Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Schematic of approach to design, express, and characterize a panel of immunologically-silenced mAbs against cfDNA components (dsDNA, mononucleosomes, histones). Priming agent mAbs were generated using VH and VL amino acid sequences of mAbs that bind cfDNA components fused to a murine IgG2a backbone with L234A/L235A/P329G silencing mutations. B) EMSA of mAbs binding to free dsDNA or MNs, and quantification of binding interaction with biolayer interferometry. Free dsDNA binding was determined by the presence of shifted bands in the DNA lane. MN-only binding was determined by shift or disappearance of the MN band in the MN lane and no evidence of a shifted band in the DNA lane. For mAbs that bound free dsDNA or MN, the binding interaction was subsequently quantified with BLI. C) Measurement of binding kinetics (k on and k off ) and avidity (apparent K d ) to free dsDNA (left) and MN (right) of intact mAbs. D) Comparison of K d of dsDNA binders and MN binders to free dsDNA and MN. E) Interaction of MN binders with individual components of mononucleosomes and different dsDNA topologies, including individual histones, H2A/H2B dimer, H3/H4 tetramer, H2A/H2B/H3/H4 octamer, bent dsDNA (supercoiled pUC18), linear dsDNA (linearized pUC18, lin. pUC18), and intact MN (histone octamer + 147bp dsDNA). NB - no binding.

Article Snippet: Widom601 dsDNA, either free (cat. 16-0006, Epicypher) or histone bound in mononucleosomes (18-0005, Epicypher), was mixed to a final concentration of 0.2 ng/μL with each mAb binder to a concentration of 0.4 mg/mL in DPBS.

Techniques: Generated, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Degradation of free dsDNA and dsDNA in mononucleosomes in presence of 0.2U/mL of DNase I and mAb priming agents. IgG2a is an unrelated control antibody. Each dot represents the mean of two replicates. B) Experimental approach for testing impact of priming agents on clearance of free dsDNA and MN. C) Percentage of W601 DNA remaining in plasma 1 hour after injection of W601 either in free dsDNA form (left) or as MN (right), with or without dsDNA binding and MN binding priming agents. D) Percentage of cfDNA isolated from mouse plasma by various priming agents via immunoprecipitation using mAb-coupled magnetic beads, adjusted for background binding to beads alone. E) Percentage of cfDNA, W601 free dsDNA, W601 MN, and W601 MN with mild DNase I treatment isolated from mouse plasma using each MN-only mAb priming agent. F) cfDNA fragment length distribution in mice (n=8) with dashed lines at 167bp and 147bp (left) and percent of fragments <=147 bp and <=167 bp (right). * p < 0.05, ** p < 0.01, *** p < 0.001, ns - not significant.

Article Snippet: Widom601 dsDNA, either free (cat. 16-0006, Epicypher) or histone bound in mononucleosomes (18-0005, Epicypher), was mixed to a final concentration of 0.2 ng/μL with each mAb binder to a concentration of 0.4 mg/mL in DPBS.

Techniques: Control, Clinical Proteomics, Injection, Binding Assay, Isolation, Immunoprecipitation, Magnetic Beads

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: Antibody-based priming agents bind cfDNA in the bloodstream and protect it from clearance, enabling more to be collected in a subsequent blood draw. This study identified the key molecular determinants of priming activity. The optimal target binder was dsDNA, rather than mononucleosomes, and the priming activity was correlated with strength of binding to dsDNA, with the best priming agents having K d dsDNA < 10nM. The best dsDNA-binding priming agents had different magnitudes of DNase protection ability and impact on cfDNA fragmentation. In particular, one agent, DNA1, best preserved the endogenous fragmentation profile in cfDNA and protected short, informative cfDNA fragments at transcription factor binding sites from clearance. The Fc domain was found to be dispensable for the priming effect, suggesting that agents with more rapid clearance can still elicit a priming effect. Finally, we leveraged some of the principles identified in this study to engineer new single chain molecules that can similarly elicit a priming effect in tumor bearing mice, extending the space of priming agents to non-immunoglobulin dsDNA binding domains.

Article Snippet: Widom601 dsDNA, either free (cat. 16-0006, Epicypher) or histone bound in mononucleosomes (18-0005, Epicypher), was mixed to a final concentration of 0.2 ng/μL with each mAb binder to a concentration of 0.4 mg/mL in DPBS.

Techniques: Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Schematic of approach to design, express, and characterize a panel of immunologically-silenced mAbs against cfDNA components (dsDNA, mononucleosomes, histones). Priming agent mAbs were generated using VH and VL amino acid sequences of mAbs that bind cfDNA components fused to a murine IgG2a backbone with L234A/L235A/P329G silencing mutations. B) EMSA of mAbs binding to free dsDNA or MNs, and quantification of binding interaction with biolayer interferometry. Free dsDNA binding was determined by the presence of shifted bands in the DNA lane. MN-only binding was determined by shift or disappearance of the MN band in the MN lane and no evidence of a shifted band in the DNA lane. For mAbs that bound free dsDNA or MN, the binding interaction was subsequently quantified with BLI. C) Measurement of binding kinetics (k on and k off ) and avidity (apparent K d ) to free dsDNA (left) and MN (right) of intact mAbs. D) Comparison of K d of dsDNA binders and MN binders to free dsDNA and MN. E) Interaction of MN binders with individual components of mononucleosomes and different dsDNA topologies, including individual histones, H2A/H2B dimer, H3/H4 tetramer, H2A/H2B/H3/H4 octamer, bent dsDNA (supercoiled pUC18), linear dsDNA (linearized pUC18, lin. pUC18), and intact MN (histone octamer + 147bp dsDNA). NB - no binding.

Article Snippet: Streptavidin biosensor tips (cat. 18-5019, Sartorius) were used to immobilize mononucleosomes (16-0006, Epicypher), free dsDNA (18-0005, Epicypher) with the Widom601 sequence and a biotin tag, or biotinylated supercoiled or linearized pUC18 plasmid DNA, diluted to a final concentration of 8nM in kinetics buffer (5mg/ml BSA, 0.05% Tween-20 in PBS).

Techniques: Generated, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Degradation of free dsDNA and dsDNA in mononucleosomes in presence of 0.2U/mL of DNase I and mAb priming agents. IgG2a is an unrelated control antibody. Each dot represents the mean of two replicates. B) Experimental approach for testing impact of priming agents on clearance of free dsDNA and MN. C) Percentage of W601 DNA remaining in plasma 1 hour after injection of W601 either in free dsDNA form (left) or as MN (right), with or without dsDNA binding and MN binding priming agents. D) Percentage of cfDNA isolated from mouse plasma by various priming agents via immunoprecipitation using mAb-coupled magnetic beads, adjusted for background binding to beads alone. E) Percentage of cfDNA, W601 free dsDNA, W601 MN, and W601 MN with mild DNase I treatment isolated from mouse plasma using each MN-only mAb priming agent. F) cfDNA fragment length distribution in mice (n=8) with dashed lines at 167bp and 147bp (left) and percent of fragments <=147 bp and <=167 bp (right). * p < 0.05, ** p < 0.01, *** p < 0.001, ns - not significant.

Article Snippet: Streptavidin biosensor tips (cat. 18-5019, Sartorius) were used to immobilize mononucleosomes (16-0006, Epicypher), free dsDNA (18-0005, Epicypher) with the Widom601 sequence and a biotin tag, or biotinylated supercoiled or linearized pUC18 plasmid DNA, diluted to a final concentration of 8nM in kinetics buffer (5mg/ml BSA, 0.05% Tween-20 in PBS).

Techniques: Control, Clinical Proteomics, Injection, Binding Assay, Isolation, Immunoprecipitation, Magnetic Beads

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: A) Design of priming agents using the 7 kDa dsDNA binding protein sso7d. Mono-valent, bi-valent, and tetra-valent constructs were designed using flexible short (G4S) 3 and long (G4S) 5 linkers fused to murine IgG2a CH2-CH3 domain carrying the LALAPG silencing domain, with addition on knob-in-hole mutations to promote heterodimerization for the mono-sso7d construct. B) Interferometry recordings of the interaction of sso7d priming agents with free dsDNA (“dsDNA binding”) and MN (“MN binding”). For binding to dsDNA, the range of priming agent concentrations tested was 1.56-50nM, whereas for binding to MN, the range of concentrations tested was 12.5-400nM in order to capture weaker interactions. C) Estimated avidity (K d ) for binding to dsDNA and MN for sso7d priming agents compared to aST3. D) Experimental approach for testing the effect of sso7d priming agents on cfDNA and ctDNA recovery. E) Fold-change in plasma cfDNA concentration after administration of each priming agent, based on qPCR quantification. F) Fold-change in number of ctDNA molecules detected in plasma after administration of priming agents. G) Priming effect versus K d to dsDNA and MN for sso7d priming agents. * p < 0.05, ** p < 0.01, *** p < 0.001, ns - not significant. Points represent mean and intervals represent 95% confidence intervals in G. Box plots represent median and interquartile range.

Article Snippet: Streptavidin biosensor tips (cat. 18-5019, Sartorius) were used to immobilize mononucleosomes (16-0006, Epicypher), free dsDNA (18-0005, Epicypher) with the Widom601 sequence and a biotin tag, or biotinylated supercoiled or linearized pUC18 plasmid DNA, diluted to a final concentration of 8nM in kinetics buffer (5mg/ml BSA, 0.05% Tween-20 in PBS).

Techniques: Binding Assay, Construct, Clinical Proteomics, Concentration Assay

Journal: bioRxiv

Article Title: Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

doi: 10.64898/2026.01.27.701975

Figure Lengend Snippet: Antibody-based priming agents bind cfDNA in the bloodstream and protect it from clearance, enabling more to be collected in a subsequent blood draw. This study identified the key molecular determinants of priming activity. The optimal target binder was dsDNA, rather than mononucleosomes, and the priming activity was correlated with strength of binding to dsDNA, with the best priming agents having K d dsDNA < 10nM. The best dsDNA-binding priming agents had different magnitudes of DNase protection ability and impact on cfDNA fragmentation. In particular, one agent, DNA1, best preserved the endogenous fragmentation profile in cfDNA and protected short, informative cfDNA fragments at transcription factor binding sites from clearance. The Fc domain was found to be dispensable for the priming effect, suggesting that agents with more rapid clearance can still elicit a priming effect. Finally, we leveraged some of the principles identified in this study to engineer new single chain molecules that can similarly elicit a priming effect in tumor bearing mice, extending the space of priming agents to non-immunoglobulin dsDNA binding domains.

Article Snippet: Streptavidin biosensor tips (cat. 18-5019, Sartorius) were used to immobilize mononucleosomes (16-0006, Epicypher), free dsDNA (18-0005, Epicypher) with the Widom601 sequence and a biotin tag, or biotinylated supercoiled or linearized pUC18 plasmid DNA, diluted to a final concentration of 8nM in kinetics buffer (5mg/ml BSA, 0.05% Tween-20 in PBS).

Techniques: Activity Assay, Binding Assay

Journal: bioRxiv

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

doi: 10.64898/2026.01.21.700667

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

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

Techniques: Western Blot, Concentration Assay

Journal: bioRxiv

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

doi: 10.64898/2026.01.21.700667

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

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

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

Journal: bioRxiv

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

doi: 10.64898/2026.01.21.700667

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

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

Techniques: Mass Spectrometry, Electrophoresis, Western Blot

Journal: bioRxiv

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

doi: 10.64898/2026.01.21.700667

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

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

Techniques: Western Blot, Concentration Assay

Journal: bioRxiv

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

doi: 10.64898/2026.01.21.700667

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

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

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

Journal: bioRxiv

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

doi: 10.64898/2026.01.21.700667

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

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

Techniques: Mass Spectrometry, Electrophoresis, Western Blot

Journal: bioRxiv

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

doi: 10.64898/2026.01.21.700667

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

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

Techniques: Cryo-EM Sample Prep, Sequencing, Generated

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2025.110997

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

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

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

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2025.110997

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

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

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

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2025.110997

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

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

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

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2025.110997

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

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

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

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2025.110997

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

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

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

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2025.110997

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

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

Techniques: Modification, Methylation, Binding Assay