Journal: American Journal of Human Genetics

Article Title: DNA Methylation Signature for EZH2 Functionally Classifies Sequence Variants in Three PRC2 Complex Genes

doi: 10.1016/j.ajhg.2020.03.008

Figure Lengend Snippet: Comprehensive Visualization of EZH2 Sequence Variants using ProteinPaint Schematic representation of EZH2 sequence variants included in the study. Each distinct variant in EZH2 is represented by a disc sized in proportion to the number of samples and filled with the color representing its class based on the legend. Missense variants which constitute the large proportion of variants in EZH2 are colored in red, nonsense variants in blue, and indels in green. Sequence variants are positioned by their amino acid coordinates based on EZH2 (GenBank: NM_004456.4 , hg19). The dotted vertical lines inside the protein delineate the boundaries of coding exons and the filled colors within the protein correspond to known protein domains.

Article Snippet: The luminescence assay to determine EZH2 enzymatic activity for the p.Ala738Thr sequence variant was performed at BPS Bioscience as follows: a 50 μl reaction mix containing 50 μM S-adenosylmethionine, EZH2 enzyme (as part of an artificially assembled PRC2 complex derived from baculovirus expression vectors), and 20 mM phosphate buffer (pH 7.4), 0.05% Tween-20 HMT buffer 2 (BPS #52170) was added to wells coated with the substrate.

Techniques: Sequencing, Variant Assay

Journal: American Journal of Human Genetics

Article Title: DNA Methylation Signature for EZH2 Functionally Classifies Sequence Variants in Three PRC2 Complex Genes

doi: 10.1016/j.ajhg.2020.03.008

Figure Lengend Snippet: EZH2 -Specific DNAm Signature Principal components analysis (PCA) plot (A) and corresponding hierarchical clustering (B) (Eucledian distance metrics) and representative heatmap of 31 samples (n = 8 WS; n = 23 controls) using the differentially methylated CpG sites comprising the EZH2 -specific DNAm signature (229 CpG sites). In both (A) and (B), samples labeled with red represent Weaver syndrome, blue samples are controls. On the heatmap, yellow indicates high DNAm and blue indicates low DNAm. For the heatmap, data are normalized for visualization (mean = 0, variance = 1).

Article Snippet: The luminescence assay to determine EZH2 enzymatic activity for the p.Ala738Thr sequence variant was performed at BPS Bioscience as follows: a 50 μl reaction mix containing 50 μM S-adenosylmethionine, EZH2 enzyme (as part of an artificially assembled PRC2 complex derived from baculovirus expression vectors), and 20 mM phosphate buffer (pH 7.4), 0.05% Tween-20 HMT buffer 2 (BPS #52170) was added to wells coated with the substrate.

Techniques: Methylation, Labeling

Journal: American Journal of Human Genetics

Article Title: DNA Methylation Signature for EZH2 Functionally Classifies Sequence Variants in Three PRC2 Complex Genes

doi: 10.1016/j.ajhg.2020.03.008

Figure Lengend Snippet: Testing the Sensitivity and Specificity of the EZH2- Specific Signature (A) Plot representing the median-methylation profiles of WS-affected individuals (y axis) and control subjects (x axis) using the EZH2 DNAm signature. The dashed line is set to represent the decision boundary for which individuals above the dashed lines have DNAm profiles more similar to EZH2 signature and below the dashed line have DNAm profiles more similar to control subjects. A set of independent WS-affected individuals (validation cohort, purple circles, n = 8) as well as a WS-affected family (light orange circles, n = 5 affected members) with EZH2 pathogenic variants were classified as “WS” (i.e., all individuals classified as more similar to the EZH2 signature than control subjects) indicating high accuracy of the EZH2 DNAm signature. The specificity of EZH2 signature was estimated on an independent control validation set of 148 control samples (green crossed boxes); all subjects classified as more similar to control subjects (specificity 100%). (B) Performance of the EZH2 signature on data generated on 450k array including overlapping WS-affected subjects from the discovery cohort n = 7 (red circles), controls n = 80 (blue squares), and GEO controls n = 718 (brown squares) generated on 450k array. All control subjects had DNAm profiles more similar to the control profile and were therefore classified as “not-WS.” WS, Weaver syndrome.

Article Snippet: The luminescence assay to determine EZH2 enzymatic activity for the p.Ala738Thr sequence variant was performed at BPS Bioscience as follows: a 50 μl reaction mix containing 50 μM S-adenosylmethionine, EZH2 enzyme (as part of an artificially assembled PRC2 complex derived from baculovirus expression vectors), and 20 mM phosphate buffer (pH 7.4), 0.05% Tween-20 HMT buffer 2 (BPS #52170) was added to wells coated with the substrate.

Techniques: Methylation, Generated

Journal: American Journal of Human Genetics

Article Title: DNA Methylation Signature for EZH2 Functionally Classifies Sequence Variants in Three PRC2 Complex Genes

doi: 10.1016/j.ajhg.2020.03.008

Figure Lengend Snippet: Testing the Ability of the EZH2 -Specific Signature to Classify EZH2 Variants (A) Plot representing the median-methylation profiles of WS-affected (y axis) and control subjects (x axis) using the EZH2 DNAm signature. A set of independent individuals with EZH2 sequence variants (pink squares, n = 19) were classified using the EZH2 signature. Of the 19 variants, ten classified as more similar to the EZH2 DNAm profile than controls. The remaining nine variants classified as more similar to the control profile. (B) Plot representing the Support Vector machine (SVM) scores (y axis). The SVM prediction model was used to predict pathogenicity of EZH2 variants based on the DNAm signature. All ten variants predicted as pathogenic in (A) had also very high SVM scores > 70% and the remaining nine variants had very low SVM scores < 20% except one variant with an SVM score of 49%. Blue arrow represents sample MDL#67845 (p.Ser669Asn). Orange arrow represents sample S126694 ( EZH2 _c.2196-2_2211dupAGATACAGCCAGGCTGAT). Green arrow represents sample A1646 (p.Ala738Thr). WS, Weaver syndrome.

Article Snippet: The luminescence assay to determine EZH2 enzymatic activity for the p.Ala738Thr sequence variant was performed at BPS Bioscience as follows: a 50 μl reaction mix containing 50 μM S-adenosylmethionine, EZH2 enzyme (as part of an artificially assembled PRC2 complex derived from baculovirus expression vectors), and 20 mM phosphate buffer (pH 7.4), 0.05% Tween-20 HMT buffer 2 (BPS #52170) was added to wells coated with the substrate.

Techniques: Methylation, Sequencing, Plasmid Preparation, Variant Assay

Journal: American Journal of Human Genetics

Article Title: DNA Methylation Signature for EZH2 Functionally Classifies Sequence Variants in Three PRC2 Complex Genes

doi: 10.1016/j.ajhg.2020.03.008

Figure Lengend Snippet: Gain-of-Function Variant in EZH2 Have Opposite DNA Methylation Profile at the EZH2 Signature (A) Heatmap showing the hierarchical clustering of the DNAm profile of WS individuals (n = 8, red) with LoF (hypomorphic) variants in EZH2 , controls (n = 23, blue) and EZH2 GoF variant (p.Ala738Thr; pink) using the EZH2 signature. On the heatmap, yellow indicates high DNAm and blue indicates low DNAm. The subject with an EZH2 variant in pink display opposite DNAm profile when compared to WS DNAm profiles. For the heatmap, data are normalized for visualization (mean = 0, variance = 1). (B) Enzymatic activity of EZH2 GoF variant using an in vitro luminescence assay. Mutant EZH2 (p.Ala738Thr) pre-assembled into PRC2 showed increased EZH2-mediated H3K27 methylation activity. WS, Weaver syndrome; GoF, gain of function; LoF, loss of function.

Article Snippet: The luminescence assay to determine EZH2 enzymatic activity for the p.Ala738Thr sequence variant was performed at BPS Bioscience as follows: a 50 μl reaction mix containing 50 μM S-adenosylmethionine, EZH2 enzyme (as part of an artificially assembled PRC2 complex derived from baculovirus expression vectors), and 20 mM phosphate buffer (pH 7.4), 0.05% Tween-20 HMT buffer 2 (BPS #52170) was added to wells coated with the substrate.

Techniques: Variant Assay, DNA Methylation Assay, Activity Assay, In Vitro, Luminescence Assay, Mutagenesis, Methylation

Journal: American Journal of Human Genetics

Article Title: DNA Methylation Signature for EZH2 Functionally Classifies Sequence Variants in Three PRC2 Complex Genes

doi: 10.1016/j.ajhg.2020.03.008

Figure Lengend Snippet: Testing the Utility of the EZH2 Signature in Classifying Sequence Variants in Other Components of the PRC2 Complex Using the EZH2 signature, we compared the DNAm profiles of three subjects with EED sequence variants (yellow triangles) and those with SUZ12 variants (green diamonds) to the DNAm profiles of controls (blue crossed boxes) and EZH2 pathogenic variants (red circles). All three subjects with pathogenic variants in EED had DNAm profiles more similar to the EZH2 profile than control subjects. Two subjects with SUZ12 pathogenic variants also classified with Weaver syndrome and the three remaining SUZ12 variants showed DNAm profiles more similar to controls.

Article Snippet: The luminescence assay to determine EZH2 enzymatic activity for the p.Ala738Thr sequence variant was performed at BPS Bioscience as follows: a 50 μl reaction mix containing 50 μM S-adenosylmethionine, EZH2 enzyme (as part of an artificially assembled PRC2 complex derived from baculovirus expression vectors), and 20 mM phosphate buffer (pH 7.4), 0.05% Tween-20 HMT buffer 2 (BPS #52170) was added to wells coated with the substrate.

Techniques: Sequencing

Journal: American Journal of Human Genetics

Article Title: DNA Methylation Signature for EZH2 Functionally Classifies Sequence Variants in Three PRC2 Complex Genes

doi: 10.1016/j.ajhg.2020.03.008

Figure Lengend Snippet: Classification of Subjects with Syndromic Overgrowth using EZH2 Signature Plot representing samples with sequence variants in epigenes associated with other overgrowth syndromes. These included subjects with: NSD1 pathogenic variants associated with Sotos syndrome (n = 49, GEO: GSE74432 ), DNMT3A pathogenic variants associated with Tatton-Brown Rahman syndrome (n = 5; GEO: GSE128801 ), and CHD8 pathogenic variants associated with macrocephaly and susceptibility to autism (n = 10; GEO: GSE113967 ). These data were compared to seven WS-affected subjects from the discovery cohort and five test individuals with pathogenic mutations in EZH2 which were run on the Illumina 450k. All overgrowth syndrome individuals had DNAm profiles more similar to control subjects and distinguishable from WS.

Article Snippet: The luminescence assay to determine EZH2 enzymatic activity for the p.Ala738Thr sequence variant was performed at BPS Bioscience as follows: a 50 μl reaction mix containing 50 μM S-adenosylmethionine, EZH2 enzyme (as part of an artificially assembled PRC2 complex derived from baculovirus expression vectors), and 20 mM phosphate buffer (pH 7.4), 0.05% Tween-20 HMT buffer 2 (BPS #52170) was added to wells coated with the substrate.

Techniques: Sequencing

Journal: American Journal of Human Genetics

Article Title: DNA Methylation Signature for EZH2 Functionally Classifies Sequence Variants in Three PRC2 Complex Genes

doi: 10.1016/j.ajhg.2020.03.008

Figure Lengend Snippet: Testing the Ability of the EZH2 Signature in Classifying Undiagnosed OGID-Affected Subjects Based on Their DNAm Profiles OGID-affected subjects included in this analysis were previously tested negative for targeted mutations screening in NSD1 and EZH2 . Out of the 73 subjects with OGID (brown triangles), we identified that most had DNAm profiles similar to control subjects (blue squares). Interestingly, we identified two subjects with DNAm profiles more similar to the EZH2 profile than control samples. OGID, overgrowth and intellectual disability.

Article Snippet: The luminescence assay to determine EZH2 enzymatic activity for the p.Ala738Thr sequence variant was performed at BPS Bioscience as follows: a 50 μl reaction mix containing 50 μM S-adenosylmethionine, EZH2 enzyme (as part of an artificially assembled PRC2 complex derived from baculovirus expression vectors), and 20 mM phosphate buffer (pH 7.4), 0.05% Tween-20 HMT buffer 2 (BPS #52170) was added to wells coated with the substrate.

Techniques:

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) PARylation of EZH2 by PARP1 in vitro . Human EZH2/PRC2 complex (EZH2, EED, SUZ12, RbAP48 and AEBP2) was incubated alone (lane 1) or with the agents indicated at the top (250 nM of olaparib (PARP inhibitor) was used and NAD+ is necessary for PARP1 activity). After 1 hour, PARylation was blocked by adding olaparib to all samples and PARylated proteins were pulled-down by PAR-affinity resin and analyzed by western blot with anti-EZH2 and anti-PAR antibodies. PARylation appears as a smear due to the different sizes of the various PAR polymers. Input corresponds to 1/10 th the amount of protein used for immunoprecipitation. Input was immunoblotted with anti-PARP1 and anti-EZH2 antibodies. (B) Schematic of the experimental strategy for C) and D). Briefly, the EZH2/PRC2 complex was incubated with PARP1 in the presence or absence of NAD+ as in A). After 1 hour, PARylation was stopped with olaparib and PARP1 was removed by immunoprecipitation with an anti-PARP1 antibody. The EZH2/PRC2 complex was incubated with EZH2 substrates histone H3 and S-adenosyl methionine (SAM) to allow histone methylation to occur. After 30 minutes, histone methytransferase activity was determined by assessing H3K27me3 levels. (C) In vitro histone methyltransferase assay. As indicated in B), purified histone H3 and SAM were incubated with the agents indicated at the top. After 30 minutes, proteins were analyzed by western blot using anti-Histone H3, anti-H3K27me3 and anti-PAR antibodies. Input corresponds to 1/20 th the amount of the protein used for immunoblotting. Input was probed with an anti-EZH2 antibody. (D) Levels of EZH2 activity with (black) and without (grey) PARP1 activity. Extracts from the EZH2/PRC2 complex incubated with histone H3 and SAM as in lanes 2 and 4 from C) were assessed for H3K27me3 levels by ELISA. N=3 ± SD.

Article Snippet: After 30 minutes, samples were incubated at RT for 10 minutes and the enzymatic activity of EZH2/PRC2 complex was measured by the EpiQuick Histone Methyltransferase Activity/Inhibition assay (EpiGentek) according to manufacturer's protocol.

Techniques: In Vitro, Incubation, Activity Assay, Western Blot, Immunoprecipitation, Methylation, HMT Assay, Purification, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) Time course of in vitro PARylation assay. EZH2/PRC2 complex was incubated with PARP1, NAD+ and DNA fragments to allow in vitro PARylation. The reaction was blocked at different time points by adding the PARP inhibitor olaparib. After removing PARP1 from the reaction, PARylated proteins were pulled down with a PAR-affinity resin and analyzed by western blot with an anti-EZH2 antibody. Input corresponds to 1/10 th the amount of protein used for PAR pulldown. Input was probed with an anti-EZH2 antibody. (B) In vitro histone methylation assay. EZH2/PRC2 complex treated as in A) was incubated with histone H3 and SAM to allow methylation of lysine 27 of histone H3. After 30 minutes, histone H3 was extracted and H3K27me3 levels were measured by ELISA. EZH2 activity was calculated by setting H3K27me3 levels at time 0 as 100% EZH2 activity. N=3 mean ± SD. (C) In vitro histone methyltransferase activity assay. EZH2/PRC2 complex was incubated with PARP1 in the presence (PARylated) or absence (unmodified) of NAD+. After 1 hour, the reaction was blocked as in A) and EZH2/PRC2 complex was incubated with SAM and different concentrations of histone H3 to allow histone H3-K27 methylation to occur. After 30 minutes, the reaction was blocked and the amount of methylated histone H3-K27 generated by EZH2 activity was measured using an H3K27me3 ELISA kit. N=3, mean ± SD. (D) Time course of in vitro histone methyltransferase (HMT) activity. EZH2/PRC2 complex was treated as in C) and incubated with SAM and histone H3 to allow methylation of H3-K27. The reaction was blocked at different time points and the amount of methylated histone H3-K27 generated by EZH2 activity was measured by an H3K27me3 ELISA. N=3, mean ± SD.

Article Snippet: After 30 minutes, samples were incubated at RT for 10 minutes and the enzymatic activity of EZH2/PRC2 complex was measured by the EpiQuick Histone Methyltransferase Activity/Inhibition assay (EpiGentek) according to manufacturer's protocol.

Techniques: In Vitro, Incubation, Western Blot, Methylation, Enzyme-linked Immunosorbent Assay, Activity Assay, Generated

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) In vitro PARG assay. EZH2/PRC2 complex and PARP1 were incubated with or without PARG as indicated (Note: NAD+ is required for PARylation). After 1 hour, the reaction was blocked by addition of the PARP inhibitor olaparib and the EZH2/PRC2 complex was incubated with (lanes 2 and 4) or without (lanes 1 and 3) PARG to allow degradation of PAR polymers. After 1 hour, the reaction was stopped by adding Laemmli buffer and the proteins were analyzed by western blot using anti-EZH2 and anti-PAR antibodies. The upper band in the top panel represents PARylated EZH2. PARG activity was confirmed by reduction of PAR smear. (B) In vitro histone methyltransferase (HMT) activity assay. EZH2/PRC2 complex treated as in A) was subsequently assayed for histone methyltransferase activity using an HMT assay kit. The activity of EZH2 under the indicated conditions was calculated based on the amount of H3-K27 converted in the assay. As a control, the activity of EZH2/PRC2 complex alone was also determined. N=3, mean ± SD.

Article Snippet: After 30 minutes, samples were incubated at RT for 10 minutes and the enzymatic activity of EZH2/PRC2 complex was measured by the EpiQuick Histone Methyltransferase Activity/Inhibition assay (EpiGentek) according to manufacturer's protocol.

Techniques: In Vitro, PARG Assay, Incubation, Western Blot, Activity Assay, HMT Assay, Control

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) Schematic of experimental approach that couples histone PARylation and methylation in vitro . First, histone H3 was incubated with PARP1 in the presence or absence of NAD+ and olaparib to allow for PARylation. After 60 minutes, the reaction was blocked by addition of olaparib, PARP1 was removed by immunoprecipitation and the remaining histone H3 was either assessed for PARylation by PAR-resin pulldown or incubated with EZH2/PRC2 and SAM to allow H3-K27 methylation in vitro . After 30 minutes, the histone methyltransferase reaction was blocked and H3K27me3 levels were determined by different approaches. (B) Histone H3 PARylation decreases subsequent H3-K27 methylation. Histone H3 proteins treated as in A) were analyzed by western blot using an anti-H3K27me3 antibody and an anti-histone H3 antibody as a control. The signal intensity of H3K27me3 relative to H3 was measured using ImageJ software and normalized to the signal from unmodified histone H3 (H3 incubated with PARP1 in the absence of NAD+, lane 1). PARP1 activity was confirmed by western blot using an anti-PAR antibody. The western blot is representative of three independent experiments. (C) PARylation reduces histone methylation in vitro . Histone H3 samples treated as in A) were used to determine EZH2 activity toward unmodified and PARylated histone H3 by measuring H3K27me3 levels using an ELISA kit. H3K27me3 levels from unmodified histone H3 were set as 100% EZH2 activity. N=3, mean ± SD. (D) In vitro PARylation of histone H3. Histone H3 proteins treated as in A) were immunoprecipitated using the PAR-affinity resin and PARylation of H3 was confirmed by western blot using an anti-H3 antibody. PAR-resin specificity and PARylation levels were determined by western blot analysis of purified proteins with an anti-PAR antibody. The smear observed in lane 2 indicated PARylation. H. (E) PARP1 Immunoprecipitation. PARP1 removal after PARylation of histone H3 treated as above was confirmed by western blot analysis of proteins immunoprecipitated with an anti-PARP1 antibody.

Article Snippet: After 30 minutes, samples were incubated at RT for 10 minutes and the enzymatic activity of EZH2/PRC2 complex was measured by the EpiQuick Histone Methyltransferase Activity/Inhibition assay (EpiGentek) according to manufacturer's protocol.

Techniques: Methylation, In Vitro, Incubation, Immunoprecipitation, Western Blot, Control, Software, Activity Assay, Enzyme-linked Immunosorbent Assay, Purification

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) Schematic of histone peptide pull-down after PARylation. (B) Histone peptide pull-down for EZH2. Synthesized histone H3 peptide, corresponding to residues 21-44 of human histone H3, was conjugated with biotin and incubated with PARP1 in the presence or absence of NAD+ to allow for PARylation. After 1 hour, the reaction was blocked with 250 nM olaparib and the H3 peptide was immunopurified with streptavidin-magnetic beads and subsequently incubated with EZH2/PRC2 complex. After 4 hours, the peptide-coated, streptavidin-conjugated beads were washed to remove unbound proteins and bound proteins were analyzed by western blot using an anti-EZH2 antibody. PARylation of the peptide was confirmed by western blot using an anti-PAR antibody. Top panel shows short film exposure; lower panel longer film exposure. (C) Schematic of histone peptide pull-down after in vitro histone methyltransferase assay. (D) Histone peptide pull-down assay for PARP1. Synthesized histone H3 peptide containing tri-methylated lysine 27 was conjugated with streptavidin magnetic beads followed by incubation with purified PARP1. After 4 hours, the peptide-coated, streptavidin-conjugated beads were washed to remove unbound proteins and bound proteins were analyzed by western blot using an anti-PARP1 antibody.

Article Snippet: After 30 minutes, samples were incubated at RT for 10 minutes and the enzymatic activity of EZH2/PRC2 complex was measured by the EpiQuick Histone Methyltransferase Activity/Inhibition assay (EpiGentek) according to manufacturer's protocol.

Techniques: Synthesized, Incubation, Magnetic Beads, Western Blot, In Vitro, HMT Assay, Pull Down Assay, Methylation, Purification