Journal: eLife

Article Title: An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability

doi: 10.7554/eLife.82596

Figure Lengend Snippet: dCypher assay alpha counts for the interaction of GST-PHD queries (9.5 nM KDM7A (Uniprot #Q6ZMT4; residues 1–100); 2.4 nM DIDO1 (Uniprot #Q9BTC0; residues 250–340); 18 nM MLL5 (Uniprot #Q8IZD2; residues 100–180)) with PTM-defined peptides ( A ) vs . nucleosomes ( B ) (the potential targets). All error bars represent the range of two replicates. Key: H3.1 ∆2, H3.1 ∆32, and H4 ∆15 are nucleosomes assembled with histones lacking the indicated N-terminal residues of H3.1 or H4. All data were plotted using GraphPad Prism 9.0. Of note, each reader query showed minimal interaction-free DNA (147 bp or 199 bp: ).

Article Snippet: Briefly, 5 μL of GST-tagged reader domain was incubated with 5 μL of 10 nM biotinylated nucleosomes (e.g. EpiCypher #16–9001) for 30 min at room temperature in 20 mM HEPES pH 7.5, 250 mM NaCl, 0.01% BSA, 0.01% NP-40, 1 mM DTT in a 384-well plate.

Techniques:

Journal: eLife

Article Title: An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability

doi: 10.7554/eLife.82596

Figure Lengend Snippet: ( A ) Binding curves to determine the optimal concentration for screening reader queries (GST-KDM7A PHD, GST-DIDO1 PHD, and GST-MLL5 PHD) to indicate post-translational modification (PTM)-defined nucleosome targets . ( B ) Library binding screen with indicated GST-PHD queries and PTM-defined nucleosome or free DNA (147 bp (Widom 601 sequence) or 199 bp (Widom 601 sequence flanked by 21 bp linkers)) targets. Error bars represent the range of two replicates. Key: H3.1N ∆2, H3.1N ∆32, and H4N ∆15 are nucleosomes assembled with histones lacking the indicated N-terminal residues of H3.1 or H4. ( C ) Relative EC 50 (EC 50 Rel ) binding values (in nM) (calculated as in Methods ) between GST-PHD reader proteins and select histone peptides; 95% confidence intervals are represented parenthetically. ( D ) EC 50 Rel binding values (in nM) between GST-PHD proteins and select nucleosomes; 95% confidence intervals are represented parenthetically. ND = not determined. Figure 1—figure supplement 1—source data 1. EC 50 Rel binding values (in nM) for GST-PHD proteins with histone peptides and nucleosomes.

Article Snippet: Briefly, 5 μL of GST-tagged reader domain was incubated with 5 μL of 10 nM biotinylated nucleosomes (e.g. EpiCypher #16–9001) for 30 min at room temperature in 20 mM HEPES pH 7.5, 250 mM NaCl, 0.01% BSA, 0.01% NP-40, 1 mM DTT in a 384-well plate.

Techniques: Binding Assay, Concentration Assay, Modification, Sequencing

Journal: eLife

Article Title: An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability

doi: 10.7554/eLife.82596

Figure Lengend Snippet: ( A ) Endpoint methylation assays of H3K4me0 [H3 • H3]-me1-me2-me3 nucleosomes and their cognate H3K9ac14ac18ac (H3tri ac ) partners (all 100 nM) with MLL1 Complex (MLL1C; 4 nM). Reactions performed in triplicate with error bars as SEM. p- values were determined using a two-tailed t-test: ****=<0.0001, ***=0.0008, **=0.0038. ( B ) MLL1C (4 nM) methylation activity on H3K4me0-me1-me2-me3 nucleosomes and their cognate H3tri ac partners (all substrates: 1.5-fold serial dilution, 0–0.4 μM). Reactions performed in triplicate with error bars as SEM (see also ). ( C ) MLL1C does not differentially associate with post-translational modification (PTM)-defined nucleosome substrates under study conditions. dCypher binding curves of hexahistidine (6HIS)-tagged MLL1C (concentrations noted) with PTM-defined nucleosomes (20 nM). Error bars represent the range of two replicates. ( D ) MLL1C-mediated methylation is enhanced on cis but not trans- acetylated nucleosomal H3 tails. Endpoint methylation assays of MLL1C (4 nM) with homotypic [H3 • H3] vs . heterotypic (e.g., [H3 • H3tetra ac ]; see Methods ) nucleosome substrates (all 100 nM). Reactions performed in triplicate with error bars as SEM. p- values were determined using a two-tailed t-test: ****=<0.0001. Key: H3tri ac = H3K9acK14acK18ac. H3tetra ac = H3K4acK9acK14acK18ac.

Article Snippet: Briefly, 5 μL of GST-tagged reader domain was incubated with 5 μL of 10 nM biotinylated nucleosomes (e.g. EpiCypher #16–9001) for 30 min at room temperature in 20 mM HEPES pH 7.5, 250 mM NaCl, 0.01% BSA, 0.01% NP-40, 1 mM DTT in a 384-well plate.

Techniques: Methylation, Two Tailed Test, Activity Assay, Serial Dilution, Modification, Binding Assay

Journal: eLife

Article Title: An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability

doi: 10.7554/eLife.82596

Figure Lengend Snippet: ( A ) MLL1C activity on 2 μg chicken oligonucleosomes (2 μg) was 2D-titration tested by enzyme [0, 4, and 40 nM] and salt [0, 50, and 300 mM NaCl] in the presence of [ methyl - 3 H]-SAM donor. Methylation (in fmol) is graphed as a function of [MLL1C]. N=2 and error bars are SD. ( B ) Replicate SDS-PAGE Coomassie-stained gels for methylation data from panel ( A ). Resolved subunits of MLL1C and nucleosomes are indicated. ( C ) Steady-state kinetic data for MLL1C activity with nucleosome substrates as shown in but fit to the Michaelis-Menten equation. ( D ) Endpoint methylation activity of MLL1C (4 nM) with MLA (methyl-lysine analog) nucleosome substrates (100 nM) in triplicate; error bars are SD. ( E ) Replicate SDS-PAGE Coomassie-stained gels for methylation data from panel ( D ); Lanes marked with ‘*’ and ‘+’ in the Replicate three gel are switched. ( F ) Replicate SDS-PAGE Coomassie-stained gels for methylation data from and ( C ). 0.5 μg BSA was loaded as a lane marker to each quenched reaction (except for H3K4me3tri ac ). ( G ) SDS-PAGE Coomassie-stained gel for methylation data from . ( H ) EC 50 rel binding values (in nM) between MLL1C and select nucleosomes; 95% confidence intervals (CI) are represented parenthetically. Note: gel images in grayscale and blue are both Coomassie-stained gels. All data were plotted using GraphPad Prism 9.0. Figure 2—figure supplement 1—source data 1. Files of raw Coomassie gel images are used in . Individual files, as well as a composite labeled file, of all raw Coomassie gels for images in . Each raw image file name is labeled with the panel to which it corresponds in except for the composite labeled image, which has each image labeled with the corresponding panel within the composite image. Figure 2—figure supplement 1—source data 2. EC 50 rel binding values (in nM) for MLL1C and nucleosomes.

Article Snippet: Briefly, 5 μL of GST-tagged reader domain was incubated with 5 μL of 10 nM biotinylated nucleosomes (e.g. EpiCypher #16–9001) for 30 min at room temperature in 20 mM HEPES pH 7.5, 250 mM NaCl, 0.01% BSA, 0.01% NP-40, 1 mM DTT in a 384-well plate.

Techniques: Activity Assay, Titration, Methylation, SDS Page, Staining, Marker, Binding Assay, Labeling