human nucleosomes  (EpiCypher)

Journal: bioRxiv

Article Title: Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

doi: 10.64898/2026.01.21.700667

Figure Lengend Snippet: A. Diagram of H3K9 mono- and dimethylation preferences on nucleosomes by G9a-G9a homodimer and G9a-GLP heterodimer, as shown in Sanchez et al. G9a-G9a is depressed in its ability to convert H3K9me1 to H3K9me2. B. Western blot of H3K9me1 and me2 production over time from H3K9me0 nucleosomes with wildtype G9a-GLP, and SET domain catalytic mutants G9a SETm -GLP and G9a-GLP SETm . C. As above, but for wildtype and ANK domain aromatic cage mutants G9a ANKm -GLP, G9a-GLP ANKm , and G9a ANKm -GLP ANKm . Note, the H3K9me1 antibody evidences some background in the t=0 timepoint. D. As above but for wildtype and deletion of G9a and GLP ANK domains E. Measurement of turnover rates on H3K9me0 (left) or H3K9me1 (right) nucleosomes under kcat/KM conditions (G9a-GLP concentration: 500 nM) with wildtype G9a-GLP or G9a ANKm -GLP ANKm . In this regime, the slope is roughly equivalent to the specificity constant.

Article Snippet: Incorporation of histone modifications was verified by western blotting and LC-MS. Commercial mononucleosomes containing H3K9me1 (SKU 16-0325) and H3K9me2 (SKU 16-0324-20) were purchased from EpiCypher and used directly.

Techniques: Western Blot, Concentration Assay

Journal: bioRxiv

Article Title: Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

doi: 10.64898/2026.01.21.700667

Figure Lengend Snippet: A. Electrophoretic mobility shift assay (EMSA) with H3K9me0 and me2 nucleosomes and G9a-GLP. Nucleosomes (15 nM) were incubated with G9a–GLP at concentrations (0, 0.15, 0.32, 0.63, 1.25, 2.5, and 5 µM) and subsequently crosslinked with 0.1% glutaraldehyde. The fitted K 1/2 curves are shown below, indicating a specificity of 6.5X. A slight nonspecific band overlaps the G9a-GLP:nucleosome complex (asterisk). B. EMSA with H3K9me1 and me2 nucleosomes and G9a-GLP as in A. (including concentration regime). C. As above, but for H3K9me2 nucleosomes with wildtype G9a-GLPor G9a ANKm -GLP ANKm as in A. (including concentration regime) D. TOP: Swap of the ANK domains of G9a and GLP in the heterodimer. BOTTOM LEFT: EMSA with me2 nucleosomes and wildtype G9a-GLP or G9a-GLP SWAP . G9a-GLP concentrations were 0, 0.067, 0.135, 0.27, and 0.55 µM. BOTTOM RIGHT: EMSA with me2 nucleosomes and G9a-GLP SWAP . G9a-GLP SWAP concentrations for me1 nucleosomes were 0, 0.09, 0.19, 0.38, 0.75, 1.5, 3, and 6 µM. E. Western blot of H3K9me1 and me2 production over time from H3K9me0 nucleosomes with wildtype G9a-GLP or G9a-GLP SWAP as in . We note that in this experiment, the primary antibody detected a lower level of H3K9me1.

Article Snippet: Incorporation of histone modifications was verified by western blotting and LC-MS. Commercial mononucleosomes containing H3K9me1 (SKU 16-0325) and H3K9me2 (SKU 16-0324-20) were purchased from EpiCypher and used directly.

Techniques: Electrophoretic Mobility Shift Assay, Incubation, Concentration Assay, Western Blot

Journal: bioRxiv

Article Title: Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

doi: 10.64898/2026.01.21.700667

Figure Lengend Snippet: A. diagram of initial substrate and reaction intermediate mimics. The reaction intermediate mimic is asymmetric H3K9Nle/H3K9me2. B. complex formation for crosslinking mass-spectrometry (CLMS). Lane 1, H3K9Ecx mononucleosomes; lane 2, G9a–GLP bound to H3K9Ecx “initial substrate” mononucleosomes; lane 3, G9a–GLP bound to asymmetric H3K9me2/H3K9Nle “reaction intermediate” mononucleosomes. Samples were crosslinked with DSSO (800 µM) prior to electrophoresis (see Methods). C. CLMS overview crosslinked peptides from the initial substrate mimic (black) and reaction intermediate mimic (red) to GLP. Only crosslinks to H3 tail are shown. D. As in C., but for crosslinks to G9a. E. EMSA with reaction intermediate mimic nucleosomes and wildtype G9a-GLP or G9aANK8 GS -GLP. E. Western blot of H3K9me1 and me2 production over time from H3K9me0 nucleosomes with wildtype G9a-GLP or G9aANK8 GS -GLP as in .

Article Snippet: Incorporation of histone modifications was verified by western blotting and LC-MS. Commercial mononucleosomes containing H3K9me1 (SKU 16-0325) and H3K9me2 (SKU 16-0324-20) were purchased from EpiCypher and used directly.

Techniques: Mass Spectrometry, Electrophoresis, Western Blot

Journal: bioRxiv

Article Title: Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

doi: 10.64898/2026.01.21.700667

Figure Lengend Snippet: A. Diagram of H3K9 mono- and dimethylation preferences on nucleosomes by G9a-G9a homodimer and G9a-GLP heterodimer, as shown in Sanchez et al. G9a-G9a is depressed in its ability to convert H3K9me1 to H3K9me2. B. Western blot of H3K9me1 and me2 production over time from H3K9me0 nucleosomes with wildtype G9a-GLP, and SET domain catalytic mutants G9a SETm -GLP and G9a-GLP SETm . C. As above, but for wildtype and ANK domain aromatic cage mutants G9a ANKm -GLP, G9a-GLP ANKm , and G9a ANKm -GLP ANKm . Note, the H3K9me1 antibody evidences some background in the t=0 timepoint. D. As above but for wildtype and deletion of G9a and GLP ANK domains E. Measurement of turnover rates on H3K9me0 (left) or H3K9me1 (right) nucleosomes under kcat/KM conditions (G9a-GLP concentration: 500 nM) with wildtype G9a-GLP or G9a ANKm -GLP ANKm . In this regime, the slope is roughly equivalent to the specificity constant.

Article Snippet: Incorporation of histone modifications was verified by western blotting and LC-MS. Commercial mononucleosomes containing H3K9me1 (SKU 16-0325) and H3K9me2 (SKU 16-0324-20) were purchased from EpiCypher and used directly.

Techniques: Western Blot, Concentration Assay

Journal: bioRxiv

Article Title: Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

doi: 10.64898/2026.01.21.700667

Figure Lengend Snippet: A. Electrophoretic mobility shift assay (EMSA) with H3K9me0 and me2 nucleosomes and G9a-GLP. Nucleosomes (15 nM) were incubated with G9a–GLP at concentrations (0, 0.15, 0.32, 0.63, 1.25, 2.5, and 5 µM) and subsequently crosslinked with 0.1% glutaraldehyde. The fitted K 1/2 curves are shown below, indicating a specificity of 6.5X. A slight nonspecific band overlaps the G9a-GLP:nucleosome complex (asterisk). B. EMSA with H3K9me1 and me2 nucleosomes and G9a-GLP as in A. (including concentration regime). C. As above, but for H3K9me2 nucleosomes with wildtype G9a-GLPor G9a ANKm -GLP ANKm as in A. (including concentration regime) D. TOP: Swap of the ANK domains of G9a and GLP in the heterodimer. BOTTOM LEFT: EMSA with me2 nucleosomes and wildtype G9a-GLP or G9a-GLP SWAP . G9a-GLP concentrations were 0, 0.067, 0.135, 0.27, and 0.55 µM. BOTTOM RIGHT: EMSA with me2 nucleosomes and G9a-GLP SWAP . G9a-GLP SWAP concentrations for me1 nucleosomes were 0, 0.09, 0.19, 0.38, 0.75, 1.5, 3, and 6 µM. E. Western blot of H3K9me1 and me2 production over time from H3K9me0 nucleosomes with wildtype G9a-GLP or G9a-GLP SWAP as in . We note that in this experiment, the primary antibody detected a lower level of H3K9me1.

Article Snippet: Incorporation of histone modifications was verified by western blotting and LC-MS. Commercial mononucleosomes containing H3K9me1 (SKU 16-0325) and H3K9me2 (SKU 16-0324-20) were purchased from EpiCypher and used directly.

Techniques: Electrophoretic Mobility Shift Assay, Incubation, Concentration Assay, Western Blot

Journal: bioRxiv

Article Title: Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

doi: 10.64898/2026.01.21.700667

Figure Lengend Snippet: A. diagram of initial substrate and reaction intermediate mimics. The reaction intermediate mimic is asymmetric H3K9Nle/H3K9me2. B. complex formation for crosslinking mass-spectrometry (CLMS). Lane 1, H3K9Ecx mononucleosomes; lane 2, G9a–GLP bound to H3K9Ecx “initial substrate” mononucleosomes; lane 3, G9a–GLP bound to asymmetric H3K9me2/H3K9Nle “reaction intermediate” mononucleosomes. Samples were crosslinked with DSSO (800 µM) prior to electrophoresis (see Methods). C. CLMS overview crosslinked peptides from the initial substrate mimic (black) and reaction intermediate mimic (red) to GLP. Only crosslinks to H3 tail are shown. D. As in C., but for crosslinks to G9a. E. EMSA with reaction intermediate mimic nucleosomes and wildtype G9a-GLP or G9aANK8 GS -GLP. E. Western blot of H3K9me1 and me2 production over time from H3K9me0 nucleosomes with wildtype G9a-GLP or G9aANK8 GS -GLP as in .

Article Snippet: Incorporation of histone modifications was verified by western blotting and LC-MS. Commercial mononucleosomes containing H3K9me1 (SKU 16-0325) and H3K9me2 (SKU 16-0324-20) were purchased from EpiCypher and used directly.

Techniques: Mass Spectrometry, Electrophoresis, Western Blot

Journal: bioRxiv

Article Title: Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

doi: 10.64898/2026.01.21.700667

Figure Lengend Snippet: A. Side-view of cryo-EM density (map 709) of intermediate (H3K9Nle/H3K9me2) nucleosome bound by G9a ANK domain, nucleosome is colored in gray and G9a in orange. B. Detail of A. showing the H2A N-terminus:G9a ANK interaction. C. Side-view of cryo-EM density (map 808) of intermediate (H3K9Nle/H3K9me2) nucleosome bound by G9a ANK domain, colors as in A. D. Detail of B. showing the H4 N-terminus:G9a ANK7/8 interaction. The ANK8 unique sequence in G9a is colored in light blue. E. model combining all electron density maps of G9a and the ANK intermediate (H3K9Nle/H3K9me2) nucleosome. The ANK8 unique sequence in G9a is colored in light blue, and the ANK aromatic cage in green. F. AlphaLink2 model of the ANK-SET G9a-GLP heterodimer. Crosslinks are indicated with a unique color per crosslink. Inset: A rotated view of the G9a and GLP ANK domains. G. Structural model of G9a/GLP dimer bound intermediate (H3K9Nle/H3K9me2) nucleosome generated by X-MS and cryo-EM models, nucleosome is colored in gray, G9a colored in dark orange and GLP in purple.

Article Snippet: Incorporation of histone modifications was verified by western blotting and LC-MS. Commercial mononucleosomes containing H3K9me1 (SKU 16-0325) and H3K9me2 (SKU 16-0324-20) were purchased from EpiCypher and used directly.

Techniques: Cryo-EM Sample Prep, Sequencing, Generated

Journal: The Journal of Biological Chemistry

Article Title: HMGN1 and HMGN2 are recruited to acetylated and histone variant H2A.Z-containing nucleosomes to regulate chromatin state and transcription

doi: 10.1016/j.jbc.2025.110997

Figure Lengend Snippet: HMGN proteins localize to transcriptionally active regions of the genome . A , genome browser tracks of HMGN1, HMGN2, H3K27ac, H3K4me3, H2A.Z, RAD21, and CTCF ChIP-Seq signal at the promoter of Sox2 and the super-enhancer domain downstream of Sox2 in WT mESCs. B , Pearson’s correlation hierarchical clustering heatmap of genome-wide signal of HMGN1, HMGN2, H3K27ac, H3K4me3, H2A.Z, RAD21, and CTCF ChIP-Seq datasets in WT mESCs. C , bar graph of the number of expressed genes and non-expressed genes in the mouse embryonic stem cell (mESC) genome bound and not bound by HMGN1 and HMGN2. Active genes are defined as genes with a RPKM value ≥22 as defined by the EMBL Expression Atlas. D , UpSet plot of HMGN1 ChIP-Seq peaks in WT mESCs displaying intersection of sets of peaks at H3K27ac, H3K4me3, transcription start sites (TSSs), H2A.Z, RAD21, CTCF, and other sites. E , bar graph of the number of HMGN1 peaks that overlap with H3K4me3, H3K27ac, CTCF, H2A.Z, TSSs, RAD21, and other peaks in WT mESCs. F , average signal plot of HMGN1, HMGN2, H3K27ac, H3K4me3, H2A.Z, RAD21, and CTCF ChIP-Seq signal at a union list of all HMGN1 and HMGN2 peaks (Z-score normalized). G , clustered heatmaps of HMGN1, HMGN2, H3K27ac, H3K4me3, H2A.Z, RAD21, and CTCF ChIP-Seq signal at active enhancers, active promoters, and insulator sites, ordered by HMGN2 signal (Z-score normalized). H , average signal plots of HMGN1, HMGN2, H3K27ac, H3K4me3, H2A.Z, RAD21, and CTCF ChIP-Seq signal in WT mESCs at active enhancers, active promoters, and insulator sites (Z-score normalized). ChIP-Seq, chromatin immunoprecipitation followed by sequencing; HMGN, High Mobility Nucleosome-binding protein; mESC, mouse embryonic stem cell; RPKM, reads per kilobase of transcript per million mapped reads.

Article Snippet: HAT assays were performed at 37 ̊C for 30 min with 500 nM purified p300 (BPS Biosciences; catalog no.: 50071), 300 nM nucleosome substrate (Epicypher 16-0009, 16-1014, or 16-1014), 100 μM acetyl-CoA (Sigma; catalog no.: A2056), and 100 nM, 300 nM, or 900 nM purified GST-HMGN protein (HMGN1, HMGN2, HMGN1ΔC, or HMGN2ΔC) in a reaction volume of 10 μl (in HAT buffer: 50 nM Tris–HCl [pH 8.0], 10% glycerol, 1 mM DTT, 0.1 mM EDTA, 1 mM PMSF, and 10 mM sodium butyrate).

Techniques: ChIP-sequencing, Genome Wide, Expressing, Chromatin Immunoprecipitation, Sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: HMGN1 and HMGN2 are recruited to acetylated and histone variant H2A.Z-containing nucleosomes to regulate chromatin state and transcription

doi: 10.1016/j.jbc.2025.110997

Figure Lengend Snippet: HMGN1 and HMGN2 are required for maintenance of cell identity gene expression programs . A , bar graphs of average fold change (FC) relative to Tbp in transcript levels of Hmgn1 and Hmgn2 in WT mESCs, Hmgn1 −/− mESCs, Hmgn2 −/− mESCs, and Hmgn1 −/− Hmgn2 −/− mESCs. Error bars represent the standard deviation calculated from two biological replicates, each consisting of three technical replicates, with two outliers removed from the dataset. A t test was used to assess statistical significance, with one asterisk (∗) denoting a p value less than 0.05, ∗∗ indicating a p value less than 0.01, and ∗∗∗ representing a p value less than 0.001. B , Western blot analysis of nuclear lysates of HMGN2 protein levels in WT mESCs, Hmgn1 −/− mESCs, Hmgn2 −/− mESCs, and Hmgn1 −/− Hmgn2 −/− mESCs relative to H3 loading control. C , overlap of differentially expressed genes (DEGs) in Hmgn1 −/− mESCs, Hmgn2 −/− mESCs, and Hmgn1 −/− Hmgn2 −/− mESCs relative to WT mESCs. DEGs shared between all three genotypes are highlighted as common. D , clustered heatmap of -log2 FC in expression for a combined list of DEGs in Hmgn1 −/− mESCs, Hmgn2 −/− mESCs, and Hmgn1 −/− Hmgn2 −/− mESCs, all relative to WT mESCs. E , bar graphs of -log2 FC in expression of HMGN genes ( Hmgn1 , Hmgn2 , Hmgn3 , Hmgn4 , and Hmgn5 ), HMGB genes ( Hmgb1 , Hmgb2 , Hmgb3 , and Hmgb4 ), and HMGA genes ( Hmga1 and Hmga2 ) in Hmgn1 −/− mESCs, Hmgn2 −/− mESCs, and Hmgn1 −/− Hmgn2 −/− mESCs relative to WT mESCs. Asterisks indicate significant differences from WT determined using DESeq2 ( p -adjusted < 0.01, L 2 FC ≥|1|). F , bar graphs of -log2 FC in expression of pluripotency genes ( Pou5f1 , Sox2 , and Nanog ), ectodermal lineage genes ( Pax6 and Nestin ), endodermal lineage genes ( Gata6 and Sox17 ), and mesodermal genes ( Kdr and Pdgfra ) in Hmgn1 −/− mESCs, Hmgn2 −/− mESCs, and Hmgn1 −/− Hmgn2 −/− mESCs relative to WT mESCs. Asterisks indicate significant differences from WT determined using DESeq2 ( p -adjusted < 0.01, L 2 FC ≥|1|). G , Gene Ontology (GO) analysis for biological processes correlated with DEGs that are upregulated and downregulated in Hmgn1 −/− Hmgn2 −/− mESCs relative to WT mESCs. HMGN, High Mobility Nucleosome-binding protein; mESC, mouse embryonic stem cell.

Article Snippet: HAT assays were performed at 37 ̊C for 30 min with 500 nM purified p300 (BPS Biosciences; catalog no.: 50071), 300 nM nucleosome substrate (Epicypher 16-0009, 16-1014, or 16-1014), 100 μM acetyl-CoA (Sigma; catalog no.: A2056), and 100 nM, 300 nM, or 900 nM purified GST-HMGN protein (HMGN1, HMGN2, HMGN1ΔC, or HMGN2ΔC) in a reaction volume of 10 μl (in HAT buffer: 50 nM Tris–HCl [pH 8.0], 10% glycerol, 1 mM DTT, 0.1 mM EDTA, 1 mM PMSF, and 10 mM sodium butyrate).

Techniques: Gene Expression, Standard Deviation, Western Blot, Control, Expressing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: HMGN1 and HMGN2 are recruited to acetylated and histone variant H2A.Z-containing nucleosomes to regulate chromatin state and transcription

doi: 10.1016/j.jbc.2025.110997

Figure Lengend Snippet: Cohesin and CTCF localization on chromatin is not dependent on HMGN1 or HMGN2 . A , genome browser tracks of RAD21 and CTCF ChIP-Seq signal near the promoter of Zbp1 (differentially expressed gene in Hmgn1 −/− Hmgn2 −/− mESCs) in WT mESCs and Hmgn1 −/− Hmgn2 −/− mESCs. B , MA plot showing differential enrichment of RAD21 signal between WT mESCs and Hmgn1 −/− Hmgn2 −/− mESCs at conserved binding sites. C , MA plot showing differential enrichment of CTCF signal between WT mESCs and Hmgn1 −/− Hmgn2 −/− mESCs at conserved binding sites. D , average signal plots of RAD21 and CTCF ChIP-Seq signal at a union list of all HMGN1 and HMGN2 peaks in WT mESCs and Hmgn1 −/− Hmgn2 −/− mESCs (Z-score normalized). E , average signal plots of RAD21 and CTCF ChIP-Seq signal in WT mESCs and Hmgn1 −/− Hmgn2 −/− mESCs at CTCF sites, cohesin sites, active enhancers, and transcription start sites (TSSs). F , ChIP-Seq signal of RAD21 and CTCF in WT mESCs and Hmgn1 −/− Hmgn2 −/− mESCs shown at the promoters of upregulated and downregulated differently expressed genes in either Hmgn1 −/− mESCs, Hmgn2 −/− mESCs, or Hmgn1 −/− Hmgn2 −/− mESCs. ChIP-Seq, chromatin immunoprecipitation followed by sequencing; HMGN, High Mobility Nucleosome-binding protein; mESC, mouse embryonic stem cell.

Article Snippet: HAT assays were performed at 37 ̊C for 30 min with 500 nM purified p300 (BPS Biosciences; catalog no.: 50071), 300 nM nucleosome substrate (Epicypher 16-0009, 16-1014, or 16-1014), 100 μM acetyl-CoA (Sigma; catalog no.: A2056), and 100 nM, 300 nM, or 900 nM purified GST-HMGN protein (HMGN1, HMGN2, HMGN1ΔC, or HMGN2ΔC) in a reaction volume of 10 μl (in HAT buffer: 50 nM Tris–HCl [pH 8.0], 10% glycerol, 1 mM DTT, 0.1 mM EDTA, 1 mM PMSF, and 10 mM sodium butyrate).

Techniques: ChIP-sequencing, Binding Assay, Chromatin Immunoprecipitation, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: HMGN1 and HMGN2 are recruited to acetylated and histone variant H2A.Z-containing nucleosomes to regulate chromatin state and transcription

doi: 10.1016/j.jbc.2025.110997

Figure Lengend Snippet: HMGN1 and HMGN2 preferentially bind to nucleosomes containing H2A.Z and acetylated histone tails . A , titration of GST-HMGN1 protein with each nucleosome-bead conjugate, expressed as relative fluorescence units before normalization. B , titration of GST-HMGN2 protein with each nucleosome-bead conjugate, expressed as relative fluorescence units before normalization. One outlier data point was excluded from the H2A.Z variant at the 5 nM protein concentration. C , GST-HMGN1 binding relative to the canonical nucleosome with background subtracted. Background signal captured by negative control bead conjugates (average signal of 50 mM BSA-bead, 100 mM BSA-bead, and 200 mM BSA-bead conjugates) and wells containing 0 mM GST-HMGN1 protein were subtracted from raw values for each nucleosome-bead conjugate at 0.625 nM GST-HMGN1 concentration. A t test was used to assess statistical significance, with one asterisk (∗) denoting a p value less than 0.05, ∗∗ indicating a p value less than 0.01, and ∗∗∗ representing a p value less than 0.001. Error bars represent the standard deviation calculated from three technical replicates. D , GST-HMGN2 binding relative to the canonical nucleosome with background subtracted. Background signal captured by negative control bead conjugates (average signal of 50 mM BSA-bead, 100 mM BSA-bead, and 200 mM BSA-bead conjugates) and wells containing 0 mM GST-HMGN2 protein were subtracted from raw values for each nucleosome-bead conjugate at 0.625 nM GST-HMGN2 concentration. A t test was used to assess statistical significance, with one asterisk (∗) denoting a p value less than 0.05, ∗∗ indicating a p value less than 0.01, and ∗∗∗ representing a p value less than 0.001. Error bars represent the standard deviation calculated from three technical replicates. E , GST-HMGN1ΔC binding relative to the canonical nucleosome with background subtracted. Background signal captured by negative control bead conjugates (average signal of 50 mM BSA-bead, 100 mM BSA-bead, and 200 mM BSA-bead conjugates) and wells containing 0 mM GST-HMGN1ΔC protein were subtracted from raw values for each nucleosome-bead conjugate at 0.625 nM GST-HMGN1ΔC concentration. A t test was used to assess statistical significance, with one asterisk (∗) denoting a p value less than 0.05, ∗∗ indicating a p value less than 0.01, and ∗∗∗ representing a p value less than 0.001. Error bars represent the standard deviation calculated from three technical replicates. F , GST-HMGN2ΔC binding relative to the canonical nucleosome with background subtracted. Background signal captured by negative control bead conjugates (average signal of 50 mM BSA-bead, 100 mM BSA-bead, and 200 mM BSA-bead conjugates) and wells containing 0 mM GST-HMGN2ΔC protein were subtracted from raw values for each nucleosome-bead conjugate at 0.625 nM GST-HMGN2ΔC concentration. A t test was used to assess statistical significance, with one asterisk (∗) denoting a p value less than 0.05, ∗∗ indicating a p value less than 0.01, and ∗∗∗ representing a p value less than 0.001. Error bars represent the standard deviation calculated from three technical replicates. G , bar graph of normalized GST-HMGN1ΔC nucleosome binding data over GST-HMGN1 nucleosome binding data relative to unmodified H3.1 mononucleosome-bead conjugate. H , bar graph of normalized GST-HMGN2ΔC nucleosome binding data over GST-HMGN2 nucleosome binding data relative to unmodified H3.1 mononucleosome-bead conjugate. BSA, bovine serum albumin; GST, glutathione- S -transferase; HMGN, High Mobility Nucleosome-binding protein.

Article Snippet: HAT assays were performed at 37 ̊C for 30 min with 500 nM purified p300 (BPS Biosciences; catalog no.: 50071), 300 nM nucleosome substrate (Epicypher 16-0009, 16-1014, or 16-1014), 100 μM acetyl-CoA (Sigma; catalog no.: A2056), and 100 nM, 300 nM, or 900 nM purified GST-HMGN protein (HMGN1, HMGN2, HMGN1ΔC, or HMGN2ΔC) in a reaction volume of 10 μl (in HAT buffer: 50 nM Tris–HCl [pH 8.0], 10% glycerol, 1 mM DTT, 0.1 mM EDTA, 1 mM PMSF, and 10 mM sodium butyrate).

Techniques: Titration, Fluorescence, Variant Assay, Protein Concentration, Binding Assay, Negative Control, Concentration Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: HMGN1 and HMGN2 are recruited to acetylated and histone variant H2A.Z-containing nucleosomes to regulate chromatin state and transcription

doi: 10.1016/j.jbc.2025.110997

Figure Lengend Snippet: HMGN1 and HMGN2 reduce p300-mediated acetylation of the H3 tail . A , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN1 or GST-HMGN1ΔC protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K18, K23, and K27 was imaged via PTM-specific antibodies. B , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN2 or GST-HMGN2ΔC protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K18, K23, and K27 was imaged via PTM-specific antibodies. C , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) or recombinant H2A.Z-containing mononucleosomes with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN1 protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K27 was imaged via PTM-specific antibodies. D , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) or recombinant H2A.Z-containing mononucleosomes with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN2 protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K27 was imaged via PTM-specific antibodies. E , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) or recombinant H2AE61A mononucleosomes with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN1 protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K27 was imaged via PTM-specific antibodies. F , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) or recombinant H2AE61A mononucleosomes with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN2 protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K27 was imaged via PTM-specific antibodies. GST, glutathione- S -transferase; HAT, histone acetyltransferase; HMGN, High Mobility Nucleosome-binding protein; PTM, post-translational modification.

Article Snippet: HAT assays were performed at 37 ̊C for 30 min with 500 nM purified p300 (BPS Biosciences; catalog no.: 50071), 300 nM nucleosome substrate (Epicypher 16-0009, 16-1014, or 16-1014), 100 μM acetyl-CoA (Sigma; catalog no.: A2056), and 100 nM, 300 nM, or 900 nM purified GST-HMGN protein (HMGN1, HMGN2, HMGN1ΔC, or HMGN2ΔC) in a reaction volume of 10 μl (in HAT buffer: 50 nM Tris–HCl [pH 8.0], 10% glycerol, 1 mM DTT, 0.1 mM EDTA, 1 mM PMSF, and 10 mM sodium butyrate).

Techniques: Western Blot, Recombinant, Sequencing, Incubation, Binding Assay, Modification

Journal: The Journal of Biological Chemistry

Article Title: HMGN1 and HMGN2 are recruited to acetylated and histone variant H2A.Z-containing nucleosomes to regulate chromatin state and transcription

doi: 10.1016/j.jbc.2025.110997

Figure Lengend Snippet: Loss of HMGN1 and HMGN2 increases steady-state H3K27me2/3 . A , stacked bar chart showing the relative abundance of different modification states for histone H3 lysine residues in WT mESCs. Colors indicate modification types: trimethylated ( dark blue ), dimethylated ( medium blue ), monomethylated ( light blue ), acetylated ( purple ), and unmodified ( gray ). B , bar graph showing the relative abundance of unmodified, acetylated, and methylated H3K27 states in WT ( gray ) and Hmgn1 −/− Hmgn2 −/− ( dark orange ) mESCs. Values represent the percentage of total H3.1K27 in each modification state (mean ± SD, n = 3 biological replicates). Loss of HMGN1 and HMGN2 results in a significant decrease in unmodified H3K27, accompanied by an increase in H3K27me2 and H3K27me3 ( p < 0.05, Student’s t test). C , bar graph showing the relative abundance of unmodified, acetylated, and methylated H3K4 states in WT ( gray ) and Hmgn1 −/− Hmgn2 −/− ( dark orange ) mESCs. Values represent the percentage of total H3.1K4 in each modification state (mean ± SD, n = 3 biological replicates). D , bar graph showing the relative abundance of unmodified, acetylated, and methylated H3K9 states in WT ( gray ) and Hmgn1 −/− Hmgn2 −/− ( dark orange ) mESCs. Values represent the percentage of total H3.1K9 in each modification state (mean ± SD, n = 3 biological replicates). Loss of HMGN1 and HMGN2 results in a significant decrease in unmodified H3K9 ( p < 0.05, Student’s t test). E , bar graph showing the relative abundance of unmodified and acetylated H3K14 states in WT ( gray ) and Hmgn1 −/− Hmgn2 −/− ( dark orange ) mESCs. Values represent the percentage of total H3.1K4 in each modification state (mean ± SD, n = 3). F , bar graph showing the relative abundance of unmodified, acetylated, and methylated H3K18 states in WT ( gray ) and Hmgn1 −/− Hmgn2 −/− ( dark orange ) mESCs. Values represent the percentage of total H3.1K18 in each modification state (mean ± SD, n = 3 biological replicates). G , bar graph showing the relative abundance of unmodified, acetylated, and methylated H3K23 states in WT ( gray ) and Hmgn1 −/− Hmgn2 −/− ( dark orange ) mESCs. Values represent the percentage of total H3.1K23 in each modification state (mean ± SD, n = 3 biological replicates). Loss of HMGN1 and HMGN2 results in a significant increase in H3K23me1 ( p < 0.05, Student’s t test). HMGN, High Mobility Nucleosome-binding protein; mESC, mouse embryonic stem cell.

Article Snippet: HAT assays were performed at 37 ̊C for 30 min with 500 nM purified p300 (BPS Biosciences; catalog no.: 50071), 300 nM nucleosome substrate (Epicypher 16-0009, 16-1014, or 16-1014), 100 μM acetyl-CoA (Sigma; catalog no.: A2056), and 100 nM, 300 nM, or 900 nM purified GST-HMGN protein (HMGN1, HMGN2, HMGN1ΔC, or HMGN2ΔC) in a reaction volume of 10 μl (in HAT buffer: 50 nM Tris–HCl [pH 8.0], 10% glycerol, 1 mM DTT, 0.1 mM EDTA, 1 mM PMSF, and 10 mM sodium butyrate).

Techniques: Modification, Methylation, Binding Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: Conversion from H3K9me2 to H3K9me3 is compromised outside nucleation centers in S18 and S24 Swi6 mutants. ( A ) Overview of the ChIP-seq experiments. ( B–D ) ChIP-seq signal visualization plots. The solid ChIP/input line for each genotype represents the mean of three repeats, while the shading represents the 95% confidence interval. ( B ) Plots of H3K9me2 (top) and H3K9me3 (bottom) ChIP signal over input at the MAT ΔREIII HSS mating type locus for wild-type (black), swi6 S18/24A (blue), and Δswi6 (gold). Signal over “green” and “orange” reporters are grayed out. Reads from these reporters map to multiple locations within the reference sequence, as all reporters contain control elements derived from the ura4 and ade6 genes. ( C ) H3K9me2 (top) and H3K9me3 (bottom) plots as in (A) for subtelomere IIR for wildtype and swi6 S18/24A . The red bar on the H3K9me2 plot indicates the distance from tlh2 to where H3K9me2 levels drop in swi6 S18/24A relative to wild-type. Insets: a zoomed-in view proximal to tlh2 is shown for H3K9me2 and me3. The red arrows in the insets indicate the point of separation of the 95% confidence intervals, which is significantly further telomere-proximal for H3K9me3. ( D ) H3K9me2 (top) and H3K9me3 (bottom) plots as in (A) for centromere II for wild-type and swi6 S18/24A . Insets: the left side of the pericentromere.

Article Snippet: H3K9me2 nucleosomes were purchased from Epicypher (#16-0324), and pSwi6 and unpSwi6 were purified as above.

Techniques: ChIP-sequencing, Sequencing, Control, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: Swi6 phosphorylation mitigates inhibition of the Clr4-mediated conversion of H3K9me2 to H3K9me3. ( A ) Most Swi6 molecules in the cell are phosphorylated at S18 and S24. Quantitative western blots against total Swi6 and phosphorylated Swi6 at S18 and/or S24. A standard curve of pSwi6 isolated as in Fig. is included in both blots. Total protein lysates from wild-type swi6 and swi6 S18/24A strains were probed with a polyclonal anti-Swi6 antibody (α-Swi6) or an antibody raised against a phosphorylated S18/S24 peptide (α-pS18-pS24). α-Tubulin was used as a loading control. One of two independent experiments is shown. L; ladder. Total fraction of Swi6 phosphorylated in vivo at S18 and/or S24 is adjusted by the prevalence of phosphorylation in the in vitro produced standard (∼0.76, see ). ( B ) Experimental scheme to probe the impact of Swi6 on H3K9 trimethylation. ( C ) Quantitative western blots on the time-dependent formation of H3K9me3 from H3K9me2 mononucleosomes in the presence of pSwi6 or unpSwi6 under single turnover conditions. The same blots were probed with α-H3K9me3 and α-H4 antibodies as a loading and normalization control. ( D ) Single exponential fits of production of H3K9me3 tails over time for indicated concentrations of unpSwi6 or pSwi6. ( E ) Plot of the observed single turnover rate constant measured from exponential fits ( k obs ) against the Swi6 concentration in µM.

Article Snippet: H3K9me2 nucleosomes were purchased from Epicypher (#16-0324), and pSwi6 and unpSwi6 were purified as above.

Techniques: Phospho-proteomics, Inhibition, Western Blot, Isolation, Control, In Vivo, In Vitro, Produced, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: Swi6 phosphorylation focuses Swi6 onto heterochromatin nucleation sites and away from euchromatin. ( A ) Schematic of Swi6 ChIP-seq experiments. The anti-pS18-pS24 experiment was carried out with wild-type swi6 (and swi6 S18/24A as a negative control, see ). For the anti-FLAG experiments, the endogenous swi6 locus was 3XFLAG tagged in the context of wild-type swi6 (black), swi6 S18/24A (blue), or swi6 S18/24/117–220A ( 6S/A , red). For quantitative normalization, ChIP reactions were supplemented with Drosophila chromatin spike-in. ( B ) Heat maps of spike-in normalized FLAG ChIP-seq signal (in counts per million, CPM) for swi6, swi6 S18/24A , or swi6 6S/A at features previously classified as nucleators . ( C ) As in (B) but for features classified as regions of H3K9me2 spreading . ( D ) FLAG ChIP-seq signal visualization plot for the MAT ΔREIII HSS mating type locus. The solid ChIP/input line for each genotype represents the mean of three repeats, while the shading represents the 95% confidence interval. ( E ) As in (D) but for cen II with zoom-in of the left and right pericentromeric region. The brown dashed boxes indicate siRNAi-generating centers as mapped in . ( F ) Heat map as in (B) and (C), but for H3K9me2 negative regions. ( G ) ChIP-seq signal visualization plots for H3K9me2 negative regions. The solid ChIP/input line for each genotype represents the mean of two repeats, while the shading represents the 95% confidence interval. A high signal replicate was removed for both genotypes. ChIP-seq signal visualization for all three genotypes with all three repeats in .

Article Snippet: H3K9me2 nucleosomes were purchased from Epicypher (#16-0324), and pSwi6 and unpSwi6 were purified as above.

Techniques: Phospho-proteomics, ChIP-sequencing, Negative Control

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: S18 and/or S24 phosphorylation contributes to pSwi6’s biochemical behaviors. ( A ) Schematic of phosphorylated Swi6 molecules used in this figure. ( B ) pSwi6 S18/24A is defective in oligomerization. pSwi6 or pSwi6 S18/24A was crosslinked or not (-) at indicated concentrations, separated on SDS–PAGE, and probed with a polyclonal anti-Swi6 antibody. M, monomer; D, dimer; T, tetramer; O, octamer; O + , higher molecular weight species. Below: Quantification of oligomer signal divided by dimer signal for crosslinked species. ( C ) FP with H3K9me0 (open circles) or H3K c 9me3 (MLA, filled circles) mononucleosomes as in Fig. , with pSwi6 (green) and pSwi6 S18/24A (magenta). Error bars represent the standard deviation of three repeats. Relative dissociation constant ( K d ) values in . ( D ) Quantitative western blots on the time-dependent formation of H3K9me3 from H3K9me2 mononucleosomes in the presence of pSwi6 or pSwi6 S18/24A , as in Fig. . Quantification of the signal below. Note that the reactions were not fast enough in this experiment to derive a single exponential observed rate. ( E ) Model of the impact of pSwi6 on Clr4 activity. Left: pSwi6 does not engage with H3K9me0 nucleosomes, clearing the substrate for Clr4, and has reduced interactions with the nucleosome core. The pS18/pS24 NTE releases the ARK loop (light blue), allowing CD–CD contacts. Right: unpSwi6 binds H3K9me3 and me0 nucleosomes, occluding Clr4 access. The S18/S24 unphosphorylated NTE blocks the ARK from engaging CDs and additionally may contribute to increased nucleosome affinity by contacting DNA or octamer.

Article Snippet: H3K9me2 nucleosomes were purchased from Epicypher (#16-0324), and pSwi6 and unpSwi6 were purified as above.

Techniques: Phospho-proteomics, SDS Page, Molecular Weight, Standard Deviation, Western Blot, Activity Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: Swi6 phosphorylation decreases nucleosome affinity without affecting specificity. ( A ) Overview of FP experiments with fluorescein (star)-labeled H3 tail peptides [ – ] and nucleosomes to assess pSwi6 and unpSwi6 substrate affinity and specificity. ( B ) FP of H3K9me0 (open circles) and H3K9me3 (filled circles) tail peptides with pSwi6 (green) or unpSwi6 (black). The binding affinity was too low to be fit for unpSwi6 and H3K9me0 peptides. ( C ) FP with H3K9me0 (open circles) or H3K c 9me3 (MLA, filled circles) mononucleosomes. Fluorescein (green star) is attached by a flexible linker at one end of the 147 bp DNA template. For (B and C), the average of three independent fluorescent polarization experiments for each substrate is shown. Error bars represent standard deviation. ( D ) Summary table of affinities and specificities for (B and C). ( E ) Representative maximum projection live microscopy images of indicated Swi6-GFP/ Sad1-mKO2 strains. ( F ) Analysis of signal intensity in Swi6-GFP foci in indicated strains. Wt Swi6, n = 242; Swi6 S18/24A , n = 251; Swi6 S18/24/117–220A (6S/A), n = 145; Swi6 S46/52/117–220A , n = 192; n , number of foci analyzed.

Article Snippet: Signals were quantified on a Li-Cor imager by using a dilution of H3K9me3 nucleosomes (Epicypher, #16-0315), establishing standard curves for H4 and H3K9me3.

Techniques: Phospho-proteomics, Labeling, Binding Assay, Standard Deviation, Microscopy

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: Conversion from H3K9me2 to H3K9me3 is compromised outside nucleation centers in S18 and S24 Swi6 mutants. ( A ) Overview of the ChIP-seq experiments. ( B–D ) ChIP-seq signal visualization plots. The solid ChIP/input line for each genotype represents the mean of three repeats, while the shading represents the 95% confidence interval. ( B ) Plots of H3K9me2 (top) and H3K9me3 (bottom) ChIP signal over input at the MAT ΔREIII HSS mating type locus for wild-type (black), swi6 S18/24A (blue), and Δswi6 (gold). Signal over “green” and “orange” reporters are grayed out. Reads from these reporters map to multiple locations within the reference sequence, as all reporters contain control elements derived from the ura4 and ade6 genes. ( C ) H3K9me2 (top) and H3K9me3 (bottom) plots as in (A) for subtelomere IIR for wildtype and swi6 S18/24A . The red bar on the H3K9me2 plot indicates the distance from tlh2 to where H3K9me2 levels drop in swi6 S18/24A relative to wild-type. Insets: a zoomed-in view proximal to tlh2 is shown for H3K9me2 and me3. The red arrows in the insets indicate the point of separation of the 95% confidence intervals, which is significantly further telomere-proximal for H3K9me3. ( D ) H3K9me2 (top) and H3K9me3 (bottom) plots as in (A) for centromere II for wild-type and swi6 S18/24A . Insets: the left side of the pericentromere.

Article Snippet: Signals were quantified on a Li-Cor imager by using a dilution of H3K9me3 nucleosomes (Epicypher, #16-0315), establishing standard curves for H4 and H3K9me3.

Techniques: ChIP-sequencing, Sequencing, Control, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: Swi6 phosphorylation mitigates inhibition of the Clr4-mediated conversion of H3K9me2 to H3K9me3. ( A ) Most Swi6 molecules in the cell are phosphorylated at S18 and S24. Quantitative western blots against total Swi6 and phosphorylated Swi6 at S18 and/or S24. A standard curve of pSwi6 isolated as in Fig. is included in both blots. Total protein lysates from wild-type swi6 and swi6 S18/24A strains were probed with a polyclonal anti-Swi6 antibody (α-Swi6) or an antibody raised against a phosphorylated S18/S24 peptide (α-pS18-pS24). α-Tubulin was used as a loading control. One of two independent experiments is shown. L; ladder. Total fraction of Swi6 phosphorylated in vivo at S18 and/or S24 is adjusted by the prevalence of phosphorylation in the in vitro produced standard (∼0.76, see ). ( B ) Experimental scheme to probe the impact of Swi6 on H3K9 trimethylation. ( C ) Quantitative western blots on the time-dependent formation of H3K9me3 from H3K9me2 mononucleosomes in the presence of pSwi6 or unpSwi6 under single turnover conditions. The same blots were probed with α-H3K9me3 and α-H4 antibodies as a loading and normalization control. ( D ) Single exponential fits of production of H3K9me3 tails over time for indicated concentrations of unpSwi6 or pSwi6. ( E ) Plot of the observed single turnover rate constant measured from exponential fits ( k obs ) against the Swi6 concentration in µM.

Article Snippet: Signals were quantified on a Li-Cor imager by using a dilution of H3K9me3 nucleosomes (Epicypher, #16-0315), establishing standard curves for H4 and H3K9me3.

Techniques: Phospho-proteomics, Inhibition, Western Blot, Isolation, Control, In Vivo, In Vitro, Produced, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: S18 and/or S24 phosphorylation contributes to pSwi6’s biochemical behaviors. ( A ) Schematic of phosphorylated Swi6 molecules used in this figure. ( B ) pSwi6 S18/24A is defective in oligomerization. pSwi6 or pSwi6 S18/24A was crosslinked or not (-) at indicated concentrations, separated on SDS–PAGE, and probed with a polyclonal anti-Swi6 antibody. M, monomer; D, dimer; T, tetramer; O, octamer; O + , higher molecular weight species. Below: Quantification of oligomer signal divided by dimer signal for crosslinked species. ( C ) FP with H3K9me0 (open circles) or H3K c 9me3 (MLA, filled circles) mononucleosomes as in Fig. , with pSwi6 (green) and pSwi6 S18/24A (magenta). Error bars represent the standard deviation of three repeats. Relative dissociation constant ( K d ) values in . ( D ) Quantitative western blots on the time-dependent formation of H3K9me3 from H3K9me2 mononucleosomes in the presence of pSwi6 or pSwi6 S18/24A , as in Fig. . Quantification of the signal below. Note that the reactions were not fast enough in this experiment to derive a single exponential observed rate. ( E ) Model of the impact of pSwi6 on Clr4 activity. Left: pSwi6 does not engage with H3K9me0 nucleosomes, clearing the substrate for Clr4, and has reduced interactions with the nucleosome core. The pS18/pS24 NTE releases the ARK loop (light blue), allowing CD–CD contacts. Right: unpSwi6 binds H3K9me3 and me0 nucleosomes, occluding Clr4 access. The S18/S24 unphosphorylated NTE blocks the ARK from engaging CDs and additionally may contribute to increased nucleosome affinity by contacting DNA or octamer.

Article Snippet: Signals were quantified on a Li-Cor imager by using a dilution of H3K9me3 nucleosomes (Epicypher, #16-0315), establishing standard curves for H4 and H3K9me3.

Techniques: Phospho-proteomics, SDS Page, Molecular Weight, Standard Deviation, Western Blot, Activity Assay