Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a EZH2 ChIP peak profiles of gene Otx2 for WT, Mtf2 null, and Jarid2 null cells. Peaks were RPKM normalized and scaled across backgrounds. b Phase-contrast of Embryoid Bodies from different background and time point of differentiation. Each differentiation time point and background were performed in replicates. c , d Single-cell UMAPs of 4949 cells over all backgrounds and time points. e Single-cell UMAP of cells colored by predicted clusters. f – i Feature maps of selected lineage genes, color intensity based on normalized expression of individual gene.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Expressing

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a Heatmap of differentially expressed genes comparing different mutants against WT in undifferentiated mESCs. Z-score normalized counts (row scaling) are shown in heatmap. b Venn diagram depicting overlap of a number of genes which were up/downregulated in Mtf2 null and Jarid2 null cells, with PRC2 targets (as determined from EZH2 ChIP targets; next panel). Significance of overlap calculated using hyper-geometrical test. c Heatmap of EZH2 binding peaks (RPKM normalized) for upregulated genes (from Mtf2 and Jarid2 null ESCs) for different genetic backgrounds. Heatmap depicts a window of +/−400 bp from the transcription start site (TSS) of the genes. d Dot plot showing the enriched Gene Ontology terms for the differentially expressed genes in different genetic backgrounds.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a ChIP peak profiles of histone mark H3K27me3 and H3K4me3 for selected upregulated ( Mtf2 null) lineage transcription factors for WT and Mtf2 null cells at pluripotent stage. ChIP profiles were RPKM normalized and scaled between two merged profiles per histone ChIP. b Boxplots depicting the RPKM values of promoter (as defined by +/−500 bp from TSS) H3K27me3, H3K4me3, and EZH2 for all PRC2 targets and the upregulated genes in Mtf2 null and Jarid2 null cells. Asterisk(*) represents a Two-sample Kolmogorov–Smirnov test p -value <0.05. n = 2878 for all PRC2 targets, n = 242 for upregulated Mtf2 null genes and n = 58 for upregulated Jarid2 null genes. Whisker ends of boxplot represent the maximum (top) and minimum values, respectively. Top and bottom of boxplots represent 75th and 25th percentile values, respectively, and finally, median values are shown as colored lines within the boxplots. c Transcription factor motif activity that explains part of the variance in transcript levels, based on motifs in the promoters (as defined by +/−500 bp from TSS) of all upregulated PRC2-bound genes (“Methods”; upregulated genes in all four cell lines, cf. Fig. ). Motifs are shown in aggregated z-scores. d – f Heatmaps showing the mRNA fold change, H3K27me3 and H3K4me3 levels for a set of transcription factors (left, identified in panel c ) and signaling factors (right, identified from Mtf2 and Jarid2 null DEGs list). Genes shown are all PRC2 targets. g Barplot depicting the GATA2 ChIP recovery relative to input. Control is a gene desert region (“Methods”). Dots in bars represent 4 replicates per sample.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Whisker Assay, Activity Assay, Control

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: a Schematic of directed differentiation for monolayer cells from different genetic backgrounds (WT, Jarid2 null, and Mtf2 null). b Line plots showing the expression (normalized counts from RNA-seq) of selected temporally regulated genes during early lineage specification processes for different genetic backgrounds and differentiation directions. c , d Heatmap of differentially expressed genes across all time points and between genetic backgrounds. Data was k-means clustered and the normalized counts were shown. e , f Dot plots showing the enrichment of biological pathways for each cluster in ( c , d ), selected by p -value and gene-ratios of the terms. Top selected pathways were picked for each cluster and shown here. g , h Barplots of of H3K27me3 and H3K4me3 ChIP for selected targets ( Eomes and Gata6) . Each bar represents the average of the percentage of input recovered in the ChIP experiment of replicate experiments. Mtf2 WT and Jarid2 WT represent the background-matched wild-type lines of, respectively, Mtf2 null and Jarid2 null cells. Each dot represents a qPCR technical replicate for the sample.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Expressing, RNA Sequencing

Journal: Nature Communications

Article Title: Loss of PRC2 subunits primes lineage choice during exit of pluripotency

doi: 10.1038/s41467-021-27314-4

Figure Lengend Snippet: Model of the role of PRC2 in the exit of pluripotency. PRC2 contributes to a repressive threshold for activation of key regulators of differentiation. This affects the exit of pluripotency and the differentiation to lineages of the three germ layers. One example is the upregulation of GATA2 upon MTF2 loss which results in increasing expression of other factors such as WNT7B and IRX3, as part of a feedforward loop that regulates the exit of pluripotency.

Article Snippet: ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Techniques: Activation Assay, Expressing

Journal: European Journal of Human Genetics

Article Title: An MTF1 binding site disrupted by a homozygous variant in the promoter of ATP7B likely causes Wilson Disease

doi: 10.1038/s41431-018-0221-4

Figure Lengend Snippet: Genomic context of the candidate causative variant. a UCSC Genome Browser (hg19 minus strand) view of the region near the candidate causative variant positioned 676 bp upstream of the canonical ATP7B translation start site (chr13:g.52,586,149A>G, cyan highlight and asterisk). The variant lies in a 100-way vertebrate alignment conserved element and region of high cross-species sequence conservation as computed by PhastCons and PhyloP [30]. The chromatin surrounding chr13:g.52,586,149T>C is hypersensitive to DNaseI (and, therefore, open and accessible) in HepG2 cells. The sequence cloned into the luciferase reporter vector is indicated by the black bar. b Genotype verification for chr13:g.52,586,149T>C (red asterisk) by Sanger sequencing. Representative chromatograms show the indicated number (n=x/y) of amplification products containing the reference (T) or alternate (C) allele for each individual. Box indicates a predicted binding site for MTF1. c The candidate causative variant (bolded, pink) in the Wilson Disease (WD) patient disrupts a key base in the MTF1 position weight matrix. This exact base (boxed) is unaltered in all primates and in dozens of other mammals, even as distant as Tasmanian devil (Supplementary Fig. 1b) (color figure online).

Article Snippet: ChIP-qPCR Polyclonal rabbit anti-MTF1 antibody (Proteintech, Rosemont, IL, USA: 25383-1-AP) was used for chromatin immunoprecipitation.

Techniques: Variant Assay, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Amplification, Binding Assay

Journal: European Journal of Human Genetics

Article Title: An MTF1 binding site disrupted by a homozygous variant in the promoter of ATP7B likely causes Wilson Disease

doi: 10.1038/s41431-018-0221-4

Figure Lengend Snippet: Functional interrogation of chr13:g.52,586,149T>C. a Luciferase reporter assays in HepG2 cells were performed to quantify differences in transactivation by a 379 bp fragment of the ATP7B promoter with the chr13:g.52,586,149T (Ref) or chr13:g.52,586,149C (Alt) allele, in the presence (+) or absence (−) of MTF1 overexpression (OE). Both with and without MTF1 OE, the reference allele drove significantly higher expression compared to the alternate allele (comparison 1 and 2, respectively). Both alleles experienced dramatic increases in activity with MTF1 OE (comparisons 3 and 4), but the reference allele yielded a greater increase in expression than did the alternate allele in this context (comparison 5). Bars represent mean ± SD. Two-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. b ChIP-qPCR was performed to determine the extent of MTF1 binding at the SNV locus (ATP7B) compared to a computationally predicted negative control (Neg Ctrl, in an intron of WDPCP) and to a previously published [32] experimentally validated locus (Pos Ctrl, in the 5′ UTR of SELENOH). Enrichment of DNA immunoprecipitated by MTF1-specific vs. non-specific isotype-matched control antibodies was measured in technical triplicate by quantitative PCR analysis. Bars represent mean ± SD. One-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. c Proposed model for disease-causing mechanism of chr13:g.52,586,149T>C: (1) Excess intracellular accumulation of copper, Cu2+, increases (2) expression or nuclear translocation of MTF1 [12, 13]. In wildtype individuals, MTF1 then binds at chr13:g.52,586,149T (3) to recruit transcriptional machinery for upregulating ATP7B expression [34] and eliminating copper through serum ceruloplasmin and, ultimately, through the bile. We propose that the Wilson Disease (WD) patient’s homozygous single nucleotide promoter variant chr13:g.52,586,149T>C exhibits reduced affinity to MTF1. (4) The result is an insufficient ATP7B transcriptional response, consequent copper accumulation, and symptoms characteristic of Wilson Disease

Article Snippet: ChIP-qPCR Polyclonal rabbit anti-MTF1 antibody (Proteintech, Rosemont, IL, USA: 25383-1-AP) was used for chromatin immunoprecipitation.

Techniques: Functional Assay, Luciferase, Over Expression, Expressing, Comparison, Activity Assay, Two Tailed Test, ChIP-qPCR, Binding Assay, Negative Control, Immunoprecipitation, Control, Real-time Polymerase Chain Reaction, One-tailed Test, Translocation Assay, Variant Assay

Journal: Sensors and Actuators B: Chemical

Article Title: Optogenetic STING clustering system through nanobody-fused photoreceptor for innate immune regulation

doi: 10.1016/j.snb.2023.134822

Figure Lengend Snippet: Fig. 3. Blue light-induced clustering of FL-OptoSTING and its subcellular localization. (A) Schematic of mCherry fused αGFP-CRY2 construct and FP-fused FL- OptoSTING construct. (B) Fluorescence images of HeLa cells co-expressing an ER marker with either STING(1−378)-EYFP or EYFP-STING(1−378). (C) Fluorescence images of HeLa cells co-expressing #7 (αGFP-mCherry-CRY2) and #8 (EYFP-STING(1−378)). Cells were illuminated by blue light for 3 min at 20-s intervals. Power density of light was fixed at 1 mW mm−2. Enlarged images of indicated white box in right bottom. (D) Fluorescence images of HeLa cells expressing #8 with either mTFP1-fused Golgi or ER marker. (E) Analysis of co-localization of EYFP-STING(1−378) and indicated organelle markers from D. Graph represents the mean and SD. Scale bars, 20 µm in B, C, and D and 2 µm in enlarged image in C, respectively.

Article Snippet: Chemical 399 (2024) 134822 (Clontech) was PCR-amplified using mCherry-F and mCherry-R and inserted between αGFP and CRY2 at the AgeI site using InFusion cloning. mTFP1 sequence from mTFP1-C1 (Addgene plasmid #54613) was digested via AgeI and BsrGI and ligated into pmTurquoise2-ER (Addgene plasmid #36204) and pmTurquoise2-Golgi (Addgene plasmid #36205) after the excision of mTurquoise2 to generate mTFP1-ER and mTFP1-Golgi, respectively.

Techniques: Construct, Fluorescence, Expressing, Marker

Journal: bioRxiv

Article Title: DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

doi: 10.1101/354134

Figure Lengend Snippet: (A) Titration ELISA: DARPins 1238_E11 (left panel) and 1238_G01 (right panel) show specific binding to mTFP1 over control surfaces (GFP, mCherry and MBP). (B) Fluorescence anisotropy measurements of 1238_E11 (left panel) and 1238_G01 (right panel) reveal high affinities with K D values of 3 nM and 88 nM, respectively. (C) Epitope blocking ELISA. Immobilized mTFP1 was incubated with a mixture of HA-and FLAG-tagged DARPins with a relative 1:5 ratio (100 nM of the FLAG-tagged DARPins and 500 nM of the HA-tagged competitor) to analyse the influence of the competitor on the original signal (shown on left side, named “no competition“).

Article Snippet: The mitochondrial bait mito-mTFP1, containing an N-terminal anchor sequence from the human CISD1 protein (the first 59 amino acids) fused to the N-terminus of mTFP1, was generated from pcDNA4TO-mito-mCherry-10xGCN4_v4 (Addgene plasmid 60914 ( )) by substituting the mCherry coding sequence with that of mTFP1 and substituting the 10xGCN4_v4 tags with 1xGCN4_v4 tag. mTFP1-CAAX and HIST2BJ-mTFP1 were cloned into CMV expression vectors (pmKate2-N, Evrogen,) replacing mKate2 with mTFP1 coding sequences.

Techniques: Titration, Enzyme-linked Immunosorbent Assay, Binding Assay, Control, Fluorescence, Blocking Assay, Incubation

Journal: bioRxiv

Article Title: DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

doi: 10.1101/354134

Figure Lengend Snippet: (A) Cartoon representation of mTFP1 (green) and DARPin 1238_E11 (grey). The mTFP1 chromophore is shown in sphere representation. (B) Close-up view of the binding interface of mTFP1 (green) and DARPin 1238_E11 (grey). DARPin residues are labelled in bold; hydrogen bonding interactions are indicated by black dashed lines.

Article Snippet: The mitochondrial bait mito-mTFP1, containing an N-terminal anchor sequence from the human CISD1 protein (the first 59 amino acids) fused to the N-terminus of mTFP1, was generated from pcDNA4TO-mito-mCherry-10xGCN4_v4 (Addgene plasmid 60914 ( )) by substituting the mCherry coding sequence with that of mTFP1 and substituting the 10xGCN4_v4 tags with 1xGCN4_v4 tag. mTFP1-CAAX and HIST2BJ-mTFP1 were cloned into CMV expression vectors (pmKate2-N, Evrogen,) replacing mKate2 with mTFP1 coding sequences.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

doi: 10.1101/354134

Figure Lengend Snippet: Confocal images of HeLa cells transiently transfected (A) with pCMV-DARPin 1238_E11-mCherry alone, (B) pCMV-DARPin 1238_G01-mCherry alone, (C) pMITO-mTFP1 alone, (D) the combination of pMITO-mTFP1 and pCMV-DARPin 1238_E11-mCherry , (E) pMITO-mTFP1 and pCMV-DARPin 1238_G01-mCherry. The first column represents the mTFP1 channel (green), the second column is the mCherry channel (red), the third column is the overlay of the two channels, showing the mitochondrial colocalization (indicated in yellow) of the mito-mTFP1 bait with the respective DARPin, the fourth column represents the nuclear Hoechst staining (blue) and the fifth column is the merge of all three channels (with the scale bar in white (15 μm) on the bottom right corner). Images were taken 24 hours post transfection. Transfected constructs are indicated on top of each row and the different channels are indicated inside the panels of the first row. The figures are from a representative experiment, performed at least three times.

Article Snippet: The mitochondrial bait mito-mTFP1, containing an N-terminal anchor sequence from the human CISD1 protein (the first 59 amino acids) fused to the N-terminus of mTFP1, was generated from pcDNA4TO-mito-mCherry-10xGCN4_v4 (Addgene plasmid 60914 ( )) by substituting the mCherry coding sequence with that of mTFP1 and substituting the 10xGCN4_v4 tags with 1xGCN4_v4 tag. mTFP1-CAAX and HIST2BJ-mTFP1 were cloned into CMV expression vectors (pmKate2-N, Evrogen,) replacing mKate2 with mTFP1 coding sequences.

Techniques: Transfection, Staining, Construct

Journal: bioRxiv

Article Title: DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

doi: 10.1101/354134

Figure Lengend Snippet: Confocal images of HeLa cells transiently transfected (A) with pcDNA3-mTFP1-Rab5c alone, (B) pCMV-DARPin 1238_E11-YPet-CAAX alone , (C) pCMV-DARPin 1238_G01-YPet-CAAX alone, (D) the combination of pcDNA3-mTFP1-Rab5c and pCMV-DARPin 1238_E11-YPet-CAAX, (E) pcDNA3-mTFP1-Rab5c and pCMV-DARPin 1238_G01-YPet-CAAX. The first column represents the mTFP1 channel (green), the second column is the YPet channel (magenta), the third column is the overlay of the two channels, showing the recruitment of mTFP1-Rab5c to the plasma membranes, the fourth column represents the nuclear Hoechst staining (blue) and the fifth column is the merge of all three channels (with the scale bar in white (15 μm) on the bottom right corner). Images were taken 24 hours post transfection. Transfected constructs are indicated on top of each row and the different channels are indicated inside the panels of the first row. The figures are from a representative experiment, performed at least three times.

Article Snippet: The mitochondrial bait mito-mTFP1, containing an N-terminal anchor sequence from the human CISD1 protein (the first 59 amino acids) fused to the N-terminus of mTFP1, was generated from pcDNA4TO-mito-mCherry-10xGCN4_v4 (Addgene plasmid 60914 ( )) by substituting the mCherry coding sequence with that of mTFP1 and substituting the 10xGCN4_v4 tags with 1xGCN4_v4 tag. mTFP1-CAAX and HIST2BJ-mTFP1 were cloned into CMV expression vectors (pmKate2-N, Evrogen,) replacing mKate2 with mTFP1 coding sequences.

Techniques: Transfection, Clinical Proteomics, Staining, Construct

Journal: bioRxiv

Article Title: DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

doi: 10.1101/354134

Figure Lengend Snippet: Confocal images of HeLa cells transiently transfected with (A) pcDNA3-mTFP1-Rab5c alone, (B) pCMV-DARPin 1238_E11-YPet-H2B alone, (C) pCMV-DARPin 1238_G01-YPet-H2B alone, (D) the combination of pcDNA3-mTFP1-Rab5c and pCMV-DARPin 1238_E11-YPet-H2B, (E) pcDNA3-mTFP1-Rab5c and pCMV-DARPin 1238_G01-YPet-H2B. The first column represents the mTFP1 channel (green), the second column is the YPet channel (magenta), the third column is the overlay of the two channels, showing the recruitment of mTFP1-Rab5c to the nuclei, the fourth column represents the nuclear Hoechst staining (blue) and the fifth column is the merge of all three channels (with the scale bar in white (15 μm) on the bottom right corner). Images were taken 24 hours post transfection. Transfected constructs are indicated on top of each row and the different channels are indicated inside the panels of the first row. The figures are from a representative experiment, performed at least three times.

Article Snippet: The mitochondrial bait mito-mTFP1, containing an N-terminal anchor sequence from the human CISD1 protein (the first 59 amino acids) fused to the N-terminus of mTFP1, was generated from pcDNA4TO-mito-mCherry-10xGCN4_v4 (Addgene plasmid 60914 ( )) by substituting the mCherry coding sequence with that of mTFP1 and substituting the 10xGCN4_v4 tags with 1xGCN4_v4 tag. mTFP1-CAAX and HIST2BJ-mTFP1 were cloned into CMV expression vectors (pmKate2-N, Evrogen,) replacing mKate2 with mTFP1 coding sequences.

Techniques: Transfection, Staining, Construct

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: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

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: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

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: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

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: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

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: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

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) Heatmap of ChIP-seq signal for EZH2 in multiple conditions including Mtf2 cells with double inhibition (d.i.) using EED226 (to remove H3K27me3) and MG132 (to remove H2AK119ub). b) Example loci of the data shown in (a). c) Average profiles of the ChIP signal shown in (a), centred on called peaks. Lower panels represent the same data with cropped y axis, for better visualization.

Article Snippet: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

Techniques: ChIP-sequencing, Inhibition

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: Primary antibodies used were rabbit anti-MTF2 (ProteinTech; 16208-1-AP), rabbit anti-JARID2 (Novus Bio; NB100-2214), rabbit anti-H3K27me3 (Millipore; 07-449), rabbit anti-H3 (Abcam;1791).

Techniques: Binding Assay