Journal: Cancer gene therapy

Article Title: Novel pharmacologic inhibition of lysine-specific demethylase 1 as a potential therapeutic for glioblastoma.

doi: 10.1038/s41417-024-00847-8

Figure Lengend Snippet: Fig. 2 S2172 affected H3K4me2 modifications. A Western blot analysis of H3K4me1, H3K4me2, H3K4me3, H3K9me2, and H3 after treatment with either DMSO or S2172 (5 μM) for 96 h in GSC1228, GSC222, and GSC316. Histone H3 was used as the loading control. B Quantification of band signal intensities from the western blot of A. The y-axis in the panel indicates relative signal intensity of each protein normalized to histone H3. Error bars indicate the SD. C Upper panel, ChIP-seq peaks (3 kb upstream and downstream of the H3K4me2-binding site) of H3K4me1, H3K4me2, H3K4me3 and H3K9me2. Signal intensity is shown on the right. Lower panel, Distribution of H3K4me2 and H3K4me3 signals at H3K4me2-binding site in GSC1228 treated with DMSO or S2172 (5 μM). D Peaks of H3K4me1, H3K4me2, H3K4me3 were identified using HOMER findPeaks function with histone setting. Venn diagrams for each binding peak are shown. E Distribution of H3K4me2 peaks at the ‘increased’ and ‘de novo’ regions. F Representative ChIP-seq peaks at the MYC and Nestin loci.

Article Snippet: Lysates were incubated overnight with 50 μL of Dynabeads Protein G (Thermo Fisher Scientific) which were pre-coated with 4 μL of anti-H3K4me1 antibody (ab8895, Abcam, Cambridge, UK), 3 μL of anti-H3K4me2 antibody (#39141, Active Motif), 2.5 μL of anti-H3K4me3 antibody (#39159, Active Motif), 4 μL of anti-H3K9me2 antibody (ab1220, Abcam).

Techniques: Western Blot, Control, ChIP-sequencing, Binding Assay

Journal: Algal Research

Article Title: Stress priming using X-irradiation or H2O2 treatment enhances growth and metabolite production in Chlamydomonas reinhardtii cells

doi: 10.1016/j.algal.2024.103594

Figure Lengend Snippet: Fig. 8. Transcription levels and chromatin modifications of cell cycle- associated genes in C. reinhardtii cells after transient H2O2 treatment. Mid- exponential phase C. reinhardtii cells were harvested. The cells were incu bated in the 110 μM H2O2-treated TAP medium for 1 h and then cultivated in the normal TAP medium for 24 h. Mock represents no treatment, whereas HP- 0H, HP-6H, HP-12H, and HP-24H are 0, 6, 12, and 24 h after treatment with H2O2, respectively. (A) The transcription levels of the cell cycle-associated genes CrCDKA1, CrDP1, CrE2F1, and CrMAT3 as measured by reverse tran scription and quantitative polymerase chain reaction (RT-qPCR) after H2O2 treatment. (B, C) The percentage input immunoprecipitation of the active chromatin markers H3K9ac and H3K4me3 on the cell cycle-associated genes, respectively. Data represent the mean ± standard error (SE) with n = 9 (A) or n = 12 (B, C) from three independent experiments. Invisible error bars are smaller than the symbol size. Different letters indicate significant differences at p < 0.05 (one-way analysis of variance followed by Tukey's honestly significant difference test).

Article Snippet: Then, ChIP was performed for specific chromatin modifications using antibodies against histone 3 lysine 9 acetylation (H3K9ac) and histone 3 lysine 4 trimethylation (H3K4me3) (Epigentek).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunoprecipitation

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) dCypher is a no-wash bead-based proximity assay on the Alpha platform to interrogate chromatin readers (see ’Materials and methods’). PTM-defined biotinylated peptides or NCPs (the potential Targets) are immobilized to streptavidin donor beads and tagged reader domain(s) (the Queries) to anti-tag Acceptor beads. Donor beads are excited at 680 nm causing the release of singlet oxygen and quantitative fluorescence emission (520–620 nm) from proximal acceptor beads (i.e., bridged by a [Query:Target] interaction). ( b,c ) GST-PHD-BD selectivity (Query concentration as noted; chosen from dCypher curves in [ EC 80 rel for H3K4me3 peptide; EC 50 rel for H3K4me3tri ac NCP]) for a panel of histone peptides (~100 nM final; b ) and NCP (10 nM final; c ). ( d,e ) GST-PHD-BD binding to histone H3 lysine 4 (H3K4) methyl states (me0 /me1/me2/me3) on peptides ( d ) or NCPs ( e ). Alpha Counts are plotted as a function of Query concentration. ( f ) dCypher assay alpha counts plotted as a function of GST-PHD-BD concentration to NCPs. ( g ) EC 50 rel values with 95% confidence intervals (CI95) (see ’Materials and methods’ for calculation of same) from dCypher curves in ( f ) with Targets color coded as per legend (see also ). ND, Not Detected.

Article Snippet: The resulting products (e.g. H3K4me3; EpiCypher #16 – 0316 ) contained full-length ‘scarless’ histones and minimal free-DNA (<5%).

Techniques: Proximity Assay, Fluorescence, Concentration Assay, Binding Assay

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) The PHD-H3K4me3 (top) and BD-Kac (bottom) binding pockets on previously solved structures of the individual domains in complex with histone peptides (PDB: 2FUU and 3QZT). Binding pockets are circled/labeled: on PHD for A1, R2, and K4me3; on BD for Kac. Relative location of PTM-binding residues W2891 (PHD) and N3007 (BD) also indicated and mutated to alanine in ( b, c ). ( b ) dCypher assay Alpha counts plotted as a function of GST-PHD-BD N3007A (GST-PHD-BD mut ; top) or GST-PHD W2891A -BD (GST-PHD mut -BD; bottom) Query concentration to NCP Targets. ( c ) EC 50 rel (CI95) values from dCypher curves in ( b ). Targets color coded as per legends. ND, Not Detected.

Article Snippet: The resulting products (e.g. H3K4me3; EpiCypher #16 – 0316 ) contained full-length ‘scarless’ histones and minimal free-DNA (<5%).

Techniques: Binding Assay, Labeling, Concentration Assay

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) 1 H, 15 N-HSQC overlays of 15 N-PHD-BD apo (black) or in the presence of H3tri ac (green), H3tetra ac (blue), or H3K4me3tri ac (red) peptides. Arrows denote trajectory of chemical shift perturbation (CSP) and are colored by peptide. Shown are representative resonances for the bromodomain (BD) (left) and plant homeodomain (PHD) (right) binding pockets. ( b ) Histone H3-A1 is essential for 6His-PHD-BD binding to nucleosome core particles (NCPs) (compare H3K4me3tri ac to NΔ1H3K4me3tri ac [integrity of each target confirmed with anti-H3K4me3 ]). dCypher assay Alpha counts are plotted as a function of Query concentration to indicated NCP Targets.

Article Snippet: The resulting products (e.g. H3K4me3; EpiCypher #16 – 0316 ) contained full-length ‘scarless’ histones and minimal free-DNA (<5%).

Techniques: Binding Assay, Concentration Assay

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a–c ) Full spectral overlays of 15 N-PHD-BD on titration with histone H3 tail peptides (aa 1–34): H3K4me3tri ac ( a ); H3tri ac ( b ); or H3tetra ac ( c ). Molar ratios of each peptide are denoted by color in panel legends. ( d ) Validation of N-terminally truncated H3 NCP (NΔ1H3K4me3tri ac ). Alpha counts plotted as a function of anti-H3K4me3 ( RevMab Cat# 31-1039-00, lot # P-09–00676) concentration: binding of this antibody is dependent on H4K4me3 (compare H3K4me3 and NCP-unmodified) but independent of residue A1 (NΔ1H3K4me3tri ac ) or acetylation at K9, K14 or K18 (H3K4me3tri ac ). Table shows EC 50 rel (with CI95) values calculated from dCypher curves. Targets color coded as per legend. ND, not detected.

Article Snippet: The resulting products (e.g. H3K4me3; EpiCypher #16 – 0316 ) contained full-length ‘scarless’ histones and minimal free-DNA (<5%).

Techniques: Titration, Biomarker Discovery, Concentration Assay, Binding Assay, Residue

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) dCypher assay Alpha counts plotted as a function of GST-PHD-BD Query concentration to homotypic (e.g. [H3 • H3]) or heterotypic (e.g. [H3 • H3K4me3]) NCP Targets (created as in ’Materials and methods’). ( b ) Relative EC 50 (EC 50 rel ) and 95% confidence interval (CI95) values from dCypher curves (in a ; for calculation see ’Materials and methods’). • indicates a heterotypic Target. Limited testing data from a 6His-PHD-BD query added for comparison. Targets are color coded as per legends. ND, Not detected; -, Not tested.

Article Snippet: The resulting products (e.g. H3K4me3; EpiCypher #16 – 0316 ) contained full-length ‘scarless’ histones and minimal free-DNA (<5%).

Techniques: Concentration Assay, Comparison

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) Heatmap of CUT&RUN signal (see ’Materials and methods’) aligned to the transcription start site (TSS, +/−2 kb) of 18,793 genes in K562 cells. High and low signal (red and blue, respectively) are ranked by / linked to H3K4me3 (top to bottom). ( b–c ) CUT&RUN RPKM normalized tracks at representative loci using Integrative Genomics Viewer (IGV, Broad Institute). Note the general co-localization of BTPF (endogenous) with H3K4me3 and H3K18ac. ( d ) Venn overlap for H3K4me3, H3K18ac and BPTF peaks. Peaks were called using SEACR, designed for the S parse E nrichment A nalysis of C UT& R UN data , and intersected using bedtools . To compare data sets, the top peaks (number noted for each) were considered. ( e ) BPTF signal distribution for SEACR peaks represented as a Violin plot, and derived from Venn diagram groups. Column 1 depicts the signal distribution for all 8,405 BPTF peaks. Other columns depict BPTF peaks that overlap with peaks for H3K4me3, H3K18ac, or both PTMs (as noted). The Fisher exact test was used to determine if BPTF co-occupancy with specific PTMs or combinations were statistically significant (**** denotes p-value <10 –4 ). See for sequencing statistics; all project files available at Gene Expression Omnibus (GEO) accession GSE150617 .

Article Snippet: The resulting products (e.g. H3K4me3; EpiCypher #16 – 0316 ) contained full-length ‘scarless’ histones and minimal free-DNA (<5%).

Techniques: Derivative Assay, Sequencing, Gene Expression

Journal: eLife

Article Title: Nucleosome conformation dictates the histone code

doi: 10.7554/eLife.78866

Figure Lengend Snippet: ( a ) CUTANA nucleosome spike-ins contain a 5’biotin for immobilization to magnetic beads and a DNA barcode to define post-translational modification (PTM) status/monitor release into the CUT&RUN eluate. A four-member panel was assembled to explore GST-PHD-BD binding (unmodified, H3K4me3, H3tetra ac , H3K4me3tri ac ; on 80- N -25 DNA containing a central 147 bp 601 N ucleosome positioning sequence with embedded 22 bp DNA barcode). ( b ) GST-PHD-BD shows strong preference for spike-in nucleosome containing H3K4me3tri ac . Table shows relative release of spike-ins (percent barcoded nucleosome/total barcode reads) in Reader CUT&RUN (’Materials and methods’). Antibodies are noted by column; GST-BPTF (PHD-BD) is detected by α-GST to facilitate pAG-MNase recruitment. ‘Nucleosome bandwidth’ is the percentage of total sequence reads taken up by spike-in standards. ( c ) Heatmap of CUT&RUN signal aligned to the transcription start site (TSS, +/−2 kb) of 18,793 genes in K562 cells. Rows were k-means clustered into four groups (boxed) using ChAsE chromatin analysis tool . High and low signal (red and blue, respectively) are ranked by / linked to H3K4me3 (top to bottom). ( d ) CUT&RUN RPKM normalized tracks at representative loci using Integrative Genomics Viewer (IGV, Broad Institute). Note the co-localization of BTPF (endogenous) or GST-PHD-BD (exogenous) with H3K4me3 and H3K18ac; that H3K18ac alone is insufficient to recruit BTPF or GST-PHD-BD; and that GST-PHD-BD shows robust recruitment at some locations where BPTF is absent (e.g. B4GALT2 promoter; see ‘Discussion’).

Article Snippet: The resulting products (e.g. H3K4me3; EpiCypher #16 – 0316 ) contained full-length ‘scarless’ histones and minimal free-DNA (<5%).

Techniques: Magnetic Beads, Modification, Binding Assay, Sequencing

Journal: Science (New York, N.Y.)

Article Title: JARID2 and AEBP2 regulate PRC2 in the presence of H2AK119ub1 and other histone modifications

doi: 10.1126/science.abc3393

Figure Lengend Snippet: (A) Schematic representation of the proteins in the PRC2-AEBP2-JARID2 complex. Newly modeled regions in EZH2, JARID2, and AEBP2 that contribute to the interaction with the nucleosome are marked by dashed boxes. (B) Coomassie-stained gel showing all of the PRC2-core subunits, cofactors JARID2 and AEBP2, and histone proteins in the sample used for structural studies. (C) (Top) Cryo-EM density map for PRC2-AJ1–450 bound to an H2AK119ub1-containing nucleosome. (Bottom) Atomic model of PRC2-AJ1–450 bound to an H2AK119ub1-containing nucleosome, with EZH2 (SANT1) highlighted in gold, EZH2 (SANT2) in hot pink, EZH2 (SET) in blue, EED in light blue, RBAP48 in light purple, SUZ12 in green, JARID2 in magenta, AEBP2 in red, ubiquitin in orange, histone H3 in light pink, H2A in salmon, H4 and H2B in khaki, and nucleosome DNA in cyan. The image shown is a composite map where the PRC2 and nucleosome core correspond to the local resolution-filtered map from multibody refinement, whereas the ubiquitin, JARID2 UIM, and AEBP2 zinc finger densities correspond to the local resolution-filtered map from the consensus refinement. This color coding also applies to movie S1.

Article Snippet: All recombinant Xenopus nucleosomes containing H3K4me3 or H3K36me3 were generated as MLA analogs, whereas all human nucleosomes purchased from Epicypher contained native ligated H3K4me3 or H3K36me3.

Techniques: Staining, Cryo-EM Sample Prep

Journal: Science (New York, N.Y.)

Article Title: JARID2 and AEBP2 regulate PRC2 in the presence of H2AK119ub1 and other histone modifications

doi: 10.1126/science.abc3393

Figure Lengend Snippet: (A) (Top) Cartoon representation of the newly defined EZH2 bridge helix (amino acids 497 to 513) that interacts with nucleosomal DNA and the H3 tail. (Left) Cryo-EM structure of the PRC2-AEBP2-JARID2 complex bound to an H2AK119ub1-containing nucleosome. The zoom out shows the bridge helix, with residues interacting with the EZH2 (SET) domain, nucleosomal DNA, and the histone tail depicted in stick representation. (Right) Helix wheel diagram for the bridge helix that shows the distribution of positively charged residues on the nucleosomal DNA interacting face (cyan), positive and hydrophobic residues on the EZH2 (SET) interacting face (blue), and the residues interacting with the backbone of the H3 tail (pink). aa, amino acids. (B) (Left) Density map of the histone H3 tail (amino acids 21 to 40) (contour level: 0.024) with the corresponding atomic model. Clear density for residues R26 and K27, which have been previously observed, as well as density for K23, K36, K37, and V35 allow us to define the full extent of interaction between EZH2 (SET) and the histone H3 tail. (Right) Extensive electrostatic and van der Waals interactions between the residues in EZH2 (SET) (blue; shown in green stick representation) and the histone H3 tail (black; shown in pink stick representation) guide the H3 tail into the catalytic site. (C) Close-up view of the interaction of EZH2 (SET and CXC) with the histone H3 tail (stick representation with corresponding cryo-EM density in transparency), which shows residues involved in either direct or indirect interaction with the histone H3 tail and nucleosome DNA that are also frequently found mutated in cancers as orange spheres [COSMIC database (57)]. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Article Snippet: All recombinant Xenopus nucleosomes containing H3K4me3 or H3K36me3 were generated as MLA analogs, whereas all human nucleosomes purchased from Epicypher contained native ligated H3K4me3 or H3K36me3.

Techniques: Cryo-EM Sample Prep

Journal: Science (New York, N.Y.)

Article Title: JARID2 and AEBP2 regulate PRC2 in the presence of H2AK119ub1 and other histone modifications

doi: 10.1126/science.abc3393

Figure Lengend Snippet: (A) Bar graph showing the comparison of end-point, cumulative HMTase activity (H3K27me1/me2/me3) of PRC2-core, PRC2-AEBP2, PRC2-JARID2 (amino acids 106 to 450), PRC2-AEBP2ΔZn-JARID2 (amino acids 1 to 450), and PRC2-AEBP2-JARID2 (amino acids 1 to 450) on unmodified nucleosomes (gray), H3K4me3-containing (orange), H3K36me3-containing (green), and H2AK119ub1-containing nucleosomes (magenta). The differences in end-point measurements that are statistically significant are indicated by the P values. (B) Western blot analysis comparing the H3K27me3 production for unmodified nucleosomes versus the H3K4me3-containing, H3K36me3-containing, and H2AK119ub1-containing nucleosomes.

Article Snippet: All recombinant Xenopus nucleosomes containing H3K4me3 or H3K36me3 were generated as MLA analogs, whereas all human nucleosomes purchased from Epicypher contained native ligated H3K4me3 or H3K36me3.

Techniques: Activity Assay, Western Blot