016).
Hwang, W. W. et al. A conserved RING finger protein required for histone H2B monoubiquitination and cell size control. Mol. Cell 11, 261–266 (2003).
Kim, J. et al. RAD6-mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells. Cell 137, 459–471 (2009).
Wood, A. et al. Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Mol. Cell 11, 267–274 (2003).
Van Oss, S. B. et al. The histone modification domain of Paf1 complex subunit Rtf1 directly stimulates H2B ubiquitylation through an interaction with Rad6. Mol. Cell 64, 815–825 (2016).
Zhang, F. & Yu, X. WAC, a functional partner of RNF20/40, regulates histone H2B ubiquitination and gene transcription. Mol. Cell 41, 384–397 (2011).
Alberti, S. Phase separation in biology. Curr. Biol. 27, R1097–R1102 (2017).
Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357, eaaf4382 (2017).
Gibson, B. A. et al. Organization of chromatin by intrinsic and regulated phase separation. Cell 179, 470–484 (2019).
Larson, A. G. et al. Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 547, 236–240 (2017).
Boija, A. et al. Transcription factors activate genes through the phase-separation capacity of their activation domains. Cell 175, 1842–1855 (2018).
Cho, W. K. et al. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361, 412–415 (2018).
Chong, S. et al. Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science 361, eaar2555 (2018).
Sabari, B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science 361, eaar3958 (2018).
Turco, E., Gallego, L. D., Schneider, M. & Köhler, A. Monoubiquitination of histone H2B is intrinsic to the Bre1 RING domain–Rad6 interaction and augmented by a second Rad6-binding site on Bre1. J. Biol. Chem. 290, 5298–5310 (2015).
Wei, M. T. et al. Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles. Nat. Chem. 9, 1118–1125 (2017).
Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 174, 688–699 (2018).
Rossi, M. J., Lai, W. K. M. & Pugh, B. F. Simplified ChIP-exo assays. Nat. Commun. 9, 2842 (2018).
Huisinga, K. L. & Pugh, B. F. A genome-wide housekeeping role for TFIID and a highly regulated stress-related role for SAGA in Saccharomyces cerevisiae. Mol. Cell 13, 573–585 (2004).
Reja, R., Vinayachandran, V., Ghosh, S. & Pugh, B. F. Molecular mechanisms of ribosomal protein gene coregulation. Genes Dev. 29, 1942–1954 (2015).
Elbaum-Garfinkle, S. et al. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc. Natl Acad. Sci. USA 112, 7189–7194 (2015).
Lai, W. K. M. & Pugh, B. F. Understanding nucleosome dynamics and their links to gene expression and DNA replication. Nat. Rev. Mol. Cell Biol. 18, 548–562 (2017).
DeSanto, C. et al. WAC loss-of-function mutations cause a recognisable syndrome characterised by dysmorphic features, developmental delay and hypotonia and recapitulate 10p11.23 microdeletion syndrome. J. Med. Genet. 52, 754–761 (2015).
Vinayachandran, V. et al. Widespread and precise reprogramming of yeast protein-genome interactions in response to heat shock. Genome Res. 28, 357–366 (2018).
Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://arxiv.org/abs/1303.3997 (2013).
Bartonek, L. & Zagrovic, B. VOLPES: an interactive web-based tool for visualizing and comparing physicochemical properties of biological sequences. Nucleic Acids Res. 47, W632–W635 (2019).
Armstrong, J. K., Wenby, R. B., Meiselman, H. J. & Fisher, T. C. The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation. Biophys. J. 87, 4259–4270 (2004).
Yassour, M. et al. Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing. Proc. Natl Acad. Sci. USA 106, 3264–3269 (2009).
We thank B. Zagrovic and A. Polyansky for critical insights into the multivalency of Lge1 LLPS, and Lge1 and WAC sequence analyses; O. R. Abdi for technical support; and G. Warren and D. Gerlich for discussions. A.K. was funded in part by a NOMIS Pioneering Research Grant, L.D.G. by a L’Oréal-UNESCO-OeAW Austria Fellowship and A.R. by an OeAW DOC Fellowship. B.F.P. and C.M. were funded by the National Institutes of Health grant HG004160.
B.F.P. has a financial interest in Peconic LLC, which uses the ChIP-exo technology implemented in this study and could potentially benefit from the outcomes of this research. The remaining authors declare no competing interests.
Peer review information Nature thanks Fred van Leeuwen, Tanja Mittag and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Bre1 copurifies with Lge1. Lge1–TAP was tandem-affinity-purified from yeast. Bre1 and CBP (calmodulin binding peptide)-tagged Lge1 (Lge1–CBP; a remnant after protease cleavage) were confirmed by mass spectrometry. Asterisk indicates a Bre1 degradation product. b, Bre1 interacts with Lge1 in vitro. Binding assay using recombinant GST–Lge1–Strep as bait (black dot) with Strep–Bre1 constructs (arrowheads) (1:3 molar ratio). Proteins were analysed by Coomassie staining and anti-Strep immunoblotting. c, Input proteins for b. d, Same setup as in b. Black dots indicate immobilized Lge1 constructs; asterisk indicates a degradation product. e, Sucrose gradient sedimentation assays (5–45%) of recombinant Strep–Bre1 and GST–Lge1 proteins. Peak fractions are highlighted (arrowheads). f, Input protein for 6×His–Lge1 constructs used in phase-separation assays. g, Quantification of condensate sizes in Fig. 1c. n, number of condensates. Dot plots with median and interquartile range. **P = 0.0189, ****P < 0.0001, determined by two-sided Mann–Whitney test. n.d., not determinable. h, Analysis of condensate growth using DIC imaging (6×His–Lge1, 5 μM) (Supplementary Video 1). Scale bar, 2 μm. i, Increasing amounts of mGFP–Bre1 were added to preformed 6×His–Lge1(IDR) condensates (which lack the coiled-coil domain of Lge1). The unrestricted diffusion of Bre1 (5 μM) into Lge1(IDR) condensates caused their interior to collapse into coarse aggregates. Scale bar, 2 μm. j, Quantification of mGFP–Bre1 shell thickness in i and Fig. 1d. Box-and-whisker plot shows median, interquartile range, and minimum and maximum values. ***P value < 0.001, determined with two-sided t-test (1.5 μm–3 μm, t = 7.6, degrees of freedom = 67; 3 μm–5 μm, t = 14, degrees of freedom = 69). n, number of condensates; n.d. not determinable. k, GST–Lge1 used in Fig. 1b phase-separates in vitro. Recombinant GST–Lge1 (7 μM), GST (7 μM) and buffer-only (20 mM Tris pH 7.5, 10 mM KCl and 5 mM MgCl2) were visualized by DIC microscopy at 20 °C for the indicated times (min). Scale bar, 10 μm. See Supplementary Fig. 2 for uncropped gels and western blots.
a, Amino acid sequence of S. cerevisiae Lge1. The IDR (amino acids 1–242) is highlighted in light orange; the predicted coiled-coil domain (amino acids 281–332) is highlighted in in dark orange; and the Y/R-rich sticker region (amino acids 1–80) is underlined. The mutated Y and R residues are labelled in magenta and blue, respectively. b, Comparative sequence analyses of yeast Lge1 (left) and human WAC (WW-domain-containing adaptor protein with coiled-coil) (right). Cartoon shows predicted Lge1 and WAC domain organization; boundaries are drawn to scale. WW, protein–protein interaction domain with two tryptophans (W). Asterisks indicate residues the mutation of which has previously been implicated in the pathogenesis of DeSanto–Shinawi syndrome28. This neurodevelopmental disorder causes a developmental delay and dysmorphic facial features. It is caused by mutations that are predicted to truncate the IDR of WAC, and therefore disrupt the WAC interaction with RNF20/RNF40 (that is, nonsense and frameshift mutations leading to nonsense-mediated decay or protein truncation)28. For disorder prediction, disorder scores were calculated with the IUPred algorithm. Both Lge1 and WAC show extensive intrinsic disorder. For coiled-coil domain predictions, the COILS software was used, showing putative coiled-coil elements at the C termini of both proteins. For hydrophobicity analysis, the VOLPES web server was used to plot hydrophobicity profiles of protein sequences31 (further details are provided in the Methods). Lge1 and WAC display a similar hydrophobicity pattern in their N-terminal regions. The region with the best match is highlighted with a grey rectangle. Similarity in this region is indicated by Pearson correlation coefficient (R = 0.68) and root-mean-squared deviation (r.m.s.d. = 0.16). In their charge distribution profile, both Lge1 and WAC exhibit alternating blocks of negative and positive charge. The sequence charge density was calculated using a custom-made script. A window of ten residues was used for smoothing. For condensate formation, the catGRANULE algorithm was used to predict the propensity for condensate formation. Top scores are indicated. The high-scoring sequence in Lge1 corresponds approximately to the Y/R-rich region.
a, b, Reconstitution of condensates with core–shell architecture. Recombinant mGFP-tagged proteins (1.5 μM) were added to preformed 6×His–Lge1 condensates or 6×His–Lge1(IDR). Samples were incubated for 15 min before imaging by DIC and fluorescence microscopy. Scale bar, 2 μm. c, Reconstitution of hybrid condensates, with varying ratios of 6×His–Lge1:6×His–Lge1(IDR) show a differential partitioning of mGFP–Bre1 into the core. Proteins were mixed at the indicated molar ratios and incubated for 15 min at 20 °C. mGFP–Bre1 (0.5 μM) was added to the preformed condensates and incubated for 15 min before imaging by DIC and fluorescent microscopy. Fluorescent intensities were quantified across single condensates. Cartoons indicate putative assembly state of hybrid condensates, with a deterioration of the core–shell structure upon reduction of available Lge1 coiled-coil domains. n, number of condensates. Scale bar, 2 μm. d, Quantification of mGFP–Bre1 shell thickness in c. Box-and-whisker plot shows median, interquartile range, and minimum and maximum values. ****P < 0.0001, determined with two-sided t-test (t = 9.4, degrees of freedom = 62). n, number of condensates; n.d. not determinable. e, Analysis of condensate fusion. 6×His–Lge1 condensates (1.5 μM) with an mGFP–Bre1 shell (1.5 μM) were followed over time by microscopy. Condensates collide but do not fuse (Supplementary Video 2). Compare to Extended Data Fig. 1h for fusion dynamics. Scale bar, 2 μm.
a, Quantification of 6×His–Lge1 condensate sizes at different protein concentrations after 5 min of incubation at 20 °C. Quantification was done using ImageJ. n, number of condensates. Dot plots show median and interquartile range. **P (for 1 versus 1.5) = 0.0046, **P (for 1.5 versus 2) = 0.0029, ****P < 0.0001, determined by two-sided Mann–Whitney test. n.d. not determinable. b, Quantification of 6×His–Lge1 condensate size in the presence of 1,6-hexanediol indicates an inhibition of LLPS. Concentrated Lge1 protein was diluted to 1.5 μM in buffer with 1,6-hexanediol (%, w/v) and incubated for 15 min before imaging. n, number of condensates. Dot plots show median and interquartile range. **P = 0.0032, ****P < 0.0001, determined by two-sided Mann–Whitney test. n.d. not determinable. c, Strep–mGFP–Bre1 does not phase-separate under the conditions we tested. Concentrated proteins were diluted and incubated at 20 °C for 5 min before DIC microscopy. Scale bar, 10 μm. d, Turbidity measurements of 6×His–Lge1 at 450 nm, with or without Strep–Bre1. Proteins were mixed at the indicated molar ratios. LLPS of Lge1 occurred already at 0.1 μM, Strep–Bre1 shows no LLPS under the conditions we tested. Mean and s.d. are indicated. e, Condensates of 6×His–Lge1, 6×His–Lge1(IDR) or 6×His–Lge1 with an mGFP–Bre1 shell were incubated with TRITC-labelled dextran of different sizes (final dextran concentration 0.05 mg ml−1) for 15 min at 20 °C. Samples were imaged by DIC and fluorescence microscopy. Scale bar, 10 μm. The table below shows the hydrodynamic radius (Rh) for different dextrans in aqueous buffer, and is adapted from previously published work32. f, Lge1–Bre1 condensates are permeable to dextran of different sizes. Dextran is never excluded (partition ratios ≥ 1). Mean and s.d. are indicated; n = 60 condensates. g, Average Rh of recombinant proteins used in this work as measured by dynamic light scattering at 20 °C. The expected molecular mass was calculated according to the amino acid composition of the protein, and compared to the experimental molecular mass obtained by dynamic light scattering. Final data correspond to the average of at least two independent measures. n.d. not determinable.
a, Phase-separation assay with the indicated 6×His–Lge1 constructs (10 μM). Scale bar, 10 μm. Cartoons are drawn to scale. Lge1(Y>A, 1–102) contains three additional mutations within amino acids 1–102 besides the Y>A mutations in the sticker region (amino acids 1–80) (Extended Data Fig. 2a), which increases the disruption of Lge1 LLPS in vitro. b, Quantification of condensate sizes (6×His–Lge1 constructs, 10 μM). n, quantified condensates. Dot plot shows median and interquartile range. ****P value < 0.0001, determined by two-sided Mann–Whitney test. n.d. not determinable. c, Input gel for a. Asterisk indicates degradation product, arrowheads indicate Lge1 constructs. See Supplementary Fig. 2 for uncropped gels.
a, Recruitment of Pacific-Blue-labelled 6×His–Rad6 (here denoted ‘Rad6*’) (3 μM) or mGFP–Bre1 (3 μM) to Lge1 condensates. Experimental conditions as in Fig. 2a. Fluorescent intensities were quantified across single condensates. n, number of condensates. Scale bar, 5 μm. b, The recruitment of Rad6 to condensates was examined by adding Rad6* to 6×His–Lge1 condensates in the presence and absence of an mGFP–Bre1 (1.5 μM) or mGFP–Bre1(LBD) (15 μM) shell. The Bre1(LBD) construct has a weaker affinity to Lge1 than full-length Bre1, and therefore requires a higher concentration in the assay. Microscopy was performed immediately after adding Rad6* (3 μM) and followed over time. Fluorescent intensities were quantified across single condensates. n, number of condensates. Scale bar, 5 μm. c, Reconstituted mononucleosomes (1×NCP) and oligonucleosomes (16×NCP) were analysed by SDS–PAGE and Coomassie staining to assess purity and stoichiometry. d, Negative-stain electron microscopy was performed to assess the structure of oligonucleosomes. White arrowheads label individual nucleosomes, blue arrowheads indicate the linker DNA. Scale bar, 100 nm. e, Recruitment of mononucleosomes to Lge1 condensates with an mGFP–Bre1 shell. Hoechst-labelled, reconstituted mononucleosomes (1×NCP*, 0.5 μM) were added to 6×His–Lge1 condensates with an mGFP–Bre1 shell (3 μM), and imaged over time (min). n, number of condensates. Scale bar, 5 μm. f, Recruitment of oligonucleosomes to Lge1 condensates. The 16×NCPs* (0.5 μM) were added to 6×His–Lge1 condensates and imaged over time (min). n, number of condensates. Scale bar, 5 μm. g, Diffusion and retention of 601 Widom DNA into Lge1 condensates. Same setup as in f but with Hoechst-labelled 16× 601 Widom DNA (16×DNA*) added to 6×His–Lge1 condensates. n, number of condensates. Scale bar, 5 μm. h, Traces (optical density at 254 nm) of the sucrose gradient sedimentation assays (5–45%) show a reproducible fractionation pattern for cell extracts prepared from the indicated strains. The different ribosomal species and fractions are indicated and correspond to the fractions in Fig. 3a. i, Protein levels in cell extracts used for sucrose gradient assays. Anti-Pgk1 serves as a loading control. See Supplementary Fig. 2 for uncropped gels and western blots.
a, Gallery of representative images of bre1∆ lge1∆ cells co-expressing the indicated constructs from the strong GPD promoter. Scale bar, 2 μm. b, 1,6-Hexanediol treatment (10%, w/v) reduces the formation of Lge1–Bre1 puncta, but also affects nuclear import and thus complicates the interpretation of hexanediol effects in cells. White asterisk labels the vacuole; dashed white line shows the cell contour. Scale bar, 2 μm. c, d, Live imaging of bre1∆ lge1∆ cells expressing mGFP–Bre1 (c) or an empty vector (d), and the indicated Lge1–mCherry constructs. Dashed white line shows the cell contour. The fluorescence intensity of Lge1–mCherry constructs was quantified across a line spanning the nucleus. For comparison, the arbitrary fluorescence unit value = 1 is marked with a horizontal dashed line (except for d, in which the dashed line indicates a value of 0.5). n, number of randomly selected cells. Scale bar, 2 μm. e, Quantification of background-corrected total cell fluorescence (CTCF) of mCherry in c, d. Dot plots show median and interquartile range. f, Comparison of protein-expression levels of different Lge1–mCherry constructs in c. Cell lysates were analysed by SDS–PAGE and immunoblotting with anti-mCherry antibody. Anti-Pgk1 serves as a loading control. Asterisk indicates a degradation product. Red arrowheads indicate Lge1 constructs according to their predicted sizes. See Supplementary Fig. 2 for uncropped western blot. g, Gallery of representative images of bre1∆ lge1∆ cells expressing VC–Bre1 and Lge1–VN or Lge1(CC)–VN. Nup188–mCherry marks the nuclear envelope. Scale bar, 2 μm. h, Live imaging of bre1∆ lge1∆ cells expressing VC–Bre1 and the indicated Lge1–VN constructs from their endogenous promoters. Nup188–mCherry marks the nuclear envelope, and the dashed line marks the cell contour. Histograms represent pixel frequencies of fluorescence intensity values. Scale bar, 2 μm. i, Quantification of mean nuclear BiFC intensity in h and Fig. 3d. Median and interquartile range are indicated. n, number of cells. ***P < 0.001, determined by two-sided Mann–Whitney test. ns, not significant.
Extended Data Fig. 8 Effects of the IDR of Lge1 on global H2B ubiquitination, and additional ChIP-exo analyses.
a, Global levels of H2BK123ub. A lge1∆ Flag-H2B strain was transformed with plasmid-based variants of LGE1-mCherry or an empty plasmid. Cell lysates were subjected to anti-Flag immunoprecipitation, and analysed by SDS–PAGE and immunoblotting with anti-Flag antibody. Different exposures of the monoubiquitinated H2B band are shown. See Supplementary Fig. 2 for uncropped western blots. Expression levels of the Lge1–mCherry constructs in cell extracts are shown in Extended Data Figs. 7f, 10e. b, H2B (left) or H2BK123ub (right) ChIP-exo tag 5′ ends were plotted relative to the +1 nucleosome of all genes for wild type (grey trace) and the indicated lge1 mutants (red traces). Sequencing tags were normalized across datasets to a 30-bp window centred in the nucleosome-free region (NFR) of all genes, representing unbound background regions. The first three genic nucleosomes are labelled +1, +2 and +3. Two H2B peaks are observed per nucleosome position. H2BK123ub patterns are shown separately for ribosomal-protein genes, and SAGA- and TFIID-dominated genes. Analyses were performed as in Fig. 4a. c, ChIP-exo analysis shows that Lge1 function is not compromised by the plasmid-based approach used in this study (top). H2B (left) or H2BK123ub (right) occupancy in strains with plasmid-based expression of LGE1 under its endogenous promoter (grey trace) was compared to strains with LGE1 in its chromosomal context (blue trace). Analysis of ChIP-exo background using a non-specific IgG antibody (bottom). H2BK123ub enrichment in an lge1∆ bre1∆ strain (red trace) is reduced to background levels (IgG negative control, black trace). Analyses were performed as described in b.
Extended Data Fig. 9 Enrichment of Lge1 along gene bodies coincides with establishment of H2B ubiquitination pattern.
a, TAP-tagging does not impair Lge1 function under the conditions we tested (related to ChIP-exo in b). C- or N- terminally TAP-tagged versions of LGE1 were transformed into lge1∆ cells, and their growth was compared to an untagged BY4741 control strain. Lge1 proteins were expressed at similar levels. Cell lysates were analysed by SDS–PAGE and immunoblotting with anti-protein A antibody. Anti-Pgk1 serves as a loading control. See Supplementary Fig. 2 for uncropped western blots. b, Genome-wide binding profiles of Lge1. Frequency distribution of 5′ tags of Lge1 ChIP-exo were mapped to the midpoint between gene transcript start (TSS) and end (TES) for ribosomal-protein, SAGA-dominated and TFIID-dominated gene classes. Each class was sorted by gene length, thus generating bell plots. Lge1 binding profiles in wild-type and bre1∆ backgrounds are depicted. Insertion of the TAP tag at the N or C terminus gave similar enrichment patterns. TAP-tagged Reb1 and ‘No TAP tag’ serve as positive (site-specific binding) and negative controls, respectively. c, Lge1 binding is tied to the expression of the target gene, and is largely independent of Bre1 (black versus red traces). Composite plots of Lge1 enrichment at top- and bottom-15% expressed genes33 are shown in the top and bottom panels, respectively. The IgG negative control is depicted as a grey trace. ChIP-exo tag 5′ ends were mapped to the +1 nucleosome dyad (as defined by micrococcal nuclease (MNase) H3 ChIP sequencing). H2B occupancy is depicted as a filled light grey trace. The +1, +2, +3 and +4 nucleosome positions are highlighted. The table represents the percentage of the members of each gene class included in the top and bottom 15%. d, Quantification of H2BK123ub density for the first three genic nucleosome positions in various Lge1 mutants. Table depicts the fold enrichment of H2BK123ub density (ChIP-exo H2BK123ub/H2B) at canonical nucleosome positions +1, +2, and +3 for the indicated mutants relative to wild type. Data for two biological replicates are shown. BY4741 is a positive control and correlates with the strain carrying wild-type LGE1 on a plasmid. Ratios for each gene class are indicated, to directly compare with graphs shown in Fig. 4a, Extended Data Fig. 8b. e, Correlation between two biological replicates for H2B and H2BK123ub datasets are shown. ChIP-exo tag 5′ ends were binned in 500-bp intervals, and the coefficient of correlation between the two datasets was calculated.
Extended Data Fig. 10 Specific features of yeast Lge1 and human WAC contribute to H2BK123ub and cell viability.
a, The IDRs of WAC and LAF1 promote LLPS. Phase-separation assay of recombinant 6×His–Lge1 and the fusion constructs 6×His–WAC(1–318)–Lge1(CC) and 6×His–LAF1(1–169)–Lge1(CC) (both proteins, 5 μM; buffer, 20 mM Tris pH 7.5, 100 mM NaCl and 1 mM DTT, 20 °C). Scale bar, 10 μm. Protein inputs are shown on the right (black arrows). b, Quantification of condensate sizes (in μm2) in a. n, number of condensates. Dot plot showing median and interquartile range. **P = 0.004, ****P < 0.0001, determined by two-sided Mann–Whitney test. c, A synthetic genetic approach was used to investigate the functionality of Lge1 LLPS in vivo. Cells were inviable when LGE1 and HTZ1 were deleted together, indicating a functional relationship. Double-deletion strains containing a wild-type LGE1 cover plasmid (Ura marker) were cotransformed with the indicated plasmids (His marker). Growth was followed on SDC–His (loading control) and on SDC + 5-FOA, which shuffles out the Ura cover plasmid. Cells were spotted in tenfold serial dilutions and incubated for two (SDC–His) or three days (5-FOA) at 30 °C. d, Live imaging of bre1∆ lge1∆ cells expressing mGFP–Bre1 and WAC(1–318)–Lge1(CC)–mCherry or LAF1(1–169)–Lge1(CC)–mCherry shows protein import into the nucleus. Dashed white line indicates the cell contour. Fluorescence intensity of the mCherry construct was quantified across a line spanning the nucleus. For comparison, the arbitrary fluorescence unit value = 1 is marked with a horizontal dashed line. n, number of cells. Scale bar, 2 μm. e, Cell lysates of strains in c and d were analysed by SDS–PAGE and immunoblotting with anti-mCherry antibody. Anti-Pgk1 serves as a loading control; asterisks indicate degradation products. Red arrowheads indicate Lge1 constructs according to their predicted sizes. f, Live imaging of bre1∆ lge1∆ cells expressing VC–Bre1 and WAC (1–318)–Lge1(CC)–VN or LAF1(1–169)–Lge1(CC)–VN constructs from LGE1 endogenous promoter. Arrowheads label nuclear BiFC puncta, Nup188–mCherry labels the nuclear envelope and dashed lines indicate the cell contours. Histograms represent pixel frequency of fluorescent intensity values. Scale bar, 2 μm. g, Coefficient of variation of the fluorescence-intensity distribution of BiFC signals in f and Fig. 3e. The higher the coefficient of variation, the greater the heterogeneity of the BiFC signal. A propensity for LLPS is suggested by an increased coefficient of variation of the WAC(1–318)–Lge1(CC)–VN construct. Dot plot showing median and interquartile range. n, number of cells. **P = 0.0024, ***P < 0.001, determined by two-sided Mann–Whitney test. h, Expression levels of Lge1–mCherry constructs used in i. Cells lysates were analysed by SDS–PAGE and immunoblotting with anti-mCherry antibody. Anti-Pgk1 serves as a loading control. Asterisk indicates degradation products. Red arrowhead indicates Lge1 constructs. i, Genetic interaction analysis, set up as in c with the indicated plasmids. See Supplementary Fig. 2 for uncropped gels and western blots.
This file contains Supplementary Table 1, a list of strains and plasmids used, and Supplementary Figures 1-2 showing the uncropped blots with size marker indications.
Lge1 condensates grow by fusion. DIC imaging of 6xHis-Lge1 (5 μM) diluted in 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT. Protein solution was pipetted into a 16-well μ-slide chamber and incubated at 20 °C. Imaging was started after 15 mins and images were taken every 2 sec for 2 mins. Scale bar, 5 μm.
Bre1 shell prevents Lge1 condensate fusion. DIC imaging of 6xHis-Lge1 (1.5 μM) with Bre1 shell (1.5 μM). Proteins were mixed and treated as in Video S1. Scale bar, 5 μm.
About this article
Cite this article
Gallego, L.D., Schneider, M., Mittal, C. et al. Phase separation directs ubiquitination of gene-body nucleosomes. Nature 579, 592–597 (2020). https://doi.org/10.1038/s41586-020-2097-z
Biomolecular Condensates: Sequence Determinants of Phase Separation, Microstructural Organization, Enzymatic Activity, and Material Properties
The Journal of Physical Chemistry B (2021)
Biomedicine & Pharmacotherapy (2021)
Discovery of Arginine Methylation, Phosphorylation, and Their Co-occurrence in Condensate-Associated Proteins in Saccharomyces cerevisiae
Journal of Proteome Research (2021)
EMBO reports (2021)