Journal: Journal of Biological Chemistry

Article Title: Identification and characterization of second-generation EZH2 inhibitors with extended residence times and improved biological activity

doi: 10.1016/j.jbc.2021.100349

Figure Lengend Snippet: Figure 3. Exploration of allosteric modulation of EZH2i potencies. A, effect of allosteric modulation on inhibitor residence times for EZH2 inhibitors used to develop kinetic methodologies. Error bars represent propagated error from measured values used in calculation. B, modulation of CPI-1205 residence time by MAK683 (EEDi). Representative data are plotted as the average of two technical replicates (±) standard deviation. The residence time of CPI-1205 for basal PRC2 is 1.2 ± 0.3 h (mean ± SD of nine biological replicates each in duplicate). For basal PRC2 in the presence of 10 μM MAK683, the residence time of CPI-1205 is 0.97 ± 0.1 h (average ±SD of three biological replicates each in duplicate). C, MAK683 (10 μM) inhibition of CPI-1205 residence time enhancement by H3K27me3 activator peptide (10 μM). Representative data are plotted as the average of two technical replicates ± standard deviation. The residence time of CPI-1205 PRC2 in the presence of H3K27me3 activator peptide and MAK-683 (10 mM each) is 1.3 ± 0.2 h (mean ± SD of four biological replicates each in duplicate). D, comparison of association rate constant values of EZH2i (±) 10 μM H3K27me3 activator peptide. Data are individual replicates with error bars representing the standard deviation of the mean. E and F, plots of residence time values for basal (E; R2 0.78) and activated (F; R2

Article Snippet: Twenty-five microliters of αH3K27me3 (0.25 μg/ml, Cell Signaling 9733), which had previously been validated in-house (48) or anti-Histone H3 (0.125 μg/ml, Cell Signaling 4499) in 1% BSA in TBST, was added for 30 min at RT with shaking.

Techniques: Standard Deviation, Inhibition, Comparison

Journal: Journal of Biological Chemistry

Article Title: Identification and characterization of second-generation EZH2 inhibitors with extended residence times and improved biological activity

doi: 10.1016/j.jbc.2021.100349

Figure Lengend Snippet: Figure 7. Biological characterization of second-generation EZH2i. A, plot of inhibitor residence time values versus EC50 values for H3K27me3 reduction in HeLa cells. B, plot of inhibitor residence time values versus GI50 values for KARPAS-422 cell line. C, plot of inhibitor pKi values versus GI50 values in KARPAS- 422 cell line. D, modulation of global H3K27me3 levels (Day 12) in a Karpas-422 xenograft model by CPI-1328 and EPZ-6438. Data are individual replicates with error bars representing the standard deviation of the mean. E and F, efficacy of CPI-1328 and EPZ-6438 in a Karpas-422 xenograft model, respectively. Data are the average of three or more animals with error bars representing the standard error of the mean.

Article Snippet: Twenty-five microliters of αH3K27me3 (0.25 μg/ml, Cell Signaling 9733), which had previously been validated in-house (48) or anti-Histone H3 (0.125 μg/ml, Cell Signaling 4499) in 1% BSA in TBST, was added for 30 min at RT with shaking.

Techniques: Standard Deviation

Journal: International journal of oral science

Article Title: NUP62 alleviates senescence and promotes the stemness of human dental pulp stem cells via NSD2-dependent epigenetic reprogramming.

doi: 10.1038/s41368-025-00362-y

Figure Lengend Snippet: Fig. 5 NUP62 mediates epigenetic reprogramming by upregulating the global levels of H3K36me2 and H3K36me3. a, b Western blot analyses of H3K36me2, H3K36me3, H3K27ac, H3K4me3, and H3K9me3 in human dental pulp stem cells (HDPSCs) transfected with lentiviral vectors or overexpressing NUP62. c, d Immunofluorescence staining and quantification of H3K36me2, respectively (n = 5). Scale bar, 50 μm. e, f Immunofluorescence staining and quantification of H3K36me3, respectively (n = 5). Scale bar, 50 μm. g Heatmap of global H3K36me2 and H3K36me3 CUT&Tag-seq signals from HDPSCs transfected with empty lentiviral vector or NUP62 overexpression lentivirus (n = 2). h Gene track view of normalized bigWig reads at the promoters of SIRT6, HMGA1 and HMGA2 in HDPSCs transfected with a lentiviral vector or overexpressing (n = 2). *P < 0.05, **P < 0.01 and ***P < 0.001

Article Snippet: After blocking in 5% BSA for 30 min, the cells were incubated overnight at 4 °C with primary antibodies against NUP62 (Abcam, ab96134, 1:200), γH2AX (Cell Signaling Technology, #9718T, 1:200), H3K36me2 (Active Motif, 39056, 1:200), H3K36me3 (Cell Signaling Technology, #4909S, 1:200) or E2F1 (Cohesion, CQA8351, 1:100).

Techniques: Western Blot, Transfection, Staining, Plasmid Preparation, Over Expression

Journal: bioRxiv

Article Title: Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery

doi: 10.1101/2022.04.28.489608

Figure Lengend Snippet: Identification of topologically distinct Polycomb sub-compartments with LIMe-Hi-C a) Density plot depicting the relative density ( y -axis) of the average fraction lamina GpC methylation ( x -axis) for 50 kb bins across A and B compartments in K562. The density function for each compartment is independently scaled. b) Heatmap of 50 kb genomic regions clustered into sub-compartments identified by k -means clustering in K562. Heatmap color represents the z -score transformed value for lamina GpC methylation, CpG methylation, and principal component 1 of each genomic bin. c) Boxplot showing levels of histone modifications ( y -axis, fold-change over input/median signal) for 50 kb bins across sub-compartments ( x -axis) in K562. Outlier points are excluded. Published datasets are specified in Supplementary Table 1. Significance markers calculated by a Mann-Whitney-Wilcoxon two-sided test are as follows: ns: not significant; *: 0.01 < p ≤ 0.05; **: 0.001 < p ≤ 0.01; ***: 0.0001 < p ≤ 0.001; ****: p ≤ 0.0001. d) Aggregate plot depicting the fraction of a specified sub-compartment type ( y -axis) across compartment regions ( x -axis) for the B compartment (top) and A compartment (bottom). e) Representative genome browser tracks depicting H3K27ac, H3K27me3, and H3K9me3 ChIP-seq signal along with CpG methylation (replicate 1), lamina GpC methylation (replicate 1), and principal component 1 (replicate 1) data near the HOXD gene cluster. The sub-compartment designation is included below. Published ChIP-seq datasets are specified in Supplementary Table 1. f) Normalized average lamina GpC methylation for loci as a function of the sub-compartment status of the loci’s interaction partner for chromosome 1 (see Methods). Curved lines represent the compartment identity of the DNA within the interaction pair. g) Boxplot showing average log2(observed/expected contact frequency) for 50 kb bin level interactions ( y -axis) among all sub-compartments ( x -axis). O/E denotes observed/expected. Outlier points are excluded. Significance markers calculated by a Mann-Whitney-Wilcoxon two-sided test are as follows: ns: not significant; *: 0.01 < p ≤ 0.05; **: 0.001 < p ≤ 0.01; ***: 0.0001 < p ≤ 0.001; ****: p ≤ 0.0001. h) Summary interaction heatmap ordered by sub-compartment and H3K27me3 ChIP-seq signal of the log2(observed/expected contact frequency). Replicate-averaged z -score lamina GpC methylation (left) and H3K27me3 levels (top) per quantile are displayed alongside the interaction heatmap. The genome was divided into 100 quantiles with the number of quantiles within a sub-compartment reflective of the relative size of the sub-compartment. O/E denotes observed/expected. i) LIMe-Hi-C contact map for K562 cells merged across the three replicates. Example PcG-A and PcG-B regions are highlighted with black boxes.

Article Snippet: H3K9me3 immunoprecipitation was performed on 3 million cells with 8 μL of H3K9me3 antibody (Cell Signaling, 13969S Lot #3).

Techniques: Hi-C, Methylation, Transformation Assay, CpG Methylation Assay, MANN-WHITNEY, ChIP-sequencing

Journal: bioRxiv

Article Title: Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery

doi: 10.1101/2022.04.28.489608

Figure Lengend Snippet: H3K27me3 antagonizes constitutive heterochromatin spreading a) Boxplot showing log 2 fold-change H3K9me3 ChIP-seq signal between EZH2 inhibition and vehicle treatment for 50 kb bins ( y -axis) across sub-compartments ( x -axis). Outlier points are excluded. Significance markers calculated by a Mann-Whitney-Wilcoxon two-sided test are as follows: ns: not significant; *: 0.01 < p ≤ 0.05; **: 0.001 < p ≤ 0.01; ***: 0.0001 < p ≤ 0.001; ****: p ≤ 0.0001. b) Boxplot showing log 2 fold-change in H3K9me3 levels between EZH2i and vehicle treatment for 50 kb bins ( y -axis) segregated into three equally sized quantiles by change in z -score normalized lamina GpC methylation ( x -axis) for PcG-B domains. Outlier points are excluded. Significance markers calculated by a Mann-Whitney-Wilcoxon two-sided test are as follows: ns: not significant; *: 0.01 < p ≤ 0.05; **: 0.001 < p ≤ 0.01; ***: 0.0001 < p ≤ 0.001; ****: p ≤ 0.0001. c) Boxplot showing log 2 fold-change in H3K27me3 levels between EZH2i and vehicle treatment for 50 kb bins ( y -axis) segregated into three equally sized quantiles by log 2 fold-change in H3K9me3 ( x -axis) for PcG-B domains. Outlier points are excluded. Significance markers calculated by a Mann-Whitney-Wilcoxon two-sided test are as follows: ns: not significant; *: 0.01 < p ≤ 0.05; **: 0.001 < p ≤ 0.01; ***: 0.0001 < p ≤ 0.001; ****: p ≤ 0.0001. d) Aggregate profile plot of log 2 fold-change H3K9me3 ChIP-seq signal between EZH2 inhibition and vehicle treatment ( y -axis) across B compartment regions ( x -axis). e) Genome browser tracks of z -score normalized lamina GpC methylation levels averaged across replicates for the LIMe-Hi-C data, log 2 fold-change H3K9me3 ChIP-seq, and log 2 fold-change H3K27me3 ChIP-seq signal between EZH2i and vehicle treatment near the HOXD locus. f) Boxplot of log 2 (TRIP expression) ( y -axis) across sub-compartments for promoters ( x -axis). Outlier points are excluded. Published TRIP data is specified in Supplementary Table 1. Significance markers calculated by a Mann-Whitney-Wilcoxon two-sided test are as follows: ns: not significant; *: 0.01 < p ≤ 0.05; **: 0.001 < p ≤ 0.01; ***: 0.0001 < p ≤ 0.001; ****: p ≤ 0.0001. g) Boxplot of log 2 (fold-change expression) ( y -axis) between EZH2 inhibition and vehicle treatment for genes that overlap with H3K27me3 peaks across sub-compartments ( x -axis). Outlier points are excluded. Significance markers calculated by a Mann-Whitney-Wilcoxon two-sided test are as follows: ns: not significant; *: 0.01 < p ≤ 0.05; **: 0.001 < p ≤ 0.01; ***: 0.0001 < p ≤ 0.001; ****: p ≤ 0.0001.

Article Snippet: H3K9me3 immunoprecipitation was performed on 3 million cells with 8 μL of H3K9me3 antibody (Cell Signaling, 13969S Lot #3).

Techniques: ChIP-sequencing, Inhibition, MANN-WHITNEY, Methylation, Hi-C, Expressing

Journal: bioRxiv

Article Title: Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery

doi: 10.1101/2022.04.28.489608

Figure Lengend Snippet: Polycomb sub-compartments represent distinct transcriptional environments a) Genome browser tracks of z -score normalized lamina GpC methylation levels averaged across replicates for the LIMe-Hi-C data, log 2 fold-change H3K9me3 ChIP-seq, and log 2 fold-change H3K27me3 ChIP-seq signal between EZH2i and vehicle treatment near the HOXA locus. b) Aggregate profile plot of log 2 fold-change H3K9me3 ChIP-seq signal between EZH2 inhibition and vehicle treatment ( y -axis) across LIMe LADs ( x -axis). c) Boxplot with individual points overlaid of log 2 (TRIP expression) ( y -axis) across the PcG-A and PcG-B sub-compartments ( x -axis) for promoter integrations that overlap with H3K27me3 peaks. Significance markers calculated by a Mann-Whitney-Wilcoxon two-sided test are as follows: ns: not significant; *: 0.01 < p ≤ 0.05; **: 0.001 < p ≤ 0.01; ***: 0.0001 < p ≤ 0.001; ****: p ≤ 0.0001. d) Bar charts depicting number of genes within each sub-compartment for those that are detected by RNA-seq (non-zero) and those that are either upregulated or downregulated upon EZH2 inhibition. e) Volcano plot depicting -log 10 (adjusted p -value) ( y -axis) relative to log 2 (fold-change expression) ( x -axis) between EZH2 inhibition and vehicle treatment for H3K27me3-positive PcG-A and PcG-B genes. f) Boxplot of baseline expression ( y -axis, fragment per million mapped fragments (FPM)) for vehicle treatment of non-zero genes that overlap with H3K27me3 peaks across PcG-A and PcG-B ( x -axis). Outlier points are excluded. Significance markers calculated by a Mann-Whitney-Wilcoxon two-sided test are as follows: ns: not significant; *: 0.01 < p ≤ 0.05; **: 0.001 < p ≤ 0.01; ***: 0.0001 < p ≤ 0.001; ****: p ≤ 0.0001.

Article Snippet: H3K9me3 immunoprecipitation was performed on 3 million cells with 8 μL of H3K9me3 antibody (Cell Signaling, 13969S Lot #3).

Techniques: Methylation, Hi-C, ChIP-sequencing, Inhibition, Expressing, MANN-WHITNEY, RNA Sequencing