nucleosome substrate  (EpiCypher)

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: bioRxiv

Article Title: Hydra domain drives SNF2L multimerization and marks ISWI diversification in parasites

doi: 10.1101/2025.09.03.673926

Figure Lengend Snippet: a . Recombinant Tg SNF2L and its hydra domain deletion variant (Δhydra) were purified and analyzed by 4-12% NuPAGE, followed by Coomassie blue staining and anti-His tag Western blotting. b . Nucleosome remodeling assay using restriction enzyme accessibility confirms that both full-length and Δhydra recombinant Tg SNF2L retain catalytic activity. Commercial Hs SNF2h (top), recombinant full-length Tg SNF2L (middle), and truncated Tg SNF2L lacking the Hydra domain (bottom) were incubated with EpiDyne nucleosome remodeling substrates. In this assay, remodeling exposes previously occluded GATC sites, enabling cleavage by the restriction enzyme DpnII. The upper band corresponds to intact nucleosomes; the appearance of the lower band indicates successful remodeling. The first lane serves as a -DpnII control, subsequent lanes represent increasing reaction times and the final lane is - ATP control. c . Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALLS) shows that removing the hydra domain decreases the higher oligomeric forms of Tg SNF2L in the micromolar range. With the loss of the hydra domain, two new forms are detected, corresponding to a Tg SNF2L and Tg SNF2LΔhydra SEC-MALLS (Superose 6 Increase) chromatograms shown as the refractive index curves in blue and orange, respectively. Point measurements of the molecular weight in kDa are displayed as black curves with average masses within the peak regions. d . Mass photometry demonstrates a decrease in tetramer and higher oligomeric forms in the nanomolar range upon hydra domain deletion. The data, shown as normalized counts per molecular weight bin (one representative experiment), compares Tg SNF2L and Tg SNF2LΔhydra in blue and orange, respectively. Monomer, dimer and tetramer peaks are fitted using Gaussian distribution model while higher oligomeric forms are delimited by a dotted line. The relative quantifications of these peaks or windows are shown on the right with the mean and standard deviations shown from duplicate measurements. e . Proposed model: The hydra domain acts as a multimerization module, facilitating Tg SNF2L storage in a functionally primed state. In this model, Tg SNF2L’s multi-oligomeric forms may rapidly release Tg SNF2L and its associated proteins in response to DNA damage or replication fork progression.

Article Snippet: Reaction components were added sequentially in the following order: 10 nM remodeler enzyme (2.5 μL), 20 nM nucleosome substrate (2.5 μL), 10 units DpnII (New England Biolabs, R0543S; 2.5 μL), and 2 mM ATP (2.5 μL).

Techniques: Recombinant, Variant Assay, Purification, Staining, Western Blot, Activity Assay, Incubation, Control, Size-exclusion Chromatography, Multi-Angle Light Scattering, Refractive Index, Molecular Weight

Journal: bioRxiv

Article Title: Hydra domain drives SNF2L multimerization and marks ISWI diversification in parasites

doi: 10.1101/2025.09.03.673926

Figure Lengend Snippet: a . Recombinant Tg SNF2L and its hydra domain deletion variant (Δhydra) were purified and analyzed by 4-12% NuPAGE, followed by Coomassie blue staining and anti-His tag Western blotting. b . Nucleosome remodeling assay using restriction enzyme accessibility confirms that both full-length and Δhydra recombinant Tg SNF2L retain catalytic activity. Commercial Hs SNF2h (top), recombinant full-length Tg SNF2L (middle), and truncated Tg SNF2L lacking the Hydra domain (bottom) were incubated with EpiDyne nucleosome remodeling substrates. In this assay, remodeling exposes previously occluded GATC sites, enabling cleavage by the restriction enzyme DpnII. The upper band corresponds to intact nucleosomes; the appearance of the lower band indicates successful remodeling. The first lane serves as a -DpnII control, subsequent lanes represent increasing reaction times and the final lane is - ATP control. c . Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALLS) shows that removing the hydra domain decreases the higher oligomeric forms of Tg SNF2L in the micromolar range. With the loss of the hydra domain, two new forms are detected, corresponding to a Tg SNF2L and Tg SNF2LΔhydra SEC-MALLS (Superose 6 Increase) chromatograms shown as the refractive index curves in blue and orange, respectively. Point measurements of the molecular weight in kDa are displayed as black curves with average masses within the peak regions. d . Mass photometry demonstrates a decrease in tetramer and higher oligomeric forms in the nanomolar range upon hydra domain deletion. The data, shown as normalized counts per molecular weight bin (one representative experiment), compares Tg SNF2L and Tg SNF2LΔhydra in blue and orange, respectively. Monomer, dimer and tetramer peaks are fitted using Gaussian distribution model while higher oligomeric forms are delimited by a dotted line. The relative quantifications of these peaks or windows are shown on the right with the mean and standard deviations shown from duplicate measurements. e . Proposed model: The hydra domain acts as a multimerization module, facilitating Tg SNF2L storage in a functionally primed state. In this model, Tg SNF2L’s multi-oligomeric forms may rapidly release Tg SNF2L and its associated proteins in response to DNA damage or replication fork progression.

Article Snippet: Nucleosome remodeling reactions were performed using the EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1 (EpiCypher, SKU: 16-4101).

Techniques: Recombinant, Variant Assay, Purification, Staining, Western Blot, Activity Assay, Incubation, Control, Size-exclusion Chromatography, Multi-Angle Light Scattering, Refractive Index, Molecular Weight

Journal: bioRxiv

Article Title: Catalytic pocket of Clr4 (Suv39h) methyltransferase serves as a substrate receptor for Cullin 4-dependent histone H3 ubiquitination

doi: 10.1101/2025.08.28.672867

Figure Lengend Snippet: (A) Strategy for expression and purification of the CLRC E3 complex. The S. pombe CLRC complex consists of Cullin 4 (Cul4), the central E3 ligase, Rik1 (DDB1 homolog), Raf1 (DDB2 homolog, Raf2, and the histone methyltransferase Clr4. (B) Purification of the five-subunit CLRC complex. SDS-PAGE showing the Ni-NTA purified CLRC complex lacking Clr4 (CLRC -Clr4 )(left), bacterially expressed and purified Clr4 (middle), and isolation of holo CLRC by size-exclusion chromatography (SEC) on a Superose 6 Increase 3.2/300 column (right). Elution fractions between 1.45-1.55 mL contained all five subunits. *, MBP-Raf1 degradation products. (C) Comparison of AlphaFold-predicted structure of CLRC -Raf2 with the crystal structure of mammalian CRL4 complex. Crystal structure of the mammalian CRL4 complex (PDB 4A0K) (left); AlphaFold3-predicted structure of the S. pombe CLRC -Raf2 complex (right). In a pairwise search for interactions, AF3 predicted an interaction between the N-terminal region of Clr4 and Raf1. Protein components are color-coded based on sequence conservation and homology. (D) Predicted Aligned Error (PAE) Plot of the AlphaFold3 model of the CLRC complex. PAE plot of the AlphaFold3 model showing predicted alignment confidence between residue pairs, where blue indicates low expected positional error (high confidence), white indicates high expected error (low confidence), and red denotes no predicted interaction between regions. (E) Identification of Clr4 domains required for incorporation into CLRC. MBP pulldown assays of CLRC subunits with Clr4 full length (FL) or truncation mutants (light blue arrows). MBP-Raf1 was immobilized on amylose beads and incubated with the indicated Clr4 fragments. After washing and elution, bound proteins were analyzed by SDSPAGE to identify Clr4 domains that are necessary for its binding to MBP-Raf1. *, MBP-Raf1 degradation products. (F) Specific ubiquitination of H3K14 by CLRC requires Clr4. SDS-PAGE and Western blot analysis of in vitro ubiquitination assays using nucleosome substrates with the indicated histone mutations. Full ubiquitination reactions contained CLRC, E1 (UBE1), E2 (UbcH5c/UBE2D3), ubiquitin (Ub), and ATP. No H3 ubiquitination was observed in the absence of CLRC (lane 1) or without Clr4 (lanes 2-3). Mono-ubiquitination (~25 kDa band) was detected with WT (lane 4) and H3K9M (lane 6) nucleosomes, but not with H3K14R (lanes 3, 5, 7) or H3K9me3 nucleosomes (lane 8). HO, histone octamer.

Article Snippet: Where indicated, methylated (H3K9me3) and/or ubiquitinated (H3K14ub) nucleosomes used as substrates were purchased from EpiCypher (SKU: 16-0315 and 16-0398, respectively) 28– 30,32,33 .

Techniques: Expressing, Purification, SDS Page, Isolation, Size-exclusion Chromatography, Comparison, Sequencing, Residue, Incubation, Binding Assay, Ubiquitin Proteomics, Western Blot, In Vitro

Journal: bioRxiv

Article Title: Catalytic pocket of Clr4 (Suv39h) methyltransferase serves as a substrate receptor for Cullin 4-dependent histone H3 ubiquitination

doi: 10.1101/2025.08.28.672867

Figure Lengend Snippet: (A) H3K14ub activates Clr4 for intranucleosomal methylation independently of its automethylation. Methyltransferase assays with wild-type (WT) and an automethylation-deficient mutant (K455,472R) Clr4 proteins were performed using unmodified and/or H3K14ub-modified nucleosomes. The automethylation mutant showed reduced activity on an unmodified nucleosome (lane 3), but both enzymes displayed similar activity on H3K14ub substrates (lanes 4–7). H3K14ub markedly enhanced Clr4 activity compared to unmodified substrates, despite longer exposure for lanes 2-3. No methylation of unmodified H3 was observed in reactions containing both unmodified and H3K14ub nucleosomes (lanes 4, 6), indicating that under these reaction conditions H3K14ub stimulates intranucleosomal cis H3K9 methylation. HO, histone octamer. (B) Clr4 in vitro methyltransferase assay using radioactively labeled [ H]-SAM. Methyltransferase assays using Clr4 WT and nucleosomes modified with H3K9me3 or double-modified with H3K9me3 and H3K14ub. No H3 methylation signal was observed with H3K9me3 or doubly modified H3K9me3K14ub nucleosomes (lanes 3 and 5), but Clr4 was automethylated in the doubly modified nucleosome (lane 5). HO, histone octamer. (C) H3K14ub promotes Clr4-mediated methylation of K9 on an unmodified H3 tail in an automethylationdependent manner. In vitro methylation and ubiquitination assays were reconstituted using Clr4 WT and Clr4 K455,472R proteins. Both WT and K455,472R Clr4 proteins ubiquitinated H3 (~25 kDa band detected by Coomassie staining, middle panel, lanes 5 and 6). Both WT Clr4 and Clr4 K455, 472R efficiently methylated a ubiquitinated form of H3, but only WT Clr4 methylated unmodified H3 (autoradiography, lower panel, lanes 5 and 6). HO, histone octamer. (D) Schematic summary based on the results in panels A-C.

Article Snippet: Where indicated, methylated (H3K9me3) and/or ubiquitinated (H3K14ub) nucleosomes used as substrates were purchased from EpiCypher (SKU: 16-0315 and 16-0398, respectively) 28– 30,32,33 .

Techniques: Methylation, Mutagenesis, Modification, Activity Assay, In Vitro, Labeling, Ubiquitin Proteomics, Staining, Autoradiography

Journal: medRxiv

Article Title: Androgens mediate sexual dimorphism in Pilarowski-Bjornsson Syndrome

doi: 10.1101/2025.05.06.25326635

Figure Lengend Snippet: (A) A schematic representation of the CHD1 protein. Location of variants shown as stars (LOF variants) and circles (missense variants). Male variants are above and female variants below the baseline. Color coding of missense variants represents likelihood of the variant leading to loss of function according to AlphaMissense. ChEx: Chd1 Exit-side binding domain, DBD: DNA-binding domain, CHCT: CHD C-terminal domain. (B) The distribution of missense and LOF variants in both sexes. (C) Key phenotypic aspects of individuals carrying missense variants predicted to cause loss of protein function and individuals carrying loss of function variants. (D) Phenotypic scores for females and males with curated missense and LOF variants (male, n = 24, female, n = 12). (E) Expected interactions for human CHD1 R618, based on a yeast Chd1-nucleosome structure , which would be potentially disrupted by the missense variant p.R618Q. (F) A schematic of the in vitro nucleosome remodeling assay. (G) A representative image from three biological replicates of the chromatin remodeling assay. The CHD1-WT and CHD1-R618Q proteins are denoted by “+” and “-” symbols. The upper band ∼220bp represents the uncut nucleosome-wrapped DNA substrate. The lower band at ∼180bp represents remodeled DpnII-digested DNA-nucleosome substrate. The negative control comprised the DNA alone, with no DpnII restriction site, and the positive control comprised DNA alone with the DpnII restriction site. DpnII was added to all conditions. (H) An overview of the CHD1 protein based on a yeast Chd1-nucleosome structure , with highlighted residues harboring missense variants. * p < 0.05, Welch’s two-tailed t-test, ns = not significant.

Article Snippet: Reactions were prepared by combining the previously synthesized human CHD1 proteins with N-terminal deletions (either WT or R618Q) at a concentration of 20 nM with 40 nM Epidyne Nucleosome Remodeling Assay Substrate (Epicypher, 16-4101) and 25 units of DpnII enzyme (New England Biolabs) in assay buffer (20 mM Tris pH 7.5, 50 mM KCl, 3 mM MgCl 2 , 0.1 mg/mL bovine serum albumin) to a final volume of 20 µL.

Techniques: Variant Assay, Binding Assay, In Vitro, Negative Control, Positive Control, Two Tailed Test