doi: 10.1038/s41586-020-2097-z.
Epub 2020 Mar 11.
Phase separation directs ubiquitination of gene-body nucleosomes
Affiliations
- PMID: 32214243
- PMCID: PMC7481934
- DOI: 10.1038/s41586-020-2097-z
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Phase separation directs ubiquitination of gene-body nucleosomes
Nature.
2020 Mar.
Abstract
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.
Conflict of interest statement
Declaration of interests
B.F.P. has a financial interest in Peconic, LLC, which utilizes 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.
Figures
a. Bre1 co-purifies with Lge1. Lge1-TAP was tandem affinity-purified from yeast. Bre1 and Lge1-CBP 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 analyzed by Coomassie staining and anti-Strep immunoblotting. Input proteins are shown in c. d. Same setup as in b. Black dots indicate immobilized Lge1 constructs, asterisk a degradation product. e. Sucrose gradient sedimentation assays (5 %−45 %) of recombinant Strep-Bre1 and GST-Lge1 proteins. Main peak fractions are highlighted (arrowheads). f. Input protein for 6His-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-value = 0.0189, ****p-value < 0.0001 determined by two-sided Mann-Whitney test. n.d., not determinable. h. Analysis of condensate growth using DIC imaging (6His-Lge1; 5 μM). Scale bar, 2 μm. See also Video S1. i. Increasing amounts of mGFP-Bre1 were added to preformed 6His-Lge1 IDR condensates, lacking Lge1 CC. Note that 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 Fig. 1d and Extended Fig. 1i. Box-whisker plot shows median, interquartile range, minimum and maximum values. ***p-value < 0.001 determined with two-sided T-test (1.5 μm-3 μm: t= 7.6, df= 67; 3 μm-5 μm: t=14, df=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, 5 mM MgCl2) were visualized by DIC microscopy at 20 °C for the indicated times (min). Scale bar, 10 μm. See SF2 for uncropped gels and western blots.
a. Amino-acid sequence of S.cerevisiae Lge1. The Intrinsically Disordered Region (IDR; aa1-242) is highlighted in light orange; the predicted Coiled-Coil (CC; aa281-332) in dark orange; the Y/R rich ‘sticker’ (aa1-80) is underlined. The mutated Y and R residues are labelled in magenta and blue, respectively. b. Comparative sequence analyses of yeast Lge1 (left panels) and human WAC (WW domain-containing adapter protein with coiled-coil) (right panels). Cartoon shows predicted Lge1 and WAC domain organization, boundaries are drawn to scale. IDR, Intrinsically Disordered Region; CC, Coiled-Coil; WW, protein-protein interaction domain with two tryptophans (W). Asterisks indicate residues whose mutation has been implicated in DeSanto-Shinawi syndrome pathogenesis. This neurodevelopmental disorder, which causes a developmental delay and dysmorphic facial features. It is caused by mutations, which are predicted to truncate the WAC IDR and hence disconnect it from the RNF20/40-interacting CC domain (i.e. nonsense and frameshift mutations leading to nonsense mediated decay or protein truncation). Disorder prediction: disorder scores were calculated with the IUPred algorithm. Both Lge1 and WAC show extensive intrinsic disorder. Coiled-Coils: prediction was performed with the COILS software, showing putative CC elements at the C-termini of both proteins. Hydrophobicity: the VOLPES web-server was used to plot hydrophobicity profiles of protein sequences, see Methods for further details. Lge1 and WAC display a similar hydrophobicity pattern in their N-terminal regions. The region with the best match is highlighted with a gray rectangle. Similarity in this region is indicated by Pearson correlation coefficient (R, 0.68) and Root-Mean-Squared Deviation (RMSD, 0.16). 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 10 residues was used for smoothing. Condensate formation: the catGRANULE algorithm was used to predict the propensity for condensate formation. Top scores are indicated. Note that 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 6His-Lge1 condensates or 6His-Lge1 IDR. Samples were incubated for 15 min prior to imaging by DIC and fluorescence microscopy. Scale bar, 2 μm. c. Reconstitution of ‘hybrid condensates’ with varying ratios of 6His-Lge1 FL: 6His-Lge1 IDR show 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 prior to 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 CCs. FU, arbitrary fluorescent units. n = number of condensates. Scale bar, 2 μm. d. Quantification of mGFP-Bre1 shell thickness in c. Box-whisker plot shows median, interquartile range, minimum and maximum values. ****p-value < 0.0001 determined with two-sided T-test (t= 9.4, df= 62). n = number of condensates. n.d., not determinable. e. Analysis of condensate fusion. 6His-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. Compare to Extended Data Fig. 1h. Scale bar, 2 μm. See also Video S2.
a. Quantification of 6His-Lge1 condensate sizes at different protein concentrations after 5 min of incubation at 20 °C. Quantification was done with ImageJ. n = number of condensates. Dot plots show median and interquartile range. **p-value 1 vs. 1.5 = 0.0046, **p-value 1.5 vs. 2 = 0.0029, ****p-value < 0.0001 determined by two-sided Mann-Whitney test, n.d., not determinable, b. Quantification of 6His-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-value = 0.0032, ****p-value < 0.0001 determined by two-sided Mann-Whitney test, n.d., not determinable, c. Strep-mGFP-Bre1 does not phase-separate under the conditions tested. Concentrated proteins were diluted and incubated at 20 °C for 5 min prior to DIC microscopy. Scale bar, 10 μm. d. Turbidity measurements of 6His-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 tested. Mean and standard deviation are indicated, e. Condensates of 6His-Lge1, 6His-Lge1 IDR or 6His-Lge1 with an mGFP-Bre1 shell were incubated with TRITC-labelled dextran of different sizes (final dextran concentration 0.05 mg/ml) 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 dextrans in aqueous buffer; adapted from published work, f. Lge1-Bre1 condensates are permeable to dextrans of different sizes. Dextran is never excluded (partition ratios ≥ 1). Mean and standard deviation are indicated; n = 60 condensates, g. Average Rh of recombinant proteins used in this work as measured by dynamic light scattering (DLS) at 20 °C. The expected molecular mass was calculated according to the protein’s amino-acid composition and compared to the experimental molecular mass obtained by DLS. Final data correspond to the average of at least two independent measures, n.d., not determinable.
a. Phase-separation assay with the indicated 6His-Lge1 constructs (10 μM). Scale bar, 10 μm. Cartoons drawn to scale. Lge1 Y>A 1-102 contains three additional mutations within aa1-102 besides the Y>A mutations in the sticker (aa1-80) (see Extended Data Fig. 2a), which increases the disruption of Lge1 LLPS in vitro. b. Quantification of condensate sizes (6His-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. Lge1 Y>A 1-102 contains three additional mutations within aa1-102 besides the Y>A mutations in the sticker (aa1-80). c. Input gel for a. Asterisk indicates degradation product, arrowheads Lge1 constructs. See SF2 for uncropped gels.
a. Recruitment of Pacific Blue-labelled 6His-Rad6 (His-Rad6*, 3 μM) or mGFP-Bre1 (3 μM) to Lge1 condensates. Experimental conditions as in Fig. 2a. Fluorescent intensities were quantified across single condensates. FU, arbitrary fluorescent units, n = number of condensates. Scale bar, 5 μm. b. Recruitment of Rad6 to condensates was examined by adding Pacific Blue-labelled 6His-Rad6 (His-Rad6*) to 6His-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, hence requiring a higher concentration. Microscopy was performed immediately after adding His-Rad6* (3 μM) and followed over time. Fluorescent intensities were quantified across single condensates. FU, arbitrary fluorescent units, n = number of condensates. Scale bar, 5 μm. c. Reconstituted mononucleosomes (1x NCP) and oligonucleosomes (16x NCP) were analyzed 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 the linker DNA. Scale bar, 100 nm. e. Recruitment of mononucleosomes to Lge1 condensates with an mGFP-Bre1 shell. Hoechst-labelled, reconstituted mononucleosomes (1xNCP*; 0.5 μM) were added to 6His-Lge1 condensates with an mGFP-Bre1 shell (3 μM) and imaged over time (min). FU, arbitrary fluorescent units, n = number of condensates. Scale bar, 5 μm. f. Recruitment of oligonucleosomes to Lge1 condensates. Hoechst-labelled, reconstituted 16x nucleosomes (16xNCP*; 0.5 μM) were added to 6His-Lge1 condensates and imaged overtime (min). FU, arbitrary fluorescent units, 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-labeled 16x 601 Widom DNA (16xDNA*) added to 6His-Lge1 condensates, n = number of condensates. Scale bar, 5 μm. h. OD 254 nm traces 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 SF2 for uncropped gels and western blots.
a. Gallery of representative images of bre1Δ lge1Δ 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, thus, complicating 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 bre1Δ lge1Δ 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 FU value = 1 is marked with a horizontal dashed line, except for d where the dashed lines indicates FU = 0.5. n = number of randomly selected cells. FU, arbitrary fluorescence units. 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 analyzed by SDS-PAGE and immunoblotting with anti-mCherry antibody. Anti-Pgk1 serves as a loading control. Asterisk indicates degradation product. Red arrowheads indicate Lge1 constructs according to their predicted sizes. See SF2 for uncropped western blot. g. Gallery of representative images of bre1Δ lge1Δ 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 bre1Δ Ige1Δ cells expressing VC-Bre1 and the indicated Lge1-VN constructs from their endogenous promoters. Nup188-mCherry marks the nuclear envelope, dashed line 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-value < 0.001 determined by two-sided Mann-Whitney test, n.s., not significant.
a. Global levels of H2BK123 ubiquitination. A lge1Δ FLAG-H2B strain was transformed with plasmid-based variants of LGE-mCherry or an empty plasmid. Cell lysates were subjected to anti-FLAG immunoprecipitation and analyzed by SDS-PAGE and immunoblotting with anti-FLAG antibody. Different exposures of the H2Bub band are shown. See SF2 for uncropped western blots. Expression levels of the Lge1-mCherry constructs in cell extracts are shown in Extended Data Fig. 7f and Extended Data Fig. 10e. b. H2B (left) or H2BK123ub (right) ChIP-exo tag 5’ ends were plotted relative to the +1 nucleosome of all genes for WT (grey trace) and the indicated Ige1 mutants (red traces). Sequencing tags were normalized across datasets to a 30 bp window centered in the nucleosome-free region of all genes, representing unbound background regions. The first three genic nucleosomes are labeled +1, +2 and +3. Two H2B peaks are observed per nucleosome position. H2BK123ub patterns are shown separately for Ribosomal protein genes, 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 (upper panel). 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 (lower panel). H2BK123ub enrichment in a lge1Δbre1Δ strain (red trace) is reduced to background levels (IgG negative control, black trace). Analyses were performed as described in b.
a. TAP-tagging does not impair Lge1 function under the conditions tested (related to ChIP-exo in b). C- or N- terminally TAP tagged versions of LGE1 were transformed into lge1Δ cells and their growth was compared to an untagged BY4741 control strain. Lge1 proteins were expressed at similar levels. Cell lysates were analyzed by SDS-PAGE and immunoblotting with anti-Protein A antibody. Anti-Pgk1 serves as a loading control. See SF2 for uncropped western blots, b. Genome wide binding profiles of Lge1. Frequency distribution of 5’ tags of Lge1 ChIP-exo are mapped to the midpoint between gene transcript start (TSS) and end (TES) for RP, SAGA and TFIID gene classes. Each class was sorted by gene length, thus generating bell plots. Lge1 binding profiles in WT and bre1Δ 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 expression of the target gene and is largely independent of Bre1 (compare black and red traces). Composite plots of Lge1 enrichment at top and bottom 15% expressed genes are shown in the upper and lower panels, respectively. IgG negative control is depicted as a grey trace. ChIP-exo tag 5’ ends were mapped to the +1 nucleosome dyad (as defined by MNase H3 ChIP-seq). H2B occupancy is depicted as a filled light grey trace. +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 nucleosomes positions in various Lge1 mutants. Table depicts fold enrichment of H2BK123ub density (ChIP-exo H2BK123ub/H2B) at canonical nucleosome positions +1, +2, and +3 for the indicated mutants relative to WT. Data for two biological replicates are shown. BY4741 is a positive control and correlates with WT Lge1 strain. Ratios for each gene class are indicated, to directly compare with graphs shown in Fig. 4a and Extended 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 calculated.
a. The WAC and LAF1 IDRs promote LLPS. Phase-separation assay of recombinant 6His-Lge1 and the fusion constructs 6His-WAC(1-318)-Lge1 CC and 6His-LAF1(1-169)-Lge1 CC (both proteins 5 μM; buffer 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 20 °C). Scale bar, 10 μm. Protein inputs are shown on the right (black arrows), b. Quantification of condensate sizes (μm2) in a. n = number of condensates. Dot plot showing median and interquartile range. **p-value = 0.004, ****p-value < 0.0001 determined by two-sided Mann-Whitney test. c. A synthetic genetic approach was used to interrogate the functionality of Lge1 LLPS in vivo. Cells were inviable when LGE1 and HTZ1 were deleted, indicating a functional relationship. Double deletion strains harboring a wild-type LGE1 cover plasmid (URA marker) were co-transformed with the indicated plasmids (HIS marker). Growth was followed on SDC-HIS (loading control) and on SDC+5-fluoroorotic acid (5-FOA), which shuffles out the URA cover plasmid. Cells were spotted in 10-fold serial dilutions and incubated for two (SDC-HIS) or three days (5-FOA) at 30 °C. d. Live imaging of bre1Δ lge1Δ 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 mCherry construct was quantified across a line spanning the nucleus. For comparison, the FU value = 1 is marked with a horizontal dashed line, n = number of cells. FU, arbitrary fluorescence units. Scale bar, 2 μm. e. Cell lysates of strains in c and d were analyzed 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 bre1Δ lge1Δcells 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 the nuclear envelope, dashed lines the cell contours. Histograms represent pixel frequency of fluorescent intensity values. Scale bar, 2 μm. g. Coefficient of variation (CV) of the fluorescence intensity distribution of BiFC signals in f and Fig. 3e. The higher the CV, the greater the heterogeneity of the BiFC signal. A propensity for LLPS is suggested by an increased CV of the WAC (1-318)- Lge1 CC-VN construct. Dot plot showing median and interquartile range.n= number of cells. **p-value = 0.0024, ***p-value < 0.001 determined by two-sided Mann-Whitney test. h. Expression levels of Lge1-mCherry constructs used in i. Cells lysates were analyzed 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 SF2 for uncropped gels and western blots.
a. Bre1 and Lge1 organization. CC, Coiled-Coil domain (green), non-CC regions (grey); RBD, Rad6 Binding Domain; LBD, Lge1 Binding Domain; RING, Really Interesting New Gene domain; IDR, Intrinsically Disordered Region, b. Sucrose gradient analysis of Strep-Bre1 and GST-Lge1. Arrowheads label peak fractions. See SF1 for uncropped gels. c. LLPS assay with 6His-Lge1 constructs (1.5 μM) (cartoons). Scale bar, 10 μm. See also Video S1. d. Reconstitution of core-shell condensates with increasing amounts of mGFP-Bre1 added to preformed 6His-Lge1 condensates. Arrowheads label shell in DIC images. Scale bar, 2 μm.
a. Recruitment of Pacific Blue-labelled 6His-Rad6 (His-Rad6*) to Lge1 condensates with a Bre1 shell. Fluorescent intensities were quantified across single condensates. FU, arbitrary fluorescent units, n = number of condensates. Scale bar, 5 μm. b. Partitioning of Hoechst-labeled, reconstituted yeast oligonucleosome arrays (16xNCP*) into Lge1-Bre1 condensates. Scale bar, 5 μm. c. Time-resolved in vitro H2B ubiquitination assay performed under LLPS conditions. +/− refers to the indicated Lge1 constructs used at equimolar concentration. Intensity of the H2B~Ub band was quantified and normalized to 12 min timepoint without Lge1 for each assay, n = independent experiments. Mean and standard deviation are indicated.
a. Sucrose gradient analysis of cell extracts from Ige1Δ cells transformed with the indicated plasmids. Ribosomal species serve as size markers. Gradient fractions were analyzed by immunoblotting. Peak fractions are highlighted (arrowheads), b. Live imaging of bre1Δ lge1Δ cells co-expressing the indicated constructs from a strong GPD promoter. Scale bar, 2 μm. c. Bimolecular Fluorescence Complementation (BiFC) design. Complementary Venus fragments: VN, VC. d. Live imaging of bre1Δ lge1Δ cells expressing VC-Bre1 and the indicated Lge1-VN constructs from their endogenous promoters. Arrowheads label nuclear BiFC puncta, Nup188-mCherry the nuclear envelope, dashed line the cell contour. Histograms represent pixel frequencies of fluorescence intensity values. Scale bar, 2 μm. e. Coefficient of variation (CV) of nuclear BiFC fluorescence intensity profiles (see Extended Data Fig. 7i). The higher the CV, the greater the heterogeneity of the BiFC signal. Median and interquartile range are indicated, n = number of analyzed cells. **p-value = 0.0037 and ***p-value < 0.001 determined by two-sided Mann-Whitney test.
a. H2B (left) or H2BK123ub (right) ChIP-exo tag 5’ ends were plotted relative to the + 1 nucleosome of all genes for WT (gray trace) and the indicated lge1 mutants (red traces). The first three genic nucleosomes are labeled +1, +2 and +3. Two H2B peaks are observed per nucleosome position. H2BK123ub patterns are shown separately for each gene class. b. Genetic interaction analysis. Double deletion strains harboring a wild-type LGE1 cover plasmid (URA marker) were co-transformed with the indicated plasmids (HIS marker). Growth (10-fold serial dilutions) was followed on SDC-HIS (control) and on SDC+5-fluoroorotic acid (5-FOA), which shuffles out the URA cover plasmid. c. LLPS-based ubiquitination model. The Htz1-containing +1 NCP is depicted outside of the ‘reaction chamber’ as it is ubiquitinated independently of Lge1 LLPS. For simplicity, Rad6 and the E1 are omitted and only a single layer of Bre1 molecules in the shell is shown. TSS, transcription start site; NFR, nucleosome free region.
Comment in
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Chromatin modified in a molecular reaction chamber.Nature. 2020 Mar;579(7800):503-504. doi: 10.1038/d41586-020-00638-9. Nature. 2020. PMID: 32161343 No abstract available.
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Liquid phase condensation directs nucleosome epigenetic modifications.Signal Transduct Target Ther. 2020 May 6;5(1):64. doi: 10.1038/s41392-020-0166-2. Signal Transduct Target Ther. 2020. PMID: 32376821 Free PMC article. No abstract available.
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