Journal: Nature Communications

Article Title: Clr4 SUV39H1 ubiquitination and non-coding RNA mediate transcriptional silencing of heterochromatin via Swi6 phase separation

doi: 10.1038/s41467-024-53417-9

Figure Lengend Snippet: a Chromatin immunoprecipitation and sequencing (ChIP-seq) reads for H3K9me2 (blue), H3K9me3 (green) and RNA Pol II (Rpb1, red) were mapped to centromere 3 ( cnt3 ) right arm in the indicated strains. Relative enrichments of ChIP-seq reads from each strain were divided by the total count in WT. Scales for dcr1Δ ubc4-1 and clr4Δ H3K9me2 and H3K9me3 were expanded to visualize low reads. A schematic diagram of chromosome centromere 3 right arm showing innermost repeat ( imr ), outermost repeats ( dg , dh ) and boundary region ( irc ) is shown. Vertical red lines and pink boxes represent tRNA and euchromatic genes respectively. b , d Western blot (WB) analyses of immunoprecipitated Flag-Clr4 (Flag-Clr4 IP) in the indicated strains. The ratio of ubiquitinated Clr4 (Clr4-Ub) compared to unmodified Clr4 is indicated below each lane. Bottom, WB analysis of Flag-Clr4 from whole cell extract (WCE). Molecular weight markers are shown and uncropped images are provided in Source Data. c Schematic diagram of domain structure of Clr4 protein and the position of lysine residues. Ovals show the position of all 36 lysines of Clr4, which are arranged in 6 sub-groups. The second sub-group (2–8 K) is highlighted and amino acid sequences are shown. 2–4 K sub-group includes K109-K114. Red dots represent putative mono-ubiquitination sites identified by in vitro ubiquitination and Mass Spec analyses. CD: chromodomain, PS: PostSET. e ChIP-seq reads of H3K9me2 (blue) and H3K9me3 (green) mapped to chromosome 3 centromere right arm in the indicated strains. f sRNA-seq reads mapped to centromere 3 right arm in indicated strains. g Left, schematic diagram showing the presumptive subunit arrangement in the complex of Ubc4-CLRC and its substrate Clr4 for mono-ubiquitination. Red circle (U), ubiquitin. Right, model for the binding of Clr4 and Swi6 to existing H3K9me3 and the activity of Clr4 for converting H3K9me2 to H3K9me3. Blue circle, H3K9me2 and green circle, H3K9me3. Created in BioRender. Kim, H. (2024) BioRender.com/r28w184.

Article Snippet: For in vitro ubiquitination, recombinant 6xHis-Clr4 protein or 3xFlag-Clr4 purified form S. pombe cells were mixed with human Ubiquitin-activating Enzyme (UBE1, R&D systems E-305), human UbcH5c/UBE2D3 (R&D systems E2-627), human Ubiquitin (R&D systems U-100H) or human HA-Ubiquitin (R&D systems U-110), Mg-ATP (R&D systems B-20) and 10x E3 Ligase Reaction Buffer (R&D systems B-71) with or without CLRC complex purified from S. pombe cells.

Techniques: Chromatin Immunoprecipitation, Sequencing, ChIP-sequencing, Western Blot, Immunoprecipitation, Molecular Weight, In Vitro, Mass Spectrometry, Binding Assay, Activity Assay

Journal: Nature Communications

Article Title: Clr4 SUV39H1 ubiquitination and non-coding RNA mediate transcriptional silencing of heterochromatin via Swi6 phase separation

doi: 10.1038/s41467-024-53417-9

Figure Lengend Snippet: a ChIP-seq reads of Flag-Clr4 and RNA Pol II (Rpb1) and sRNA-seq reads mapped to chromosome 3 centromere ( cnt3 ) right arm in indicated strains. N/A, not available. b ChIP-qPCR assay showing enrichment of Flag-Clr4 at centromeric dh repeat in the indicated strains. Data are presented as mean ± SD ( n = 3). P values are from one-way ANOVA test (Dunnett’s multiple comparisons test) without any adjustment. P = 0.0271 for ubc4-1 , P = 0.0081 for cul4-1 and P < 0.0001 for rik1Δ, raf1Δ and raf2Δ . c RNA immunoprecipitation (RNA IP) and qPCR of Flag-Clr4 to centromeric dg and act1 transcript in the indicated strains. Data are presented as mean ± SD ( n = 3). P values are from one-way ANOVA test (Dunnett’s multiple comparisons test) without any adjustment. P < 0.0001 for ubc4-1 and cul4-1 ( dg , left). ns, not significant ( act1 , right). d Binding assay of in vitro ubiquitinated His-Clr4 protein with nucleosomes with unmodified histone H3 (me0) or H3K9me3 histone (me3) anchored to streptavidin beads. Input and His-Clr4 proteins from pull-down were analyzed by WB analyses using antibodies as shown. U, ubiquitin. HA-Ub, HA-ubiquitin. Schematic diagram (left). Molecular weight markers (KDa) are shown on left side of panels (right) and uncropped images are provided in a Source Data file. e Schematic diagram for tethering of GBD-Clr4- ΔCD to 5xUAS-ade6 locus with free Clr4 protein. Primers for ChIP-qPCR are indicated (left). Right, assay for silencing of ade6 on Low Ade medium in the indicated strains. Silencing (red color) depends on Ubc4 and Cul4. Bottom, ChIP-qPCR assays showing enrichment of H3K9me2 and H3K9me3 at ade6 in the indicated strains. Data are presented as mean ± SD (n = 3). P values are from one-way ANOVA test (Dunnett’s multiple comparisons test) without any adjustment. P < 0.0001 for 2, 3 and 4 (H3K9me2, Left). P < 0.0001 for 2, 3 and 4 (H3K9me3, Right). Created in BioRender. Kim, H. (2024) BioRender.com/r28w184.

Article Snippet: For in vitro ubiquitination, recombinant 6xHis-Clr4 protein or 3xFlag-Clr4 purified form S. pombe cells were mixed with human Ubiquitin-activating Enzyme (UBE1, R&D systems E-305), human UbcH5c/UBE2D3 (R&D systems E2-627), human Ubiquitin (R&D systems U-100H) or human HA-Ubiquitin (R&D systems U-110), Mg-ATP (R&D systems B-20) and 10x E3 Ligase Reaction Buffer (R&D systems B-71) with or without CLRC complex purified from S. pombe cells.

Techniques: ChIP-sequencing, RNA Immunoprecipitation, Binding Assay, In Vitro, Molecular Weight