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: Recombinant nucleosomes containing monoubiquitinated histone H3K14 (H3K14ub) were purchased from EpiCypher (SKU: 16-0398).

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) Clr4 SET domain is required for H3K14 ubiquitination. In vitro ubiquitination assays with full-length (FL) and the indicated Clr4 fragments (light blue arrows). Only Clr4 fragments retaining both the hinge region and the SET domain allowed H3K14 ubiquitination (lanes 3 and 5). HO, histone octamer. (B) Effect of Clr4 mutations and H3K9 methylation on H3K14 ubiquitination. Comparison of WT Clr4 with mutants defective in H3K9me3 binding (W31G, chromodomain) or catalytically dead (Y451N, SET domain). The Y451N mutation markedly impaired H3K14 ubiquitination (lane 9), while W31G caused only a moderate reduction in H3K14 ubiquitination (lane 6). H3K9me3 inhibited H3K14 ubiquitination by both WT and W31G Clr4 proteins. HO, histone octamer. (C) Clr4N-Set2 fusion fails to promote H3K14ub. Ubiquitination assays using WT Clr4 or a chimera consisting of the Clr4 chromodomainhinge fused to the SET domain of S. pombe Set2. WT Clr4 promoted H3K14 ubiquitination (lane 2), whereas the Set2 fusion failed to promote ubiquitination (lane 3), suggesting s specific role for the Clr4 SET domain as a substrate receptor for H3K14 ubiquitination. HO, histone octamer.

Article Snippet: Recombinant nucleosomes containing monoubiquitinated histone H3K14 (H3K14ub) were purchased from EpiCypher (SKU: 16-0398).

Techniques: Ubiquitin Proteomics, In Vitro, Methylation, Comparison, Binding Assay, Mutagenesis

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: Recombinant nucleosomes containing monoubiquitinated histone H3K14 (H3K14ub) were purchased from EpiCypher (SKU: 16-0398).

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

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) The chromodomain promotes Clr4 binding to H3K14ub nucleosomes. Size-exclusion chromatography (SEC) analysis of complex formation between the indicated full-length (FL) Clr4 or its subfragments and H3K14ub-modified nucleosomes. Full-length Clr4 and a hinge domain-deleted variant (Clr4Δhinge) co-migrated with H3K14ub nucleosomes, suggesting the formation of stable complexes. By contrast, the Clr4-SET, which lacks both the chromodomain and hinge region (ΔCDΔhinge), did not co-migrate with H3K14ub nucleosomes, suggesting that the chromodomain is essential for stable nucleosome binding in the context of H3K14 ubiquitination. HO, histone octamer. (B) Cross-linking mass spectrometry reveals a shared interface between ubiquitin, Clr4 chromodomain, and the autoregulatory loop (ARL). Cross-linking mass spectrometry (XL-MS) was performed on the full-length Clr4-H3K14ub nucleosome complex. Cross-linked lysine residue pairs were mapped onto the linear domain architecture of Clr4, histones, and ubiquitin, and visualized as connecting lines. Intermolecular cross-links between the Clr4 chromodomain and ubiquitin (green lines) or Clr4 autoregulatory loop (ARL, red), and between the chromodomain, hinge region, and ubiquitin (red lines) are highlighted. The histone H3 tail (highlighted in violet) displayed numerous cross-links with both Clr4 chromodomain and ARL. For clarity, only crosslinks involving Clr4 are shown; all crosslinks, including those within the nucleosome, are listed in Table S5. (C) Representative 2D class averages show extranucleosomal density on top of the nucleosomal disk (light blue arrows, scale bar 100 Å). An extended set of 2D classes is shown in . Nucleosome highlighted by dark-pink arrows. (D) XL-MS-guided model of Clr4 in complex with two ubiquitin molecules via ubiquitin interactions site 1 (UBS1, blue) and ubiquitin interaction site 2 (UBS2, yellow). The Clr4ubiquitin complex model was generated using HADDOCK, with input from an AlphaFold3 model of pairwise Clr4-ubiquitin interactions (see ), guided by crosslinking mass spectrometry (XL-MS) restraints. The model satisfies 33 crosslinks within a 30 Å threshold, with one mild violation (31-34 Å) in a flexible region and one significant violation (>35 Å). (E) Model of Clr4-H3K14ub nucleosome interaction guided by XL-MS restraints. HADDOCK docking driven by XL-MS distance constraints was used to model the interaction between Clr4 and the H3K14ub nucleosome, using AF3 and XL-MS guided HADDOC model of Clr4 (panel C) and the nucleosome (PDB 1KX5) as input. The resulting complex was refined by molecular dynamics in YASARA. The model suggests that the Clr4 chromodomain (CD, yellow) and autoregulatory loop (ARL, red) interact directly with the nucleosome core and with H3K14 ubiquitinated H3 tail via UBS1 and UBS2; Ub, green, SET domain, blue. (F) CryoEM map of the Clr4-H3K14ub nucleosome complex. The Clr4-H3K14ub nucleosome model was fitted into a low-pass filtered cryo-EM map (purple surface representation, 50% transparency) using COOT (see ). The density observed at the top of the nucleosome likely corresponds to a composite of the Clr4 chromodomain, one ubiquitin moiety (UBS2), and the ARL region (color coding as in panel E). The dynamic nature of these regions likely contributes to the low-resolution features of the map. The second ubiquitin and the unresolved portions of the SET domain (UBS1, docked from PDB 9ISZ), which could not be confidently fitted due to flexibility, are outlined in gray. (G) Mapping of XL-MS crosslinks onto the Clr4-H3K14ub nucleosome model. Of the 86 crosslinks identified in the XL-MS analysis, the majority were satisfied within expected distance thresholds, with distances ≤30 Å highlighted in green and 31-34 Å in pink. Ten crosslinks exceeded 35 Å (dark red). Six of these longer-distance crosslinks involved the flexible N-terminal linker and hinge regions of Clr4, while the remaining four connected flexible histone tails to Clr4. These outliers likely reflect conformational flexibility and dynamic regions not fully represented in the static model. (H) Methyltransferase activity of full length and truncated Clr4 proteins (light blue arrows) with unmodified and H3K14ub nucleosomes. In vitro methyltransferase assays performed using the indicated full-length or truncated Clr4 proteins and reconstituted nucleosomes containing either unmodified H3 (WT, lanes 5-8) or ubiquitinated H3K14 (H3K14ub, lanes 9-12). Deletion of the Nterminal chromodomain (CD) enhanced Clr4 automethylation (lanes 2 and 6) and H3K9 methylation (lane 6), suggesting that the chromodomain inhibits automethylation and H3 substrate methylation. HO, histone octamer.

Article Snippet: Recombinant nucleosomes containing monoubiquitinated histone H3K14 (H3K14ub) were purchased from EpiCypher (SKU: 16-0398).

Techniques: Binding Assay, Size-exclusion Chromatography, Modification, Variant Assay, Ubiquitin Proteomics, Structural Proteomics, Residue, Generated, Mass Spectrometry, Cryo-EM Sample Prep, Activity Assay, In Vitro, Methylation

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) Predicted Aligned Error (PAE) plots for AlphaFold3 (AF3) model of Clr4-ubiquitin. PAE plot show the predicted alignment error between residue pairs for the Clr4ubiquitin complex. Low predicted error (blue) indicates high confidence in residue placement; high error (white/red) reflects flexibility or lack of interaction. (B) Overlay of the AF3-predicted Clr4–ubiquitin complex with the crystal structure of Clr4-SET bound to an H3K14ub peptide. The predicted interface between the Clr4 SET domain (blue) and ubiquitin (green) closely matches the crystal structure (PDB 9ISZ, grey). The corresponding PAE plot is shown in panel A. (C) Per-residue model confidence (pLDDT) for the AF3 prediction in panel B. High-confidence regions are indicated in blue; lower-confidence regions are shown in green to red. (D) AF3-predicted intramolecular interaction between Clr4’s autoregulatory loop (ARL, red) and chromodomain (CD, yellow). (E) pLDDT scores for the predicted structure in panel D. (F, G) Overlay of the predicted Clr4 SET domain structure with the crystal structure of open automethylated Clr4 SET domain. The AF3-predicted structure (blue) aligns closely with the SET domain in its open conformation (PDB 6BP4, grey). pLDDT confidence scores are shown in panel G and the PAE plot in panel A. (H) Predicted Aligned Error (PAE) plots for AlphaFold3 (AF3) models of Clr4-H3 tail. (I, J) Overlay of the AF3-predicted interactions between Clr4 and H3 tails with experimental structures. AF3-predicted binding of the Clr4 CD (yellow) and SET domain (blue) to the H3 N-terminal tail (salmon) is in close agreement with respective crystal structures of CD-H3 (PDB 3G7L, grey) and SET–H3 (PDB 9ISZ, grey) (I). pLDDT confidence scores are shown in panel J and the PAE plot in panel H.

Article Snippet: Recombinant nucleosomes containing monoubiquitinated histone H3K14 (H3K14ub) were purchased from EpiCypher (SKU: 16-0398).

Techniques: Ubiquitin Proteomics, Residue, Binding Assay

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) Clr4 as a dual substrate receptor within the CLRC complex. Left: The catalytic SET domain of Clr4 acts as a substrate receptor for histone H3, enabling Cul4mediated monoubiquitination of H3K14. Middle: The N-terminus of Clr4 interacts directly with Raf1 that functions as a substrate receptor for Clr4 ubiquitination on multiple surface-exposed lysines, predominantly located within the hinge region of Clr4. Right: Cul4-mediated ubiquitination of Clr4 promotes its dissociation from the CLRC complex, a step required for the spreading of H3K9 methylation. (B) Ubiquitin-binding site 1 (UBS1) drives cis methylation. H3K14 monoubiquitination strongly stimulates Clr4 to methylate H3K9 on the same histone tail in cis. This is mediated by UBS1, an interaction surface between ubiquitin and the SET domain that positions H3K9 in the catalytic site. (C) Ubiquitin-binding site 2 (UBS2) and automethylation enable trans methylation. UBS2, identified in this study, involves interactions between H3K14ub and the chromodomain-ARL interface within Clr4. Together with Clr4 automethylation of the ARL, UBS2 licenses Clr4 to methylate H3K9 on an unmodified histone tail in trans, thereby facilitating methylation spreading.

Article Snippet: Recombinant nucleosomes containing monoubiquitinated histone H3K14 (H3K14ub) were purchased from EpiCypher (SKU: 16-0398).

Techniques: Ubiquitin Proteomics, 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: Science (New York, N.Y.)

Article Title: Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

doi: 10.1126/science.aar2781

Figure Lengend Snippet: (A) Cryo-EM density map of the hCENP-N1–286/CENP-A nucleosome complex viewed down theaxis of the DNA supercoil. (B) Schematicof the functional domains of CENP-N known to bind the CENP-A nucleosome (gray) and CENP-L (black) (top panel). The CENP-N construct used for the present structural analysis (hCENP-N1–286) and the regions of the sequence whose structure we report here [N-terminal domain: residues 1 to 81, and central domain: residues 101 to 185; hCENP-N(1–185)] are shown in the middle and bottom panels, respectively. (C) Cryo-EM density mapof the hCENP-N1–286/CENP-A nucleosome complex as viewed from the side, at an orientation 90° to the view shownin (A). This view also depicts the extra density connected to the N-terminal domain that we assign to MBP, shown with lighter shading. (D) Representative regions of the cryo-EM density mapto illustrate map quality (from left to right) for canonical histones H2A, H2B, and H4, centromere-specific H3 variant CENP-A, nucleosomal DNA, and CENP-N.

Article Snippet: We refined this population to obtain a 3D reconstruction at an overall resolution of 3.9 Å for the complex formed between hCENP-N 1–286 and the CENP-A nucleosome ( ; figs. S5 and S6; and table S1, data set 2 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 caption a8 Structure of the human CENP-N/CENP-A nucleosome complex. (A) Cryo-EM density map of the hCENP-N 1–286 /CENP-A nucleosome complex viewed down theaxis of the DNA supercoil. (B) Schematicof the functional domains of CENP-N known to bind the CENP-A nucleosome (gray) and CENP-L (black) (top panel).

Techniques: Cryo-EM Sample Prep, Functional Assay, Construct, Sequencing, Variant Assay

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

Article Title: Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

doi: 10.1126/science.aar2781

Figure Lengend Snippet: (A) Cut-away view of the hCENP-N1–286/CENP-A nucleosome model to highlight interfaces involved in complex formation (see also fig. S11, A and B). For the CENP-N/DNA interface (labeled “1a” and “1b”), nucleosomal DNAis shown as a red ribbon, whereas positively charged residues of CENP-N that are proposed to interact with it are shown as blue spheres. Forthe CENP-N/CENP-A interface (labeled “2”), CENP-A residues (R80, G81, and V82) are marked by the short yellow ribbon, whereas interacting CENP-N residues (E3, T4, and E7) are shown as yellow spheres. (B) View of the CENP-N/DNA interface at different magnifications to highlight details of interactions between the nucleosomal DNA and positively charged residues of CENP-N. (C) Gel mobility shift experiment to examine the effects of CENP-N mutations (indicated atop the gel) on binding to the CENP-A nucleosome. Impaired binding is reflected by increased intensity of the free nucleosome (Nuc) band, concomitant with the disappearance of defined 1:1 and 2:1 bands. “N” indicates the migration position of the free CENP-A nucleosome; “1” and “2” denote the migration positions of CENP-A nucleosomes bound with either one or two molecules of CENP-N, respectively. WT, wild type. (D) Similar analysis to that in (C), carried out with a set of CENP-N mutations involving residues distal from the binding interface. (E) Images of interphase nuclei in Xenopus egg extracts with exogenous MBP-xCENP-N and xCENP-L proteins containing the indicated mutations (with analogous human mutations in parentheses), stained with an antibody forMBP (green) and Hoechst (blue). (F) Centromeric MBP fluorescence intensity normalized as a percentage of that observed for wild-type MBP-xCENP-N. Error bars represent SEM (n > 200 centromeres). A.U., arbitrary units.

Article Snippet: We refined this population to obtain a 3D reconstruction at an overall resolution of 3.9 Å for the complex formed between hCENP-N 1–286 and the CENP-A nucleosome ( ; figs. S5 and S6; and table S1, data set 2 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 caption a8 Structure of the human CENP-N/CENP-A nucleosome complex. (A) Cryo-EM density map of the hCENP-N 1–286 /CENP-A nucleosome complex viewed down theaxis of the DNA supercoil. (B) Schematicof the functional domains of CENP-N known to bind the CENP-A nucleosome (gray) and CENP-L (black) (top panel).

Techniques: Labeling, Mobility Shift, Binding Assay, Migration, Staining, Fluorescence

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

Article Title: Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

doi: 10.1126/science.aar2781

Figure Lengend Snippet: (A and B) Overall (A) and close-up (B) view of the hCENP-N1–286/CENP-A interface formed betweenR80, G81, and V82 on the L1 loop of CENP-A and E3, T4, and E7 on helix 1of CENP-N. (C) Gel mobility shift experiment to examine the effects of CENP-N mutations (indicated atopthe gel) on binding to the CENP-A nucleosome. (D) Images of interphase nuclei in Xenopus egg extracts with exogenous MBP-xCENP-L andxCENP-L proteins containing the indicated mutations of xCENP-N residues E21 and E25 (correspondingto residues E3 and E7 in hCENP-N), stained with an antibody forMBP (green) and Hoechst (blue).(E) Centromeric MBP fluorescence intensity normalized as a percentageof that observed for wild-type MBP-xCENP-N. Error bars representSEM (n > 200 centromeres). (F) Alignment of human and Xenopus laevis sequences corresponding to the L1 loop of CENP-A and helix 1 of CENP-N. Closely interacting segments of the L1 loop of CENP-A and helix 1 of CENP-N are highlighted bythe shaded areas. The asterisks indicate conserved glutamic acid residues(black asterisks) and variability in the hydrophobic residue correspondingto position T4 (red asterisk) of human CENP-N. (G) Images of interphase nuclei in Xenopus egg extracts with exogenous MBP-xCENP-N and xCENP-L proteins containing the indicated mutations of xCENP-N, as in (D).(H) Centromeric MBP fluorescence intensity, determined as in (E). (I) Gel mobility shift experiment to examine the effects of correlated amino acid substitutions between the L1 loop of CENP-A and helix 1 of CENP-N.

Article Snippet: We refined this population to obtain a 3D reconstruction at an overall resolution of 3.9 Å for the complex formed between hCENP-N 1–286 and the CENP-A nucleosome ( ; figs. S5 and S6; and table S1, data set 2 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 caption a8 Structure of the human CENP-N/CENP-A nucleosome complex. (A) Cryo-EM density map of the hCENP-N 1–286 /CENP-A nucleosome complex viewed down theaxis of the DNA supercoil. (B) Schematicof the functional domains of CENP-N known to bind the CENP-A nucleosome (gray) and CENP-L (black) (top panel).

Techniques: Mobility Shift, Binding Assay, Staining, Fluorescence

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

Article Title: Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

doi: 10.1126/science.aar2781

Figure Lengend Snippet: (A) Sequence alignment between humanH3.1 and CENP-A to highlight distinct CENP-A motifs involvedin deposition and recognition of CENP-A at centromeric chromatin (see also fig. S11, C and D). 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. (B) Two different views of the CENP-A nucleosome bound to hCENP-N and a modeled CENP-C motif peptide (5) to highlight potential dual binding of full-length CENP-C and CENP-N proteins on the CENP-A nucleosome. The second CENP-N (shown with lighter shading) is modeled on the basis of the cryo-EM density map obtained in the presence of excess hCENP-N1–286 (fig. S4), whereas the CENP-C motif peptides (human numbering shown for clarity) on each face of the nucleosome are positioned according to the crystal structure of the nucleosome in complex with the rat CENP-C motif (5). (C) Schematic view to highlight recognition and possible enrichment of CENP-A nucleosomes by the CCAN proteins CENP-C, CENP-N, and CENP-L. Other kinetochore proteins and the dimerization of CENP-C have been omitted for clarity.

Article Snippet: We refined this population to obtain a 3D reconstruction at an overall resolution of 3.9 Å for the complex formed between hCENP-N 1–286 and the CENP-A nucleosome ( ; figs. S5 and S6; and table S1, data set 2 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 caption a8 Structure of the human CENP-N/CENP-A nucleosome complex. (A) Cryo-EM density map of the hCENP-N 1–286 /CENP-A nucleosome complex viewed down theaxis of the DNA supercoil. (B) Schematicof the functional domains of CENP-N known to bind the CENP-A nucleosome (gray) and CENP-L (black) (top panel).

Techniques: Sequencing, Binding Assay, Cryo-EM Sample Prep

Journal: The Journal of Biological Chemistry

Article Title: High Mobility Group Nucleosomal Binding Domain 2 (HMGN2) SUMOylation by the SUMO E3 Ligase PIAS1 Decreases the Binding Affinity to Nucleosome Core Particles *

doi: 10.1074/jbc.M114.555425

Figure Lengend Snippet: Assessing SUMOylation of HMGN2 in an E. coli system. A, schematic diagram of modification of the HMGN2 protein and the five predicted SUMOylation sites in the nucleosome binding domain region of HMGN2. Dark circles, phosphorylation sites; dark and open triangles, in vivo and in vitro acetylation sites, respectively; arrows, predicted SUMOylation sites; open circles, nuclear localization sequences. Dot boxes, nucleosome binding domain and chromatin unfolding domain, respectively. B, His-tagged HMGN2 plasmid was transformed into E. coli with or without pT-E1E2S1, and 4 μg of each E. coli lysate was fractionated by 12% SDS-PAGE. The membrane was analyzed with anti-HMGN2 antibody. C, bacterial whole cell lysates, which were from co-transformation with pGEX-4T-HMGN2 and pT-E1E2S1, were loaded on 10% SDS-PAGE and analyzed with Coomassie Blue staining. The membrane was immunoblotted with anti-SUMO1 antibody. pGEX-4T-expressing GST protein was used as a control protein.

Article Snippet: The cells were permeabilized with 1% Triton X-100 and then incubated overnight with a primary antibody pair of different species directed to HMGN2 (mouse IgG2a) and SUMO1 (rabbit polyclonal, Cell signaling).

Techniques: Modification, Binding Assay, Phospho-proteomics, In Vivo, In Vitro, Plasmid Preparation, Transformation Assay, SDS Page, Membrane, Staining, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: High Mobility Group Nucleosomal Binding Domain 2 (HMGN2) SUMOylation by the SUMO E3 Ligase PIAS1 Decreases the Binding Affinity to Nucleosome Core Particles *

doi: 10.1074/jbc.M114.555425

Figure Lengend Snippet: HMGN2 is SUMOylated in HEK293T cells. A, HEK293T cells were co-transfected with pMyc-HMGN2 in the presence of pFLAG-SUMO1 or pFLAG-SUMO2 as indicated. Whole cell lysates were separated and analyzed by immunoblotting (IB) using an anti-Myc antibody after Western blotting. B, HEK293T cells were co-transfected with pMyc-HMGN2 and pFLAG-SUMO1 plasmids, and the whole cell lysates were immunoprecipitated (IP) with FLAG M2 beads. Immunoblotting was performed using anti-Myc and anti-HMGN2 antibodies. C, HEK293T cells were co-transfected with pMyc-HMGN2 in the presence of wild-type SUMO1 (GG) or mutant SUMO1 (GA) plasmid, and immunoblotting analysis were performed using anti-Myc after immunoprecipitation (left panel). pMyc-HMGN2 were co-transfected with pFLAG-SUMO1 (GG) or pFLAG-SUMO1 (GA) in HEK293T cells, and pMyc-HMGN2 were co-transfected with increasing amounts of pFLAG-SUMO1 (GG) as indicated (right panel). Cell lysates were immunoprecipitated with anti-FLAG antibodies and subsequently immunoblotted with their respective antibodies as indicated. The dotted line is the cut line of the same membrane. D, HMGN2 can be deSUMOylated by SENP1. HEK293T cells were co-transfected with 2 μg of a mixture of plasmids of HMGN2 and SUMO1 in the presence of wild-type (WT) SENP1 or mutant (mt) SENP1C603S plasmid. Whole cell lysates were immunoblotted with anti-HMGN2 antibody after immunoprecipitation. E, 293T cells were transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with their individual antibodies as indicated. F, HEK293T cells were co-transfected with plasmids expressing Myc-HMGN2 and FLAG-SUMO1 in the presence of a candidate plasmid of E3 ligase of FLAG-tagged PIAS1, PIAS3, and PIASy. The cells were immunoprecipitated, and SUMO modification of HMGN2 was detected by anti-HMGN2 antibody. G, HEK293T cells were co-transfected with a mixture of Myc-HMGN2 and FLAG-SUMO1 plasmids in the presence of wild-type or mutant type PIAS1 plasmid. HMGN2 SUMOylation was observed. Arrows, SUMOylated HMGN2. In A and C, relative levels of SUMO-HMGN2 are indicated after normalization.

Article Snippet: The cells were permeabilized with 1% Triton X-100 and then incubated overnight with a primary antibody pair of different species directed to HMGN2 (mouse IgG2a) and SUMO1 (rabbit polyclonal, Cell signaling).

Techniques: Transfection, Western Blot, Immunoprecipitation, Mutagenesis, Plasmid Preparation, Membrane, Expressing, Modification

Journal: The Journal of Biological Chemistry

Article Title: High Mobility Group Nucleosomal Binding Domain 2 (HMGN2) SUMOylation by the SUMO E3 Ligase PIAS1 Decreases the Binding Affinity to Nucleosome Core Particles *

doi: 10.1074/jbc.M114.555425

Figure Lengend Snippet: HMGN2 Lys-17 and Lys-35 are involved in SUMOylation. A, amino acid alignment of HMGN2 from different mammalian species. Lysine residues within potential SUMOylation motifs are indicated by asterisks. B, SUMOylation analysis of mutant HMGN2 in E. coli. E. coli were co-transformed with Myc-HMGN2 variant (WT, K10R, K17R, K35R, K58R, K75R, and double mutant of K17R/K35R) plasmid and pT-E1E2S1, and the whole cell lysates were immunoblotted (IB) for SUMOylation. C, HEK293T cells were co-transfected with plasmid coding for different Myc-HMGN2 variants (WT, K10R, K17R, K35R, K58R, and K17R/K35R) and SUMO1 plasmid, and cell lysates were immunoprecipitated (IP) with M2 FLAG beads. The membrane was immunoblotted for SUMOylated HMGN2. D, for the in situ PLA analysis, HEK293T cells were co-transfected with Myc-HMGN2 or Myc-HMGN2K17R/K35R and FLAG-SUMO1 plasmids as indicated. After incubation with anti-Myc and anti-SUMO1 antibodies, PLA probes were added according to the manufacturer's instructions. A positive signal was observed using confocal microscopy. E, HEK293T cells were transfected with wild type Myc-HMGN2 and mutant Myc-HMGN2K17R/K35R plasmids and then treated with 100 μm H2O2 for 1 h. After incubation with anti-Myc and anti-SUMO1 antibodies, in situ PLA was performed. Myc-HMGN2-transfected HEK293T cells were SUMOylated by H2O2 treatment, but mutant Myc-HMGN2K17R/K35R-transfected cells showed no SUMOylation. Bar: 20 μm.

Article Snippet: The cells were permeabilized with 1% Triton X-100 and then incubated overnight with a primary antibody pair of different species directed to HMGN2 (mouse IgG2a) and SUMO1 (rabbit polyclonal, Cell signaling).

Techniques: Mutagenesis, Transformation Assay, Variant Assay, Plasmid Preparation, Transfection, Immunoprecipitation, Membrane, In Situ, Incubation, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: High Mobility Group Nucleosomal Binding Domain 2 (HMGN2) SUMOylation by the SUMO E3 Ligase PIAS1 Decreases the Binding Affinity to Nucleosome Core Particles *

doi: 10.1074/jbc.M114.555425

Figure Lengend Snippet: Assay for endogenous HMGN2 SUMOylation. A, THP1 cells were differentiated with 500 nm PMA followed by treatment with LPS for 1 h. Differentiated THP1 cells were probed with primary antibody pairs of anti-SUMO1 and anti-HMGN2 antibodies. Dual binding was performed by a pair of corresponding proximity probes of secondary antibodies conjugated with complementary oligonucleotides generates spots. B, normal human PBMCs were cultured with 30 nm PMA and 100 units/ml of rIL-2 for 24 h, and in situ PLA was performed. C, recombinant His-HMGN2 protein was incubated with GST-SAE1/SAE2, His-UBC9, and GST-SUMO1 at 37 °C for 1 h. SUMOylated proteins were detected by immunoblotting (IB) with anti-HMGN2 antibody. D, Myc-HMGN2 (wt) or mutant Myc-HMGN2K17R/K35R (mt) over-expressed HEK293T cells were treated with 1 mm H2O2 for 1 h. The cells were immunoprecipitated (IP) with anti-Myc antibody, and SUMO modification of HMGN2 was detected by anti-SUMO1 antibody. A SUMO-conjugated band is indicated by an asterisk (*). Bar, 10 μm.

Article Snippet: The cells were permeabilized with 1% Triton X-100 and then incubated overnight with a primary antibody pair of different species directed to HMGN2 (mouse IgG2a) and SUMO1 (rabbit polyclonal, Cell signaling).

Techniques: Binding Assay, Cell Culture, In Situ, Recombinant, Incubation, Western Blot, Mutagenesis, Immunoprecipitation, Modification

Journal: The Journal of Biological Chemistry

Article Title: High Mobility Group Nucleosomal Binding Domain 2 (HMGN2) SUMOylation by the SUMO E3 Ligase PIAS1 Decreases the Binding Affinity to Nucleosome Core Particles *

doi: 10.1074/jbc.M114.555425

Figure Lengend Snippet: SUMOylation of HMGN2 reduces its binding affinity to NCP. A, E. coli were co-transformed with pRset-HMGN2 and pT-E1E2S1 plasmids, and SUMOylated HMGN2 protein was purified using nickel-nitrilotriacetic acid-agarose beads and loaded in 12% SDS-PAGE for Coomassie Blue staining and Western blotting with anti-HMGN2 antibody. The mono- or di-SUMOylated form of HMGN2 (fraction number around 19–22) was collected and re-purified from non-SUMOylated HMGN2 using CM-Sepharose column chromatography, and then immunoblotted (IB) with anti-SUMO1 antibody. B and C, mobility shift assay of binding affinity of wild-type and SUMOylated HMGN2 to purified NCPs was tested two times at various molar ratios of HMGN2 protein to NCPs. The band intensities (*) of test #1 were measured as a representative, and the ratio of HMGN2 to SUMO1-HMGN2 at each molar ratio to NCPs was drawn. Arrow, HMGN2 and NCP complexes. D and E, mobility shift assay of binding of wild-type and SUMOylated HMGN2 to nuclear deproteined DNA was measured, and the ratio of HMGN2 to SUMO1-HMGN2 at each molar ratio to nuclear DNA binding was drawn. Arrow, HMGN2 and DNA complex.

Article Snippet: The cells were permeabilized with 1% Triton X-100 and then incubated overnight with a primary antibody pair of different species directed to HMGN2 (mouse IgG2a) and SUMO1 (rabbit polyclonal, Cell signaling).

Techniques: Binding Assay, Transformation Assay, Purification, SDS Page, Staining, Western Blot, Column Chromatography, Mobility Shift

Journal: The Journal of Biological Chemistry

Article Title: High Mobility Group Nucleosomal Binding Domain 2 (HMGN2) SUMOylation by the SUMO E3 Ligase PIAS1 Decreases the Binding Affinity to Nucleosome Core Particles *

doi: 10.1074/jbc.M114.555425

Figure Lengend Snippet: SUMO1-conjugated HMGN2 binding to chromatin in living cells. A, plasmids coding for SUMO1-ΔN16-HMGN2-EGFP were constructed, in which the N-terminal 16 amino acids are deleted to mimic SUMOylated HMGN2-EGFP, ΔN16-HMGN2-EGFP, and HMGN2-EGFP, and the mammalian expression was assessed by Western blot analysis. B, confocal images of live HeLa cells expressing the indicated GFP fusion proteins after transfection are shown. C, quantitative FRAP analysis of SUMOylated HMGN2. HeLa cells expressed the indicated EGFP-tagged HMGN2 proteins and the recovery percentage of fluorescence was measured in the area of photobleaching for the protein mobility. The binding property of each protein is indicated on the right.

Article Snippet: The cells were permeabilized with 1% Triton X-100 and then incubated overnight with a primary antibody pair of different species directed to HMGN2 (mouse IgG2a) and SUMO1 (rabbit polyclonal, Cell signaling).

Techniques: Binding Assay, Construct, Expressing, Western Blot, Transfection, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: High Mobility Group Nucleosomal Binding Domain 2 (HMGN2) SUMOylation by the SUMO E3 Ligase PIAS1 Decreases the Binding Affinity to Nucleosome Core Particles *

doi: 10.1074/jbc.M114.555425

Figure Lengend Snippet: SUMOylation of HMGN2 enhances NF-κB-mediated transcriptional activity. A and B, HEK293T cells were co-transfected with vectors encoding a luciferase reporter for the NF-κB, pCMV-β-galactosidase and Myc-HMGN2, FLAG-SUMO, or Myc-SENP1 as indicated (A). NF-κB reporter and pCMV-β-galactosidase were co-transfected into HEK293T cells together with Myc-HMGN2 (wt), mutant Myc-HMGN2K17R/K35R (mt), or FLAG-SUMO plasmids (B). One day after co-transfection, the cell lysates were harvested and assessed for luciferase reporter gene activity. Data are expressed as mean ± S.D. relative fold-increase to the basal activity (NF-κB reporter transfected) from at least three independent experiments. C, HMGN2-EGFP, ΔN16-HMGN2-EGFP, or SUMO1-ΔN16-HMGN2-EGFP was co-transfected into HEK293T cells together with NF-κB reporter and pCMV-β-galactosidase for 24 h. The cells were harvested, and luciferase activity was determined. D, HEK293T cells were transiently transfected with the indicated plasmid. After total RNA was prepared from each sample, induction of IL-6, TNF-α, and IFN-β mRNA was measured by quantitative RT-PCR. Data are presented as mean ± S.D. from three independent experiments.

Article Snippet: The cells were permeabilized with 1% Triton X-100 and then incubated overnight with a primary antibody pair of different species directed to HMGN2 (mouse IgG2a) and SUMO1 (rabbit polyclonal, Cell signaling).

Techniques: Activity Assay, Transfection, Luciferase, Mutagenesis, Cotransfection, Plasmid Preparation, Quantitative RT-PCR

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

Journal: Nucleic Acids Research

Article Title: Post-translational modification of H2B C-terminal helix regulates nucleosome interactions and chromatin signaling

doi: 10.1093/nar/gkaf897

Figure Lengend Snippet: Nucleosome library design. ( A ) Electrostatic surface of nucleosome (PDBID: 1KX5) with percentage of nucleosome disk binding proteins with decreased nucleosome binding upon mutations of labeled patches indicated. ( B ) Nucleosome with location of H2B (pink) with the H2B αC helix highlighted (red). N- and C-terminal ends of the helix and positions of PTMs annotated. ( C ) Three views (top, side, end) of H2B αC helix with residues mutated or modified in the nucleosome library. ( D ) Semisynthesis scheme for preparation of H2B K108ac, K116ac, K120ac, K125ac, and K120ub. EPL = expressed protein ligation, R = CH 2 CH 2 SO 3 H.

Article Snippet: Briefly, 20 μg of biotinylated nucleosomes were immobilized on streptavidin T1 magnetic Dynabeads (MyOne, Thermo Scientific) in BB120 buffer (20 mM HEPES, pH 7.5, 120 mM NaCl, 10% glycerol, 1 mM dithiothreitol (DTT), and 0.1% NP-40).

Techniques: Binding Assay, Labeling, Modification, Ligation

Journal: Nucleic Acids Research

Article Title: Post-translational modification of H2B C-terminal helix regulates nucleosome interactions and chromatin signaling

doi: 10.1093/nar/gkaf897

Figure Lengend Snippet: Nucleosome affinity proteomics screen. ( A ) Schematic of nucleosome affinity proteomics screen. ( B ) PCA plot, left, of triplicate pulldowns for WT and H2B αC helix mutant or modified nucleosomes. Zoomed view of PCA plot region shown in gray, right.

Article Snippet: Briefly, 20 μg of biotinylated nucleosomes were immobilized on streptavidin T1 magnetic Dynabeads (MyOne, Thermo Scientific) in BB120 buffer (20 mM HEPES, pH 7.5, 120 mM NaCl, 10% glycerol, 1 mM dithiothreitol (DTT), and 0.1% NP-40).

Techniques: Mutagenesis, Modification

Journal: Nucleic Acids Research

Article Title: Post-translational modification of H2B C-terminal helix regulates nucleosome interactions and chromatin signaling

doi: 10.1093/nar/gkaf897

Figure Lengend Snippet: Analysis of effects of H2B αC helix mutations and PTMs on nucleosome interactome. ( A ) Volcano plots for nucleosome binding changes of each mutant or modified nucleosome relative to WT controls. Horizontal and vertical lines designate P -value <.01 and 1.4-fold change significance thresholds, respectively. ( B ) Histogram illustrating number of proteins with significantly increased or decreased binding relative to WT nucleosomes. ( C ) Correlation analysis of each mutant or modified nucleosome with each other. Pearson R 2 values are indicated.

Article Snippet: Briefly, 20 μg of biotinylated nucleosomes were immobilized on streptavidin T1 magnetic Dynabeads (MyOne, Thermo Scientific) in BB120 buffer (20 mM HEPES, pH 7.5, 120 mM NaCl, 10% glycerol, 1 mM dithiothreitol (DTT), and 0.1% NP-40).

Techniques: Binding Assay, Mutagenesis, Modification

Journal: Nucleic Acids Research

Article Title: Post-translational modification of H2B C-terminal helix regulates nucleosome interactions and chromatin signaling

doi: 10.1093/nar/gkaf897

Figure Lengend Snippet: Cross-correlation of nucleosome affinity proteomics studies. ( A ) Zoomed view of H2A/H2B structure (from PDBID: 1KX5) showing key positions mutated or modified in datasets compared in panel (B). Approximate binding positions of arginine anchors (Anchor) and type 1 and 2 variant arginines (V1, V2) are indicated. ( B ) Correlation analysis of each mutant or modified nucleosome with published acidic patch mutation dataset . Pearson R 2 values are indicated. E113A’ represents the same mutant in nucleosome acidic patch mutation dataset.

Article Snippet: Briefly, 20 μg of biotinylated nucleosomes were immobilized on streptavidin T1 magnetic Dynabeads (MyOne, Thermo Scientific) in BB120 buffer (20 mM HEPES, pH 7.5, 120 mM NaCl, 10% glycerol, 1 mM dithiothreitol (DTT), and 0.1% NP-40).

Techniques: Modification, Binding Assay, Variant Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Post-translational modification of H2B C-terminal helix regulates nucleosome interactions and chromatin signaling

doi: 10.1093/nar/gkaf897

Figure Lengend Snippet: Clustered heat maps highlight patterns of nucleosome binding. ( A ) Clustered heat map of all proteins with significant binding gains or losses to modified nucleosomes. ( B ) Clustered heat map of all proteins with significant binding gains or losses to nucleosomes containing H2B H109A, E113A, of K120ub illustrating decreased binding for selected acidic patch-dependent proteins. ( C ) Clustered heat map of select proteins binding to H2B K108A, K108ac, S112A, T115A, K120ac, and K125ac nucleosomes. APC/C and SRCAP subunits are shown in orange and purple, respectively.

Article Snippet: Briefly, 20 μg of biotinylated nucleosomes were immobilized on streptavidin T1 magnetic Dynabeads (MyOne, Thermo Scientific) in BB120 buffer (20 mM HEPES, pH 7.5, 120 mM NaCl, 10% glycerol, 1 mM dithiothreitol (DTT), and 0.1% NP-40).

Techniques: Binding Assay, Modification

Journal: Nucleic Acids Research

Article Title: Post-translational modification of H2B C-terminal helix regulates nucleosome interactions and chromatin signaling

doi: 10.1093/nar/gkaf897

Figure Lengend Snippet: Validation of nucleosome affinity screen with RCC1-nucleosome TR-FRET binding assays. ( A ) RCC1–Ran-nucleosome complex structure (PDBID: 8UX1) ( B ) Zoomed view of structure, highlighting interaction of RCC1 and Ran with H2B αC helix. ( C ) Affinity constants ( K d ) from TR-FRET nucleosome-binding experiments with human RCC1 with indicated nucleosomes. ( D ) K d values from TR-FRET nucleosome-binding experiments with human RCC1 and Ran with indicated nucleosomes. Means ± standard error for triplicate measurements are shown in panels (C) and (D) with dashed horizontal line indicating K d for interactions with WT nucleosome.

Article Snippet: Briefly, 20 μg of biotinylated nucleosomes were immobilized on streptavidin T1 magnetic Dynabeads (MyOne, Thermo Scientific) in BB120 buffer (20 mM HEPES, pH 7.5, 120 mM NaCl, 10% glycerol, 1 mM dithiothreitol (DTT), and 0.1% NP-40).

Techniques: Biomarker Discovery, Binding Assay

Journal: Nucleic Acids Research

Article Title: Post-translational modification of H2B C-terminal helix regulates nucleosome interactions and chromatin signaling

doi: 10.1093/nar/gkaf897

Figure Lengend Snippet: Ubiquitylation assays of RING1B–BMI1, BRCA1–BARD1, RNF168 showing different structural requirements and regulation by PTMs. ( A ) Structure of nucleosome (PDBID: 1KX5) indicating H2A lysines that are ubiquitylated by RING1B–BMI1, BRCA1–BARD1, RNF168, left. Position of H2B αC helix is highlighted in red. Primary and secondary structure of H2A with ubiquitylated lysines in boxes, right. ( B ) RING1B–BMI1-E2-nucleosome structure with H2B αC helix shown in red (PDBID: 4R8P). ( C ) BRCA1–BARD1-E2-nucleosome structure with H2B αC helix shown in red (PDBID: 7JZV). ( D ) RNF168-E2-nucleosome structure with H2B αC helix shown in red (PDBID: 8UPF). ( E ) RING1B–BMI1 ubiquitylation assays using mutant or modified nucleosomes. Means ± standard deviations for triplicate measurements are shown for all assays. Statistical significance is denoted as **** P <.0001, *** P <.001, ** P <.01, * P <.05 for all assays. Negative control reactions performed without ubiquitin (-ubq). ( F ) Zoomed view of RING1B–BMI1-E2 interacting with H2B αC helix. ( G ) BRCA1–BARD1 ubiquitylation assays using mutant or modified nucleosomes. ( H ) Zoomed view of BRCA1–BARD1-E2 interacting with H2B αC helix. ( I ) RNF168 ubiquitylation assays using mutant or modified nucleosomes. ( J ) Zoomed view of RNF168-E2 interacting with H2B αC helix.

Article Snippet: Briefly, 20 μg of biotinylated nucleosomes were immobilized on streptavidin T1 magnetic Dynabeads (MyOne, Thermo Scientific) in BB120 buffer (20 mM HEPES, pH 7.5, 120 mM NaCl, 10% glycerol, 1 mM dithiothreitol (DTT), and 0.1% NP-40).

Techniques: Mutagenesis, Modification, Negative Control, Ubiquitin Proteomics

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