Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Schematic representation of the recruitment of PRC2.1 and PRC2.2. MTF2 binds to DNA, while the EED subunit of core PRC2 (orange) binds to H3K27me3 as part of an allosteric feedback loop. The EZH2 subunit of core PRC2 catalyses H3K27 methylation. The PRC2.2 complex contains JARID2 but not MTF2. Both contain the core PRC2 subunits, however the interactions of the PRC2.1 and PRC2.2-specific subunits with chromatin are different. The arrow from JARID2 to DNA is dashed as DNA binding has been shown in vitro but not in vivo b) PRC2.1 (MTF2) and PRC2.2 (JARID2) co-localize to all EZH2 targets. c-f) Heatmap and rpkm quantification (boxplots) of PRC2 subunits and the catalytic product H3K27me3. EZH2 recruitment is heavily affected by the absence of MTF2, while JARID2 and H3K27me3 absence have minor effects (c). The effect of MTF2 and JARID2 on EZH2 recruitment is reflected on H3K27me3 deposition (d). MTF2 is marginally affected by H3K27me3 removal, but its binding is reduced to approximately half the WT level in the absence of JARID2 (e). JARID2 recruitment is strongly reduced in the absence of either H3K27me3 or MTF2 (f). ChIP profiles are highly reproducible g) Genome browser examples of PRC2 binding to classical Polycomb targets. Box plots represent median and interquartile range (IQR; whiskers, 1.5 IQR).

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Methylation, Binding Assay, In Vitro, In Vivo

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a-d) Heatmap of WT ChIP-seq signal on the indicated peak set. H3K27me3-negative JARID2 peaks were excluded from further analysis. e) Venn diagram showing the overlap of peaks called for the ChIP-Seq of each protein independently. f) Mass spectrometry quantification of PRC2 subunits in the different cell lines. Detection of JARID2 and MTF2 in the respective mutants (asterisks) is due to value imputation in Perseus. g) Western blot validation of EED226 depletion of H3K27me3, for the ChIP shown in Figs 1 and 2. h) Scatterplot of peak RPKM showing high reproducibility of ChIP replicates.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: ChIP-sequencing, Mass Spectrometry, Western Blot

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Clustering of all PRC2 targets using ChIPseq data in multiple PRC2 mutants. Cluster 1-4 are unmethylated CpG islands (strong BioCap) signal, showing bivalent marks in WT (H3K4me3 and H3K27me3). These regions display heavy reduction of EZH2 recruitment in the MTF2 mutant, milder effects of H3K27me3 absence (EED226 treatment), and little or no effect of JARID2 absence. The intensity of MTF2 binding depends on both H3K27me3 and JARID2 but binding is still clearly detectable even in the absence of PRC2 core ( Eed -/- ) indicating a primary binding to DNA, reinforced by other mechanisms, such as JARID2-mediated recruitment, which in turn also depends on both H3K27me3 and MTF2. Cluster 5 and 6 have lower BioCap and H3K4me3 signal, and, while still affected by the absence of MTF2, this has a much less marked effect on recruitment of both EZH2 and JARID2, and on H3K27me3 deposition. b) WT-normalized, input-subtracted RPKM quantification of signal shown in (a). c) Quantification of GCG trinucleotides matching DNA shape requirement for MTF recruitments as defined in . Cluster 1-4 are strongly enriched in shape-matching GCGs, indicating potential for strong DNA-mediated MTF2 recruitment. d) Enrichment of anatomical terms in the genes associated with peaks in the six clusters. Enrichment within PRC2 targets. Cluster 4 show strong enrichment for CNS structures, cluster 5 and 6 for limb and branchial arches tissues and mesenchyme. See for the full overview.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Heatmap showing the cluster specific effect of H3K27me3 depletion on the binding of EZH2. WT and MTF2 GT/GT show mild reduction of EZH2 binding when treated with EED226 inhibitor, while the treatment is highly synergistic with the depletion of JARID2. b) Bootstrapping-based RPKM quantification (methods) of the signal in (a). Each coloured dot represent the median of one round of bootstrapping, grey bar represent 99.9% confidence interval for the mean of bootstrapped values in each condition and cluster. c) Treatment with EED226 further affected MTF2 recruitment in Jarid2 -/- and JARID2 recruitment in Mtf2 GT/GT , with the former leading to recruitment patter closely resembling the Eed -/- line (cf. ), highlighting the recruitment differences between cluster 1-4 and 5-6. d) Bootstrapping-based RPKM quantification (methods) of the signal in (c) similar as in 3b. e) Genome browser view of example Polycomb targets. For each genotype two tracks are overlaid: the darker colour represent EED226 treated samples, the lighter colour untreated cells.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Heatmap showing EZH2, MTF2 and JARID2 binding in the absence of H3K27me3 in PRC2 and PRC1 mutant lines. In the absence of H3K27me3, JARID2 and RING1A/B mutant phenocopy each other with regard to EZH2 and MTF2 binding, suggesting JARID2 and RING1B act in the same PRC2 recruitment mechanism. JARID2 recruitment is also strongly affected by the absence of RING1A/B, in line with the JARID2-mediated PRC2 recruitment via binding to PRC1-deposited H2AK119ub. b-d) Average plot of the ChIP signal shown in (a), for EZH2 (b) MTF2 (c) and JARID2 (d) centred on called peaks. Lower panels represent the same data with cropped y axis, for better visualization. e) Heatmap showing Ring1b binding in the discussed conditions. Ring1b is only mildly affected by removing H3K27me3 using EED226 (~40%). Binding is further attenuated in MTF2 and JARID2 mutant ESCs. f) Average plot of the ChIP signal shown in (e), centred on called peaks. g) Examples of loci of the data as shown in (e).

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) On PRC2.1 main targets (clusters 1-4) relatively little MTF2 binding is sufficient to kick start the EED positive feedback loop which heavily relies on JARID2. As primary recruitment is mediated to a large extent via MTF2, such a loop can still exist in the absence JARID2. In the absence of H3K27me3, an alternative route can take over that requires JARID2 binding to H2AK119ub. b) On PRC2.2/PRC1 targets (clusters 5-6), instead, Polycomb binding is initiated by PRC1 that, upon H2AK119ub deposition, is followed by JARID2-containing PRC2.2. These regions also see the presence of MTF2 in physiological conditions, but this is the result of indirect recruitment via the PRC2 core binding to PRC2.2-initiated H3K27me3 deposition.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Binding Assay

Journal: eLife

Article Title: A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation

doi: 10.7554/eLife.08536

Figure Lengend Snippet: ( A ) In vitro ubiquitination of TBP by HeLa cell lysates. In vitro transcribed and translated HA-TBP was incubated with indicated HeLa cellular fractions for ubiquitination assays. After the reaction, immunopurified HA-TBP was analyzed western blot using an antibody against ubiquitin. ( B ) Fractions eluted from the ion exchange (D52) column were assayed in the presence of E1 (UBE1), E2 (UbcH5b), ubiquitin, ubiquitin aldehyde (deubiquitinase inhibitor) and MG132 (proteasome inhibitor). TBP ubiquitination was analyzed by western blots using anti-TBP antibody. ( C ) Chromatography scheme of purification of TBP E3 ligase. Hela cytoplasmic fraction (S100) was subjected to a series of chromatographic fractionation as indicated. ( D ) TBP E3 ligase migrates at a size around 440–660 kDa. Input (In) and Superose 6 fractions were assayed as in ( B ). Motilities of the peak activity (440–660 kDa) are shown (bottom). ( E ) TBP E3 ligase activity after the final Mono Q chromatography step. Reactions containing Input (In) and Mono Q fractions were performed as in ( B ). ( F ) The abundance of Huwe1 protein positively correlates with levels of TBP E3 ligase activity within selected MonoQ fractions. Protein compositions of selected MonoQ fractions were determined by Mudpit (Multidimensional Protein Identification Technology). Shown are numbers of detected peptides and percentage of coverage for Huwe1 protein in indicated fractions. DOI: http://dx.doi.org/10.7554/eLife.08536.005

Article Snippet: 2 μg of ubiquitin (U-100, Boston Biochem, Cambridge, MA), 50 ng E1 (E-305, Boston Biochem), 100 ng UbcH5b (E2-622, Boston Biochem), 2 μM ubiquitin aldehyde (U-201, Boston Biochem), 10 μM MG132 (474790, EMD Millipore) and 10 μg of S100, partial purified fractions or purified recombinant Huwe1 protein for one and a half hour.

Techniques: In Vitro, Ubiquitin Proteomics, Incubation, Western Blot, Chromatography, Purification, Fractionation, Activity Assay

Journal: eLife

Article Title: A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation

doi: 10.7554/eLife.08536

Figure Lengend Snippet: ( A ) PageBlue SDS-PAGE Gel of purified recombinant wild-type (WT), catalytic domain truncation (ΔC) and catalytic site C4341S mutant (C → S) Huwe1 proteins are shown. HiMark pre-stained protein marker (Invitrogen, highest molecular weight 460 kDa) was used to ensure the purification of full-length proteins. ( B ) Huwe1 ubiquitinates TBP in a dose-dependent manner. Titrations (twofold concentration range) of recombinant His-tag Huwe1 protein were incubated with GST-TBP supplemented with E1, UbcH5b, methyl-ubiquitin, ATP regenerating mix, and ubiquitin aldehyde. After the reaction, TBP ubiquitination was analyzed through western blot using an anti-TBP antibody. ( C ) Hect domain of Huwe1 is required for the TBP ubiquitination activity. Wildtype (WT), Hect domain truncated (ΔC) and catalytic site C4341S (C → S) Huwe1 mutants were used in the ubiquitination assay as in ( B ). ( D ) Hect domain alone is insufficient for Huwe1's TBP ubiquitination activity. Two different doses of His-tag Huwe1 (over twofold range) and Hect-domain containing E3 E6AP (over twofold range) were used in the ubiquitination assay. ( E ) Huwe1 E3 activity can be activated by UbcH5 family E2s and UbcH7. A panel of different E2 conjugating enzymes were tested by the in vitro ubiquitination assays. DOI: http://dx.doi.org/10.7554/eLife.08536.008

Article Snippet: 2 μg of ubiquitin (U-100, Boston Biochem, Cambridge, MA), 50 ng E1 (E-305, Boston Biochem), 100 ng UbcH5b (E2-622, Boston Biochem), 2 μM ubiquitin aldehyde (U-201, Boston Biochem), 10 μM MG132 (474790, EMD Millipore) and 10 μg of S100, partial purified fractions or purified recombinant Huwe1 protein for one and a half hour.

Techniques: SDS Page, Purification, Recombinant, Mutagenesis, Staining, Marker, Molecular Weight, Concentration Assay, Incubation, Ubiquitin Proteomics, Western Blot, Activity Assay, In Vitro

Journal: eLife

Article Title: A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation

doi: 10.7554/eLife.08536

Figure Lengend Snippet: ( A ) Recombinant Huwe1 protein ubiquitinates TBP in vitro. Titrations (twofold concentration range) of recombinant His-tag Huwe1 protein are incubated with GST-TBP supplemented with E1, UbcH5b, wild-type ubiquitin and ATP regenerating mix, ubiquitin aldehyde in in vitro ubiquitination assays. After the reaction, TBP ubiquitination was analyzed through western blot using the anti-TBP antibody. ( B ) Hect domain of Huwe1 is required for its TBP ubiquitination activity. Wildtype (WT), Hect domain truncation (ΔC) and catalytic site mutant (C → S) Huwe1 were assayed as in ( A ) to test their activities to ubiquitinate TBP. ( C ) Hect domain alone is insufficient for Huwe1's TBP ubiquitination activity. Two different doses of His-tag Huwe1 (twofold range) and recombinant E6AP protein (twofold range) are used in the ubiquitination assay as in ( A ). ( D ) Huwe1 E3 activity can be supported by UbcH5 family E2s and UbcH7. A panel of different E2 conjugating enzymes is used in the in vitro ubiquitination assays. ( E ) Huwe1 mediated the K48-linked ubiquitination of TBP. Wild-type ubiquitin (WT), lysine 11 to arginine mutant (K11R), lysine48 to arginine mutant (K48R), lysine63 to arginine mutant (K63R) and lysine-methylated ubiquitin (Methyl-Ub) are used in the ubiquitination assays. DOI: http://dx.doi.org/10.7554/eLife.08536.007

Article Snippet: 2 μg of ubiquitin (U-100, Boston Biochem, Cambridge, MA), 50 ng E1 (E-305, Boston Biochem), 100 ng UbcH5b (E2-622, Boston Biochem), 2 μM ubiquitin aldehyde (U-201, Boston Biochem), 10 μM MG132 (474790, EMD Millipore) and 10 μg of S100, partial purified fractions or purified recombinant Huwe1 protein for one and a half hour.

Techniques: Recombinant, In Vitro, Concentration Assay, Incubation, Ubiquitin Proteomics, Western Blot, Activity Assay, Mutagenesis, Methylation

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: UBE2D family proteins redundantly promote the ubiquitination of GSPT1 (A) Sequence alignment of human UBE2D family proteins using Clustal W 2.1. Note that the amino acid sequence identity among all 4 family proteins is close to 90%. (B) In vitro ubiquitination of GSPT1 MBP fusion protein by recombinant CRL4CRBN complex in the presence of UBE2G1, UBE2D1, UBE2D2, or UBE2D3, alone or in combination. Recombinant protein products as indicated were incubated with or without 80 μM CC-885 in the ubiquitination assay buffer at 30 °C for 2 hours, and then analyzed by immunoblotting. SE, short exposure; LE, long exposure

Article Snippet: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

Techniques: Sequencing, In Vitro, Recombinant, Incubation, Ubiquitin Assay, Western Blot